Fibrotic Rat Liver Research Articles

The Upregulation of Leucine-Rich Repeat Containing 1 Expression Activates Hepatic Stellate Cells and Promotes Liver Fibrosis by Stabilizing Phosphorylated Smad2/3.

Liver fibrosis poses a significant global health risk due to its association with hepatocellular carcinoma (HCC) and the lack of effective treatments. Thus, the need to discover additional novel therapeutic targets to attenuate liver diseases is urgent. Leucine-rich repeat containing 1 (LRRC1) reportedly promotes HCC development. Previously, we found that LRRC1 was significantly upregulated in rat fibrotic liver according to the transcriptome sequencing data. Herein, in the current work, we aimed to explore the role of LRRC1 in liver fibrosis and the underlying mechanisms involved. LRRC1 expression was positively correlated with liver fibrosis severity and significantly elevated in both human and murine fibrotic liver tissues. LRRC1 knockdown or overexpression inhibited or enhanced the proliferation, migration, and expression of fibrogenic genes in the human hepatic stellate cell line LX-2. More importantly, LRRC1 inhibition in vivo significantly alleviated CCl4-induced liver fibrosis by reducing collagen accumulation and hepatic stellate cells' (HSCs) activation in mice. Mechanistically, LRRC1 promoted HSC activation and liver fibrogenesis by preventing the ubiquitin-mediated degradation of phosphorylated mothers against decapentaplegic homolog (Smad) 2/3 (p-Smad2/3), thereby activating the TGF-β1/Smad pathway. Collectively, these results clarify a novel role for LRRC1 as a regulator of liver fibrosis and indicate that LRRC1 is a promising target for antifibrotic therapies.

Protective effect of combination therapy with ischemic preconditioning and rapamycin in fibrotic rat livers

Protective effect of combination therapy with ischemic preconditioning and rapamycin in fibrotic rat livers

Ciplukan (Physalis Angulata Linn.) Extract Compounds Potential on High-Fat Diet Induced Nonalcoholic Fatty Liver Disease (NAFLD) for Liver Anti-Fibrotic Drug Development

An increasing percentage of people have or are at risk to develop the non-alcoholic fatty liver disease (NAFLD) worldwide. Fibrosis has also been identified as the most important predictor of prognosis in patients with NAFLD. Ciplukan (Physalis angulata Linn.) was reported to have antifibrotic potency in CCl4-induced liver fibrotic rats. This study was conducted to evaluate the antifibrotic effect of ciplukan extract through repairing the liver function, anti-inflammatory, and lowering cholesterol. The liver fibrosis model using 20% margarine was injected subcutaneously 8 times with a frequency of twice a week for 4 weeks in 35 male and 35 female Wistar strains which were divided into 7 groups. Furthermore, the rats were given Ciplukan extract (CPL) orally starting the 6th week of treatment with 2 different doses, namely 13.5 mg (CPL-1) and 27 mg (CPL-2) every day for 4 weeks. The histopathological changes of liver fibrosis was analyzed using Haematoxylin Eosin (HE) staining. Determination of serum IL-6 and TGF-β1 levels was carried out by the ELISA method. ALT and cholesterol levels were tested using a diagnostic kit. Single and multiple doses of ciplukan extract with or without standard therapy (Vitamin E) can reduce fibrotic scores up to 1.30±0.95 (p=0.001), TGF-β1 levels up to 24.20±2.02 ng/mL (p = 0.000), IL-6 levels up to 1.68±0.52 pg/mL (p=0.156), ALT levels up to 104.57±2.02 U/mL (p=0.001), and cholesterol levels up to 81, 07±2.02 mg/dL (p=0.000). Ciplukan herb ethanol extract was proven to have liver antifibrotic activity, which means it has potential as a liver fibrotic drug. The liver antifibrotic effect of Ciplukan herb was shown by a histopathological decrease in liver fibrosis scores accompanied by a decrease in TGF-β1, IL-6, ALT, and cholesterol levels.
 Keywords: Physalis angulata Linn (Ciplukan), liver fibrosis, NAFLD, ALT, Cholesterol, IL-6

Nicorandil/ morphine crosstalk accounts for antinociception and hepatoprotection in hepatic fibrosis in rats: Distinct roles of opioid/cGMP and NO/KATP pathways

Previous report indicated that nicorandil potentiated morphine antinociception and attenuated hepatic injury in liver fibrotic rats. Herein, the underlying mechanisms of nicorandil/morphine interaction were investigated using pharmacological, biochemical, histopathological, and molecular docking studies. Male Wistar rats were injected intraperitoneally (i.p.) with carbon tetrachloride (CCl4, 40%, 2 ml/kg) twice weekly for 5 weeks to induce hepatic fibrosis. Nicorandil (15 mg/kg/day) was administered per os (p.o.) for 14 days in presence of the blockers; glibenclamide (KATP channel blocker, 5 mg/kg, p.o.), L-NG-nitro-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor, 15 mg/kg, p.o.), methylene blue (MB, guanylyl cyclase inhibitor, 2 mg/kg, i.p.) and naltrexone (opioid antagonist, 20 mg/kg, i.p.). At the end of the 5th week, analgesia was evaluated using tail flick and formalin tests along with biochemical determinations of liver function tests, oxidative stress markers and histopathological examination of liver tissues. Naltrexone and MB inhibited the antinociceptive activity of the combination. Furthermore, combined nicorandil/morphine regimen attenuated the release of endogenous peptides. Docking studies revealed a possible interaction of nicorandil on µ, κ and δ opioid receptors. Nicorandil/morphine combination protected against liver damage as evident by decreased liver enzymes, liver index, hyaluronic acid, lipid peroxidation, fibrotic insults, and increased superoxide dismutase activity. Nicorandil/morphine hepatoprotection and antioxidant activity were inhibited by glibenclamide and L-NAME but not by naltrexone or MB. These findings implicate opioid activation/cGMP versus NO/KATP channels in the augmented antinociception, and hepatoprotection, respectively, of the combined therapy and implicate provoked cross talk by nicorandil and morphine on opioid receptors and cGMP signaling pathway. That said, nicorandil/morphine combination provides a potential multitargeted therapy to alleviate pain and preserve liver function.

The Role of Pyrazolo[3,4-d]pyrimidine-Based Kinase Inhibitors in The Attenuation of CCl4-Induced Liver Fibrosis in Rats

Liver Fibrosis can be life-threatening if left untreated as it may lead to serious, incurable complications. The common therapeutic approach is to reverse the fibrosis while the intervention is still applicable. Celecoxib was shown to exhibit some antifibrotic properties in the induced fibrotic liver in rats. The present study aimed to investigate the possible antifibrotic properties in CCl4-induced liver fibrosis in male Sprague-Dawley rats compared to celecoxib of three novel methoxylated pyrazolo[3,4-d]pyrimidines. The three newly synthesized compounds were proved to be safe candidates. They showed a therapeutic effect against severe CCl4-induced fibrosis but at different degrees. The three compounds were able to partially reverse hepatic architectural distortion and reduce the fibrotic severity by showing antioxidant properties reducing MDA with increasing GSH and SOD levels, remodeling the extracellular matrix proteins and liver enzymes balance, and reducing the level of proinflammatory (TNF-α and IL-6) and profibrogenic (TGF-β) cytokines. The results revealed that the dimethoxy-analog exhibited the greatest activity in all the previously mentioned parameters compared to celecoxib and the other two analogs which could be attributed to the different methoxylation patterns of the derivatives. Collectively, the dimethoxy-derivative could be considered a safe promising antifibrotic candidate.

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.

Transplantation of SDF-1α-loaded liver extracellular matrix repopulated with autologous cells attenuated liver fibrosis in a rat model

Cell-based therapy and tissue engineering are promising substitutes for liver transplantation to cure end-stage liver disorders. However, the limited sources for healthy and functional cells and poor engraftment rate are main challenges to the cell-based therapy approach. On the other hand, feasibility of production and size of bioengineered tissues are primary bottlenecks in tissue engineering. Here, we induce regeneration in a rat fibrotic liver model by transplanting a natural bioengineered scaffold with a native microenvironment repopulated with autologous stem/progenitor cells. In the main experimental group, a 1 mm3 stromal derived factor-1α (SDF-1α; S) loaded scaffold from decellularized liver extracellular matrix (LEM) was transplanted (Tx) into a fibrotic liver and the endogenous stem/progenitor cells were mobilized via granulocyte colony stimulating factor (G-CSF; G) therapy. Four weeks after transplantation, changes in liver fibrosis and necrosis, efficacy of cell engraftment and differentiation, vasculogenesis, and liver function recovery were assessed in this (LEM-TxSG) group and compared to the other groups. We found significant reduction in liver fibrosis stage in the LEM-TxSG, LEM-TxS and LEM-TxG groups compared to the control (fibrotic) group. Liver necrosis grade, and alanine transaminase (ALT) and aspartate transaminase (AST) levels dramatically reduced in all experimental groups compared to the control group. However, the number of engrafted cells into the transplanted scaffold and ratio of albumin (Alb) positive cells per total incorporated cells were considerably higher in the LEM-TxSG group compared to the LEM-Tx, LEM-TxS and LEM-TxG groups. Serum Alb levels increased in the LEM-Tx, LEM-TxS, and LEM-TxG groups, and was highest in the LEM-TxSG group, which was significantly more than the fibrotic group. Small vessel formation in the LEM-TxSG group was significantly higher than the LEM-Tx and LEM-TxS groups. Totally, these findings support application of the in vivo tissue engineering approach as a possible novel therapeutic strategy for liver fibrosis.

Interleukin-10 regulates starvation-induced autophagy through the STAT3-mTOR-p70s6k axis in hepatic stellate cells.

The degree of activation of hepatic stellate cells (HSCs) is closely related to the level of autophagy in HSCs. We previously showed that interleukin-10 (IL-10) strongly inhibits HSC activation in rat fibrotic liver. However, little is known about the effect of IL-10 on HSC autophagy. For investigation of the effect of IL-10 on starvation-induced autophagy in immortal rat hepatic stellate cells (HSC-T6) and the molecular mechanism, HSC-T6 cells were incubated with serum-free DMEM for different periods and treated with IL-10 at different concentrations. Transmission electron microscopy (TEM), analysis of autophagic flux and Western blotting (WB) assays were used to observe changes in autophagosome morphology and number and autophagy-related protein expression in HSC-T6 cells and to evaluate the regulatory effect of IL-10 on starvation-induced autophagy. Cryptotanshinone (CPT) and rapamycin (Rapa) were used to block activation of the signal transducer and activator of transcription 3 (STAT3) and mTOR signaling pathways, respectively. STAT3-mTOR-p70s6k signaling pathway proteins were analyzed by WB to assess the signaling pathway by which IL-10 regulates autophagy. WB showed an increased LC3II/I ratio, increased Beclin1 expression, and decreased p62 expression in HSC-T6 cells starved for 3 h (p < 0.05). IL-10 inhibited the increases in the LC3II/I ratio and Beclin1 expression and upregulated p62 expression (p < 0.05), and the optimal IL-10 concentration was 20 ng/mL. TEM and double-labeled immunofluorescence analysis showed that IL-10 inhibited autophagosome formation and autophagic flux, as indicated by the decreased numbers of double-membrane autophagosomes and yellow autophagic puncta. Further examination of signaling pathway molecules showed that phosphorylation of the mTOR, STAT3, and p70s6k proteins was significantly decreased during starvation-induced autophagy, but IL-10 could increase mTOR, STAT3, and p70s6k protein phosphorylation (p < 0.05). Blocking either the mTOR or STAT3 pathway reversed the inhibitory effect of IL-10 on starvation-induced autophagy in HSC-T6 cells (p < 0.05). IL-10 suppresses starvation-induced autophagosome formation through activation of the STAT3-mTOR-p70s6k axis in HSC-T6 cells.

Identification of differentially expressed long noncoding RNAs and pathways in liver tissues from rats with hepatic fibrosis.

To identify long non-coding RNAs (lncRNAs) and their potential roles in hepatic fibrosis in rat liver issues induced by CCl4, lncRNAs and genes were analyzed in fibrotic rat liver tissues by RNA sequencing and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Differentially expressed (DE) lncRNAs (DE-lncRNAs) and genes were subjected to bioinformatics analysis and used to construct a co-expression network. We identified 10 novel DE-lncRNAs that were downregulated during the hepatic fibrosis process. The cis target gene of DE-lncRNA, XLOC118358, was Met, and the cis target gene of the other nine DE-lncRNAs, XLOC004600, XLOC004605, XLOC004610, XLOC004611, XLOC004568, XLOC004580 XLOC004598, XLOC004601, and XLOC004602 was Nox4. The results of construction of a pathway-DEG co-expression network show that lncRNA-Met and lncRNAs-Nox4 were involved in oxidation-reduction processes and PI3K/Akt signaling pathway. Our results identified 10 DE-lncRNAs related to hepatic fibrosis, and the potential roles of DE-lncRNAs and target genes in hepatic fibrosis might provide new therapeutic strategies for hepatic fibrosis.

The Novel Chinese Medicine JY5 Formula Alleviates Hepatic Fibrosis by Inhibiting the Notch Signaling Pathway.

Advanced liver fibrosis can lead to cirrhosis, resulting in an accelerated risk of hepatocellular carcinoma and liver failure. Fuzheng Huayu formula (FZHY) is a traditional Chinese medicine formula treated liver fibrosis in China approved by a Chinese State Food and Drug Administration (NO: Z20050546), composed of Salvia Miltiorrhiza bge., Prunus davidiana (Carr.) Franch., cultured Cordyceps sinensis (BerK.) Sacc. Mycelia, Schisandra chinensis (Turcz.) Baill., Pinus massoniana Lamb., and Gynostemma pentaphyllum (Thunb.) Makino. However, the main active substances and mechanism of FZHY are unclear. The aim of this study is to identify a novel anti-fibrotic compound, which consists of the main active ingredients of FZHY, and investigate its mechanism of pharmacological action. The main active ingredients of FZHY were investigated by quantitative analysis of FZHY extracts and FZHY-treated plasma and liver in rats. The anti-fibrotic composition of the main active ingredients was studied through uniform design in vivo, and its mechanism was evaluated in carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced liver fibrosis models in rats and mice, and transforming growth factor beta 1-induced LX-2 cell activation model in vitro. A novel Chinese medicine, namely JY5 formula, consisting of salvianolic acid B, schisantherin A, and amygdalin, the main active ingredients of FZHY, significantly alleviated hepatic hydroxyproline content and collagen deposition in CCl4-and BDL-induced fibrotic liver in rats and mice. In addition, JY5 inhibited the activation of hepatic stellate cells (HSCs) by inactivating Notch signaling in vitro and in vivo. In this study, we found a novel JY5 formula, which exerted anti-hepatic fibrotic effects by inhibiting the Notch signaling pathway, consequently suppressing HSCs activation. These results provide an adequate scientific basis for clinical research and application of the JY5 formula, which may be a potential novel therapeutic candidate for liver fibrosis.

Dynamic expression of protein tyrosine phosphatase SHP2 in liver tissue of rats with carbon tetrachloride-induced liver fibrosis

Objective: To investigate the dynamic expression of protein tyrosine phosphatase SHP2 in liver tissue of rats with carbon tetrachloride (CCl(4))-induced liver fibrosis. Methods: Rat liver fibrosis model was established by intraperitoneal injection of CCl(4). Rat liver tissue histopathological changes were detected by HE and Masson-trichrome staining. Immunohistochemical staining, Western blot and real-time fluorescent quantitative PCR were used to detect SHP2 protein and mRNA expression in rat liver tissue. One-way analysis of variance was used for the comparison of means between multiple groups, and the LSD test was used for further inter-group comparison. Results: CCl(4)-induced rat liver fibrosis model was successfully constructed, and with the extension of modeling time, the degree of liver fibrosis in rats were aggravated gradually. Immunohistochemical staining results showed that SHP2 was mainly expressed in the cytoplasm of rat liver tissues. With the aggravation of liver fibrosis, the number of cells with positive expression of SHP2 was aggravated gradually (P < 0.05). Western blot and real-time fluorescent quantitative PCR results showed that the expressions of SHP2 protein and mRNA in rat fibrotic liver tissues at different times in week 2, 4, 6, and 8 were higher in modeling than control group (P < 0.05), and was aggravated gradually with the liver fibrosis aggravation (P < 0.05). Conclusion: The expression of SHP2 protein and mRNA in the liver tissue of rats with CCl(4)-induced liver fibrosis increased gradually with the degree of liver fibrosis, and the degree of increase was consistent with the degree of liver fibrosis.

Modulation of vascular contraction via soluble guanylate cyclase signaling in a novel ex vivo method using rat precision-cut liver slices.

Fibrotic processes in the liver of non‐alcoholic steatohepatitis (NASH) patients cause microcirculatory dysfunction in the organ which increases blood vessel resistance and causes portal hypertension. Assessing blood vessel function in the liver is challenging, necessitating the development of novel methods in normal and fibrotic tissue that allow for drug screening and translation toward pre‐clinical settings. Cultures of precision cut liver slices (PCLS) from normal and fibrotic rat livers were used for blood vessel function analysis. Live recording of vessel diameter was used to assess the response to endothelin‐1, serotonin and soluble guanylate cyclase (sGC) activation. A cascade of contraction and relaxation events in response to serotonin, endothelin‐1, Ketanserin and sGC activity could be established using vessel diameter analysis of rat PCLS. Both the sGC activator BI 703704 and the sGC stimulator Riociguat prevented serotonin‐induced contraction in PCLS from naive rats. By contrast, PCLS cultures from the rat CCl4 NASH model were only responsive to the sGC activator, thus establishing that the sGC enzyme is rendered non‐responsive to nitric oxide under oxidative stress found in fibrotic livers. The role of the sGC pathway for vessel relaxation of fibrotic liver tissue was identified in our model. The obtained data shows that the inhibitory capacities on vessel contraction of sGC compounds can be translated to published preclinical data. Altogether, this novel ex vivo PCLS method allows for the differentiation of drug candidates and the translation of therapeutic approaches towards the clinical use.

Inhibition of TGFβ1 accelerates regeneration of fibrotic rat liver elicited by a novel two-staged hepatectomy.

Aims: Emerging evidence is demonstrating that rapid regeneration of remnant liver elicited by associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) may be attenuated in fibrotic livers. However, the molecular mechanisms responsible for this process are largely unknown. It is widely acknowledged that the TGFβ1 signaling axis plays a major role in liver fibrosis. Therefore, the aims of this study were to elucidate the underlying mechanism of liver regeneration during ALPPS with or without fibrosis, specifically focusing on TGFβ1 signaling.Approach: ALPPS was performed in rat models with N-diethylnitrosamine-induced liver fibrosis and no fibrosis. Functional liver remnant regeneration and expression of TGFβ1 were analyzed during the ALPPS procedures. Adeno-associated virus-shTGFβ1 and the small molecule inhibitor LY2157299 (galunisertib) were used separately or in combination to inhibit TGFβ1 signaling in fibrotic rats.Results: Liver regeneration following ALPPS was lower in fibrotic rats than non-fibrotic rats. TGFβ1 was a key mediator of postoperative regeneration in fibrotic liver. Interestingly, AAV-shTGFβ1 accelerated the regeneration of fibrotic functional liver remnant and improved fibrosis, while LY2157299 only enhanced liver regeneration. Moreover, combination treatment elicited a stronger effect.Conclusions: Inhibition of TGFβ1 accelerated regeneration of fibrotic liver, ameliorated liver fibrosis, and improved liver function following ALPPS. Therefore, TGFβ1 is a promising therapeutic target in ALPPS to improve fibrotic liver reserve function and prognosis.

Ultrasound-targeted microbubble destruction optimized HGF-overexpressing bone marrow stem cells to repair fibrotic liver in rats

Background/aimsBone marrow mesenchymal stem cells (BMSCs) have shown their therapeutic potential in cytotherapy for liver fibrosis. However, the insufficient homing of BMSCs and undefined proliferation of BMSCs represent a significant challenge and largely limit the effective implementation. The aims of the present study were to determine whether stable expression of hepatic growth factor (HGF) in BMSCs coupled with ultrasound-targeted microbubble destruction (UTMD) technique could effectively and definitely alleviating carbon tetrachloride (CCl4)-induced liver fibrosis in rats.Materials and methodsA rat model of liver fibrosis was acquired by injection of carbon tetrachloride (CCl4). The experimental rats were randomly assigned to the four groups: normal, CCl4, BMSCs-HGF/US, and BMSCs-HGF/UTMD groups. The BMSCs, transfected by recombinant adeno-associated virus vector encoding human genome sequence of HGF (BMSCs-HGF), were transplanted in rat via the tail vein. The homing efficiency of BMSCs was observed by immunofluorescence staining. The liver function and its morphological changes were analyzed by biochemical tests and liver histology. The expression of liver fibrosis markers including α-smooth muscle actin (α-SMA), collagen I, and vimentin were examined by immunohistochemistry and quantitative real-time polymerase chain reaction.ResultsThe homing efficiency of BMSCs in the fibrotic liver was significantly greater with the application of UTMD. The biochemical markers of liver function and histopathological results showed significantly better improvement in BMSCs-HGF/UTMD group than the other groups, and the serum levels of biochemical markers returned to normal ranges in 12 weeks in this group. Furthermore, the expression levels of liver fibrosis markers (α-SMA, collagen I, and Vimentin) were all significantly lower in BMSCs-HGF/UTMD group in comparison with other groups.ConclusionsOur findings have demonstrated that stable expression of HGF in BMSCs and application of the UTMD technique facilitate the homing of BMSCs, and more importantly, which could further improve their alleviation of liver fibrosis. Therefore, these findings have an important clinical implication that AAV-BMSCs-HGF and UTMD hold promise as a novel therapeutic approach for liver fibrosis.

MiR-375 Impairs the Invasive Capabilities of Hepatoma Cells by Targeting HIF1α Under Hypoxia.

Hypoxia represents one of the most pervasive microenvironmental stresses in HCC due to the overwhelming growth and inadequate blood supply. HIF1α as an important transcription factor participates in the regulation of various biological behaviors of HCC cells under hypoxia. Our previous study indicated that miR-375 is a hypoxia-associated miRNA. However, the interaction between miR-375 and HIF1α remains unclear. Bioinformatic analysis was performed for miRNA screening. qRT-PCR, western blotting, and immunohistochemical staining were used to detect the expression of related molecules. Bioinformatic analysis and dual luciferase assay were used to predict and further confirm the target association. Transwell chamber assay and flow cytometry were, respectively, used to detect migration, invasion and apoptosis of hepatoma cells. MiR-375 presented an obviously differential expression in human HCCs versus background livers (BLs) and HCCs versus normal liver tissues (NLTs). In rat models, miR-375 was gradually declined during hepatocarcinogenesis. HIF1α was remarkably upregulated at protein level rather than at mRNA level in human HCCs versus BLs, HCCs versus NLTs, BLs versus NLTs, and in rat fibrotic livers versus NLTs. HIF1α was determined to be a target of miR-375. MiR-375 inhibitor induced the migration and invasive capabilities and attenuated apoptosis of hepatoma cells under hypoxia. Depriving HIF1α by siRNA could partially reverse the function of miR-375 inhibitor under hypoxia. MiR-375 impairs the invasive capabilities of HCC cells by targeting HIF1α under hypoxia.

Comparative Pharmacokinetic Study of Taxifolin after Oral Administration of Fructus Polygoni Orientalis Extract in Normal and Fibrotic Rats by UPLC-MS/MS

Fructus polygoni orientalis (FPO) is widely used in clinical practice in China, especially in treatment of liver diseases including viral hepatitis, liver fibrosis, and liver cirrhosis. However, its pharmacokinetic (PK) alterations in liver fibrotic rats have rarely been reported. To study whether taxifolin, one of the main flavonoids in FPO can be absorbed into blood after oral administration of FPO extract and to compare the differences in pharmacokinetic parameters of taxifolin to normal and liver fibrotic rats induced by porcine serum (PS), a UPLC-MS/MS method was developed and validated for determination of taxifolin in rat plasma using puerarin as the internal standard (IS). All validation parameters met the acceptance criteria according to regulatory guidelines. The results indicated that after treatment of rats with PS alone for 12 weeks, the liver fibrotic model group was built successfully. The taxifolin can be absorbed into the blood after oral administration of the FPO extract. The Cmax of taxifolin was 1940 ± 502.2 ng/mL and 2648 ± 208.5 ng/mL (p < 0.05), the AUC0∼t of taxifolin was 4949.7 ± 764.89 h·ng/mL and 6679.9 ± 734.26 h·ng/mL (p < 0.05), the AUC0∼∞ of taxifolin was 5049.4 ± 760.7 and 7095.2 ± 962.3 h·ng/mL (p < 0.05), and the mean residence time (MRT) of taxifolin was 2.46 ± 0.412 h and 3.17 ± 0.039 h (p < 0.05) in the normal and fibrotic model groups, respectively. These results confirmed that the pharmacokinetic parameters of taxifolin are altered in liver fibrosis, manifested as Cmax, AUC0∼t, AUC0∼∞, and the mean residence time (MRT). It suggested that it is essential to consider the characteristics of pharmacokinetics after oral administration of FPO in liver disease patients.

Effect of SEPT6 on the biological behavior of hepatic stellate cells and liver fibrosis in rats and its mechanism

Hepatic stellate cells (HSCs) are key effectors during the development of liver fibrosis. Septin 6 (SEPT6) is a highly evolutionarily conserved GTP-binding protein that regulates various cell biological behaviors. The expression and function of SEPT6 in HSCs remain unknown. Here we demonstrate that SEPT6 expression is significantly elevated following the activation of primary rat HSCs, the human hepatic stellate cell line LX-2 and the rat hepatic stellate cell line HSC-T6, as well as in both human and rat fibrotic liver tissue. In vitro, the overexpression of SEPT6 promoted HSCs activation, proliferation, cell cycle progression and migration and inhibited HSCs apoptosis. In contrast, knockdown of SEPT6 exerted the opposite effects on HSCs. Mechanistically, SEPT6 exerted its pro-fibrogenic effect by promoting the expression of TGF-β1 and the phosphorylation of Smad2, Smad3, extracellular-signal-regulated kinase, c-Jun NH2-terminal kinase, stress-activated protein kinase-2, and protein kinase B. However, in HSC-T6 cells, blockade of the TGF-β1/Smad signaling pathway by SB431542 significantly decreased the expression of α-smooth muscle actin, cyclin D1, BCL2, and matrix metalloproteinase-2 and -9, which had been enhanced by SEPT6 overexpression. In vivo, adenovirus-mediated SEPT6 inhibition attenuated thioacetamide (TAA)-induced liver fibrosis in rats by decreasing the deposition of the extracellular matrix (ECM). SEPT6 inhibition decreased the proliferation capacity of HSCs and induced apoptosis of HSCs. Collectively, our results reveal that SEPT6 regulates various biological behaviors in HSCs through TGF-β1/Smad, mitogen-activated protein kinases and phosphatidylinositol-3-kinase/protein kinase B signaling pathways, thus promoting liver fibrosis.The cytoskeletal GTPase SEPT6 is elevated during hepatic stellate cell (HSC) activation and in liver fibrosis. SEPT6 promotes HSC activation, proliferation, cell cycle progression, survival and migration through the TGF-β1/Smad, MAPK and PI3K/AKT signaling pathways. Adenovirus-mediated SEPT6 inhibition attenuates thioacetamide-induced liver fibrosis.

Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds.

Precision-cut liver tissue slice (PCLS) contains all major cell types of the liver parenchyma and preserves the original cell-cell and cell-matrix contacts. It represents a promising ex vivo model to study liver fibrosis and test the antifibrotic effect of experimental compounds in a physiological environment. In this study using RNA sequencing, we demonstrated that various pathways functionally related to fibrotic mechanisms were dysregulated in PCLSs derived from rats subjected to bile duct ligation. The activin receptor-like kinase-5 (Alk5) inhibitor SB525334, nintedanib, and sorafenib each reversed a subset of genes dysregulated in fibrotic PCLSs, and of those genes we identified 608 genes whose expression was reversed by all three compounds. These genes define a molecular signature characterizing many aspects of liver fibrosis pathology and its attenuation in the model. A panel of 12 genes and 4 secreted biomarkers including procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) were further validated as efficacy end points for the evaluation of antifibrotic activity of experimental compounds. Finally, we showed that blockade of αV-integrins with a small molecule inhibitor attenuated the fibrotic phenotype in the model. Overall, our results suggest that the rat fibrotic PCLS model may represent a valuable system for target validation and determining the efficacy of experimental compounds. NEW & NOTEWORTHY We investigated the antifibrotic activity of three compounds, the activin receptor-like kinase-5 (Alk5) inhibitor SB525334, nintedanib, and sorafenib, in a rat fibrotic precision-cut liver tissue slice model using RNA sequencing analysis. A panel of 12 genes and 4 secreted biomarkers including procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) were then established as efficacy end points to validate the antifibrotic activity of the αV-integrin inhibitor CWHM12. This study demonstrated the value of the rat fibrotic PCLS model for the evaluation of antifibrotic drugs.

Hepatic regeneration by associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is feasible but attenuated in rat liver with thioacetamide-induced fibrosis

BackgroundThe associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) procedure promotes the proliferation of the future liver remnant, but evidence to support the feasibility of ALPPS in livers with fibrosis is needed. Therefore the aim of this study was to establish a fibrotic ALPPS model in the rat to compare the capacity of regeneration in the remnant liver with or without fibrosis. MethodsIn our study we first established a thioacetamide-induced fibrotic ALPPS model in rats. Then the ALPPS-induced regenerative capacities of normal and fibrotic liver were compared in this animal model. In addition, markers of regeneration, including the proliferative index and cyclin D1 and proliferating cell nuclear antigen levels, as well as various indicators of liver function were determined to evaluate the quality of the hepatic regeneration. ResultsCompared with that of the sham group (opening of the peritoneal cavity with no further operative manipulation), the proliferation of the future liver remnant in fibrotic rat liver after the ALPPS procedure was increased on postoperative days 1, 2, and 5 (P < .039 each). In addition, the proliferative response was greater in the ALPPS group than in the ligation group subjected only to portal vein ligation of the left lateral, left middle, right, and caudate lobes (P = .099, P = .006, and P = .020 on postoperative days 1, 2, and 5, respectively). In contrast, the ALPPS-induced regenerative capacity in the fibrotic rat livers was attenuated compared with that in the normal liver on postoperative days 1, 2, and 5 (P < .031 for each) after stage I and on postoperative day 5 after stage II of the ALPPS procedure (P < .005). This attenuated the recovery of liver function, and the greater mortality rate indicated that functional proliferation was either delayed or not as extensive in the fibrotic rat livers. ConclusionThrough establishing a rat model of thioacetamide-induced liver fibrosis, we found that ALPPS-derived liver regeneration was present and feasible in fibrotic livers, but this effect was attenuated compared with that in normal liver.

Histone deacetylase inhibitor suberoylanilide hydroxamic acid alleviates liver fibrosis by suppressing the transforming growth factor-β1 signal pathway

BackgroundHistone deacetylases (HDACs) inhibitors are new anti-fibrotic drugs that inhibit the activity of hepatic stellate cells. The present study focused on the anti-fibrotic function of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) by suppressing transforming growth factor-β1 (TGF-β1) signaling. MethodsMale Sprague-Dawley rats were used to induce liver fibrosis with carbon tetrachloride (CCl4) and LX2 cell (human hepatic stellate cell line) was stimulated by TGF-β1. Both animals and cells were treated with SAHA. The Smad7 and connective tissue growth factor (CTGF) mRNA levels were detected by real-time polymerase chain reaction (PCR). Western blotting was used to examine the protein levels of CTGF, Histone H3 (H3), Smad7, Smad2/3, Acetyl-Histone H3 (AH3), HDAC2, α-smooth muscle actin (α-SMA), HDAC6, p-Smad2/3 and HDAC8. In addition, the TGF-β1 and liver enzyme levels from rat serum were detected. Histopathological changes were examined by hematoxylin and eosin (HE), Sirius red and Masson trichrome staining. The α-SMA expression was detected by immumohistochemical staining. ResultsCompared with control group, the TGF-β1 and liver enzyme levels from rat serum, together with the mRNA levels of CTGF and protein levels of CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were elevated in fibrotic rats (P < 0.01). But the Smad7 mRNA and AH3 protein levels were notably suppressed in the fibrotic rats (P < 0.01). Pathological examination showed the typical changes of liver fibrosis in the fibrotic rats. After the treatment with SAHA, the levels of liver enzymes, TGF-β1, CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were reduced (P < 0.01) and Smad7 and AH3 protein contents were elevated in liver fibrotic rats (P < 0.01). Moreover, immumohistochemistry showed that SAHA significantly suppressed the α-SMA protein content in fibrotic liver (P < 0.01). ConclusionThe HDAC inhibitor SAHA alleviated liver fibrosis by suppressing the TGF-β1 signaling.