The effects of Shenmai injection (SMI) and am inophylline on apoptosis of sm all airway smooth muscle cells (SASMC) and the Fas/ Fas L expression in rats with papain- induced em physe- ma were investigated.Rat emphy...The effects of Shenmai injection (SMI) and am inophylline on apoptosis of sm all airway smooth muscle cells (SASMC) and the Fas/ Fas L expression in rats with papain- induced em physe- ma were investigated.Rat emphysema model was established by a single intratracheal instillation of papain.Apoptosis and Fas/Fas L expression of SASMC were detected by im munohistochemistry SABC and TU NEL assay at day1,3,5 ,7,15 ,30 after modeling,and the effect of SMI and am inophylline on them were observed.The results indicated that the Fas/Fas L expression positive rate in SASMC was2 .31± 0 .5 5 /1.2 8± 0 .4 7respectively.After a single intratracheal instillation of papain,the expression of Fas/Fas L positive rate in the placebo group was increased in a tim e- dependent manner.SMI could inhibit the expression of Fas/ Fas L ,but aminophylline couldn't. The positive rate of apoptosis in the control group was 0 .87± 0 .32 .After a single intratracheal in- stillation of papain,the SASMC apoptosis positive rate in the placebo group was increased in a tim e- dependent manner.The SASMC apoptosis rate in all groups was declined after treatment with SMI,but the effect of am inophylline was notobvious.Itwas dem onstrated thatin the patho- genesis of emphysem a Fas/Fas L played an important role in the regulation of SASMC apoptosis. SMI influenced the expression of Fas/ Fas L and declined SASMC apoptosis by inhibiting the releas- ing of inflamm atory m edia and played an im portant role in the therapy of em physema.展开更多
The effects of protein kinase C (PKC) on the tension and the activity of voltage-dependent delayed rectifier potassium channel (K,,) were examined in normal and passively sensitized human airway smooth muscle (H...The effects of protein kinase C (PKC) on the tension and the activity of voltage-dependent delayed rectifier potassium channel (K,,) were examined in normal and passively sensitized human airway smooth muscle (HASM), by measuring tones and whole-cell patch clamp techniques, and the Kv activities and membrane potential (Em) were also detected. The results showed that phorbol 12-myristate 13-acetate (PMA), a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group (P〈0.05), and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. Kv activities of HASM cells were significantly inhibited by PMA, and the Em became more positive, as compared with the DMSO (a PMA menstruum)-treated group (P〈0.01). This effect could be blocked by Ro31-8220 (P〈0.01 ). It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced Kv activity. In passively sensitized HASM rings, this effect was more notable.展开更多
Objective: To investigate the role of large Ca^2+-activated, delayed-rectifier and ATP-sensitive potassium channel in regulating the relaxation induced by nitric oxide (NO) in normal and passively sensitized human...Objective: To investigate the role of large Ca^2+-activated, delayed-rectifier and ATP-sensitive potassium channel in regulating the relaxation induced by nitric oxide (NO) in normal and passively sensitized human airway smooth muscle (HASM) with serum from asthmatic patients. Methods: The effects of NO or/and potassium channel blockers on the tensions of normal and passively sensitized HASM were measured by using nitric oxide donor and potassium blockers, with the isometric tension recording technique. Results: Showed that(1)In the control group and passively sensitized group, Kv blocker(4-AP) cause concentration-dependent augmentation in the contraction induced by histamine ( 1 ×10^-4 mol/L ), (P 〈 0.05), but Glib ( 1 × 10^-2 mol/L ) and TEA (1×10^-4 mol/L) have no significant effects on the contraction induced by histamine (1×10^-4 mol/L). The maximum tension induced by histamine in passively sensitized group is higher than that in the control group (P 〈 0.05). (2) NO-donor Sodium Nitroprusside (SNP) bring about significant relaxation in normal and passively sensitized HASM rings (P 〈 0.05). Relaxations of passively sensitized airway rings [ (29.4 ± 3.3)% ] were significant less than those of normal HASM rings [ (44.1 ± 10.2)% ], (P 〈 0.05).(3) Glib(1×10^-2 mol/L)have no significant effect on the relaxations induced by SNP(1×10^-4 mol/L). 4-AP(1×10^-2 mol/L) inhibited relaxation induced by SNP (1×10^-4 mol/L), (P 〈 0.01). TEA (1×10^-3 tool/L) inhibited relaxation induced by SNP (1×10^-4 mol/L) (P 〈 0.05), and the inhibiting effect in passively sensitized HASM rings were significant less than in normal HASM, (P 〈 0.05). Conclusion: It was concluded that SNP(NO-donor) relaxed the contraction of HASM partly via BKca channel opening. In passively sensitized HASM in vitro, the relaxation of SNP decreased compared with control group, which might be associated with the down-regulating activity of BKca in passively sensitized HASM.展开更多
Airway remodeling is an important pathological feature of asthma and the basis of severe asthma. Proliferation of airway smooth muscle cells (ASMCs) is a major contributor to airway remod- eling. As an important Ca2...Airway remodeling is an important pathological feature of asthma and the basis of severe asthma. Proliferation of airway smooth muscle cells (ASMCs) is a major contributor to airway remod- eling. As an important Ca2+ channel, transient receptor potential vanilloid 1 (TRPV1) plays the key role in the cell pathological and physiological processes. This study investigated the expression and activity of TRPV1 channel, and further clarified the effect of TRPV1 channel on the ASMCs proliferation and apoptosis in order to provide the scientific basis to treat asthmatic airway remodeling in clinical practice Immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of TRPVI in rat ASMCs. Intracellular Ca2+ was detected using the single cell confocal fluorescence microscopy measurement loaded with Fluo-4/AM. The cell cycles were observed by flow cytometry. MTT assay and Hoechst 33258 staining were used to detect the proliferation and apoptosis of ASMCs in rats respectively. The data showed that: (1) TRPV1 channel was present in rat ASMCs. (2) TRPV1 channel agonist, capsaicin, increased the Ca2~ influx in a concentration-dependent manner (EC50=284.3+58 nmol/L). TRPV1 channel antagonist, capsazepine, inhibited Ca2+ influx in rat ASMCs. (3) Capsaicin significantly increased the percentage of S+G2M ASMCs and the absorbance of MTT assay. Capsazepine had the opposite effect. (4) Capsaicin significantly inhibited the apoptosis, whereas capsazepine had the opposite effect. These results suggest that TRPV1 is present and mediates Ca2+ influx in rat ASMCs. TRPV1 activity stimulates proliferation of ASMCs in rats.展开更多
The effects of ATP-sensitive mitochondrial K + channel(mitoK ATP) on mitochondrial membrane potential(Δψm),cell proliferation and protein kinase C alpha(PKCα) expression in airway smooth muscle cells(ASMCs) were in...The effects of ATP-sensitive mitochondrial K + channel(mitoK ATP) on mitochondrial membrane potential(Δψm),cell proliferation and protein kinase C alpha(PKCα) expression in airway smooth muscle cells(ASMCs) were investigated.Thirty-six Sprague-Dawley(SD) rats were immunized with saline(controls) or ovalbumin(OVA) with alum(asthma models).ASMCs were cultured from the lung of control and asthma rats.ASMCs were treated with diazoxide(the potent activator of mitoK ATP) or 5-hydroxydencanote(5-HD,the inhibitor of mitoK ATP).Rhodamine-123(R-123) was used to detect Δψm.The expression of PKCα protein was examined by using Western blotting,while PKCα mRNA expression was detected by using real-time PCR.The proliferation of ASMCs was measured by MTT assay and cell cycle analysis.In diazoxide-treated normal ASMCs,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls.The ratio of G 0 /G 1 cells was decreased(P<0.05) in diazoxide-treated ASMCs from normal rats.However,there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group.In untreated and diazoxide-treated ASMCs of asthmatic rats,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group.Furthermore,in comparison to ASMCs from asthmatic rats,these values were considerably increased in asthmatic group treated with diazoxide(P<0.05).After exposure to 5-HD for 24 h,these values were decreased as compared with asthma control group(P<0.05).In ASMCs of asthma,the signal transduction pathway of PKCα may be involved in cell proliferation,which is induced by the opening of mitoK ATP and the depolarization of Δψm.展开更多
To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of culture...To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A_1, A_2 and A_3 and the group B was sub-divided into the group of B_1, B_2, B_3, B_4 and B_5. No other agents were added to the group A_1 and B_1. The cells of group A_2 and B_2 were stimulated with 5 % CSE for 24 h. HASMCs from group A_3 and B_3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5 %) for 24 h. PKC inhibitor Ro-31-8220 (5 μmol/L) was added to the HASMCs of group B_4 for 24 h. The cells from group B_5 were stimulated with Ro-31-8220 (5 μmol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-α in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_1, B_2 and B_3 were significantly increased compared to those of group A_1, A_2 and A_3 correspondingly and respectively (P<0.01). The proliferation of HASMCs of group A_2 and B_2 stimulated with CSE and group A_3 and B_3 stimulated with CSE and PMA were also significantly enhanced when group A_1, A_2 and A_3 and group B_1, B_2 and B_3 compared to each other (P<0.05, P<0.01, respectively). The percentage of S phase, absorbency (A) value, 3H-TdR incorporation rate, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_4 treated with Ro-31-8220 and group B_5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B_1 and B_2 correspondingly and respectively (P<0.05, P<0.01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.展开更多
Objective. To investigate the characteristics of urotensin II (U II) receptor in the rat airway smooth muscle and the effect and signal transduction pathway of U II on the proliferation of airway smooth muscle cells. ...Objective. To investigate the characteristics of urotensin II (U II) receptor in the rat airway smooth muscle and the effect and signal transduction pathway of U II on the proliferation of airway smooth muscle cells. Methods. Using 125I UII binding assay to measure the Bmax and Kd of U II receptor. Using the 3H TdR incorporation to determine the effect of U II on the proliferation of airway smooth muscle cells and its signal transduction pathway. Using Fura 2/AM to measure the effect of U II on the cytosolic free calcium concentration. Results. 1. 125I UII binding increased with the time and reached saturation at 45min. The Bmax was (11.36±0.37)fmol/mg pr and Kd was (4.46±0.61)nmol/L. 2. U II increased 3H TdR incorporation of the airway smooth muscle cells in a dose dependent manner. 3. H7, PD98059 and nicardipine, inhibitors of PKC, MAPK, calcium channel, respectively, significantly inhibited U II stimulated 3H TdR incorporation of airway smooth muscle cells. W7, inhibitor of CaM PK, had no effect. 4. Cyclosporin A, inhibitor of CaN, inhibited 3H TdR incorporation of the airway smooth muscle cells induced by U II in a dose dependent manner. 5. U II promoted cytosolic free calcium concentration increase by 18%. Conclusions. 1. There was U II receptor in the rat airway smooth muscle. 2. The effect of U II stimulated 3H TdR incorporation of airway smooth muscle cells was mediated by such signal transduction pathway as Ca2+, PKC, MAPK and CaN, etc.展开更多
Cigarette smoke is associated with the development of several diseases, such as chronic ob- structive pulmonary disease (COPD). The purpose of this study was to investigate genotoxicity and heat shock protein 70 (H...Cigarette smoke is associated with the development of several diseases, such as chronic ob- structive pulmonary disease (COPD). The purpose of this study was to investigate genotoxicity and heat shock protein 70 (Hsp70) in human airway smooth muscle cells (HASMCs) exposed to cigarette smoke extract (CSE). HASMCs was exposed to CSE with different doses for 24 h. The level of 8-hydroxydeoxyguanosine (8-OHdG) was determined by using HPLC-ECD, the DNA damage was ana- lyzed by using comet assay, and apoptosis was examined by using Annexin-FITC/PI staining. The pro- duction of Hsp70 after CSE stimulation was tested. Results indicated that CSE significantly increased the level of 8-OHdG, DNA damage and cell apoptosis, and reduced the production of Hsp70. In par- ticular, levels of Hsp70 were inversely correlated with 8-OHdG, DNA damage and cell apoptosis. It was concluded that cigarette smoke induced genotoxicity and decreased the production of cell protective protein Hsp70, which may contribute to the development of some airway diseases.展开更多
Store-operated Ca2+ channels (SOCs) are plasma membrane Ca2+ permeable channels activated by depletion of intracellular Ca2+ store. Ca2+ entry through SOCs is known as store-operated Ca2+ entry (SOCE), which ...Store-operated Ca2+ channels (SOCs) are plasma membrane Ca2+ permeable channels activated by depletion of intracellular Ca2+ store. Ca2+ entry through SOCs is known as store-operated Ca2+ entry (SOCE), which plays an important role in the functional regulation of airway smooth muscle cells (ASMCs). Protein kinase C (PKC) has been shown to have an activating or inhibiting effect on SOCE, depending on cell types and PKC isoforms that are involved. In ASMCs, the effect of PKC on SOCE has not been elucidated so far. In this study, the role of PKC in the activation of SOCE in rat ASMCs was examined by using Ca2+ fluorescence imaging technique. The results showed that acute application of PKC activators PMA and PDBu did not affect SOCE induced by the sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin. The non-selective PKC inhibitor chelerythrine significantly inhibited thapsigargin- and bradykinin-induced SOCE. RT-PCR assay identified PKCα, δ and ε isoforms in rat ASMCs. PKCα-selective inhibitor G6976 and PKCε-inhibiting peptide Epsilon-V1-2 had no effect on SOCE; by contrast, PKCδ-selective inhibitor rottlerin attenuated SOCE dramatically, suggesting that PKCδ was the major PKC isoform involved in the activation of SOCE in ASMCs. Moreover, PKC down-regulation by extended exposure to high doses of PMA or PDBu also reduced SOCE, confirming the essential role of PKC in the activation of SOCE in ASMCs. In addition, PKC down-regulation did not influence the expression of stromal interaction molecule 1 (STIM1) and Orai1, two elementary molecules in the regulation and activation of SOCs. These results identified PKCδ as an essential PKC isoform involved in the activation of SOCE, and confirmed that PKC regulates the function of ASMCs in a SOCE-dependent manner.展开更多
Objective S100A11 is a member of the S100 calcium-binding protein family and has intracellular and extracellular regulatory activities.We previously reported that S100A11 was differentially expressed in the respirator...Objective S100A11 is a member of the S100 calcium-binding protein family and has intracellular and extracellular regulatory activities.We previously reported that S100A11 was differentially expressed in the respiratory tracts of asthmatic rats as compared with normal controls.Here,we aimed to analyze the potential of S100A11 to regulate both allergen-induced airway hyperresponsiveness(AHR)as well as acetylcholine(ACh)-induced hypercontractility of airway smooth muscle(ASM)and contraction of ASM cells(ASMCs).Methods Purified recombinant rat S100A11 protein(rS100A11)was administered to OVA-sensitized and challenged rats and then the AHR of animals was measured.The relaxation effects of rS100A11 on ASM were detected using isolated tracheal rings and primary ASMCs.The expression levels of un-phosphorylated myosin light chain(MLC)and phosphorylated MLC in ASMCs were analyzed using Western blotting.Results Treatment with rS100A11 attenuated AHR in the rats.ASM contraction assays showed that rS100A11 reduced the contractile responses of isolated tracheal rings and primary ASMCs treated with ACh.In addition,rS100A11 markedly decreased the ACh-induced phosphorylation of the myosin light chain in ASMCs.Moreover,rS100A11 also suppressed the contractile response of tracheal rings in calcium-free buffer medium.Conclusion These results indicate that S100A11 protein can relieve AHR by relaxing ASM independently of extracellular calcium.Our data support the idea that S100A11 is a potential therapeutic target for reducing airway resistance in asthma patients.展开更多
Objective:To evaluate the effect of midkine on lipopolysaccharide(LPS)-induced airway smooth muscle cells(ASMCs).Methods:LPS-stimulated acute lung injury model was used to analyze the effect of midkine on ASMCs in vit...Objective:To evaluate the effect of midkine on lipopolysaccharide(LPS)-induced airway smooth muscle cells(ASMCs).Methods:LPS-stimulated acute lung injury model was used to analyze the effect of midkine on ASMCs in vitro.Recombinant midkine and midkine siRNA were used to investigate the role of Notch2 signaling pathway.Cell proliferation was assessed using Cell Counting Kit-8 assay.Additionally,apoptosis was measured by flow cytometry and protein and mRNA expression of midkine and Notch2 was assessed by Western blotting and qPCR,respectively.Immunofluorescence analysis was also conducted.Results:LPS increased the mRNA and protein expression of midkine and Notch2.Midkine silencing reduced LPS-induced midkine and Notch2 expression.In addition,midkine silencing further reduced the viability and increased apoptosis of ASMCs induced by LPS,which was attenuated by recombinant midkine.Conclusions:The midkine/Notch2 signaling pathway plays a regulatory role in ASMC proliferation and apoptosis in airway inflammation.展开更多
Background:The Huashanshen(HSS)dripping pill has been widely used in asthma for a long time in China.However,the relaxant mechanism of HSS is not well understood.Methods:In this report,high performance liquid chromato...Background:The Huashanshen(HSS)dripping pill has been widely used in asthma for a long time in China.However,the relaxant mechanism of HSS is not well understood.Methods:In this report,high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to identify the constituents in rat plasma after oral administration of HSS.Ovalbumin-sensitized allergic asthma and isolated trachea were studied for the anti-asthmatic mechanism of HSS.Results:D-anisodamine,L-anisodamine,scopolamine and atropine were detected in the rat plasma containing HSS.It was clear that the HSS inhibited the release of inflammatory mediators,regulated the balance of T-helper 1 and T-helper 2 to reduce the airway inflammation,and relaxed the tracheal smooth muscle by controlling the KCa channel,Ca^(2+)influx and release to reduce the airway hyperresponsiveness.Conclusion:Atropine,anisodamine and scopolamine might be active compounds of HSS which inhibited the release of inflammatory mediators,regulated the balance of Th1/Th2,and relaxed the tracheal smooth muscle to reduce airway hyperresponsiveness.展开更多
Objective:To investigate the effect of inhibiting miR-155 expression on the proliferation and migration of airway smooth muscle cells(ASMCs)in patients with chronic obstructive pulmonary disease(COPD).Methods:ASMCs we...Objective:To investigate the effect of inhibiting miR-155 expression on the proliferation and migration of airway smooth muscle cells(ASMCs)in patients with chronic obstructive pulmonary disease(COPD).Methods:ASMCs were isolated and cultured from 8 patients with COPD(observation group)and 3 patients with benign lung cancer without COPD(control group).The ASMCs were transfected with miR-155 suppression expression plasmid(to detect the expression of miR-155;flow cytometry was used to detect the cell cycler of cell clones;Transwell was used to detect cell migration and invasion;Enzyme-linked immunosorbent aanti-miR-155)and the negative contre;clone formation experiment was used to detect the numbol plasmid(anti-miR-NC),and blank control group was set.Real-time quantitative PCR(RT-qPCR)was usedssay(ELISA)method was used to detect tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)level.Results:The expression level of miR-155 in ASMCs of observation group was significantly higher than that in the control group(P<0.05).The miR-155 expression level in inhibited miR-155 expression group was significantly lower,compared with the negative control group and the blank group(P<0.05).In the inhibited miR-155 expression group,the proportion of G0-G1 phase cells was increased,the proportion of S phase cells was decreased,the number of cell clones,migration,and the number of invasive cells were decreased,and the levels of TNF-αand IL-6 were increased(P<0.05).Conclusions:Inhibiting the expression of miR-155 can inhibit the proliferation and migration of airway smooth muscle cells in COPD patients,and inhibit the release of proinflammatory factors.展开更多
Airway hyperresponsiveness (AHR) is a characteristic feature of asthma, and generally correlates with severity of asthma. Understanding the protection mechanism against excessive airway narrowing and how it breaks dow...Airway hyperresponsiveness (AHR) is a characteristic feature of asthma, and generally correlates with severity of asthma. Understanding the protection mechanism against excessive airway narrowing and how it breaks down is fundamental to solving the problem of asthma. In this paper we have proposed a stochastic modeling the airway smooth muscle bundle for reproducing AHR such as an increased sensitivity of the airways to an inhaled constrictor agonist, a steeper slope of the dose-response curve, and a greater maximal response to agonist. A large number N of contractile muscle cells was assumed to repeat themselves in between contraction and relaxation asynchronously. Dynamic equilibrium of statistic physics was applied to the system of ASM bundle. Thus, the relation of dose to response of a piece of ASM bundle was described by Φ=tanh(βH) , where β was Boltzman factor and H represented energy of contraction induced by constrictor agents. Each of adjacent pair contractile cells was assumed to have Ising-type of antimagnetic interactions of preference energy J (for the condition of contraction-relaxation) between them. A motion equation for a piece of ASM bundle was described by Φ=N(H-zJΦ , which explained existence of combined tonic and phasic contractions. Based on observations of Venegas et al. [4], airway responsiveness was assumed to be assessable by total volume of the ventilation defects (TVD) of 13NN PET-CT images. Interactions via propagation of Ca ion waves between ASM bundles would cause percolation probability by PΦ=(1+tanh(βH))2/4 along the tree, then the relation of dose βH to TVD was described by TVD=PΦ[1-(1-PΦ)3/PΦ3]-TVD0. TVD0 represented the protection mechanism against excessive airway narrowing, which was determined by the ratio of amplitudes between tonic and phasic contractions, thus the balance of amplitudes between tonic and phasic contractions of peripheral lobular smooth muscles would be the determinant of AHR.展开更多
Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as a worldwide pathogen, and the incidence of community-acquired infections (CA-MRSA) is increased. A virulence factor has been found in most CA-MRSA in...Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as a worldwide pathogen, and the incidence of community-acquired infections (CA-MRSA) is increased. A virulence factor has been found in most CA-MRSA infections, the Panton-Valentin leukocidin (PVL), which causes polymorphonuclear leukocytes lysis and acute uncontrolled inflammation and tissue injury. In this study we investigated the effect of bacterial supernatant of PVL positive or negative strains on airway smooth muscle obtained from rabbit trachea. MRSA that carry the PVL-gene, confirmed by PCR, is cultured on GP agar and colonies were transferred into casein casein yeast extract medium. The culture supernatants were removed after centrifugation and the presence of PVL was confirmed using an immunochromatographic test. Rabbit tracheal ASMC were isolated and incubated with PVL positive or negative bacterial supernatant (1:20 - 1:2000) for 1 - 3 days. The effect of PVL on the ASMC morphology or viability was estimated using microscope observations or indirect immunofluores- cence with anti-Smooth muscle α-actin antibody and Dapi for DNA staining, and Trypan blue staining, respectively. ASMC incubated with PVL exhibit increased cell size, granular cytoplasm, and ruptured nuclei. Furthermore, PVL reduces cell number mainly in ASMC incubated in the presence of 10% FBS, therefore actively proliferating cells. These results show that apart from the known effect of PVL on immune cells and inflammation process, PVL has a direct toxic effect on airway smooth muscle cells.展开更多
Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT<sub>1</sub> and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including func...Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT<sub>1</sub> and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α<sub>1</sub>-adrenergic receptors (α<sub>1</sub>-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10<sup>−5</sup> M), and in some experiments, 5-Methylurapidil (α<sub>1A</sub>-AR antagonist), AH11110A (α<sub>1B</sub>-AR antagonist), or BMY-7378 (α<sub>1D</sub>-AR antagonist), were used to identify the α<sub>1</sub>-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α<sub>1D</sub>-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α<sub>1</sub>-ARs proteins were evaluated. α<sub>1D</sub>-AR increased at 30 and 60 min post Ang II exposure, the α<sub>1A</sub>-AR diminished from 1 to 4 h, while α<sub>1B</sub>-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α<sub>1D</sub>-AR at short times, and BMY-7378 protected α<sub>1D</sub>-AR from desensitization.展开更多
studies have shown that the R- and S-enantiomers of racemic albuterol, a β2-adrenergic receptor agonist used in asthma treatment, have differential effects on the contractile properties of airway smooth muscle. Howev...studies have shown that the R- and S-enantiomers of racemic albuterol, a β2-adrenergic receptor agonist used in asthma treatment, have differential effects on the contractile properties of airway smooth muscle. However, the effect of albuterol on the proliferation and migration has never been tested. Since (R)-, but not (S)-albuterol enhances bronchodilation, we expect the two racemic isomers would also affect proliferation and migration of tracheal cells differentially. By monitoring migratory properties of airway smooth muscle cells in the presence of albuterol isomers, the different effect of albuterol on proliferation and migration of airway smooth muscle cells is probed. The results show Both of R- and S-albuterol could inhibit the proliferation of smooth muscle cells and the inhibition ratio of these two isomers had no significant difference;R-albuterol, but not S-albuterol, inhibited cell migration.展开更多
Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis...Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.展开更多
Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,...Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods PASMCs were incubated with different concentrations of GW501516(10,30,100 nmol/L)under the hypoxic condition.The proliferation was determined by a CCK-8 assay.The cell cycle progression was analyzed by flow cytometry.The expression of PPARδ,S phase kinase-associated protein 2(Skp2),and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting.Then PASMCs were treated with 100 nmol/L GW501516,100 nmol/L mammalian target of rapamycin(mTOR)inhibitor rapamycin and/or 2µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs.Results The presented data demonstrated that hypoxia reduced the expression of PPARδin an oxygen concentration-and time-dependent manner,and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle.In accordance with these findings,GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs.Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation,arresting the cell cycle,regulating the expression of Skp2 and p27,and inactivating mTOR in hypoxia-exposed PASMCs.Moreover,MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs.Conclusion GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.展开更多
Background:Vascular smooth muscle cells(VSMCs)undergo a conversion from a contractile phenotype to a proliferative synthetic phenotype,contributing to the pathogenesis of cardiovascular diseases.Semaphorin 7A(SEMA7A)i...Background:Vascular smooth muscle cells(VSMCs)undergo a conversion from a contractile phenotype to a proliferative synthetic phenotype,contributing to the pathogenesis of cardiovascular diseases.Semaphorin 7A(SEMA7A)is a glycosylphosphatidylinositol-anchored membrane protein that plays an important role in vascular homeostasis by regulating endothelial cell behaviors.However,the expression and role of SEMA7A in VSMCs remain unclear.Methods:In this study,we screened for VSMC-regulating genes in publicly available datasets and analyzed the expression of SEMA7A in human coronary artery smooth muscle cells(hCASMCs)treated with platelet-derived growth factor-BB(PDGF-BB).The effects of SEMA7A overexpression and knockdown on hCASMC proliferation and migration were examined.The signaling pathways involved in the action of SEMA7A in hCASMCs were determined.Results:Bioinformatic analysis showed that SEMA7A was significantly dysregulated in VSMCs treated with oxidized low-density lipoprotein or overexpressing progerin,a pro-atherogenic gene.The PDGF-BB stimulation led to a concentration-and time-dependent induction of SEMA7A.Depletion of SEMA7A attenuated PDGF-BB-induced hCASMC proliferation and migration.Conversely,overexpression of SEMA7A enhanced hCASMC proliferation and migration.Mechanistically,SEMA7A stimulated the activation of theβ-catenin pathway and upregulated c-Myc,CCND1,and MMP7.Knockdown ofβ-catenin impaired SEMA7A-induced hCASMC proliferation and migration.Conclusions:SEMA7A triggers phenotype switching in VSMCs through theβ-catenin signaling pathway and may serve as a potential therapeutic target for cardiovascular diseases.展开更多
基金This project was supported by a grant from Natural Sci-ences Foundation of China(No.396 70 338)
文摘The effects of Shenmai injection (SMI) and am inophylline on apoptosis of sm all airway smooth muscle cells (SASMC) and the Fas/ Fas L expression in rats with papain- induced em physe- ma were investigated.Rat emphysema model was established by a single intratracheal instillation of papain.Apoptosis and Fas/Fas L expression of SASMC were detected by im munohistochemistry SABC and TU NEL assay at day1,3,5 ,7,15 ,30 after modeling,and the effect of SMI and am inophylline on them were observed.The results indicated that the Fas/Fas L expression positive rate in SASMC was2 .31± 0 .5 5 /1.2 8± 0 .4 7respectively.After a single intratracheal instillation of papain,the expression of Fas/Fas L positive rate in the placebo group was increased in a tim e- dependent manner.SMI could inhibit the expression of Fas/ Fas L ,but aminophylline couldn't. The positive rate of apoptosis in the control group was 0 .87± 0 .32 .After a single intratracheal in- stillation of papain,the SASMC apoptosis positive rate in the placebo group was increased in a tim e- dependent manner.The SASMC apoptosis rate in all groups was declined after treatment with SMI,but the effect of am inophylline was notobvious.Itwas dem onstrated thatin the patho- genesis of emphysem a Fas/Fas L played an important role in the regulation of SASMC apoptosis. SMI influenced the expression of Fas/ Fas L and declined SASMC apoptosis by inhibiting the releas- ing of inflamm atory m edia and played an im portant role in the therapy of em physema.
基金This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30270583)
文摘The effects of protein kinase C (PKC) on the tension and the activity of voltage-dependent delayed rectifier potassium channel (K,,) were examined in normal and passively sensitized human airway smooth muscle (HASM), by measuring tones and whole-cell patch clamp techniques, and the Kv activities and membrane potential (Em) were also detected. The results showed that phorbol 12-myristate 13-acetate (PMA), a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group (P〈0.05), and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. Kv activities of HASM cells were significantly inhibited by PMA, and the Em became more positive, as compared with the DMSO (a PMA menstruum)-treated group (P〈0.01). This effect could be blocked by Ro31-8220 (P〈0.01 ). It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced Kv activity. In passively sensitized HASM rings, this effect was more notable.
文摘Objective: To investigate the role of large Ca^2+-activated, delayed-rectifier and ATP-sensitive potassium channel in regulating the relaxation induced by nitric oxide (NO) in normal and passively sensitized human airway smooth muscle (HASM) with serum from asthmatic patients. Methods: The effects of NO or/and potassium channel blockers on the tensions of normal and passively sensitized HASM were measured by using nitric oxide donor and potassium blockers, with the isometric tension recording technique. Results: Showed that(1)In the control group and passively sensitized group, Kv blocker(4-AP) cause concentration-dependent augmentation in the contraction induced by histamine ( 1 ×10^-4 mol/L ), (P 〈 0.05), but Glib ( 1 × 10^-2 mol/L ) and TEA (1×10^-4 mol/L) have no significant effects on the contraction induced by histamine (1×10^-4 mol/L). The maximum tension induced by histamine in passively sensitized group is higher than that in the control group (P 〈 0.05). (2) NO-donor Sodium Nitroprusside (SNP) bring about significant relaxation in normal and passively sensitized HASM rings (P 〈 0.05). Relaxations of passively sensitized airway rings [ (29.4 ± 3.3)% ] were significant less than those of normal HASM rings [ (44.1 ± 10.2)% ], (P 〈 0.05).(3) Glib(1×10^-2 mol/L)have no significant effect on the relaxations induced by SNP(1×10^-4 mol/L). 4-AP(1×10^-2 mol/L) inhibited relaxation induced by SNP (1×10^-4 mol/L), (P 〈 0.01). TEA (1×10^-3 tool/L) inhibited relaxation induced by SNP (1×10^-4 mol/L) (P 〈 0.05), and the inhibiting effect in passively sensitized HASM rings were significant less than in normal HASM, (P 〈 0.05). Conclusion: It was concluded that SNP(NO-donor) relaxed the contraction of HASM partly via BKca channel opening. In passively sensitized HASM in vitro, the relaxation of SNP decreased compared with control group, which might be associated with the down-regulating activity of BKca in passively sensitized HASM.
基金supported by the National Natural Science Foundation of China(No.81100029)
文摘Airway remodeling is an important pathological feature of asthma and the basis of severe asthma. Proliferation of airway smooth muscle cells (ASMCs) is a major contributor to airway remod- eling. As an important Ca2+ channel, transient receptor potential vanilloid 1 (TRPV1) plays the key role in the cell pathological and physiological processes. This study investigated the expression and activity of TRPV1 channel, and further clarified the effect of TRPV1 channel on the ASMCs proliferation and apoptosis in order to provide the scientific basis to treat asthmatic airway remodeling in clinical practice Immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of TRPVI in rat ASMCs. Intracellular Ca2+ was detected using the single cell confocal fluorescence microscopy measurement loaded with Fluo-4/AM. The cell cycles were observed by flow cytometry. MTT assay and Hoechst 33258 staining were used to detect the proliferation and apoptosis of ASMCs in rats respectively. The data showed that: (1) TRPV1 channel was present in rat ASMCs. (2) TRPV1 channel agonist, capsaicin, increased the Ca2~ influx in a concentration-dependent manner (EC50=284.3+58 nmol/L). TRPV1 channel antagonist, capsazepine, inhibited Ca2+ influx in rat ASMCs. (3) Capsaicin significantly increased the percentage of S+G2M ASMCs and the absorbance of MTT assay. Capsazepine had the opposite effect. (4) Capsaicin significantly inhibited the apoptosis, whereas capsazepine had the opposite effect. These results suggest that TRPV1 is present and mediates Ca2+ influx in rat ASMCs. TRPV1 activity stimulates proliferation of ASMCs in rats.
基金supported by grants from Natural Science Foundation of Hubei Province,China (No. 2010CDB096)the National Key Technology R&D Program of the 12th National Five-year Development Plan of China (No. 2012BAI05B01)
文摘The effects of ATP-sensitive mitochondrial K + channel(mitoK ATP) on mitochondrial membrane potential(Δψm),cell proliferation and protein kinase C alpha(PKCα) expression in airway smooth muscle cells(ASMCs) were investigated.Thirty-six Sprague-Dawley(SD) rats were immunized with saline(controls) or ovalbumin(OVA) with alum(asthma models).ASMCs were cultured from the lung of control and asthma rats.ASMCs were treated with diazoxide(the potent activator of mitoK ATP) or 5-hydroxydencanote(5-HD,the inhibitor of mitoK ATP).Rhodamine-123(R-123) was used to detect Δψm.The expression of PKCα protein was examined by using Western blotting,while PKCα mRNA expression was detected by using real-time PCR.The proliferation of ASMCs was measured by MTT assay and cell cycle analysis.In diazoxide-treated normal ASMCs,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls.The ratio of G 0 /G 1 cells was decreased(P<0.05) in diazoxide-treated ASMCs from normal rats.However,there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group.In untreated and diazoxide-treated ASMCs of asthmatic rats,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group.Furthermore,in comparison to ASMCs from asthmatic rats,these values were considerably increased in asthmatic group treated with diazoxide(P<0.05).After exposure to 5-HD for 24 h,these values were decreased as compared with asthma control group(P<0.05).In ASMCs of asthma,the signal transduction pathway of PKCα may be involved in cell proliferation,which is induced by the opening of mitoK ATP and the depolarization of Δψm.
文摘To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A_1, A_2 and A_3 and the group B was sub-divided into the group of B_1, B_2, B_3, B_4 and B_5. No other agents were added to the group A_1 and B_1. The cells of group A_2 and B_2 were stimulated with 5 % CSE for 24 h. HASMCs from group A_3 and B_3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5 %) for 24 h. PKC inhibitor Ro-31-8220 (5 μmol/L) was added to the HASMCs of group B_4 for 24 h. The cells from group B_5 were stimulated with Ro-31-8220 (5 μmol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-α in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_1, B_2 and B_3 were significantly increased compared to those of group A_1, A_2 and A_3 correspondingly and respectively (P<0.01). The proliferation of HASMCs of group A_2 and B_2 stimulated with CSE and group A_3 and B_3 stimulated with CSE and PMA were also significantly enhanced when group A_1, A_2 and A_3 and group B_1, B_2 and B_3 compared to each other (P<0.05, P<0.01, respectively). The percentage of S phase, absorbency (A) value, 3H-TdR incorporation rate, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_4 treated with Ro-31-8220 and group B_5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B_1 and B_2 correspondingly and respectively (P<0.05, P<0.01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.
文摘Objective. To investigate the characteristics of urotensin II (U II) receptor in the rat airway smooth muscle and the effect and signal transduction pathway of U II on the proliferation of airway smooth muscle cells. Methods. Using 125I UII binding assay to measure the Bmax and Kd of U II receptor. Using the 3H TdR incorporation to determine the effect of U II on the proliferation of airway smooth muscle cells and its signal transduction pathway. Using Fura 2/AM to measure the effect of U II on the cytosolic free calcium concentration. Results. 1. 125I UII binding increased with the time and reached saturation at 45min. The Bmax was (11.36±0.37)fmol/mg pr and Kd was (4.46±0.61)nmol/L. 2. U II increased 3H TdR incorporation of the airway smooth muscle cells in a dose dependent manner. 3. H7, PD98059 and nicardipine, inhibitors of PKC, MAPK, calcium channel, respectively, significantly inhibited U II stimulated 3H TdR incorporation of airway smooth muscle cells. W7, inhibitor of CaM PK, had no effect. 4. Cyclosporin A, inhibitor of CaN, inhibited 3H TdR incorporation of the airway smooth muscle cells induced by U II in a dose dependent manner. 5. U II promoted cytosolic free calcium concentration increase by 18%. Conclusions. 1. There was U II receptor in the rat airway smooth muscle. 2. The effect of U II stimulated 3H TdR incorporation of airway smooth muscle cells was mediated by such signal transduction pathway as Ca2+, PKC, MAPK and CaN, etc.
基金supported by grants from the National Natural Science Foundation of China(No.8107003681370145+3 种基金81370156 and 81070021)The National Key Technology R&D Program of the 12th National Five-year Development Plan:Clinical Study on Translational Medicine of Respiratory Disease(No.212BA105B01)The Specific Project of National Health Research Project of Ministry of Health of China(No.201002008)Program for Changjiang Scholars and Innovative Research Team in University(No.PCSIRT1131)
文摘Cigarette smoke is associated with the development of several diseases, such as chronic ob- structive pulmonary disease (COPD). The purpose of this study was to investigate genotoxicity and heat shock protein 70 (Hsp70) in human airway smooth muscle cells (HASMCs) exposed to cigarette smoke extract (CSE). HASMCs was exposed to CSE with different doses for 24 h. The level of 8-hydroxydeoxyguanosine (8-OHdG) was determined by using HPLC-ECD, the DNA damage was ana- lyzed by using comet assay, and apoptosis was examined by using Annexin-FITC/PI staining. The pro- duction of Hsp70 after CSE stimulation was tested. Results indicated that CSE significantly increased the level of 8-OHdG, DNA damage and cell apoptosis, and reduced the production of Hsp70. In par- ticular, levels of Hsp70 were inversely correlated with 8-OHdG, DNA damage and cell apoptosis. It was concluded that cigarette smoke induced genotoxicity and decreased the production of cell protective protein Hsp70, which may contribute to the development of some airway diseases.
基金supported by grants from the National Natural Science Foundation of China(No.30871122,No.81072684)
文摘Store-operated Ca2+ channels (SOCs) are plasma membrane Ca2+ permeable channels activated by depletion of intracellular Ca2+ store. Ca2+ entry through SOCs is known as store-operated Ca2+ entry (SOCE), which plays an important role in the functional regulation of airway smooth muscle cells (ASMCs). Protein kinase C (PKC) has been shown to have an activating or inhibiting effect on SOCE, depending on cell types and PKC isoforms that are involved. In ASMCs, the effect of PKC on SOCE has not been elucidated so far. In this study, the role of PKC in the activation of SOCE in rat ASMCs was examined by using Ca2+ fluorescence imaging technique. The results showed that acute application of PKC activators PMA and PDBu did not affect SOCE induced by the sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin. The non-selective PKC inhibitor chelerythrine significantly inhibited thapsigargin- and bradykinin-induced SOCE. RT-PCR assay identified PKCα, δ and ε isoforms in rat ASMCs. PKCα-selective inhibitor G6976 and PKCε-inhibiting peptide Epsilon-V1-2 had no effect on SOCE; by contrast, PKCδ-selective inhibitor rottlerin attenuated SOCE dramatically, suggesting that PKCδ was the major PKC isoform involved in the activation of SOCE in ASMCs. Moreover, PKC down-regulation by extended exposure to high doses of PMA or PDBu also reduced SOCE, confirming the essential role of PKC in the activation of SOCE in ASMCs. In addition, PKC down-regulation did not influence the expression of stromal interaction molecule 1 (STIM1) and Orai1, two elementary molecules in the regulation and activation of SOCs. These results identified PKCδ as an essential PKC isoform involved in the activation of SOCE, and confirmed that PKC regulates the function of ASMCs in a SOCE-dependent manner.
基金This work was supported by the National Natural Science Foundation of China(No.81973952 and No.81774429)the Natural Science Foundation of Shanghai(No.19ZR1451500),and the Yangfan Innovation Project(No.20YF1445300).
文摘Objective S100A11 is a member of the S100 calcium-binding protein family and has intracellular and extracellular regulatory activities.We previously reported that S100A11 was differentially expressed in the respiratory tracts of asthmatic rats as compared with normal controls.Here,we aimed to analyze the potential of S100A11 to regulate both allergen-induced airway hyperresponsiveness(AHR)as well as acetylcholine(ACh)-induced hypercontractility of airway smooth muscle(ASM)and contraction of ASM cells(ASMCs).Methods Purified recombinant rat S100A11 protein(rS100A11)was administered to OVA-sensitized and challenged rats and then the AHR of animals was measured.The relaxation effects of rS100A11 on ASM were detected using isolated tracheal rings and primary ASMCs.The expression levels of un-phosphorylated myosin light chain(MLC)and phosphorylated MLC in ASMCs were analyzed using Western blotting.Results Treatment with rS100A11 attenuated AHR in the rats.ASM contraction assays showed that rS100A11 reduced the contractile responses of isolated tracheal rings and primary ASMCs treated with ACh.In addition,rS100A11 markedly decreased the ACh-induced phosphorylation of the myosin light chain in ASMCs.Moreover,rS100A11 also suppressed the contractile response of tracheal rings in calcium-free buffer medium.Conclusion These results indicate that S100A11 protein can relieve AHR by relaxing ASM independently of extracellular calcium.Our data support the idea that S100A11 is a potential therapeutic target for reducing airway resistance in asthma patients.
基金supported by the National Natural Science Foundation of China(NO.81660011,81960351)Hainan Provincial Social Development Foundation(NO.ZDYFXGFY2020004)Hainan Provincial Medical and Health Research Project(NO.22A200036).
文摘Objective:To evaluate the effect of midkine on lipopolysaccharide(LPS)-induced airway smooth muscle cells(ASMCs).Methods:LPS-stimulated acute lung injury model was used to analyze the effect of midkine on ASMCs in vitro.Recombinant midkine and midkine siRNA were used to investigate the role of Notch2 signaling pathway.Cell proliferation was assessed using Cell Counting Kit-8 assay.Additionally,apoptosis was measured by flow cytometry and protein and mRNA expression of midkine and Notch2 was assessed by Western blotting and qPCR,respectively.Immunofluorescence analysis was also conducted.Results:LPS increased the mRNA and protein expression of midkine and Notch2.Midkine silencing reduced LPS-induced midkine and Notch2 expression.In addition,midkine silencing further reduced the viability and increased apoptosis of ASMCs induced by LPS,which was attenuated by recombinant midkine.Conclusions:The midkine/Notch2 signaling pathway plays a regulatory role in ASMC proliferation and apoptosis in airway inflammation.
基金supported by grants 81673647,81673535,81503086 from National Natural Science Foundation of China.
文摘Background:The Huashanshen(HSS)dripping pill has been widely used in asthma for a long time in China.However,the relaxant mechanism of HSS is not well understood.Methods:In this report,high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to identify the constituents in rat plasma after oral administration of HSS.Ovalbumin-sensitized allergic asthma and isolated trachea were studied for the anti-asthmatic mechanism of HSS.Results:D-anisodamine,L-anisodamine,scopolamine and atropine were detected in the rat plasma containing HSS.It was clear that the HSS inhibited the release of inflammatory mediators,regulated the balance of T-helper 1 and T-helper 2 to reduce the airway inflammation,and relaxed the tracheal smooth muscle by controlling the KCa channel,Ca^(2+)influx and release to reduce the airway hyperresponsiveness.Conclusion:Atropine,anisodamine and scopolamine might be active compounds of HSS which inhibited the release of inflammatory mediators,regulated the balance of Th1/Th2,and relaxed the tracheal smooth muscle to reduce airway hyperresponsiveness.
基金Hunan provincial traditional Chinese medicine scientific research project(No.201838)。
文摘Objective:To investigate the effect of inhibiting miR-155 expression on the proliferation and migration of airway smooth muscle cells(ASMCs)in patients with chronic obstructive pulmonary disease(COPD).Methods:ASMCs were isolated and cultured from 8 patients with COPD(observation group)and 3 patients with benign lung cancer without COPD(control group).The ASMCs were transfected with miR-155 suppression expression plasmid(to detect the expression of miR-155;flow cytometry was used to detect the cell cycler of cell clones;Transwell was used to detect cell migration and invasion;Enzyme-linked immunosorbent aanti-miR-155)and the negative contre;clone formation experiment was used to detect the numbol plasmid(anti-miR-NC),and blank control group was set.Real-time quantitative PCR(RT-qPCR)was usedssay(ELISA)method was used to detect tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)level.Results:The expression level of miR-155 in ASMCs of observation group was significantly higher than that in the control group(P<0.05).The miR-155 expression level in inhibited miR-155 expression group was significantly lower,compared with the negative control group and the blank group(P<0.05).In the inhibited miR-155 expression group,the proportion of G0-G1 phase cells was increased,the proportion of S phase cells was decreased,the number of cell clones,migration,and the number of invasive cells were decreased,and the levels of TNF-αand IL-6 were increased(P<0.05).Conclusions:Inhibiting the expression of miR-155 can inhibit the proliferation and migration of airway smooth muscle cells in COPD patients,and inhibit the release of proinflammatory factors.
文摘Airway hyperresponsiveness (AHR) is a characteristic feature of asthma, and generally correlates with severity of asthma. Understanding the protection mechanism against excessive airway narrowing and how it breaks down is fundamental to solving the problem of asthma. In this paper we have proposed a stochastic modeling the airway smooth muscle bundle for reproducing AHR such as an increased sensitivity of the airways to an inhaled constrictor agonist, a steeper slope of the dose-response curve, and a greater maximal response to agonist. A large number N of contractile muscle cells was assumed to repeat themselves in between contraction and relaxation asynchronously. Dynamic equilibrium of statistic physics was applied to the system of ASM bundle. Thus, the relation of dose to response of a piece of ASM bundle was described by Φ=tanh(βH) , where β was Boltzman factor and H represented energy of contraction induced by constrictor agents. Each of adjacent pair contractile cells was assumed to have Ising-type of antimagnetic interactions of preference energy J (for the condition of contraction-relaxation) between them. A motion equation for a piece of ASM bundle was described by Φ=N(H-zJΦ , which explained existence of combined tonic and phasic contractions. Based on observations of Venegas et al. [4], airway responsiveness was assumed to be assessable by total volume of the ventilation defects (TVD) of 13NN PET-CT images. Interactions via propagation of Ca ion waves between ASM bundles would cause percolation probability by PΦ=(1+tanh(βH))2/4 along the tree, then the relation of dose βH to TVD was described by TVD=PΦ[1-(1-PΦ)3/PΦ3]-TVD0. TVD0 represented the protection mechanism against excessive airway narrowing, which was determined by the ratio of amplitudes between tonic and phasic contractions, thus the balance of amplitudes between tonic and phasic contractions of peripheral lobular smooth muscles would be the determinant of AHR.
文摘Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as a worldwide pathogen, and the incidence of community-acquired infections (CA-MRSA) is increased. A virulence factor has been found in most CA-MRSA infections, the Panton-Valentin leukocidin (PVL), which causes polymorphonuclear leukocytes lysis and acute uncontrolled inflammation and tissue injury. In this study we investigated the effect of bacterial supernatant of PVL positive or negative strains on airway smooth muscle obtained from rabbit trachea. MRSA that carry the PVL-gene, confirmed by PCR, is cultured on GP agar and colonies were transferred into casein casein yeast extract medium. The culture supernatants were removed after centrifugation and the presence of PVL was confirmed using an immunochromatographic test. Rabbit tracheal ASMC were isolated and incubated with PVL positive or negative bacterial supernatant (1:20 - 1:2000) for 1 - 3 days. The effect of PVL on the ASMC morphology or viability was estimated using microscope observations or indirect immunofluores- cence with anti-Smooth muscle α-actin antibody and Dapi for DNA staining, and Trypan blue staining, respectively. ASMC incubated with PVL exhibit increased cell size, granular cytoplasm, and ruptured nuclei. Furthermore, PVL reduces cell number mainly in ASMC incubated in the presence of 10% FBS, therefore actively proliferating cells. These results show that apart from the known effect of PVL on immune cells and inflammation process, PVL has a direct toxic effect on airway smooth muscle cells.
文摘Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT<sub>1</sub> and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α<sub>1</sub>-adrenergic receptors (α<sub>1</sub>-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10<sup>−5</sup> M), and in some experiments, 5-Methylurapidil (α<sub>1A</sub>-AR antagonist), AH11110A (α<sub>1B</sub>-AR antagonist), or BMY-7378 (α<sub>1D</sub>-AR antagonist), were used to identify the α<sub>1</sub>-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α<sub>1D</sub>-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α<sub>1</sub>-ARs proteins were evaluated. α<sub>1D</sub>-AR increased at 30 and 60 min post Ang II exposure, the α<sub>1A</sub>-AR diminished from 1 to 4 h, while α<sub>1B</sub>-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α<sub>1D</sub>-AR at short times, and BMY-7378 protected α<sub>1D</sub>-AR from desensitization.
文摘studies have shown that the R- and S-enantiomers of racemic albuterol, a β2-adrenergic receptor agonist used in asthma treatment, have differential effects on the contractile properties of airway smooth muscle. However, the effect of albuterol on the proliferation and migration has never been tested. Since (R)-, but not (S)-albuterol enhances bronchodilation, we expect the two racemic isomers would also affect proliferation and migration of tracheal cells differentially. By monitoring migratory properties of airway smooth muscle cells in the presence of albuterol isomers, the different effect of albuterol on proliferation and migration of airway smooth muscle cells is probed. The results show Both of R- and S-albuterol could inhibit the proliferation of smooth muscle cells and the inhibition ratio of these two isomers had no significant difference;R-albuterol, but not S-albuterol, inhibited cell migration.
基金funded by the National Natural Science Foundation of China(No.82070376 and No.81873491)the Natural Science Foundation of Zhejiang Province(No.LY21H020005)+1 种基金the Zhejiang Medical Science and Technology Project(No.2019KY376 and No.2018KY071)a Ningbo Science and Technology Project(No.202002N3173).
文摘Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.
基金supported by the National Natural Science Foundation of Hubei Province(No.2018CFC801).
文摘Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods PASMCs were incubated with different concentrations of GW501516(10,30,100 nmol/L)under the hypoxic condition.The proliferation was determined by a CCK-8 assay.The cell cycle progression was analyzed by flow cytometry.The expression of PPARδ,S phase kinase-associated protein 2(Skp2),and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting.Then PASMCs were treated with 100 nmol/L GW501516,100 nmol/L mammalian target of rapamycin(mTOR)inhibitor rapamycin and/or 2µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs.Results The presented data demonstrated that hypoxia reduced the expression of PPARδin an oxygen concentration-and time-dependent manner,and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle.In accordance with these findings,GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs.Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation,arresting the cell cycle,regulating the expression of Skp2 and p27,and inactivating mTOR in hypoxia-exposed PASMCs.Moreover,MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs.Conclusion GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.
基金supported by the Basic Research Program of Shanxi Province(Free Exploration)of China(20210302124416)Science and Technology Grant for Selected Returned Chinese Scholars of Shanxi Province of China(20220043)Four“Batches”Innovation Project of Invigorating Medical through Science and Technology of Shanxi Province of China(2022XM08).
文摘Background:Vascular smooth muscle cells(VSMCs)undergo a conversion from a contractile phenotype to a proliferative synthetic phenotype,contributing to the pathogenesis of cardiovascular diseases.Semaphorin 7A(SEMA7A)is a glycosylphosphatidylinositol-anchored membrane protein that plays an important role in vascular homeostasis by regulating endothelial cell behaviors.However,the expression and role of SEMA7A in VSMCs remain unclear.Methods:In this study,we screened for VSMC-regulating genes in publicly available datasets and analyzed the expression of SEMA7A in human coronary artery smooth muscle cells(hCASMCs)treated with platelet-derived growth factor-BB(PDGF-BB).The effects of SEMA7A overexpression and knockdown on hCASMC proliferation and migration were examined.The signaling pathways involved in the action of SEMA7A in hCASMCs were determined.Results:Bioinformatic analysis showed that SEMA7A was significantly dysregulated in VSMCs treated with oxidized low-density lipoprotein or overexpressing progerin,a pro-atherogenic gene.The PDGF-BB stimulation led to a concentration-and time-dependent induction of SEMA7A.Depletion of SEMA7A attenuated PDGF-BB-induced hCASMC proliferation and migration.Conversely,overexpression of SEMA7A enhanced hCASMC proliferation and migration.Mechanistically,SEMA7A stimulated the activation of theβ-catenin pathway and upregulated c-Myc,CCND1,and MMP7.Knockdown ofβ-catenin impaired SEMA7A-induced hCASMC proliferation and migration.Conclusions:SEMA7A triggers phenotype switching in VSMCs through theβ-catenin signaling pathway and may serve as a potential therapeutic target for cardiovascular diseases.