Electroacupuncture improves myocardial fibrosis in heart failure rats by attenuating ECM collagen deposition through modulation of TGF-β1/Smads signaling pathway

Background: To explore the effects of electroacupuncture on cardiac function and myocardial fibrosis in rat models of heart failure, and to elucidate the underlying mechanism of electroacupuncture in heart failure treatment. Methods: Healthy male Sprague-Dawley rats were allocated into three groups: Sham group, Model group, and electroacupuncture (Model + EA) group, with each group comprising 8 rats. The model underwent a procedure involving the ligation of the left anterior descending coronary artery to induce a model of heart failure. The Model + EA group was used for 7 consecutive days for electroacupuncture of bilateral Shenmen (HT7) and Tongli (HT5), once a day for 30 min each time. Left ventricular parameters in rats were assessed using a small-animal ultrasound machine to analyze changes in left ventricular end-diastolic volume, left ventricular end-systolic volume, left ventricular ejection fraction, and left ventricular fractional shortening. Serum interleukin-1β (IL-1β), cardiac troponin (cTn), and N-terminal brain natriuretic peptide precursor levels were measured using ELISA. Histopathological changes in rat myocardium were observed through HE staining, while collagen deposition in rat myocardial tissue was assessed using the Masson staining method. Picro sirius red staining, immunohistochemical staining, and RT-qPCR were utilized to distinguish between the various types of collagen deposition. The expression level of TGF-β1 and SMAD2/3/4/7 mRNA in rat myocardial tissues was determined using RT-qPCR. Additionally, western blot analysis was conducted to assess the protein expression levels of TGF-β1, SMAD3/7, and p-SMAD3 in rat myocardial tissues. Results: Compared with the Sham group, the left ventricular ejection fraction and left ventricular fractional shortening values of the Model group were significantly decreased (P < 0.01);the left ventricular end-diastolic volume and left ventricular end-systolic volume values were remarkably increased (P < 0.01);serum N-terminal brain natriuretic peptide precursor content was increased (P < 0.01);serum IL-1β and cTn levels were increased (P < 0.01);myocardial collagen volume fraction were increased (P < 0.01);and those of the expression of TGF-β1 and SMAD2/3/4 mRNA was increased (P < 0.01);the expression of SMAD7 mRNA was decreased (P < 0.01);the protein expression levels of TGF-β1, SMAD3, and p-Smad3 were increased (P < 0.01);the protein expression level of SMAD7 was decreased (P < 0.01) in the Model group. Compared to the Model group, the expression levels of the proteins TGF-β1, SMAD3, and p-Smad3 in myocardial tissue were found to be decreased (P < 0.01), and the expression level of the protein SMAD7 was found to be increased (P < 0.01) in the Model + EA group;the collagen volume fraction and deposition of type Ⅰ /Ⅲ collagen were decreased (P < 0.01) in the Model + EA group. Conclusion: Electroacupuncture alleviates myocardial fibrosis in rats with heart failure, and this effect is likely due to attributed to the modulation of the TGF-β1/Smads signaling pathway, which helps reduce collagen deposition in the extracellular matrix.

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.

Total flavonoids of Scutellaria barbata improving podocyte injury induced by high glucose through Smad4/PKM2/HIF‑1α pathway

Objective: To observe the effect of total flavonoids of Scutellaria barbata (TF‑SB) on the injury of high glucose induced podocytes (MPC‑5) and the influence of Smad4/PKM2/HIF‑1α pathway. Methods: Firstly, CCK8 was used to analyze the safety and efficacy concentration of TF‑SB on MPC‑5 cells. Then, MPC‑5 was then divided into the control group, model group and TF‑SB group. In addition to the control group, model group and TF‑SB group were induced by high glucose to establish MPC‑5 cell injury model. The effects of TF‑SB on ATP, apoptosis and ROS levels of MPC‑5 cells were detected respectively. The contents of IL‑1β, TNF‑α, and MCP‑1 were determined by ELISA, the expression abundance of glycolytic genes (GLU1, PFK1 and HK1) were detected by RT‑PCR. Western blot method was used to detect the expression level of related proteins in Smad4/PKM2/HIF‑1α pathway. Results: Compared with the blank group, ATP content, GLU1, PKF1 and HK1 expression abundance of MPC‑5 cells in the model group decreased significantly, apoptosis, ROS level and IL‑1 β、 TNF‑ α And MCP‑1 significantly increased (P<0.01);Compared with model group, ATP content, GLU1, PKF1 and HK1 expression abundance, apoptosis, ROS level and IL‑1β in TF‑SB group were significantly increased , TNF‑ α The contents of MCP‑1 and MCP‑1 decreased significantly (P<0.01). In addition, compared with the blank group, the model group Smad4 and HIF‑1 α The protein expression and PKM2 expression in nucleus were significantly increased, while PKM2 expression in cytoplasm was significantly decreased (P<0.01);Compared with model group, TF‑SB group Smad4, HIF‑1 α The expression of PKM2 in the nucleus and expression of PKM2 were significantly decreased, while the expression of PKM2 in the cytoplasm was significantly increased (P<0.01). Conclusion: TF‑SB promotes the mitochondrial activity of MPC‑5 cells to induce glycolysis, and then inhibits the secretion of inflammation, which may play a role in treating diabetes nephropathy by inhibiting Smad4/PKM2/HIF‑1α signaling pathway.

MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.

Berberine Attenuates Cigarette Smoke Extract-induced Airway Inflammation in Mice:Involvement of TGF-β1/Smads Signaling Pathway

Although several studies confirmed that berberine may attenuate airway inflammation in mice with chronic obstructive pulmonary disease(COPD),its underlying mechanisms were not clear until now.We aimed to establish an experiment mouse model for COPD and to investigate the effects of berberine on airway inflammation and its possible mechanism in COPD model mice induced by cigarette smoke extract(CSE).Twenty SPF C57BL/6 mice were randomly divided into PBS control group,COPD model group,low-dose berberine group and high-dose berberine group,5 mice in each group.The neutrophils and macrophages were examined by Wright's staining.The levels of inflammatory cytokines TNF-α and IL-6 in bronchoalveolar lavage fluid(BALF)were detennined by enzyme-linked immunosorbent assay.The expression levels of TGF-β1,Smad2 and Smad3 mRNA and proteins in lung tissues were respectively detected by quantitative real-time polymerase chain reaction and Western blotting.It was found that CSE increased the number of inflammation cells in BALF,elevated lung inflammation scores,and enhanced the TGF-β1/Smads signaling activity in mice.High-dose berberine restrained the alterations in the COPD mice induced by CSE.It was concluded that high-dose berberine ameliorated CSE-induced airway inflammation in COPD mice.TGF-β1/Smads signaling pathway might be involved in the mechanism.These findings suggested a therapeutic potential of high-dose berberine on the CSE-induced airway inflammation.

Cetirizine regulates scleroderma skin fibrosis in mice via the TGF-β1/Smad3 signaling pathway

Objective:To investigate the effect of cetirizine on the fibrosis of skin tissue in systemic sclerosis(SSc)mice and its mechanism of action.Methods:Thirty-two BALB/C mice were randomly divided into a blank group,a model group,a cetirizine low-dose group,and a cetirizine high-dose group,with eight in each group.The blank group was injected with normal saline on the back,and the other three groups were injected with bleomycin on the back to prepare SSc mouse models.The mice were injected once a day for 28 consecutive days,while the normal group and the model group were given saline.The dose group was administrated intragastrically at 2 mg/kg and 5 mg/kg,respectively,for 28 consecutive days.Detect the thickness of the dermis by taking the skin tissue in the back injection area of each group.Hematoxylin-eosin staining(HE)and Masson staining.Sample hydrolysis method to detect hydroxyproline(HYP)content in skin tissue.Immunohistochemical detection ofα-smooth muscle actin(α-SMA)expression in skin tissues.Enzyme-linked immunosorbent assay(ELISA)to detect serum interleukin(IL-6,IL-10)and transforming growth factor(TGF-αand TGF-β1).Quantitative real-time PCR(qRT-PCR)was used to detect the expression levels of collagen type I(COL1A1),type III collagen(COL3A1),Smad homolog 3(Smad3),and TGF-β1 mRNA.Western blot was used to detect the expression levels of COL1A1,COL3A1 and p-Smad3.Results:Compared with the blank group,the dermis thickness and HYP content of the model group increased,the skin tissue lesions and fibrosis were more severe,theα-SMA positive expression intensity in the skin tissue was higher,and the serum IL-6,IL-10,TGF-α,TGF-β1 content increased,COL1A1,COL3A1,Smad3,TGF-β1 mRNA expression levels increased in skin tissues,COL1A1,COL3A1,p-Smad3 protein expression increased,the differences were statistically significant(P<0.05).Compared with the model group,the dermal thickness and HYP content of the low and high dose cetirizine groups were reduced,the degree of skin tissue lesions and fibrosis was improved,the expression ofα-SMA in skin tissues was weakened,the levels of IL-6,IL-10,TGF-α,TGF-β1 in serum were reduced,the expression levels of COL1A1,COL3A1,Smad3 and TGF-β1 in skin tissues were reduced,and the expression levels of COL1A1,COL3A1,and p-Smad3 proteins were reduced,the decrease in the high-dose group was more significant,and the differences were statistically significant(P<0.05).Conclusion:Cetirizine can improve the degree of fibrosis of skin tissue in SSc mice and reduce the immune inflammation response.The mechanism of action is related to the TGF-β1/Smad3 signaling pathway.

Role of transforming growth factor-beta1-smad signal transduction pathway in patients with hepatocellular carcinoma

AIM： To explore the role of transforming growth factorbeta1 （TGF-β1）-smad signal transduction pathway in patients with hepatocellular carcinoma. METHODS： Thirty-six hepatocellular carcinoma specimens were obtained from Qidong Liver Cancer Institute and Department of Pathology of the Second Affiliated Hospital of Nanjing Medical University. All primary antibodies （polyclonal antibodies） to TGF-β1, type H Transforming growth factor-beta receptor （TβR-Ⅱ）, nuclear factor-kappaB （NF-KB）, CD34, smad4 and smad7, secondary antibodies and immunohistochemical kit were purchased from Zhongshan Biotechnology Limited Company （Beijing, China）. The expressions of TGF-β1, TβR-Ⅱ, NF- KB, smad4 and smad7 proteins in 36 specimens of hepatocellular carcinoma （HCC） and its adjacent tissue were separately detected by immunohistochemistry to observe the relationship between TGF-β1 and TβR-Ⅱ, between NF-KB and TGF-β1, between smad4 and smad7 and between TGF-β1 or TβR-Ⅱ and microvessel density （MVD）. MVD was determined by labelling the vessel endothelial cells with CD34. RESULTS： The expression of TGF-β1, smad7 and MVD was higher in HCC tissue than in adjacent HCC tissue （P〈0.01, P〈0.05,P〈0.01 respectively）. The expression of TβR-Ⅱ and smad4 was lower in HCC tissue than in its adjacent tissue （P〈0.01, P〈0.05 respectively）. The expression of TGF-β1 protein and NF-KB protein was consistent in HCC tissue. The expression of TGF-β1 and MVD was also consistent in HCC tissue. The expression of TIER- Ⅱ was negatively correlated with that of MVD in HCC tissue. CONCLUSION： The expressions of TGF-IB1, TβR- Ⅱ, NF-KB, smad4 and smad7 in HCC tissue, which are major up and down stream factors of TGF-β1-smad signal transduction pathway, are abnormal. These factors are closely related with NVD and may play an important role in HCC angiogenesis. The inhibitory action of TGF-β1 is weakened in hepatic carcinoma cells because of abnormality of TGF-β1 receptors （such as TIBR- Ⅱ） and postreceptors （such as smad4 and smad7）. NF-KB may cause activation and production of TGF-β1.

Elevated retinol binding protein 4 levels are associated with atherosclerosis in diabetic rats via JAK2/STAT3 signaling pathway

BACKGROUND Atherosclerosis is a major cause of mortality worldwide and is driven by multiple risk factors,including diabetes,which results in an increased atherosclerotic burden,but the precise mechanisms for the occurrence and development of diabetic atheroscerosis have not been fully elucidated.AIM To summarize the potential role of retinol binding protein 4(RBP4) in the pathogenesis of diabetic atheroscerosis,particularly in relation to the RBP4-Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway.METHODS Male Wistar rats were randomly divided into three groups,including a control group(NC group),diabetic rat group(DM group),and diabetic atherosclerotic rat group(DA group).The contents of total cholesterol(TC), high-density lipoprotein cholesterol(HDL-c), triglycerides(TG), low-density lipoprotein cholesterol(LDLc), fasting insulin(FINS),fasting plasma glucose,and hemoglobin A1 c(HbA1 c)were measured.Moreover,the adipose and serum levels of RBP4,along with the expression levels of JAK2, phosphorylated JAK2(p-JAK2), STAT3,phosphorylated STAT3(p-STAT3), B-cell lymphoma-2(Bcl-2), and Cyclin D1 in aortic tissues were also measured.Besides,homeostasis model assessment of insulin resistance(HOMA-IR) and atherogenic indexes(AI) were calculated.RESULTS Compared with the NC and DM groups,the levels LDL-c,TG,TC,FINS,HOMAIR,RBP4,and AI were upregulated,whereas that of HDL-c was downregulated in the DA group(P <0.05);the mRNA levels of JAK2,STAT3,Cyclin D1,and Bcl-2 in the DA group were significantly increased compared with the NC group and the DM group;P-JAK2,p-JAK2/JAK2 ratio,p-STAT3,p-STAT3/STAT3 ratio,Cyclin D1,and Bcl-2 at protein levels were significantly upregulated in the DA group compared with the NC group and DM group.In addition,as shown by Pearson analysis,serum RBP4 had a positive correlation with TG,TC,LDL-c,FINS,HbA1 C,p-JAK2,p-STAT3,Bcl-2,Cyclin D1,AI,and HOMA-IR but a negative correlation with HDL-c.In addition,multivariable logistic regression analysis showed that serum RBP4,p-JAK2,p-STAT3,and LDL-c were predictors of the presence of diabetic atherosclerosis.CONCLUSION RBP4 could be involved in the initiation or progression of diabetic atherosclerosis by regulating the JAK2/STAT3 signaling pathway.

Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/MyD88 signaling pathway in BV2 cells

Ramulus Cinnamomi （RC）, a traditional Chinese herb, has been used to attenuate inflammatory responses. The purpose of this study was to investigate the effect of RC extract on lipopolysaccharide （LPS）-induced neuroinflammation in BV2 microglial cells and the underlying mechanisms involved. BV2 cells were incubated with normal medium （control group）, LPS, LPS plus 30 pg/mL RC extract, or LPS plus 100 pg/mL RC extract. The BV2 cell morphology was observed under an optical microscope and cell viability was detected by MTT assay. Nitric oxide level in BV2 cells was detected using Griess regents, and the levels of interleukin-6, interleukin-1 β, and tumor necrosis factor u in BV2 cells were determined by ELISA. The expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 proteins were detected by western blot assay. Compared with the LPS group, both 30 and 100 μg/mL RC extract had no significant effect on the viability of BV2 cells. The levels of nitric oxide, interleukin-6, interleukin-1β and tumor necrosis factor ct in BV2 cells were all significantly increased after LPS induction, and the levels were significantly reversed after treatment with 30 and 100 μg/mL RC extract. Furthermore, RC extract significantly inhibited the protein expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 in LPS-induced BV2 cells. Our findings suggest that RC extract alleviates neuroinflammation by downregulating the TLR4/MyD88 signaling pathway.

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells

Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

Cross-talk between Smad4 and P38 Proteins in Non-small Cell Lung Cancer

Objective： Impaired signal transduction is associated with tumorigenesis and progression of various kinds of human cancers. Transforming growth factor （TGF）-beta/Smad and ras-mitogen activated protein kinase （MAPK） are two major signal transduction pathways for adjusting cell proliferation and differentiation. Little is known about TGF-beta/Smad4 in non-small cell lung cancer （NSCLC）. Hereby, we investigated the expression of Smad4 in NSCLC, its correlation with MAPK proteins （including p38, ERK1 and JNK1 proteins） and their clinical significance in NSCLC. Methods： The expressions of Smad4, p38, ERK1 and JNK1 were detected at protein level with Western blotting and immunohistochemistry, at transcription level with RT-PCR. Statistical analysis was performed for the comparisons of expressions of Smad4, p38, ERK1 and JNK1, and their correlation with various clinicopathological parameters and the prognosis of NSCLC. Results： The levels of protein and mRNA expression of Smad4 in lung cancer tissues were significantly lower than in normal tissues （P〈0.05）. All these four proteins were associated with TNM staging. There was a strongly negative correlation between p38 and Smad4. Expressions of Smad4, p38 and JNK1, as well as tumor differentiation and staging were significantly correlated with the prognosis of NSCLC by univariate analysis. By multivariate analysis, only Smad4, p38, tumor differentiation and staging were correlated with the prognosis. Taken together, the negative expression of p38 and positive expression of Smad4 were associated with a better prognosis of NSCLC. Conclusion： Smad4 could be of vital importance for the initiation and development of NSCLC. The expression of Smad4 might be inhibited by p38, supporting a cross-talk between main proteins of TGF-beta/Smad and ras-MAPK signal transduction pathways. Smad4 and p38 could be possible prognostic factors for NSCLC.

Cytokine receptor-like factor 1(CRLF1)promotes cardiac fibrosis via ERK1/2 signaling pathway

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease.Anti-fibrosis treatment is a significant therapy for heart disease,but there is still no thorough understanding of fibrotic mechanisms.This study was carried out to ascertain the functions of cytokine receptor-like factor 1(CRLF1)in cardiac fibrosis and clarify its regulatory mechanisms.We found that CRLF1 was expressed predominantly in cardiac fibroblasts.Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction,but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-β1(TGF-β1).Gain-and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts(NMCFs)with or without TGF-β1 stimulation.CRLF1 overexpression increased cell viability,collagen production,cell proliferation capacity,and myofibroblast transformation of NMCFs with or without TGF-β1 stimulation,while silencing of CRLF1 had the opposite effects.An inhibitor of the extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway and different inhibitors of TGF-β1 signaling cascades,comprising mothers against decapentaplegic homolog(SMAD)-dependent and SMAD-independent pathways,were applied to investigate the mechanisms involved.CRLF1 exerted its functions by activating the ERK1/2 signaling pathway.Furthermore,the SMAD-dependent pathway,not the SMAD-independent pathway,was responsible for CRLF1 up-regulation in NMCFs treated with TGF-β1.In summary,activation of the TGF-β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression.CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway.CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.

Linggui Zhugan Decoction(苓桂术甘汤)Inhibits Ventricular Remodeling after Acute Myocardial Infarction in Mice by Suppressing TGF-β1 Smad Signaling Pathway

Objective:To investigate the inhibitory effect of Linggui Zhugan Decoction(LZD,苓桂术甘汤)on the ventricular remodeling(VR)after acute myocardial infarction(AMI)and related mRNA and proteins expression in transforming growth factor-beta 1(TGF-β1)/Smad signaling pathway,and explain its putative mechanism.Methods:A VR model was generated by ligation of coronary artery in mice.Two weeks after surgery,60 mice were randomly divided into the model group,the sham-operation group(distilled water),the positive control group(2.4 mg/kg simvastatin),and the low-,medium-and high-dose LZD groups(2.1,4.2,8.4 g crude drug/kg,respectively)by a random number table,10 mice in each group.Mice in each group was treated for 4 weeks.Changes of hemodynamics indices and cardiac weight index were detected by the PowerLab data acquisition and analysis recording instrument.Morphology changes of myocardial tissue were observed by hematoxylin-eosin and Masson staining.The expressions of TGF-β1,Smad2,Smad3,p-Smad2 and p-Smad3 in myocardial tissue were detected by Western blotting.The mRNA expressions of TGF-β1,Smad2 and Smad3 were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR).The expressions of matrix metalloprotein 2(MMP2),MMP9,collagenⅠand collagen Ⅲ were observed by immunohistochemical methods.Results:VR mice showed significant dysfunction in hemodynamic indices and cardiac structure and function.Compared with the shamoperation group,myocardial tissue damage,interstitial fibrosis occurred in the model mice,left ventricular systolic pressure(LVSP),left ventricular pressure maximum contraction rate(+dp/dtmax)and left ventricular pressure maximum relaxation rate(-dp/dtmax)decreased significantly(all P<0.01),while left ventricular end-diastolic pressure(LVEDP),cardiac weight index and left ventricular weight index elevated significantly,meanwhile TGF-β1,p-Smad2,p-Smad3,Smad2,Smad3,MMP2,MMP9,collagen Ⅰ,collagen Ⅲ protein expressions in myocardial tissue and TGF-β1 Smad2 and Smad3 mRNA expressions increased significantly(all P<0.01).Compared with the model group,LZD could significantly improve the pathological changes of myocardial tissue,increase LVSP,+dp/dtmax and-dp/dtmaxf lower LVEDP,reduce the whole heart weight index and left ventricular weight index and inhibit the over-expressions of TGF-β1f p-Smad2,p-Smad3,Smad2,Smad3,MMP2,MMP9,collagenⅠand collagenⅢproteins in myocardial tissue and mRNA expressions of TGF-β1 Smad2 and Smad3(P<0.05 or P<0.01).Conclusion:LZD can significantly suppress VR induced by AMI,and its underlying mechanism may be associated with its inhibitory effect on the TGF-β1/Smad signaling pathway.

Association of overexpression of TIF1γ with colorectal carcinogenesis and advanced colorectal adenocarcinoma

AIM:To determine the expression and clinical significance of transcriptional intermediary factor 1 gamma (TIF1γ),Smad4 and transforming growth factor-beta (TGFβR) across a spectrum representing colorectal cancer (CRC) development.METHODS:Tissue microarrays were prepared from archival paraffin embedded tissue,including 51 colorectal carcinomas,25 tubular adenomas (TA) and 26 HPs,each with matched normal colonic epithelium.Immunohistochemistry was performed using antibodies against TIF1γ,Smad4 and TGFβ RⅡ.The levels of expression were scored semi-quantitatively (score 0-3 or loss and retention for Smad4).RESULTS:Overexpression of TIF1γ was detected in 5/26 (19%) HP;however,it was seen in a significantly higher proportion of neoplasms,15/25 (60%) TAs and 24/51 (47%) CRCs (P<0.05).Normal colonic mucosa,HP,and TAs showed strong Smad4 expression,while its expression was absent in 22/51 (43%) CRCs.Over-expression of TGFβ RⅡ was more commonly seen in neoplasms,13/25 (52%) TAs and 29/51 (57%) CRCs compared to 9/26 (35%) HP (P<0.05).Furthermore,there was a correlation between TIF1γ overexpression and Smad4 loss in CRC (Kendall tau rank correlation value=0.35,P<0.05).The levels of TIF1γ overexpression were significantly higher in stage Ⅲ than in stage Ⅰ and Ⅱ CRC (P<0.05).CONCLUSION:The findings suggest that over-expression of TIF1γ occurs in early stages of colorectal carcinogenesis,is inversely related with Smad4 loss,and may be a prognostic indicator for poor outcome.

Celastrol Protects TGF-β1-induced Endothelial-mesenchymal Transition

The endothelial-to-mesenchymal transition（End MT） in endothelial cells contributes to the development of cardiac fibrosis,ultimately leading to cardiac remodeling.In this study,the effects and molecular mechanisms of celastrol（CEL） on transforming growth factor-β1（TGF-β1）-induced End MT in human umbilical vein endothelial（HUVEC-12） cells were investigated.The presented data demonstrated that CEL significantly blocked the morphology change of HUVEC-12 cells induced by TGF-β1 without cell cytotoxicity.In accordance with these findings,CEL blocked TGF-β1-induced EndM T as evidenced by the inhibition of the mesenchymal markers,including collagen Ⅰ,Ⅲ,α-SMA,fibronectin m RNA expression,and the increase in the m RNA expression of endothelial cell marker CD31.These changes were also confirmed by double immunofluorescence staining of CD31 and vimentin.The in vitro scratch assay showed that CEL inhibited the migration capacity of the transitioned endothelial cells induced by TGF-β1.Further experiments showed that the beneficial effect of CEL on blocking the End MT in HUVEC-12 cells was associated with the suppression of the TGF-β1/Smads signalling pathway,which was also confirmed by the inhibition of its downstream transcription factor snail1,twist1,twist2,ZEB1 and ZEB2.These results indicate that CEL blocks TGF-β1-induced End MT through TGF-β1/Smads signalling pathway and suggest that it may be a feasible therapy for cardiac fibrosis diseases.

Qingxuan Jiangya Decoction(清眩降压汤) Prevents Blood Pressure Elevation and Ameliorates Vascular Structural Remodeling via Modulating TGF-β 1/Smad Pathway in Spontaneously Hypertensive Rats

Objective:To elevate the effects of Qingxuan Jiangya Decoction(清眩降压汤,QXJYD)on hypertension and vascular structural remodeling(VSR)in spontaneously hypertensive rats(SHRs),and investigate the underlying mechanisms.Methods:SHRs(n=8)were given intra-gastric administration with 60 mg/kg of QXJYD or saline,daily for 8 weeks,while rats in SHR-control(n=8)and WKY(n=8)groups were received equal volumes of saline solution.Systolic blood pressures(SBP),diastolic blood pressures(DBP)and mean blood pressures(MBP)were measured once a week.The levels of angiotensinⅡ(AngⅡ),endothelin 1(ET-1)and plasma renin activity(PRA)were tested by enzyme-linked immunosorbent assay(ELISA)and radioimmunoassay,respectively.The effect of QXJYD on VSR was determined by examining the media thickness and the ex vivo contractility of thoracic aortic.The proliferation and fibrosis of vascular smooth muscle cells(VSMCs)were examined via immunohistochemical(IHC)staining for proliferating cell nuclear antigen(PCNA),collagen Ⅰ and collagen Ⅲ,respectively.The mRNA and protein expressions of transforming growth factor β1(TGF-β1),Smad3 and phosphorylation of Smad3 in thoracic aorta tissues were determined by real-time polymerase chain reaction(PCR)and Western blot assay,respectively.Results:QXJYD treatment led to a significant decrease of the elevation of blood pressure in SHRs and reduced the levels of AngⅡ,ET-1 and PRA in the serum(P<0.05).In addition,QXJYD treatment remarkably ameliorated VSR and vascular function in SHRs.Moreover,QXJYD inhibited VSMC proliferation and fibrosis by suppressing the expression of PCNA,collagen Ⅰ and collagen Ⅲ in thoracic aortic.Furthermore,QXJYD inhibited the expression of TGF-β1,Smad3 and the phosphorylation of Smad3,respectively(P<0.05).Conclusion:QXJYD reversed VSR by inhibiting VSMC proliferation and collagen deposition via regulation of TGF-β1/Smad signaling pathway,which may,in part,illuminate its anti-hypertensive activities.

Pharmacological mechanisms of Yishen Xingyang capsule in the treatment of oligoasthenospermia in rats

Objective:To investigate the therapeutic effects and pharmacological mechanisms of Yishen Xingyang capsule(YXC)in oligoasthenospermia(OA)rats.Methods:Forty-eight male SpragueeDawley rats were randomly divided into eight groups of six rats each:normal control(NC);model control(MC);three different positive drug(PD);and low-,medium-,and high-dose YXC groups.A rat model of OA was established by administering glucosides of Tripterygium wilfordii Hook.F(GTW).After YXC administration,penile erectile function was observed.The epididymis,blood,and testes of the rats were harvested for analysis of sperm quality,sex hormone levels,mitochondrial membrane potential,and the transforming growth factor(TGF)-b1/Smad signaling pathway.Results:Compared with that in the MC group,penile erectile function in the YXC groups and three PD groups increased(all P<.01).Moreover,sperm quality in the YXC groups and three PD groups improved(all P<.001).The levels of testosterone,follicle stimulating hormone,and luteinizing hormone in the three PD and YXC groups increased(all P<.05).The mitochondrial membrane potential in the three PD and YXC groups significantly improved(all P<.001).Furthermore,the YXC and three PD groups showed decreased TGF-b1 expression(all P<.05)compared with the MC group.The high-dose YXC group and three PD groups improved Smad2 and Smad4 expression(all P<.05).Conclusion:YXC improved penile erectile function and sperm quality in OA rats,and the underlying mechanism included increase in sex hormones,inhibition of sperm apoptosis,and regulation of the TGFb1/Smad signaling pathway.Meanwhile,this study provides a new effective drug option for the treatment of OA,which is beneficial to male reproductive health and social harmony.

Anti-ageing effects of a new Dimethylaminoethanol-based formulation on DGalactose induced skin ageing model of rat

Background Dimethylaminoethanol has been widely used to fight against wrinkles, in the field of aesthetic medicine there is an increasing demand for safe and effective Dimethylaminoethanol-based products to counteract the ageing process. Objective To evaluate the anti- ageing effects of a new DMAE- based formulation. Methods 30 male rats were randomly allocated into treatment,D-gal ageing modeland control groups, each of which contained ten rats.Treatment group and D- gal ageing model group were subcutaneously injected with D- galactose prepared in normal saline 125mg·kg-1·d-1for 42 d. Control groups were injected with normal saline for42 d with same method and dose. From the 18 th day,after shaving their hair,the treatment grouprats were injected thisnew DMAE-based formulation at a dose of 1ml per week for 4 weeks in the Dermis of two sides hip skin mark zone.Meanwhile,D-gal ageing model group rats were administrated the same volume of normal saline with same method. Skin specimens were obtained 3days after the last treatment. Dermal collagen density and dermal thickness were evaluated by H&E and Massontrichrome staining. And m RNA expressions of TGFβ1, Smad3, Type I,Type III Pro-collagen,TIMP-1,MMP- 1,were assessed by Real- time quantitative polymerase chain reaction. Results Dermal thickness, dermal collagen density and hydroxyproline content in treatment group increased significantly comparing with D- gal ageing model group. No differences were found in m RNA expression of MMP- 1 and Type III Pro- collagen between the treatment group and D- gal ageing model group. In addition, m RNA expression of TGFβ1, Type I Pre-collagen, TIMP1 and smad3 in treatment group were significantly up- regulated in contrast with D- gal ageing model and control group. Conclusion This new DMAE- based formulationcould generate anti- ageing effects by activating collagen synthesisthrough TGF-β1/Smads signaling pathway.

Use of network pharmacology and molecular docking to explore the potential mechanism governing the efficacy of Jinhuaqinggan granules in the treatment of novel coronavirus-induced pneumonia

Background:Now that the epidemic of new coronavirus pneumonia(corona virus disease 2019)is spreading all over the world,Jinhuaqinggan granules in the Chinese treatment plan has been proved to be an effective Chinese patent medicine for the treatment of corona virus disease 2019.Methods:This study aims to clarify the possible therapeutic mechanism governing the efficacy of Jinhuaqinggan granules in the treatment of corona virus disease 2019,through using network pharmacology and molecular docking.During the analysis,227 active components were obtained and screened by using the ADME method.Furthermore,282 Jinhuaqinggan granule targets and 56 common targets with corona virus disease 2019 were gathered from various databases.Then the protein-protein interaction network of Jinhuaqinggan granules and corona virus disease 2019 targets were constructed and 6 core targets were selected through network topology analysis.In addition,A total of 262 biological function annotation entries(P<0.01)and 101 pathways(P<0.01)were obtained by gene ontology functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis.Results:Molecular docking showed that quercetin,luteolin,kaempferol,wogonin and naringin had an affinity for SARS-CoV-23CL hydrolase and angiotensin-converting enzyme 2.Conclusion:corona virus disease 2019 can be prevented by the primary targets of Jinhuaqinggan granules.The most important bioactive components in Jinhuaqinggan granules-quercetin,naringenin,luteolin and wogonin-can play antiviral effect,anti-inflammatory storm,regulate immunity by regulating signal transducers and activators of transcription 1,interleukin 4,interferon-γ,heme oxygenase 1 and acting on the lipopolysaccharide response,toll-like receptor signaling pathway,mitogen-activated protein kinase signaling pathway,etc.

Transferrin receptor-targeted immunostimulant for photodynamic immunotherapy against metastatic tumors through β-catenin/CREB interruption

The immunosuppressive phenotype of tumor cells extensively attenuates the immune activation effects of traditional treatments.In this work,a transferrin receptor(TfR)targeted immunostimulant(PTI)is fabricated for photodynamic immunotherapy against metastatic tumors by interrupting β-catenin signal pathway.To synthesize PTI,the photosensitizer conjugated TfR targeting peptide moiety(Palmitic-K(PpIX)-HAIYPRH)is unitized to encapsulate the transcription interrupter of ICG-001.On the one hand,the recognition of PTI and TfR can promote drug delivery into tumor cells to destruct primary tumors through photodynamic therapy and initiate an immunogenic cell death with the release of tumorassociated antigens.On the other hand,PTI will interrupt the binding between b-catenin andcAMP response element-binding protein(CREB),regulating the gene transcription to downregulate programmed death ligand 1(PD-L1)while upregulating CeC motif chemokine ligand 4(CCL4).Furthermore,the elevated CCL4 can recruit the dendritic cells to present tumor-specific antigens and promote T cells activation and infiltration,and the downregulated PD-L1 can avoid the immune evasion of tumor cells and activate systemic anti-tumor immunity to eradicate lung metastasis.This work may inspire the development of antibody antibody-free strategy to activate systemic immune response in consideration of immunosuppressive conditions.