Trigonelline protects the cardiocyte from hydrogen peroxide induced apoptosis in H9c2 cells

Objective:To elucidate the key parameters associated with hydrogen peroxide induced oxidative stress and investigates the mechanism of trigonelline(TG)for reducing the H_2O_2induced toxicity in H9c2 cells.Methods:Cytotoxicity and antioxidant activity of TG was assessed by EZ-CYTOX kit.RNA extraction and cDNA synthesized according to the kit manufacture protocol.Apoptosis was measured by the Flowcytometry,general PCR and qPCR.Results:It was found that the TG significantly rescued the morphology of the H9c2 cells.Treatment of cells with TG attenuated H_2O_2 induced cell deaths and improved the antioxidant activity.In addition,TG regulated the apoptotic gene caspase-3,caspase-9 and anti-apoptotic gene Bcl-2.Bcl-XL during H_2O_2 induced oxidative stress in H9c2 cells.These results were comparable with quercetin treatment.For evident,flow cytometer results also confirmed the TG significantly reduced the H_2O_2 induced necrosis and apoptosis in H9c2 cells.However,further increment of TG concentration against H_2O_2 could induce the necrosis and apoptosis along with H_2O_2.Conclusions:It is suggested that less than 125μM of TG could protect the cells from H_2O_2 induced cell damage by down regulating the caspases and up regulating the Bcl-2 and Bcl-XL expression.Therefore,we suggest the trigonelline could be useful for treatment of oxidative stress mediated cardiovascular diseases in future.

Paris chinensis dioscin induces G2/M cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells

AIM:To investigate the anti-tumor effects of Paris chinensis dioscin(PCD)and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells.METHODS:Cell viability was analyzed by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope(LSCM)using Annexin-V/propidium iodide(PI)staining,and the cell cycle was evaluated using PI staining with flow cytom-etry.Intracellular calcium ions were detected under fluorescence microscope.The expression of cell cycle and apoptosis-related proteins cyclin B1,CDK1,cytochrome C and caspase-3 was measured by immunohistochemical staining.RESULTS:PCD had an anti-proliferation effect on human gastric cancer SGC-7901 cells in a dose-and time-de-pendent manner.After treatment of SGC-7901 cells with PCD,apoptosis appeared in SGC-7901 cells.Morpho-logical changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining,and the cell number of the G0/G1 phase was decreased,while the number of cells in the G2/M phase was increased.Cell cycle-related proteins,such as cyclin B1 and CDK1,were all down-regulated,but caspase-3 and cytochrome C were up-regulated.Moreover,intracellular calcium accumulation occurred in PCD-treated cells.CONCLUSION:G2/M phase arrest and apoptosis induced by PCD are associated with the inhibition of CDK-activating kinase activity and the activation of Ca2+-related mitochondrion pathway in SGC-7901 cells.

Modulation of TGFβ_2 and dopamine by PKC in retinal Müller cells of guinea pig myopic eye

AIM: To investigate the effect of protein kinase C (PKC) on transforming growth factor-β2 (TGFβ2) and dopamine in retinal Müller cells of guinea pig myopic eye. METHODS: Myopia was induced by translucent goggles in guinea pig, whose retinal Müller cells were cultured using the enzyme-digesting method. Retinal Müller cells were divided into 5 groups: normal control, myopia, myopia plus GF109203X, myopia plus PMA, myopia plus DMSO. PKC activities were detected by the non-radioactive methods. TGFβ2 and tyrosine hydroxylase (TH) proteins were analyzed by Western Blotting in retinal Müller cells. Dopamine was determined by the high-performance liquid chromatography- electrochemical detection in suspensions. RESULTS: After 14 days deprived, the occluded eyes became myopic with ocular axle elongating. Müller cells of guinea pigs were obtained using enzyme digestion. Compared with normal control group, the increase in PKC activity and the up-regulation in TGFβ2 expression were found in retinal Müller cells of myopic eyes, with the decrease of TH and dopamine content (P <0.05). After PKC activated by PMA, TGFβ2 and TH content were up-regulated with the increase of dopamine content (P <0.05). While the PKC activities was inhibited by GF109203X, proteins of TGFβ2 and TH were down-regulated in the myopic eyes, with the decrease of dopamine content (P <0.05). CONCLUSION: TGFβ2 and dopamine are modulated by PKC in Müller cells of the myopic eyes in guinea pig.

Nardosinone Protects H9c2 Cardiac Cells from Angiotensin Ⅱ-induced Hypertrophy

Pathological cardiac hypertrophy induced by angiotensin Ⅱ （Ang Ⅱ ） can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots ofNardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on Ang Ⅱ-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosi- none （25, 50, 100, and 200μmol/L） or Ang Ⅱ （1 μmol/L）. Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by Ang Ⅱ. The mRNA expression of ANP, BNP and 13-MHC was obviously elevated in Ang Ⅱ-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang Ⅱ-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways.

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

AIM： TO establish a cell culture system with longterm replication of hepatitis C virus （HCV） genome and expression of viral antigens in vitro. METHODS： HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect na＇ive cells. The presence of minus （antisense） RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques （flow cytometry and Western blot） respectively. RESULTS： The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies （anti-core, and anti-E1）. Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION： HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

HBsAg stimulates NKG2D receptor expression on natural killer cellsand inhibits hepatitis C virus replication

Background： Higher hepatitis B surface antigen （HBsAg） facilitates hepatitis C virus （HCV） clearance inpatients with hepatitis B virus （HBV）/HCV co-infection. We investigated the effect of exogenous HBsAgon the inhibition of HCV replication mediated by natural killer （NK） cells.

Human umbilical cord blood mesenchymal stem cells conditioned media inhibits hypoxia-induced apoptosis in H9c2 cells by activation of the survival protein Akt

This work aimed to study the beneficial role of human umbilical cord blood-derived mesenchymal stem cellconditioned medium(MSC-CM)in hypoxia-induced apoptosis in H9c2 cardiomyoblasts,in which the serine/heroine kinases(Akt)pathway would be involved.For this,CM was collected by culturing MSCs in serum-free DMEM medium for 24 h,and paracrine factors were analyzed by protein chip.H9c2 cells were divided into the following groups:control group,hypoxia group,MSC-CM intervention group(CM group),MSC-CM+Akt phosphorylation inhibitor(LY294002)group(LY group).Apoptosis of the H9c2 cells was tested with chromatin dye Hoechst 33342 and FITC-conjugated Annexin V apoptosis detection kit by flow cytometer after a hypoxia/serum deprivation(H/SD)for 24 h.The apoptosis-related proteins were evaluated by Western blot.MSC-CM displayed significantly elevated levels of growth factors,anti-inflammatory,and anti-apoptosis cytokines.On Hoechst 33342 apoptosis staining,the H9c2 cell morphology displayed a lower proportion of apoptosis in the CM group than those in the hypoxia group,while apoptosis was increased in LY group.Flow cytometer analysis revealed the apoptosis ratio in the CM group was lower than the hypoxia group(12.34±2.00%vs.21.73±2.58%,p<0.05),while the LY group was significantly higher(22.54±3.89%).Active caspase-3 expression was increased in hypoxia group than control group(p<0.05),but decreased in CM group(p<0.01).Umbilical cord blood-derived mesenchymal stem cell-conditioned media secrete multiple paracrine factors that are able to inhibit hypoxia-induced H9c2 cardiomyoblasts apoptosis,and in which the activation of Akt phosphorylation is involved to achieve the protective effect.

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Cross-talk between microRNA-let7c and transforming growth factor-β2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells

AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

Erbin Interacts with Sema4C and Inhibits Sema4C-induced Epithelial-mesenchymal Transition in HK2 Cells

Erbin, a member of Leucine-rich repeat and PDZ-containing protein family, was found to inhibit TGF-β-induced epithelial-mesenchymal transition （EMT） in our previous study. However, the mechanism of Erbin in regulating EMT is unclear. Semaphorin protein Sema4C, with PDZ binding site at C-terminal has been recognized as a positive regulator of EMT. Here, we aimed to examine the inter- action between Erbin and Sema4C. HK2 cells were treated with TGF-β1, or transfected with Erbin and （or） Sema4C. Interaction of Erbin and Sema4C was identified by immunoprecipitation. RT-PCR was used to detect the expression of Erbin and Sema4C at mRNA level after transfection. The expression levels of Erbin, Sema4C, and markers of EMT were measured by using Western blotting or ELISA. Af- ter HK2 cells were stimulated with 10 ng/mL TGF-β1 for 72 h, the protein expression levels of Erbin and Sema4C were both up-regulated, and immunoprecipitation results showed Erbin interacted with Sema4C in HK2 cells both at endogenous and exogenous levels. Furthermore, overexpression of Sema4C suppressed E-cadherin, induced vimentin and promoted fibronectin secretion, indicating Sema4C promotes the process of EMT. However, HK2 cells overexpressing Erbin were resistant to Sema4C-induced EMT. In contrast, Erbin specific siRNA promoted EMT induced by Sema4C. Taken together, these results suggest that Erbin can interact with Sema4C, and co-expression of Erbin blocks the process of Sema4C-induced EMT.

Inhibition of the α-mannosidase Man2c1 gene expression enhances adhesion of Jurkat cells

Protein N-glycosylation plays very important roles in immunity and α-mannosidase is one of the key enzymes in Nglycosylation. This paper reports that inhibition of α-mannosidase Man2c1 gene expression enhances adhesion of Jurkat T cells. In comparison to the controls with normal expression of the enzyme, Jurkat cells with the inhibition of Man2c1 gene expression （AS cell） formed larger aggregates in culture, indicating an enhancement of adhesion between the cells. mRNA differential display analysis discovered up-regulation of several adhesion molecule genes in the AS cell. Because of the pivotal role played by CD54-LFA-1 interaction in immune cell interaction, this study focused on the contribution of enhanced expression of CD54 and LFA-1 to the enhanced adhesion of AS Jurkat cells. These facts, including increased binding of AS cells to ICAM-1-Fc, Mg^2＋ activation of the binding of AS cells to ICAM-1-Fc and enhanced aggregation of AS cells, together with the inhibiting effect of a blocking CD1 la mAb on the binding to ICAM-1-Fc and aggregation of the cells demonstrate an important contribution of enhanced CD54-LFA-1 interaction to increased adhesion between AS cells. The enhanced CD54-LFA-1 interaction also resulted in increased adhesion between AS Jurkat T cells and Raji B cells. In addition, AS cells showed cytoskeletal rearrangement. The data imply a biological significance of MAN2C1 in T-cell functioning.

Role of Protein Kinase C in the Activation of Store-operated Ca^(2+) Entry in Airway Smooth Muscle Cells

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.

Co-regulation of Dectin-1 and TLR2 in inflammatory response of human corneal epithelial cells induced by Aspergillus fumigates

AIM： To investigate the co-regulation of dendritic cell- associated C-type lectin-1 （Dectin-1）, Toll-like receptor 2 （TLR2）, and relative chemotactic factors in the Telomease- immortalized human corneal epithelial （THCE） cells after exposure to ,Aspergillus fumigatus （Af） hyphae. METHODS： The normal THCE cells were investigated as control. After cultured in vitro with Af hyphae, with or without laminarin and anti-TLR2 antibody for 4, 8, 16 and 24h, THCE cells were harvested. The expression of Dectin-1, TLR2, CXCL1 and CXCL8 mRNA were measured by real-time quantitative polymerase chain reaction at the stimulation of 4, 8 and 16h separately. The protein expression of Dectin-1 and TLR2 were analyzed at 8, 16, and 24h by Western blot. ~ RESULTS： The mRNA CXCL8 increased in THCE expression of CXCL1 and cells after stimulated by Af hyphae. The stimulatory effects on these inflammatory chemokines were shown in a dose-dependent manner and reached the peak at 8h. Af hyphae significantly stimulated the production of Dectin-1 and TLR2 in THCE cells at both mRNA and protein levels. The protein of Dectin-1 and TLR2 gradually increased till 16h. While pretreated with laminarin （a Dectin-1 inhibitor）, the expression of TLR2, CXCL1 and CXCL8 all decreased dramatically at the peak point, interestingly, when pretreated with TLR2 neutralizing antibody, the expression of Dectin -1, CXCL1 and CXCL8 also decreased dramatically at the peak point. CONCLUSION： These findings suggest that Dectin-1 and TLR2 co-regulated with each other after treated with inactive Af hyphae in the THCE cells, and they contribute together to the inflammatory responses by induction of chemokines CXCL1 and CXCL8.

Effects of progesterone on T-type-Ca^(2+)-channel expression in Purkinje cells

Plasticity of cerebellar Purkinje cells(PC)is influenced by progeste rone via the classical progeste rone receptors PR-A and PR-B by stimulating dendritogenesis,spinogenesis,and synaptogenesis in these cells.Dissociated PC cultures were used to analyze progeste rone effects at a molecular level on the voltage-gated T-type-Ca^(2+)-channels Ca_(v)3.1,Ca_(v)3.2,and Ca_(v)3.3 as they helped determine neuronal plasticity by regulating Ca^(2+)-influx in neuronal cells.The results showed direct effects of progesterone on the mRNA expression of T-type-Ca^(2+)-channels,as well as on the protein kinases A and C being involved in downstream signaling pathways that play an important role in neuronal plasticity.For the mRNA expression studies of T-type-Ca^(2+)-channels and protein kinases of the signaling cascade,laser microdissection and purified PC cultures of diffe rent maturation stages were used.Immunohistochemical staining was also performed to characte rize the localization of T-type-Ca^(2+)-channels in PC.Expe rimental progesterone treatment was performed on the purified PC culture for 24 and 48 hours.Our results show that progesterone increases the expression of Ca_(v)3.1 and Ca_(v)3.3 and associated protein kinases A and Cin PC at the mRNA level within 48 hours after treatment at latest.These effects extend the current knowledge of the function of progesterone in the central nervous system and provide an explanatory approach for its influence on neuronal plasticity.

Cladogynos orientalis Zipp. extracts inhibit cell culture-derived hepatitis C virus genotype 2a replication in Huh-7 cells through NS5B inhibition

Objective: To evaluate the potential anti-hepatitis C virus (HCV) activities of Cladogynos orientalis Zipp. ex Span and to investigate the molecular mode of action. Methods: Ethanolic and water extracts from various parts of Cladogynos orientalis were examined for cytotoxicity by MTT assay. Sub-cytotoxic concentrations of the extracts were used for further determining anti-HCV activity using cell culture-derived HCV genotype 2a propagated in HepaRG cell line. Immunofluorescence assay was performed to observe the effect on viruses at the pre-entry step. Mode of action at the post-entry step was investigated for the viral RNA and protein expressions by real time RT-PCR and Western blotting assays, respectively. Results: Although Cladogynos orientalis water extracts exhibited lower cytotoxicity than ethanolic extracts, all ethanolic extracts from roots, stems, and leaves of Cladogynos orientalis exhibited higher anti-HCV activities than water extracts. The highest anti-HCV activity was observed in infected cells treated with the extracts 5 h after absorption. No extracts showed pre-viral entry effect. At the post-viral entry step, only leaf ethanolic extracts inhibited NS5B expression, while all extracts did not inhibit HCV NS3 expression. Conclusions: Cladogynos orientalis ethanolic extracts could be further studied and the major active compound needs to be identified as a promising source for anti-HCV agents.

Liguzinediol protects against apoptosis induced by doxorubicin in H9C2 cells

OBJECTIVE Liguzinediol is a derivative of the natural active ingredient ligustrazine,and we found that liguzinediol has significant positive inotropic effects,which are stronger than that of TMP.Besides,it does not lead to arrhythmia,hypotension and other side effects.This study aims to investigate the anti-apoptotic effects of liguzinediolon H9C2 cells.METHODS Apoptotic H9C2 cells induced by DOX were observed by electron microscope and FCM analysis.The protein expressions of Bax,Bcl-2,caspases 3 and NF-κB were detected by Western blotting.RESULTS Apoptotic H9C2 cells induced by DOX were observed,but without apoptotic bodies in liguzinediol group.Declined peak of H9C2 cell apoptosis was seen in liguzinediol group by FCM analysis.And downregulation of Bax,caspases 3,NF-κB and upregulation of Bcl-2 were found by Western blotting.CONCLUSION Liguzinediol protected cardiomyocytes against apoptosis through downregulation of Bax and caspases 3 and upregulation of Bcl-2.Liguzinediol can inhibited cardiomyocyte apoptosis through the NF-κB signal pathway.

Paeoniflorin reduces cardiotoxicity of aconitine in H9c2 cells

OBJECTIVE Aconitine(ACO)as the main active component in Aconitum carmichaelii debeaux(family Ranunlaceae),has highly toxicity in heart and the mechanisms are not clear yet.Paeoniflorin(PF),the main chemical ingredient in Herbaceous peony,can protect heart hurt by antioxidant,vasodilator effect and other effects.In this study,we focused on investigating the mechanism of PF reducing the cardiotoxicity of ACO.METHODS We chose H9c2 cells as experimental subject.MTT,Western blotting and real-time PCR were used to measure cell proliferation,apoptosis,ion channels and oxidative stress.RESULTS Cell proliferation in ACO+PF group was significantly increased compared with ACO group;the ratio with Bcl-2 and Bax and the level of p53 were upregulated by PF,while the level of caspase-3 was lightly reduced.The expression of SCN5A mRNA significantly was increased in ACO+PF group,while the expres⁃sion of RyR2 and Cx43 mRNA was dropped.Compared with ACO group,extracellular LDH and intracellular MDA were highly decreased,while intracellular SOD was regulated.CONCLUSION Cardiotoxicity of ACO in H9c2 cells was signifi⁃cantly decreased by PF.

Antibody to El peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro

AIM： To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus （HCV）. Specific polydonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS： Hyper-immune HCV E1 antibodies were incubated over night at 4 ℃ with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RTPCR and flow cytometry. RESULTS： Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera （72%） showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION： In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.

Polysaccharide extracts of Cirsium japonicum protect rat H9c2 myocardial cells from oxidative stress induced by hydrogen peroxide

Daji （Cirsium japonicum） has been applied against gastric disorders, lung diseases, and cardiovascular problems in thetraditional Chinese medicinal system. The present study was to investigate the protective effects of Daji （Cirsiumjaponicum） polysaccharide extracts （CJP） against hydrogen peroxide （H2O2） shock in rat H9c2 myocardial cells. First,CJP was isolated by hot water extraction and ethanol precipitation; it was then characterized by high performance liquidchromatography and infrared spectrum analysis. Rat H9c2 cells were subjected to H2O2 treatment to establish a cellinjury model. The 3- （4,5- dimethylthiazol- 2-yl）-2,5- diphenyltetrazolium bromide assay showed that CJP pretreatmentsignificantly ameliorated the H2O2 injury in a dose-dependent manner. Furthermore, the cell apoptosis induced by H2O2was markedly inhibited by CJP pretreatment, whereas the cleavage level of caspase-3, -8, and -9 was reduced. Inaddition, the p38 mitogen-activated protein kinase pathway might be involved in the protective effect of CJP onmyocardial cells. Therefore, we conclude that polysaccharide extracts of Daji （Cirsium japonicum） protect rat H9c2myocardial cells from oxidative stress induced by H2O2.