Th17 cells are involved in liver fibrosis by activating hepatic stellate cells (HSCs). We aimed to investigate whether HSCs are able to regulate the function of Th17 cells and to determine the relevant mechanism. Sixty-five patients diagnosed with chronic hepatitis B (CHB) were enrolled in this study. To determine the effect of HSCs on T cells, naïve CD4+T cells and Th17 cells were sorted from CHB patients and cultured with or without activated-HSCs, and cytokine expression and gene transcription were analyzed. In addition, the regulatory mechanism of HSCs was investigated. ELISA and qRT-PCR showed that Th17 cells from CHB patients were more pathogenic, on the basis of the expression of IL-17A, IL-23R, RORC, CCL20 and CCR6, and meanwhile, they could activate the primary HSCs. Co-culture experiments indicated that activated HSCs dramatically promoted proliferation of CD4+T cells in a time- and dose-dependent manner. In addition, they could induce naïve CD4+T cells to become Th17 cells which had a more pathogenic phenotype. Moreover, activated HSCs-mediated induction of Th17 cells might depend on the release of IL-1β and IL-6 as well as on the COX-PGE2 pathway. Th17 cells cooperated with HSCs in a proinflammatory feedback loop might provide a better understanding of the pathogenic role of Th17 cells in the chronicity of HBV infection.

Hypoxia is a common environmental stress factor and is associated with fibrogenesis. Matrix metalloproteinase-2 (MMP-2), produced by hepatic stellate cells (HSCs), plays an important role in liver fibrogenesis. However, inconsistent results have been reported on the impact of hypoxia on MMP-2 expression and activity in HSCs. We speculated that cell-cell interaction is involved in the regulation of MMP-2 expression and activity at low oxygen level in vivo. Therefore, in this report we investigated the mechanism by which hypoxic hepatocytes regulates MMP-2 expression in HSCs. Our results showed that the conditioned medium from hypoxia-treated rat hepatocytes strongly induced the expression of MMP-2 mRNA and protein in rat HSC-T6 cells. Reduced glutathione neutralized ROS released from hypoxic hepatocytes, leading to reduced MMP-2 expression in HSC-T6 cells. In addition, phospho-IκB-α protein level was increased in HSC-T6 cells treated with hypoxia conditioned medium, and NF-κB signaling inhibitor inhibited MMP-2 expression in HSC-T6 cells. Taken together, our data suggest that ROS is an important factor released by hypoxic hepatocytes to regulate MMP-2 expression in HSCs, and NF-κB signaling is crucially involved in ROS-induced MMP-2 expression in HSCs. Our findings suggest that strategies aimed at antagonizing the generation of ROS in hypoxic hepatocytes and inhibiting NF-κB signaling in HSCs may represent novel therapeutic options for liver fibrosis.

Recently, emerging evidence shows that dysregulation of circadian genes is closely associated with liver fibrosis. However, how dysregulation of circadian genes promotes liver fibrosis is unknown. In this study, we show that neuronal PAS domain protein 2 (NPAS2), one of the core circadian molecules that has been shown to promote hepatocarcinoma cell proliferation, significantly contributed to liver fibrogenesis. NPAS2 is upregulated in hepatic stellate cells (HSCs) after fibrogenic injury, which subsequently contributes to the activation of HSCs. Mechanistically, NPAS2 plays a profibrotic role via direct transcriptional activation of hairy and enhancer of split 1 (Hes1), a critical transcriptor of Notch signaling for the fibrogenesis process, in HSCs. Our findings demonstrate that NPAS2 plays a critical role in liver fibrosis through direct transcriptional activation of Hes1, indicating that NPAS2 may serve as an important therapeutic target to reverse the progression of liver fibrosis.

Reverting activated hepatic stellate cells (HSCs) to less activation or quiescent status is a promising strategy for liver fibrosis. Histone deacetylase inhibitor (HDACI) could suppress HSCs activation. Our previous study demonstrated a critical role of miRNAs in HSCs activation. Here, we explored the involvement of miRNAs in HDACI induced HSCs deactivation. Human cell line LX2 that resembled activated HSCs was treated with an HDACI - valproic acid (VPA). The effects of VPA on the protein and miRNA profile of LX2 were comprehensively analyzed by iTraq quantitative proteomics and miRNA microarray. The interaction between miRNA and proteins was investigated systematically. The biofunctions of differentially expressed proteins and miRNA targeted proteins were annotated. VPA treatment attenuated the activation phenotype of LX2. In VPA treated LX2, among 1548 quantified proteins, only 86 proteins were differentially expressed (VPA-proteins). While among 282 high-abundance miRNAs, 123 were differentially expressed (VPA-miRNAs), with 104 down-regulated and 19 up-regulated. The top biofunctions of VPA-proteins were closely related to HSCs activation, including cell death and survival, cell movement, cellular growth and proliferation. Furthermore, 22 out of the 36 VPA-proteins involved in cell death and survival, and 19 out of the 30 VPA-proteins involved in cellular movement were predicted targets of VPA-miRNAs. A direct regulatory effect of histone acetylation on miRNA expression was also established. In conclusion, our data provided the molecular mechanisms for VPA induced HSCs deactivation at the protein level and suggested crosstalk between histone acetylation and miRNAs in the inhibitory effects of HDACI on HSCs activation.

Icaritin is an active ingredient extracted from the plant Herba Epimedium Sagittatum (Sieb. et Zucc.) Maxim. The purpose of this study is to investigate the effects and mechanisms of icaritin-induced cell death in activated hepatic stellate cells (HSCs) and ameliorating the development of liver fibrosis in rats.

The activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis, however non-alcoholic fatty liver disease (NAFLD) associated liver fibrogenesis have been poorly understood. We aimed to determine the significance of mineralocorticoid receptor (MR)/osteopontin (OPN)/high-mobility group box-1 (HMGB1) axis in this setting.

Hepatic fibrosis is a public health problem characterized by activation of hepatic stellate cells (HSCs), which triggers excessive production of extracellular matrix (ECM). Inhibition of HSC activation may be an effective treatment. Since various pathways control HSC activation, a combination of drugs with different mechanisms may be more effective than monotherapy.

Various stimuli, including Clonorchis sinensis infection, can cause liver fibrosis. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) with massive production of extracellular matrix (ECM). Our previous study showed that the TGF-β1-induced Smad signaling pathway played a critical role in the activation of HSCs during liver fibrosis induced by worm infection; however, the mechanisms that modulate the TGF-β/Smad signaling pathway are still poorly understood. Accumulating evidence demonstrates that miRNAs act as an important regulator of activation of HSCs during liver fibrosis.

The activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis progression. Phospholipase D (PLD) enzymes participate in multiple cellular activities. However, whether and how PLD regulates HSCs activation remain elusive.

Chronic liver allograft dysfunction (CLAD) remains the most common cause of patient morbidity and allograft loss in liver transplant patients. However, the pathogenesis of CLAD has not been completely elucidated. By establishing rat CLAD models, in this study, we identified the informative CLAD-associated genes using isobaric tags for relative and absolute quantification (iTRAQ) proteomics analysis and validated these results in recipient rat liver allografts. CXCL4, CXCR3, EGFR, JAK2, STAT3, and Collagen IV were associated with CLAD pathogenesis. We validated that CXCL4 is upstream of these informative genes in the isolated hepatic stellate cells (HSC). Blocking CXCL4 protects against CLAD by reducing liver fibrosis. Therefore, our results indicated that therapeutic approaches that neutralize CXCL4, a newly identified target of fibrosis, may represent a novel strategy for preventing and treating CLAD after liver transplantation.

The severe shortage of donor liver organs requires the development of alternative methods to provide transplantable liver tissues such as stem cell-derived organoids. Despite several studies describing the generation of vascularized and functional liver tissues, none have succeeded in assembling human liver buds containing hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs). Here, we report a reproducible, easy-to-follow, and comprehensive self-assembly protocol to generate three-dimensional (3D) human liver buds from naïve mesenchymal stem cells (MSCs), MSC-derived hepatocytes, and HSC- and LSEC-like cells. By optimizing the ratio between these different cell lineages, the cell mixture self-assembled into 3D human liver buds within 72 h in vitro, and exhibited similar characteristics with early-stage murine liver buds. In a murine model of acute liver failure, the mesenteric transplantation of self-assembled human liver buds effectively rescued animal death, and triggered hepatic ameliorative effects that were better than the ones observed after splenic transplantation of human hepatocytes or naïve MSCs. In addition, transplanted human liver buds underwent maturation during injury alleviation, after which they exhibited a gene expression profile signature similar to the one of adult human livers. Collectively, our protocol provides a promising new approach for the in vitro construction of functional 3D human liver buds from multiple human MSC-derived hepatic cell lineages; this new technique would be useful for clinical transplantation and regenerative medicine research.

A pyrimidine moiety exhibiting a wide range of pharmacological activities has been employed in the design of privileged structures in medicinal chemistry. To prepare libraries of novel heterocyclic compounds with potential biological activities, a series of novel 2-(pyridin-2-yl) pyrimidine derivatives were designed, synthesized and their biological activities were evaluated against immortalized rat hepatic stellate cells (HSC-T6). Fourteen compounds were found to present better anti-fibrotic activities than Pirfenidone and Bipy55'DC. Among them, compounds ethyl 6-(5-(p-tolylcarbamoyl)pyrimidin-2-yl)nicotinate (12m) and ethyl 6-(5-((3,4-difluorophenyl)carbamoyl)pyrimidin-2-yl)nicotinate (12q) show the best activities with IC50 values of 45.69 μM and 45.81 μM, respectively. Furthermore, the study of anti-fibrosis activity was evaluated by Picro-Sirius red staining, hydroxyproline assay and ELISA detection of Collagen type I alpha 1 (COL1A1) protein expression. Our study showed that compounds 12m and 12q effectively inhibited the expression of collagen, and the content of hydroxyproline in cell culture medium in vitro, indicating that compounds 12m and 12q might be developed the novel anti-fibrotic drugs.

Liver fibrosis is a severe disease characterized by excessive deposition of extracellular matrix (ECM) components in the liver. Activated hepatic stellate cells (HSCs) are a major source of ECM and a key regulator of liver fibrosis. Collagen type I alpha I (COL1A1) is one of the main components of ECM and is a major component in fibrotic tissues. Previously, we demonstrated that soluble egg antigen from Schistosoma japonicum could inhibit the expression of COL1A1 in activated HSCs. In addition, studies have found that Ets proto-oncogene 1 (Ets-1) suppresses the production of ECM by down-regulating matrix related genes such as COL1A1 induced by transforming growth factor β, and ultimately inhibits liver fibrosis. In this study, the major aim was to investigate the effect and mechanism of Ets-1 on inhibiting COL1A1 gene promoter activity in HSCs by recombinant Schistosoma japonicum protein P40 (rSjP40). We observed the rSjP40 inhibited the expression of COL1A1 by inhibiting the activity of the COL1A1 promoter, and the core region of rSjP40 acting on COL1A1 promoter was located at -1,722/-1,592. In addition, we also demonstrated that rSjP40 could promote the expression of Ets-1, and Ets-1 has a negative regulation effect on the COL1A1 promoter in human LX-2 cells. These data suggest that rSjP40 might inhibit the activity of COL1A1 promoter and inhibit the activation of HSCs by increasing the expression of transcription factor Ets-1, which will provide a new experimental basis for the prevention and treatment of liver fibrosis.

Fibrosis of lung tissue can induce the occurrence and development of numerous types of lung disease. The expression levels of the long non‑coding RNA (lncRNA) HOXA distal transcript antisense RNA (HOTTIP) have been reported to be upregulated during the development of fibrosis in liver tissues, which subsequently activated hepatic stellate cells. However, whether the lncRNA HOTTIP participates in the occurrence and development of lung fibrosis remains unknown. The present study aimed to investigate the role of lncRNA HOTTIP in lung fibrosis and its potential mechanism. In the present study, A549 cells were stimulated with TGF‑β1 to induce lung fibrosis in vitro. A549 was transfected with short hairpin RNA‑HOTTP, overexpression‑polypyrimidine tract binding protein 1 (PTBP1), microRNA (miR)‑744‑5p mimic or miR‑744‑5p to regulate gene expression. Cell proliferation and migration were determined using 5'‑ethynl‑2'‑deoxyuridine and wound healing assays, respectively. The expression levels of α‑smooth muscle actin, collagen I, collagen III and fibronectin 1 were analyzed using western blotting. starBase was used to identify molecules that may interact with the lncRNA HOTTIP and dual luciferase reporter assays were used to validate the findings. Moreover, an in vivo lung fibrosis model was established by bleomycin induction in mice. Histological injury was observed using hematoxylin and eosin and masson staining. The results of the present study revealed that the proliferation and migration of A549 cells were both suppressed following the knockdown of HOTTIP. The lncRNA HOTTIP was found to target and downregulate the expression levels of miR‑744‑5p. The overexpression of miR‑744‑5p inhibited the proliferation and migration of A549 cells. Furthermore, miR‑744‑5p targeted and downregulated the expression levels of PTBP1. It was subsequently demonstrated that the overexpression of PTBP1 rescued miR‑744‑5p‑induced suppression of the proliferation and migration of A549 cells. The knockdown of lncRNA HOTTIP expression also relieved the fibrosis of the lung tissues of mice. In conclusion, the results of the present study suggested that the lncRNA HOTTIP may promote the fibrosis of lung tissues by downregulating the expression levels of miR‑744‑5p and upregulating the expression levels of PTBP1.