Roles of transforming growth factor-βsignaling in liver disease

In this editorial we expand the discussion on the article by Zhang et al published in the recent issue of the World Journal of Hepatology.We focus on the diagnostic and therapeutic targets identified on the basis of the current understanding of the molecular mechanisms of liver disease.Transforming growth factor-β(TGF-β)belongs to a structurally related cytokine super family.The family members display different time-and tissue-specific expression patterns associated with autoimmunity,inflammation,fibrosis,and tumorigenesis;and,they participate in the pathogenesis of many diseases.TGF-βand its related signaling pathways have been shown to participate in the progression of liver diseases,such as injury,inflammation,fibrosis,cirrhosis,and cancer.The often studied TGF-β/Smad signaling pathway has been shown to promote or inhibit liver fibrosis under different circumstances.Similarly,the early immature TGF-βmolecule functions as a tumor suppressor,inducing apoptosis;but,its interaction with the mitogenic molecule epidermal growth factor alters this effect,activating anti-apoptotic signals that promote liver cancer development.Overall,TGF-βsignaling displays contradictory effects in different liver disease stages.Therefore,the use of TGF-βand related signaling pathway molecules for diagnosis and treatment of liver diseases remains a challenge and needs further study.In this editorial,we aim to review the evidence for the use of TGF-βsignaling pathway molecules as diagnostic or therapeutic targets for different liver disease stages.

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.

Total flavone of Abelmoschus manihot suppresses epithelial-mesenchymal transition via interfering transforming growth factor-β1 signaling in Crohn's disease intestinal fibrosis

AIM To explore the role and mechanism of total flavone of Abelmoschus manihot(TFA) on epithelial-mesenchymal transition(EMT) progress of Crohn's disease(CD) intestinal fibrosis.METHODS First,CCK-8 assay was performed to assess TFA on the viability of intestinal epithelial(IEC-6) cells and select the optimal concentrations of TFA for our further studies.Then cell morphology,wound healing and transwell assays were performed to examine the effect of TFA on morphology,migration and invasion of IEC-6 cells treated with TGF-β1.In addition,immunofluorescence,real-time PCR analysis(q RT-PCR) and western blotting assays were carried out to detect the impact of TFA on EMT progress.Moreover,western blotting assay was performed to evaluate the function of TFA on the Smad and MAPK signaling pathways.Further,the role of co-treatment of TFA and si-Smad or MAPK inhibitors has been examined by q RTPCR,western blotting,morphology,wound healing andtranswell assays.RESULTS In this study,TFA promoted transforming growth factor-β1(TGF-β1)-induced(IEC-6) morphological change,migration and invasion,and increased the expression of epithelial markers and reduced the levels of mesenchymal markers,along with the inactivation of Smad and MAPK signaling pathways.Moreover,we revealed that si-Smad and MAPK inhibitors effectively attenuated TGF-β1-induced EMT in IEC-6 cells.Importantly,co-treatment of TFA and si-Smad or MAPK inhibitors had better inhibitory effects on TGF-β1-induced EMT in IEC-6 cells than either one of them.CONCLUSION These findings could provide new insight into the molecular mechanisms of TFA on TGF-β1-induced EMT in IEC-6 cells and TFA is expected to advance as a new therapy to treat CD intestinal fibrosis.

Spatial signalling mediated by the transforming growth factor-β signalling pathway during tooth formation

Tooth development relies on sequential and reciprocal interactions between the epithelial and mesenchymal tissues, and it is continuously regulated by a variety of conserved and specific temporal-spatial signalling pathways. It is well known that suspensions of tooth germ cells can form tooth-like structures after losing the positional information provided by the epithelial and mesenchymal tissues. However, the particular stage in which the tooth germ cells start to form tooth-like structures after losing their positional information remains unclear. In this study, we investigated the reassociation of tooth germ cells suspension from different morphological stages during tooth development and the phosphorylation of Smad2/3 in this process. Four tooth morphological stages were designed in this study. The results showed that tooth germ cells formed odontogenic tissue at embryonic day （E） 14.5, which is referred to as the cap stage, and they formed tooth-like structures at E16.5, which is referred to as the early bell stage, and E18.5, which is referred to as the late bell stage. Moreover, the transforming growth factor-β signalling pathway might play a role in this process.

Activation of phospholipase D activity in transforming growth factor-beta-induced cell growth inhibition

Cells regulate phospholipase D (PLD) activity in response to numerous extracellular signals. Here, we investigated the involvement of PLD activity in transforming growth factor-β(TGF-β1)-mediated growth inhibition of epithelial cells. TGFβ1 inhibits the growth of MDCK, Mv1Lu, and A-549 cells. In the presence of 0.4 % butanol, TGF-β1 induces an increase in the formation of phosp hat idylbutanol, a unique product catalyzed by PLD. TGF-β1 also induces an increase in phosphatidic acid (PA) level in A-549 and MDCK cells. TGF-β1 induces an increase in the levels of DAG labeled with [~3H]-myristic acid in A-549 and MDCK cells but not in Mv1Lu cells- No increase of DAG was observed in cells prelabeled with [~3H]-arachidonic acid.The data presented suggest that PLD activation is involved in the TGF-β1-induced cell growth inhibition.

Glycine Attenuates Myocardial Fibrosis in Myocardial Infarction in Rats Partly through Modulating Signal Transducer and Activator of Transcription 3/Nuclear Factor-κB/Transforming Growth Factor-β axis

Objective: Inflammation and fibrosis are strongly associated with each other. Glycine is present in various traditional Chinese medicines and exhibits anti-inflammatory activity. However, the effects of glycine on myocardial fibrosis(MF) in rats with myocardial infarction(MI) have not been reported. The purpose of this study is to investigate the effects of glycine therapy on MF and comprehend its underlying mechanisms. Materials and Methods: Left anterior descending artery ligation-induced MI in Sprague Dawley rats was leveraged to assess the therapeutic effects of Glycine. Rats received either normal saline or glycine(0.5 mg/g bodyweight) for 7 days. Results: Glycine upregulated cardiac ejection fraction and fractional shortening to improve cardiac function, as evaluated by echocardiography. Histological and immunohistochemical analyses demonstrated that glycine could decrease inflammatory cell infiltration and alleviate collagen deposition. Western blotting revealed that nuclear factor-κB(NF-κB)-mediated inflammatory signaling was also downregulated by glycine treatment. The expression of signal transducer and activator of transcription 3(STAT3), tumor necrosis factor-α, and transforming growth factor-β(TGF-β) was decreased significantly in the glycine-treated group compared to the model group. Thus, glycine plays a protective role against myocardial ischemia and subsequent MF. Conclusion: The protective effects of glycine were achieved partly through STAT3/NF-κB/TGF-β signaling pathway.

Wulong Xiaozheng Wan medicated serum inhibits epithelial-mesenchymal transition in human gastric carcinoma cell line BGC823 by modulation of transforming growth factor-β1/Smad signaling

OBJECTIVE:To evaluate the effect of Wulong Xiaozheng Wan medicated serum on the epithelial-mesenchymal transition (EMT) of BGC823 cell induced by transforming growth factor-β,(TGF-β,) and to explore its mechanism.METHODS:EMT model of BGC823 was stimulated by TGF-β1.Wulong Xiaozheng Wan medicated serum and LY-364947 were used as intervention.The proliferation and adhesion of BGC823 were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and flow cytometry was used to detect the apoptosis.The invasion and migration were detected by Transwell.The level of matrix metalloproteins was detected by enzyme-linked immunosorbent assay.The expressions of related proteins and mRNA of EMT marker and TGF-β1/Smad signal pathway were detected by Western blot and reverse transcription-polymerase chain reaction.RESULTS:Compared with the TGF-β1 group,Wulong Xiaozheng Wan medicated serum could inhibit the ability of proliferation,heterogeneous adhesion,invasion,and migration.It also promotes apoptosis and homotypic adhesion in BGC823,with a dose-dependent manner.Meanwhile,Wulong Xiaozheng Wan medicated serum could regulate the expression of related proteins and mRNA of TGF-β1/Smad signaling pathway,and inhibit the expressions of EMT transcription factors and EMT markers.CONCLUSION:Wulong Xiaozheng Wan medicated serum inhibited epithelial-mesenchymal transition by down-regulated the expression of TβRI and the activation of TGF-β1/Smad signaling pathway.

Shugan Huoxue Huayu Fang(疏肝活血化瘀方)attenuates carbon tetrachloride-induced hepatic fibrosis in rats by inhibiting transforming growth factor-β1/Smad signaling

OBJECTIVE:To investigate the potential mechanism by which Shugan Huoxue Huayu Fang(疏肝活血化瘀方,SGHXHYF)ameliorates liver fibrosis.METHODS:Liver fibrosis was induced in rats by intraperitoneal injection of carbon tetrachloride(CCl_(4))in peanut oil solution(40%,3 m L/kg body weight)twice a week for 8 weeks.A normal control group received the same volume of peanut oil alone.During weeks 5-8,the CCl_(4)-injected rat groups were administered saline(vehicle control),colchicine(0.1 mg/mL,1 mg/kg,positive control),or SGHXHYF(0.1 mg/mL;0.3,0.6 and 1.2 mg/kg)once daily by oral gavage.Rats were sacrificed 24 h after the last treatment.Blood samples were collected for measurement of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),albumin(ALB),collagenⅠ,and collagenⅢlevels.Liver samples were analyzed by histopathological staining,Masson's staining of extracellular matrix proteins,and immune-ohistochemical staining ofα-smooth muscle actin(α-SMA).TGF-β1/Smad protein and mRNA levels were analyzed by Western blot and quantitative reverse transcription-polymerase chain reaction analysis,respectively.In vitro experiments were also performed using rat hepatic stellate cells(HSCs).RESULTS:Compared with the control animals,CCl_(4)-exposed rats exhibited elevated serum levels of ALT,AST,ALP,collagenⅠ,and collagenⅢ;reduced serum levels of ALB;and increased collagen deposition andα-SMA expression in liver sections,reflecting liver fibrosis.CCl_(4) also increased expression of TGF-β1 and the activated(phosphorylated)forms of Smad2 and Smad3 but reduced expression of the negative regulator Smad7 in the liver.Notably,concomitant administration of SGHXHYF to CCl_(4)-exposed rats was found to significantly reverse or abolish the pro-fibrotic effects of CCl_(4) in the liver and reduced serum transferase levels.Analysis of HSCs in vitro confirmed that,mechanistically,SGHXHYF inhibited activation of the TGF-β1/Smad signaling pathway by downregulating phosphorylated Smad2 and Smad3 and upregulating Smad7 levels.CONCLUSION:SGHXHYF ameliorated CCl_(4)-induced liver fibrosis by inhibiting the TGF-β1/Smad signaling pathway.These findings suggest that SGHXHYF may have clinical utility for the treatment or prevention of liver fibrosis.

Huangqi decoction(黄芪汤) attenuates renal interstitial fibrosis via transforming growth factor-β1/mitogen-activated protein kinase signaling pathways in 5/6 nephrectomy mice

OBJECTIVE: To investigate the effect of Huangqi decoction( 黄芪汤) on renal interstitial fibrosis and its association with the transforming growth factor-β1(TGF-β1)/mitogen-activated protein kinase(MAPK) signaling pathway. METHODS: 120 C57/BL mice were randomly divided into six groups: sham group, Enalapril(20 mg/kg) group, 5/6 nephrectomy model group, and 5/6 nephrectomy model plus Huangqicoction(0.12, 0.36 and 1.08 g/kg respectively) groups. Detecting 24hours urinary protein, blood pressure, serum creatinine, urea nitrogen content changes. Periodic Acid-Schiff stain(PAS) and Masson’s trichrome staining was used to observe the renal tissue pathological changes. Protein expression of TGF-β1, Phosphorylated P38 mitogen activated protein kinases(P-P38), Phosphorylated c-jun N-terminal kinase(P-JNK), Phosphorylated extracellular regulated proteinhnase(PERK), Fibroblast-specific protein-1(FSP-1), Alpha smooth muscle actin(α-SMA), Type Ⅲ collagen(Collagen Ⅲ), Connective tissue growth factor(CTGF),Bcl-2 Assaciated X protein(Bax) and B cell lymphoma 2(Bcl-2) were measured with western blot and immunohistochemical. RESULTS: Both Huangqi decoction and Enalapril improved the kidney function, 24 h urinary protein and the fibrosis in 5/6 nephrectomy mice, Huangqi decoction downregulated the expressions of TGF-β1, FSP-1, α-SMA, Collagen Ⅲ and CTGF in a dose-dependent manner, and it has a significant difference(P < 0.01) compared with model group.Huangqi decoction downregulated the expressions of P-P38, P-JNK, P-ERK and Bcl-2 in a dose-dependent manner, while upregulated the expression of Bax. CONCLUSIONS: The protective effect of Huangqi decoction for renal interstitial fibrosis in 5/6 nephrectomized mice via the inhibition of EpithelialMesenchymal Transitions and downregulating the TGF-β1/MAPK signaling pathway.

大麻二酚对转化生长因子β1诱导肝星状细胞活化和肝纤维化的作用

背景:大麻二酚具有抗炎、抗氧化等药理学作用,并且不具有精神活性,在肝脏疾病中的研究日渐增加,但其对肝星状细胞转化生长因子β1/Smad信号转导通路的作用尚未明确。目的:探讨大麻二酚对肝星状细胞中转化生长因子β1/Smad信号转导通路的影响,研究其抗肝纤维化的可能机制。方法:(1)体外实验:选取大鼠肝星状细胞株(HSC-T6),分6组培养:对照组常规培养24 h,单纯药物组加入大麻二酚培养24 h,造模组加入转化生长因子β1培养24 h,造模+低剂量药物组、造模+高剂量药物组、造模+阳性对照组均加入转化生长因子β1培养24 h后分别加入1,5μmol/L大麻二酚或水飞蓟素培养24 h。培养结束后,检测各组细胞α-平滑肌肌动蛋白与Ⅰ型胶原mRNA表达、白细胞介素1β与肿瘤坏死因子α水平及Ⅰ型胶原、转化生长因子β1/Smad信号转导通路蛋白表达。(2)动物体内实验:将C57BL/6J小鼠随机分为5组,每组8只:假手术组不造模,造模组、造模+低剂量药物组、造模+高剂量药物组、造模+阳性对照组通过胆管结扎法建立肝纤维化模型,造模3周后分别腹腔注射4,8 mg/kg大麻二酚或水飞蓟素,1次/d,连续给药7 d。给药结束后,检测各组小鼠肝功能、肝脏病理形态及α-平滑肌肌动蛋白、Ⅰ型胶原、转化生长因子β1/Smad信号转导通路相关蛋白表达。结果与结论:(1)体外实验:与对照组比较,造模组HSC-T6细胞α-平滑肌肌动蛋白与Ⅰ型胶原mRNA表达、白细胞介素1β与肿瘤坏死因子α水平以及Ⅰ型胶原、转化生长因子β1、p-Smad2/3蛋白表达均升高(P<0.05),Smad7蛋白表达降低(P<0.05);两种剂量大麻二酚处理可改善转化生长因子β1诱导的HSC-T6细胞上述指标的变化,并且以造模+高剂量药物组改善作用更明显;(2)体内实验:与假手术组比较,模型组小鼠血清丙氨酸氨基转移酶、天门冬氨酸氨基转移酶活性升高(P<0.05),肝组织中炎性细胞浸润及胶原含量增加(P<0.05),转化生长因子β1/Smad信号转导通路被激活,α-平滑肌肌动蛋白、Ⅰ型胶原蛋白表达升高(P<0.05);两种剂量大麻二酚干预可减轻造模小鼠上述指标的变化,并且以造模+高剂量药物组效果更明显;(3)结果表明,大麻二酚通过抑制肝星状细胞转化生长因子β1/Smad信号传导通路的激活抑制肝纤维化。

TGF-β1 signal pathway in the regulation of inflammation in patients with atrial fibrillation

Objective:To observe the expression changes of inflammatory markers TGF-β1,Smad3 and IL-6 in patients with atrial fibrillation(AF),and to explore the significance of TGF-β1 signaling pathway in the structural remodeling of AF.Methods:The expression of TGF-β1,Smad3 and IL-6 in 50 cases with AF and 30 normal cases were detected by RT-PCR and ELISA.Results:The TGF-β1,Smad3 and IL-6 mRNA and protein expression levels in patients with AF were significantly higher than that in the control group(P<0.05),but there was no significantly different between the paroxysmal AF group and the persistent AF group(P>0.05).The TGF-β1mRNA expression in the≥50 years subgroup was significantly higher than that in the<50years subgroups,and it was higher in the NYHAⅢsubgroup than in theⅠ/Ⅱgrade subgroup.It was also higher in the left ventricular ejection fraction(LVEF)<50%subgroup than in LVEF≥50%group,and it was significantly higher in the AF time≥36 months subgroup than that in<36months subgroup(P<0.05).The Smad3 and IL-6 expressions in the in the LVEF<50%subgroup were both high that than that in LVEF≥50%group,and higher in the AF time≥36 months subgroup than that in<36 months subgroup(P<0.05).There were a positive correlation between TGF-β1,Smad3 and IL-6(r=0.687,r=0.547).There were also a positive correlation between Smad3 and IL-6 mRNA(r=0.823).Conclusions:AF is associated with inflammation,and the inflammation is also involved in the fibrillation and sustain of AF.The TGF-β1 signal pathway may be involved in the process of atrial structural remodeling in patients with AF,and iss related with the occurrence and maintenance of AF.

Targeting key signalling pathways in oesophageal adenocarcinoma:A reality for personalised medicine?

Cancer treatments are rapidly changing.Curative treatment for oesophageal adenocarcinoma currently involves surgery and cytotoxic chemotherapy or chemoradiotherapy.Outcomes for both regimes are generally poor as a result of tumor recurrence.We have reviewed the key signalling pathways associated with oesophageal adenocarcinomas and discussed the recent trials of novel agents that attempt to target these pathways.There are many trials underway with the aim of improving survival in oesophageal cancer.Currently,phase 2 and 3 trials are focused on MAP kinase inhibition,either through inhibition of growth factor receptors or signal transducer proteins.In order to avoid tumor resistance,it appears to be clear that targeted therapy will be needed to combat the multiple signalling pathways that are in operation in oesophageal adenocarcinomas.This may be achievable in the future with the advent of gene signatures and a combinatorial approach.

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.

Toxicarioside A,isolated from tropical Antiaris toxicaria,blocks endoglin/TGF-βsignaling in a bone marrow stromal cell line

Objective:To investigate possible mechanism of toxicarioside A in HS-5 bone stromal cells.Methods:HS-5 bone stromal cells were cultured in media supplemented with various concentrations of toxicarioside A or control DMSO(not treatment).Endoglin and TGF-βwere detected by Northern and Western blot analysis and quantified in a standard method. Downstream molecules of endoglin and TGF-β(Smad1,Smad2 and their active phosphorylated counterparts,pSmad1 and pSmad2) were also detected and quantified by Western blot analysis. In addition,cell proliferation assay and small interfering RNA(siRNA) against endoglin were used to certificate the function of endolgin in the HS-5 cells.Results:Compared with the not treated(0μg/mL) or DMSO treated control HS-5 cells,HS-5 cells treated with toxicarioside A were found significant attenuation of endolgin and TGF-βexpression.Significant inhibition of cell proliferation was also found in the HS-5 cells treated with toxicarioside A.ALK1-related Smad1 and ALK5-related Smad2 were decreased in HS-5 cells treated with toxicarioside A.In addition,phosphorylated Smad1(pSmad1) and Smad2(pSmad2) were also found attenuation in toxicarioside A-treated HS-5 cells.RNA interference showed that blockage of endoglin by siRNA also decreased Smad1 and Smad2 expression in HS-5 cells.Conclusions:Our results indicate that toxicarioside A can influence bone marrow stromal HS-5’s function and inhibit HS-5 cell proliferation by alteration of endoglin-related ALK1(Smad1) and ALK5(Smad2) signaling.

Moxibustion enables protective effects on rheumatoid arthritis-induced myocardial injury via transforming growth factor beta1 signaling and metabolic reprogramming

OBJECTIVE:To examine the effects of moxibustion on myocardial injury and myocardial metabolomics in rats with rheumatoid arthritis(RA)based on the transforming growth factor beta1(TGF-β1)/Smads signaling pathway.METHODS:One hundred rats were treated with saline[normal control(NC)group]or complete Freund’s adjuvant(CFA)by right plantar injection for the RA model group,and the latter were randomly divided into 4 groups.Tripterygium wilfordii polyglycoside tablets(雷公藤多苷片,TPT)have anti-inflammatory and are widely used in the clinical treatment of RA,therefore serving as a positive control group.Three days post injection rats were given TPT tablet(TPT group),acupuncture therapy(APT group),and moxibustion treatment(MOX group)for 15 consecutive days,while NC group and model group were equally grasped and fixed and received normal saline.Rat joint swelling scores and arthritis index(AI)were evaluated in each group before the CFA challenge,therapy and after receiving therapy.Myocardial ultrastructure was observed by electron microscope.Enzyme-linked immunosorbent assay was used to detect cardiac troponin I(cTnI)levels in rat myocardial tissue.Quantitative reverse transcription polymerase chain reaction and Western blotting analysis were used to measure the mRNA and protein levels of TGF-βsignaling molecules including TGF-β1,Smad2,Smad3,Smad4,and Smad7.Myocardial metabolomics was analyzed using gas chromatography-mass spectrometer.RESULTS:Compared with model group,RA model rats receiving TPT,acupuncture,or moxibustion therapy all showed reduced joint swelling scores and AI(all P<0.01)and improved myocardial damage,whereas rats treated with moxibustion were found to be more marked.Consistently,the expressions of cTnI,TGF-β1,Smad2,Smad3,and Smad4 were found to be elevated in model rat group in contrast to NC rats and were significantly downregulated in TPT,APT and MOX group when compared with model group,while the levels of Smad7 showed the opposite result(all P<0.01).Moreover,the dissection of metabolomics suggested a novel metabolite biomarker panel including D-Xylulose 5-phosphate,dihydroxyacetone phosphate,arachidonic acid,etc was defined and implicated in amino acid,glucose,and fatty acid metabolic processes as revealed by principal component analysis and partial least squares discriminant analysis.CONCLUSION:Moxibustion prevents RA-induced inflammatory response and offers potent therapeutic effects on myocardial dysfunctions.The protective effects might be associated with its role in TGF-β1 inactivation and metabolic reprogramming.

Regulation Mechanism of TFP on TGF-β1/STAT3 Signaling Pathway in Immune-mediated Liver Injury in Mice

[Objectives]To study the effect of total flavonoids extracted from Polygonum perfoliatum L.(TFP)on immune-mediated liver injury induced by bacillus Calmette-Guerin plus lipopolysaccharide(BCG+LPS)in mice,and to explore its action mechanism.[Methods]60 Kunming mice were divided into normal group,model group,control group(bifendate)and TFP low,medium and high dose groups according to random number table method,with 10 mice in each group.On the first day of modeling,mice were injected with 0.2 mL of BCG solution(12.5 mg/mL)through the tail vein,and on the eleventh day,0.2 mL of LPS(37.5μg/mL)were injected into the tail vein to prepare a mouse model of immune-mediated liver injury;from the first day of modeling,the normal group and the model group were administered intragastrically with the corresponding volume of distilled water,and the bifendate group and the TFP high,medium,and low dose groups were administered intragastrically with the corresponding doses once a day for 11 d.After the last time administration,fasting but giving water for 16 h,took blood from eyes,then collected the liver tissue.The levels of alanine transaminase(ALT)and aspartate transaminase(AST)in serum were detected by biochemical method;transforming growth factor-β1(TGF-β1),intercellular adhesion molecule-1(ICAM-1),interleukin-6(IL-6)and interleukin-1β(IL-1β)expression levels in liver tissue were detected by enzyme-linked immunosorbent assay(ELISA);phosphorylated protein tyrosine kinase JAK-2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)protein expression levels were detected by Western Blot method;the degree of liver tissue lesions was detected by HE staining.[Results]Compared with the model group,the levels of ALT and AST in the serum of mice in each dose group of TFP(high dose 600 mg/kg,medium dose 400 mg/kg,and low dose 200 mg/kg)were reduced,and the activities of T-SOD and GSH-Px were increased;the content or expression ofβ1,ICAM-1,IL-6,IL-1βdecreased,and the expression of p-JAK2 and p-STAT3 protein decreased;pathological sections showed that the degree of inflammatory necrosis and the degree of lesions in the liver tissues of each dose group of TFP were reduced by varying degrees.[Conclusions]TFP has a protective effect on BCG+LPS-induced immune-mediated liver injury in mice.The mechanism may be related to regulating the phosphorylation level of JAK2 and inhibiting the inflammatory reaction,thereby regulating the TGF-β1/STAT3 signaling pathway and improving the immune-mediated liver injury.

TGF-β信号通路在骨髓增生异常综合征中作用机制的研究进展

骨髓增生异常综合征(MDS)是一种细胞遗传学与分子遗传学高度异质性的恶性克隆性疾病,且进展为急性髓系白血病(AML)的风险极高。转化生长因子-β(TGF-β)与MDS的发病密切相关,是免疫稳态及免疫耐受的关键执行者,可抑制免疫系统许多组成部分的扩张和功能。TGF-β信号通路能调控微环境中的造血细胞及免疫细胞,抑制其发挥正常生物学功能,从而加快MDS的疾病进展。就近年来TGF-β信号通路对MDS红细胞、T淋巴细胞、自然杀伤细细胞、巨噬细胞的调控作用进行综述,以期为MDS的发病机制及治疗提供新的视角。

硫酸吲哚酚对心肌梗死模型小鼠心肌重构的影响

目的探讨硫酸吲哚酚(IS)对心肌梗死(myocardial infarction,MI)模型小鼠心肌重构的影响。方法C57BL/6J成年雄性小鼠50只,随机分为假手术组(Sham组)10只,硫酸吲哚酚组(Sham+IS组)10只,心肌梗死组(MI组)15只,心肌梗死+硫酸吲哚酚组(MI+IS组)15只。采用左前降支冠状动脉结扎术构建小鼠MI模型,术后第24小时,Sham+IS组和MI+IS组小鼠每天腹腔注射IS 100 mg/kg,Sham组和MI组每天腹腔注射等体积PBS,连续28 d,期间记录各组小鼠存活情况。实验第30天,心脏超声检查评估各组小鼠心脏功能,超高效液相色谱法检测各组小鼠血清中IS浓度,Masson染色评估各组小鼠梗死区心肌纤维化程度,RT-qPCR技术检测心肌组织α-sma、CollagenⅠ基因的表达水平,Western blot技术检测心肌组织TGF-β信号通路标记蛋白TGF-β、p-Smad2、p-Smad3的表达水平。结果实验第30天时,与MI组相比,MI+IS组小鼠存活率显著下降(χ^(2)=5.02,P<0.05)。与Sham组相比,Sham+IS组小鼠血清中IS浓度显著升高(t=54.87,P<0.05),与MI组相比,MI+IS组小鼠血清中IS浓度显著升高(t=38.55,P<0.05)。心脏超声检查示,Sham组和Sham+IS组的小鼠左心室舒张末期内径(LVIDd)、左心室收缩末期内径(LVIDs)、左心室射血分数(LVEF)、左心室短轴缩短率(LVFS)无显著差异(P>0.05);与MI组相比,MI+IS组小鼠LVIDd、LVIDs显著增大(t=3.96、4.31,P<0.05),LVEF、LVFS显著降低(t=5.68、4.07,P<0.05)。Masson染色示,MI+IS组与MI组比较小鼠心肌间质胶原纤维沉积明显增多。RT-qPCR技术检测显示,MI+IS组与MI组相比,小鼠心肌组织α-sma、CollagenⅠ基因的表达水平显著升高(t=8.74、4.78,P<0.05)。Western blot方法检测显示,MI+IS组与MI组相比小鼠心肌组织中TGF-β、p-Smad2、p-Smad3蛋白的表达水平均显著升高(t=4.04~5.64,P<0.05)。结论IS可加重小鼠MI后病理性心肌重构,其机制可能与TGF-β信号通路激活相关。

转化生长因子胞内信号蛋白Smad2/3在糖尿病大鼠肾脏表达的动态观察及意义研究

目的:动态观察大鼠糖尿病肾病发生发展过程中TGF-β1及其下游信号分子Sm ad2/3在肾脏的表达及定位变化,探讨TGF-β1-Sm ad2/3信号通路在糖尿病肾病(DN)肾纤维化发病机制中的作用。方法:链脲菌素诱导大鼠糖尿病肾病,以免疫组化、W estern b lotting及荧光实时定量PCR(RT-PCR)的方法动态观察糖尿病肾病发生发展过程中大鼠肾脏TGF-β1、Sm ad2/3蛋白及mRNA表达,并以RT-PCR方法观察结缔组织生长因子(CTGF)、胶原-3(COL-Ⅲ)、纤溶酶原激活剂抑制因子-1(PAI-1)mRNA表达。结果:TGF-β1在糖尿病肾病(DN)大鼠肾小管表达明显多于正常组(P<0.05);Sm ad2/3蛋白主要以胞浆表达阳性为主,部分呈胞核表达阳性;Sm ad2/3蛋白及mRNA从2周起表达明显多于正常对照组(P<0.05),且随着糖尿病进展有逐渐增加的趋势。16周DN组大鼠肾脏TGF-β1、Sm ad2、CTGF、COL-Ⅲ、PAI mRNA表达显著高于正常对照组(P<0.05)。结论:TGF-β1-Sm ad2/3信号转导通路参与了糖尿病肾病肾脏纤维化形成的信号转导过程,伴随着Sm ad2/3信号蛋白表达的增多及入核增加,CTGF、COL-Ⅲ、PAI-1等基因转录增加,导致肾脏细胞外基质的大量沉积,这是细胞因子TGF-β1导致糖尿病肾病肾纤维化进展可能的信号转导途径之一。

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。