Here, we employed human embryonic stem cell spheroid (3D hESCs)-derived exosomes to assess inflammatory responses of macrophages in liver fibrosis, and found that 3D hESC-exosomes promoted the transformation of macrophages from M1 to M2 phenotype in vitro. The transplantation of 3D hESC-exosomes exhibited a notable decrease of pro-inflammatory factors, and significant increase of anti-inflammatory factors, and led to a reduction of CD86+M1 macrophages and an increase of Arg-1+M2 macrophages in the livers of treated fibrotic mice, restricting the advancement of liver fibrosis. hsa_circ_0076798, derived from 3D hESC-exosomes, acts as a molecular sponge that sequesters miR-1184, thereby mitigating miR-1184-mediated repression of DICER1 expression within macrophages. Further investigation revealed that DICER1 ultimately mitigated macrophage inflammation by deactivating TNF/NF-κB signaling pathway. Therefore, our findings demonstrated that 3D hESC-exosomes alleviated inflammatory responses by suppressing TNF/NF-κB pathway through hsa_circ_0076798/miR-1184/DICER1 axis, and offers insights into the targeted treatment of liver fibrosis via exosome-based cell-free therapy.

Ethnopharmacological relevance: The Chinese herbal medicine Shugan Jianpi Formula (SGJPF) has traditionally been used to treat various chronic liver disorders. Previous studies have indicated that SGJPF inhibits hepatic stellate cells (HSCs) activation in rats with liver fibrosis (LF) and that miR-193a-3p may be a crucial molecule in LF. However, the mechanisms by which SGJPF regulates HSCs activation through miR-193a-3p remain unclear. Aim of the study: This study aimed to determine whether the effect of SGJPF on LF is related to its regulation of miR-193a-3p and TGF-(32, both in a carbon tetrachloride (CCl4)-induced LF mouse model and in TGF-(31-induced JS-1 cells. Materials and methods: A CCl4-induced LF mouse model was established to evaluate the anti-fibrotic efficacy of SGJPF by examining liver histopathological changes, collagen deposition, and the expression of a-smooth muscle actin (a-SMA) and collagen-I. To investigate the role of miR-193a-3p in HSCs activation, miR-193a-3p mimics and inhibitors were transfected into TGF-(31-induced JS-1 cells. The potential targets of miR-193a-3p were identified using miRDB, TargetScan 8.0, RNA-seq, and dual-luciferase reporter assays. Finally, the effects of SGJPF on HSCs activation and the miR-193a-3p/TGF-(32 axis were assessed in TGF-(31-treated JS-1 cells using CCK-8, EDU, scratch, RT-qPCR, and Western blotting assays. Results: SGJPF significantly reduced liver damage and fibrosis, inhibited HSCs activation, decreased TGF-(32 levels, and increased miR-193a-3p expression in CCl4-induced LF tissue. Additionally, miR-193a-3p was upregulated in HSCs transfected with miR-193a-3p mimics and downregulated in those with miR-193a-3p inhibitors. High levels of miR-193a-3p, combined with miRNA mimics, inhibited HSCs activation, proliferation, and migration. TGF-(32, a target negatively regulated by miR-193a-3p, partially reversed the effects of miR-193a-3p on TGF-(31-induced HSCs activation. SGJPF also reduced HSCs activation, proliferation, and migration in TGF-(31- treated JS-1 cells. Moreover, treatment with SGJPF-containing serum and miR-193a-3p inhibition restored HSCs activation, proliferation, and migration in TGF-(31-induced JS-1 cells. Conclusions: This study demonstrates that SGJPF ameliorates CCl4-induced liver fibrosis, which is associated with the regulation of miR-193a-3p and TGF-(32 in HSCs. These findings provide a new pharmacological basis for SGJPF and suggest a novel strategy for treating LF through TCM by regulating miRNAs.

The hepatic copper accumulation characteristic of Wilson's disease (WD) leads to activation of hepatic stellate cells, extracellular matrix deposition, and the progression to liver fibrosis (LF), with specific therapeutic interventions for this condition notably deficient.Increasing evidence suggests the potential utility of GanDouLing (GDL) in addressing WD-related LF.In this study, the pharmacological effects of GDL on WD and LF were assessed by establishing a copper-induced human hepatic stellate cell (LX-2) model and elucidating potential mechanisms underlying GDL treatment.The findings indicate that GDL treatment significantly attenuates the viability of copper-induced LX-2 cells and reduces the expression of fibrotic markers.This inhibitory effect is mediated through the PAM pathway, as it can be reversed by activators such as IGF-1 or MHY1485.Furthermore, GDL treatment downregulates critical components of the PAM pathway at both mRNA and protein levels.Molecular docking simulations further confirm the strong affinity and stable binding between active constituents of GDL, such as luteolin and vestitol, and their target proteins (AKT1, PIK3CA, mTOR).In copper-induced LX-2 cells, GDL treatment upregulates autophagic indicators, including LC3, Beclin-1, and AO, while downregulating p62.TEM experiments reveal that GDL treatment increases the presence of autophagosomes in copper-induced LX-2 cells, thereby ameliorating mitochondrial damage.In conclusion, GDL exerts its inhibitory effects on copper-induced LX-2 cell activation by downregulating the PAM pathway.These findings provide a novel approach for addressing LF related to WD and support the utilization of herbal-based supplements and alternative therapies.

Background: Liver fibrosis (LF) is a kind of progressive liver injury reaction. The goal of this study was to achieve a more detailed understanding of the molecular changes in response to CCl4-induced LF through the identification of a differentially expressed liver transcriptomic and proteomic.Results: A total of 1224 differentially expressed genes (DEGs) and 302 differentially expressed proteins (DEPs) were significantly identified at the transcriptomic and proteomic level, respectively, and 69 genes (hereafter "cor-DEGs-DEPs" genes) were detected at both levels. Pathway enrichment analysis showed that these cor-DEGs-DEPs genes were significantly enriched in 133 pathways. Importantly, among the cor-DEGs-DEPs genes, Gstm1, Gstm3, Ephx1 and Gstp1 were shown to be associated with metabolic pathways, and confirmed by RT-qPCR and parallel reaction monitoring (PRM) verification.Conclusions: Through the combined analysis of transcriptomic and proteomic data, this study provides valuable insights into the potential mechanism of the pathogenesis of LF, and lays a theoretical foundation for the further development of targeted therapy for LF.

Liver fibrosis (LF), commonly associated with chronic liver diseases, is a major public health problem worldwide. Protein phosphorylation is not only an important form of protein modification in organisms but also the most important mechanism to regulate and control the activity and function of proteins, affecting the occurrence and development of many diseases. However, comprehensive phosphoproteomic profiling in LF has not been fully elucidated. In this study, data-independent acquisition (DIA) was used to analyse the phosphoproteomics of mice with LF. A total of 553 phosphopeptides (representing 440 phosphoproteins) had significant phosphorylation levels. Among these phosphoproteins, 49 were upregulated and 401 were downregulated, and 5 phosphoserine (P-Ser) motifs and 2 phosphothreonine (P-Thr) motifs were conserved in LF. GO and KEGG pathway enrichment analyses identified 769 significant GO terms and 49 significant KEGG pathways. Four phosphorylated proteins were selected for parallel reaction monitoring (PRM) verification, and the results were consistent with DIA data. Together, there were significantly different phosphoproteomic profiles in LF, suggesting that protein phosphorylation was related to the occurrence and progression of LF, which could pave the way for further investigation into the related regulatory mechanisms. SIGNIFICANCE: LF is a necessary stage in the development of chronic liver disease to liver cirrhosis and has attracted wide attention. To the best of our knowledge, there are few reports on the phosphorylated proteomics of LF. In this study, DIA and PRM techniques were used to study the liver tissue of mice induced by CCl4. The results showed that phosphorylation had a significant effect on the activity and function of proteins, and the PRM results were consistent with the trend observed in DIA analysis. This study will help to better reveal the relationship of phosphorylated proteins in LF and lay a foundation for further study of related regulatory mechanisms.

The Jiedu Huazhuo Quyu formula (JHQ) shows significant beneficial effects against liver fibrosis caused by Wilson's disease (WD). Hence, this study aimed to clarify the mechanisms of the JHQ treatment in WD-associated liver fibrosis. First, we collected 103 active compounds and 527 related targets of JHQ and 1187 targets related to WD-associated liver fibrosis from multiple databases. Next, 113 overlapping genes (OGEs) were obtained. Then, we built a protein-protein interaction (PPI) network with Cytoscape 3.7.2 software and performed the Gene Ontology (GO) term and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analyses with GENE DENOVO online sites. Furthermore, module analysis was performed, and the core target genes in the JHQ treatment of WD-associated liver fibrosis were obtained. Pathway and functional enrichment analyses, molecular docking studies, molecular dynamic (MD) simulation, and Western blot (WB) were then performed. The results indicated that 8 key active compounds including quercetin, luteolin, and obacunone in JHQ might affect the 6 core proteins including CXCL8, MAPK1, and AKT1 and 107 related signaling pathways including EGFR tyrosine kinase inhibitor resistance, Kaposi sarcoma-associated herpesvirus infection, and human cytomegalovirus infection signaling pathways to exhibit curative effects on WD-associated liver fibrosis. Mechanistically, JHQ might inhibit liver inflammatory processes and vascular hyperplasia, regulate the cell cycle, and suppress both the activation and proliferation of hepatic stellate cells (HSCs). This study provides novel insights for researchers to systematically explore the mechanism of JHQ in treating WD-associated liver fibrosis.

目的:基于TLR4/MyD88/NLRP3信号轴探究疏肝健脾方对肝纤维化小鼠的治疗作用及机制.方法:采用四氯化碳(CCl 4)与橄榄油混合液背部皮下注射的方法,复制化学性肝纤维化小鼠模型.造模首日,灌胃给予疏肝健脾方,每天1次,连续12周;HE、Masson和天狼猩红染色观察小鼠肝组织病理学损伤程度,透射电镜观察肝组织超微结构的变化及焦亡小体情况;RT-qPCR、Western blot和免疫组织化学染色检测肝组织中α-SMA、CollagenⅠ、Caspase-1、IL-1β、IL-18以及TLR4/MyD88/NLRP3信号轴表达的变化情况.结果:病理分析显示模型组小鼠肝脏肝小叶结构模糊,肝细胞索排列紊乱,胶原沉积增加,焦亡小体数量明显增加,肝组织中α-SMA、CollagenⅠ、Caspase-1、IL-1β、IL-18以及TLR4、MyD88和NLRP3的mRNA和蛋白表达水平相较于正常组肝脏均显著升高.与模型组相比,疏肝健脾方给药后可改善肝脏病理组织学损伤程度,抑制细胞焦亡,显著下调α-SMA、CollagenⅠ、Caspase-1、IL-1β、IL-18以及TLR4、MyD88和NLRP3 mRNA与蛋白表达水平.结论:疏肝健脾方抗肝纤维化作用可能与调控TLR4/MyD88/NLRP3信号轴,减少细胞焦亡,抑制肝星状细胞活化有关.

N6-Methyladenosine (m6A), a unique and common mRNA modification method in eukaryotes, is involved in the occurrence and development of many diseases. Liver fibrosis (LF) is a common response to chronic liver injury and may lead to cirrhosis and even liver cancer. However, the involvement of m6A methylation in the development of LF is still unknown. In this study, we performed a systematic evaluation of hepatic genome-wide m6A modification and mRNA expression by m6A-seq and RNA-seq using LF mice. There were 3,315 genes with significant differential m6A levels, of which 2,498 were hypermethylated and 817 hypomethylated. GO and KEGG analyses illustrated that differentially expressed m6A genes were closely correlated with processes such as the endoplasmic reticulum stress response, PPAR signaling pathway and TGF-β signaling pathway. Moreover, a total of 90 genes had both a significant change in the m6A level and mRNA expression shown by joint analysis of m6A-seq and RNA-seq. Hence, the critical elements of m6A modification, including methyltransferase WTAP, demethylases ALKBH5 and binding proteins YTHDF1 were confirmed by RT-qPCR and Western blot. In an additional cell experiment, we also observed that the decreased expression of WTAP induced the development of LF as a result of promoting hepatic stellate cell (HSC) activation. Therefore, this study revealed unique differential m6A methylation patterns in LF mice and suggested that m6A methylation was associated with the occurrence and course of LF to some extent.

N6-甲基腺嘌呤(m6A)是存在于多种RNAs中的化学修饰方式,最常见于mRNA.肝脏是机体重要的代谢和消化器官,m6 A甲基化在肝脏生理病理过程中发挥着重要作用.简述了m6 A甲基化在肝脏生理和病毒性肝炎、非酒精性脂肪性肝病、肝纤维化以及肝细胞癌等肝脏疾病中的生物学作用及潜在应用价值,指出m6 A甲基化可调控相关因子,参与肝脏疾病的发生发展,为其临床诊疗提供新的思路和靶点.

Rheumatoid arthritis (RA) is a common autoimmune disease and characterized by chronic inflammation, abnormal synovial cell proliferation, and joint swelling and tenderness, and it causes patients substantial pain. To date, the pathogenesis of RA remains unclear, and specific treatment is still lacking in the clinic. Evidence from previous research indicated that the long noncoding RNA (lncRNA) LOC100912373 is a key lncRNA and involved in RA. However, our understanding of the specific mechanism of lncRNA LOC100912373 in RA development and progression is still in its infancy. In this study, fibroblast-like synoviocytes (FLSs) were cultured by enzyme-dispersed and substrate-attached explant methods. The MTT method, flow cytometry and transmission electron microscopy were used to determine the effect of lncRNA LOC100912373 on FLSs. The expression of key genes such as lncRNA LOC100912373, miR-17-5p, PDK1 and AKT in FLSs was detected by RT-qPCR, immunofluorescence and Western blot. The localization of lncRNA LOC100912373 was determined by fluorescence in situ hybridization. The specific targeting relationship between lncRNA LOC100912373 and miR-17-5p/PDK1 was verified by RNA immunoprecipitation and luciferase reporter gene analysis. The results showed that lncRNA LOC100912373 localized in the cytoplasm and was highly expressed in the synovial tissues and FLSs of AA rats. LncRNA LOC100912373 overexpression promoted the proliferation of FLSs. In addition, lncRNA LOC100912373 could bind to miR-17-5p, and the expression of lncRNA LOC100912373 was negatively correlated with miR-17-5p and positively correlated with PDK1/AKT. In conclusion, lncRNA LOC100912373 may upregulate the expression of PDK1 by sponging miR-17-5p, accelerating the phosphorylation of AKT and inducing the proliferation of FLSs, thus promoting the occurrence and development of RA.