Effects of Tanshinone ⅡA on Transforming Growth Factor β1-Smads Signal Pathway in Renal Interstitial Fibroblasts of Rats

The effects of tanshinone ⅡA （TSN） on transforming growth factor β1 （TGFβ1） signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFβ1 and pretreated with 10-6, 10-5, 10-4 mol/L TSN respectively. The mRNA levels of fibronectin （FN） were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFβ1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively （P〈0.01, P〈0.01）, and the protein expression of phosphorylated Smad2/3 （p-Smad2/3） increased by 7 times at the end of TGFβ1 stimulation （P〈0.01）. TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10-6 mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression （both P〉0.05）. After pretreatment with 10-5 and 10-4 mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively （P〈0.05, P〈0.01）, the FN protein levels were decreased by 40% and 44% respectively （P〈0.05, P〈0.05）, and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively （P〈0.05, P〈0.01）. The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFβ1-Smads signal pathway in renal intersti- tial fibroblasts.

PVT1通过TGF-β1/SMAD通路促进子宫内膜基质细胞过度纤维化的研究

目的探讨长链非编码RNA(lncRNA)浆细胞瘤多样异位基因1(PVT1)在调控子宫内膜基质细胞纤维化中的病理生理作用及分子机理。方法通过实时荧光定量聚合酶链反应(qRT-PCR)检测人子宫内膜基质细胞(HESCs)中PVT1、胶原蛋白Ⅰ、胶原蛋白Ⅲ、转化生长因子-β1(TGF-β1)、SMAD2及SMAD3的表达水平。结果PVT1过表达促进HESCs增殖(P<0.05),而PVT1沉默抑制HESCs增殖(P<0.05)。过表达PVT1在HESCs中上调胶原蛋白Ⅰ和胶原蛋白ⅢmRNA的表达(P<0.05),而PVT1被敲除时,胶原蛋白Ⅰ和胶原蛋白ⅢmRNA的表达水平被显著下调(P<0.05)。过表达PVT1在HESCs中可以进一步上调TGF-β1/SMAD信号相关基因(TGF-β1、SMAD2、SMAD3)的表达(P<0.05)。当PVT1被敲除时,TGF-β1/SMAD信号相关基因胶原蛋白Ⅰ和胶原蛋白ⅢmRNA的表达水平被显著下调(P<0.05)。PVT1可诱导HESCs上清液中TGF-β1的产生(P<0.05)。结论PVT1可通过TGF-β1/SMAD通路促进子宫内膜基质细胞过度纤维化而产生胶原蛋白。同时,PVT1可能是一个用于治疗子宫内膜粘连的新靶点。

Roles of Smad3 and Smad7 in rat pancreatic stellate cells activated by transforming growth factor-beta 1

BACKGROUND: Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor beta 1 (TGF-beta 1) is a critical mediator of this process. This study aimed to determine the expression of the Smad3 and Smad7 genes in the process of PSC activation, and explore the mechanisms of chronic pancreatitis. METHODS: The expressions of Smad3 and Smad7 in PSCs before and after TGF-beta 1 treatment were detected by reverse transcription-polymerase chain reaction and Western blotting analysis. Smad3 expression was detected in PSCs after treatment with 5 ng/ml of TGF-beta 1 for 24 hours. RESULTS: Smad7 expression was decreased in TGF-beta 1 -activated PSCs (P<0.05) in a dose-dependent manner. When TGF-beta 1 concentration reached 10 ng/ml, the expression of p-Smad3, Smad3, and Smad7 was inhibited (P<0.05). CONCLUSIONS: TGF-beta 1 promotes the expression of Smad3 and inhibits the expression of Smad7 during the activation of PSCs. In contrast, high-dose TGF-beta 1 downregulates the expression of Smad3 in completely activated PSCs.

柴芩肾安方对IgA肾病大鼠肾组织TGF-β_1和Smad 7的影响

目的:探讨柴芩肾安方对IgAN大鼠肾组织TGF-β1和Smad 7表达的影响。方法:采用灌服并定时静脉注射BSA复合感染SEB的方法制成大鼠IgAN模型,并用柴芩肾安方治疗,以氯沙坦为对照。第15周末,观察肾组织形态学变化和免疫复合物的沉积情况;采用免疫组化和RT-PCR的方法,分别检测肾组织TGF-β1和Smad 7蛋白及其mRNA的表达。结果:与正常组相比,模型组大鼠肾小球系膜细胞和系膜基质轻度增生,伴见弥漫的IgA和少量IgG沉积;TGF-β1和Smad 7蛋白及其mRNA表达均明显升高(P<0.01)。柴芩肾安方治疗后上述指标均明显减轻(P<0.01),且作用效果与氯沙坦类似。结论:抑制肾组织TGF-β1和Smad 7蛋白及其mRNA的表达,可能是柴芩肾安方延缓IgAN肾脏病变进展的机制之一。

加味当归补血汤对肾小管上皮细胞TGF-β_1/SMADs信号转导通路的影响

目的：观察加味当归补血汤对转化生长因子β1（TGF-β1）刺激的小鼠肾小管上皮细胞Smad3、Smad4、Smad7信号通路的影响，探讨该方防治肾间质纤维化的细胞分子生物学机制。方法：原代培养BalB／C小鼠肾小管上皮细胞．用TGF-β（1ng／ml）刺激24h，加味当归补血汤及洛汀新含药血清干预。共分6组：正常组、模型组、加味当归补血汤低中高剂量组（含药血清浓度分别为2．5％、5％、10％）、洛汀新组，观察各组细胞形态学变化，检测细胞Smad3、Smad4、Smad7的mRNA和蛋白质表达情况（PT—PCR、Western-Blotting）。结果：（1）TGF-β1刺激后，肾小管上皮细胞部分形态发生变大，伸长，呈梭形，经中药加味当归补血汤和洛汀新干预后，细胞形态又恢复正常；（2）TGF-β1刺激肾小管上皮细胞后，Smad3、Smad4 mRNA和蛋白质表达显著增加，Smad7 mRNA和蛋白质则显著减少（P〈0、05～0、01）；（3）各浓度加味当归补血汤能显著下调异常增高的Smad3，上调Smad7的基因和蛋白质表达水平（P〈0．05～0．01），能显著下调Smad4基因表达。结论：加味当归补血汤防治肾间质纤维化可能与调控肾小管上皮细胞TGF-β1／Smads信号转导途径相关。

TGF-β_1及其信号蛋白Smad2,3在大鼠心肌细胞肥大中的作用

目的 探讨转化生长因子 β1(transforminggrowthfactor β1,TGF β1)及其信号蛋白Smad2 ,3在诱导大鼠心肌细胞肥大中的作用。方法 培养新生大鼠心肌细胞 ,用不同剂量TGF β1刺激 ,检测 [3 H] Leu掺入量 ,RT PCR检测信号蛋白Smad2 ,3mRNA的表达。结果 不同剂量的TGF β1均能明显增加心肌细胞 [3 H] Leu掺入量 ,TGF β1增加肥大心肌细胞Smad2 ,3mRNA的表达。结论 TGF β1能诱导大鼠心肌细胞肥大 ,信号蛋白Smad2 ,3在其中发挥了重要作用。

Smad 1/5和TGF-β_1 mRNA在舌鳞状细胞癌组织中的表达

目的研究舌鳞状细胞癌(SCC)组织中Smad 1/5和转化生长因子β1(TGF-β1) mRNA的表达,探讨其与肿瘤临床分期、病理分级及淋巴结转移的关系。方法采用原位杂交法检测49例舌SCC、20例上皮异常增生以及10例正常口腔舌黏膜组织中Smad 1/5和TGF-β1 mRNA的表达。运用χ2检验,分析Smad 1/5和TGF-β1 mRNA表达与临床病理指标的关系;应用Spearman等级相关分析,检验Smad 1/5 mRNA与TGF-β1 mRNA表达间的相关性。结果舌SCC、上皮异常增生和正常黏膜组织中,Smad 1/5 mRNA的表达阳性率分别为73.5%、35%和30%;TGF-β1 mRNA表达阳性率分别为67.3%、25%和20%。Smad 1/5和TGF-β1 mRNA在癌组织中的表达显著高于正常黏膜组织(P<0.05)。Smad 1/5 mRNA表达阳性率,在有或无淋巴结转移及不同TNM分期间有显著差异(P<0.05)。Smad 1/5 mRNA与TGF-β1 mRNA表达呈显著正相关(r=0.405,P=0.039)。结论舌SCC的发生和发展可能与TGF-β mRNA及其下游信号通路相关。

补肾除湿联合富血小板血浆调控血清TGF-β1及Smad-1水平治疗膝骨关节炎

目的:探讨补肾除湿联合富血小板血浆(platelet-rich plasma,PRP)治疗早中期膝骨关节炎(knee osteoarthritis,KOA)的临床疗效及对患者血清中转化生长因子-β1(transforming growth factor-β1,TGF-β1)和Smad-1水平的影响。方法:选取2020年5月至2022年4月收治的45例早中期KOA患者,分为对照组和观察组。对照组30例,男12例,女18例;年龄43~69(57.3±6.5)岁;Kellgren-Lawrence(K-L)分级Ⅰ级8例,Ⅱ级13例,Ⅲ级9例;病程1.5~5.0(3.8±1.7)年;分别于1、3周时向患膝关节腔注射5 ml的PRP 1次,共2次。观察组15例,男7例,女8例;年龄45~70(56.7±6.2)岁;K-L分级Ⅰ级4例,Ⅱ级9例,Ⅲ级2例;病程1.8~5.7(4.0±1.8)年;注射PRP 5 ml,时间、次数与对照组相同,同时口服补肾除湿方水煎剂,每日1剂,共28剂。两组疗程均为4周。分别于治疗前后采用疼痛视觉模拟评分(visual analogue scale,VAS)和Lequesne MG评分评估患膝疼痛及关节功能改善情况。分别于治疗前、治疗结束后1 d采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测TGF-β1、Smad-1的含量,并观察两组患者并发症发生情况。结果:45例患者获得随访,时间26~30(28.0±0.6)d。治疗前两组VAS和膝关节Lequesne MG评分比较,差异无统计学意义(P>0.05);治疗结束后1 d,两组VAS和膝关节Lequesne MG评分均低于治疗前(P<0.05);治疗结束后1 d,观察组VAS和膝关节Lequesne MG评分低于对照组(P<0.05)。两组治疗结束后1 d,TGF-β1含量均较治疗前降低(P<0.05);治疗结束后1 d,观察组TGF-β1含量低于对照组(P<0.05)。两组治疗结束后1 d的Smad-1含量与治疗前比较,差异无统计学意义(P>0.05);治疗结束后1 d,观察组Smad-1含量高于对照组(P<0.05)。两组并发症情况比较,差异无统计学意义(P>0.05)。结论:补肾除湿联合关节腔注射PRP治疗早中期KOA疗效确切,优于单纯关节腔内注射PRP,并在一定程度上降低患者血清TGF-β1水平,升高Smad-1水平,抑制炎症反应,促进软骨修复,这可能是补肾除湿联合PRP治疗KOA的作用机制之一。

Vascular endothelial growth factor A, secreted in response to transforming growth factor-β1 under hypoxic conditions, induces autocrine effects on migration of prostate cancer cells

Hypoxia and transforming growth factor-β1 （TGF-β1） increase vascular endothelial growth factor A （VEGFA） expression in a number of malignancies. This effect of hypoxia and TGF-β1 might be responsible for tumor progression and metastasis of advanced prostate cancer. In the present study, TGF-β1 was shown to induce VEGFA165 secretion from both normal cell lines （HPV7 and RWPE1） and prostate cancer cell lines （DU 145 and PC3）. Conversely, hypoxia-stimulated VEGFA165 secretion was observed only in prostate cancer cell lines. Hypoxia induced TGF-β1 expression in PC3 prostate cancer cells, and the TGF-β1 type I receptor （ALK5） kinase inhibitor partially blocked hypoxia-mediated VEGFA16s secretion. This effect of hypoxia provides a novel mechanism to increase VEGFA expression in prostate cancer cells. Although autocrine signaling of VEGFA has been implicated in prostate cancer progression and metastasis, the associated mechanism is poorly characterized. VEGFA activity is mediated via VEGF receptor （VEGFR） 1 （Fit-l） and 2 （FIk-I/KDR）. Whereas VEGFR-1 mRNA was detected in normal prostate epithelial cells, VEGFR-2 mRNA and VEGFR protein were expressed only in PC3 cells. VEGFA165 treatment induced phosphorylation of extracellular signal-regulated kinase 1/2 （ERKI/2） in PC3 cells but not in HPV7 cells, suggesting that the autocrine function of VEGFA may be uniquely associated with prostate cancer. Activation of VEGFR-2 by VEGFA165 was shown to enhance migration of PC3 cells. A similar effect was also observed with endogenous VEGFA induced by TGF-β1 and hypoxia. These findings illustrate that an autocrine loop of VEGFA via VEGFR-2 is critical for the tumorigenic effects of TGF-β1 and hypoxia on metastatic prostate cancers.

The Effect of Simvastatin on mRNA Expression of Transforming Growth Factor-β1,Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor in Tooth Extraction Socket

Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （VEGF） in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups （n=24）. Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-131 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.

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 (TGF beta 1) is known as the strongest effector of liver fibrosis. Therefore, we aimed to investigate the detailed interaction between IGFBPrP1 and TGF beta 1 in primary hepatic stellate cells (HSCs). METHODS: We overexpressed TGF beta 1 or IGFBPrP1 and inhibited TGF beta 1 expression in primary HSCs for 6, 12, 24, 48, 72, and 96 hours to investigate their interaction and observe the accompanying expressions of a-smooth muscle actin (alpha-SMA), collagen I, fibronectin, and phosphorylated-mothers against decapentaplegic homolog 2/3 (p-Smad2/3). RESULTS: We found that the adenovirus vector encoding the TGF beta 1 gene (AdTGF beta 1) induced IGFBPrP1 expression while that of alpha-SMA, collagen I, fibronectin, and TGF beta 1 increased gradually. Concomitantly, AdIGFBPrP1 upregulated TGF beta 1, alpha-SMA, collagen I, fibronectin, and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours. Inhibition of TGF beta 1 expression reduced the IGFBPrP1-stimulated expression of alpha-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 TGF beta 1, which likely accelerates liver fibrosis progression. Furthermore, IGFBPrP1 likely participates in liver fibrosis in a TGF beta 1-depedent manner, and may act as an upstream regulatory factor of TGF beta 1 in the Smad pathway.

参附汤联合茯苓四逆汤治疗心阳亏虚型慢性心力衰竭患者的疗效及对其转化生长因子β1、Smad同源物3表达的影响

目的探讨参附汤联合茯苓四逆汤治疗心阳亏虚型慢性心力衰竭患者的疗效及对其转化生长因子β1(transforming growth factor-β,TGF-β1)、Smad同源物3(Smad homolog 3,Smad3)表达的影响。方法选取2020年1月—2023年1月期间北京市怀柔区中医医院收治的90例心阳亏虚型慢性心力衰竭患者,按随机数字表法分为对照组和研究组,每组各45例。对照组予常规西医治疗,研究组在对照组的基础上加用参附汤合茯苓四逆汤治疗。4周为1个疗程,两组患者均治疗4个疗程。观察比较两组患者治疗前后心功能[左室舒张末期内径(Left ventricular end diastolic diameter,LVEDD)、左室收缩末期内径(Left ventricular end systolic diameter,LVESD)、左室射血分数(Left ventricular ejection fraction,LVEEF)、每搏输血量(Blood transfusion volume per stroke,SV)]、心衰因子[心型脂肪酸结合蛋白(Heart-type Fatty Acid Binding Protein,H-FABP)、脑钠肽(Natriuretic peptide,BNP)]、心肌纤维化指标[Ⅰ型前胶原氨基端前肽(Type I procollagen amino terminal peptide,PICP)、Ⅲ型前胶原氨基端末肽(TypeⅢprocollagen amino terminal peptide,PⅢNP)、心肌肌钙蛋白I(cardiac troponin IcTnI)]、TGF-β1、Smad3表达量变化,评价活动耐力、心衰评分、明尼苏达评分、中医证候积分,并统计临床疗效及主要心血管不良事件(MACE)发生情况。结果治疗后两组患者心功能LVEDD、LVESD指标均较治疗前降低,LVEF、SV指标较治疗前升高,差异有统计学意义(P<0.05);且研究组心功能LVEDD、LVESD指标明显低于对照组,LVEF、SV指标明显高于对照组,差异有统计学意义(P<0.05)。治疗后两组患者心衰因子H-FABP、cTnI、BNP水平均较治疗前降低,差异有统计学意义(P<0.05);且研究组心衰因子H-FABP、cTnI、BNP水平均明显低于对照组,差异有统计学意义(P<0.05)。治疗后两组患者心肌纤维化PICP、PⅢNP水平均较治疗前降低,差异有统计学意义(P<0.05);且研究组心肌纤维化PICP、PⅢNP水平明显低于对照组,差异有统计学意义(P<0.05)。治疗后两组患者TGF-β1、Smad3表达量均较治疗前降低,差异有统计学意义(P<0.05);且研究组TGF-β1、Smad3表达量均明显低于对照组,差异有统计学意义(P<0.05)。治疗后两组患者活动耐力较治疗前升高,心衰、明尼苏达评分较治疗前降低,差异有统计学意义(P<0.05);且研究组活动耐力评分明显高于对照组,心衰、明尼苏达评分均明显低于对照组,差异有统计学意义(P<0.05)。治疗后两组患者中医证候积分较治疗前降低,差异有统计学意义(P<0.05);且研究组中医证候积分明显低于对照组,差异有统计学意义(P<0.05)。治疗后研究组临床总有效率95.56%(43/45)明显高于对照组80.00%(36/45),差异有统计学意义(P<0.05)。治疗期间,研究组MACE发生率4.44%(2/45)明显低于对照组17.78%(8/45),差异有统计学意义(P<0.05)。结论参附汤合茯苓四逆汤可显著改善心阳亏虚型慢性心力衰竭患者临床症状及体征,降低TGF-β1、Smad3表达,抑制心肌纤维化,促进心脏功能恢复,效果理想。

Feedback regulation between phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 and transforming growth factor β1 and prognostic value in gastric cancer

BACKGROUND Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1(PREX1)was reported to be overexpressed in some cancers and involved in cancer development,but its expression and significance in gastric cancer remain unclear.AIM To evaluate the expression of PREX1 in gastric cancer and its significance in the development of gastric cancer,especially to evaluate the potential mechanism of PREX1 in gastric cancer.METHODS Bioinformatic analysis was performed in order to examine the expression of PREX1 in gastric cancer.The relationship between the survival rate of gastric cancer patients and PREX1 expression was assessed by Kaplan Meier portal.The Gene Set Enrichment Analysis and the correlation between PREX1 and transforming growth factor(TGF)β1 pathway-related mediators were evaluated by cBioPortal for Cancer Genomics.Western blotting and reverse transcriptase polymerase chain reaction assay were used to test the role of TGFβ1 on the expression of PREX1.Western blotting and dual-luciferase reporter system was used to evaluate the effect of PREX1 on the activation of TGFβ1 pathway.Wound healing and Transwell assay were used to assess the effect of PREX1 on the metastasis activity of gastric cancer cells.RESULTS PREX1 was overexpressed in the gastric tumors,and the expression levels were positively associated with the development of gastric cancer.Also,the high expression of PREX1 revealed poor prognosis,especially for those advanced and specific intestinal gastric cancer patients.PREX1 was closely involved in the positive regulation of cell adhesion and positively correlated with TGFβ1-related mediators.Furthermore,TGFβ1 could induce the expression of PREX1 at both the protein and mRNA level.Also,PREX1 could activate the TGFβ1 pathway.The induced PREX1 could increase the migration and invasion activity of gastric cancer cells.CONCLUSION PREX1 is overexpressed in gastric cancer,and the high level of PREX1 predicts poor prognosis.PREX1 is closely associated with TGFβsignaling and promotes the metastasis of gastric cancer cells.

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。

积雪草对早期糖尿病肾病大鼠TGF-β_1表达及相关下游信号的影响

目的:通过研究积雪草(CA)对早期糖尿病肾病大鼠转化生长因子β1(TGF-β1)表达及相关下游信号的影响,阐明积雪草防治早期糖尿病肾病(DN)的分子机制。方法:60只雄性SD大鼠,按体重随机分为假手术组(n=10)和造模组(n=50)。造模组大鼠进行右肾切除术,1周后给以腹腔注射链脲佐菌素(STZ)30 mg/kg,连续给3d;72 h后测血糖,以≥16.7 mmol/L,尿糖+++以上及尿量大于对照组的50%为DN模型成模标准。假手术组进行右肾被膜损伤,并注射相应量生理盐水。造模组通过灌胃给药,分为:DN模型组(模型组)、DN+福辛普利组(蒙组1.6 mg/kg·d)、DN+积雪草高剂量组(高剂量组16.8 mg/kg·d)、DN+积雪草中剂量组(中剂量组11.2 mg/kg·d)和DN+积雪草低剂量组(低剂量组5.6 mg/kg·d)(n=10),连续给药16周,每日上午1次灌胃。利用实时荧光定量PCR和Western blot分别检测肾组织中TGF-β1、TβR1、TβR2、Smad2/3、p-Smad2/3及Smad7 mRNA和蛋白的表达。结果:与假手术组相比,DN组TGF-β1、TβR1、TβR2、Smad2/3 mRNA和蛋白表达及Smad2/3蛋白的磷酸化水平显著增加(P<0.05)、Smad7 mRNA和蛋白表达明显减少(P<0.05),而福辛普利和高剂量积雪草能倒转DN引起的TGF-β1、TβR1、TβR2、Smad2/3 mRNA和蛋白表达增加(P<0.05)及Smad7 mRNA和蛋白表达降低(P<0.05)。结论:积雪草可能通过调控TGF-β1/Smad信号通路起到防治DN的作用。

Saponin Ⅰ from Shuitianqi(Rhizoma Schizocapasae Plantagineae) inhibits metastasis by negatively regulating the transforming growth factor-β1/Smad7 network and epithelial-mesenchymal transition in the intrahepatic metastasis Bagg's Albino/c mouse model

OBJECTIVE:To examine the influence of SaponinⅠfrom Shuitianqi(Rhizoma Schizocapasae Plantagineae)(SSPHⅠ)on hepatocellular carcinoma(HCC)metastasis,and to elucidate the underlying mechanism.METHODS:The intrahepatic metastasis Bagg's Albino/c(BALB/c)mouse model was established with human hepatocellular carcinomas(HepG2)cells,then treated with normal saline(once per day),cisplatin(2 mg/kg,once every 2 d),and SSPHⅠ(25,50,and 75 mg/kg,once per day).Then,we assessed alterations in the hepatic pathology and target protein expressions in the intrahepatic metastasis BALB/c mouse model using a series of molecular biology techniques.RESULTS:Based on our analysis,SSPHⅠsignificantly alleviated hepatocyte necrosis and tumor cells infiltration.Moreover,SSPHⅠsuppressed extracellular matrix(ECM)degradation and angiogenesis via a decrease in matrix etalloproteinase-2(MMP-2),MMP-9,CD31,CD34,and vascular endothelial growth factor(VEGF)levels.Furthermore,SSPHⅠrepressed invasion and metastasis by suppressing the transforming growth factor-β1(TGF-β1)/Smad7 axis and epithelial-mesenchymal transition(EMT),as evidenced by the scarce TGF-β1,Ncadherin,and Vimentin expressions,and elevated Smad7 and E-cadherin expressions.CONCLUSION:The SSPHⅠ-mediated negative regulation of the TGF-β1/Smad7 axis and EMT are critical for the inhibition of HCC invasion and metastasis.

Role of transforming growth factor-beta signaling pathway in pathogenesis of benign biliary stricture

AIM: To characterize the expression of members of the transforming growth factor-beta (TGF-β)/Smad/ connective tissue growth factor (CTGF) signaling pathway in the tissue of benign biliary stricture, and to investigate the effect of TGF-β signaling pathway in the pathogenesis of benign biliary stricture. METHODS: Paraffin embedded materials from 23 cases of benign biliary stricture were analyzed for members of the TGF-β/Smad/CTGF signaling pathway. TGF-β_1, TβRⅠ, TβRⅡ, Smad4, Smad7 and CTGF protein were detected by immunohistochemical strepto-advidinbiotin complex method, and CTGF mRNA was evaluated by hybridization in situ, while 6 cases of normal bile duct served as controls. The percentages of positive cells were counted. The correlation between TGF-β_1, Smad4 and CTGF was analyzed. RESULTS: The positive expression ratios of TGF-β_1, TβRⅠ , TβRⅡ , Smad4, CTGF and CTGF mRNA in 23 cases with benign biliary stricture were 91.3%, 82.6%, 87.0%, 78.3%, 82.6% and 65.2%, respectively, signifi cantly higher than that in 6 cases of normal bile duct respectively (vs 33.3%, 16.7%, 50.0%, 33.3%, 50.0%, 16.7%, respectively, P < 0.05). The positiveexpression ratio of Smad7 in cases with benign biliary stricture was 70.0%, higher than that in normal bile duct, but this difference is not statistically signifi cant 70.0% vs 50%, P > 0.05). There was a positive correlation between positive expression of TGF-β_1, Smad4 and CTGF in cases with benign biliary stricture. CONCLUSION: The high expression of TGF-β/Smad/ CTGF signaling pathway plays an important role in the pathogenesis of benign biliary stricture.

Influence of Exogenous TGFβ_1 on the Expression of Smad2 and Smad3 in Rat Bone Marrow-derived Mesenchymal Stem Cells

The expression of Smad2 and Smad3 and the influence of exogenous transforming growth factorβ 1 (TGFβ 1) on them in rat bone marrow-derived mesenchymal stem cells (MSCs) cultured in vitro were investigated. The effects of different concentrations of TGFβ 1 on cell proliferation and ALP activity were detected by MTT and PNPP in MSCs respectively. The expression of Smad2 and Smad3 and the influence of exogenous TGFβ 1 on them were also examined by immunocytochemistry and Western blot assays. The exogenous TGFβ 1 induced a dose-dependent decrease in cell proliferation and a dose-dependent increase in ALP activity, which plateaued at 5 ng/ml. Smad2 and Smad3 proteins were detected only in the cytoplasm in the absence of TGFβ 1 and TGFβ 1 could stimulate the translocation of them from the cytoplasm to the nucleus. The total amount of Smad2 protein remained unchanged before and after TGFβ 1 treatment (P>0.05). The expression levels of Smad3 remained unchanged after 3 h and 6 h treatment (P>0.05), but decreased markedly after 24 h treatment (P<0.05). It was concluded that TGFβ 1 is a latent osteoinductive factor involved in osteoblastic differentiation. Both Samd2 and Smad3 mediate TGFβ 1 signaling as downstream mediators in MSCs. The biological output of TGFβ 1 triggering the osteoblastic differentiation could be entirely determined by Smad3 in MSCs.

积雪草对转染TGF-β1基因的大鼠肾小球系膜细胞Smad 2/3、Smad 7和胶原蛋白Ⅳ表达的影响

目的:通过细胞转基因技术,获得稳定表达转化生长因子β1(TGF-β1)的系膜细胞(MC)克隆,观察积雪草(CA)对Smad 2/3、Smad 7和胶原蛋白1V表达及Smad 2/3磷酸化的影响。方法:采用脂质体的方法将TGF-β1表达质粒转入MC细胞,采用G418筛选并建立稳定表达TGF-β1的细胞株。将MC细胞株分为3组:对照组(未转染TGF-β1的MC+RPMI 1640+10%正常大鼠血清),TGF-β1转染组(稳定表达TGF-β1的MC+RPMI 1640+10%正常大鼠血清),积雪草(CA)组:稳定表达TGF-β1的MC+RPMI 1640+10%含高浓度CA的大鼠血清。实验重复5次。ELISA法检测各组培养上清中TGF-β1和胶原Ⅳ的含量;RT-PCR方法检测各组细胞TGF-β1、Smad 2/3、Smad7的mRNA表达水平;Western印迹法检测各组细胞TGF-β1、Smad 2/3、p-Smad 2/3、Smad 7、胶原Ⅳ的蛋白质水平。结果:TGF-β1转染组细胞上清液中的TGF-β1和胶原蛋白Ⅳ水平显著升高,积雪草能显著降低TGF-β1和胶原蛋白Ⅳ水平;TGF-β1转染组细胞中TGF-β1、Smad 2/3的mRNA和蛋白质表达水平及Smad 2/3的磷酸化水平均显著升高,而CA可显著降低MC细胞中TGF-β1、Smad 2/3的mRNA和蛋白质表达水平及Smad 2/3的磷酸化水平;TGF-β1转染组细胞中的Smad 7 mRNA水平显著降低,而CA能使Smad 7的mRNA水平显著升高。结论:稳定表达TGF-β1的MC细胞能激活TGF-β1/Smad信号通路,并引起胶原蛋白Ⅳ表达增加,而CA通过抑制此通路的激活,进而抑制胶原蛋白Ⅳ的表达而减缓糖尿病肾病(DN)的发生。

依那普利对大鼠肾间质纤维化中TGF-β1,p-Smad2/3及Smad7的作用

目的:观察依那普利对肾间质纤维化大鼠肾组织中TGF-β1,p-Smad2/3及Smad7的影响,探讨依那普利抗肾间质纤维化的作用机制。方法:将30只雌性SD大鼠随机分为假手术组、模型组和依那普利治疗组。对治疗组和模型组大鼠行左侧输尿管结扎术建立单侧输尿管梗阻的动物模型。术后14d处死大鼠,留取梗阻侧肾组织行HE和Masson染色以观察各组大鼠肾组织病理改变,用免疫组织化学方法检测ColⅠ,TGF-β1,p-Smad2/3和Smad7的蛋白水平表达,用RT-PCR方法检测TGF-β1和Smad7mRNA水平表达。结果:模型组肾间质损伤指数、胶原相对面积及间质内ColⅠ表达均较假手术组增高(P<0.01),依那普利治疗后好转。假手术组肾组织TGF-β1蛋白及mRNA表达、p-Smad2/3蛋白表达较低,模型组表达明显增强(P<0.01),依那普利治疗后明显减弱(P<0.01)。假手术组肾组织可见较多的Smad7蛋白及mRNA表达,模型组肾组织Smad7表达明显减弱(P<0.01),依那普利治疗后Smad7表达较模型组增强(P<0.01)。结论:依那普利可以通过影响单侧输尿管梗阻大鼠肾组织中TGF-β/Smads信号通路中的TGF-β1,p-Smad2/3和Smad7而起到抑制纤维化的作用。