Si-Wu-Tang attenuates liver fibrosis via regulating lncRNA H19-dependent pathways involving cytoskeleton remodeling and ECM deposition / 中国天然药物

Liver fibrosis is a dynamic wound-healing response characterized by the agglutination of the extracellular matrix (ECM). Si-Wu-Tang (SWT), a traditional Chinese medicine (TCM) formula, is known for treating gynecological diseases and liver fibrosis. Our previous studies demonstrated that long non-coding RNA H19 (H19) was markedly upregulated in fibrotic livers while its deficiency markedly reversed fibrogenesis. However, the mechanisms by which SWT influences H19 remain unclear. Thus, we established a bile duct ligation (BDL)-induced liver fibrosis model to evaluate the hepatoprotective effects of SWT on various cells in the liver. Our results showed that SWT markedly improved ECM deposition and bile duct reactions in the liver. Notably, SWT relieved liver fibrosis by regulating the transcription of genes involved in the cytoskeleton remodeling, primarily in hepatic stellate cells (HSCs), and influencing cytoskeleton-related angiogenesis and hepatocellular injury. This modulation collectively led to reduced ECM deposition. Through extensive bioinformatics analyses, we determined that H19 acted as a miRNA sponge and mainly inhibited miR-200, miR-211, and let7b, thereby regulating the above cellular regulatory pathways. Meanwhile, SWT reversed H19-related miRNAs and signaling pathways, diminishing ECM deposition and liver fibrosis. However, these protective effects of SWT were diminished with the overexpression of H19 in vivo. In conclusion, our study elucidates the underlying mechanisms of SWT from the perspective of H19-related signal networks and proposes a potential SWT-based therapeutic strategy for the treatment of liver fibrosis.

Vitamin D receptor (VDR) mediates the quiescence of activated hepatic stellate cells (aHSCs) by regulating M2 macrophage exosomal smooth muscle cell-associated protein 5 (SMAP-5) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

An effective therapeutic regimen for hepatic fibrosis requires a deep understanding of the pathogenesis mechanism. Hepatic fibrosis is characterized by activated hepatic stellate cells (aHSCs) with an excessive production of extracellular matrix. Although promoted activation of HSCs by M2 macrophages has been demonstrated, the molecular mechanism involved remains ambiguous. Herein, we propose that the vitamin D receptor (VDR) involved in macrophage polarization may regulate the communication between macrophages and HSCs by changing the functions of exosomes. We confirm that activating the VDR can inhibit the effect of M2 macrophages on HSC activation. The exosomes derived from M2 macrophages can promote HSC activation, while stimulating VDR alters the protein profiles and reverses their roles in M2 macrophage exosomes. Smooth muscle cell-associated protein 5 (SMAP-5) was found to be the key effector protein in promoting HSC activation by regulating autophagy flux. Building on these results, we show that a combined treatment of a VDR agonist and a macrophage-targeted exosomal secretion inhibitor achieves an excellent anti-hepatic fibrosis effect. In this study, we aim to elucidate the association between VDR and macrophages in HSC activation. The results contribute to our understanding of the pathogenesis mechanism of hepatic fibrosis, and provide potential therapeutic targets for its treatment.

Huangqi Decoction, a compound Chinese herbal medicine, inhibits the proliferation and activation of hepatic stellate cells by regulating the long noncoding RNA-C18orf26-1/microRNA-663a/transforming growth factor-β axis / 中西医结合学报

OBJECTIVE@#Huangqi Decoction (HQD), a classical traditional Chinese medicine formula, has been used as a valid treatment for alleviating liver fibrosis; however, the underlying molecular mechanism is still unknown. Although our previous studies showed that microRNA-663a (miR-663a) suppresses the proliferation and activation of hepatic stellate cells (HSCs) and the transforming growth factor-β/small mothers against decapentaplegic (TGF-β/Smad) pathway, whether long noncoding RNAs (lncRNAs) are involved in HSC activation via the miR-663a/TGF-β/Smad signaling pathway has not yet reported. The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis.@*METHODS@#The expression levels of lnc-C18orf26-1, miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction. HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs. The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs. Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs. RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs. Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA. The expression levels of collagen α-2(I) chain (COL1A2), α-smooth muscle actin (α-SMA) and TGF-β/Smad signaling pathway-related proteins were determined using Western blotting.@*RESULTS@#Lnc-C18orf26-1 was upregulated in TGF-β1-activated HSCs and competitively bound to miR-663a. Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation, downregulated TGF-β1-stimulated α-SMA and COL1A2 expression, and inhibited the TGF-β1/Smad signaling pathway. HQD suppressed the proliferation and activation of HSCs. HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs. Further studies showed that HQD inhibited the expression of COL1A2, α-SMA, TGF-β1, TGF-β type I receptor (TGF-βRI) and phosphorylated Smad2 (p-Smad2) in HSCs, and these effects were reversed by miR-663a inhibitor treatment.@*CONCLUSION@#Our study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis. HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-β1/TGF-βRI/p-Smad2 axis.

Establishment of a reporter system for estimating activation of human hepatic stellate cells based on COL1A1 promoter and enhanced green fluorescent protein / 北京大学学报(医学版)

OBJECTIVE@#To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs.@*METHODS@#The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-β1 (TGF-β1) or co-treated with TGF-β1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot.@*RESULTS@#The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-β1 and 5 μmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-β1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-β1 single treatment group (P < 0.05).@*CONCLUSION@#A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.

Geniposide inhibits hepatic fibrosis and hepatic stellate cell activation through blocking the TGF-β1/Smad signaling pathway / 生理学报

The purpose of this study was to investigate the effect of Geniposide on hepatic fibrosis and activation of hepatic stellate cells (HSCs) and to explore possible underlying mechanism. Human HSCs (LX-2) were treated with 5 ng/mL transforming growth factor-β1 (TGF-β1), followed by co-culture with Geniposide at various concentrations (0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100 μmol/L). Cell viability was determined by MTT assay. Then, LX-2 cells were divided into control, TGF-β1 (5 ng/mL) and TGF-β1 + Geniposide (20 μmol/L) groups, and the gene and protein expression of collagen I, fibronectin, α-smooth muscle actin (α-SMA), p-Smad2 and p-Smad3 was detected by qPCR and Western blot, respectively. BALB/c mice were treated with CCl4 (25%, 1 mL/kg) to generate a model of hepatic fibrosis (CCl4 group), and the control group and CCl4 + Geniposide group were administered with olive oil and CCl4 + 40 mg/kg Geniposide, respectively. After 4 weeks of treatment, the liver function and serum hepatic fibrosis indexes of mice were detected, histological observation was performed by HE and Masson staining, and α-SMA expression in the tissue was analyzed by immunohistochemistry. Western blot was utilized for the determination of the protein expression of α-SMA, TGF-β1, p-Smad2 and p-Smad3. The results showed that Geniposide inhibited LX-2 cell proliferation. In addition, Geniposide significantly downregulated the gene and protein expression of collagen I, fibronectin and α-SMA and the expression of TGF-β1/Smad signaling-related proteins induced by TGF-β1 in vitro. Histological observations showed that Geniposide significantly inhibited CCl4-induced hepatic fibrosis, HSC activation and expression of TGF-β1/Smad signaling-related proteins in mice. In summary, Geniposide prevents the hepatic fibrosis and HSC activation possibly through the inhibition of the TGF-β1/Smad signaling pathway.

Management Strategies for Liver Fibrosis

Abstract: Liver fibrosis resulting from chronic liver injury are major causes of morbidity and mortality worldwide. Among causes of hepatic fibrosis, viral infection is most common (hepatitis B and C). In addition, obesity rates worldwide have accelerated the risk of liver injury due to nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Also liver fibrosis is associated with the consumption of alcohol, or autoimmune hepatitis and chronic cholangiophaties. The response of hepatocytes to inflammation plays a decisive role in the physiopathology of hepatic fibrosis, which involves the recruitment of both pro- and anti-inflammatory cells such as monocytes and macrophages. As well as the production of other cytokines and chemokines, which increase the stimulus of hepatic stellate cells by activating proinflammatory cells. The aim of this review is to identify the therapeutic options available for the treatment of the liver fibrosis, enabling the prevention of progression when is detected in time.

Effect of curcumin on the proliferation and apoptosis of hepatic stellate cells

This study was designed to investigate the effect of curcumin (diferuloylmethane) on the proliferation and apoptosis of hepatic stellate cells (HSC). The cell line HSC-T6 (1.25 x 10(5) cells/mL) was incubated with curcumin and HSC proliferation was detected by a methyl thiazolyl tetrazolium colorimetric assay. HSC apoptosis was detected by flow cytometry, transmission electron microscope and agarose gel electrophoresis. HSC proliferation was significantly inhibited in a concentration-dependent manner (10.6 to 63.5 percent) after incubation with 20-100 ìM curcumin, compared with a control group. At 20, 40, and 60 ìM, after 24 h of incubation, curcumin was associated with a significant increase in the number of HSC in the G2/M phase, and a significant decrease in cell numbers in the S phase (P < 0.05). At these concentrations, curcumin was also associated with an increase in the apoptosis index of 15.3 ± 1.9, 26.7 ± 2.8, and 37.6 ± 4.4 percent, respectively, compared to control (1.9 ± 0.6 percent, P < 0.01). At 40 ìM, the curcumin-induced apoptosis index at 12, 24, 36, and 48 h of incubation was 12.0 ± 2.4, 26.7 ± 3.5, 33.8 ± 1.8, and 49.3 ± 1.6 percent, respectively (P < 0.01). In conclusion, curcumin inhibits the in vitro proliferation of HSCs in the G2/M phase of the cell cycle and also induces apoptosis in a concentration- and time-dependent manner. The in vivo effect of curcumin on HSCs requires further investigation.

Immunohistochemical studies of stellate cells in experimental cholestasis in newborn and adult rats

BACKGROUND AND AIMS: Although there is much known about liver diseases, some aspects remain unclear, such as the nature of the differences between the diseases observed in newborn infants and those in adults. For example, how do newborns respond to duct epithelial cell injury? Do the stellate cells in newborns respond similarly to those in adults during biliary obstruction? METHODS: Ninety newborn Wistar rats aged six days, weighing 8.0 - 13.9 g each, and 90 adult rats weighing 199.7 - 357.0 g each, were submitted to bile duct ligation. After surgery, they were randomly divided and sacrificed on the 3rd, 5th, 7th, 14th, 21st or 28th day post-bile duct ligation. Hepatic biopsies were obtained and immunohistochemical semi-quantification of desmin and á-SMA expression was performed in hepatic stellate cells and in myofibroblasts in the portal space, and between the portal space and the liver lobule. RESULTS: Desmin expression in the myofibroblast cells post-bile duct ligation was higher in young rats, reaching its peak level in a shorter time when compared to the adult animals. The differences between the groups for á-SMA expression were less significant than for desmin. CONCLUSIONS: These findings indicate that there is an increase in the number of collagen-producing myofibroblast cells in young animals, suggesting that there is more intense fibrosis in this population. This finding may explain why young animals with bile duct obstruction experience more intense portal fibrosis that is similar to the pathology observed in the livers of newborns with biliary atresia.