大鼠肾间质纤维化动物模型的实验研究

目的　观察单侧输尿管结扎 (UUO)大鼠模型中肾脏病理改变 ,肾组织α 平滑肌肌动蛋白 (α SMA)和纤维连接蛋白 (FN)表达的变化 ,证明UUO方法可快速制作理想的肾脏细胞转分化和间质纤维化动物模型。方法　大鼠随机分为假手术组 (SOR)和UUO组。随机选取各组中的 6只大鼠分别于单侧输尿管结扎术后 3天、 7天、 14天、 2 1天和 2 8天处死 ,收集血清与肾组织供生化及病理分析。结果　① 2 4小时尿蛋白定量 ,UUO组与假手术组无统计学差异。UUO术后 7～ 14天 ,大鼠出现明显血尿素氮、肌酐值升高 ;术后 2 1～ 2 8天 ,血尿素氮、肌酐值反而下降 ,但 2 8天时血肌酐值仍然高于假手术组 (P <0 0 5 )。②UUO组肾小管间质损伤评分 (TIS)明显高于假手术组。UUO术后 3天 ,肾脏组织出现了早期纤维化的病理改变。随着梗阻时间延长 ,小管间质纤维化程度和间质细胞增殖、炎细胞浸润逐渐加重 ,皮质变薄十分明显。术后 2 8天 ,Masson染色见皮质区、皮髓交界处纤维化明显 ,但肾小球仍无明显病变。③免疫组化结果显示 ,UUO术后 3天肾间质α SMA阳性细胞数和FN表达增加 ,并随时间递增。UUO术后 7天 ,可见少许α SMA阳性的转分化小管上皮细胞 ;术后14天转分化的小管上皮细胞明显增多。α SMA表达与FN表达成正相关 (r=0 996 ,P <     

Deletion of Smad3 protects against C-reactive protein-induced renal fibrosis and inflammation in obstructive nephropathy

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism.Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E).Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80⁺ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling.Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.     

Protective Effect of the Traditional Chinese Patent Medicine Qing‐Xuan Granule against Bleomycin‐Induced Pulmonary Fibrosis in Mice

Pulmonary fibrosis (PF) is a chronic obstructive pulmonary disease without effective clinical drug treatment. Qing‐Xuan Granule (QX) as a traditional Chinese patent medicine is clinically used to cure children's cough. This study was designed to investigate the effects of QX and possible molecular mechanisms for bleomycin‐induced PF. The work used Western blotting and Q‐PCR to explore the vitro and vivo mechanisms of QX treatment, while using HPLC‐TOF/MS to explore the composition of QX. QX was given daily orally for two weeks after bleomycin intratracheal instillation. The protective effects of QX on lung function, inflammation, growth factors, hydroxyproline content and deposition of extracellular matrix were investigated. QX decreased expression of Col I and α‐SMA in lung tissues by down‐regulating TGF‐β1‐Smad2/3 signaling and suppressed epithelial‐mesenchymal transition and effectively reversed abnormal mRNA levels of MMP‐1and TIMP‐1 as well as LOXL‐2 in lung tissues. HPLC‐TOF/MS indicate that six substances could be the main active components, which were reported to protect against experimental lung disease.     

Itch E3 Ubiquitin Ligase Positively Regulates TGF-β Signaling to EMT via Smad7 Ubiquitination

TGF-β regulates pleiotropic cellular responses including cell growth, differentiation, migration, apoptosis, extracellular matrix production, and many other biological processes. Although non-Smad signaling pathways are being increasingly reported to play many roles in TGF-β-mediated biological processes, Smads, especially receptor-regulated Smads (R-Smads), still play a central mediatory role in TGF-β signaling for epithelial-mesenchymal transition. Thus, the biological activities of R-Smads are tightly regulated at multiple points. Inhibitory Smad (I-Smad also called Smad7) acts as a critical endogenous negative feedback regulator of Smad-signaling pathways by inhibiting R-Smad phosphorylation and by inducing activated type I TGF-β receptor degradation. Roles played by Smad7 in health and disease are being increasingly reported, but the molecular mechanisms that regulate Smad7 are not well understood. In this study, we show that E3 ubiquitin ligase Itch acts as a positive regulator of TGF-β signaling and of subsequent EMT-related gene expression. Interestingly, the Itch-mediated positive regulation of TGF-β signaling was found to be dependent on Smad7 ubiquitination and its subsequent degradation. Further study revealed Itch acts as an E3 ubiquitin ligase for Smad7 polyubiquitination, and thus, that Itch is an important regulator of Smad7 activity and a positive regulator of TGF-β signaling and of TGF-β-mediated biological processes. Accordingly, the study uncovers a novel regulatory mechanism whereby Smad7 is controlled by Itch.     

TGF-β1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner

Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-β1 can induce the EMT and the role of TGF-β1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-β1 and EMT markers were assessed. TGF-β1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-β1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-β1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-β1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-β1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-β receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-β1 dependent and is mediated through Smad2 and Smad3. TGF-β1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.     

Anp32e promotes renal interstitial fibrosis by upregulating the expression of fibrosis-related proteins

Acidic nuclear phosphoprotein 32 family member e (Anp32e) has been reported to contribute to early mammalian development and cancer metastasis. However, the pathophysiological role of Anp32e in renal interstitial fibrosis (RIF) is poorly understood. Here, we demonstrated that Anp32e was highly expressed in the region of RIF in patients with IgA nephropathy, unilateral ureteral obstruction (UUO) mouse kidneys, and Boston University mouse proximal tubular (BUMPT) cells when treated with TGF-β1; this upregulation was positively correlated with the total fibrotic area of the kidneys. The overexpression of Anp32e enhanced the TGF-β1-induced production of fibrosis-related proteins (fibronectin (Fn) and collagen type I (Col-I)) in BUMPT cells whereas the knockdown of Anp32e suppressed the deposition of these fibrosis-related proteins in UUO mice and TGF-β1-stimulated BUMPT cells. In particular, Anp32e overexpression alone induced the deposition of Fn and Col-I in both mouse kidneys and BUMPT cells without TGF-β1 stimulation. Furthermore, we revealed that the overexpression of Anp32e induced the expression of TGF-β1 and p-Smad3 while TGF-β1 inhibitor SB431542 reversed the Anp32e-induced upregulation of Fn and Col-I in BUMPT cells without TGF-β1 stimulation. Collectively, our data demonstrate that Anp32e promotes the deposition of fibrosis-related proteins by regulating the TGF-β1/Smad3 pathway.     

Phospho-specific Smad3 signaling: Impact on breast oncogenesis

Members of the TGFβ superfamily are known to exert a myriad of physiologic and pathologic growth controlling influences on mammary development and oncogenesis. In epithelial cells, TGFβ signaling inhibits cell growth through cytostatic and pro-apoptotic activities but can also induce cancer cell EMT and, thus, has a dichotomous role in breast cancer biology. Mechanisms governing this switch are the subject of active investigation. Smad3 is a critical intracellular mediator of TGFβ signaling regulated through phosphorylation by the TGFβ receptor complex at the C terminus. Smad3 is also a substrate for several other kinases that phosphorylate additional sites within the Smad protein. This discovery has expanded the understanding of the significance and complexity of TGFβ signaling through Smads. This review highlights recent advances revealing the critical role of phospho-specific Smad3 in malignancy and illustrates the potential prognostic and therapeutic impact of Smad3 phospho-isoforms in breast cancer.     

Suppression of ADAM8 attenuates angiotensin II-induced cardiac fibrosis and endothelial-mesenchymal transition via inhibiting TGF-β1/Smad2/Smad3 pathways

Endothelial-to-mesenchymal transition (EndMT) is involved in cardiac fibrosis induced by angiotensin II (Ang II). A disintegrin and metalloproteinase 8 (ADAM8), a member of ADAMs family, participates in cell adhesion, proteolysis and various signaling. However, its effects on the development of cardiac fibrosis remain completely unknown. This study aimed to reveal whether ADAM8 aggravates cardiac fibrosis induced by Ang II in vivo and in vitro. The C57BL/6J mice or cardiac endothelial cells were subjected to Ang II infusion to induce fibrosis. The results showed that systolic blood pressure and diastolic blood pressure were significantly increased under Ang II infusion, and ADAM8 was up-regulated. ADAM8 inhibition attenuated Ang II-induced cardiac dysfunction. ADAM8 knockdown suppressed Ang II-induced cardiac fibrosis as evidenced by the down-regulation of CTGF, collagen I, and collagen III. In addition, the endothelial marker (VE-cadherin) was decreased, whilst mesenchymal markers (α-SMA and FSP1) were increased following Ang II infusion. However, ADAM8 repression inhibited Ang II-induced EndMT. Moreover, ADAM8 silencing repressed the activation of TGF-β1/Smad2/Smad3 pathways. Consistent with the results in vivo, we also found the inhibitory effects of ADAM8 inhibition on EndMT in vitro. All data suggest that ADAM8 promotes Ang II-induced cardiac fibrosis and EndMT via activating TGF-β1/Smad2/Smad3 pathways.     

Functions of exogenous FGF signals in regulation of fibroblast to myofibroblast differentiation and extracellular matrix protein expression

Fibroblasts are widely distributed cells found in most tissues and upon tissue injury, they are able to differentiate into myofibroblasts, which express abundant extracellular matrix (ECM) proteins. Overexpression and unordered organization of ECM proteins cause tissue fibrosis in damaged tissue. Fibroblast growth factor (FGF) family proteins are well known to promote angiogenesis and tissue repair, but their activities in fibroblast differentiation and fibrosis have not been systematically reviewed. Here we summarize the effects of FGFs in fibroblast to myofibroblast differentiation and ECM protein expression and discuss the underlying potential regulatory mechanisms, to provide a basis for the clinical application of recombinant FGF protein drugs in treatment of tissue damage.     

Tight junction protein 1 is regulated by transforming growth factor-β and contributes to cell motility in NSCLC cells

Tight junction protein 1 (TJP1), a component of tight junction, has been reported to play a role in protein networks as an adaptor protein, and TJP1 expression is altered during tumor development. Here, we found that TJP1 expression was increased at the RNA and protein levels in TGF-β-stimulated lung cancer cells, A549. SB431542, a type-I TGF-β receptor inhibitor, as well as SB203580, a p38 kinase inhibitor, significantly abrogated the effect of TGF-β on TJP1 expression. Diphenyleneiodonium, an NADPH oxidase inhibitor, also attenuated TJP1 expression in response to TGF-β in lung cancer cells. When TJP1 expression was reduced by shRNA lentiviral particles in A549 cells (A549-sh TJP1), wound healing was much lower than in cells infected with control viral particles. Taken together, these data suggest that TGF-β enhances TJP1 expression, which may play a role beyond structural support in tight junctions during cancer development. [BMB Reports 2015; 48(2): 115-120]     

Latent TGF-β1 protects against diabetic kidney disease via Arkadia/Smad7 signaling

TGF-β1 has long been considered as a key mediator in diabetic kidney disease (DKD) but anti-TGF-β1 treatment fails clinically, suggesting a diverse role for TGF-β1 in DKD. In the present study, we examined a novel hypothesis that latent TGF-β1 may be protective in DKD mice overexpressing human latent TGF-β1. Streptozotocin-induced Type 1 diabetes was induced in latent TGF-β1 transgenic (Tg) and wild-type (WT) mice. Surprisingly, compared to WT diabetic mice, mice overexpressing latent TGF-β1 were protected from the development of DKD as demonstrated by lowing microalbuminuria and inhibiting renal fibrosis and inflammation, although blood glucose levels were not altered. Mechanistically, the renal protective effects of latent TGF-β1 on DKD were associated with inactivation of both TGF-β/Smad and nuclear factor-κB (NF-κB) signaling pathways. These protective effects were associated with the prevention of renal Smad7 from the Arkadia-induced ubiquitin proteasomal degradation in the diabetic kidney, suggesting protection of renal Smad7 from Arkadia-mediated degradation may be a key mechanism through which latent TGF-β1 inhibits DKD. This was further confirmed in vitro in mesangial cells that knockdown of Arkadia failed but overexpression of Arkadia reversed the protective effects of latent TGF-β1 on high glucose-treated mesangial cells. Latent TGF-β1 may protect kidneys from TGF-β1/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in diabetes through inhibiting Arkadia-mediated Smad7 ubiquitin degradation.     

Correlation between TGF-β2/3 promoter DNA methylation and Smad signaling during palatal fusion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant that is strongly associated with a number of human diseases and birth defects, including cleft palate. Transforming growth factor (TGF) plays a significant role during mammalian palatogenesis. However, the epigenetic mechanism of transforming growth factors in the process of TCDD-induced cleft palate is unclear. The purpose of this research was to investigate the relationship and potential mechanism between TGF-β2/3 promoter DNA methylation and Smad signaling during TCDD-induced cleft palate. Pregnant C57BL/6N mice were exposed to 64 µg/kg TCDD on gestational day 10 (GD10) to establish the cleft palate model and palatal tissues of embryos were collected on GD13, GD14, and GD15 for subsequent experiments. TGF-β2/3 mRNA expression, TGF-β2/3 promoter methylation, and Smad signaling molecules expression were assessed in the palate of the two groups. The results showed that the incidence of cleft palate was 94.7% in the TCDD-treated group whereas no cleft palate was found in the control group. TCDD-treated group altered specific CpG sites of TGF-β2/3 promoter methylation. Compared to the control group, the proliferation of mouse embryonic palate mesenchymal stromal cells (MEPM), the expressions of TGF-β2/3, p-Smad2, and Smad4 were all reduced, while the expression of Smad7 was significantly increased in the atAR group. Smad signaling was downregulated by TCDD. Therefore, we suggest that TGF-β2/3 promoter methylation and Smad signaling may be involved in TCDD-induced cleft palate formation in fetal mice.     

MiR-92d-3p suppresses the progression of diabetic nephropathy renal fibrosis by inhibiting the C3/HMGB1/TGF-β1 pathway

The pathogenesis of diabetic nephropathy (DN) has not been fully elucidated. MicroRNAs (miRNAs) play an important role in the onset and development of DN renal fibrosis. Thus, the present study aimed to investigate the effect of miR-92d-3p on the progression of DN renal fibrosis. We used qRT-PCR to detect the expression levels of miR-92d-3p in the kidneys of patients with DN. Then, after transfecting lentiviruses containing miR-92d-3p into the kidneys of a DN mouse model and HK-2 cell line, we used qRT-PCR to detect the expression levels of miR-92d-3p, C3, HMGB1, TGF-β1, α-SMA, E-cadherin, and Col I. The expression levels of interleukin (IL) 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the HK-2 cells were detected through enzyme-linked immunosorbent assay (ELISA), and Western blotting and immunofluorescence were used in detecting the expression levels of fibronectin, α-SMA, E-cadherin, and vimentin. Results showed that the expression levels of miR-92d-3p in the kidney tissues of patients with DN and DN animal model mice decreased, and C3 stimulated HK-2 cells to produce inflammatory cytokines. The C3/HMGB1/TGF-β1 pathway was activated, and epithelial-to-interstitial transition (EMT) was induced in the HK-2 cells after human recombinant C3 and TGF-β1 protein were added. miR-92d-3p inhibited inflammatory factor production by C3 in the HK-2 cells and the activation of the C3/HMGB1/TGF-β1 pathway and EMT by C3 and TGF-β1. miR-92d-3p suppressed the progression of DN renal fibrosis by inhibiting the activation of the C3/HMGB1/TGF-β1 pathway and EMT.