Human Hepatic Stellate Cell Line Research Articles

Novel protein C6ORF120 promotes liver fibrosis by activating hepatic stellate cells through the PI3K/Akt/mTOR pathway.

The role of C6ORF120 in promoting CCL4-induced hepatic fibrosis and its possible mechanisms were explored in C6orf120 knockout rats (C6orf120-/-) and LX-2 cells (a type of human hepatic stellate cell line). In vivo experiments, wild-type and C6orf120-/- rats were used to investigate the function of C6ORF120. In the in vitro experiments, C6ORF120 recombinant protein (rC6ORF120) at a concentration of 200ng/mL was used to stimulate LX-2 cells. Sirius Red staining, Masson staining, western blotting, polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to explore fibrosis-associated factors. C6orf120-/- rats showed mild fibrosis and liver injury in the CCL4-induced liver fibrosis model. Furthermore, RNA-seq revealed that C6orf120-/- rats had less extracellular matrix deposition and activated stellate cells. Consistent with the in vivo, the rC6ORF120 induced LX-2 cell activation. Moreover, mechanistic studies revealed that the p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR levels were significantly elevated and LY294002 (a PI3K/Akt/mTOR typical pathway inhibitor) reversed the function of C6ORF120 in activating LX-2 cells. C6ORF120 could activate hepatic stellate cells and promote hepatic fibrosis via the PI3K/Akt/mTOR signaling pathway.

Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation

ObjectiveThis study explores the mechanism of action of Danhongqing formula (DHQ), a compound-based Chinese medicine formula, in the treatment of cholestatic liver fibrosis. MethodsIn vivo experiments were conducted using 8-week-old multidrug resistance protein 2 knockout (Mdr2-/-) mice as an animal model of cholestatic liver fibrosis. DHQ was administered orally for 8 weeks, and its impact on cholestatic liver fibrosis was evaluated by assessing liver function, liver histopathology, and the expression of liver fibrosis-related proteins. Real-time polymerase chain reaction, Western blot, immunohistochemistry and other methods were used to observe the effects of DHQ on long non-coding RNA H19 (H19) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in the liver tissue of Mdr2-/- mice. In addition, cholangiocytes and hepatic stellate cells (HSCs) were cultured in vitro to measure the effects of bile acids on cholangiocyte injury and H19 expression. Cholangiocytes overexpressing H19 were constructed, and a conditioned medium containing H19 was collected to measure its effects on STAT3 protein expression and cell activation. The intervention effect of DHQ on these processes was also investigated. HSCs overexpressing H19 were constructed to measure the impact of H19 on cell activation and assess the intervention effect of DHQ. ResultsDHQ alleviated liver injury, ductular reaction, and fibrosis in Mdr2-/- mice, and inhibited H19 expression, STAT3 expression and STAT3 phosphorylation. This formula also reduced hydrophobic bile acid-induced cholangiocyte injury and the upregulation of H19, inhibited the activation of HSCs induced by cholangiocyte-derived conditioned medium, and decreased the expression of activation markers in HSCs. The overexpression of H19 in a human HSC line confirmed that H19 promoted STAT3 phosphorylation and HSC activation, and DHQ was able to successfully inhibit these effects. ConclusionDHQ effectively alleviated spontaneous cholestatic liver fibrosis in Mdr2-/- mice by inhibiting H19 upregulation in cholangiocytes and preventing the inhibition of STAT3 phosphorylation in HSC, thereby suppressing cell activation.Please cite this article as: Li M, Zhou Y, Zhu H, Xu LM, Ping J. Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation. J Integr Med. 2024; Epub ahead of print.

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.

Melatonin ameliorates hepatic fibrosis via the melatonin receptor 2-mediated upregulation of BMAL1 and anti-oxidative enzymes

Hepatic fibrosis, when left untreated, causes serious health problems that progress to cirrhosis and, in some cases, liver cancer. Activation of hepatic stellate cells may be a key characteristic in the development of hepatic fibrosis. Melatonin, a pineal hormone, exerts anti-fibrotic effects; however, the exact mechanisms remain unclear. In this study, the beneficial effects of melatonin against hepatic fibrosis and the underlying mechanisms were investigated using the human hepatic stellate cell line, LX-2, and in vivo murine animal models. The results showed that melatonin suppressed the expression of transforming growth factor (TGF)-β1-induced fibrosis markers and production of reactive oxygen species in LX-2 cells. Either 4-phenyl-2-propionamidotetralin, a melatonin receptor 2 selective antagonist, or melatonin receptor 2 small interfering RNA abolished the suppressive effects of melatonin, suggesting the involvement of melatonin receptor 2 in melatonin-induced anti-fibrotic and anti-oxidative actions. Melatonin increased the expression of the brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1), a positive circadian clock gene. BMAL1 knockdown reduced the anti-fibrotic and anti-oxidative effects of melatonin, demonstrating the protective effects of melatonin against TGF-β1-induced hepatic stellate cell activation by exhibiting melatonin receptor 2-BMAL1-anti-oxidative effects. In high-fat diet-induced and carbon tetrachloride-induced hepatic fibrosis models, oral melatonin administration decreased the expression of hepatic fibrosis markers and increased the expression of messenger RNA and levels of proteins of BMAL1 and melatonin receptor 2. Thus, melatonin exerted protective effects against hepatic fibrosis through melatonin receptor 2 activation, followed by an upregulation of the BMAL1-anti-oxidative enzyme pathways.

An RORα agonist, ODH-08, inhibits fibrogenic activation of hepatic stellate cells via suppression of SMAD3

AimsHepatic fibrosis is a dynamic process characterized by the net accumulation of an extracellular matrix resulting from chronic liver injury such as nonalcoholic steatohepatitis. Activation of hepatic stellate cells (HSCs) plays a role in transdifferentiation of quiescent cells into fibrogenic myofibroblasts. We aimed to examine the function of retinoic acid receptor-related orphan receptor alpha (RORα) and its novel agonistic ligand, 1-(4-benzyloxybenzyl)-3-(2-dimethylaminoethyl)-thiourea (ODH-08) against activation of HSCs using hepatic fibrosis mouse models. Main methodsChemical synthesis, a reporter gene assay, surface plasmon resonance analysis, and a docking study were performed to evaluate ODH-08 as a ligand of RORα. In vivo experiments with mice fed a Western diet were performed to evaluate the effect of ODH-08. The human HSC line, Lx-2, and primary mouse HSCs were employed to identify the molecular mechanisms underlying the antifibrogenic effect of ODH-08. Key findingsA novel RORα-selective ligand, ODH-08, was developed based on modification of JC1-40, an analog of N-methylthiourea. Administration of ODH-08 to the Western diet-fed mice reduced hepatic collagen deposition and expression levels of fibrogenic markers such as α-smooth muscle actin and collagen type I alpha 1 chain. Activation of RORα—either by transient overexpression of RORα or treatment with ODH-08—suppressed the expression of fibrogenic proteins in HSCs. The activation of RORα suppressed the activity of SMAD2 and 3, which are the primary downstream proteins of transforming growth factor β. SignificanceRORα and its agonist ODH-08 have a potent antifibrotic effect, which could provide a novel antifibrotic strategy against hepatic fibrosis.

The inhibition of YAP Signaling Prevents Chronic Biliary Fibrosis in the Abcb4-/- Model by Modulation of Hepatic Stellate Cell and Bile Duct Epithelium Cell Pathophysiology.

Primary sclerosing cholangitis (PSC) represents a chronic liver disease characterized by poor prognosis and lacking causal treatment options. Yes-associated protein (YAP) functions as a critical mediator of fibrogenesis; however, its therapeutic potential in chronic biliary diseases such as PSC remains unestablished. The objective of this study is to elucidate the possible significance of YAP inhibition in biliary fibrosis by examining the pathophysiology of hepatic stellate cells (HSC) and biliary epithelial cells (BEC). Human liver tissue samples from PSC patients were analyzed to assess the expression of YAP/connective tissue growth factor (CTGF) relative to non-fibrotic control samples. The pathophysiological relevance of YAP/CTGF in HSC and BEC was investigated in primary human HSC (phHSC), LX-2, H69, and TFK-1 cell lines through siRNA or pharmacological inhibition utilizing verteporfin (VP) and metformin (MF). The Abcb4-/- mouse model was employed to evaluate the protective effects of pharmacological YAP inhibition. Hanging droplet and 3D matrigel culture techniques were utilized to investigate YAP expression and activation status of phHSC under various physical conditions. YAP/CTGF upregulation was observed in PSC patients. Silencing YAP/CTGF led to inhibition of phHSC activation and reduced contractility of LX-2 cells, as well as suppression of epithelial-mesenchymal transition (EMT) in H69 cells and proliferation of TFK-1 cells. Pharmacological inhibition of YAP mitigated chronic liver fibrosis in vivo and diminished ductular reaction and EMT. YAP expression in phHSC was effectively modulated by altering extracellular stiffness, highlighting YAP's role as a mechanotransducer. In conclusion, YAP regulates the activation of HSC and EMT in BEC, thereby functioning as a checkpoint of fibrogenesis in chronic cholestasis. Both VP and MF demonstrate effectiveness as YAP inhibitors, capable of inhibiting biliary fibrosis. These findings suggest that VP and MF warrant further investigation as potential therapeutic options for the treatment of PSC.

Portulaca oleracea L. extract relieve mice liver fibrosis by inhibiting TLR-4/NF-κB, Bcl-2/Bax and TGF-β1/Smad2 signalling transduction

Portulaca oleracea L. are annual herb, which has various pharmacological effects including hepatoprotective property. However, the effect of Portulaca oleracea L. (POL-1) in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and its mechanism of action have not been clarified. POL-1 ameliorated the CCl4-induced liver fibrosis in mice, as shown by decreased collagen deposition and the decreased expression of liver fibrosis marker collagen I and α-smooth muscle actin (α-SMA) mRNA. In addition, treatment with POL-1 suppressed the proliferation of activated human hepatic stellate cell line (LX-2). POL-1 inhibited the oxidative stress and inflammation in fibrotic livers of mice. Mechanistically, POL-1 inhibited the CCl4-induced expression of toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor kappa-B (NF-κBp65) p65, Bcl2-associated X (Bax), transforming growth factor-β1 (TGF-β1) and drosophila mothers against decapentaplegic 2 (Smad2) proteins, upregulated B-cell lymphoma −2 (Bcl-2) proteins in livers of mice. These findings suggested that POL-1 attenuated liver fibrosis.

Development of an optimized, non-stem cell line for intranasal delivery of therapeutic cargo to the central nervous system.

Neural stem cells (NSCs) are considered to be valuable candidates for delivering a variety of anti-cancer agents, including oncolytic viruses, to brain tumors. However, owing to the previously reported tumorigenic potential of NSC cell lines after intranasal administration (INA), here we identified the human hepatic stellate cell line LX-2 as a cell type capable of longer resistance to replication of oncolytic adenoviruses (OAVs) as a therapeutic cargo, and that is non-tumorigenic after INA. Our data show that LX-2 cells can longer withstand the OAV XVir-N-31 replication and oncolysis than NSCs. By selecting the highly migratory cell population out of LX-2, an offspring cell line with a higher and more stable capability to migrate was generated. Additionally, as a safety backup, we applied genomic herpes simplex virus thymidine kinase (HSV-TK) integration into LX-2, leading to high vulnerability to ganciclovir (GCV). Histopathological analyses confirmed the absence of neoplasia in the respiratory tracts and brains of immuno-compromised mice 3 months after INA of LX-2 cells. Our data suggest that LX-2 is a novel, robust, and safe cell line for delivering anti-cancer and other therapeutic agents to the brain.

Serum Exosome-Derived microRNA-193a-5p and miR-381-3p Regulate Adenosine 5'-Monophosphate-Activated Protein Kinase/Transforming Growth Factor Beta/Smad2/3 Signaling Pathway and Promote Fibrogenesis.

Liver fibrosis results from chronic liver injury and inflammation, often leading to cirrhosis, liver failure, portal hypertension, and hepatocellular carcinoma. Progress has been made in understanding the molecular mechanisms underlying hepatic fibrosis; however, translating this knowledge into effective therapies for disease regression remains a challenge, with considerably few interventions having entered clinical validation. The roles of exosomes during fibrogenesis and their potential as a therapeutic approach for reversing fibrosis have gained significant interest. This study aimed to investigate the association between microRNAs (miRNAs) derived from serum exosomes and liver fibrosis and to evaluate the effect of serum exosomes on fibrogenesis and fibrosis reversal, while identifying the underlying mechanism. Using serum samples collected from healthy adults and paired histologic patients with advanced fibrosis or cirrhosis, we extracted human serum exosomes by ultrahigh-speed centrifugation. Transcriptomic analysis was conducted to identify dysregulated exosome-derived miRNAs. Liver fibrosis-related molecules were determined by qRT-PCR, Western blot, Masson staining, and immunohistochemical staining. In addition, we analyzed the importance of serum exosome-derived miRNA expression levels in 42 patients with advanced fibrosis or cirrhosis. Exosome-derived miR-193a-5p and miR-381-3p were associated with fibrogenesis, as determined by transcriptomic screening. Compared with healthy control group, the high expression of serum exosome-derived miR-193a-5p and miR-381-3 in chronic hepatitis B (n = 42) was closely associated with advanced liver fibrosis and cirrhosis. In vitro , exosome-derived miRNA-193a-5p and miR-381-3p upregulated the expression of α-smooth muscle actin, collagen 1a1, and tissue inhibitors of metalloproteinase 1 in the human hepatic stellate cell line at both mRNA and protein levels. Serum exosome-derived miR-193a-5p and miR-381-3p regulated the adenosine 5'-monophosphate-activated protein kinase/transforming growth factor beta/Smad2/3 signaling pathway and promoted fibrogenesis.

Study on the molecular mechanism of dihydromyricetin in alleviating liver cirrhosis based on network pharmacology.

Dihydromyricetin (DHM) is a bioactive flavonoid extracted from Hovenia dulcis, which has various activities. In the present study, the molecular mechanism of dihydromyricetin (DHM) in relieving liver cirrhosis was investigated through network pharmacology and experimental verification. The cell model was induced by TGF-β1 activating the human hepatic stellate cell line (HSC; LX-2). The protein levels of α-SMA, collagen I, and collagen III and pathway-related proteins within LX-2 cells were detected using Western blot. EdU staining was conducted to detect cell proliferation. Immunofluorescence staining was performed to detect the expression levels of α-SMA and collagen I. Next, the drug targets of DHM were screened from the PubChem database. The differentially expressed genes in the liver cirrhosis dataset GSE14323 were identified. The expression of the identified drug targets in LX-2 cells was verified using qRT-PCR. The results showed that TGF-β1 treatment notably increased LX-2 cell viability, promoted cell proliferation, and elevated α-SMA, collagen I, and collagen III protein contents. DHM treatment could partially eliminate TGF-β1 effects, as evidenced by the inhibited cell viability and proliferation and reduced α-SMA, collagen I, and collagen III contents. After network pharmacology analysis, nine differentially expressed target genes (MMP2, PDGFRB, PARP1, BCL2L2, ABCB1, TYR, CYP2E1, SQSTM1, and IL6) in liver cirrhosis were identified. According to qRT-PCR verification, DHM could inhibit the expression of MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, and IL6, and enhance ABCB1 expression levels within LX-2 cells. Moreover, DHM inhibited mTOR and MAPK signaling pathways in TGF-β1-induced HSCs. In conclusion, DHM could inhibit HSC activation, which may be achieved via acting on MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, IL6, and ABCB1 genes and their downstream signaling pathways, including mTOR and MAPK signaling pathway.

Predictive toxicological effects of Artemisia absinthium essential oil on hepatic stellate cells

Medicinal plants are important worldwide, considering their properties for treating diseases; however, few studies have evaluated their toxicological potential. Among them, Artemisia absinthium is frequently used to treat liver diseases, because its essential oil has several popular therapeutic properties. Based on this information, in the present study, we investigated molecular connectors of physiological effects of the Artemisia absinthium essential oil on human hepatic stellate cell line, LX-2, to explore the potential toxicity of the plant on liver cells. LX-2 is a cellular model to investigate mechanisms of liver fibrosis; then, to analyze the essential oil effects LX-2 was cultured under different conditions, treated or not with the essential oil at 0.4 μg/μL for 24 h. Next, fluorescence microscopy analyses, gene expression measurements, and biochemical approaches revealed that the essential oil reduced pro-fibrogenic markers; however, disrupt lipid metabolism, and cause cellular stress, by the activation of cellular detoxification and pro-inflammatory processes. In conclusion, the hepatic stellate cells incubated with the essential oil present an antifibrotic potential, supporting its popular use; however, the combined results suggest that the essential oil of Artemisia absinthium should be used with caution.

Calcitriol Inhibits NaAsO2 Triggered Hepatic Stellate Cells Activation and Extracellular Matrix Oversecretion by Activating Nrf2 Signaling Pathway Through Vitamin D Receptor.

Previous studies, including our own, have demonstrated that arsenic exposure can induce liver fibrosis, while the underlying mechanism remains unclear and there is currently no effective pharmacological intervention available. Recent research has demonstrated that vitamin D supplementation can ameliorate liver fibrosis caused by various etiologies, potentially through modulation of the Nrf2 signaling pathways. However, it remains unclear whether vitamin D intervention can mitigate arsenic-caused liver fibrosis. As is known hepatic stellate cells (HSCs) activation and extracellular matrix (ECM) deposition are pivotal in the pathogenesis of liver fibrosis. In this study, we investigated the intervention effect of calcitriol (a form of active vitamin D) on arsenite-triggered Lx-2 cells (a human hepatic stellate cell line) activation and ECM oversecretion. Additionally, we also elucidated the role and mechanism of Nrf2 antioxidant signaling pathway. Our results demonstrated that calcitriol intervention significantly inhibits Lx-2 cell activation and ECM oversecretion induced by arsenite exposure. Additionally, calcitriol activates Nrf2 and its downstream antioxidant enzyme expression in Lx-2 cells, thereby reducing ROS overproduction caused by arsenite exposure. Further investigation reveals that calcitriol activates the Nrf2 signaling pathway and inhibits arsenite-triggered Lx-2 cell activation and ECM oversecretion by targeting vitamin D receptor (VDR). In conclusion, this study has demonstrated that vitamin D intervention can effectively inhibit HSC activation and ECM oversecretion triggered by arsenite exposure through its antioxidant activity. This provides a novel strategy for targeted nutritional intervention in the treatment of arsenic-induced liver fibrosis.

Curcumin inhibits the activity and induces apoptosis of activated hepatic stellate cell by suppressing autophagy.

Curcumin, a kind of natural compound, has been previously proven to inhibit the autophagy in hepatic stellate cells (HSCs) and induce their apoptosis. However, it is not clear whether the enhanced apoptosis of activated HSCs (aHSCs) caused by curcumin depends on autophagy inhibition. We aim to verify this hypothesis and explore the potential mechanisms in this study. Immortalized human HSC line LX-2 was used as an experimental specimen and pretreated with transforming growth factor β1(TGF-β1) for 24 h to activate it before drug application. The levels of autophagy, apoptosis, cell activity, lipid metabolism, and the activity of the PI3K/Akt/mTOR signal pathway were evaluated by multiple methods, such as Western blotting, mcherry-EGFP-LC3B adenoviruses transfection, immunofluorescence, Nile Red staining, flow cytometry among others. Our results showed that rapamycin, an autophagy activator, could partly offset the effects of curcumin on autophagy and apoptosis of LX-2 cells, while 3-Methyladenine (3-MA), an autophagy inhibitor, could enhance these effects. Furthermore, curcumin could promote the activity of the PI3K/Akt/mTOR signal pathway in LX-2 cells, while PI3K inhibitor could partly offset this effect and increase the autophagy level. Overall, we demonstrated that curcumin could inhibit the activity and promote LX-2 cells apoptosis by suppressing autophagy by activating the PI3K/Akt/mTOR signal pathway. In addition, lipid recovery and energy deprivation due to autophagy inhibition may be the exact mechanism by which curcumin attenuates the pro-fibrotic activity of LX-2.

Polyenylphosphatidylcholine as bioactive excipient in tablets for the treatment of liver fibrosis

Liver fibrosis is a condition characterized by the accumulation of extracellular matrix (ECM) arising from the myofibroblastic transdifferentiation of hepatic stellate cells (HSCs) occurring as the natural response to liver damage. To date, no pharmacological treatments have been specifically approved for liver fibrosis. We recently reported a beneficial effect of polyenylphosphatidylcholines (PPCs)-rich formulations in reverting fibrogenic features of HSCs. However, unsaturated phospholipids’ properties pose a constant challenge to the development of tablets as preferred patient-centric dosage form. Profiting from the advantageous physical properties of the PPCs-rich Soluthin® S 80 M, we developed a tablet formulation incorporating 70% w/w of this bioactive lipid. Tablets were characterized via X-ray powder diffraction, thermogravimetry, and Raman confocal imaging, and passed the major compendial requirements. To mimic physiological absorption after oral intake, phospholipids extracted from tablets were reconstituted as protein-free chylomicron (PFC)-like emulsions and tested on the fibrogenic human HSC line LX-2 and on primary cirrhotic rat hepatic stellate cells (PRHSC). Lipids extracted from tablets and reconstituted in buffer or as PFC-like emulsions exerted the same antifibrotic effect on both activated LX-2 and PRHSCs as observed with plain S 80 M liposomes, showing that the manufacturing process did not interfere with the bioactivity of PPCs.

Formation and Investigation of Cell-Derived Nanovesicles as Potential Therapeutics against Chronic Liver Disease.

A new therapeutic approach using cell-derived nanovesicles (cdNVs) is offered here to overcome the lack of effective treatments for liver fibrosis, a reversible chronic liver disease. To achieve this goal the formation and purification of cdNVs from untreated, quiescent-like, or activated LX-2 cells, an immortalized human hepatic stellate cell (HSC) line with key features of transdifferentiated HSCs are established. Analysis of the genotype and phenotype of naïve and transdifferentiated LX-2 cells activated through transforming growth factor beta 1, following treatment with cdNVs, reveals a concentration-dependent fibrosis regression. The beneficial fibrosis-resolving effects of cdNVs are linked to their biomolecular corona. Liposomes generated using lipids extracted from cdNVs exhibit a reduced antifibrotic response in perpetuated LX-2 cells and show a reduced cellular uptake. However, incubation with soluble factors collected during purification results in a new corona, thereby restoring fibrosis regression activity. Overall, cdNVs display encouraging therapeutic properties, making them a promising candidate for the development of liver fibrosis resolving therapeutics.

Anlotinib Attenuates Liver Fibrosis by Regulating the Transforming Growth Factor β1/Smad3 Signaling Pathway.

Hepatic stellate cell hyperactivation is a central link in liver fibrosis development, transforming growth factor β1 (TGF-β1) is a key activator of HSCs. This study investigated whether anlotinib attenuates CCl4 induced liver fibrosis in mice and explored its antifibrotic mechanism. We used the human hepatic stellate cell line LX-2 for in vitro assays and used TGF-β1 to induce hepatic fibrosis in LX-2 cells. We analyzed cytotoxicity using a cell-counting kit-8 and transwell chambers to detect the migratory ability of LX-2 cells. Western blotting was used to detect the protein levels of collagen type I, α-smooth muscle actin, and p-Smad3. In addition, mice with CCl4-induced hepatic fibrosis were used as in vivo models. Histopathological examination was performed using H&E staining, Masson's trichrome staining, and immunohistochemistry. Anlotinib significantly reversed TGF-β1-induced protein levels of Col I, α-SMA and p-Smad3 and inhibits migratory and proliferative abilities in vitro using LX-2 cells. CCl4 cause F4 grade (Ishak) hepatic fibrosis, liver inflammatory scores ranged from 12 to 14 (Ishak), a mean ALT measurement of 130 U/L and a mean measurement AST value of 119 U/L in mice. However, the CCl4-induced changes were markedly attenuated by anlotinib treatment, which returned to F2 grade (Ishak) hepatic fibrosis, liver inflammatory scores ranged from 4 to 6 (Ishak), a mean ALT measurement of 40 U/L and a mean measurement AST value of 56 U/L in mice. Our results suggest that anlotinib-mediated suppression of liver fibrosis is related to the inhibition of TGF-β1 signaling pathway. Hepatic stellate cell hyper activation is a central link in liver fibrosis development, transforming growth factor β1 is a key activator of HSCs. Anlotinib is a multi-targeted tyrosine kinase inhibitor that has similar targets to nintedanib, a clinically used anti-pulmonary fibrosis drug. Our study demonstrates an FDA-approved drug-anlotinib-that could prevent liver fibrosis and inflammation. Experiments in cell cultures and mice show that anlotinib can inhibit the activation of hepatic stellate cells by down-regulating the TGFβ1/smad3 pathway, thereby reversing liver fibrosis. In animal experiments, anlotinib showed protective effects on the CCl4-induced liver damage, including ameliorating liver inflammation, reversing liver fibrosis and reducing liver enzymes. This is a very good signal, anlotinib may be useful for halting or reversing the progression of liver fibrosis and could be employed in the development of novel therapeutic drugs for the management of chronic liver diseases.

CREKA-modified liposomes target activated hepatic stellate cells to alleviate liver fibrosis by inhibiting collagen synthesis and angiogenesis.

Activated hepatic stellate cells (HSCs) are considered the key driver of excessive extracellular matrix and abnormal angiogenesis, which are the main pathological manifestations of hepatic fibrosis. However, the absence of specific targeting moieties has rendered the development of HSC-targeted drug delivery systems a significant obstacle in the treatment of liver fibrosis. Here we have identified a notable increase in fibronectin expression on HSCs, which positively correlates with the progression of hepatic fibrosis. Thus, we decorated PEGylated liposomes with CREKA, a peptide with high affinity for fibronectin, to facilitate the targeted delivery of sorafenib to activated HSCs. The CREKA-coupled liposomes exhibited enhanced cellular uptake in the human hepatic stellate cell line LX2 and selective accumulation in CCl4-induced fibrotic liver through the recognition of fibronectin. When loaded with sorafenib, the CREKA-modified liposomes effectively suppressed HSC activation and collagen accumulation in vitro. Furthermore. in vivo results demonstrated that the administration of sorafenib-loaded CREKA-liposomes at a low dose significantly mitigated CCl4-induced hepatic fibrosis, prevented inflammatory infiltration and reduced angiogenesis in mice. These findings suggest that CREKA-coupled liposomes have promising potential as a targeted delivery system for therapeutic agents to activated HSCs, thereby providing an efficient treatment option for hepatic fibrosis. STATEMENT OF SIGNIFICANCE: In liver fibrosis, activated hepatic stellate cells (aHSCs) are the key driver of extracellular matrix and abnormal angiogenesis. Our investigation has revealed a significant elevation in fibronectin expression on aHSCs, which is positively associated with the progression of hepatic fibrosis. Thus, we developed PEGylated liposomes decorated with CREKA, a molecule with a high affinity for fibronectin, to facilitate the targeted delivery of sorafenib to aHSCs.The CREKA-coupled liposomes can specifically target aHSCs both in vitro and in vivo. Loading sorafenib into CREKA-Lip significantly alleviated CCl4-induced liver fibrosis, angiogenesis and inflammation at low doses. These findings suggest that our drug delivery system holds promise as a viable therapeutic option for liver fibrosis with minimal risk of adverse effects.

Nano-calcipotriol as a potent anti-hepatic fibrosis agent.

Calcipotriol (CAL) has been widely studied as a fibrosis inhibitor and used to treat plaque psoriasis via transdermal administration. The clinical application of CAL to treat liver fibrosis is bottlenecked by its unsatisfactory pharmacokinetics, biodistribution, and side effects, such as hypercalcemia in patients. The exploration of CAL as a safe and effective antifibrotic agent remains a major challenge. Therefore, we rationally designed and synthesized a self-assembled drug nanoparticle encapsulating CAL in its internal hydrophobic core for systematic injection (termed NPs/CAL) and further investigated the beneficial effect of the nanomaterial on liver fibrosis. C57BL/6 mice were used as the animal model, and human hepatic stellate cell line LX-2 was used as the cellular model of hepatic fibrogenesis. Immunofluorescence staining, flow cytometry, western blotting, immunohistochemical staining, and in vitro imaging were used for evaluating the efficacy of NPs/CAL treatment. We found NPs/CAL can be quickly internalized in vitro, thus potently deactivating LX-2 cells. In addition, NPs/CAL improved blood circulation and the accumulation of CAL in liver tissue. Importantly, NPs/CAL strongly contributed to the remission of liver fibrosis without inducing hypercalcemia. Overall, our work identifies a promising paradigm for the development of nanomaterial-based agents for liver fibrosis therapy.

Aucubin inhibits hepatic stellate cell activation through stimulating Nrf2/Smad7 axis

AimLiver fibrosis may develop into end-stage liver disease if left unprevented. The study is attempting to identify a compound to ameliorate liver fibrosis progression with high efficiency and low toxicity, as well as to analyze its potential molecular mechanism. MethodsThe drug screening was performed using human hepatic stellate cell line LX-2 for identifying the compound as collagen I inhibitor. Primary Human hepatic stellate cells and LX-2 cell line were used to detect the antifibrotic function activity and molecular mechanism analysis in vitro. The CCl4-induced mouse experimental model was used to measure the amelioration in liver fibrosis. ResultsThis study identified Aucubin, a natural compound, as a candidate for anti-liver fibrosis. Besides, Aucubin could inhibit the collagen I and α-SMA expressions in LX-2 cells and primary human hepatic stellate cells, as well as the cell proliferation. In terms of mechanism, Aucubin could upregulate Smad7 in hepatic stellate cells in a dose-dependent manner and block TGF-β signaling. We also found that Nrf2 might be a direct target for the action of Aucubin, whose activation was necessary for Smad7 upregulation. In an in-vivo mouse model, Aucubin efficiency ameliorated the progression of CCl4-induced liver fibrosis, and reduced the hepatic levels of collagen deposition, transaminase and inflammatory cytokines. ConclusionCapable of inhibiting the activation of hepatic stellate cells in vitro and in vivo, Aucubin may be a potential therapeutic candidate for liver fibrosis, which is dependent on the suppression of TGF-β signaling through stimulating Nrf2/Smad7 axis.

Kinetin inhibits hepatic stellate cell activation and induces apoptosis via interactions with the TGF-β1/Smad signaling pathway

Hepatic fibrosis is the pathological repair response of the liver to chronic injury; hepatic stellate cell (HSC) activation is the central link in the pathogenesis of hepatic fibrosis. Previously, we showed that kinetin, a plant cytokinin hormone, has a protective effect on CCl4-induced liver injury in mice. However, the role of kinetin in liver fibrosis remains unclear. We aimed to study these protective effects and to determine the mechanisms by which kinetin mediates HSC activation and apoptosis. For this purpose, the human HSC line LX-2 was treated with 10 ng/ml transforming growth factor-β1 (TGF-β1) for 24 h to stimulate activation. We found that treatment with kinetin at the sub-cytotoxic dose of 40 μg/ml for 48 h reduced the expression of the HSC activation marker α-SMA and inhibited the secretion of extracellular matrix proteins. In addition, kinetin was found to inhibit the proliferation and migration of LX-2 cells. We found that kinetin induced apoptosis in LX-2 cells by increasing the level of cleaved-caspase 3 and the Bax-to-Bcl-2 ratio. Interestingly, these effect were not observed in quiescent HSCs, suggesting that they are activation-dependent. Further study showed that kinetin attenuates activation and promotes apoptosis of LX-2 cells in vitro in part by suppressing the TGF-β1/Smad signaling pathway.