Thymoquinone alleviates liver fibrosis via miR-30a-mediated epithelial-mesenchymal transition.

Thymoquinone (TQ), the main active constituent of Nigella sativa seeds, has been shown to play a role in antioxidation, anti-inflammation, and antitumor. Recent studies have demonstrated that TQ contributes to the suppression of liver fibrosis. Abnormal activated epithelial-mesenchymal transition (EMT) promotes the activation of hepatic stellate cells (HSCs). However, whether the antifibrotic effects of TQ occur through inhibiting EMT is largely unknown. In this study, it was found that TQ ameliorated liver fibrosis and collagen accumulation in carbon tetrachloride (CCl4) mice. In vitro, TQ inhibited HSC activation including reduced proliferation, α-smooth muscle actin, and collagen. In addition, TQ markedly suppressed the EMT process, with enhanced E-cadherin and reduced desmin. Notably, snail family transcriptional repressor 1 (Snai1), the EMT master transcription factor, was obviously inhibited by TQ in vivo and in vitro. Further studies demonstrated that Snai1 was a target of microRNA-30a (miR-30a), which was upregulated by TQ. Interestingly, the effects of TQ on HSC activation and EMT were almost inhibited by miR-30a inhibitor. Collectively, we demonstrate that TQ inhibits HSC activation, at least in part, via regulation of miR-30a and Snai1. TQ upregulates miR-30a expression, resulting in a reduced Snai1 level as well as EMT process inactivation, which contributes to the inhibition of HSC activation. TQ may be a potential therapeutic agent for liver fibrosis.

Geng W ，Li C ，Zhan Y ，Zhang R ，Zheng J ... -  《-》

MicroRNA-30a Suppresses the Activation of Hepatic Stellate Cells by Inhibiting Epithelial-to-Mesenchymal Transition.

The activation of hepatic stellate cells (HSCs) is considered as a pivotal event in liver fibrosis and epithelial-mesenchymal transition (EMT) process has been reported to be involved in HSC activation. It is known that microRNAs (miRNAs) play a pro-fibrotic or anti-fibrotic role in HSC activation. Recently, emerging studies show that miR-30a is down-regulated in human cancers and over-expression of miR-30a inhibits tumor growth and invasion via suppressing EMT process. However, whether miR-30a could regulate EMT process in HSC activation is still unclear. miR-30a expression was quantified using real-time PCR in carbon tetrachloride (CCl4)-induced rat liver fibrosis, activated HSCs and patients with cirrhosis. Roles of miR-30a in liver fibrosis in vivo and in vitro were also analyzed. Luciferase activity assays were performed to examine the binding of miR-30a to the 3'-untranslated region of snail family transcriptional repressor 1 (Snai1). miR-30a was down-regulated in human cirrhotic tissues. In CCl4 rats, reduced miR-30a was found in fibrotic liver tissues as well as isolated HSCs. There was a significant reduction in miR-30a in primary HSCs during culture days. miR-30a over-expression resulted in the suppression of CCl4-induced liver fibrosis. Restoration of miR-30a led to the inhibition of HSC activation including cell proliferation, α-SMA and collagen expression. Notably, miR-30a inhibited EMT process, with a reduction in TGF-β1 and Vimentin as well as an increase in GFAP and E-cadherin. miR-30a induced a significant reduction in Snai1 protein expression when compared with the control. Interestingly, Snail protein expression was increased during liver fibrosis, indicating that there may be a negative correlation between miR-30a level and Snai1 protein expression. Further studies demonstrated that Snai1 was a target of miR-30a. Our results suggest that miR-30a inhibits EMT process, at least in part, via reduction of Snai1, leading to the suppression of HSC activation in liver fibrosis.

Zheng J ，Wang W ，Yu F ，Dong P ，Chen B ，Zhou MT ... -  《-》

miR-30a negatively regulates TGF-β1-induced epithelial-mesenchymal transition and peritoneal fibrosis by targeting Snai1.

Although epithelial-mesenchymal transition (EMT) and the subsequent development of peritoneal fibrosis are key processes leading to the peritoneal failure related to peritoneal dialysis (PD), mechanisms underlying these processes remain largely unclear. In the present study, we found that miR-30a was significantly down-regulated in peritoneal tissues, with progressive fibrosis in patients with continuous ambulatory peritoneal dialysis and in a rat model of PD. In vitro, transforming growth factor (TGF)-β1-induced EMT, identified by de novo expression of α-smooth muscle actin and a loss of E-cadherin in both human and rat peritoneal mesothelial cells, was associated with down-regulation of miR-30a but up-regulation of Snai1, suggesting a close link between miR-30a and Snai1 in TGF-β1-induced peritoneal fibrosis. It was further demonstrated in vitro that miR-30a was able to bind the 3' untranslated region of Snai1 and overexpression of miR-30a blocked TGF-β1-induced up-regulation of Snai1 and, therefore, inhibited EMT and collagen expression. To determine the functional role of miR-30a, we overexpressed miR-30a in the peritoneal tissue in a rat model of PD and found that overexpression of miR-30a blocked both Snai1 and EMT and inhibited peritoneal fibrosis, with improvement of peritoneal dysfunction. In conclusion, miR-30a negatively regulates Snai1-mediated EMT during peritoneal fibrosis in vitro and in vivo. Blockade of peritoneal fibrosis by overexpressing miR-30a in a rat model of PD reveals a therapeutic potential of miR-30a for peritoneal fibrosis associated with PD.

Zhou Q ，Yang M ，Lan H ，Yu X ... -  《-》

Hepatocyte-derived exosomal miR-146a-5p inhibits hepatic stellate cell EMT process: a crosstalk between hepatocytes and hepatic stellate cells.

Recently, Salidroside (Sal) has been demonstrated to suppress hepatic stellate cell (HSC) activation, a crucial event for liver fibrosis. Moreover, Sal has been reported to decrease hepatocyte injury. A growing number of reports have indicated that the crosstalk between hepatocytes and HSCs is very crucial for liver fibrosis development. Whether Sal-treated hepatocytes could inhibit HSC activation is unclear. Exosomes, as vital vehicles of intercellular communication, have been shown to transfer cargos between hepatocytes and HSCs. Herein, we aimed to investigate the roles of exosomal miRNAs from Sal-treated hepatocytes in HSC activation as well as liver fibrosis. Our results showed that Sal suppressed carbon tetrachloride (CCl4)-induced liver fibrosis in vivo. HSC activation as well as cell proliferation was repressed in HSCs co-cultured with Sal-treated hepatocytes. Interestingly, miR-146a-5p was up-regulated by Sal in CCl4-treated mice. Also, enhanced miR-146a-5p was found in hepatocytes isolated from Sal-treated CCl4 mice and hepatocyte-derived exosomes. Notably, hepatocyte exosomal miR-146a-5p contributed to HSC inactivation. Inhibiting miR-146a-5p in hepatocyte exosomes resulted in reduced E-cadherin (E-cad) and increased desmin in HSCs, indicating that miR-146a-5p caused HSC inactivation via epithelial-mesenchymal transition (EMT). miR-146a-5p inhibition-mediated HSC activation and EMT process were blocked down by loss of EIF5A2. Further studies revealed that EIF5A2 was a target of miR-146a-5p. Furthermore, exosomes with miR-146a-5p overexpression inhibited liver fibrosis in CCl4 mice. Collectively, exosomal miR-146a-5p from Sal-treated hepatocytes inhibits HSC activation and liver fibrosis, at least in part, by suppressing EIF5A2 and EMT process.

Lang Z ，Li Y ，Lin L ，Li X ，Tao Q ，Hu Y ，Bao M ，Zheng L ，Yu Z ，Zheng J ... -  《Cell Death Discovery》

Anti-fibrotic effect of thymoquinone on hepatic stellate cells.

Hepatic stellate cells (HSCs) are the major cell type involved in the production of extracellular matrix in liver. After liver injury, HSCs undergo transdifferentiation process from quiescent state to activated state, which plays an important role in liver fibrosis. Previous studies have shown that thymoquinone (TQ) might have protective effect against liver fibrosis in animal models; however, the underlying mechanism of action is not fully understood. The aim of this study is to examine whether TQ has any direct effect on HSCs. Our results showed that pretreatment of mice with TQ has protective effect against CCl4-induced liver injury compared to control group (untreated), which is consistent with previous studies. Moreover, our in vivo study showed that COL1A1 and α-SMA mRNA levels were significantly downregulated by TQ treatment. Similarly, in vitro study confirmed that TQ downregulated COL1A1, COL3A1 and α-SMA mRNA levels in activated rat HSCs and LX2 cells, an immortalized human hepatic stellate cell line. Pretreatment with TQ also inhibited the LPS-induced proinflammatory response in LX2 cells as demonstrated by reduced mRNA expression of IL-6 and MCP-1. Mechanistically, inactivation of NF-κB pathway is likely to play a role in the TQ-mediated inhibition of proinflammatory response in HSCs. Finally, we have shown that TQ inhibited the culture-triggered transdifferentiation of freshly isolated rat HSCs as shown by significant downregulation of mRNA expression of several fibrosis-related genes. In conclusion, our study suggests that TQ has a direct effect on HSCs, which may contribute to its overall antifibrotic effect.

Ghazwani M ，Zhang Y ，Gao X ，Fan J ，Li J ，Li S ... -  《-》