The liver is one of the most important organs in the human body. It performs many important functions, including being responsible for the metabolism of most drugs, which is often associated with its drug-induced damage. Currently, there are no ideal pharmacological models that would allow the evaluation of the effect of newly tested drugs on the liver in preclinical studies. Moreover, the influence of hepatic metabolism on the effectiveness of the tested drugs is rarely evaluated. Therefore, in this work we present an advanced model of the liver, which reflects most of the morphologically and metabolically important features of the liver in vivo, namely: three-dimensionality, cellular composition, presence of extracellular matrix, distribution of individual cell types in the structure of the liver model, high urea and albumin synthesis efficiency, high cytochrome p450 activity. In addition, the work, based on the example of commonly used anticancer drugs, shows how important it is to take into account hepatic metabolism in the effective assessment of their impact on the target organ, in this case cancer. In our research, we have shown that the most similar to liver in vivo are 3D cellular aggregates composed of three important liver cells, namely hepatocytes (HepG2), hepatic stellate cells (HSCs), and hepatic sinusoidal endothelial cells (HSECs). Moreover, we showed that the cells in 3D aggregate structure need time (cell-cell interactions) to improve proper liver characteristic. The triculture model additionally showed the greatest ability to metabolize selected anticancer drugs.

This study aims to investigate the mitigating effect and mechanism of Cichorium glandulosum n-butanol extraction site(CGE) on the disease in carbon tetrachloride(CCl_4)-induced chronic liver injury model in rats. A chronic liver injury model was constructed by subcutaneous injection of CCl_4 olive oil solution, and after four weeks of CGE treatment, serum levels of aspartate aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatase(AKP), hydroxyproline(HYP), interleukin-4(IL-4), interleukin-6(IL-6), malondialdehyde(MDA), superoxide dismutase(SOD), and tumor necrosis factor-α(TNF-α) were detected. Liver tissue was processed by hematoxylin-eosin(HE) staining and Masson staining to observe the structure of the rat liver. qPCR and Western blot were used to examine the expression of transforming growth factor-β1(TGF-β1)/small mothers against decapentaplegic(Smad), Toll-like receptor 4(TLR4), α-smooth muscle actin(α-SMA), and fibronectin(Fn) in rat liver tissue and hepatic stellate-T6(HSC-T6) and evaluate the inhibitory effect of CGE on HSC activation. The results showed that CGE could significantly reduce the serum levels of AST, ALT, AKP, HYP, and affect the levels of related inflammatory indexes including IL-4, IL-6, and TNF-α, and MDA in CCl_4-induced chronic liver injury in rats and had no effect on SOD activity, which could delay the process of liver injury, alleviate the hepatic collagen deposition and inflammatory infiltration, and had significant efficacy in mitigating chronic liver injury in rats. CGE could inhibit α-SMA and TLR4 protein expression in the liver tissue and reverse the increased TGF-β1/Smad, Fn, and TLR4-related expression in HSC-T6 in vitro. The above results indicated that CGE exerted hepatoprotective effects in rats by inhibiting HSC activation and alleviated CCl_4-induced chronic liver injury in rats and could ameliorate inflammatory response and slight liver fibrosis in rat liver tissue. Its pharmacodynamic mechanism might be related to TGF-β1/Smad and TLR4-related expression.

OBJECTIVE: Metabolic dysfunction-associated steatohepatitis (MASH) is linked to insulin resistance and type 2 diabetes and marked by hepatic inflammation, microvascular dysfunction, and fibrosis, impairing liver function and aggravating metabolic derangements. The liver homeostatic interactions disrupted in MASH are still poorly understood. We aimed to elucidate the plasticity and changing interactions of non-parenchymal cells associated with advanced MASH.
METHODS: We characterized a diet-induced mouse model of advanced MASH at single-cell resolution and validated findings by assaying chromatin accessibility, bioimaging murine and human livers, and via functional experiments in vivo and in vitro.
RESULTS: The fibrogenic activation of hepatic stellate cells (HSCs) led to deterioration of a signaling module consisting of the bile acid receptor NR1H4/FXR and HSC-specific GS-protein-coupled receptors (GSPCRs) capable of preserving stellate cell quiescence. Accompanying HSC activation, we further observed the attenuation of HSC Gdf2 expression, and a MASH-associated expansion of a CD207-positive macrophage population likely derived from both incoming monocytes and Kupffer cells.
CONCLUSIONS: We conclude that HSC-expressed NR1H4 and GSPCRs of the healthy liver integrate postprandial cues, which sustain HSC quiescence and, through paracrine signals, overall sinusoidal health. Hence HSC activation in MASH not only drives fibrogenesis but may desensitize the hepatic sinusoid to liver homeostatic signals.
UNASSIGNED: Homeostatic interactions between hepatic cell types and their deterioration in metabolic dysfunction-associated steatohepatitis are poorly characterized. In our current single cell-resolved study of advanced murine metabolic dysfunction-associated steatohepatitis, we identified a quiescence-associated hepatic stellate cell-signaling module with potential to preserve normal sinusoid function. As expression levels of its constituents are conserved in the human liver, stimulation of the identified signaling module is a promising therapeutic strategy to restore sinusoid function in chronic liver disease.

To analyze miR-223-3p expression in patients with hepatitis B virus (HBV) live fibrosis and its effects on proliferation, activation, and apoptosis of human hepatic stellate cell line. One hundred patients with HBV-associated liver fibrosis were divided into S0 to 1, S2 to 3, and S4 groups according to Scheuer histological staging; healthy individuals during the same period were enrolled as healthy group. Relative expressions of miR-223-3p in healthy, S0 to 1, S2 to 3, and S4 groups were 0.56 ± 0.11, 1.08 ± 0.27, 2.16 ± 0.42, and 3.59 ± 1.06, respectively. Absorbance values of human hepatic stellate cell line cells at 24, 48, and 72 hours were higher in miR-223-3p-mimic group than in control group (CG) and NC-mimic group and were lower in miR-223-3p-inhibitor group than in CG and NC-inhibitor group (P < .05). mRNA miR-223-3p, α-smooth muscle actin, collagen 1A1, collagen 1A2, collagen 3A1, and transforming growth factor (TGF)-β1 levels were higher in miR-223-3p-mimic group than in CG and NC-mimic group and lower in miR-223-3p-inhibitor group than in CG and NC-inhibitor group (P < .05). Protein expressions of α-smooth muscle actin, transforming growth factor-β1, collagen I, collagen III, p-Smad3, p-Smad2, and B-cell lymphoma 2 were higher in miR-223-3p-mimic group than in CG and NC-mimic groups and lower in miR-223-3p-inhibitor group than in CG and NC-inhibitor group, whereas those of B-cell lymphoma 2-associated death promoter, B-cell lymphoma 2 associated X protein, cleaved caspase3, cleaved caspase9, poly ADP-ribose polymerase were lower in miR-223-3p-mimic group than in CG and NC-mimic group and higher in miR-223-3p-inhibitor group than in CG and NC-inhibitor group (P < .05). HBV liver fibrosis patients had elevated expression of miR-223-3p in plasma. Upregulation of miR-223-3p expression may be related to transforming growth factor-β1/Smad signaling pathway activation.

The aim of this study is to determine whether activated hepatic stellate cells (HSCs) may represent a prognostic marker of progressive liver fibrosis in chronic viral hepatitis C (VHC) before antiviral therapy. The possible correlation between HSCs immunohistochemical features, histopathological aspects and clinical data before therapy were also studied.
This retrospective pilot study was conducted on 27 liver biopsies from VHC patients before antiviral therapy. HSCs\'s immunohistochemical analysis used the antibodies alpha-smooth muscle actin (α-SMA), glial fibrillary acidic protein (GFAP) and vinculin. We correlated immunopositive HSCs with HCV load, liver stiffness (LS), fibrosis stage and necro-inflammatory degree before treatment. Also, we assessed the association between liver fibrosis after therapy, the sustained virological response at 12 weeks after therapy (SVR 12) and the type of therapy.
HSCs were increased in VHC patients compared to controls, mainly in the intermediate and periportal lobular regions. α-SMA and vinculin HSCs correlated positively with fibrosis stage (p=0.044), (p=0.028). Furthermore, α-SMA and vinculin HSCs were associated with LS (p=0.027), (p=0.002) and viral load (p=0.021), (p=0.006), but not with necro-inflammation degree. GFAP HSCs inversely correlated with fibrosis stage (r= -0.475), LS (r= -0.422) and HCV load (r= -0.517), but positively with necro-inflammation degree (p=0.038). Liver fibrosis post therapy correlated positively with SVR12 (p<0.001) and the type of therapy (p=0.006) and SVR12 correlated positively with treatment\'s type (p=0.002).
Activated HSCs may represent a marker of increased liver fibrosis in VHC. Different immunohistochemical markers can detect various HSCs subpopulations involved in the evolution of VHC and liver fibrosis.

Coronavirus disease 19 (COVID-19) has affected over 112 million people and killed more than 2.5 million worldwide. When the pandemic was declared, Spain and Italy accounted for 29% of the total COVID-19 related deaths in Europe, while most infected patients did not present severe illness. We hypothesised that shared genomic characteristics, distinct from the rest of Europe, could be a contributor factor to a poor prognosis in these two populations. To identify pathways related to COVID-19 severity, we shortlisted 437 candidate genes associated with host viral intake and immune evasion from SARS-like viruses. From these, 21 were associated specifically with clinically aggressive COVID-19. To determine the potential mechanism of viral infections, we performed signalling pathway analysis with either the full list (n = 437) or the subset group (n = 21) of genes. Four pathways were significantly associated with the full gene list (Caveolar-mediated Endocytosis and the MSP-RON Signalling) or with the aggressive gene list (Hepatic Fibrosis/Hepatic Stellate Cell (HSC) Activation and the Communication between Innate and Adaptive Immune Cells). Single nucleotide polymorphisms (SNPs) from the ±1 Mb window of all genes related to these four pathways were retrieved from the dbSNP database. We then performed Principal Component analysis for these SNPs in individuals from the 1000 Genomes of European ancestry. Only the Hepatic Fibrosis/HSC Activation pathway showed population-specific segregation. The Spanish and Italian populations clustered together and away from the rest of the European ancestries, with the first segregating further from the rest. Additional in silico analysis identified potential genetic markers and clinically actionable therapeutic targets in this pathway, that may explain the severe disease.

Objective: To observe the effect of miR-217 on angiotensin II (AngII)-induced hepatic stellate cells (HSCs) activation, and carbon tetrachloride (CCl4)-induced overexpression in mice, so as to clarify miR-217 role in liver fibrosis. Methods: HSCs were stimulated with AngⅡ and the changes condition in the expression level of miR-217 were detected. HSCs were divided into control group, AngII-treated group and AngⅡ+miR-217-treated group. The expression levels of alpha-smooth muscle actin, fibroblast-specific protein 1 and collagen Ⅰ (Collagen Ⅰ) in each group were detected. The target gene of mir-217 was screened and verified by Targetscan and Dual luciferase gene reporter assay. Real-time quantitative PCR and Western blot were used to detect the effect of miR-217 on the expression level of transforming growth factor beta type Ⅱ receptor (TGFBR2). A CCl4-induced mouse liver fibrosis model was constructed. Masson staining and Sirius red staining were used to detect the effect of miR-217 overexpression on the progression of liver fibrosis in CCl4 mice. Data of two groups were compared using t-test. Data of multiple groups were statistically analyzed with one-way ANOVA. Results: The expression level of miR-217 was downregulated by AngⅡ-stimulated HSC cells. The expression levels of α-smooth muscle actin, fibroblast-specific protein 1 and Collagen Ⅰ induced by AngⅡ was inhibited by miR-217 mimics transfection. The 3\'-UTR of TGFBR2 had specifically bind miR-217. The mRNA and protein expression levels of TGFBR2 was inhibited with miR-217 mimics transfection in HSCs. The overexpression of miR-217 had inhibited the expression levels of Collagen Ⅰ and Ⅲ in CCl4 mice and alleviated the progression of liver fibrosis . Conclusion: miR-217 regulates liver fibrosis by targeting TGFBR2, inhibits AngII-induced HSC activation, and slows down the process of liver fibrosis in CCl4 mice, suggesting that miR-217 may have an inhibitory effect on liver fibrosis.
目的： 观察miR-217对血管紧张素Ⅱ（AngⅡ）诱导的肝星状细胞（HSC）活化的影响，观察四氯化碳（CCl4）诱导小鼠中miR-217过表达产生的效果，阐明miR-217在肝纤维化中的作用。 方法： 使用AngⅡ刺激HSC并检测miR-217表达水平的变化情况。将HSC分为对照组、AngⅡ处理组和AngⅡ+miR-217处理组，检测各组中α-平滑肌肌动蛋白，成纤维细胞特异蛋白1和Ⅰ型胶原蛋白（Collagen I）的表达水平。采用TargetScan和双荧光素酶报告基因实验对miR-217的靶基因进行筛选及验证。荧光实时定量PCR和Western blot检测miR-217对转化生长因子β受体Ⅱ（TGFBR2）表达水平的影响。构建CCl4诱导的小鼠肝纤维化模型，Masson染色和天狼星红染色检测过表达miR-217对CCl4小鼠肝纤维化进程的影响。2组数据间的比较采用t检验，多组数据比较采用One-Way ANOVA进行统计分析。 结果： AngⅡ刺激HSC细胞能下调miR-217的表达水平，转染miR-217 mimics能抑制AngⅡ诱导的α-平滑肌肌动蛋白，成纤维细胞特异蛋白1和Collagen I的表达水平。miR-217能特异性结合TGFBR2的3’-UTR，转染miR-217 mimic能抑制HSC中TGFBR2的mRNA和蛋白表达水平。CCl4小鼠中过表达miR-217能抑制Collagen I和Collagen Ⅲ的表达水平，缓解肝纤维化进程。 结论： miR-217通过靶向调控TGFBR2调节肝纤维化，抑制AngⅡ诱导的HSC活化，减缓CCl4小鼠的肝纤维化进程，表明miR-217可能具有抑制肝纤维化的功能。.

Huangqi decoction, also known as Huangqi Liuyi decoction, was first recorded in the prescriptions of the Bureau of Taiping People\'s Welfare Pharmacy. It comprises astragalus and licorice, which is a commonly used prescription in traditional Chinese medicine for the clinical treatment of chronic liver disease, especially liver cirrhosis. Total astragalus saponins (AST) is the main component of astragalus, and glycyrrhizic acid (GA) is the main component of licorice. In this study, normal macrophage exosomes were extracted, and the exosomes incubated with lipopolysaccharides (LPS) and those incubated with LPS + AST + GA were co-cultured with JS1 cells (hepatic stellate cell line). The survival rate and the activation of key signaling pathways of JS1 cells in each group were detected and compared. We found that the co-culture of LPS-induced macrophage exosomes with JS1 cells could significantly increase the expression levels of Collagen-1 (Col-1) and Alpha smooth muscle actin (α-SMA)in JS1 cells. However, a significant reversal effect was observed after pretreatment with AST combined with GA. Further evaluation found that the expression levels of phospho (p)-Smad2 and p-Smad3 in the JS1 cells were significantly increased after macrophages were induced with LPS, whereas pretreatment with AST + GA could significantly decrease the expression levels of p-Smad2 and p-Smad3. Preliminary results of this study indicated that LPS-induced macrophage exosomes can promote the activation of hepatic stellate cells, and the pretreatment of AST combined with GA can exert a significant intervention effect. In this study, the new mechanism of anti-hepatic fibrosis effect of traditional Chinese medicine components of Huangqi Decoction was analyzed from the perspective of exosomes.
摘要:黄芪汤, 又名黄芪六一汤, 始载于《太平惠民和剂局方》, 由黄芪和甘草组成, 是中医临床治疗慢性肝病的常用方剂, 尤其在肝硬化的治疗中有重要作用。黄芪总皂苷(AST)是黄芪的主要成分, 甘草酸(GA)是甘草的主要成分。本研究通过LPS 孵育建立巨噬细胞活化模型, 观察其分泌的外泌体对肝星状细胞 (JS1) 的影响, 进一步采用 AST 联合 GA 预处理巨噬细胞, 观察其干预效应。将各组外泌体分别与 JS1 细胞共培养, 检测各组 JS1 细胞的增殖情况、活化状态及关键信号通路激活情况。研究发现 LPS 诱导的巨噬细胞外泌体可以显著增加 JS1 细胞Col1、α-SMA 表达水平, 促进 JS1 细胞的活化。然而, 对巨噬细胞进行 AST 联合 GA 的预处理后具有显著的逆转作用。同时, LPS 诱导巨噬细胞后 JS1 细胞的 p-Smad2、p-Smad3 表达水平显着升高, 而 AST 联合 GA 预处理可降低 p-Smad2、 p-Smad3 的表达。本研究结果表明:脂多糖诱导的巨噬细胞外泌体可以促进肝星状细胞活化, AST 联合 GA 预处理能发挥显著的干预效应。本研究从外泌体角度解析了黄芪汤中药组分抗肝纤维化的新机制。.

Liver fibrosis is currently a global health challenge with no approved therapy, with the activation of hepatic stellate cells being a principal factor. Lipophilic constituents in Salvia miltiorrhiza (LS) have been reported to improve liver function and reduce the indicators of liver fibrosis for patients with chronic hepatitis B induced hepatic fibrosis. However, the pharmacological mechanisms of LS on liver fibrosis have not been clarified. In this study, 71 active compounds, 342 potential target proteins and 22 signaling pathways of LS were identified through a network pharmacology strategy. Through text mining and data analysis, the JAK1/STAT3 signaling pathway was representatively selected for further experimental validation. We firstly confirmed the protective effect of LS on liver fibrosis in vivo by animal experiments. Hepatic stellate cells, which proliferated and displayed a fibroblast-like morphology similar to activated primary stellate cells, were applied to evaluate its underlying mechanisms. The results showed that LS could inhibit the cell viability, promote the cell apoptosis, decrease the expression of liver fibrosis markers, and downregulate the JAK1/STAT3 signaling pathway. These results demonstrated that LS could exert anti-liver-fibrosis effects by inhibiting the activation of HSCs and regulating the JAK1/STAT3 signaling pathway, which is expected to benefit its clinical application.

The present study clarified the molecular mechanism of curcumol against liver fibrosis based on its effects on the autopha-gy and apoptosis of hepatic stellate cells. The hepatic stellate cells were divided into a blank control group, a transforming growth factor-β1(TGF-β1)(10 ng·mL~(-1)) group, and low-(12.5 mg·L~(-1)), medium-(25 mg·L~(-1)), and high-dose(50 mg·L~(-1)) curcumol groups. The effect of curcumol on the viability of hepatic stellate cells induced by TGF-β1 was detected by the MTT assay kit. The apo-ptosis in each group was determined by flow cytometry. Real-time fluorescence-based quantitative PCR(RT-PCR) was employed for the detection of mRNA expression of α-smooth muscle actin(α-SMA), type Ⅰ collagen(collagen Ⅰ), and type Ⅲ collagen(collagen Ⅲ). Western blot was used to detect the protein expression of p62, microtubule-associated protein 1 light chain 3(LC3), beclin1, B cell lymphoma 2(Bcl-2), and Bcl-2-associated X protein(Bax). Transmission electron microscopy(TEM) was used to observe cell morphology and autophagosome formation in each group. The autophagic flux was observed after cell infection with adenovirus under double fluorescence labeling. The cell viability assay revealed that compared with the TGF-β1 group, the curcumol groups showed significantly decreased cell viability. The apoptosis assay showed that the apoptosis rates of the curcumol groups were significantly higher than that of the TGF-β1 group. RT-PCR indicated that the mRNA expression of α-SMA, collagenⅠ, and collagen Ⅲ in the curcumol groups was significantly lower than that of the TGF-β1 group. Western blot showed that the expression of p62, LC3, beclin1, Bcl-2, and Bax in the curcumol groups was significantly different from that in the TGF-β1 group. As demonstrated by TEM, compared with the TGF-β1 group, the curcumol groups showed significantly increased autophagosomes. The detection of autophagic flow by the adenovirus under double fluorescence labeling showed that autolysosomes in the curcumol groups were significantly increased compared with those in the TGF-β1 group. Curcumol can induce the autophagy and apoptosis of hepatic stellate cells, which may be one of its anti-liver fibrosis mechanisms.