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.

Effects of Cigu Xiaozhi Formula on miR-378a-3p Expression and Hh Signaling Pathway in TGF-β1 Induced LX2 Cells

[Objectives]To observe the effects of Cigu Xiaozhi Formula on miR-378a-3p expression and Hh signaling pathway in TGF-β1 induced and activated LX2 cells.[Methods]Cells were divided into control group,induction group,drug-containing serum group,miR-378a-3p inhibitor group,and miR inhibitor NC group.CCK-8 method was used to detect the cell viability of each group,and flow cytometry was used to detect the apoptosis rate of each group.RT-qPCR was used to detect the expression of miR-378a-3p in each group s cells,and RT-qPCR and Western blot were used to detect mRNA and protein expression of Shh,Gli1,Gli2,Col-I,andα-SMA in each group s cells.[Results]Compared with the control group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,andα-SMA mRNA and protein in induction group increased(P<0.01),while the expression of miR-378a-3p decreased(P<0.01).Compared with the induction group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA andα-SMA and Gli2 protein decreased in drug-containing serum group(P<0.05),while cell apoptosis rate and miR-378a-3p expression increased(P<0.01).In miR-378a-3p inhibitor group,cell viability and the expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA and Gli1,Gli2,α-SMA protein increased(P<0.05,P<0.01),while the apoptosis rate and miR-378a-3p expression decreased(P<0.05,P<0.01).[Conclusions]Cigu Xiaozhi Formula containing serum can upregulate miR-378a-3p expression and downregulate the expression of Gli2 andα-SMA in TGF-β1 induced LX2 cells,thereby inhibiting the activation of LX2 cells and exerting the effects of anti liver fibrosis.

TGF-β1/SMAD SIGNALING PATHWAY MEDIATES p53-DEPENDENT APOPTOSIS IN HEPATOMA CELL LINES

Objective To determine whether transforming growth factor betal (TGF-β1)/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines.Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study.TGF-β1-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay.For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements.After transfection, cells were treated with TGF-β1, then assayed for luciferase activity.Results The apoptosis rate of HepG2 cell lines (48.51%± 8.21%) was significantly higher than control ( 12.72%±2.18%, P<0.05).But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines.The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4.38) was significantly higher than control (1.00, P< 0.05).But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control.Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines.Smad4 is a central mediator of TGF-β1 signaling transdution pathway.TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.

Involvement of M3 Cholinergic Receptor Signal Transduction Pathway in Regulation of the Expression of Chemokine MOB-1, MCP-1 Genes in Pancreatic Acinar Cells

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-κB and the expression of chemokine MOB-1, MCP-1genes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-κB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10 -3 mol/L, 10 -4 mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10 -3 mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-κB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10 -3 mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10 -5 mol/L atropine) or NF-κB inhibitor (10 -2 mol/L PDTC) could obviously inhibit the activation of NF-κB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-1genes in pancreatic acinar cells in vitro through the activation of NF-κB.

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.

YWHAH activates the HMGA1/PI3K/AKT/mTOR signaling pathway by positively regulating Fra-1 to affect the proliferation of gastric cancer cells

Fos-related antigen 1(Fra-1)is a nuclear transcription factor that regulates cell growth,differentiation,and apoptosis.It is involved in the proliferation,invasion,apoptosis and epithelial mesenchymal transformation of malignant tumor cells.Fra-1 is highly expressed in gastric cancer(GC),affects the cycle distribution and apoptosis of GC cells,and participates in GC occurrence and development.However,the detailed mechanism of Fra-1 in GC is unclear,such as the identification of Fra-1-interacting proteins and their role in GC pathogenesis.In this study,we identified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta(YWHAH)as a Fra-1-interacting protein in GC cells using co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry.Experiments showed that YWHAH positively regulated Fra-1 mRNA and protein expression,and affected GC cell proliferation.Whole proteome analysis showed that Fra-1 affected the activity of the high mobility group AT-hook 1(HMGA1)/phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(AKT)/mechanistic target of rapamycin(mTOR)signaling pathway in GC cells.Western blotting and flow cytometry confirmed that YWHAH activated HMGA1/PI3K/AKT/mTOR signaling pathway by positively regulating Fra-1 to affect GC cell proliferation.These results will help to discover new molecular targets for the early diagnosis,treatment,and prognosis prediction of GC.

Xinfuli Granule improves post-myocardial infarction ventricular remodelingand myocardial fibrosis in rats by regulating TGF-β/Smads signaling pathway

Background Recent clinical and experimental studies have confirmed the effects of Xinfuli Granule （XG）, a compound Chinesemedicine in the prevention and treatment of heart failure （HF）. This study aimed to investigate the effects and the mechanisms of XG onventricular reconstruction in rats with acute myocardial infarction （AMI）. Methods Sprague-Dawley rats were subjected to left anteriordescending branch ligation. The rats that survived 24 h were randomly assigned to five groups： medium-dose of XG group （MI＋XGM）,high-dose of XG group （MI＋XGH）, carvedilol group （MI＋C）, medium-dose of XG ＋ carvedilol group （MI＋C＋XGM）. Fourteen rats under-went identical surgical procedures without artery ligation, serving as sham controls. At 28 days, left ventricular weight to body weight（LVW/BW） and heart weight to body weight （HW/BW） were calculated; left ventricular ejection fraction （LVEF）, left ventricular shorteningfraction （LVFS）, left ventricular internal diameter at systole （LVIDS） were measured by ultrasound; HE staining, Masson staining, and Siriusred staining were used to assess the myocardial pathological and physiological changes as well as myocardial fibrosis area and non-infarctzone Ⅰ/Ⅲ collagen ratio. Expression of Smad3 were detected and analyzed by Western blot, immunohistochemistry and immunofluorescenceP-Smad3, Smad2 and Smad7 in the TGF-13/Smads signaling pathway were also analyzed by Western blot. Results The LVIDS （P 〈 0.01）,HW/BW （P 〈 0.05）, type UIII collagen ratio （P 〈 0.01） and myocardial collagen （P 〈 0.01） decreased significantly while the LVW/BW,LVFS （P 〈 0.05） increased significantly in MI＋XGM group as compared with those in other groups. The expression of key signal moleculesof the TGF-β/Smads signaling pathway, including Smad3, P-Smad3 and Smad2 protein were decreased, while the expression of Smad7 in-creased in both XG and carvedilol treatment groups as compared to those of the MI group （all P 〈 0.01）. Immunohistochemistry and im-munofluorescence further confirmed the down-regulated Smad3 expression. Conclusion XG can improve ventricular reconstruction andinhibit myocardial fibrosis in rats with AMI by regulating TGF-β/Smads 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.

Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells

Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.

Study of Celastrol on Akt Signaling Pathway and Its Roles in the Apoptosis of K562 Cells

The purpose Celastrol, the main active compound of the Celastrus genus plants, belonging to Celastraceae, has recently marked antitumour potency on solid tumours of various derivations, Methods: We demonstrate here that Celastrol also present powerful antileukaemic potency through both growth arrest and apoptosis induction in K562 cells, which was accompanied by typical apoptotic morphological and sharp decreased expression of phosphorylation level of Caspase family members and Akt signaling pathway related proteins were determined by western blot before and after celastrol treatment, and further the effect of AKT signaling pathway on celastrol-induced-apoptosis was analyzed. However, in vitro treatment with Celastrol resulted in significantly reduced expression of phophorylation of Akt, Survivin and Bcl-2 significantly in K562 cells. Results: 25 nmol/L WORT (PI3K-Akt inhibitor) can significantly augmented cell apoptosis induced by Celastrol in K562 cells in dose-dependent manner, Moreover, most Caspase3,8,6 were activated in K562 cells during Celastrol treatment, 50 μmol/Lz-VAD-fmk (Caspase inhibitor) can to enhance the apoptosis induced by Celastrol. Discussion: These results suggest that the fact that Akt signaling pathway might act as new targets of Celastrol, correlates well with the sensitivity to Celastrol, as well as the rate of apoptosis induced by Celastrol, Mechanisms that regulate Akt signaling pathway may be provide novel opportunities for drug development.

青光安缓释剂对抗青光眼术后滤过道瘢痕化TGF-β/Smad信号传导通路相关mRNA的影响

目的:观察青光安缓释剂对抗青光眼术后滤过道瘢痕化过程中TGF-β/Smad信号传导通路相关mRNA的影响,探讨青光安缓释剂对滤过道瘢痕化的效果及作用机制,并判断其安全性。方法:通过高通量中药筛选体系提取4种青光安有效组份,并制成缓释药膜。将48只实验用兔分为8组,每组6只。分别为:A:空白组;B:模型组;C:MMC对照组;D:有效组份1组;E:有效组份2组;F:有效组份3组;G:有效组份4组;H:安慰剂组。除A组外,其余各组兔眼行常规小梁切除+虹膜根部切除术;C组在术中联合应用MMC;D、E、F、G 4组分别在结膜下植入相对应的青光安缓释药膜;H组植入安慰剂膜片。术后各组进行眼部常规检查。用药4周后处死实验用兔,取术眼滤过道巩膜组织,进行HE染和Real-time PCR法检测,并进行统计学分析。结果:(1)术后C、E两组眼压回升缓慢,第4周时两组眼压低于其他各组(P<0.05)。(2)C、E组术后都能维持有效滤过泡,B组滤过泡在术后两周内基本都变为无功能滤过泡或消失,D、F、G、H组在术后第4周都有少量功能性滤过泡存在。(3)HE染色见C、E两组滤过道轮廓清晰,纤维细胞及炎症细胞少,其他各手术组滤过道轮廓消失,被大量纤维细胞和炎症细胞填充。(4)C组Smad3、Smad4的mRNA表达值明显低于其他各组(P<0.01);C组和E组CollagenⅠ的mRNA表达值明显低于其他各组(P<0.01)。结论:青光安有效组份2可能与TGF-β竞争性结合TGF-β受体,或者可能通过影响炎症因子的生成减少TGF-β的产生,使TGF-β/Smad细胞信号传导通路的信号传导减弱,抑制滤过道瘢痕形成。且通过局部给药其不良反应小,安全性优于MMC。

扶正抑癌方含药血清对结肠癌Lovo细胞TGF-/Smad信号转导通路的影响及意义

目的通过观察扶正抑癌方含药血清对结肠癌Lovo细胞TGF-β/smad信号转导通路中TGF-β1、Smad3和p-Smad3蛋白表达的影响,明确扶正抑癌方抑制结肠癌细胞间质化转变的作用机制。方法 Lovo细胞体外培养,制备大鼠不同浓度含药血清后,MTT法测定不同浓度含药血清对细胞增殖的影响,以5-Fu作为阳性对照;通过Western blot检测TGF-β1、Smad3和p-Smad3蛋白的表达。结果大剂量含药血清组24 h对Lovo细胞抑制率较中剂量、小剂量组作用更加明显,差异具有统计学意义(P<0.01)。Western blot检测结果表明扶正抑癌方含药血清可以下调Lovo细胞TGF-β1、Smad3和p-Smad3的蛋白表达,与正常组和空白组相比,差异具有统计学意义(P<0.05)。结论扶正抑癌方含药血清可能通过下调TGF-β/smad信号转导通路中TGF-β1、Smad3和p-Smad3的蛋白表达,抑制Lo-vo细胞间质化转变。

BMP signaling in mesenchymal stem cell differentiation and bone formation

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and have diverse functions during development and organogenesis. BMPs play a major role in skeletal development and bone formation, and disruptions in BMP signaling cause a variety of skeletal and extraskeletal anomalies. Several knockout models have provided insight into the mechanisms responsible for these phenotypes. Proper bone formation requires the differentiation of osteoblasts from mesenchymal stem cell (MSC) precursors, a process mediated in part by BMP signaling. Multiple BMPs, including BMP2, BMP6, BMP7 and BMP9, promote osteoblastic differentiation of MSCs both in vitro and in vivo. BMP9 is one of the most osteogenic BMPs, yet it is a poorly characterized member of the BMP family. Several studies demonstrate that the mechanisms controlling BMP9-mediated osteogenesis differ from other osteogenic BMPs, but little is known about these specific mechanisms. Several pathways critical to BMP9-mediated osteogenesis are also important in the differentiation of other cell lineages, including adipocytes and chondrocytes. BMP9 has also demonstrated translational promise in spinal fusion and bone fracture repair. This review will summarize our current knowledge of BMP-mediated osteogenesis, with a focus on BMP9, by presenting recently completed work which may help us to further elucidate these pathways.

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro.METHODS: HSCs were isolated from rats by in situperfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 hwith DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscleα-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲprocollagen (PCⅢ) in supernatants were determined byradioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2%(control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P＜0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%,75.7% or 80.7% respectively (P＜0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P＜0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P＜0.01;r = 0.938, P＜0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L)was confirmed by Western blotting as well.CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.

盐酸氨溴索治疗大鼠肺纤维化的作用研究及其对TGF-β/Smad/ERK信号转导通路和Th17/Treg细胞失衡的影响

目的:分析盐酸氨溴索治疗大鼠肺纤维化的作用研究及其对TGF-β/Smad/ERK信号转导通路和Th17/Treg细胞失衡的影响。方法:选取由温州康宁医院实验动物中心提供SPF级Wistar雄性大鼠30只,随机数字表法分成5组:正常组,模型组,氨溴索低、中、高剂量组,每组各10只。采用5 mg/kg博来霉素0. 2 m L气管内灌注成功制备肺纤维化模型,术后第2天起氨溴索低、中、高剂量组大鼠腹腔内注射剂量为10、20、40 mg/kg盐酸氨溴索,正常组和模型组大鼠腹腔注射剂量为3. 33 m L/kg生理盐水,连续注射14 d。HE染色和Masson染色观察各组大鼠组织病理变化,ELISA法检测大鼠成纤维细胞内转化生长因子β1(TGF-β1)含量,实时荧光定量PCR检测TGF-β1蛋白表达状况,Western blot检测成纤维细胞内ERK1/2、p-ERK和Smad3、Smad4、Smad7蛋白表达状况,流式细胞术法检测各组大鼠肺组织内Treg、Th17细胞表达状况。结果:与正常组相比,模型组大鼠成纤维细胞内TGF-β1含量、TGF-β1基因表达量、ERK1/2、p-ERK和Smad3、Smad4蛋白表达、肺组织内Th17、Th17/Treg均升高(均P<0. 05);与模型组相比,氨溴索中、高剂量组大鼠均降低(均P <0. 05)。而模型组大鼠成纤维细胞Smad7蛋白表达、肺组织内Treg表达比率较正常组均降低(均P <0. 05),氨溴索中、高剂量组大鼠较模型组升高(均P <0. 05)。结论:盐酸氨溴索可维持肺纤维化大鼠肺组织Th17/Treg平衡,抑制大鼠TGF-β/Smad/ERK信号转导通路内主要细胞因子信号传递,进而起到抗肺纤维化的作用。

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.

Effect of cigarette smoke extract on lipopolysaccharide-activated mitogen-activated protein kinase signal transduction pathway in cultured cells

Background Lipopolysaccharide （LPS） forms outer membrane of the wall of Gram-negative cells. LPS can directly cause damage to epithelia of respiratory tract and is the major factor responsible for the chronic inflammation of respiratory passage. The mitogen-activated protein kinase （MAPK） signal transduction pathway of the airway epithelia is intimately associated with the action of LPS. The chronic inflammation of respiratory tract and smoking are interrelated and entwined in the development and progression of chronic lung diseases. This study was designed to examine the effects of cigarette smoke extract （CSE） and LPS on MAPK signal transduction pathway in order to further understand the roles CSE and LPS play in chronic lung inflammation. Methods Cultured primary human epithelial cells of airway were divided into four groups according to the stimulants used： blank control group, LPS-stimulation group, CSE-stimulation group and CSE plus LPS group. Western blotting was employed for the detection of phosphorylation level of extracellular-signal-regulated-kinase （ERK1/2）, p38 MAPK and c-Jun N-terminal kinase （JNK）. The expression of cytokines of MAPK transduction pathway （granulocyte-macrophage colony stimulating factor （GM-CSF） and mRNA of IL-8） in the primary epithelial cells of respiratory tract was also determined. Results Western blotting revealed that the phosphorylation levels of ERK1/2, p38 MAPK and JNK were low and 2 hours after the LPS stimulation, the phosphorylation of ERK1/2, p38 MAPK and JNK were all increased. There was a significant difference in the phosphorylation between the LPS-stimulation group and blank control group （P〈0.05）; no significant difference was found between CSE-stimulation group and blank control group （P〉0.05）; there was a significant difference between CSE ＋ LPS group and blank control group and between CSE ＋ LPS group and LPS group （P〈0.05）. The phosphorylation of CSE-LPS group was higher than that of blank control group but lower than that of LPS group. In blank control group, the expression of IL-8 and GM-CSF mRNA was low in the epithelial cells of airway and the release of IL-8 and GM-CSF was also at a low level. One hour after LPS stimulation, the level of IL-8 mRNA increased （P〈0.05） and reached a peak after 2 hours. On the other hand, GM-CSF mRNA level increased 2 hours after the stimulation （P〈0.05） and reached the highest level 4 hours after the stimulation. Two hours after LPS stimulation, IL-8 and GM-CSF protein level began to rise （P〈0.05）, and the level was the highest 8 hours after the stimulation （P〈0.01）. Stimulation with CSE alone had no effect on the release of IL-8 and GM-CSF and expression of IL-8 mRNA （P〉0.05）, but pre-treatment with CSE could delay the LPS-induced release of IL-8 and GM-CSF and the expression of IL-8 mRNA and its peak was lower. Conclusions LPS stimulation can significantly increase the phosphorylation of ERK1/2, p38 MAPK and JNK in the epithelial cells of airway and activate the MAPK transduction pathway, thereby can activate the downstream signal transduction pathway, and can ultimately result in the release of cytokines by the epithelial cells of airway. CSE can partially abolish the LPS-induced activation of MAPK signal transduction pathway and the expression of cytokines of the pathway, which might contribute to the development and progression of the inflammatory reactions in COPD patients.

粉防己碱上调Smad7表达抑制大鼠肝星状细胞活化

目的观察低浓度粉防己碱(Tetrandrine,Tet)对培养的大鼠肝星状细胞(HSC)活化和转化生长因子β1(TGFβ1)促活化作用的影响,并探讨该作用与TGFβ1受体后信号通路的关系。方法HSC原代培养,给予Tet(0.4、0.8、1.6和3.2μmol·L-1)处理,免疫细胞化学法检测α平滑肌肌动蛋白(αSMA)表达,或给予TGFβ1(质量浓度5μg·L-1)和(或)Tet(1.6μmol·L-1)干预,分别以RTPCR和Westernblot法检测TGFβ1及其Ⅰ、Ⅱ型受体、Smad3、Smad7以及αSMAmRNA和(或)蛋白表达。结果Tet(0.4～3.2μmol·L-1)能抑制培养HSC表达αSMA。Tet(1.6μmol·L-1)抑制TGFβ1诱导的HSCαSMA表达,伴有Smad7表达上调及TGFβ1表达下降,但不影响TGFβⅠ、Ⅱ型受体和Smad3表达。结论低浓度Tet抑制培养HSC的活化和TGFβ1促活化作用,该作用与上调Smad7表达并抑制TGFβ1有关而不影响TGFβ受体表达。