Expression of Connective Tissue Growth Factor in Renal Tubulointerstitial Fibrosis in Rats and Its Pathogenic Role

Summary： In order to explore the role of connective tissue growth factor （CTGF） in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction （UUO） group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor β1 （TGF-β1）. collagen [ （col I ）, and plasminogen activator inhibitor-1 （PAI 1） were detected using rexerse transcriptional-polymerase chain reaction （RT PCR）. Immunohistochemistry was performed to evaluale the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO （P〈0.01）. With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index （r =0.62, P〈0.01）, the expression of TGF-β1 （r=0.85, P〈0.01）, colI （r=0.78, P〈0.01）, and PAI-1（r=0.76, P〈0.01）. Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course （P〈0.01, as compared with sham-operated group）. It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix （ECM） synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

Insulin-like growth factor binding protein related protein 1 knockdown attenuates hepatic ?brosis via the regulation of MMPs/TIMPs in mice

Background: Previous research suggested that insulin-like growth factor binding protein related protein 1(IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases(MMP) and tissue inhibitors of metalloproteinases(TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix(ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis. Methods: Hepatic fibrosis was induced by thioacetamide(TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog(Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin( α-SMA), transforming growth factor β 1(TGF β1), collagen I, MMPs/TIMPs, Sonic Hedgehog(Shh), and glioblastoma family transcription factors(Gli1) were investigated by immunohistochemical staining and Western blotting analysis. Results: We found that hepatic expression of IGFBPrP1, TGF β1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGF β1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance. Conclusions: Our findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGF β1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.

TRANSFORMING GROWTH FACTOR-β1 AND SMAD4 SIGNALING PATHWAY DOWN-REGULATES RENAL EXTRACELLULAR MATRIX DEGRADATION IN DIABETIC RATS

Objective To investigate the role of transforming growth factor-β1(TGF-β1)/Smad4 pathway in development of renal fibrosis in streptozotocin(STZ)-induced diabetic nephropathy(DN) rats and explore its possible mechanism.Methods Male Wistar rats weighing 180-220 g were divided into 5 groups:group A(normal control),group B [diabetes mellitus(DM) 2 weeks],group C(DM 4 weeks),group D(DM 8 weeks),and group E(DM 16 weeks).Except for the normal control group,other groups were induced DM by single injection of STZ(55 mg/kg) respectively.Blood glucose level,serum creatinine,and 24-hour urine protein were examined.Expressions of TGF-β1 and Smad4 protein and mRNA in kidney were detected using immunohistochemical technique,Western blot,and real-time PCR.mRNA expressions of stromelysin-1(MMP-3),tissue inhibitor of metalloproteinase-1(TIMP-1),and collagen Ⅲ in kidney were also detected by real-time PCR..Results The levels of blood glucose,serum creatinine,and 24-hour urine protein in rats of group B,C,D,and E were higher than those of the control group.With the progression of renal fibrosis,the expressions of TGF-β1 and Smad4 protein and mRNA in kidney of diabetic rats elevated.In addition,the renal MMP-3 mRNA expression diminished in diabetic rats,while TIMP-1 and collagen Ⅲ mRNA increased.Conclusions In STZ-induced diabetic rats,the TGF-β1/Smad4 appears to play an important role in renal fibrosis of DN.The increased expression of TGF-β1 and Smad4 might result in the transcriptional regulation of downstream target genes of TGF-β1/Smad4 pathway,which contributes to the progression of renal fibrosis in diabetic rats.

Influence of ginsenoside Rg1, a panaxatriol saponin from Panax notoginseng, on renal fibrosis in rats with unilateral ureteral obstruction

Total saponins of Panax notoginseng (PNS) have been shown to ameliorate renal interstitial fibrosis. Ginsenoside Rg1, a panaxatriol saponin, is one of the major active molecules from PNS. The present study was undertaken to investigate the effect of ginsenoside Rg1 on renal fibrosis in rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into 3 groups: sham-operation (n=15), UUO (n=15) and UUO with ginsenoside Rg1 treatment (n=15, 50 mg per kg body weight, intraperito- neally (i.p.) injected). The rats were sacrificed on Days 7 and 14 after the surgery. Histological examination demonstrated that ginsenoside Rg1 significantly inhibited interstitial fibrosis including tubular injury as well as collagen deposition. α-smooth muscle actin (α-SMA) and E-cadherin are two markers of tubular epithelial-myofibroblast transition (TEMT). Interestingly, ginsenoside Rg1 notably decreased α-SMA expression and simultaneously enhanced E-cadherin expression. The messenger RNA (mRNA) of transforming growth factor-β1 (TGF-β1), a key mediator to regulate TEMT, in the obstructed kidney increased dra- matically, but was found to decrease significantly after administration of ginsenoside Rg1. Further study showed that ginsenoside Rg1 considerably decreased the levels of both active TGF-β1 and phosphorylated Smad2 (pSmad2). Moreover, ginsenoside Rg1 substantially suppressed the expression of thrombospondin-1 (TSP-1), a cytokine which can promote the transcription of TGF-β1 mRNA and the activation of latent TGF-β1. These results suggest that ginsenoside Rg1 inhibits renal interstitial fibrosis in rats with UUO. The mechanism might be partly related to the blocking of TEMT via suppressing the expression of TSP-1.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction(四逆汤)against renal fibrosis

OBJECTIVE:To investigate the mechanism by which Sini decoction(四逆汤,SND)improves renal fibrosis(Rf)in rats based on transforming growth factor β1/Smad(TGF-β1/Smad)signaling pathway.METHODS:Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf.The targets of SND drug components and the targets of Rf were obtained by searching databases,such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCSMP)and GeenCard.The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram.Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-β1/Smad signaling pathway.The Rf rat model was established by unilateral ureteral occlusion(UUO).The expression levels of transforming growth factor,matrix metalloproteinase-9(MMP-9),matrix metal protease-2(MMP-2),connective tissue growth factor(CTGF),and tissue inhibitor of metalloproteinase-1(TIMP-1)were determined by Masson staining of rat renal tissue,and immunohistochemical methods.The expression levels of Smad3,Smad2,and Smad7 in renal tissue were determined by Western blotting(WB).The mechanism of the improving effects of SND on Rf was investigated based on TGF-β1/Smad signaling pathway.RESULTS:A total of 12 drug components of Fuzi(Radix Aconiti Lateralis Preparata),5 drug components of Ganjiang(Rhizoma Zingiber),and 9 drug components of Gancao(Radix Glycy et Rhizoma)were obtained from the database search,and 207 shared targets were found.A total of 1063 Rf targets were found in the database search.According to the Venn diagram,in total,96 intersection targets were found in two database searches.The metabolic pathways involved included TGF-β signaling pathway,phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling(PI3K/Akt)pathway,and hypoxia-inducible factor-1(HIF-1)signaling pathway.Masson staining analysis showed that compared with the model group,the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower(P<0.05).Immunohistochemical analysis,compared with the control group,the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased(P<0.05,P<0.01),and the positive cell area expression levels of CTGF and TGF-β1 were significantly increased(P<0.01).Compared with the model group,the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased(P<0.05,P<0.01),and the positive cell area expression levels of CTGF and TGF-β1 in the kidney tissue were significantly decreased(P<0.05,P<0.01).WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3(P<0.05)and increase the expression of Smad7(P<0.05).

IGFBPrP1 induces liver fibrosis by inducing hepatic stellate cell activation and hepatocyte apoptosis via Smad2/3 signaling

AIM:To investigate the role and mechanism of insulinlike growth factor binding protein-related protein 1(IGFBPrP1)in the development of liver fibrosis.METHODS:An in vitro model using hepatic stellate cell(HSC)-T6 cells and an in vivo model of rat liver overexpressing IGFBPrP1 were established using an IGFBPrP1-expressing recombinant adenovirus.The expression of IGFBPrP1 was examined by immunofluorescence,and the expression of collagen?Ⅰ?and fibronectin was mea-sured by real-time reverse transcription-polymerase chain reaction and Western blot analysis.The expression of Smad2/3 and p-Smad2/3 was examined by Western blot and immunohistochemistry.A shSmad3-expressing recombinant adenovirus(AdshSmad3)was designed and used to knockdown the Smad3 gene in HSC-T6 cells and rat liver fibrosis transfected with IGFBPrP1.The expression of collagen?Ⅰ,fibronectin,andα-smooth muscle actin(α-SMA)was determined by Western blot analysis and immunohistochemistry.Hepatocyte apoptosis was assessed using TUNEL assay.RESULTS:IGFBPrP1 overexpression induced collagen deposition and up-regulated the expression ofα-SMA and p-Smad2/3,and AdshSmad3 inhibited IGFBPrP1-stimulated p-Smad2/3 activation and the expression ofα-SMA,collagen?Ⅰ?and fibronectin in HSC-T6 cells.Similarly,increased hepatocyte apoptosis(38.56%±3.42%vs 0.24%±0.03%,P<0.05),α-SMA positive stained cells(29.84%±1.36%vs 5.83%±1.47%,P<0.05),and increased numbers of Smad3(35.88%±2.15%vs10.24%±1.31%,P<0.05)and p-Smad2/3 positive cells(28.87%±2.73%vs 8.23%±0.98%,P<0.05)were detected in the livers of IGFBPrP1-overexpressing rats compared with the control group.Moreover,AdshSmad3 reduced IGFBPrP1-stimulated Smad3 expression and attenuatedα-SMA expression(29.84%±1.36%vs 8.23%±1.28%,P<0.05),hepatocyte apoptosis(38.56%±3.42%vs 6.75%±0.52%,P<0.05),and both collagen?Ⅰ?and fibronectin deposition in the livers of AdIGFBPrP1-treated rats.CONCLUSION:IGFBPrP1 induces liver fibrosis by mediating the activation of hepatic stellate cells and hepatocyte apoptosis in a Smad3-dependent mechanism.

微小RNA在肾组织纤维化中作用的研究进展

糖尿病、高血压、肾小球肾炎以及其它肾脏疾病常常发展为慢性肾病(chronic kidney disease,CKD)。预计到2030年,慢性肾病引发肾脏功能衰竭和心脏并发症将成为全球第13大死因,而到2040年将上升到第5位死因[1]。CKD的组织病理学特点,一个是持续性肾组织炎症反应,另一个是以过多细胞外基质沉积为特征的肾小管间质和肾小球纤维化[2]。

黑地黄丸通过调控IGF-1表达对慢性肾衰竭大鼠肾纤维化的抑制作用

目的 探讨黑地黄丸对慢性肾衰竭大鼠肾纤维化的影响及其作用机制。方法 Wistar大鼠随机分为空白组(正常饲养)和造模组,造模组大鼠采用5/6肾切除手术构建CRF大鼠模型。将造模成功的大鼠随机分为模型组、黑地黄丸组(10.43 g/kg)及黑地黄丸+IGF-1R阻断剂(JB1)组(连续7 d皮下注射18μg/kg JB1后灌胃10.43 g/kg黑地黄丸)。给药8周后,检测大鼠血清Scr、BUN水平,HE和Masson染色观察肾组织病理变化,免疫组化法检测肾组织TGF-β、HIF-1α、α-SMA蛋白表达,Western blot法检测肾组织IGF-1R、TGF-β蛋白表达,RT-qPCR法检测肾组织IGF-1R、TGF-β mRNA表达。结果 与空白组比较,模型组血清Scr、BUN水平升高(P<0.05),肾组织纤维化程度加重,纤维化面积增加(P<0.05),肾组织TGF-β、HIF-1α、α-SMA蛋白表达和TGF-β mRNA表达升高(P<0.05),IGF-1R mRNA和蛋白表达降低(P<0.05);与模型组比较,黑地黄丸组大鼠血清Scr、BUN水平降低(P<0.05),肾间质炎性细胞减少,纤维化程度减轻(P<0.05),肾组织TGF-β、HIF-1α、α-SMA蛋白表达和TGF-β mRNA表达降低(P<0.05),IGF-1R mRNA和蛋白表达升高(P<0.05);而给予JB1可减弱黑地黄丸对CRF大鼠肾纤维化的改善作用(P<0.05)。结论 黑地黄丸能够抑制CRF大鼠肾纤维化,且该抑制过程与上调IGF-1表达,促进IGF-1与IGF-1R结合有关。

巨噬细胞转分化在肾纤维化中的调控机制

肾纤维化是所有进展性慢性肾病发展至终末期肾病的共同病理改变。肾移植术后发生肾纤维化会严重影响移植肾功能。巨噬细胞具有高度的异质性和可塑性,在肾损伤过程中,受局部微环境刺激被募集、激活和极化,通过多种机制参与肾组织损伤、修复和纤维化的过程。近年来,多项研究表明,巨噬细胞可以转分化为肌成纤维细胞直接参与肾纤维化形成,这一过程被称为巨噬细胞-肌成纤维细胞转分化,但其调控机制尚不清楚。因此,本文就巨噬细胞在肾纤维化中的作用、巨噬细胞-肌成纤维细胞转分化的特点及可能的调控机制进行综述,以期为肾纤维化的相关研究提供参考。

五桑饮增强SIRT1和Nrf2表达并减轻糖尿病小鼠肾损伤和纤维化

目的:探讨五桑饮减轻糖尿病诱导的小鼠肾损伤及相关机制。方法:小鼠经含高脂高糖饲料喂养后,腹腔注射链脲佐菌素,从而建立糖尿病模型,并随机分为糖尿病模型组和五桑饮治疗组,另设正常对照组。采用生化分析、HE、PAS和Masson染色观察五桑饮对糖尿病肾病小鼠肾功能、肾组织形态学和纤维化等相关参数的作用。采用TUNEL染色分析小鼠肾组织凋亡,DHE染色分析肾组织细胞内活性氧水平。比色法分析肾组织氧化应激指标,包括脂质过氧化产物丙二醛(malondialdehyde,MDA),抗氧化酶超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)。RT-qPCR检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原、Ⅲ型胶原、Bax和Bcl-2的mRNA表达水平。采用Western blot法分析肾组织沉默信息调节因子1(silent information regulator 1,SIRT1)和核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)蛋白表达。结果:五桑饮给药8周后,与对照组相比,模型组小鼠肾脏肥大指数、空腹血糖、糖化血红蛋白、甘油三酯、总胆固醇、血清肌酐和血尿素氮显著升高(P<0.05)。五桑饮组小鼠的这些指标均显著低于模型组(P<0.05)。五桑饮组小鼠的24 h尿白蛋白排泄率和24 h尿总蛋白也比模型组小鼠显著升高。对小鼠肾组织进行HE、PAS和Masson染色显示:模型组小鼠肾小球肥大、基底膜增厚、出现肾纤维化改变,五桑饮组各种病变均明显减轻,肾组织纤维化相关分子α-SMA、Ⅰ型和Ⅲ型胶原的mRNA表达均较模型组显著降低(P<0.05)。与模型组相比,五桑饮组小鼠肾组织凋亡水平,细胞内活性氧水平,肾组织脂质过氧化产物MDA含量显著降低,而SOD和CAT抗氧化酶的活性显著升高。五桑饮处理后小鼠肾组织SIRT1和Nrf2蛋白的水平明显高于模型组(P<0.05)。结论:五桑饮能改善糖尿病肾病小鼠肾功能,减轻肾脏病理损伤、纤维化、肾细胞凋亡和氧化应激反应,其肾保护作用可能与SIRT1和Nrf2蛋白的表达增强有关。

血小板源性生长因子在湿性年龄相关性黄斑变性新生血管及纤维化中的作用研究进展

黄斑区脉络膜新生血管及视网膜下纤维化(SRFi)是湿性年龄相关性黄斑变性最常见的病理机制,常导致50岁以上人群不可逆性的视力丧失。尽管目前抗血管内皮生长因子已经成为临床主要有效的一线用药,但仍有近50%的病例因SRFi产生的瘢痕而最终视力丧失。血小板源性生长因子家族是从人血小板中分离出来的一类促血管生成因子,目前已证实其参与了黄斑区脉络膜新生血管形成的过程,但具体作用机制仍不清楚。本文将血小板源性生长因子在年龄相关性黄斑变性新生血管以及SRFi形成的作用机制作一综述,为未来开发新型诊疗方式提供资料支持。

银杏叶提取物对单侧输尿管梗阻大鼠肾间质纤维化的影响及机制

目的探究银杏叶提取物(GBE)对单侧输尿管梗阻(UUO)大鼠肾间质纤维化(RIF)的影响及机制。方法随机选取20只SD大鼠作为Sham组,54只SD大鼠UUO建模成功后随机均分为模型组、GBE组和厄贝沙坦组。观察各组大鼠肾组织病理变化,比较各组大鼠血肌酐(SCr)、尿素氮(BUN)、超氧化物歧化酶(SOD)、丙二醛(MDA)、活性氧(ROS)水平及肾组织纤维连接蛋白(FN)、α-平滑肌肌动蛋白(α-SMA)、I型胶原蛋白(Col-I)、肝细胞生长因子(HGF)、转化生长因子-β1(TGF-β1)的表达。结果与Sham组比较,其他组SCr、BUN、24 h尿蛋白、MDA、ROS水平及FN、α-SMA、Col-I、TGF-β1表达显著升高,SOD水平及HGF表达显著下降(P<0.05)。与模型组比较,GBE组和厄贝沙坦组SCr、BUN、24 h尿蛋白、MDA、ROS水平及FN、α-SMA、Col-I、TGF-β1表达显著下降,SOD水平及HGF表达显著升高(P<0.05)。GBE组SCr、BUN、24 h尿蛋白、MDA、ROS水平低于厄贝沙坦组,SOD高于厄贝沙坦组(P<0.05)。结论GBE可抑制UUO大鼠RIF发生及进展,保护肾功能,其机制可能与纠正HGF/TGF-β1平衡失调有关。

基于巨噬细胞-肌成纤维细胞转分化的肾间质纤维化病理机制及药物干预作用

在肾间质纤维化过程中,骨髓中募集的巨噬细胞可在损伤的肾脏中直接转化为肌成纤维细胞。巨噬细胞-肌成纤维细胞转分化(MMT)是肾间质纤维化的重要病理机制之一。在MMT过程中,巨噬细胞的活化受转化生长因子-β1、Janus激酶3/信号转导及转录活化因子6、Wnt等信号通路调控,阻断这些信号通路可抑制MMT,改善肾间质纤维化。在临床及体内外实验中,针对防治肾纤维化的药物干预研究较活跃,其中某些药物的干预作用可能与巨噬细胞及MMT相关。因此,深入研究肾脏MMT的病理机制及药物干预作用可以为相关疾病的治疗提供新思路。

吡非尼酮对肾纤维化大鼠的治疗作用及分子机制

目的探讨吡非尼酮(PFD)对肾纤维化大鼠肾脏的治疗作用及机制。方法30只SD大鼠随机均分为对照组、模型组及治疗组,后2组大鼠腹腔注射50%四氯化碳(CCl_(4))油溶液建立肾纤维化模型,对照组腹腔注射等体积橄榄油,持续5周;造模结束,治疗组大鼠PFD水溶液灌胃给药,模型组和对照组大鼠同剂量生理盐水灌胃,持续4周;干预期间每天观察大鼠活动、进食饮水、毛发颜色以及大小便情况,于干预前以及干预第2、5、7及9周最后1次给药24 h后对大鼠进行称重并记录大鼠体质量及一般情况;干预第9周末处死各组大鼠,取心脏血检测血清尿素氮(BUN)、血肌酐(Scr)及尿酸(UA)含量,取肾脏组织采用苏木素伊红染色(HE)和Masson染色观察各组大鼠肾组织损伤和纤维化程度,采用蛋白免疫印迹法检测各组大鼠肾脏组织中沉默信息调节因子3(SIRT3)、缺氧诱导因子-1α(HIF-1α)、转化生长因子-β1(TGF-β1)、α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(ColⅠ)、Ⅲ型胶原(ColⅢ)、金属蛋白酶组织抑制因子1(TIMP1)及基质金属蛋白酶2(MMP2)蛋白的表达。结果与对照组比较,模型组大鼠肾功能损伤和纤维化明显,血清BUN、Scr及UA含量降低(P<0.05),肾组织中HIF-1α、TGF-β1、α-SMA、ColⅠ、ColⅢ及TIMP1蛋白表达增高(P<0.05),MMP2和SIRT3蛋白表达降低(P<0.05);与模型组比较,治疗组大鼠肾功能损伤和纤维化程度减轻,血清肾功能BUN、Scr、UA含量增高(P<0.05),肾组织中HIF-1α、TGF-β1、α-SMA、ColⅠ、ColⅢ及TIMP1蛋白表达降低(P<0.05),MMP2和SIRT3蛋白表达增高(P<0.05)。结论PFD可减轻肾纤维化大鼠肾功能损害和纤维化程度,其机制可能与上调SIRT3蛋白表达有关。

Effects of insulin-like growth factor binding protein-related protein 1 in mice with hepatic fibrosis induced by thioacetamide

Background Insulin-like growth factor binding protein-related protein 1 （IGFBPrP1） can activate hepatic stellate cells and increase extracellular matrix （ECM） in vitro. However, the effects of IGFBPrP1 in mice with hepatic fibrosis, and the mechanisms of these effects, are currently unknown. We aim to address these issues in this study. Methods Intraperitoneal injection of thioacetamide （TAA） is a classic method for establishing a mouse model of hepatic fibrosis. Using this model, we administered anti-IGFBPrP1 antibody, again via intraperitoneal injection. The morphological changes of liver fibrosis were observed with both HE and Masson stainning. The immunohistochemical assays and Western blotting were used to measure changes in IGFBPrP1, a-smooth muscle actin （a-SMA） and ECM in liver tissues, and the expression of transforming growth factor-β1 （TGF-β1） and Smad3. Data were statistically analyzed using one-way analysis of variance （ANOVA）, the SNK-q test for inter-group differences. Results The Masson staining analysis showed that compared with normal control group, content of collagen fiber in TAA5w group was significantly increased （P 〈0.01）, and it was significantly decreased in TAA5w/alGFBPrP1 group compared with in TAA5w group （P 〈0.01）. The expression of hepatic IGFBPrP1, a-SMA, TGF-β1, Smad3, collagen 1 and fibronectin （FN） was significantly up-regulated in the TAA5w group （P 〈0.01）. Anti-IGFBPrP1 treatment reversed these changes （P 〈0.01）. Conclusions IGFBPrP1 plays an important role in the development of hepatic fibrosis. Anti-IGFBPrP1 prevents fibrosis in mice by suppressing the activation of hepatic stellate cells, inhibiting the synthesis of major components of the ECM （namely, collagen I and FN）. The mechanism for this suppression of fibrosis is associated with the TGF-β1/Smad3 signaling pathways.

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.

畲医益肾泻浊方对慢性肾衰竭患者肾脏保护作用机制研究

目的观察畲医益肾泻浊方对脾肾两虚、浊毒内蕴型早中期慢性肾衰竭患者的中医证候、肾功能、转化生长因子(transforming growth factor,TGF)-β1水平的影响,探讨该方肾脏保护机制。方法选取2019年6月至2020年12月丽水市中医院收治的早中期慢性肾衰竭的患者80例,采用随机数字表法分为对照组(n=40)和观察组(n=40),对照组给予西医常规治疗,包括生活习惯调整,饮食营养治疗,血压、血糖、血脂管理等,观察组在对照组的基础上加用畲医益肾泻浊方。观察两组患者治疗前后中医证候、肾功能、TGF-β1水平的变化。结果治疗前,两组中医证候积分比较,差异无统计学意义(P>0.05);治疗后,观察组中医证候积分明显下降且低于对照组,差异有统计学意义(P<0.05)。治疗后,对照组、观察组的总有效率分别为60%、82.50%,差异有统计学意义(P<0.05)。观察组在改善中医证候的疗效方面优于对照组,差异有统计学意义(P<0.05)。治疗前,两组的肌酐清除率(creatinine clearance rate,CCr)、血肌酐(serum creatinine,SCr)、血尿素氮(blood urea nitrogen,BUN)比较,差异无统计学意义(P>0.05);治疗后,观察组CCr高于对照组,SCr、BUN低于对照组,治疗12周后两组CCr、SCr、BUN比较,差异有统计学意义(P<0.05)。治疗前两组TGF-β1水平比较,差异无统计学意义(P>0.05),治疗后,观察组TGF-β1水平低于治疗组,差异有统计学意义(P<0.05)。观察组治疗末期未发生终末期肾病(end stage renal disease,ESRD),对照组ESRD发生3例,两组均未发生不良反应。结论畲医益肾泻浊方能缓解患者临床症状,可一定程度降低患者SCr、BUN水平及提高患者CCr而改善患者的肾功能,同时能一定程度减低TGF-β1水平,可能延缓患者的肾纤维化进程,临床疗效较好且安全,值得临床推广。

养阴祛瘀方对高尿酸性肾病大鼠保护作用及对肾脏TGF-β_(1)/Smad3信号通路的影响

目的观察养阴祛瘀方对高尿酸性肾病大鼠肾保护作用及对炎症因子和转化生长因子-β_(1)(TGF-β_(1))/Smad3通路的影响,探讨其作用机制。方法取56只雄性SD大鼠,随机选取13只作为空白组,其余大鼠采用氧嗪酸钾联合腺嘌呤灌胃法建立高尿酸性肾病模型。将造模成功的40只大鼠随机分为模型组及养阴祛瘀高、中、低剂量组,每组10只。养阴祛瘀高、中、低剂量组分别给予养阴祛瘀方37.2 g/(kg·d)、18.6 g/(kg·d)、9.3 g/(kg·d)灌胃,空白组及模型组给予等量生理盐水灌胃,均每天1次,连续灌胃3周。收集尿液检测24 h尿蛋白定量;取下腔静脉血,检测血肌酐(SCr)、尿素氮(BUN)、尿酸(UA)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;留取肾组织,HE染色观察肾组织病理形态,荧光显微镜下计算阳性细胞百分比;RT-PCR法检测肾组织中TGF-β_(1)、Smad3 mRNA相对表达量。结果模型组24 h尿蛋白定量及SCr、BUN、UA、IL-6、TNF-α水平均明显高于空白组(P均<0.05);肾小管萎缩,管内见尿酸盐晶体沉积,间质中可见较多炎性细胞浸润;肾组织中阳性细胞百分比及TGF-β_(1)、Smad3 mRNA相对表达量均明显高于空白组(P均<0.05)。养阴祛瘀各组24 h尿蛋白定量及血清IL-6、TNF-α水平和养阴祛瘀高、中剂量组SCr、BUN、UA水平均明显低于模型组(P均<0.05),且养阴祛瘀高剂量组血清UA、IL-6、TNF-α水平均明显低于养阴祛瘀中、低剂量组(P均<0.05);养阴祛瘀中、高剂量组尿酸盐沉积、炎性细胞浸润、肾小管损伤较前减轻,而养阴祛瘀低剂量组改善不明显;养阴祛瘀各组肾组织中阳性细胞百分比及TGF-β_(1)、Smad3 mRNA相对表达量均明显低于模型组(P均<0.05),且养阴祛瘀高剂量组各指标均明显低于养阴祛瘀低剂量组(P均<0.05)。结论养阴祛瘀方可保护高尿酸性肾病大鼠肾脏,减少蛋白尿,机制与下调炎症因子IL-6、TNF-α表达,抑制肾组织中TGF-β_(1)/Smad3通路激活有关。

基于转录组测序分析转化生长因子-β1诱导肾纤维化机制

目的:探究转化生长因子-β1(TGF-β1)诱导肾纤维化的机制。方法:将TGF-β1处理和未处理的肾成纤维细胞NRK-49F进行转录组测序,采用DESeq2分析测序结果,以错误发现率低于0.05且log2FC绝对值大于1为标准筛选差异表达的基因,并对差异表达的基因进行基因本体(GO)、京都基因和基因组百科全书(KEGG)通路富集分析。进一步筛选差异表达基因中编码转录因子的基因,并利用单侧输尿管梗阻诱导的小鼠肾纤维化模型和高通量基因表达数据库GSE104954数据集对这些基因在肾纤维化过程中的表达进行验证。结果:TGF-β1处理6、12和24 h后,分别有552、1209和1028个差异表达基因。GO分析表明,这些差异表达基因在发育、细胞死亡和细胞迁移过程中显著富集。KEGG分析显示,在TGF-β1诱导早期(TGF-β1处理6 h)主要表现为Hippo、TGF-β、Wnt信号通路的变化,而在TGF-β1诱导晚期(TGF-β1处理24 h)主要表现为细胞外基质受体相互作用、局灶黏附和黏附分子连接等相关通路的改变。在TGF-β1处理6 h时291个上调的差异表达基因中,Snai1、Irf8、Bhlhe40、Junb、Arid5a、Vdr、Lef1、Ahr、Foxo1、Myc、Tcf7、Foxc2、Glis1等13个基因编码转录因子。在细胞模型中验证发现,TGF-β1可以诱导其中9个编码转录因子基因(Snai1、Irf8、Bhlhe40、Junb、Arid5a、Vdr、Lef1、Myc、Tcf7)的表达,其余4个基因的表达水平在TGF-β1处理后无显著变化。在动物模型验证中发现,Snai1、Irf8、Bhlhe40、Junb、Arid5a、Myc和Tcf7显著上调,而Vdr显著下调,Lef1无显著变化。在GSE104954数据集中验证发现,IRF8在糖尿病肾病患者和IgA肾病患者的肾小管间质中显著高表达,MYC在糖尿病肾病患者中高表达,而其他7个基因的表达产物与对照组相似。结论:TGF-β1诱导肾成纤维细胞基因差异表达,Irf8和Myc可能是慢性肾脏病和肾纤维化的潜在靶点。