[This corrects the article DOI: 10.3389/fphar.2021.671152.].

BACKGROUND:MASH has become the most prevalent chronic liver disease worldwide. Hepatocyte ferroptosis is a key driver in the progression of MASH. Salvianolic acid B (Sal B), a natural antioxidant active ingredient, has an anti-MASH effect. However, the underlying mechanism by which Sal B inhibits hepatocyte ferroptosis and improves MASH remains unclear. METHODS:An in vivo MASH model was established by feeding C57BL/6J mice a HFHC diet for 30 weeks. In the 24th week, mice were treated with Sal B, obeticholic acid (OCA) or SRT1720. In vitro, over-expression and knockdown of SIRT1 in hepatocytes were constructed with lentivirus, subsequently, a hepatocyte lipid deposition model induced by palmitic acid/oleic acid (PO) or a hepatocyte ferroptosis model induced by erastin were used to investigate the efficacy and mechanism of Sal B. Furthermore, the interaction and binding kinetics of Sal B and SIRT1 were determined by molecular docking simulation, DARTS, CETSA and SPR assays. RESULTS:Sal B attenuated hepatic inflammation, lipid deposition and fibrosis in MASH mice. In situ transmission electron microscopy revealed characteristic morphological features of hepatocyte ferroptosis in the livers of MASH mice, which were alleviated by Sal B treatment. Mechanistically, Sal B up-regulated hepatic SIRT1 protein and inhibited p53 acetylation. In vitro, Sal B alleviated PO- or erastin-induced lipid deposition and ferroptosis in hepatocytes, along with up-regulating SIRT1 and inhibiting p53 acetylation. Similar protective effects were observed upon SIRT1 overexpression. Strikingly, SIRT1 knockdown reduced the inhibitory effect of Sal B on hepatocyte ferroptosis. SPR analysis confirmed moderate-affinity binding of Sal B to recombinant human SIRT1 protein, while DARTS and CETSA assays indicated that Sal B enhances SIRT1 enzymatic and thermal stability. CONCLUSION:Sal B ameliorates MASH in mice via directly regulating SIRT1/p53 signaling to suppress hepatocyte ferroptosis. Targeting hepatocyte ferroptosis through SIRT1 activation is a promising therapeutic strategy for MASH.

BACKGROUND AND AIMS:Extracellular matrix protein 1 (ECM1) is known to inhibit transforming growth factor β signalling and HSC activation, thereby attenuating liver fibrosis. RNA-seq profiling of livers from wild-type and ECM1-deficient mice revealed different enrichments in metabolic changes in fatty acid synthesis and inflammatory pathways, suggesting a regulatory role for ECM1 in liver steatosis. Here, we studied the role of ECM1 in metabolic dysfunction-associated steatotic liver disease pathogenesis and underlying mechanisms. APPROACH AND RESULTS:Hepatic ECM1 expression was evaluated and found to be significantly reduced in liver samples from patients with metabolic dysfunction-associated steatohepatitis (MASH), and in 4 established MASH mouse models (HFD, MCD, HFHC, and ob/ob-/- ). Although overexpression of ECM1 effectively blocked hepatic insulin resistance, steatosis, and inflammation, ECM1 ablation exacerbated diet-induced MASH progression. Mechanistically, ECM1 interacted with the K-homology 3 (KH3) domain of poly r(C) binding protein 1 (PCBP1) to suppress iron overload, mitigating lipid peroxidation and consequently impeding MASH advancement under metabolic stress. Re-expression of ECM1 and PCBP1 ameliorated liver disease progression. CONCLUSIONS:Our study reveals that ECM1 is a critical regulator in MASH, modulating lipid peroxidation by maintaining PCBP1-mediated intracellular iron homeostasis. Targeting ECM1 to restore PCBP1-dependent iron homeostasis may offer a novel therapeutic avenue for MASH.

Hepatocyte ferroptosis promotes the pathogenesis and progression of liver fibrosis. Salvianolic acid B (Sal B) exerts antifibrotic effects. However, the pharmacological mechanism and target has not yet been fully elucidated. In this study, liver fibrosis was induced by CCl4 in wild-type mice and hepatocyte-specific extracellular matrix protein 1 (Ecm1)-deficient mice, which were separately treated with Sal B, ferrostatin-1, sorafenib or cilengitide. Erastin- or CCl4-induced hepatocyte ferroptosis models with or without Ecm1 gene knockdown were evaluated in vitro. Subsequently, the interaction between Ecm1 and xCT and the binding kinetics of Sal B and Ecm1 were determined. We found that Sal B significantly attenuated liver fibrosis in CCl4-induced mice. Ecm1 deletion in hepatocytes abolished the antifibrotic effect of Sal B. Mechanistically, Sal B protected against hepatocyte ferroptosis by upregulating Ecm1. Further research revealed that Ecm1 as a direct target for treating liver fibrosis with Sal B. Interestingly, Ecm1 interacted with xCT to regulate hepatocyte ferroptosis. Hepatocyte ferroptosis in vitro was significantly attenuated by Sal B treatment, which was abrogated after knockdown of Ecm1 in LO2 cells. Therefore, Sal B alleviates liver fibrosis in mice by targeting up-regulation of Ecm1 and inhibiting hepatocyte ferroptosis. The interaction between Ecm1 and xCT regulates hepatocyte ferroptosis.

Ethnopharmacological relevanceTraditional Chinese medicine has great potential and advantages in the treatment of liver fibrosis, with Fuzheng Huayu formula (FZHY) serving as a prime example due to its remarkable efficacy in delaying and reversing liver fibrosis while simultaneously improving clinical symptoms for patients.Aim of the review: In this paper, we present a comprehensive review of recent studies on the therapeutic potential of FZHY and its components/ingredients in the treatment of liver fibrosis and cirrhosis, with the aim of providing insights for future research endeavors.Materials and methodsA comprehensive literature search was conducted on FZHY, TCM319, traditional Chinese medicine 319, liver fibrosis and cirrhosis using multiple internationally recognized databases including PubMed, Embase, Springer, Web of science, SciVerse ScienceDirect, Clinical Trails. Gov, CNKI, Wanfang, and VIP.ResultsFZHY is widely used clinically for liver fibrosis and cirrhosis caused by various chronic liver diseases, with the effects of improving serum liver function, liver pathological histology, serological indices related to liver fibrosis, decreasing liver stiffness values and portal hypertension, as well as reducing the incidence of hepatocellular carcinoma and morbidity/mortality in patients with cirrhosis. Numerous in vivo and in vitro experiments have demonstrated that FZHY possesses anti-fibrotic effects by inhibiting hepatic stellate cell activation, reducing inflammation, protecting hepatocytes, inhibiting hepatic sinusoidal capillarization and angiogenesis, promoting extracellular matrix degradation, and facilitating liver regeneration. In recent years, there has been a growing focus on investigating the primary active components/ingredients of FZHY, and significant strides have been made in comprehending their synergistic mechanisms that enhance efficacy.ConclusionFZHY is a safe and effective drug for treating liver fibrosis. Future research on FZHY should focus on its active components/ingredients and their synergistic effects, as well as the development of modern cocktail drugs based on its components/ingredients. This will facilitate a more comprehensive understanding of the molecular mechanisms and targets of FZHY in treating liver fibrosis, thereby further guide clinical applications and drug development.

Total astragalus saponins (TAS) are the main active components of astragali radix, and have potent anti-hepatic fibrosis effect. However, the therapeutic efficacy of TAS and their potential mechanisms in the treatment of primary sclerosing cholangitis (PSC) remain unclear. In this study, two mouse models of PSC, including 3,5-Diethoxycarbonyl-1,4-Dihydro-2,4,6-Collidine (DDC)-induced PSC and Mdr2-/- spontaneous PSC, and the Tgr5-/- mice were used to investigate the therapeutic effect and mechanisms of TAS. Treatment with TAS, particularly with a dose of 56 mg/kg, significantly ameliorated the PSC-related liver injury, cholestasis, collagen deposition, ductular reaction (DR), and fibrosis in the DDC-induced and Mdr2-/-spontaneous PSC mice. Furthermore, treatment with TAS significantly mitigated the PSC-related inflammatory responses in vivo and HIBEpiC cells by inhibiting the expression of TNF-α, IL-6, and IL-1β. Mechanistically, treatment with TAS rescued the PSC-decreased hepatic TGR5 expression to attenuate the NF-κB p65 phosphorylation. Notably, the therapeutic efficacy of TAS on PSC in DDC-induced mice was abrogated in Tgr5-/- mice, suggesting the anti-PSC effect of TAS may depend on enhancing TGR5 expression. In conclusion, TAS ameliorated DR, inflammation and liver fibrosis in both models of PSC mice by rescuing TGR5 expression. Our findings may aid in the design of new therapeutic strategies for the treatment of PSC.

Background: It is poorly understood what cellular types participate in ductular reaction (DR) and whether DR facilitates recovery from injury or accelerates hepatic fibrosis. The aim of this study is to gain insights into the role of hepatic progenitor cell (HPC)-originated DR during fibrotic progression. Methods: DR in liver specimens of PBC, chronic HBV infection (CHB) or NAFLD, and four rodent fibrotic models by different pathogenic processes was evaluated. Gli1 expression was inhibited in rodent models or cell culture and organoid models by AAV-shGli1 or treating with GANT61. Results: Severity of liver fibrosis was positively correlated with DR extent in patients with PBC, CHB or NAFLD. HPCs were activated, expanded, differentiated into reactive cholangiocytes and constituted "HPC-originated DR", accompanying with exacerbated fibrosis in rodent models of HPC activation & proliferation (CCl4/2-AAF-treated), Mu dr2-/- spontaneous PSC, BDL-cholestatic fibrosis or WD-fed/CCl4-treated NASH-fibrosis. Gli1 expression was significantly increased in enriched pathways in vivo and in vitro. Enhanced Gli1 expression was identified in KRT19+-reactive cholangiocytes. Suppressing Gli1 expression by administration of AAV-shGli1 or GANT61 ameliorated HPC-originated DR and fibrotic extent. KRT19 expression was reduced after GANT61 treatment in sodium butyrate -stimulated WB-F344 cells or organoids or in cells transduced with Gli1 knockdown lentiviral vectors. In contrast, KRT19 expression was elevated after transducing Gli1 overexpression lentiviral vectors in these cells. Conclusions: During various modes of chronic injury, Gli1 acted as an important mediator of HPC activation, expansion, differentiation into reactive cholangiocytes that formed DR, and subsequently provoked hepatic fibrogenesis.

Ethnopharmacological relevance: Cholestatic Liver Fibrosis (CLF) is a hepatobiliary disease that typically arises as a late-stage complication of cholestasis, which can have multiple underlying causes. There are no satisfactory chemical or biological drugs for CLF. Total Astragalus saponins (TAS) are considered to be the main active constituents of the traditional Chinese herb Astragali Radix (AR), which has the obvious improvement effects for treating CLF. However, the mechanism of anti-CLF effects of TAS is still unclear. Aim of the study: The present study was undertaken to investigate the therapeutic effects of TAS against bile duct ligation (BDL) and 3, 5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) -induced CLF models and to reveal the potential mechanism to support its clinic use with scientific evidence. Materials and methods: In this study, BDL-induced CLF rats were treated with TAS (20 mg/kg, 40 mg/kg) and DDC-induced CLF mice were treated with 56 mg/kg TAS. The therapeutic effects of TAS on extrahepatic and intrahepatic CLF models were evaluated by serum biochemical analysis, liver histopathology and hydroxyproline (Hyp). Thirty-nine individual bile acids (BAs) in serum and liver were quantified by using UHPLC-Q-Exactive Orbitrap HRMS. qRT-PCR, Western blot and immunohistochemistry analysis were used to measure the expression of liver fibrosis and ductular reaction markers, inflammatory factors and BAs related metabolic transporters, along with nuclear receptor farnesoid X receptor (FXR). Results: The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBiL), direct bilirubin (DBiL) and contents of liver Hyp were dose-dependently improved after treatment for TAS in BDL and DDC- induced CLF models. And the increased levels of ALT and AST were significantly improved by total extract from Astragali radix (ASE) in BDL model. The liver fibrosis and ductular reaction markers, & alpha;-smooth muscle actin (& alpha;-SMA) and cytokeratin 19 (CK19), were significantly ameliorated in TAS group. And the liver expression of inflammatory factors: interleukin 6 (IL-6), tumor necrosis factor-& alpha; (TNF-& alpha;) and interleukin 1 & beta; (IL-1 & beta;) were significantly decreased after TAS treatment. In addition, TAS significantly ameliorated taurine-conjugated BAs (tau-BAs) levels, particularly & alpha;-TMCA, & beta;-TMCA and TCA contents in serum and liver, which correlated with induced expressions of hepatic FXR and BAs secretion transporters. Furthermore, TAS significantly improved short heterodimer partner (SHP), cholesterol 7 & alpha;-hydroxylase (Cyp7a1), Na+ taurocholate cotransport peptide (NTCP) and bile-salt export pump (BSEP) mRNA and protein expression.

Introduction and ObjectivesLiver fibrosis is a common pathological change in many chronic liver diseases. Activation of hepatic stellate cells (HSCs) is the core event in liver fibrosis. This study aimed to investigate the role of testicular orphan receptor 4 (TR4) in the activation of HSCs.Materials and MethodsIn vivo, bile duct ligation (BDL)-induced rat liver fibrosis model was established, and the expressions of TR4 and α-smooth muscle actin (α-SMA) in liver tissues were detected. In vitro, TR4 knockdown and overexpression in JS-1 cells using lentiviral vectors were constructed, and the expressions of TR4, α-SMA, Col-I, and TGF-β1/smads and retinoid X receptor (RXR) pathway-related genes were detected.ResultsTR4 was highly expressed in BDL-induced fibrotic liver, accompanied by increased expression of α-SMA. Knockdown of TR4 significantly inhibited the expressions of α-SMA, Col-I, p-TβRI, and p-Smad2/3, and up-regulated the expression of RXRα in HSCs in vitro. In contrast, TR4 overexpression significantly increased the expressions of α-SMA, Col-I, p-TβRI, and p-Smad2/3, and inhibited the expression of RXRα.ConclusionsTR4 may promote the activation of HSCs by up-regulating TβR I/Smad2/3 signaling pathway and down-regulating RXRα signaling, thereby promoting the progression of liver fibrosis. Our findings may provide a new therapeutic target against hepatic fibrosis.

The newly emerged Omicron(B.1.1.529)variant of SARS-CoV-2 is quickly overtaking the Delta variant and becoming the dominant strain around the world due to its enhanced transmissibility and high immune escape potential[1,2].Compared to the Wuhan strain,the Omicron variant carries 37 spike mutations,of which 15 are within the receptor-binding domain(RBD)(Fig.1A).A high mutation rate is associated with remarkable resistance to current vaccines and RBD-specific antibody therapeutics[2-4].It is urgent to comprehen-sively understand the impacts of these RBD mutations on the neutralization capability of RBD-specific neutralizing antibodies(NAbs)targeting diverse epitopes and search for highly potent and broadly reactive NAbs against SARS-CoV-2 variants,including the Omicron strain.This information will be critical for under-standing the evolutionary mechanisms that govern SARS-CoV-2 immune escape and for guiding vaccine design.

目的：观察不同剂量野百合碱（MCT）诱导大鼠肝窦阻塞综合征（HSOS）的病理变化，探讨造模剂量-时间致肝损伤的病理特征，并解析其部分作用机制。方法：将72只雄性SD大鼠随机分为正常组（n=12）,MCT低、中、高剂量组（每组n=20）。MCT低、中、高剂量组分别采用MCT 80、120、160 mg·kg-1灌胃一次制备模型，各剂量组大鼠于造模48、120 h处死取材。观测各组大鼠的存活率，检测大鼠体质量、肝质量与血清肝功能变化，采用扫描电镜、苏木素-伊红（HE）染色及天狼星红（SR）染色观察肝组织病理变化，微板法检测肝组织匀浆谷胱甘肽S转移酶（GST）、总超氧化物歧化酶（T-SOD）活性、丙二醛（MDA）含量，实时荧光定量聚合酶链式反应（Real-time PCR）、蛋白免疫印迹法（Western blot）和免疫组化检测肝组织相关指标的表达。结果：与正常组比较，MCT各组血清丙氨酸氨基转移酶/天冬氨酸氨基转移酶（ALT/AST）活性随造模剂量增加而升高（P＜0.05,P＜0.01）；随造模时间增加，MCT低剂量组血清ALT和AST活性均下降（P＜0.01）,MCT中、高剂量组ALT和AST活性均显著升高（P＜0.01）。HE染色显示MCT低、中、高剂量组肝组织肝细胞坏死、炎性细胞浸润及红细胞淤积，电镜下可见肝窦内皮窗孔扩大，“筛状”结构消失，其损伤程度随剂量提高而加重。与正常组比较，MCT低、中、高剂量组肝窦内皮细胞标志物CD44表达减少（P＜0.05,P＜0.01）。SR染色显示造模48 h MCT各组未见阳性染色，120 h MCT各组可见汇管区及肝窦胶原沉积。与正常组比较，MCT各剂量组肝组织MDA含量及GST活性增加，T-SOD活性降低，以MCT中、高剂量组变化显著（P＜0.01）；且造模120 h MCT各组变化呈现剂量依赖性（P＜0.01）。免疫组化及Western blot结果显示，与正常组比较，促炎巨噬细胞标志物CD68蛋白表达随剂量升高而增加（P＜0.01），抗炎巨噬细胞CD163蛋白及mRNA表达随剂量升高而减少（P＜0.01）。与正常组比较，造模后MCT中、高剂量组磷酸化核转录因子-κB/核转录因子-κB（p-NF-κB/NF-κB）、磷酸化蛋白激酶B/蛋白激酶B（p-Akt/Akt）明显升高（P＜0.05,P＜0.01）。MCT造模后肝组织α-平滑肌肌动蛋白（α-SMA）蛋白表达随时间延长、剂量升高增加，α-SMA、Ⅰ型胶原蛋白基因α1（Col 1a1）和Ⅳ型胶原蛋白基因α1（Col 4a1）mRNA表达也随时间延长、剂量升高而明显增加（P＜0.05,P＜0.01）。TUNEL染色结果显示造模后凋亡细胞数量随造模剂量升高而增加，B细胞淋巴瘤-2（Bcl-2）蛋白与Bcl-2相关X蛋白（Bax）比值显著降低（P＜0.01）。结论：不同剂量MCT灌胃诱导的大鼠肝窦阻塞综合征，病变随剂量增加而加重。80 mg·kg-1MCT导致的肝脏损伤可自愈，超过120 mg·kg-1的MCT导致的肝脏损伤会随着时间的延长而加重，甚至出现纤维化、死亡。MCT导致大鼠HSOS的病理机制可能是MCT引发肝组织内强烈的氧化应激，激活促炎巨噬细胞大量分泌炎症因子，进而激活NF-κB/Akt信号通路，导致严重的细胞损伤和死亡。

Ferroptosis, an iron-dependent non-apoptotic cell death characterized by lipid peroxidation, is a cell death pathway discovered in recent years. Ferroptosis plays an important role in tumors, ischemia-reperfusion injury, neurological diseases, blood diseases, etc. Recent studies have shown the importance of ferroptosis in chronic liver disease. This article summarizes the pathological mechanisms of ferroptosis involved in System Xc−, iron metabolism, lipid metabolism, and some GPX4-independent pathways, and the latest research on ferroptosis in chronic liver diseases such as alcoholic liver disease, non-alcoholic fatty liver disease, liver fibrosis, hepatocellular carcinoma. In addition, the current bottleneck issues that restrict the research on ferroptosis are proposed to provide ideas and strategies for exploring new therapeutic targets for chronic liver diseases.

Interleukin-33 (IL-33), an epithelial cell-derived cytokine that responds rapidly to environmental insult, has a critical role in initiating airway inflammatory diseases. However, the molecular mechanism underlying IL-33 secretion following allergen exposure is not clear. Here, we found that two cell events were fundamental for IL-33 secretion after exposure to allergens. First, stress granule assembly activated by allergens licensed the nuclear-cytoplasmic transport of IL-33, but not the secretion of IL-33. Second, a neo-form murine amino-terminal p40 fragment gasdermin D (Gsdmd), whose generation was independent of inflammatory caspase-1 and caspase-11, dominated cytosolic secretion of IL-33 by forming pores in the cell membrane. Either the blockade of stress granule assembly or the abolishment of p40 production through amino acid mutation of residues 309–313 (ELRQQ) could efficiently prevent the release of IL-33 in murine epithelial cells. Our findings indicated that targeting stress granule disassembly and Gsdmd fragmentation could reduce IL-33-dependent allergic airway inflammation.

ObjectiveTo investigate the intervention effect of GDC-0449, a hedgehog signaling pathway inhibitor, on rats with liver fibrosis induced by carbon tetrachloride (CCl4) combined with 2-acetylaminofluorene (2-AAF). MethodsA total of 18 female Fisher344 rats were randomly divided into normal group, CCl4/2-AAF group, and GDC-0449 group, with 6 rats in each group. The rats in the CCl4/2-AAF group and the GDC-0449 group were given subcutaneously injected 30% CCl4-olive oil solution at a dose of 2 mL/kg twice a week for 6 weeks to induce liver fibrosis; since week 7, in addition to the injection of CCl4-olive oil solution, the rats in these two groups were given 2-AAF (100 mg/kg/d) by gavage, and the rats in the GDC-0449 group were given GDC-0449 (25 mg/kg/d) by gavage, while those in the normal group were given an equal volume of olive oil solution by injection and normal saline by gavage. All rats were sacrificed at the end of week 9, and related samples were collected. HE staining and sirius red (SR) staining were used to observe the changes in liver histopathology and collagen deposition, and the semi-quantitative analysis of SR-positive area and Ishak score were used to evaluate fibrosis degree; the alkaline hydrolysis method was used to measure the level of hydroxyproline (Hyp) in liver tissue; immunohistochemistry, Western blot, and qRT-PCR were used to measure the expression of α-smooth muscle actin (α-SMA), type I collagen (Col-I), type IV collagen (Col-IV), cytokeratin 19 (CK19), cytokeratin 7 (CK7), the epithelial cell adhesion molecule Epcam, and the hedgehog signaling pathway in liver tissue; double immunofluorescence staining was used to observe the colocalization of CK19 and the oval cell marker OV6. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the normal group, the CCl4/2-AAF group had marked inflammatory cell aggregation and collagen deposition in liver tissue, with the formation of a pseudolobular structure, as well as significant increases in Hyp level and collagen positive area ratio in liver tissue (P＜0.05), Ishak score (P＜0.05), and the expression of α-SMA, Col-I, Col-IV, Epcam, CK19, CK7, the transmembrane transporter Smoothened (Smo), Hedgehog ligand Desert Hedgehog (Dhh), the Indian Hedgehog membrane-binding receptor Patched (Ptch2), and glioma-related oncogenes Gli1, Gli2, and Gli3 (all P＜0.05); double immunofluorescence staining showed that CK19-positive cells also expressed OV6 in the liver tissue of rats in the CCl4/2-AAF group, with a significant increase compared with the normal group. Compared with the CCl4/2-AAF group, the GDC-0449 group had significant reductions in inflammatory cell aggregation and collagen deposition in liver tissue, Hyp level and collagen positive area ratio in liver tissue (P＜0.05), Ishak score (P＜0.05), and the expression of α-SMA, Epcam, CK19, CK7, Smo, Ptch2, Gli1, Gli2, and Gli3 (all P＜0.05); double immunofluorescence staining showed a significant reduction in the number of cells with co-expression of OV6 and CK19 in liver tissue. ConclusionThe Hedgehog signaling pathway inhibitor GDC-0449 can significantly inhibit the progression of liver fibrosis induced by CCl4/2-AAF in rats, possibly by inhibiting hepatic stellate cell activation, collagen deposition, activation and proliferation of hepatic progenitor cells, and differentiation of hepatic progenitor cells into biliary epithelial cells.

Backgroud and aims: Ductular reaction (DR) is a common pathological change and thought to have a key role in the pathogenesis and progression of liver fibrosis. Our previous study reported Gypenosides (GPs) ameliorated liver fibrosis, however, the anti-fibrotic mechanisms of GPs are still unclear. Methods: Liver fibrosis was induced in rats by carbon tetrachloride combining with 2-acerylaminofluorene (CCl 4 /2-AAF), and Mdr2 knockout ( Mdr2 −/− ) mice to evaluate the anti-fibrotic role of GPs. In vitro , WB-F344 cells, a hepatic progenitor cells (HPCs) line, with or without Gli1 overexpressing lentiviral vectors, were induced by sodium butyrate (SB) to validate the mechanism of GPs and NPLC0393, the main ingredient of GPs. Results: Both in CCl 4 /2-AAF-treated rats and Mdr2 −/− mice, GPs obviously reduced the deposition of collagen and hydroxyproline content, inhibited the activation of hepatic stellate cells and inflammatory cell infiltration. Notably, GPs reduced the expressions of Epcam, CK19, CK7, Dhh, Smo, Ptch2, Gli1 and Gli2. Furthermore, CK19 + cells co-expressed Gli1, while the number of CK19 + /Gli1 + cells was decreased by GPs. In vitro , GPs and NPLC0393 inhibited the differentiation of WB-F344 cells toward a biliary phenotype. Mechanistically, GPs and NPLC0393 protected against DR by inhibiting hedgehog signaling, which was supported by the results that DR, triggered directly by Gli1 overexpressing lentiviral vector was blocked by administration with GPs or NPLC0393. Conclusion: GPs attenuated DR and liver fibrosis by inhibiting hedgehog signaling, which provided more evidences and a novel mechanism of anti-fibrotic effect of GPs.

目的:观察不同剂量野百合碱(MCT)诱导大鼠肝窦阻塞综合征(HSOS)的病理变化,探讨造模剂量-时间致肝损伤的病理特征,并解析其部分作用机制.方法:将72只雄性SD大鼠随机分为正常组(n=12),MCT低、中、高剂量组(每组n=20).MCT低、中、高剂量组分别采用MCT 80、120、160 mg·kg-1灌胃一次制备模型,各剂量组大鼠于造模48、120 h处死取材.观测各组大鼠的存活率,检测大鼠体质量、肝质量与血清肝功能变化,采用扫描电镜、苏木素-伊红(HE)染色及天狼星红(SR)染色观察肝组织病理变化,微板法检测肝组织匀浆谷胱甘肽S转移酶(GST)、总超氧化物歧化酶(T-SOD)活性、丙二醛(MDA)含量,实时荧光定量聚合酶链式反应(Real-time PCR)、蛋白免疫印迹法(Western blot)和免疫组化检测肝组织相关指标的表达.结果:与正常组比较,MCT各组血清丙氨酸氨基转移酶/天冬氨酸氨基转移酶(ALT/AST)活性随造模剂量增加而升高(P＜0.05,P＜0.01);随造模时间增加,MCT低剂量组血清ALT和AST活性均下降(P＜0.01),MCT中、高剂量组ALT和AST活性均显著升高(P＜0.01).HE染色显示MCT低、中、高剂量组肝组织肝细胞坏死、炎性细胞浸润及红细胞淤积,电镜下可见肝窦内皮窗孔扩大,"筛状"结构消失,其损伤程度随剂量提高而加重.与正常组比较,MCT低、中、高剂量组肝窦内皮细胞标志物CD44表达减少(P＜0.05,P＜0.01).SR染色显示造模48 hMCT各组未见阳性染色,120 hMCT各组可见汇管区及肝窦胶原沉积.与正常组比较,MCT各剂量组肝组织MDA含量及GST活性增加,T-SOD活性降低,以MCT中、高剂量组变化显著(P＜0.01);且造模120 hMCT各组变化呈现剂量依赖性(P＜0.01).免疫组化及Western blot结果显示,与正常组比较,促炎巨噬细胞标志物CD68蛋白表达随剂量升高而增加(P＜0.01),抗炎巨噬细胞CD163蛋白及mRNA表达随剂量升高而减少(P＜0.01).与正常组比较,造模后MCT中、高剂量组磷酸化核转录因子-κB/核转录因子-κB(p-NF-κB/NF-κB)、磷酸化蛋白激酶B/蛋白激酶B(p-Akt/Akt)明显升高(P＜0.05,P＜0.01).MCT造模后肝组织α-平滑肌肌动蛋白(α-SMA)蛋白表达随时间延长、剂量升高增加,α-SMA、Ⅰ型胶原蛋白基因α1(Col 1a1)和Ⅳ型胶原蛋白基因α1(Col 4a1)mRNA表达也随时间延长、剂量升高而明显增加(P＜0.05,P＜0.01).TUNEL染色结果显示造模后凋亡细胞数量随造模剂量升高而增加,B细胞淋巴瘤-2(Bcl-2)蛋白与Bcl-2相关X蛋白(Bax)比值显著降低(P＜0.01).结论:不同剂量MCT灌胃诱导的大鼠肝窦阻塞综合征,病变随剂量增加而加重.80 mg·kg-1 MCT导致的肝脏损伤可自愈,超过120 mg·kg-1 的MCT导致的肝脏损伤会随着时间的延长而加重,甚至出现纤维化、死亡.MCT导致大鼠HSOS的病理机制可能是MCT引发肝组织内强烈的氧化应激,激活促炎巨噬细胞大量分泌炎症因子,进而激活NF-κB/Akt信号通路,导致严重的细胞损伤和死亡.

Frequent outbreaks of coronaviruses underscore the need for antivirals and vaccines that can counter a broad range of coronavirus types. We isolated a human antibody named 76E1 from a COVID-19 convalescent patient, and report that it has broad-range neutralizing activity against multiple α- and β-coronaviruses, including the SARS-CoV-2 variants. 76E1 also binds its epitope in peptides from γ- and δ-coronaviruses. 76E1 cross-protects against SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and therapeutic murine animal models. Structural and functional studies revealed that 76E1 targets a unique epitope within the spike protein that comprises the highly conserved S2’ site and the fusion peptide. The epitope that 76E1 binds is partially buried in the structure of the SARS-CoV-2 spike trimer in the prefusion state, but is exposed when the spike protein binds to ACE2. This observation suggests that 76E1 binds to the epitope at an intermediate state of the spike trimer during the transition from the prefusion to the postfusion state, thereby blocking membrane fusion and viral entry. We hope that the identification of this crucial epitope, which can be recognized by 76E1, will guide epitope-based design of next-generation pan-coronavirus vaccines and antivirals.

OBJECTIVE:To observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model, and the underlying mechanisms were partly dissected in vivo and in vitro.METHODS:Thirty-two male mice were randomly divided into 4 groups, including control, model, low- and high-dose amygdalin-treated groups, 8 mice in each group. Except the control group, mice in the other groups were injected intraperitoneally with 10% carbon tetrachloride (CCl4)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis. At the first 3 weeks, amygdalin (1.35 and 2.7 mg/kg body weight) were administered by gavage once a day. Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week. At the end of 6 weeks, liver tissue samples were harvested to detect the content of hydroxyproline (Hyp). Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue. The expressions of collagen I (Col-I), alpha-smooth muscle actin (α-SMA), CD31 and transforming growth factor β (TGF-β)/Smad signaling pathway were observed by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot, respectively. The activation models of hepatic stellate cells, JS-1 and LX-2 cells induced by TGF-β1 were used in vitro with or without different concentrations of amygdalin (0.1, 1, 10 µmol/L). LSECs. The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells (LSECs) dedifferentiation markers CD31 and CD44 were observed.RESULTS:High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area, and decreased the mRNA and protein expressions of Col-I, α-SMA, CD31 and p-Smad2/3 in liver tissues of mice compared to the model group (P<0.01). Amygdalin down-regulated the expressions of Col-I and α-SMA in JS-1 and LX-2 cells, and TGFβ R1, TGFβ R2 and p-Smad2/3 in LX-2 cells compared to the model group (P<0.05 or P<0.01). Moreover, 1 and 10 µmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group (P<0.05 or P<0.01).CONCLUSIONS:Amygdalin can dramatically alleviate liver fibrosis induced by CCl4 in mice and inhibit TGF-β/Smad signaling pathway, consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.