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.

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.

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.

Regulation of transforming growth factor-β signaling as a therapeutic approach to treating colorectal cancer

According to data from 2020,Slovakia has long been among the top five countries with the highest incidence rate of colorectal cancer(CRC)worldwide,and the rate is continuing to rise every year.In approximately 80%of CRC cases,allelic loss(loss of heterozygosity,LOH)occurs in the long arm of chromosome 18q.The most important genes that can be silenced by 18q LOH or mutations are small mothers against decapentaplegic homolog(SMAD)2 and SMAD4,which are intracellular mediators of transforming growth factor(TGF)-βsuperfamily signals.TGF-βplays an important role in the pro-oncogenic processes,including such properties as invasion,epithelial-mesenchymal transition(commonly known as EMT),promotion of angiogenesis,and immunomodulatory effects.Several recent studies have reported that activation of TGF-βsignaling is related to drug resistance in CRC.Because the mechanisms of drug resistance are different between patients in different stages of CRC,personalized treatment is more effective.Therefore,knowledge of the activation and inhibition of factors that affect the TGF-βsignaling pathway is very important.

Effects of the Nocth signaling pathway on expression of inflammatory factors and transforming growth factors in diabetic foot ulcers

Objective:persistent hyperinflammation is an important reason for the development of diabetic foot ulcer.Notch signaling is an important signaling pathway involved in the inflammatory response and cell proliferation in diabetic foot ulcer rats.This paper aims to explore the effect of Notch signaling on inflammatory factors,chemokines and growth factors through the intervention of Notch signaling in diabetic foot ulcer rats.Methods:the experimental model was made by using high-fat feed combined with streptozotocin(STZ)to cause diabetes,and the experimental model of diabetic foot ulcer was established by constant temperature and constant pressure scald apparatus.The normal ulcer model was used as a control.The intervention controls of the experimental model included normal saline,western medicine growth factor,Notch agonist Jagged1,Notch signaling inhibitor ly-411575,and the intervention of traditional Chinese medicine Zizhu ointment for 7 days.Serum il-1,il-6,TNF-radiation,and il-17 were detected by ELISA.Real-time PCR was used to detect the inflammatory factors,chemokines,and growth factors associated with Notch signaling in wound tissues:tnf-uum,il-1,il-6,il-17,interleukin-8,ip-10,McP-1,TGF-uum,TGF-livelihood.Results:serum levels of il-1,il-6,TNF-radiation and il-17 in diabetic foot ulcer rats were significantly higher than that in normal ulcer rats.The contents of il-1,il-6,TNF-radiation and il-17a in ly-411575 group and Zizhu ointment group were significantly reduced.Real-time PCR results of wound tissue showed that the levels of inflammatory cytokines il-1,il-6,TNF-radiation,il-17 and chemokines ip-10,il-8 and McP-1 in the wound tissue of diabetic foot ulcer rat model were significantly higher than that of normal ulcer model,and the levels of growth factor TGF-exposure were lower than that of normal ulcer model.LY-411575 significantly reduced il-1,il-6,TNF-maxima,il-17,and the chemokines ip-10,il-8,and McP-1 in diabetic foot ulcer rats,and reduced the expression of TGF-,TGF-earth.Jagged1 can increase the expression of TGF--,TGF---,suggesting that inhibition of the Notch signaling pathway can reduce the expression of the inflammatory factors il-1,il-6,TNF--,il-17a,il-8,and the growth factors TGF--,TGF---.Zizhu ointment can reduce the levels of il-1,il-6,TNF-benand,il-17,and the chemokines ip-10,il-8,and McP-1 on the wound surface of diabetic foot rats,and improve the expression of TGF-benand TGF-SUNS.Ly-411575 inhibited the expression of TGF-bento and TGF-promoting of Zizhu ointment.Conclusion:the expression of inflammatory cytokines and chemokines was higher and the expression of growth factors was lower in diabetic foot ulcer rats than in normal ulcer rats.Inhibition of Notch signaling pathway can reduce the expression of inflammatory factors,chemokines and growth factors in experimental model rats,and Notch signaling pathway can promote inflammation and cell proliferation.Zizhu ointment can reduce the levels of inflammatory cytokines and chemokines in diabetic foot ulcer rats,improve the expression of growth factors,and reduce wound inflammation,which may be related to the inhibition of Nocth signal expression.

Dihydroergotamine ameliorates liver fibrosis by targeting transforming growth factor β type Ⅱ receptor

BACKGROUND The transforming growth factor β(TGFβ) signaling pathway plays a crucial role in the development of liver fibrosis by activating TGFβ type Ⅱ receptor(TGFβR2), followed by the recruitment of TGFβR1 finally triggering downstream signaling pathway.AIM To find drugs targeting TGFβR2 that inhibit TGFβR1/TGFβR2 complex formation, theoretically inhibit TGFβ signaling pathway, and thereby ameliorate liver fibrosis.METHODS Food and Drug Administration-approved drugs were screened for binding affinity with TGFβR2 by virtual molecular docking. We identified 6 candidates and further explored their potential by Cell Counting Kit-8(CCK-8) cell cytotoxic experiment to validate toxicity and titrated the best cellular working concentrations. Next, we further demonstrated the detailed molecular working mechanisms using mutagenesis analysis. Finally, we used a mouse model to investigate its potential anti-liver fibrosis effect.RESULTS We identified 6 drug candidates. Among these 6 drugs, dihydroergotamine(DHE) shows great ability in reducing fibrotic gene expressions such as collagen, p-SMAD3, and α-SMA in TGFβ induced cellular model of liver fibrosis in LX-2 cells. Furthermore, we demonstrated that DHE binds to TGFβR2. Moreover, mutation of Leu27, Phe30, Thr51, Ser52, Ile53, and Glu55 of TGFβR2 disrupted the binding of TGFβR2 with DHE. In addition, DHE significantly improved liver fibrosis, as evidenced by Masson’s trichrome staining of liver sections. This is further supported by the width and the velocity of the portal vein, and serum markers of liver function. In line with those observations, DHE also decreased macrophages infiltration and extracellular matrix deposition in the liver.CONCLUSION DHE alleviates liver fibrosis by binding to TGFβR2 thereby suppressing TGFβ signaling pathway. We show here that as far as drug repurposing, DHE has great potential to treat liver fibrosis.

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

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

基于TGF‐β1/Smads信号通路探讨红花抗硬皮病小鼠皮肤纤维化的作用机制

目的探讨红花抗硬皮病小鼠皮肤纤维化的作用及对转化生长因子β1(TGF-β1)/Smads信号通路的影响。方法将64只雌性BALB/c小鼠分为对照组(16只)、模型组(16只)、泼尼松组(8只)、红花前干预组(16只)和红花后干预组(8只)。除对照组外,其他4组小鼠采用皮下注射博来霉素诱导硬皮病小鼠模型。造模当天给予泼尼松组小鼠灌胃0.45 mg/mL醋酸泼尼松混悬液,给予红花前干预组小鼠灌胃0.15 g/mL红花水提液,给予对照组、模型组小鼠灌胃生理盐水,造模第15天给予红花后干预组小鼠灌胃0.15 g/mL红花水提液,以上各组小鼠均连续灌胃4周(0.2 mL/d)。干预4周后,观察各组小鼠皮肤外观变化及皮肤病理组织学改变,比较各组小鼠的真皮厚度,采用免疫组化法检测各组小鼠皮肤组织Ⅰ型胶原蛋白(COLⅠ)、α-平滑肌肌动蛋白(α-SMA)的蛋白表达水平,采用实时荧光定量PCR检测各组小鼠皮肤组织COLⅠ-a1、COLⅠ-a2、α-SMA、TGF-β1、Smad2、Smad3的mRNA表达水平,采用Western blot检测各组小鼠皮肤组织TGF-β1、Smad2、Smad3、磷酸化Smad2、磷酸化Smad3的蛋白表达情况。结果(1)模型组小鼠背部未见新生毛发,皮肤硬化明显、有粘连,不易被提起,真皮厚度增厚,各药物干预组皮肤症状较模型组改善,真皮厚度变薄,以泼尼松组、红花前干预组改善最明显。(2)与对照组比较,模型组小鼠皮肤组织COLⅠ、α-SMA的蛋白表达水平,以及COLⅠ-a1、COLⅠ-a2、α-SMA的mRNA表达水平升高(P<0.05);与模型组比较,红花前干预组、红花后干预组和泼尼松组小鼠皮肤组织COLⅠ、α-SMA的蛋白表达水平,以及COLⅠ-a1、COLⅠ-a2、α-SMA的mRNA表达水平下降,且泼尼松组、红花前干预组小鼠皮肤组织COLⅠ、α-SMA的蛋白表达水平低于红花后干预组,红花前干预组小鼠皮肤组织COLⅠ-a1、COLⅠ-a2的mRNA表达水平低于红花后干预组(P<0.05)。(3)与对照组比较,模型组小鼠皮肤组织TGF-β1、Smad2、Smad3的蛋白及mRNA,以及磷酸化Smad2、磷酸化Smad3的蛋白表达水平升高,红花前干预组小鼠皮肤组织TGF-β1、Smad3的mRNA表达水平降低,Smad2的mRNA表达水平升高(P<0.05);与模型组比较,红花前干预组小鼠皮肤组织TGF-β1、Smad2、Smad3的蛋白及mRNA,以及磷酸化Smad2、磷酸化Smad3的蛋白表达水平降低(P<0.05)。结论红花可以抑制肌成纤维细胞的激活,减少细胞外基质的生成与堆积,从而发挥抗硬皮病小鼠皮肤纤维化的作用,其机制可能与下调硬皮病小鼠皮肤组织TGF-β1/Smads信号通路相关蛋白的表达有关。

Interaction between insulin-like growth factor binding protein-related protein 1 and transforming growth factor beta 1 in primary hepatic stellate cells

BACKGROUND:We previously showed that insulin-like growth factor binding protein-related protein 1(IGFBPrP1) is a novel mediator in liver fibrosis.Transforming growth factor beta 1(TGFβ1) is known as the strongest effector of liver fibrosis.Therefore,we aimed to investigate the detailed interaction between IGFBPrP1 and TGFβ1 in primary hepatic stellate cells(HSCs).METHODS:We overexpressed TGFβ1 or IGFBPrP1 and inhibited TGFβ1 expression in primary HSCs for 6,12,24,48,72,and 96 hours to investigate their interaction and observe the accompanying expressions of α-smooth muscle actin(α-SMA),collagen I,fibronectin,and phosphorylated-mothers against decapentaplegic homolog 2/3(p-Smad2/3).RESULTS:We found that the adenovirus vector encoding the TGFβ1 gene(Ad TGFβ1) induced IGFBPrP1 expression while that of α-SMA,collagen I,fibronectin,and TGFβ1 increased gradually.Concomitantly,Ad IGFBPrP1 upregulated TGFβ1,α-SMA,collagen I,fibronectin,and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours.Inhibition of TGFβ1 expression reduced the IGFBPrP1-stimulated expression of α-SMA,collagen I,fibronectin,and p-Smad2/3.CONCLUSIONS:These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGFβ1,which likely accelerates liver fibrosis progression.Furthermore,IGFBPrP1 likely participates in liver fibrosis in a TGFβ1-depedent manner,and may act as an upstream regulatory factor of TGFβ1 in the Smad pathway.

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.

Regulatory Effect of Shenge Yifei Capsule on TGF-β1/Smad Signaling Pathway in Rats with Chronic Obstructive Pulmonary Disease

[Objectives]This study aimed to study the effects of Shenge Yifei capsule on the TGF-β1/Smad signaling pathway in rats with chronic obstructive pulmonary disease(COPD).[Methods]Ten rats were randomly selected as the control group,and the other 40 rats were selected for modeling by fumigation combined with Klebsiella pneumoniae infection.A total of 38 rats were successfully modeled.They were randomly divided into model group(8 rats),low-dose Shenge Yifei capsule group(10 rats),high-dose Shenge Yifei capsule group(10 rats)and theophylline group(10 rats)in accordance with the principle of half male and half female.The rats in the model and control groups were given with distilled water by gavage,and the rats in the drug administration groups were given with corresponding drugs.The TGF-β1 level in the serum,and the expression levels of TGF-β1,Smad2,Smad3 and Smad7 and TGF-β1,Smad3 and Smad7 in airway tissues were detected.[Results]After 12 weeks,the serum TGF-β1 levels of the theophylline group and high-dose Shenge Yifei capsule group were lower than that of the low-dose Shenge Yifei capsule group(P<0.05).The expression levels of TGF-β1 and Smad3 in the theophylline group and high-dose Shenge Yifei capsule group were lower than that in the low-dose Shenge Yifei capsule group(P<0.05).The expression levels of TGF-β1 and Smad3 in the high-dose Shenge Yifei capsule group were lower than those in the low-dose Shenge Yifei capsule group and theophylline group(P<0.05).The expression levels of Smad7 and the proteins in the model group were lower than those in the other groups(P<0.05).The expression levels of Smad7 in the theophylline group and high-dose Shenge Yifei capsule group were higher than that in the low-dose Shenge Yifei capsule group(P<0.05).After 18 weeks,no significant difference was found in serum TGF-β1 level among the theophylline group and low and high-dose Shenge Yifei capsule groups(P>0.05).The expression levels of Smad7 and the proteins in the model group were lower than those in the other groups.The expression level of Smad7 in the high-dose Shenge Yifei capsule group was lower than that in the theophylline group(P<0.05).[Conclusions]Shenge Yifei capsule can regulate the TGF-β1/Smads signaling pathway.They can down-regulate the expression of TGF-β1,Smad2 and Smad3 and up-regulate the expression of Smad7,reducing the degree of airway modeling,delaying the development of COPD disease.Conventional high-dose Shenge Yifei capsule is more effective in inhibiting the expression of Smad2.

Interplay between micro RNA-17-5p, insulin-like growth factor-Ⅱ through binding protein-3 in hepatocellular carcinoma

AIM: To investigate the effect of microR NA on insulinlike growth factor binding protein-3(IGFBP-3) and hence on insulin-like growth factor-Ⅱ(IGF-Ⅱ) bioavailability in hepatocellular carcinoma(HCC).METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mir Vana mi RNA Isolation Kit. microR NA-17-5p(miR-17-5p) expression was mimicked and antagonized in Hu H-7 cell lines using Hi Per Fect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cD NA followed by quantification of mi R-17-5p and IGFBP-3 expression using Taq Man real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-Ⅱ protein was measured in transfected Hu H-7 cells using IGF-Ⅱ ELISA kit. RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where mi R-17-5p was extensively underexpressed in HCC tissues(P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients(P = 0.0041) compared to healthy donors. Forcing mi R-17-5p expression in Hu H-7 cell lines showed a significant downregulation of IGFBP-3 mR NA expression(P = 0.0267) and a significant increase in free IGF-Ⅱ protein(P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of mi R-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone(P = 0.0474).CONCLUSION: These data suggest that regulating IGF-Ⅱ bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miR NAs.

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.

Smad7 dependent expression signature highlights BMP2 and HK2 signaling in HSC transdifferentiation

AIM: To analyse the influence of Smad7, antagonist of transforming growth factor (TGF)-β canonical signaling pathways on hepatic stellate cell (HSC) transdifferentia-tion in detail. METHODS: We systematically analysed genes regulated by TGF-β/Smad7 in activated HSCs by microarray analy-sis and validated the results using real time polymerase chain reaction and Western blotting analysis. RESULTS: We identif ied 100 known and unknown tar-gets underlying the regulation of Smad7 expression and delineated 8 gene ontology groups. Hk2, involved in glycolysis, was one of the most downregulated proteins, while BMP2, activator of the Smad1/5/8 pathway, was extremely upregulated by Smad7. However, BMP2 de-pendent Smad1 activation could be inhibited in vitro by Smad7 overexpression in HSCs. CONCLUSION: We conclude (1) the existence of a tight crosstalk of TGF-β and BMP2 pathways in HSCs and (2) a Smad7 dependently decreased sugar metabolism ameliorates HSC activation probably by energy with-drawal.

D-tryptophan triggered epithelial-mesenchymal transition by activating TGF-βsignaling pathway

D-tryptophan is a special kind of nonprotein amino acid showing multiple physiological functions,but the detailed mechanisms are not fully revealed,impairing its further development and applications.This work was to investigate D-tryptophan physiological function and demonstrate the underlying mechanisms.D-tryptophan suppressed HaCaT cell proliferation but increased cell migration.Specifically,D-tryptophan decreased E-cadherin and increased Snail,Twist,and Slug expression,resulting in the development of an epithelial-mesenchymal transitions(EMT)phenomenon.Moreover,D-tryptophan promoted the expression of transforming growth factor-β(TGF-β)1,and Smad4 knockout damages D-tryptophan’s ability.These results indicated that D-tryptophan stimulated HaCaT cells to produce TGF-β1 and thus activated the TGF-β/Samd pathway,resulting in the triggering of EMT.This study revealed the molecular mechanisms of D-tryptophan activity,provided D-tryptophan as a potential approach for cancer treatment,wound healing,organ development and other relevant applications.

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.

干预TGF-β/Smad信号通路预防腰椎术后神经周围瘢痕形成的研究进展

转化生长因子-β(TGF-β)/Smad信号通路在机体内参与了许多细胞过程,包括多种细胞的生长、分化及凋亡,同时也是调节瘢痕形成的关键。TGF-β/Smad信号通路的失调是组织纤维化和瘢痕形成的一个主要因素。而腰椎术后神经周围纤维瘢痕形成是导致术后效果差、产生新的压迫症状甚至出现腰椎术后综合征的重要因素。这一问题的出现,极大地增加了患者的痛苦与治疗费用。因此,如何通过药物、生物材料等有效的临床治疗手段干预TGF-β/Smad信号通路预防或减少纤维瘢痕组织的形成并提高手术治疗的疗效,是目前临床研究的重点以及面临的挑战。

温针灸对卵巢功能减退大鼠TGF-β1/Smads信号通路的影响

目的探讨温针灸通过调节转化生长因子-β1(TGF-β1)/Smads信号通路对卵巢功能减退(DOR)大鼠卵巢功能的改善效果。方法采用雷公藤多苷片灌胃法复制DOR模型,温针灸给予关元、气海、中极及双侧肾俞治疗14 d后,采用阴道脱落细胞学检查及HE染色法观察大鼠动情周期和卵巢组织病理形态,采用酶联免疫吸附试验(ELISA)法检测血清雌二醇(E_(2))、黄体生成素(LH)、卵泡刺激素(FSH)、抗米勒管激素(AMH)水平,采用免疫组织化学法检测卵巢组织TGF-β1的表达,采用Western blot及实时荧光定量聚合酶链反应(qRT-PCR)法检测卵巢组织TGF-β1/Smads通路相关蛋白和mRNA的表达。结果经温针灸治疗后,DOR大鼠动情周期紊乱率显著降低,卵巢内成熟卵泡数显著增多(P<0.05);血清中E_(2)、AMH水平显著增加,LH、FSH水平显著降低(P<0.05);显著上调了卵巢组织TGF-β1的蛋白表达及p-Smad2/Smad2、p-Smad3/Smad3比值,下调了Smad7的蛋白表达(P<0.05);同时显著上调了TGF-β1、Smad2、Smad3的mRNA表达,下调了Smad7的mRNA表达(P<0.05)。结论温针灸对DOR大鼠具有明显的治疗效果,其机制可能与调控TGF-β1/Smads信号通路相关。

miR-215-5p调控YY1通过TGF-β1/Smad信号通路对PCOS大鼠卵巢颗粒细胞凋亡的影响

目的探究MicroRNA-215-5p(miR-215-5p)在多囊卵巢综合征(PCOS)大鼠中的表达及其对卵巢颗粒细胞(GC)凋亡的影响,并分析潜在机制。方法采用脱氢表雄酮(DHEA)建立PCOS大鼠模型,分离并培养原代PCOS大鼠卵巢GC,分为对照组(control组)、sh-NC组、sh-YY1组、sh-YY1+LY2109761组、miR-NC组、miR-215-5p mimic组、miR-215-5p mimic+pc-NC组、miR-215-5p mimic+pc-YY1组。TUNEL法检测PCOS大鼠卵巢组织GC凋亡;实时荧光定量PCR(RT-qPCR)检测卵巢组织/GC中miR-215-5p、阴阳-1(YY1)mRNA表达;CCK-8法和平板克隆形成实验检测细胞增殖能力;流式细胞仪检测细胞凋亡率;Western blot检测PCOS大鼠卵巢组织/GC中YY1、转化生长因子-β1(TGF-β1)、Smad4蛋白表达。结果miR-215-5p在PCOS大鼠卵巢组织中低表达,YY1 mRNA和蛋白水平在PCOS大鼠卵巢组织中显著升高(P<0.05);敲低YY1或上调miR-215-5p可显著增强PCOS大鼠卵巢GC增殖能力,抑制GC凋亡,升高TGF-β1、Smad4蛋白水平(均P<0.05);TGF-β/Smad4信号通路抑制剂LY2109761可显著减弱敲低YY1对卵巢GC凋亡的抑制作用(P<0.05);在过表达miR-215-5p的基础上,上调YY1可抑制TGF-β1/Smad信号通路的激活,显著减弱miR-215-5p mimic对PCOS大鼠卵巢GC凋亡的抑制作用(P<0.05)。结论miR-215-5p在PCOS中呈低表达,过表达miR-215-5p可能通过下调YY1,激活TGF-β1/Smad信号通路,促进PCOS大鼠卵巢GC增殖并抑制其凋亡。