CXCL6‐EGFR‐induced Kupffer cells secrete TGF‐β1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C‐MYC/EZH2 pathway in liver fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP‐2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF‐β in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC‐T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF‐β showed increased expression of α‐SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C‐MYC/EZH2 pathway by enhancing the SMAD3‐BRD4 interaction and promoting direct binding of BRD4 to the C‐MYC promoter and CMY‐C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl₄‐induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF‐β in serum and the expression of α‐SMA, SMAD3, BRD4, C‐MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF‐β by KCs and thereby activating HSCs.     

MicroRNA-326 attenuates hepatic stellate cell activation and liver fibrosis by inhibiting TLR4 signaling

Hepatic fibrosis is a chronic inflammatory and reversible repair reaction of the liver under the continuous action of virus or various injuries. In this study, we aimed at identifying the role of miR-326 in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. In this study, the liver fibrosis mouse model was developed by injecting CCl₄. Liver tissue morphology was observed and the expression level of α-smooth muscle actin, collagen1α1 and miR-326 was measured. Target gene identification was performed by loss-of-function and gain-of-function. The effect of miR-326 on the expression level of the cytokines associated with the TLR4/MyD88/nuclear factor-κB (NF-κB) pathway was assessed in vitro and in vivo. We show that miR-326 was downregulated in CCl₄-induced fibrotic mice and activated HSCs. The target gene of miR-326 is TLR4. Moreover, miR-326 inhibited the activation of HSCs in vitro through TLR4/MyD88/NF-κB signaling. miR-326 attenuated hepatic fibrosis and inflammation of CCl₄-induced mice in vivo. Our results demonstrate for the first time that miR-326 inhibits HSC activation through TLR4/MyD88/NF-κB signaling. Furthermore, miR-326 plays critical roles in attenuating liver fibrosis and inflammation, suggesting the therapeutic potential of miRNAs.     

PLK1 regulates hepatic stellate cell activation and liver fibrosis through Wnt/β‐catenin signalling pathway

As an outcome of chronic liver disease, liver fibrosis involves the activation of hepatic stellate cells (HSCs) caused by a variety of chronic liver injuries. It is important to explore approaches to inhibit the activation and proliferation of HSCs for the treatment of liver fibrosis. PLK1 is overexpressed in many human tumour cells and has become a popular drug target in tumour therapy. Therefore, further study of the function of PLK1 in the cell cycle is valid. In the present study, we found that PLK1 expression was elevated in primary HSCs isolated from CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. Knockdown of PLK1 inhibited α‐SMA and Col1α1 expression and reduced the activation of HSCs in CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. We further showed that inhibiting the expression of PLK1 reduced the proliferation of HSCs and promoted HSCs apoptosis in vivo and in vitro. Furthermore, we found that the Wnt/β‐catenin signalling pathway may be essential for PLK1‐mediated HSCs activation. Together, blocking PLK1 effectively suppressed liver fibrosis by inhibiting HSC activation, which may provide a new treatment strategy for liver fibrosis.     

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.     

大鼠纤维化肝组织中SHP1表达与肝星状细胞活化及增殖的关系*

目的: 探讨大鼠肝纤维化病理过程中肝组织及在体肝星状细胞 (HSC)的含SH2结构域的蛋白酪氨酸磷酸酶1 (SHP1)表达变化与在体HSC活化及增殖的关系。方法: 随机将50只健康雄性SD大鼠分为对照组(10只)、模型组(40只),采用腹腔注射四氯化碳法建立大鼠肝纤维化模型,Masson三色染色及HE染色检测大鼠肝脏组织的病理组织学变化,SHP1与α-平滑肌肌动蛋白 (α-SMA)免疫荧光双标记检测大鼠肝组织中活化HSC的SHP1表达,免疫组织化学染色检测大鼠肝组织的α-SMA及SHP1表达,并分别对大鼠肝组织的SHP1表达及大鼠肝组织中活化HSC的SHP1表达与大鼠肝组织的α-SMA表达进行Pearson’s相关性分析。结果: 大鼠肝纤维化模型成功构建,随着造模时间延长,大鼠肝纤维化逐渐加重。与对照组大鼠肝组织的SHP1阳性表达平均光密度值 (MOD) (0.08±0.01)比较,造模不同时间(2周、4周、6周、8周)大鼠纤维化肝组织的SHP1阳性表达MOD (0.11±0.01、0.14±0.01、0.16±0.01、0.19±0.01)显著增加(P＜0.05),并逐渐升高(P＜0.05)。与对照组大鼠肝组织的α-SMA阳性表达MOD (0.04±0.01)比较,造模不同时间(2周、4周、6周、8周)大鼠纤维化肝组织的α-SMA阳性表达MOD (0.06±0.01、 0.09±0.01、0.12±0.01、0.16±0.02)明显增加(P＜0.05),并逐渐升高(P＜0.05),即在体HSC的活化及增殖逐渐加快(α-SMA是HSC的活化标志)。SHP1与α-SMA免疫荧光双标记检测显示,造模2周、4周、6周、8周大鼠纤维化肝组织中表达SHP1的活化HSC占总的活化HSC的百分比(26.49%±3.44%、37.14%±4.57%、44.90%±2.94%、58.09%±5.33%)逐渐升高(P＜0.05)。上述大鼠纤维化肝组织的SHP1表达及大鼠纤维化肝组织中表达SHP1的活化HSC占总的活化HSC的百分比均与大鼠纤维化肝组织的α-SMA表达呈显著正相关(r值为0.926, 0.984,P＜0.05)。结论: 在大鼠肝纤维病理过程中,肝组织及在体HSC 的SHP1表达与在体HSC的活化及增殖呈显著正相关。     

DHFR silence alleviated the development of liver fibrosis by affecting the crosstalk between hepatic stellate cells and macrophages

Liver fibrogenesis is a dynamic cellular and tissue process which has the potential to progress into cirrhosis of even liver cancer and liver failure. The activation of hepatic stellate cells (HSCs) is the central event underlying liver fibrosis. Besides, hepatic macrophages have been proposed as potential targets in combatting fibrosis. As for the relationship between HSCs and hepatic macrophages in liver fibrosis, it is generally considered that macrophages promoted liver fibrosis via activating HSCs. However, whether activated HSCs could in turn affect macrophage polarization has rarely been studied. In this study, mRNAs with significant differences were explored using exosomal RNA-sequencing of activated Lx-2 cells and normal RNA-sequencing of DHFR loss-of-function Lx-2 cell models. Cell functional experiments in both Lx-2 cells and macrophages animal model experiments were performed. The results basically confirmed exosomes secreted from activated HSCs could promote M1 polarization of macrophages further. Exosome harbouring DHFR played an important role in this process. DHFR silence in HSCs could decrease Lx-2 activation and M1 polarization of M0 macrophages and then alleviate the development of liver fibrosis both in vitro and vivo. Our work brought a new insight that exosomal DHFR derived from HSCs had a crucial role in crosstalk between HSCs activation and macrophage polarization, which may be a potential therapeutic target in liver fibrosis.     

CX3CL1/fractalkine shedding by human hepatic stellate cells: contribution to chronic inflammation in the liver

Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-γ induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro . We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases.     

Lenvatinib prevents liver fibrosis by inhibiting hepatic stellate cell activation and sinusoidal capillarization in experimental liver fibrosis

Molecular targeted agents are pharmacologically used to treat liver fibrosis and have gained increased attention. The present study examined the preventive effect of lenvatinib on experimental liver fibrosis and sinusoidal capillarization as well as the in vitro phenotypes of hepatic stellate cells. LX-2, a human stellate cell line, was used for in vitro studies. In vivo liver fibrosis was induced in F344 rats using carbon tetrachloride by intraperitoneal injection for 8 weeks, and oral administration of lenvatinib was started two weeks after initial injection of carbon tetrachloride. Lenvatinib restrained proliferation and promoted apoptosis of LX-2 with suppressed phosphorylation of extracellular signal-regulated kinase 1/2 and AKT. It also down-regulated COL1A1, ACTA2 and TGFB1 expressions by inhibiting the transforming growth factor-β1/Smad2/3 pathway. Treatment with lenvatinib also suppressed platelet-derived growth factor-BB-stimulated proliferation, chemotaxis and vascular endothelial growth factor-A production, as well as basic fibroblast growth factor-induced LX-2 proliferation. In vivo study showed that lenvatinib attenuated liver fibrosis development with reduction in activated hepatic stellate cells and mRNA expression of profibrogenic markers. Intrahepatic neovascularization was ameliorated with reduced hepatic expressions of Vegf1, Vegf2 and Vegfa in lenvatinib-treated rats. Collectively, these results suggest the potential use of lenvatinib as a novel therapeutic strategy for liver fibrosis.     

Impaired integrin α5/β1‐mediated hepatocyte growth factor release by stellate cells of the aged liver

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α₅/β₁ is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α₅ and β₁ decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α₅ and β₁ knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α₅ and laminin gene expression whereas integrin β₁ remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α₅ knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α₅/β₁ in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration.     

rSJYB1 inhibits collagen type I protein expression in hepatic stellate cells via down‐regulating activity of collagen α1 (I) promoter

YB1 is a negative regulator in liver fibrosis. We wondered whether SJYB1, a homologous protein of YB1 from Schistosoma japonicum, has an effect on liver fibrosis in vitro. Recombinant SJYB1 (rSJYB1) protein was expressed in a bacterial system and purified by Ni‐NTA His·Bind Resin. A human hepatic stellate cell line, the LX‐2 cell line, was cultured and treated with rSJYB1. The role of rSJYB1 on LX‐2 cells was then analysed by Western blot and luciferase assay. We succeeded in expressing and purifying SJYB1 in a bacterial system and the purified rSJYB1 could be recognized by S japonicum‐infected rabbit sera. Western bolt analysis showed that rSJYB1 inhibited the expression of collagen type I, but had little effect on α‐smooth muscle actin (α‐SMA). Further analysis revealed that rSJYB1 inhibited the activity of collagen α1 (I) (COL1A1) promoter and functioned at ?1592/?1176 region of COL1A1 promoter. Our data demonstrate that rSJYB1‐mediated anti‐fibrotic activity involves inhibiting the activity of COL1A1 promoter and subsequently suppressing the expression of collagen type I in hepatic stellate cells.     

Methyl helicterate inhibits hepatic stellate cell activation through downregulating the ERK1/2 signaling pathway

The present study was to investigate the inhibitory effect of methyl helicterate (MH) on hepatic stellate cells (HSC-T6), primarily elucidating the underlying mechanism of MH against liver fibrosis. HSC-T6 cells were activated by platelet-derived growth factor (PDGF) stimulation, and then the effects of MH on cell viability, cytomembrane integrity, colony, migration, apoptosis, and cell cycle were detected. Moreover, the regulative mechanism of MH on HSCs was investigated by detecting the activation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. The results showed that MH significantly inhibited HSC-T6 cell viability and proliferation in a concentration-dependent manner. It notably promoted the release of lactate dehydrogenase, destroying cell membrane integrity. MH also markedly inhibited HSC-T6 cell clonogenicity and migration. Moreover, MH treatment significantly induced cell apoptosis and arrested cell cycle at the G2 phase. The further study showed that MH inhibited the expression of ERK1, ERK2, c-fos, c-myc, and Ets-1, blocking the ERK1/2 pathway. In conclusion, this study demonstrates that MH significantly inhibits HSC activation and promotes cell apoptosis via downregulation of the ERK1/2 signaling pathway.     

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.     

Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling

Bacterial translocation and lipopolysaccharide (LPS) leakage occur at a very early stage of liver fibrosis in animal models. We studied the role of LPS in hepatic stellate cell (HSC) activation and the underlying mechanisms in vitro and in vivo. Herein, we demonstrated that LPS treatment led to a dramatic increase in autophagosome formation and autophagic flux in LX-2 cells and HSCs, which was mediated through the AKT-MTOR and AMPK-ULK1 pathway. LPS significantly decreased the lipid content, including the lipid droplet (LD) number and lipid staining area in HSCs; pretreatment with macroautophagy/autophagy inhibitors or silencing ATG5 attenuated this decrease. Furthermore, lipophagy was induced by LPS through the autophagy-lysosomal pathway in LX-2 cells and HSCs. Additionally, LPS-induced autophagy further reduced retinoic acid (RA) signaling, as demonstrated by a decrease in the intracellular RA level and Rar target genes, resulting in the downregulation of Bambi and promoting the sensitization of the HSC's fibrosis response to TGFB. Compared with CCl₄ injection alone, CCl₄ plus LPS injection exaggerated liver fibrosis in mice, as demonstrated by increased Col1a1 (collagen, type I, α 1), Acta2, Tgfb and Timp1 mRNA expression, ACTA2/α-SMA and COL1A1 protein expression, and Sirius Red staining area, which could be attenuated by injection of an autophagy inhibitor. LPS also reduced lipid content in HSCs in vivo, with this change being attenuated by chloroquine (CQ) administration. In conclusion, LPS-induced autophagy resulted in LD loss, RA signaling dysfunction, and downregulation of the TGFB pseudoreceptor Bambi, thus sensitizing HSCs to TGFB signaling.