Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.     

MeCP2‐421‐mediated RPE epithelial‐mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial‐mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl‐CpG‐binding protein 2 (MeCP2), especially P‐MeCP2‐421 in the pathogenesis of PVR. The expressions of P‐MeCP2‐421, P‐MeCP2‐80, PPAR‐γ and the double labelling of P‐MeCP2‐421 with α‐SMA, cytokeratin, TGF‐β and PPAR‐γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α‐SMA, phospho‐Smad2/3, collagen I, fibronectin and PPAR‐γ; the expression of α‐SMA stimulated by recombinant MeCP2 in ARPE‐19; and the effect of TGF‐β and 5‐AZA treatment on PPAR‐γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF‐β. Our results showed that P‐MeCP2‐421 was highly expressed in PVR membranes and was double labelled with α‐SMA, cytokeratin and TGF‐β, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF‐β. TGF‐β inhibited the expression of PPAR‐γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5‐AZA) increased the expression of PPAR‐γ. α‐SMA was up‐regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF‐β as demonstrated by Chip assay. The results suggest that MeCP2 especially P‐MeCP2‐421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.     

RGS3 inhibits TGF‐β1/Smad signalling in adventitial fibroblasts

Recent evidence suggests that adventitial fibroblasts (AFs) are crucially implicated in atherosclerosis. However, the mechanisms by which AFs are dysfunctional and contribute to atherosclerosis remain unclear. This study aimed to investigate the role of regulator of G‐protein signalling 3 (RGS3) in the regulation of AFs using apoE knockout mouse as the model. Pathological changes in aortic arteries of apoE knockout mice fed with hyperlipid diet were examined by Movat staining. The expression of RGS3, α‐SMA, TGF‐β1, Smad2, and Smad3 in the adventitia was detected by immunohistochemistry. Adventitial fibroblasts were isolated from aortic arteries of apoE knockout mice and infected with RGS3 overexpression lentivirus or empty lentivirus. The expression of RGS3, α‐SMA, TGF‐β1, Smad2, and Smad3 in AFs was detected by real‐time polymerase chain reaction and Western blot analysis. We found that hyperlipidic diet caused significant aortic intima thickening and atherosclerotic plaques in 15‐week‐old apoE knockout mice. Compared to wild‐type mice, RGS3 expression was lower while α‐SMA, TGF‐β1, Smad2, and Smad3 expression was higher in the adventitia of apoE knockout mice. In addition, lentivirus mediated overexpression of RGS3 caused decreased expression of α‐SMA, TGF‐β1, Smad2, and Smad3 in AFs derived from apoE(?/?) mice. In conclusion, these results suggest that RGS3 may provide protection against pathological changes of AFs and the development of atherosclerosis by inhibiting TGF‐β1/Smad signalling. RGS3 may be a potential therapeutic target for atherosclerosis.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

Epigenetic silencing of LncRNA ANRIL enhances liver fibrosis and HSC activation through activating AMPK pathway

Long non‐coding RNAs (LncRNAs) and DNA methylation are important epigenetic mark play a key role in liver fibrosis. Currently, how DNA methylation and LncRNAs control the hepatic stellate cell (HSC) activation and fibrosis has not yet been fully characterized. Here, we explored the role of antisense non‐coding RNA in the INK4 locus (ANRIL) and DNA methylation in HSC activation and fibrosis. The expression levels of DNA methyltransferases 3A (DNMT3A), ANRIL, α‐Smooth muscle actin (α‐SMA), Type I collagen (Col1A1), adenosine monophosphate‐activated protein kinase (AMPK) and p‐AMPK in rat and human liver fibrosis were detected by immunohistochemistry, qRT‐PCR and Western blotting. Liver tissue histomorphology was examined by haematoxylin and eosin (H&E), Sirius red and Masson staining. HSC was transfected with DNMT3A‐siRNA, over‐expressing ANRIL and down‐regulating ANRIL. Moreover, cell proliferation ability was examined by CCK‐8, MTT and cell cycle assay. Here, our study demonstrated that ANRIL was significantly decreased in activated HSC and liver fibrosis tissues, while Col1A1, α‐SMA and DNMT3A were significantly increased in activated HSC and liver fibrosis tissues. Further, we found that down‐regulating DNMT3A expression leads to inhibition of HSC activation. Reduction in DNMT3A elevated ANRIL expression in activated HSC. Furthermore, we performed the over expression ANRIL suppresses HSC activation and AMPK signalling pathways. In sum, our study found that epigenetic DNMT3A silencing of ANRIL enhances liver fibrosis and HSC activation through activating AMPK pathway. Targeting epigenetic modulators DNMT3A and ANRIL, and offer a novel approach for liver fibrosis therapy.     

Effects of FGF21‐secreting adipose‐derived stem cells in thioacetamide‐induced hepatic fibrosis

Mesenchymal stem cells (MSCs) have been investigated to treat liver diseases, but the efficiency of MSCs to treat chronic liver diseases is conflicting. FGF21 can reduce inflammation and fibrosis. We established FGF21‐secreting adipose derived stem cells (FGF21_ADSCs) to enhance the effects of ADSCs and transplanted them into thioacetamide (TAA)‐induced liver fibrosis mice via the tail vein. Transplantation of FGF21_ADSCs significantly improved liver fibrosis by decreasing serum hyaluronic acid and reducing the expression of fibrosis‐related factors such as α‐smooth muscle actin (α‐SMA), collagen and tissue inhibitor of metalloproteinase‐1 (TIMP‐1) compared with the Empty_ADSCs by inhibition of p‐JNK, NF‐κB and p‐Smad2/3 signalling. α‐lactoalbumin (LA) and lactotransferrin (LTF), secretory factors produced from FGF21_ADSCs inhibited TGF‐β1‐induced expression of α‐SMA and collagen in LX‐2 cells. These results suggest that transplantation of FGF21_ADSCs inhibited liver fibrosis more effectively than Empty_ADSCs, possibly via secretion of α‐LA and LTF.     

The miR‐3127‐5p/p‐STAT3 axis up‐regulates PD‐L1 inducing chemoresistance in non‐small‐cell lung cancer

It is less known about miRNA3127‐5p induced up‐regulation of PD‐L1, immune escape and drug resistance caused by increased PD‐L1 in lung cancer. In this study, lentivirus was transduced into lung cancer cells, and quantitative PCR and Western blot were used to detect the expression of PD‐L1. Then immunofluorescence assay was applied to detect autophagy, finally we explored the relationship between PD‐L1 expressions and chemoresistance in patients. As a result, we found that microRNA‐3127‐5p promotes pSTAT3 to induce the expression of PD‐L1; microRNA‐3127‐5p promotes STAT3 phosphorylation through suppressing autophagy, and autophagy could retaine pSTAT3 into the nucleus in miRNA‐3127‐5p knocked cells, and immune escape induced by elevated level of PD‐L1 results in chemoresistance of lung cancer. In conclusion, microRNA‐3127‐5p induces PD‐L1 elevation through regulating pSTAT3 expression. We also demonstrate that immune escape induced by PD‐L1 can be dismissed by corresponding monoclonal antibody.     

Fluorofenidone attenuates pulmonary inflammation and fibrosis via inhibiting the activation of NALP3 inflammasome and IL‐1β/IL‐1R1/MyD88/NF‐κB pathway

Interleukin (IL)‐1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL‐1β is dependent upon caspase‐1‐containing multiprotein complexes called inflammasomes and IL‐1R1/MyD88/NF‐κB pathway. In this study, we explored whether a potential anti‐fibrotic agent fluorofenidone (FD) exerts its anti‐inflammatory and anti‐fibrotic effects through suppressing activation of NACHT, LRR and PYD domains‐containing protein 3 (NALP3) inflammasome and the IL‐1β/IL‐1R1/MyD88/NF‐κB pathway in vivo and in vitro. Male C57BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α‐smooth muscle actin (α‐SMA), fibronectin, collagen I, caspase‐1, IL‐1R1, MyD88 were measured by Western blot and/or RT‐PCR. The human actue monocytic leukaemia cell line (THP‐1) were incubated with monosodium urate (MSU), with or without FD pre‐treatment. The expression of caspase‐1, IL‐1β, NALP3, apoptosis‐associated speck‐like protein containing (ASC) and pro‐caspase‐1 were measured by Western blot, the reactive oxygen species (ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome‐associated molecules were measured by Co‐immunoprecipitation. RLE‐6TN (rat lung epithelial‐T‐antigen negative) cells were incubated with IL‐1β, with or without FD pre‐treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL‐1β, IL‐6, monocyte chemotactic protein‐1 (MCP‐1), myeloperoxidase (MPO), α‐SMA, fibronectin, collagen I, caspase‐1, IL‐1R1 and MyD88 in mice lung tissues. And FD inhibited MSU‐induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome‐associated molecules, decreased the level of caspase‐1 and IL‐1β in THP‐1 cells. Besides, FD inhibited IL‐1β‐induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM‐induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL‐1β/IL‐1R1/MyD88/ NF‐κB pathway.     

IL-6/STAT3信号途径介导肉芽肿性乳腺炎的作用研究

目的:探讨IL-6/STAT3信号途径介导肉芽肿性乳腺炎的机制研究及临床意义。方法:收集我院2017年1月至2017年6月收治入住的30例肉芽肿性乳腺炎患者及30例普通患者的血清样本,对肉芽肿性乳腺炎患者同时采集炎性肿块组织样本及距离炎性肿块边缘大于等于3 cm且镜检为正常乳腺组织的样本。采用ELISA法检测血清样本中的IL-6的表达水平,同时采用Western blot检测组织样本中IL-6和pSTAT3蛋白的表达水平并分析二者的相关性。结果:与普通患者血清样本相比,肉芽肿性乳腺炎患者血清中IL-6的表达水平明显增高(P0.05),肉芽肿性乳腺炎组织样本中IL-6和pSTAT3蛋白的表达水平明显高于毗邻的正常乳腺组织(P0.01),IL-6表达与组织STAT3磷酸化水平具有明显的正相关性(r=0.353,P0.05)。结论:IL-6/STAT3信号途径可能介导肉芽肿性乳腺炎的发生发展。     

Inhibition of Interleukin‐6/glycoprotein 130 signalling by Bazedoxifene ameliorates cardiac remodelling in pressure overload mice

The role of IL‐6 signalling in hypertensive heart disease and its sequelae is controversial. Our group demonstrated that Bazedoxifene suppressed IL‐6/gp130 signalling in cancer cells but its effect on myocardial pathology induced by pressure overload is still unknown. We explored whether Bazedoxifene could confer benefits in wild‐type C57BL/6J mice suffering from transverse aortic constriction (TAC) and the potential mechanisms in H9c2 myoblasts. Mice were randomized into three groups (Sham, TAC, TAC+Bazedoxifene, n = 10). Morphological and histological observations suggested TAC aggravated myocardial remodelling while long‐term intake of Bazedoxifene (5 mg/kg, intragastric) attenuated pressure overload‐induced pathology. Echocardiographic results indicated Bazedoxifene rescued cardiac function in part. We found Bazedoxifene decreased the mRNA expression of IL‐6, MMP2, Col1A1, Col3A1 and periostin in murine hearts after 8‐week surgery. By Western blot detection, we found Bazedoxifene exhibited an inhibition of STAT3 activation in mice three hours and 8 weeks after TAC. Acute TAC stress (3 hours) led to down‐regulated ratio of LC3‐Ⅱ/LC3‐Ⅰ, while in mice after long‐term (8 weeks) TAC this ratio becomes higher than that in Sham mice. Bazedoxifene inverted the autophagic alteration induced by TAC at both two time‐points. In H9c2 myoblasts, Bazedoxifene suppressed the IL‐6‐induced STAT3 activation. Moreover, IL‐6 reduced the ratio of LC3‐Ⅱ/LC3‐Ⅰ, promoted P62 expression but Bazedoxifene reversed both changes in H9c2 cells. Our data suggested Bazedoxifene inhibited IL‐6/gp130 signalling and protected against cardiac remodelling together with function deterioration in TAC mice.