Rat Model Of Hepatic Fibrosis Research Articles

Retraction: Oxymatrine exerts anti-fibrotic effects in a rat model of hepatic fibrosis by suppressing endoplasmic reticulum stress.

Retraction: Oxymatrine exerts anti-fibrotic effects in a rat model of hepatic fibrosis by suppressing endoplasmic reticulum stress.

Ameliorative effects of sildenafil against carbon tetrachloride induced hepatic fibrosis in rat model through downregulation of osteopontin gene expression

The liver carries out many essential tasks, such as synthesising cholesterol, controlling the body’s storage of glycogen, and detoxifying metabolites, in addition to performing, and regulating homeostasis. Hepatic fibrosis is a pathological state characterized by over accumulation of extracellular matrix (ECM) including collagen fibers. Sildenafil (a selective inhibitor of type 5 phosphodiesterase) has anti-inflammatory, antioxidant and anti-apoptotic properties. It is commonly used to treat erectile dysfunction in male. The purpose of the current investigation was to evaluate sildenafil’s hepatoprotective potential against liver fibrosis in rats that was caused by carbon tetrachloride (CCl4). Liver enzymes and oxidative markers as well as profibrotic genes were determined. The findings showed that sildenafil alleviates the hepatic dysfunctions caused by CCl4 by restoring normal levels of ALT, AST, and GGT as well as by restoring the antioxidant status demonstrated by increased glutathione (GSH), and catalase. In addition, a significantly down-regulated the mRNA expressions of profibrotic genes [collagen-1α, IL-1β, osteopontin (OPN), and transforming growth factor-β (TGF-β)]. Additionally, sildenafil lessens the periportal fibrosis between hepatic lobules, congestion and dilatation in the central vein, and the inflammatory cell infiltrations. As a result, it is hypothesized that sildenafil may be helpful in the management of hepatotoxicity brought on by CCl4 through suppressing OPN.

Transcriptomic analysis reveals pharmacological mechanisms mediating efficacy of Yangyinghuoxue Decoction in CCl4-induced hepatic fibrosis in rats.

As a traditional Chinese medicine formula, Yangyinghuoxue Decoction (YYHXD) is used clinically for therapy of hepatic fibrosis. The pharmacological profile of YYHXD comprises multiple components acting on many targets and pathways, but the pharmacological mechanisms underlying its efficacy have not been thoroughly elucidated. This study aimed at probing the pharmacological mechanisms of YYHXD in the treatment of hepatic fibrosis. YYHXD aqueous extract was prepared and quality control using HPLC-MS fingerprint analysis was performed. A CCl4-induced rat model of hepatic fibrosis was established, and animals were randomly assigned to six groups: control, low-dose YYHXD (L-YYHXD), medium-dose YYHXD (M-YYHXD), high-dose YYHXD (H-YYHXD), CCl4 model, and colchicine group. Rats in the treatment groups received daily oral administration of YYHXD (5, 10, or 20g/kg) or colchicine (0.2mg/kg) for 6weeks, while the control and model groups received distilled water. Histological analysis, including hematoxylin and eosin (HE) and Masson's trichrome staining, was performed to evaluate hepatic fibrosis. Serum biochemical markers, such as AST, ALT, HA, and LN, were measured. Inflammatory cytokines (IL-6 and TNF-α) and oxidative stress indicators (SOD, GSH-Px, and MDA) in hepatic tissue were also assessed. Additionally, transcriptomic analysis using RNA-sequencing was conducted to identify differentially expressed genes (DEGs) between the control, CCl4 model, and H-YYHXD groups. Bioinformatics analysis, including differential expression analysis, protein-protein interaction analysis, and functional enrichment analysis, were performed to probe the pharmacological mechanisms of YYHXD. The regulatory effects of YYHXD on fatty acid metabolism and biosynthesis were further confirmed by Oil Red O staining, enzyme activity assays, qPCR, and Western blotting. Western blotting and immunofluorescence staining also validated the involvement of the AMPK signaling pathway in the occurrence and progression of hepatic fibrosis. HE and Masson's trichrome staining revealed reduced collagen deposition and improved liver architecture in YYHXD groups compared to the CCl4 model group. Serum biochemical markers, including AST, ALT, HA, and LN, were significantly improved in the YYHXD-treated groups compared to the CCl4 model group. The levels of inflammatory cytokines (IL-6 and TNF-α) and oxidative stress indicators (decreased SOD and GSH-Px, increased MDA) in hepatic tissue were significantly ameliorated by YYHXD treatment compared to the CCl4 model group. Moreover, 96 genes implicated in YYHXD therapy of hepatic fibrosis were screened from the transcriptomic data, which were principally enriched in biological pathways such as fatty acid metabolism and biosynthesis, and the AMPK signaling pathway. Oil Red O staining showed reduced hepatic lipid accumulation by YYHXD in a dose-dependent manner, along with decreased serum TG, TC, and LDL-C levels. Additionally, qPCR and Western blot analyses demonstrated upregulated mRNA and protein expression of key enzymes involved in fatty acid metabolism and biosynthesis, Fasn and Fads2, modulated by YYHXD. YYHXD also dose-dependently enhanced phosphorylation of AMPK as evidenced by Western blotting and immunofluorescence assays. YYHXD ameliorated CCl4-induced hepatic fibrosis in rats through pharmacological mechanisms that involved manifold targets and pathways, including aliphatic acid synthesis and metabolism pathways and the AMPK signaling pathway. This study provided a reference and basis for further research and clinical utilization of YYHXD.

Mechanism of Si Ni San Combined with Astragalus in Treating Hepatic Fibrosis: A Network Pharmacology and Molecular Docking Study.

Si Ni San combined with Astragalus (SNSQ) has demonstrated significant efficacy in the treatment of hepatic fibrosis (HF), as confirmed by clinical practice. However, its pharmacological mechanism remains unclear. This study employs network pharmacology to identify key targets and proteins for molecular docking. Additionally, animal experiments were conducted to validate the network pharmacology results, providing further insights into the mechanism of SNSQ in treating HF. Effective compounds of SNSQ were screened from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and Encyclopedia of Traditional Chinese Medicine (ETCM) databases. Molecular formula structures of these effective compounds were obtained from the PubChem database. Partial target proteins with a probability greater than 0.6 were sourced from the SWISS database. Uniprot IDs corresponding to these target proteins were retrieved from the SUPERPRED database. The remaining target proteins of the compounds were obtained from the Uniprot database based on the Uniprot IDs. The drug target proteins were then summarized. Target points related to HF were selected from the GeneCards and OMIM databases. Common target points were identified in the Venn diagram and imported into Cytoscape 3.9.1 software to construct the "SNSQ-effective compound-target pathway-HF" network. AutoDock software was used for molecular docking of compounds and target proteins with high-degree values. The common target points underwent GO function enrichment and KEGG pathway enrichment analysis using the DAVID database. An HF rat model was established, and serum AST and ALT activities were measured. The Hyp assay kit was utilized to detect the Hyp content in liver tissue. To the transcription levels of pro-inflammatory factors (IL-1β, TNF-α, IL-6) and anti-inflammatory factors (IL-10, TGF-β1, IL-4) in rat serum and liver.IL-1β, TNF-α, IL-10, and TGF-β1 were chosen for validation through ELISA. Western blotting and qRT-PCR were used to assess the expression of related proteins, namely NFKB1, NF-κBp65, NF-κBp50, α-SMA, and Col-1 in liver tissue. qRT-PCR was also employed to study the expression of ECM synthesis and proliferation-related genes, including Cyclin D1, TIMP1, COL1A1 in HSC-T6 cells and rat liver tissue, as well as the inhibition of the ECM-related gene MMP13 in HSC-T6 cells and rat liver tissue. A total of 16 valid compounds were predicted, with kaempferol, sitosterol, and isorhamnetin exhibiting high-degree values. KEGG enrichment analysis revealed that the target genes of SNSQ were enriched in multiple pathological pathways, with the NF-Kappa B signaling pathway being predominant. Molecular docking simulations indicated strong affinities between SNSQ's primary components-kaempferol, sitosterol, isorhamnetin-and NFKB1. Experimental results demonstrated significant reductions in AST, ALT, and Hyp levels in the SNSQ group. Pro-inflammatory factors (IL-1β, TNF-ɑ) were markedly reduced, while anti-inflammatory factors (IL-10, TGF-β1) were substantially increased. The protein expression and transcription levels of α-SMA and Col-1 were significantly decreased, whereas those of NFKB1, NF-κBp65, and NF-κBp50 were notably elevated. mRNA expression levels of Cyclin D1, TIMP1, COL1A1 in HSC-T6 cells and rat liver tissue were significantly decreased, whereas MMP13 mRNA expression level was significantly increased. Treatment of HF with SNSQ involves multiple targets and pathways, with a close association with the overexpression of NFKB1 and activation of the NF-Kappa B signaling pathway. Its mechanism is closely linked to the activation of inflammatory responses, HSC activation, and proliferation.

Lipopolysaccharide modification enhances the inhibitory effect of clodronate liposomes on hepatic fibrosis by depletion of macrophages and hepatic stellate cells

Hepatic fibrosis is a complex chronic liver disease in which both macrophages and hepatic stellate cells (HSCs) play important roles. Many studies have shown that clodronate liposomes (CLD-lipos) effectively deplete macrophages. However, no liposomes have been developed that target both HSCs and macrophages. This study aimed to evaluate the therapeutic efficacy of lipopolysaccharide-coupled clodronate liposomes (LPS-CLD-lipos) and the effects of liposomes size on hepatic fibrosis. Three rat models of hepatic fibrosis were established in vivo; diethylnitrosamine (DEN), bile duct ligation (BDL), and carbon tetrachloride (CCl4). Hematoxylin and eosin staining and serological liver function indices were used to analyze pathological liver damage. Masson's trichrome and Sirius red staining were used to evaluate the effect of liposomes on liver collagen fibers. The hydroxyproline content in liver tissues was determined. In vitro cell counting kit-8 (CCK-8) and immunofluorescence assays were used to further explore the effects of LPS modification and liposomes size on the killing of macrophages and HSCs. Both in vitro and in vivo experiments showed that 200 nm LPS-CLD-lipos significantly inhibited hepatic fibrosis and the abnormal deposition of collagen fibers in the liver and improved the related indicators of liver function. Further results showed that 200 nm LPS-CLD-lipos increased the clearance of macrophages and induced apoptosis of hepatic stellate cells, significantly. The present study demonstrated that 200 nm LPS-CLD-lipos could significantly inhibit hepatic fibrosis and improve liver function-related indices and this study may provide novel ideas and directions for hepatic fibrosis treatment.

Resveratrol treatment ameliorates hepatic damage via the TGF-β/SMAD signaling pathway in a phenobarbital/CCl4-induced hepatic fibrosis model.

Liver fibrosis is a wound healing response characterized by excessive accumulation of extracellular matrix proteins. This study aimed to investigate the effects of resveratrol treatment on the TGF-β/SMAD signaling pathway and related biochemical parameters, apoptosis, and liver regeneration phenobarbital-CCl4 induced hepatic fibrosis rat model. This model was created through phenobarbital and CCl4 (0.2-0.35 ml/kg). Resveratrol (1 mg/kg/day) was administered to the fibrosis and control groups. Immunohistochemical staining was performed to evaluate αSMA, TGF-β1, and PCNA in liver tissue. The TUNEL method and Masson's Trichome staining were used to determine apoptosis and collagen accumulation. AST, ALP, ALT, total protein, and total bilirubin levels were measured to determine biochemical status. SMAD2, SMAD3, SMAD4, and SMAD7 expression levels were measured to determine TGF-β1 related hepatic fibrosis. The SMAD2, SMAD3, and SMAD4 mRNA expression levels were increased and the SMAD7 mRNA expression level was decreased in the fibrosis control group. The SMAD7 mRNA expression level was higher in the phenobarbital-CCl4 induced resveratrol treated group. Increased biochemical parameters indicating hepatic damage, increased number of apoptotic cells, and collagen accumulation surrounding the central vein were observed in the fibrosis group compared with the other groups. It was concluded that administration of resveratrol ameliorates the adverse effects of hepatic fibrosis by regulating biochemical parameters, controlling TGF-β1/SMAD signaling, enhancing tissue regeneration, and reducing apoptosis in liver cells. Resveratrol can be a beneficial option for the prevention of liver damage in a phenobarbital-CCl4 induced hepatic fibrosis.

Dahuang zhechong pill ameliorates hepatic fibrosis by regulating gut microbiota and metabolites

Ethnopharmacological relevanceDHZCP is a traditional Chinese medicinal formula in “The Synopsis of Prescriptions of the Golden Chamber” that has been often used in the treatment of hepatic disorders, gynecopathy and atherosclerosis. However, its underlying mechanisms in preventing hepatic fibrosis remain incompletely understood. Aim of the studyThis study aims to explore the therapeutic efficacy and potential mechanism of DHZCP in a CCL4-induced experimental hepatic fibrosis rat model. Materials and methodsDHZCP was orally administered at doses of 0.168, 0.084 and 0.042 g⋅kg-1⋅d-1 in a CCL4-induced hepatic fibrosis model using SD rats. Histopathology, immunohistochemistry and biochemical analysis, ELISA, Flow cytometry, WB, RT-PCR, 16 S rRNA, and untargeted metabolomic analysis were used to determine the therapeutic effects and mechanisms of DHZCP in the treatment of CCL4-induced hepatic fibrosis. ResultsPharmacodynamically, DHZCP inhibited ALT and AST, improved liver function, decreased NF-κB, TNF-α and IL-6 in liver tissue, indicating its role in inhibiting CCL4-induced liver inflammation. Most importantly, it reduces the level of fibrosis in serum and liver tissue. Histological analysis also showed that DHZCP could effectively inhibit inflammatory cytokine infiltration and excessive collagen deposition. Mechanistically, DHZCP regulates gut microbiota, improves the proportion of firmicutes and bacteroidota at the phylum level, and increases the abundance of beneficial bacteria at the genus level, such as muribagulaceae unclassified, prevotella, alloprevotella, closteriales unclassified, lachnospiraceae unclassified and phascolarctobacterium. Instead, it reduced the abundance of two harmful bacteria, desulfovibrio and colidextribacter. Four types of metabolites such as hydrocarbons, organic nitrogen compounds, organic oxygen compounds, and organosulfur compounds were added. Furthermore, DHZCP was found to reduce the damage of intestinal barrier caused by changes in gut microbiota and metabolites. ConclusionDHZCP is an effective inhibitor of hepatic fibrosis by regulating gut microbiota and metabolites, improving the integrity of the intestinal barrier.

Mechanism of the anti-liver fibrosis effect of Periplaneta americana extracts that promote apoptosis of HSC-T6 cells through the Bcl-2/Bax signaling pathway

Periplaneta americana L. (Blattidae) extract (PA-B) possesses anti-oxidant, anti-inflammatory, and hepatoprotective properties; however, its mechanism of action in liver fibrosis remains unclear. Herein, we investigated the hepatoprotective effect and mechanism of action of PA-B. HSC-T6 cells treated with PA-B were stimulated with TGF-β1, and immunofluorescence, western blotting, Hoechst 33342 staining, confocal laser scanning microscopy, and flow cytometry were performed. Furthermore, the effects of PA-B were verified in a rat model of hepatic fibrosis established using 40% carbon tetrachloride (CCl4). PA-B induced apoptosis of HSC-T6 cells by inhibiting proliferation (maximum inhibition ratio 99.08%), reducing mitochondrial membrane potential, and ameliorating apoptosis-related proteins (P < 0.01). PA-B (120 mg/kg) significantly alleviated CCl4-induced hepatic histopathological fibrosis and reduced the levels of aspartate aminotransferase (from 27.68 to 20.58 U/L) and alanine aminotransferase (from 16.88 to 5.93 U/L; P < 0.05 or P < 0.01). Immunohistochemistry and western blotting showed that α-SMA levels were reduced (from 0.49 to 0.23) and that the Bcl/Bax signaling pathway was significantly inhibited by PA-B (120 mg/kg; P < 0.05 or P < 0.01). PA-B exhibits anti-fibrotic effects by mediating the Bcl-2/Bax pathway and promoting HSC-T6 cell apoptosis, providing a basis for its development as a drug to treat liver fibrosis.

Revealing immune infiltrate characteristics and potential immune-related genes in hepatic fibrosis: based on bioinformatics, transcriptomics and q-PCR experiments.

The occurrence and progression of hepatic fibrosis (HF) is accompanied by inflammatory damage. Immune genes play a pivotal role in fibrogenesis and inflammatory damage in HF by regulating immune cell infiltration. However, the immune mechanisms of HF are inadequately studied. Therefore, this research aims to identify the immune genes and biological pathway which involved in fibrosis formation and inflammatory damage in HF and explore immune target-based therapeutics for HF. The expression dataset GSE84044 of HF was downloaded from the GEO database. The crucial module genes for HF were screened according to weighted gene co-expression network analysis (WGCNA). The crucial module genes were mapped to immune-related genes obtained from the ImmPort database to obtain the hepatic fibrosis immune genes (HFIGs). In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed on HFIGs. Then, the protein-protein interaction (PPI) network was conducted on HFIGs and hub genes were identified from the PPI network. Moreover, immune infiltration analysis was performed to identified correlation between hub gene and immune cell infiltration. To verify the reliability of the GSE84044 expression profile data analysis, a rat model of CCl4-induced HF was established, followed by transcriptome sequencing and immunofluorescence analysis and quantitative reverse transcription (q-PCR) experiments were performed in HF rats and normal rat liver tissues. Finally, CMAP platform was used to explore immune target-based therapeutics for HF. In the bioinformatics analysis of GSE84044 data, 98 HFIGs were screened. These genes were mainly involved in inflammation-related biological pathways such as NOD-like receptor signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway and PI3K-Akt signaling pathway. From the PPI network, 10 hub genes were identified, including CXCL8, IL18, CXCL10, CD8A, IL7, PTPRC, CCL5, IL7R, CXCL9 and CCL2. Immune infiltration analysis showed that immune cells like neutrophils, natural killer (NK) cells, macrophages M1 and macrophages M2 were significantly correlated with the hepatic fibrosis process and hub gene expression was significantly correlated with these immune cells. Notably, most of the biological pathways HFIGs riched and all the hub gene expression except CXCL8 were validated in subsequent transcriptome and qRCR experiments. Finally, 15 small molecule compounds with the potential to reverse the high expression of hub genes were screen out as potential therapeutic agents for HF. The immune genes CXCL8, IL18, CXCL10, CD8A, IL7, PTPRC, CCL5, IL7R, CXCL9 and CCL2 may play an essential role in the fibrosis formation and inflammatory damage in HF. The outcomes of this research provide a basis for the study of the immune mechanisms of HF and contribute to the diagnosis and prevention and treatment of HF in clinical practice.

Vitamin D Ameliorates the Hepatic Oxidative Damage and Fibrotic Effect Caused by Thioacetamide in Rats

Vitamin D3 (VD3) is a sunshine hormone that regulates cellular proliferation, differentiation, apoptosis, and angiogenesis related to liver parenchyma. We used a thioacetamide (TAA)-induced hepatic fibrosis rat model in our study to investigate the beneficial roles of VD3 to overcome extensive liver fibrosis. Randomly, four equal groups (eight rats per group) underwent therapy for eight successive weeks: a control group, a group treated with TAA 100 mg/kg BW IP every other day, a group treated with VD3 1000 IU/kg BW IM every day, and a TAA+VD group treated with both therapies. Treatment with VD3 after TAA-induced hepatic fibrosis was found to alleviate elevated liver function measures by decreasing ALT, AST, and ALP activity; decreasing total bilirubin, direct bilirubin, cholesterol, and triglyceride levels; and increasing glucose and 25[OH]D3. Rats treated with VD3 showed marked decreases in MDA and increased SOD, CAT, and GSH levels. In addition, CD34 and FGF23 gene expressions were reduced after dual therapy. Liver sections from the TAA+VD group showed markedly decreased hepatic lesions, and Masson's trichrome stain showed a marked decrease in dense bluish-stained fibrous tissue. The immunohistochemical expression of TGF-β and α-SMA showed markedly decreased positive brown cytoplasmic expression in a few hepatocytes, clarifying the antifibrotic effect of VD3 in hepatic fibrosis. In conclusion, VD3 alleviates hepatotoxicity and fibrosis caused by TAA.

CaM/CaMKII mediates activation and proliferation of hepatic stellate cells regulated by ASIC1a.

The activation of hepatic stellate cells (HSCs) is closely related to hepatic fibrosis and plays a key role in its occurrence and development. In the damaged liver, inhibition of the activation, proliferation, and clearance of HSCs is an important therapeutic strategy. However, the mechanism underlying the activation of HSCs is not completely clear. Acid-sensitive ion channel 1a (ASIC1a) is a cation channel activated by extracellular acid, which is responsible for the transport of Ca2+ and Na+ and participates in the activation of HSCs and the occurrence and development of many inflammatory diseases, suggesting that ASIC1a plays an important role in liver fibrosis. A previous study by the project team found that when the membrane channel protein ASIC1a was opened, intracellular Ca2+ levels increased, the expression of CaM/CaMKII in HSCs was high, and HSC was activated and proliferated. Therefore, we established an SD rat model of hepatic fibrosis and induced HSC-T6 activation by stimulating ASIC1a with acid in vitro. In vivo, CCl4 was used to induce liver fibrosis in rats, and different doses of KN93 (0.5, 1, and 2 mg/kg/d) and colchicine (0.1 mg/kg/d) were administered. Eight weeks later, the activities of ALT and AST in serum were measured and hematoxylin-eosin and Masson staining in liver tissue, and immunohistochemistry analysis were performed in SD rats. The expressions of ASIC1a, α-SMA, Collagen-1, CaM, and CaMKII were detected. In vitro, we activated HSC-T6 cells by stimulating ASIC1a with acid. The results showed that inhibition of ASIC1a could improve acid-induced HSCs activation. In addition, CaM/CaMKII was expressed in HSC of rats with hepatic fibrosis regulated by ASIC1a. After blocking or silencing the expression of CaMKII, the fibrosis marker protein can be down-regulated. KN93 also reduced inflammation and improved the activation, proliferation and fibrosis of HSC. In summary, we concluded that CaM/CaMKII participates in ASIC1a regulation of the proliferation and activation of HSC and promotes the occurrence of liver fibrosis.

Mechanism of Allicin for Improvement of Hepatic Fibrosis by Regulating Autophagy

Abstract: Objectives: To explore the effects of allicin on the expression of mitochondrial autophagyrelated proteins in liver tissues of hepatic fibrosis (HF) rats. Materials and Methods: 45 SD rats were randomly divided into Normal, Model, Low, Middle and High groups. The HF rat model was replicated intraperitoneal injection of carbon tetrachloride and ethanol gavage for 4 weeks. Detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in rat serum; HE and Masson staining to observe pathological changes; Evaluating LC 3B protein expression by Immunofluorescence; Western Blotting and qRT-PCR methods were used to detect PINK1, Parkin, Beclin 1, and LC3 gene and protein expression levels in rat liver tissues. Results: Compared with the Normal group, the degree of HF in the model group was obvious, the volume fraction of collagen increased significantly (p<0.001), the LC 3B protein expression significantly upregulation (p<0.001), and the rat serum ALT and AST increased significantly (p<0.001, respectively). Up-regulation of PINK1, Parkin, Beclin 1, and LC 3B mRNA and protein expression and LC 3II/LC 3I ratio in liver tissues (p<0.001, respectively); Compared with the model group, Allicin treated groups had reduced HF degree, collagen volume fraction decreased significantly (p<0.05, respectively), LC 3B protein was significantly reduced (p<0.05, respectively), and liver tissue PINK1, Parkin, Beclin 1, and LC3B mRNA and protein expressions, and LC 3II/LC 3I ratio were down-regulated (p<0.05, respectively). Conclusion:Allicin could improve hepatic fibrosis. The mechanism might be related to down-regulating the expression of PINK1, Parkin, Beclin 1, and LC 3 gene and proteins and interfering with the level of mitochondrial autophagy Keywords: Allicin, HF, PINK1, Parkin, Autophagy.

MiR-146b-5p activation of hepatic stellate cells contributes to the progression of fibrosis by directly targeting HIPK1.

The present study aimed to explore the biological functions of microRNA (miR)-146b-5p and homeodomain interacting protein kinase 1 (HIPK1) in the progression of hepatic fibrosis (HF) and to identify the underlying mechanism. A rat HF model was established by administering a subcutaneous injection of carbon tetrachloride (CCl4). Relative levels of miR-146b-5p and HIPK1 in fibrotic rat liver tissues and the rat hepatic stellate cell (HSC) line HSC-T6 were measured by quantitative reverse transcription PCR, western blotting and immunohistochemistry. Following activation of HSC-T6 cells by lipopolysaccharide (LPS) induction, cell viability was examined by MTT assay. Transfection of miR-146b-5p mimic or inhibitor into HSC-T6 cells was performed, with the aim to identify the influence of miR-146b-5p on HSC-T6 cell behavior. The targeting relationship between miR-146b-5p and HIPK1 was predicted by TargetScan 7.2 and StarBase 3.0 and it was later verified by a dual-luciferase reporter assay. Through lentivirus transfection, the biological function of HIPK1 in regulating the progression of HF and the underlying mechanism were investigated. The results showed that miR-146b-5p was upregulated in liver tissues of rats with HF and activated HSC-T6 cells, while HIPK1 was downregulated in liver tissues of rats with HF and activated HSC-T6 cells. miR-146b-5p was able to upregulate the activation markers of LPS-induced HSC-T6 cells, upregulate COL1A1 and TGF-β, increase cell viability and contribute to fibrosis progression. HIPK1 was validated as the direct target of miR-146b-5p and its overexpression could effectively reduce the effect of miR-146b-5p in contribution to the progression of HF. In conclusion, miR-146b-5p was significantly upregulated during the progression of HF. By targeting and downregulating HIPK1, miR-146b-5p could significantly activate HSCs, upregulate COL1A1 and TGF-β and contribute to fibrosis progression. miR-146b-5p is a potential biomarker and therapeutic target for HF.

Therapeutic Effect of Biejiaxiaozheng Pills on Carbon Tetrachloride-Induced Hepatic Fibrosis in Rats through the NF-κB/Nrf2 Pathway.

Aims To explore the effects of Biejiaxiaozheng pills on carbon tetrachloride-induced hepatic fibrosis in rats through the NF-κB/Nrf2 pathway and to explore the possible antifibrotic mechanisms of the drug. Material and Method. A rat model of hepatic fibrosis was established via CCl4 induction. Liver function and antioxidant indices were detected using commercial kits. Hematoxylin-eosin and Masson staining were used to detect pathological changes in hepatic tissues. ELISA was used to measure plasma TNF-α, IL-β, and IL-6 levels. RT-PCR was used to measure changes in TNF-α, IL-β, and IL-6 levels in hepatic tissues. Changes in p65, P-p65, Nrf2, and HO-1 protein expression were detected using western blotting. Results In rats with hepatic fibrosis, Biejiaxiaozheng pills effectively improved liver function, alleviated fibrosis in hepatic tissues, and significantly reduced collagen accumulation. The pills significantly downregulated inflammatory cytokine expression in hepatic tissues by suppressing p65 phosphorylation and reduced plasma inflammatory cytokine levels to some extent. The pills upregulated Nrf2 and HO-1 expression in hepatic tissues, enhanced antioxidant potential, and upregulated plasma antioxidant levels. Conclusion Biejiaxiaozheng pills improved hepatic fibrosis symptoms and lesions in rats, likely by inhibiting the NF-κB pathway and promoting the Nrf2 pathway.

Effects of BiejiaYugan Granule on TGF-β1/Smads pathway in rats with hepatic fibrosis caused by compound factors

Objective: To investigate the therapeutic effects of Biejia Yugan Granule on hepatic fibrosis caused by compound factors in rats and its effect on TGF-β1/Smads signaling pathway. Methods: SD rats were randomly divided into blank control group, model control group, colchicine group, Biejia Yugan Granule low, medium and high dose (1.85, 3.70, 7.40 g/kg) groups (n= 8 in each group). The rat model of hepatic fibrosis was established by treating with 5% alcohol 15 ml/kg (ig) everyday and injecting with 40% carbon tetrachloride (sc) twice a week for 42 days. The effects of Biejia Yugan Granule on liver function, liver index and water content, serum hepatic fibrosis related indicators, key proteins and gene expression of TGF-β1/Smads signaling pathway in rats were observed. Results: Biejia Yugan Granule at the doses of 1.85, 3.70 and 7.40 g/kg could decrease the serum levels of ALT, AST, ALP and HA, PCⅢ, C-Ⅳ, LN significantly, reduce the water content of liver tissue leads to the decrease of liver index, regulate the liver tissue TGF-β1, Smad3 mRNA and Smad7 mRNA expressions. Conclusion: Biejia Yugan Granule has obvious effects of reducing enzyme and protecting liver and inhibiting hepatic fibrosis, and inhibiting TGF-β1/Smads signaling pathway is one of its mechanisms of anti-hepatic fibrosis.

Anti-inflammation treatment for protection of hepatocytes and amelioration of hepatic fibrosis in rats.

Chronic inflammation is considered as an important pathophysiologic mechanism of hepatic cirrhosis, which induces hepatocyte injury and activates hepatic stellate cells (HSCs), thus resulting in hepatic fibrosis. Previous studies have reported that cyclooxygenase-2 (COX-2) inhibitor can effectively treat liver fibrosis, while somatostatin (SST) analogues inhibit the activation of HSCs. The present study aimed to investigate the effects of a COX-2 inhibitor, celecoxib, combined with a SST analogue, octreotide, for protection of hepatocytes and prevention of fibrosis in a rat model of hepatic fibrosis. Therefore, a hepatic fibrosis rat model was established following peritoneal injection of thioacetamide (TAA), and the rats were then treated with a combination of celecoxib and octreotide (TAA + C). Immunohistochemistry and western blotting assays were used to assess the expression levels of proteins associated with inflammation, epithelial-mesenchymal transition (EMT), proliferation, apoptosis and autophagy. H&E staining, transmission electron microscopy and scanning electron microscopy were used to evaluate the destruction of hepatocytes. Masson's Trichrome and Sirius Red were used to measure the degree of liver fibrosis. The results demonstrated that, compared with those of the control group, the degree of liver fibrosis and the expression of the intrahepatic inflammation factors were aggravated in the TAA group. Furthermore, the apoptosis rate, EMT and autophagy of hepatocytes were also increased in the TAA group. However, treatment with TAA + C restored the aforementioned increased levels compared with the TAA group. In conclusion, treatment of rats with the combination of celecoxib and octreotide could attenuate the progress of hepatic fibrosis via protection of hepatocytes by reducing apoptosis, EMT and autophagy in hepatocytes.

5-methoxytryptophan alleviates liver fibrosis by modulating FOXO3a/miR-21/ATG5 signaling pathway mediated autophagy

ABSTRACT Liver fibrosis is a critical health issue in the world due to its rapidly increasing prevalence. It is of great demand to develop effective drugs for the treatment of liver fibrosis. 5-methoxytryptophan (5-MTP) has been reported to play an important role in anti-inflammatory, anti-cancer, myocardial-protective effects. However, the anti-fibrotic effect of 5-MTP is never covered in liver. Here, we investigated anti-fibrotic effects of 5-MTP on liver fibrosis and its underlying mechanism. In vitro, 5-MTP treatment could inhibit TGF-β1-induced elevated levels of collagen I, collagen III, fibronectin and α-smooth muscle actin (SMA) by stimulating autophagy process. Mechanically, the expression of FOXO3a was enhanced by 5-MTP and then repressed the level of miR-21, eventually leading to a restoration of autophagy-related gene ATG5. Furthermore, rescue experiments showed 5-MTP could activate autophagy process and suppress the activation of LX-2 cells by regulating FOXO3a/miR-21/ATG5 pathway. Consistently, 5-MTP significantly attenuated CCl4-induced hepatic fibrosis in rat model. In conclusion, our research discovered that 5-MTP effectively alleviated liver fibrosis in vitro and in vivo, which provided new insights into the application of 5-MTP for liver fibrosis.

Study on the effect of Mongolian medicine Qiwei Qinggan Powder on hepatic fibrosis through JAK2/STAT3 pathway.

This research aimed to evaluate the antihepatic fibrosis effect and explore the mechanism of Qiwei Qinggan Powder (QGS-7) in vivo and in vitro. Carbon tetrachloride (CCl4)-treated rats and hepatic stellate cells (HSCs) were used. QGS-7 treatment significantly improved the liver function of rats as indicated by decreased serum enzymatic activities of alanine aminotransferase, aspartate transaminase, and alkaline phosphatase. Meanwhile, the hydroxyproline of liver was significantly decreased. Histopathological results indicated that QGS-7 alleviated liver damage and reduced the formation of fibrosis septa. Moreover, QGS-7 significantly attenuated expressions of Alpha smooth muscle actin, Collagen I, Janus kinase 2 (JAK2), phosphorylation-JAK2, signal transducer and activator of transcription 3 (STAT3), phosphorylation-STAT3 in the rat hepatic fibrosis model. QGS-7 inhibited HSC proliferation and promoted it apoptosis. QGS-7 may affect hepatic fibrosis through JAK2/STAT3 signaling pathway so as to play an antihepatic fibrosis role.

Exosomal microRNA-155 as a biomarker for hepatic fibrosis diagnosis and progression.

BackgroundPathological examination of liver biopsies remains the gold standard for evaluating the stage of hepatic fibrosis, which are a number of disadvantages associated with biopsy. The aim of the present study was to investigate the potential of exosomal microRNA (miR)-155 as a non-invasive biomarker for the diagnosis and progression of hepatic fibrosis.MethodsExosomal miR-155 quantity was analyzed by sampling serum exosomes of patients with hepatic fibrosis and a hepatic fibrosis rat model. A total of 94 patients were divided into three groups based on Child-Pugh rating. Additionally, 30 patients with primary liver fibrosis who underwent liver transplantation were divided into the low miR-155 expression group and the high expression group; 56 rats were divided into 7 groups (n=8, 0, 2, 4, 6, 8, 10, and 12 weeks). Rats in every group were intravenously injected with CCl4 (3% vol/vol in olive oil; 0.3 mL/100 g body weight) twice weekly to produce different degrees of liver necrosis and liver fibrosis.ResultsExosomal miR-155 was found to be closely associated with the progression of cirrhosis and clinical prognostic indicators of cirrhosis. Exosomal miR-155 gradually increased with the severity of hepatic necrosis and fibrosis.ConclusionsThe findings of the present study indicate that exosomal miR-155 can act as a non-invasive biomarker for the diagnosis and progression of hepatic fibrosis.

Oxymatrine exerts anti-fibrotic effects in a rat model of hepatic fibrosis by suppressing endoplasmic reticulum stress.

ObjectiveThis study evaluated the anti-fibrotic effects of oxymatrine and the role of endoplasmic reticulum (ER) stress in hepatic fibrosis (HF) in animal models.MethodsThe HF rat model was established by exposure to NaAsO2, followed by treatment with oxymatrine. Biomarkers of HF and ER stress were measured. The difference in protein expression between groups was evaluated using isobaric tag for relative and absolute quantification (iTRAQ) analysis. The mechanism by which oxymatrine modulated ER stress to alleviate arsenic-induced HF was evaluated using LX2 hepatic stellate cells in vitro.ResultsThe rat model mimicked the pathological and physical phenotypes of HF including ER stress, oxidative stress, impaired liver function, and fibrosis. Treatment with oxymatrine suppressed these responses. Moreover, apoptosis, inflammation, and hepatic stellate cell activation were also inhibited by oxymatrine treatment. The differentially expressed proteins and pathways related to ER stress were identified in the HF and oxymatrine-treated groups via iTRAQ analysis combined with liquid chromatography–mass spectrometry. LX2 cells were activated by NaAsO2 in vitro. Meanwhile, oxymatrine suppressed the activation of LX2 cells by alleviating ER stress and regulating cellular calcium homeostasis.ConclusionsOxymatrine could reverse NaAsO2-induced HF by alleviating ER stress.