Inhibition of ASCT2 induces hepatic stellate cell senescence with modified proinflammatory secretome through an IL-1α/NF-κB feedback pathway to inhibit liver fibrosis

Senescence of activated hepatic stellate cells (aHSCs) is a stable growth arrest that is implicated in liver fibrosis regression. Senescent cells often accompanied by a multi-faceted senescence-associated secretory phenotype (SASP). But little is known about how alanine-serine-cysteine transporter type-2 (ASCT2), a high affinity glutamine transporter, affects HSC senescence and SASP during liver fibrosis. Here, we identified ASCT2 is mainly elevated in aHSCs and positively correlated with liver fibrosis in human and mouse fibrotic livers. We first discovered ASCT2 inhibition induced HSCs to senescence in vitro and in vivo. The proinflammatory SASP were restricted by ASCT2 inhibition at senescence initiation to prevent paracrine migration. Mechanically, ASCT2 was a direct target of glutaminolysis-dependent proinflammatory SASP, interfering IL-1α/NF-κB feedback loop via interacting with precursor IL-1α at Lys82. From a translational perspective, atractylenolide III is identified as ASCT2 inhibitor through directly bound to Asn230 of ASCT2. The presence of –OH group in atractylenolide III is suggested to be favorable for the inhibition of ASCT2. Importantly, atractylenolide III could be utilized to treat liver fibrosis mice. Taken together, ASCT2 controlled HSC senescence while modifying the proinflammatory SASP. Targeting ASCT2 by atractylenolide III could be a therapeutic candidate for liver fibrosis.     

Efficient drug and gene delivery to liver fibrosis: rationale,recent advances,and perspectives

Liver fibrosis results from chronic damages together with an accumulation of extracellular matrix, and no specific medical therapy is approved for that until now. Due to liver metabolic capacity for drugs, the fragility of drugs, and the presence of insurmountable physiological obstacles in the way of targeting, the development of efficient drug delivery systems for anti-fibrotics seems vital. We have explored articles with a different perspective on liver fibrosis over the two decades, then collected and summarized the information by providing corresponding in vitro and in vivo cases. We have discussed the mechanism of hepatic fibrogenesis with different ways of fibrosis induction in animals. Furthermore, the critical chemical and herbal anti-fibrotics, biological molecules such as micro-RNAs, siRNAs, and growth factors, which can affect cell division and differentiation, are mentioned. Likewise, drug and gene delivery and therapeutic systems on in vitro and in vivo models are summarized in the data tables. This review article enlightens recent advances in emerging drugs and nanocarriers and represents perspectives on targeting strategies employed in liver fibrosis treatment.     

2',4',6'-Tris(methoxymethoxy) chalcone induces apoptosis by enhancing Fas-ligand in activated hepatic stellate cells

Suppression of hepatic stellate cell (HSC) activation and proliferation, and induction of apoptosis in activated HSCs have been proposed as therapeutic strategies for the treatment and prevention of the hepatic fibrosis. We previously showed that 2′,4′,6′-tris(methoxymethoxy) chalcone (TMMC), a synthesized chalcone derivative, inhibits platelet-derived growth factor-induced HSC proliferation at 5–20 μM. Here, we showed that TMMC induces apoptosis in activated HSCs at higher concentrations (30–50 μM), but is not cytotoxic to primary hepatocytes. Moreover, TMMC induces hyperacetylation of histone by inhibiting histone deacetylase (HDAC) in activated HSCs. Interestingly, TMMC treatment remarkably increased Fas-ligand (FasL) mRNA expression in a dose-dependent manner. Cycloheximide treatment reversed the induction of TMMC on apoptosis, indicating that de novo protein synthesis was required for TMMC-induced apoptosis in activated HSCs. In addition, FasL synthesis by TMMC is closely associated with maximal procaspase-3 proteolytic processing. In vivo, TMMC reduced activated HSCs in CCl₄-intoxicated rats during liver injury recovery, as demonstrated by α-smooth muscle actin expression in rat liver. TMMC treatment also resulted in apoptosis, as demonstrated by cleavage of poly(ADP-ribose) polymerase in rat liver. In conclusion, TMMC may have therapeutic potential by inducing HSC apoptosis for the treatment of hepatic fibrosis.     

Oligo-peptide I-C-F-6 inhibits hepatic stellate cell activation and ameliorates CCl4-induced liver fibrosis by suppressing NF-κB signaling and Wnt/β-catenin signaling

Oligo-peptide I-C-F-6 is a Carapax trionycis extract component that has an effect on hepatic fibrosis, however, its mechanism of action is still unclear. This study investigated whether oligo-peptide I-C-F-6 could inhibit liver fibrosis by suppressing NF-κB and Wnt/β-catenin signaling, which are important in liver fibrosis. HSC-T6 cells were treated with oligo-peptide I-C-F-6, and rats were divided randomly into five groups: control (saline), CCl₄, CCl₄ plus oligo-peptide I-C-F-6 (0.12 and 0.24 mg/kg), and CCl₄ plus colchicine (0.11 mg/kg). Here, we demonstrated that oligo-peptide I-C-F-6 ameliorated liver injury, inflammation, and hepatic fibrogenesis induced by CCl₄. Oligo-peptide I-C-F-6 also inhibited the activation of hepatic stellate cells (HSCs) in vivo and in vitro, as evaluated by the expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA), which is a specific marker of HSC activation. Moreover, oligo-peptide I-C-F-6 significantly reduced the expression and distribution of β-catenin, P-AKT, phospho (P)-GSK-3β, nuclear factor κB (NF-κB) P65, phospho-P65, and IκB kinase α/β (IKK-α/β) levels; additionally, IκB-α level was elevated both in vivo and in vitro. Together, these results indicate that oligo-peptide I-C-F-6 has hepatoprotective and anti-fibrotic effects in animal models of liver fibrosis, the mechanism of which may be related to modulating NF-κB and Wnt/β-catenin signaling.     

Effects of salvianolic acids on oxidative stress and hepatic fibrosis in rats

Enhanced oxidative stress is associated with hepatic fibrosis. Salvianolic acids A (Sal A) and B (Sal B) have been reported to be strong polyphenolic antioxidants and free radical scavengers. The present study is to investigate if Sal A and B could attenuate oxidative stress and liver fibrosis in rats. A cell line of rat hepatic stellate cells (HSCs) was stimulated with platelet-derived growth factor (PDGF, 10 ng/ml). The inhibitory effects of Sal A and B on intracellular hydrogen peroxide levels were measured with dichlorofluorescein diacetate (DCF-DA) dye assay. α-Smooth muscle actin (α-SMA), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits were measured by Western blotting. Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA, 200 mg/kg) twice per week for 6 weeks. Sal A (10 mg/kg), Sal B (50 mg/kg) or S-adenosylmethionine (SAMe, 10 mg/kg), was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. In vitro, PDGF increased the accumulation of hydrogen peroxide in HSCs, which was attenuated by Sal A (10 μM) and Sal B (200 μM). Sal A and B attenuated the PDGF-stimulated expressions of α-SMA and NADPH oxidase subunits gp91^phox and p47^phox in membrane fractions. In vivo studies showed that the hepatic levels of collagen, malondialdehyde, TNF-α, IL-6, and IL-1β, fibrosis scores and protein expressions of α-SMA, heme-oxygenase-1, iNOS, and gp91^phox, and serum levels of ALT, AST, IL-6, and IL-1β were increased in TAA-intoxicated rats, all of which were attenuated by 4-week treatment of Sal A or Sal B. Our results showed that Sal A and B attenuated PDGF-induced ROS formation in HSCs, possibly through inhibition of NADPH oxidase. Sal A and B treatments were also effective against hepatic fibrosis in TAA-intoxicated rats.     

鳖甲活性多肽的合成及对肝星状细胞增殖的抑制作用(英文)

探讨鳖甲活性多肽的固相合成及对肝星状细胞(HSC-T6)增殖的抑制作用。根据鳖甲活性多肽的序列结构,采用固相方法对多肽进行全合成,并用MALDI-TOFMS质谱对合成多肽进行分析鉴定;肝星状细胞(HSC-T6)培养至对数生长期后,加入不同浓度合成多肽作用肝星状细胞株HSC-T6,采用MTS法分析HSC-T6生长的抑制作用;使用AnnexinV-FITC/PI法检测HSC凋亡率。结果显示通过固相合成可以得到与原序列结构一致的鳖甲合成多肽;鳖甲合成多肽浓度依赖性地抑制肝星状细胞增殖,并能显著提高肝星状细胞早期凋亡率。因此,可以成功地合成鳖甲活性多肽,且对肝星状细胞增殖有明显的抑制作用。     

Receptor-specific TRAIL as a means to achieve targeted elimination of activated hepatic stellate cells

Activated hepatic stellate cells (HSCs) are known to play a central role in liver fibrosis and their elimination is a crucial step toward the resolution and reversion of liver fibrosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a molecule that may contribute to the apoptotic removal of activated HSC through binding to its dedicated receptors. In the present study, we investigated the potential application of recombinant receptor-specific TRAIL proteins in the efficient elimination of activated HSCs. Our finding revealed differential contribution of TRAIL receptors among HSCs populations with activated hepatic stellate cells expresses more TRAIL receptors DR5. In vitro treatment of activated HSCs with DR5-specific or wild-type TRAIL variants induced a significant reduction in viability and extracellular matrix production, whereas no significant decrease in viability was associated with the treatment of cells by DR4-specific TRAIL. Our analysis indicate the successful application of the DR5 receptor-specific TRAIL variant in the targeted elimination of activated HSCs via interference with collagen production and simultaneous induction of apoptosis via activation of the caspase pathway. DR5 receptor-specific TRAIL may thus represent a new therapeutic compound for the treatment of liver fibrosis.     

Paeoniflorin prevents hepatic fibrosis of Schistosomiasis japonica by inhibiting TGF-β1 production from macrophages in mice

In order to investigate the effect of paeoniflorin (PAE) on hepatic fibrosis of mice with Schistosomiasis japonica in vivo and in vitro, a model of hepatic fibrosis caused by schistosomiasis was established in mice infected with cercariae of Schistosoma japonicum. Then, PAE was orally administered before and after praziquantel treatment and both therapeutics were given simultaneously at different time points after the infection. The concentration of serum hyaluronic acid (HA) was determined by radioimmunoassay (RIA). Hepatic granuloma and fibrosis were evaluated via HE and Masson staining. The expression of α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1) and collagen I (Col I) protein was detected by immunohistochemistry. The effect of soluble egg antigen (SEA) and PAE on the production of TGF-β1 from mouse peritoneal macrophages (PMϕs) was investigated by RT-PCR, Western blotting and ELISA. The effect of TGF-β1 in optimum macrophage-conditioned medium (OPMCM) on the proliferation of hepatic stellate cells (HSCs) and collagen secretion from HSCs with anti-TGF-β1 antibody was explored by MTT assay and ELISA. The results show that PAE could significantly reduce the concentration of serum HA, the size of egg granuloma, the severity of hepatic fibrosis and the expression of α-SMA, TGF-β1 and Col I protein in the pre-treatment group. However, in sim-or post-treatment group, PAE did not have any significant therapeutic effect. TGF-β1 could be secreted from PMϕs stimulated by SEA. Meanwhile, the production of TGF-β1 from PMϕs could be depressed significantly by PAE in a concentration-dependent manner. TGF-β1 could promote the proliferation of HSCs and the secretion of collagens. In a word, PAE can prevent hepatic granuloma and fibrosis caused by schistosomiasis japonica through the inhibition of the secretion of TGF-β1 from PMϕs, the proliferation and activation of HSCs and the secretion of collagens from HSCs. Equally contributed to this work     

[18F]MAGL-4-11 positron emission tomography molecular imaging of monoacylglycerol lipase changes in preclinical liver fibrosis models

Monoacylglycerol lipase (MAGL) is a pivotal enzyme in the endocannabinoid system, which metabolizes 2-arachidonoylglycerol (2-AG) into the proinflammatory eicosanoid precursor arachidonic acid (AA). MAGL and other endogenous cannabinoid (EC) degrading enzymes are involved in the fibrogenic signaling pathways that induce hepatic stellate cell (HSC) activation and ECM accumulation during chronic liver disease. Our group recently developed an ¹⁸F-labeled MAGL inhibitor ([¹⁸F]MAGL-4-11) for PET imaging and demonstrated highly specific binding in vitro and in vivo. In this study, we determined [¹⁸F]MAGL-4-11 PET enabled imaging MAGL levels in the bile duct ligation (BDL) and carbon tetrachloride (CCl₄) models of liver cirrhosis; we also assessed the hepatic gene expression of the enzymes involved with EC system including MAGL, NAPE-PLD, FAAH and DAGL that as a function of disease severity in these models; [¹⁸F]MAGL-4-11 autoradiography was performed to assess tracer binding in frozen liver sections both in animal and human. [¹⁸F]MAGL-4-11 demonstrated reduced PET signals in early stages of fibrosis and further significantly decreased with disease progression compared with control mice. We confirmed MAGL and FAAH expression decreases with fibrosis severity, while its levels in normal liver tissue are high; in contrast, the EC synthetic enzymes NAPE-PLD and DAGL are enhanced in these different fibrosis models. In vitro autoradiography further supported that [¹⁸F]MAGL-4-11 bound specifically to MAGL in both animal and human fibrotic liver tissues. Our PET ligand [¹⁸F]MAGL-4-11 shows excellent sensitivity and specificity for MAGL visualization in vivo and accurately reflects the histological stages of liver fibrosis in preclinical models and human liver tissues.     

Probucol ameliorates hepatic stellate cell activation and autophagy is associated with farnesoid X receptor

Probucol has antioxidant effects and inhibits inflammation. Farnesoid X receptor (FXR) is a nuclear receptor that regulates autophagy, which is regarded as the key cause of the activation of hepatic stellate cell (HSC). In this study, the effects of probucol on HSC activation and autophagy in vitro and vivo and the role of FXR in this progress were investigated. Results showed that probucol ameliorated hepatic fibrosis and autophagy, and increased the expression of FXR in liver in a mouse model of fibrosis induced by CCl₄. And probucol could alleviate lipopolysaccharide-induced autophagy and HSC activation in vitro. In addition, probucol increased FXR expression, and the Z-guggulsterone, an antagonist of FXR, could block the effects of probucol on HSC activation and autophagy. Additionally, agonists of FXR could suppress LPS-induced autophagy and activation. These results suggest that probucol could ameliorate hepatic fibrosis, and inhibit HSC autophagy and activation, and these effects are associated with FXR.     

Magnolol Attenuates Concanavalin A‐induced Hepatic Fibrosis,Inhibits CD4+ T Helper 17 (Th17) Cell Differentiation and Suppresses Hepatic Stellate Cell Activation: Blockade of Smad3/Smad4 Signalling

Magnolol is a pharmacological biphenolic compound extracted from Chinese herb Magnolia officinalis, which displays anti‐inflammatory and antioxidant effects. This study was aimed at exploring the potential effect of magnolol on immune‐related liver fibrosis. Herein, BALB/c mice were injected with concanavalin A (ConA, 8 mg/kg/week) up to 6 weeks to establish hepatic fibrosis, and magnolol (10, 20, 30 mg/kg/day) was given to these mice orally throughout the whole experiment. We found that magnolol preserved liver function and attenuated liver fibrotic injury in vivo. In response to ConA stimulation, the CD4⁺ T cells preferred to polarizing towards CD4⁺ T helper 17 (Th17) cells in liver. Magnolol was observed to inhibit Th17 cell differentiation in ConA‐treated liver in addition to suppressing interleukin (IL)‐17A generation. Hepatic stellate cells were activated in fibrotic liver as demonstrated by increased alpha smooth muscle actin (α‐SMA) and desmin. More transforming growth factor (TGF)‐β1 and activin A were secreted into the serum. Magnolol suppressed this abnormal HSC activation. Furthermore, the phosphorylation of Smad3 in its linker area (Thr179, Ser 204/208/213) was inhibited by magnolol. In vitro, the recombinant IL‐17A plus TGF‐β1 or activin A induced activation of human LX2 HSCs and promoted their collagen production. Smad3/Smad4 signalling pathway was activated in LX2 cells exposed to the fibrotic stimuli, as illustrated by the up‐regulated phospho‐Smad3 and the enhanced interaction between Smad3 and Smad4. These alterations were suppressed by magnolol. Collectively, our study reveals a novel antifibrotic effect of magnolol on Th17 cell‐mediated fibrosis.     

丹芍化纤胶囊对实验性肝纤维化大鼠肝脏Smad-7表达的影响

目的探讨Smad7在大鼠肝纤维化肝脏中的表达及丹芍化纤胶囊抗大鼠肝纤维化对其表达的影响。方法雄性Wistar大鼠80只分为正常组、肝纤维化模型组、自然恢复组、低剂量治疗组、高剂量治疗组,除正常组外,其余采用CCl4、饮酒、高脂低蛋白饮食等复合病因刺激制备肝纤维化动物模型8周,然后两治疗组分别予以低剂量(0.5g/kg)、高剂量(1.0g/kg)丹芍化纤胶囊灌胃治疗8周。实验结束后测定肝脏指数,光镜下观察肝组织纤维化程度,同时免疫组化SABC法检测肝组织中Smad7的表达。结果Smad7在肝纤维化大鼠肝脏中表达明显减少(0.98±0.20/12.80±2.32),治疗8周后,治疗组与肝纤维化模型组、自然恢复组比较,肝脏指数、肝纤维化程度显著减轻;肝脏Smad7表达显著增加(10.42±2.74/1.74±0.29)。结论丹芍化纤胶囊能显著增加肝纤维化大鼠肝组织中Smad7的表达,可能是其发挥抗纤维化作用的机制之一。     

抗肝纤维化药物研究进展

HF是各种慢性肝病的共同病理学基础,也是肝硬化的必经阶段。积极的抗HF治疗可以控制多种肝组织疾病,尤其是肝硬化的病理进程。靶点明确、疗效显著的抗HF药物是治疗的首选。本研究从HF药物作用靶点、药物研究的体内外模型及药物研究3个方面展开阐述抗HF药物的最新研究进展。     

Purification,characterization, in vitro anti-hepatic fibrosis activity of bioactive peptides derived from Carapax Trionycis hydrolysates

Anti-hepatic fibrosis peptide from Carapax Trionycis was purified, characterized, and inhibitory effect was assessed. Carapax Trionycis extract peptide hydrolysates (CTEPHs) were separated by ultrafiltration, Sephadex G-15 gel chromatography and RP-HPLC. One novel anti-hepatic fibrosis peptide (CTEPH-1: Asn-Pro-Asn-Pro-Thr) was obtained and identified. MTS assay and enzyme-linked immunosorbent assay were applied to evaluate the anti-fibrotic effect of CTEPH-1 on activated hepatic stellate cells (HSCs) in vitro. CTEPH-1 efficiently inhibited activation and proliferation of cultured HSC-T₆ cells via lowering the contents of collagen and TIMP-1 except for matrix metalloproteinase-1 (MMP-1). The purified peptide might be beneficial as functional food or potential drug for treatment of liver fibrogenesis.     

Versatile flexible micelles integrating mucosal penetration and intestinal targeting for effectively oral delivery of paclitaxel

The extremely low bioavailability of oral paclitaxel(PTX) mainly due to the complicated gastrointestinal environment,the obstruction of intestinal mucus layer and epithelium barrier.Thus,it is of great significance to construct a coordinative delivery system which can overcome multiple intestinal physicochemical obstacles simultaneously.In this work,a high-density PEGylation-based glycocholic acid-decorated micelles(PTX@GNPs) was constructed by a novel polymer,9-Fluorenylmethoxy carbonyl-poly et...     

Smad2 increases the apoptosis of activated human hepatic stellate cells induced by TRAIL

The activation of hepatic stellate cells (HSCs) plays a critical role in the development of liver fibrosis. The induction of apoptosis in activated HSCs during the recovery phase of hepatic fibrosis represents a potential anti-fibrotic therapy. We have previously shown that Smad2 protects against hepatic fibrogenesis; however, the role of Smad2 in the regulation of activated HSC apoptosis remains unknown. We hypothesized that Smad2 regulates the apoptosis of activated HSCs, leading to the resolution of liver fibrosis. To test this hypothesis, the livers of rats were harvested at 0 and 4 weeks after hepatic fibrosis was established by CCl₄ injection. Furthermore, TGF-β1-activated HSCs were treated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following the silencing or overexpression of Smad2. Both the phosphorylation of Smad2 and TRAIL were detected in fibrotic liver tissues. The results of TUNEL and α-SMA double-staining showed an increase in the apoptosis of activated HSCs during the spontaneous recovery phase. The knockdown of Smad2 reduced TRAIL-induced apoptosis in TGF-β1-activated human LX-2 cells and resulted in an increased expression of α-SMA and collagen I (Col. I). In contrast, the overexpression of Smad2 increased TRAIL-induced HSC apoptosis and reduced the expression of α-SMA and Col. I. The mechanisms underlying these findings were associated with the Smad2-mediated down-regulation of X-linked inhibitor of apoptosis protein (XIAP), resulting in enhanced caspase-3 activity and apoptosis. In conclusion, Smad2 enhances TRAIL-induced apoptosis in activated HSCs, which facilitates the resolution of hepatic fibrosis.     

抗肝纤维化药物研究进展

肝纤维化是一个可逆的动态过程,研发药物抑制、逆转肝纤维化成为治疗慢性肝病的热点问题。本文针对肝纤维化过程中的重要环节,从肝细胞、肝星状细胞和细胞外基质等不同药物作用靶点,对目前抗肝纤维化药物研究进展做一综述。     

Mesenchymal stem cell-originated exosomal circDIDO1 suppresses hepatic stellate cell activation by miR-141-3p/PTEN/AKT pathway in human liver fibrosis

Liver fibrosis is a common pathologic stage of the development of liver failure. It has showed that exosomes loaded with therapeutic circRNAs can be manufactured in bulk by exosome secreted cells in vitro, thus enabling personalized treatment. This study aimed to investigate the role of exosome-based delivery of circDIDO1 in liver fibrosis. Levels of genes and proteins were examined by qRT-PCR and Western blot. Cell proliferation, apoptosis, and cell cycle were analyzed by using cell counting kit-8 (CCK-8) assay, EdU assay, and flow cytometry, respectively. The binding between circDIDO1 and miR-141-3p was confirmed by dual-luciferase reporter, RNA pull-down and RIP assays. Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blot. CircDIDO1 overexpression or miR-141-3p inhibition suppressed the proliferation, reduced pro-fibrotic markers, and induced apoptosis as well as cell cycle arrest in hepatic stellate cells (HSCs) by blocking PTEN/AKT pathway. Mechanistically, circDIDO1 acted as an endogenous sponge for miR-141-3p, further rescue experiments showed that circDIDO1 suppressed HSC activation by targeting miR-141-3p. Extracellular circDIDO1 could be incorporated into exosomes isolated from mesenchymal stem cells (MSCs), and transmitted to HSCs to restrain HSC activation. Clinically, low levels of serum circDIDO1 in exosome were correlated with liver failure, and serum exosomal circDIDO1 had a well diagnostic value for liver fibrosis in liver failure patients. Transfer of circDIDO1 mediated by MSC-isolated exosomes suppressed HSC activation through the miR-141-3p/PTEN/AKT pathway, gaining a new insight into the prevention of liver fibrosis in liver failure patients.