Apoptosis Of Hepatic Stellate Cells Research Articles

EZH2-mediated inhibition of KLF14 expression promotes HSCs activation and liver fibrosis by downregulating PPARγ.

ObjectivesInduction of deactivation and apoptosis of hepatic stellate cells (HSCs) are principal therapeutic strategies for liver fibrosis. Krüppel‐like factor 14 (KLF14) regulates various biological processes, however, roles, mechanisms and implications of KLF14 in liver fibrosis are unknown.Materials and MethodsKLF14 expression was detected in human, rat and mouse fibrotic models, and its effects on HSCs were assessed. Chromatin immunoprecipitation assays were utilized to investigate the binding of KLF14 to peroxisome proliferator‐activated receptor γ (PPARγ) promoter, and the binding of enhancer of zeste homolog 2 (EZH2) to KLF14 promoter. In vivo, KLF14‐overexpressing adenovirus was injected via tail vein to thioacetamide (TAA)‐treated rats to investigate the role of KLF14 in liver fibrosis progression. EZH2 inhibitor EPZ‐6438 was utilized to treat TAA‐induced rat liver fibrosis.ResultsKLF14 expression was remarkably decreased in human, rat and mouse fibrotic liver tissues. Overexpression of KLF14 increased LD accumulation, inhibited HSCs activation, proliferation, migration and induced G2/M arrest and apoptosis. Mechanistically, KLF14 transactivated PPARγ promoter activity. Inhibition of PPARγ blocked the suppressive role of KLF14 overexpression in HSCs. Downregulation of KLF14 in activated HSCs was mediated by EZH2‐regulated histone H3 lysine 27 trimethylation. Adenovirus‐mediated KLF14 overexpression ameliorated TAA‐induced rat liver fibrosis in PPARγ‐dependent manner. Furthermore, EPZ‐6438 dramatically alleviated TAA‐induced rat liver fibrosis. Importantly, KLF14 expression was decreased in human with liver fibrosis, which was significantly correlated with EZH2 upregulation and PPARγ downregulation.ConclusionsKLF14 exerts a critical anti‐fibrotic role in liver fibrosis, and targeting the EZH2/KLF14/PPARγ axis might be a novel therapeutic strategy for liver fibrosis.

COX-2 Regulates the Proliferation and Apoptosis of Activated Hepatic Stellate Cells through CDC27

Cyclooxygenase-2 (COX-2) is an important rate-limiting enzyme in the synthesis of prostaglandins (PGs), which can be upregulated by various stimuli. COX-2 has been shown to be involved in the occurrence and development of hepatic fibrosis by regulating the proliferation and apoptosis of hepatic stellate cells (HSCs) in previous studies. The aims of the study are to study the mechanism of how COX-2 regulates the proliferation and apoptosis of HSCs and to provide new targets for the prevention and treatment of hepatic fibrosis. A short hairpin RNA targeting COX-2 was constructed, and the changes in proliferation and apoptosis of liver tissue cells and HSCs were observed, respectively. COX-2-shRNA-1 significantly suppressed the proliferation of HSCs in vivo. Moreover, knockdown of COX-2 significantly suppressed cell proliferation and accelerated cell cycle arrest and apoptosis in vitro. Among those differential genes related to cell proliferation and apoptosis, CDC27 and Sh3kbp1 were upregulated, but Plcd4 was suppressed. Mechanistically, the influence of COX-2 on HSCs partly depends on upregulating CDC27. Our results demonstrated that COX-2 regulates the proliferation and apoptosis of activated hepatic stellate cells through the CDC27 pathway. This study contributes to our understanding of the effect of COX-2 for the treatment of hepatic fibrosis.

Mechanism of hepatic stellate cell proliferation and apoptosis in rats by promoting circulation, removing stasis, and dredging collaterals based on the theory of liver collateral disease

This study aims to investigate the impacts on the proliferation, inhibition, apoptosis, and cell cycle of hepatic stellate cells (HSC-T6) in rats by using traditional Chinese medicine (TCM) in serum. Twenty-seven rats were randomly divided into nine groups on average: a blank control (BC) group (fed by distilled water), a positive control group (colchicine solution, 0.05 mg/mL), and seven TCM groups [with the aim of clearing the liver of choleresis with 0.24 g/mL (weight of TCM herbs per milliliter), 0.73 g/mL, 1.22 g/mL, 2.45 g/mL, 7.34 g/mL, 12.24 g/mL, 24.48 g/mL]. Each rat received gavage with a dose of 1 mL/100 g twice a day for 7 days to prepare the drug serum. The HSC-T6 were divided into a TCM serum group, a positive control group, and a BC group. The TCM group and the positive control group were given corresponding drug serum for incubation, while the BC group received the medium without drug serum. The cellular proliferation inhibition rate in the positive control and TCM groups was significantly higher (P<0.01) than that in the BC group. The number of cells in the positive, 0.24 g/mL TCM, 1.22 g/mL TCM, and 7.34 g/mL TCM groups increased significantly (P<0.05) in the G0/G1 phase compared with the BC group. The number of cells in the positive group and all the TCM groups clearly increased in phase S by 0.73 g/mL in the TCM group, 2.45 g/mL in the TCM group, 12.24 g/mL in the TCM group, and 24.48 g/mL in the TCM group compared with the BC group, and the difference was statistically significant (P<0.05). The number of cells in the G2/M phase of the positive control group and all the TCM groups decreased significantly. Compared with the BC group, the positive group, 0.24 g/mL TCM group, 0.73 g/mL TCM group, 1.22 g/mL TCM group, 2.45 g/mL TCM group have statistically difference (P<0.05). A Qinggan Lidan prescription may exert anti-hepatic fibrosis effects by blocking HSC-T6 in the G0/G1 and S phases and by suppressing the proliferation of HSC-T6.

CaMK II/Ca2+ dependent endoplasmic reticulum stress mediates apoptosis of hepatic stellate cells stimulated by transforming growth factor beta 1

Previous studies by our group have demonstrated that the calcium imbalance in rat hepatic stellate cells (HSCs) can induce endoplasmic reticulum stress (ERS) and promote cell apoptosis. KN-62, an inhibitor of Calmodulin kinase II (CaMK II), can decrease the expression of CaMK II that plays a major role in regulating the steady state of intracellular Ca2+. Uridine triphosphate (UTP) plays a biological role in increasing indirectly the level of intracellular Ca2+. In the experiment, we demonstrate that KN-62 and UTP can inhibit the proliferation and promote the apoptosis in HSCs, increase the level of intracellular Ca2+ and the expression of ERS protein GRP78, and increase the apoptosis protein Caspase-12 and Bax expression, while decrease the expression of Bcl-2 protein. Our findings indicate that the CaMK II/Ca2+ signaling pathway regulates the ERS apoptosis pathway and induces HSC apoptosis.

Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis.

Rationale: As the central hallmark of liver fibrosis, transdifferentiation of hepatic stellate cells (HSCs), the predominant contributor to fibrogenic hepatic myofibroblast responsible for extracellular matrix (ECM) deposition, is characterized with transcriptional and epigenetic remodeling. We aimed to characterize the roles of H3K27 methyltransferase EZH2 and demethylase JMJD3 and identify their effective pathways and novel target genes in HSCs activation and liver fibrosis.Methods: In primary HSCs, we analyzed effects of pharmacological inhibitions and genetic manipulations of EZH2 and JMJD3 on HSCs activation. In HSCs cell lines, we evaluated effects of EZH2 inhibition by DZNep on proliferation, cell cycling, senescence and apoptosis. In CCl4 and BDL murine models of liver fibrosis, we assessed in vivo effects of DZNep administration and Ezh2 silencing. We profiled rat primary HSCs transcriptomes with RNA-seq, screened the pathways and genes associated with DZNep treatment, analyzed EZH2 and JMJD3 regulation towards target genes by ChIP-qPCR.Results: EZH2 inhibition by DZNep resulted in retarded growth, lowered cell viability, cell cycle arrest in S and G2 phases, strengthened senescence, and enhanced apoptosis of HSCs, decreased hepatic collagen deposition and rescued the elevated serum ALT and AST activities of diseased mice, and downregulated cellular and hepatic expressions of H3K27me3, EZH2, α-SMA and COL1A. Ezh2 silencing by RNA interference in vitro and in vivo showed similar effects. JMJD3 inhibition by GSK-J4 and overexpression of wild-type but not mutant Jmjd3 enhanced or repressed HSCs activation respectively. EZH2 inhibition by DZNep transcriptionally inactivated TGF-β1 pathway, cell cycle pathways and vast ECM components in primary HSCs. EZH2 inhibition decreased H3K27me3 recruitment at target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, and increased their expressions, while Jmjd3 overexpression manifested alike effects.Conclusions: EZH2 and JMJD3 antagonistically modulate HSCs activation. The therapeutic effects of DZNep as epigenetic drug in liver fibrosis are associated with the regulation of EZH2 towards direct target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, which are also regulated by JMJD3. Our present study provides new mechanistic insight into the epigenetic modulation of EZH2 and JMJD3 in HSCs biology and hepatic fibrogenesis.

Artesunate alleviates schistosomiasis-induced liver fibrosis by downregulation of mitochondrial complex Ⅰ subunit NDUFB8 and complex Ⅲ subunit UQCRC2 in hepatic stellate cells

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. Inhibition of the HSCs activity is an ideal strategy in the treatment of fibrosis, but there is no drug yet for this strategy. Artesunate (ART) has been shown to protect liver from fibrosis through inhibition of HSCs activity. However, the mechanism of ART activity remains to be fully uncovered. In this study, we tested ART in a mouse model of hepatic fibrosis established in the schistosomiasis-infected mice. The mechanism of ART action was investigated in the HSC cell line LX-2. ART significantly inhibited hepatic fibrosis. In LX-2 cells, ART efficiently inhibited the cell activity in proliferation and mRNA expression of fibrosis marker genes including Col1a1 and Col3a1. An impact of ART on mitochondria was observed for suppression of enzymes in the citric acid cycle (TCA), such as citrate synthase (CS), isocitrate dehydrogenase (IDH2), and alpha ketoglutarate dehydrogenase (OGDH) in a dose-dependent manner. ART decreased the mitochondrial oxygen consumption rate (OCR) and the protein levels of mitochondrial complex Ⅰ subunit NDUFB8 and complex Ⅲ subunit UQCRC2 in HSCs. All of these alterations were observed with an increase in HSC apoptosis. This study suggests that ART may alleviate liver fibrosis by downregulation of HSC activity through suppression of NDUFB8 and UQCRC2 in mitochondria. This study provides a new insight into the mechanism of the ART activity in the inhibition of schistosomiasis-induced liver fibrosis.

Advances in the regulation of functions of hepatic stellate cells by exosomes

Exosomes are small vesicles with a bilayer membrane structure secreted by a variety of cells. They are widely distributed in a variety of body fluids and contain proteins, nucleic acids and other components. They can mediate information transmission between cells and participate in a variety of physiological and pathological activities of cells. Hepatocytes, hepatic sinus endothelial cells and other cells were able to communicate with hepatic stellate cells via exosomes, and regulate the activation, migration, apoptosis and other biological activities of hepatic stellate cells. In this review, the recent advances in the regulation of exosomes on the biological activity of hepatic stellate cells were reviewed.

4-Methoxy Sulfonyl Paeonol Inhibits Hepatic Stellate Cell Activation and Liver Fibrosis by Blocking the TGF-β1/Smad, PDGF-BB/MAPK and Akt Signaling Pathways

Liver fibrosis initiates the progression of cirrhosis, and, finally, hepatocellular carcinoma (HCC). The increased proliferation and activation of hepatic stellate cells (HSCs) are crucial for hepatic fibrogenesis. Paeonol is the major vigorous component of Cortex Moutan, a traditional herbal medicine widely used for treating various diseases. Here, we identified a novel paeonol derivative (4-methoxy sulfonyl paeonol, 4-MSP) that inhibits TGF-β1-induced Smad2/3 phosphorylation and collagen expression in HSCs. 4-MSP pretreatment suppressed the PDGF-BB–induced phosphorylation of MAPK pathway members (MEK/ERK, p38, JNK), Akt/p70S6K, and HSC proliferation. However, 4-MSP treatment had no effect on the induction of apoptosis in HSCs. The microarray experiments showed that 4-MSP treatment affects the TGF-β signaling, MAPK cascade, and other pathways related to HSCs activation and proliferation. The administration of 4-MSP to a liver fibrosis mouse model induced by CCl4 significantly decreased the expression of hepatic fibrosis markers (α-SMA, col1A2, TGF-β, and MMP2), and attenuated hepatic collagen deposition and liver damage. In addition, no adverse effects were observed in 4-MSP exposed mice. In conclusion, this novel paeonol-phenylsulfonyl derivative prevents the progression of liver fibrosis through blocking TGF-β1/Smad, PDGF-BB/MAPK, and Akt signaling, which suggests its use as a novel therapeutic against liver fibrosis.

Malvidin induces hepatic stellate cell apoptosis via the endoplasmic reticulum stress pathway and mitochondrial pathway.

Blueberries have great beneficial effects due to high level of anthocyanins, especially malvidin. Hepatic stellate cells (HSCs) can be activated and increase excessive extracellular matrix (ECM) components, which play a central role in liver fibrogenesis. Therefore, activated HSC’s apoptosis can be induced to recover liver fibrosis. Malvidin's effects on apoptosis in rat activated hepatic stellate T6 cells (HSC‐T6) in vitro were investigated here. High concentration of malvidin was found to significantly induce apoptosis, activate caspase‐3, increase malondialdehyde, upregulate Bax, but downregulate Bcl‐2. Moreover, malvidin upregulated the protein levels of some endoplasmic reticulum stress (ERS)‐typical markers, including caspase‐12, glucose‐regulated protein 78 (GRP78), and CCAAT/enhancer‐binding protein (C/EBP) homologous protein (CHOP), suggesting that malvidin induced HSC apoptosis by the ERS apoptosis pathway as well as the mitochondrial‐dependent pathway. These findings indicated that blueberry anthocyanins, especially malvidin, could induce activated hepatic stellate cell apoptosis and might act as one kind of functional food ingredient or a novel nutraceutical beneficial for liver health.

Kupffer Cell-Derived TNF-α Triggers the Apoptosis of Hepatic Stellate Cells through TNF-R1/Caspase 8 due to ER Stress.

Purpose To investigate the roles of ER stress in Kupffer cells (KCs) and KC-derived TNF-α in the apoptosis of hepatic stellate cells (HSCs). Methods A rat model of liver fibrosis was established. Liver and blood serum samples were collected. Liver function assays, Masson staining, Sirius Red staining, ELISAs, and TUNEL and immunohistochemical staining were performed. Liver function, liver fibrosis, KC phenotype, inflammatory factors, and number of active HSCs were investigated. KCs were isolated, treated with tunicamycin, and then, cocultured with primary hepatic stellate cells. ELISAs, immunofluorescence staining, flow cytometry, and Western blotting were performed. KC phenotype, inflammatory factors, HSC apoptosis, and TNF-R1/caspase 8 pathway activity were examined. Results. ER stress in KCs reduced the levels of liver function markers, reduced the degree of liver fibrosis, and increased the number of KCs with the M1 phenotype and the expression of TNF-α. The increase in KC-derived TNF-α reduced the number of active HSCs and increased the activity of TNF-R1/caspase 8. Furthermore, ER stress in KCs promoted the polarization of KCs towards the M1 phenotype and increased the expression of TNF-α. The increase in KC-derived TNF-α triggered the apoptosis of HSCs and the activation of TNF-R1/caspase 8 in vitro, which was consistent with the in vivo results. Conclusion ER stress in KCs promotes the polarization of these cells towards the M1 phenotype and increases the expression of TNF-α. Then, the increase in KC-derived TNF-α triggers the apoptosis of HSCs through TNF-R1/caspase 8.

Effects of Curcumin Against Matrix Metalloproteinase-2 (MMP-2) and Tissue Inhibitor Metalloproteinase-2 (TIMP-2) Serum Level on Rat Model of Liver Fibrosis Resolution Process

Background: Liver fibrosis is an effect from continuous fibrogenesis and fibrolysis process. During fibrogenesis, MMP-2 and TIMP-2 that produced by hepatic stellate cell (HSC) have a role to regulate extracellular matrix (ECM) homeostastic. Otherwise, curcumin inhibits both MMP-2 and TIMP-2 expression and enhances HSC apoptosis, thus inhibit fibrogenesis. Role of curcumin, MMP-2, and TIMP-2 in a fibrolysis process has not been widely studied. This study aimed to determine the correlation between curcumin administration and the decline of MMP-2 and TIMP-2 on rat model of liver fibrosis.Method: This is an experimental study done in male Wistar rats. There are 8 groups consist of 4 rats each. Both control and intervention group were exposed to CCl4 1 cc/kgBW intraperitoneally 2 times per week for 9 consecutive weeks to form F3 fibrosis. Negative control group was injected with normal saline. After CCl4 injection, control group was given curcumin solvent as placebo while intervention groups were given curcumin 200 mg/kgBW for 2, 5, and 9 weeks. Statistical analysis then conducted in the end of study. Results: MMP-2 and TIMP-2 were remarkably increased in positive control group, but found decreased in control group 5 and 9. There are remarkable decrease of MMP-2 and TIMP-2 serum level in intervention group 2, 5, and 9, but MMP-2 and TIMP-2 level was significantly lower in intervention group 2 compared to the control group.Conclusion: MMP-2 and TIMP-2 serum level were decreased after giving of curcumin for 2 weeks. The duration of curcumin administration correlated with decrease of TIMP-2 serum level but not correlated with MMP-2 serum level in rat model of liver fibrosis.

MiR‑148a‑3p regulates alcoholic liver fibrosis through targeting ERBB3.

Alcoholic liver disease greatly affects human health. Previous studies have identified that microRNAs (miRNAs) are associated with the pathogenesis of alcoholic liver fibrosis (ALF). Therefore, the present study explored the regulatory mechanism of miR-148a-3p in ALF. An ALF model was established in rats by alcohol gavage, followed by treatment with miR-148a-3p. Reverse transcription-quantitative (RT-q) PCR was performed to detect miR-148a-3p expression in the rat liver tissues. The levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) were determined by enzyme-labeled colorimetry. Liver damage was evaluated by liver indices and histology. The direct target gene of miR-148a-3p was predicted by a dual luciferase reporter assay. The effects of miR-148a-3p and miR-148a-3p in combination with receptor tyrosine-protein kinase erbB-3 (ERBB3) on HSC-T6 cell viability and apoptosis were detected by MTT and flow cytometry assays, respectively. Western blotting and RT-qPCR assays were performed to detect the expression levels of proteins and mRNA associated with fibrosis and apoptosis. The data showed that miR-148a-3p mimics inhibited the expression levels of AST, ALT, ALP, LDH, α-SMA and type I collagen in the model, decreased the liver indices, and improved the liver damage caused by alcohol. ERBB3, which was predicted as the direct target gene of miR-148a-3p, reversed the effects of ERBB3 on promoting cell viability and inhibiting apoptosis. Concomitantly, miR-148a-3p reversed the increased expression of Bcl-2 and inhibited the expression levels of Bax and c-cleaved-3 caused by ERBB3. These data suggested that miR-148a-3p regulated ALF and the viability and apoptosis of hepatic stellate cells through targeting ERBB3.

Kweichow Moutai ameliorates alcohol-induced liver fibrosis in mice by targeting the NFκB pathway.

Previous epidemiological and histopathological studies have demonstrated that long‐term computation of Kweichow Moutai liquor (Moutai) could induce fatty liver disease but few of these patients with fatty liver will develop hepatic fibrosis or cirrhosis. Moutai liquor has a different brewing technique from other white wine, which may generate various microorganisms in the unique geographical conditions and may produce plenty of vitamins, amino acids, and several essential microelements. In the current study, we evaluated the potential protective effect of Moutai liquor in alcohol‐induced liver fibrosis mouse model. Both in vivo and in vitro studies were performed for exploring the possible mechanisms in suppressing liver fibrosis by Moutai. We demonstrated that Moutai treatment induced hepatic stellate cell (HSC) apoptosis and suppressed collagen deposition, as well as attenuated hepatic fibrosis. The antifibrosis mechanism of Moutai was possibly related with the inhibition of Kupffer cell and HSC activation via suppressing NFκB nuclear translocation and preventing the expression of pro‐inflammatory cytokines. It is worth noting that although Moutai attenuates liver fibrosis, it still causes lipid metabolic abnormalities in mouse liver and induces fatty liver. Kweichow Moutai may ameliorate alcohol‐induced liver fibrosis in mice by targeting the NFκB pathway.

Light-triggered NO-releasing nanoparticles for treating mice with liver fibrosis

Liver fibrosis, resulting from chronic liver damage and characterized by the accumulation of extracellular matrix (ECM) proteins, is a characteristic of most types of chronic liver diseases. The activation of hepatic stellate cells (HSC) is considered an essential pathological hallmark in liver fibrosis. Although nitric oxide (NO) can effectively induce HSC apoptosis, the systemic administration of NO is ineffective and may cause severe complications such as hypotension. To overcome this limitation, nanoparticles were designed to target HSCs and release NO locally under the exposure of near infrared light (NIR). To achieve this, upconversion nanoparticle (UCNP) cores were enveloped in mesoporous silica shells (UCNP@mSiO2), which were modified with hyaluronic acid (HA-UCNP@mSiO2) and Roussin’s black salt (RBS). HA molecules recognize and bind to CD44 proteins, which are overexpressed on activated HSCs. Under exposure to a 980-nm NIR laser, the UCNP cores convert the 980-nm wavelength into ultraviolet (UV) light, which then energizes the RBS (NO donors), resulting in an efficient release of NO inside of the HSCs. Once released, NO triggers HSC apoptosis and reverses the liver fibrosis. This targeted and controlled release method provides the theoretical and experimental basis for novel therapeutic approaches to treat hepatic fibrosis.

Progress of research on effect and mechanism of Scutellariae Radix on preventing liver diseases

Scutellariae Radix is a kind of traditional Chinese medicine, which has the functions of heat-clearing and damp-drying, purging fire and detoxifying, hemostasis and miscarriage prevention. Modern pharmacological studies show that Scutellariae Radix has various effects, such as anti-oxidation, anti-inflammatory, liver protection and antiviral microorganisms. By searching the documents in the past ten years, the author has found that Scutellariae Radix and its active components play an important role in protecting the liver. It can prevent and cure liver injuries caused by different reasons, including acute or chronic hepatitis, liver fibrosis and liver cancer. Among all kinds(chemical, immunological, alcoholic, nonalcoholic, viral, ischemia-reperfusion, etc.) of acute or chronic hepatitis, most studies are on CCl_4 induced chemical liver injury. Scutellariae Radix and its active components can significantly reduce the serum transaminase level in hepatitis animals, and reduce the degree of liver pathological damage. The mechanisms include antioxidant stress, anti-inflammatory, anti-apoptosis, inhibition of immunity, anti-virus and regulation of lipid metabolism, etc. Scutellariae Radix and its active components can significantly inhibit the activation of hepatic stellate cells and reduce extracellular matrix, and its anti-fibrosis mechanism involves antioxidation, anti-inflammatory, inducing apoptosis of hepatic stellate cells and so on. Whether in vivo or in vitro, Scutellariae Radix and its active components show a good anti-hepatocarcinoma effect, especially on hepatocarcinoma. Its prevention and treatment mechanisms for hepatocarcinoma mainly include blocking cancer cell cycle, inhibiting cancer cell metastasis, promoting cancer cell apoptosis and inducing autophagy. It can be seen that Scutellariae Radix has multiple functions and mechanisms in liver protection, and has a great development potential. Therefore, this paper reviews the prevention and treatment effects and mechanism of Scutellariae Radix and its components on different liver diseases, in order to provide reference for in-depth study, development and clinical application in the prevention and treatment of liver disease.

Endoplasmic reticulum stress enhances tumor necrosis factor-α expression in rat Kupffer cells to trigger hepatic stellate apoptosis cell through TNFR/caspase-8 pathway

To investigate the role of endoplasmic reticulum (ER)-stress of Kupffer cells (KCs) and KCs-derived tumor necrosis factor-α (TNF-α) in medicating apoptosis of hepatic stellate cell (HSC). Sixty male SD rats were randomized into control group, model group, ER- stress group, depletion group and KCs block group (n=15). The 4 groups of rats were given intraperitoneal injections (twice a week for 8 weeks) of normal saline (2 mg/kg); 40% CCl4 solution (in peanut oil, 2 mg/kg); 40% CCl4 solution (2 mg/kg) and tunicamycin (1 mg/kg); and 40% CCl4 solution (2 mg/kg) and tunicamycin (1 mg/kg) followed by clodronate liposomes (50 mg/kg), respectively. After the treatments, samples of the liver tissue and serum were collected from the rats from the 4 groups to isolate KC cells, which were co-cultured with LX2 cells. In the depletion group, the rats were injected with anti-rat TNFR mAb (0.35 mg/kg) via the portal vein before isolating the KCs. Liver function examination, Eirius red staining, ELISA, immuno- histochemical staining, and RT-PCR were performed to assess the liver function, liver fibrosis, KC phenotypes, expression of the in fl ammatory factors, and the number of active HSC was detected. The isolated KCs were treated with tunicamycin before co-culture with LX2 cells, and ELISA, RT-PCR and Western blot were performed to examine KC phenotypes, in fl ammatory factors, LX2 cell apoptosis and TNFR/caspase8 pathway activity. Compared with the rats in the control group, the rats in the model group had significantly increased ALT and AST levels, Sirius red staining-positive area, and Desmin-positive cells (activated HSCs) (P &lt; 0.05) with significantly lowered number of CD16-positive KCs (M1), and TNF-α protein and mRNA expression (P &lt; 0.05). Compared with those in the model group, the rats in ER-stress group showed significantly decreased ALT and AST levels, Sirius red staining positivity and Desmin-positive cells (P &lt; 0.05) and increased number of CD16-positive KCs and TNF-α expressions (P &lt; 0.05). In the depletion group, compared with the ER-stress group, the rats had significantly increased ALT and AST levels of, Sirius red staining positivity and Desmin-positive cells (P &lt; 0.05) and reduced CD16- positive KCs and TNF-αexpressions (P &lt; 0.05). In the cell co-culture experiment, the model group showed significantly reduced TUNEL-positive LX2 cells, CD16-positive cells, and expressions of TNFR1, cleaved caspase- 8 and cleaved caspase- 3 in the KCs (P &lt; 0.05) with increased Desmin-positive LX2 cells (P &lt; 0.05). Compared with the model group, the ER- stress group exhibited significantly increased TUNEL-positive LX2 cells, CD16-positive cells and expressions of TNFR, cleaved caspase-8 and cleaved caspase-3 in the KCs (P &lt; 0.05) and decreased Desmin-positive LX2 cells (P &lt; 0.05). In the depletion group, blocking TNFR resulted in significantly decreased expressions of cleaved caspase-8 and cleaved caspase-3 compared with those in ER- stress group (P &lt; 0.05) although there was no significant changed in TNFR expression. ER stress of KCs promotes the transformation of KCs towards M1 phenotype and increases the expression of TNF-α, which triggers the apoptosis of HSCs through the TNFR/caspase-8 pathway.

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells.

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd3+ ) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca2+ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.

PLK1 regulates hepatic stellate cell activation and liver fibrosis through Wnt/β-catenin signalling pathway.

As an outcome of chronic liver disease, liver fibrosis involves the activation of hepatic stellate cells (HSCs) caused by a variety of chronic liver injuries. It is important to explore approaches to inhibit the activation and proliferation of HSCs for the treatment of liver fibrosis. PLK1 is overexpressed in many human tumour cells and has become a popular drug target in tumour therapy. Therefore, further study of the function of PLK1 in the cell cycle is valid. In the present study, we found that PLK1 expression was elevated in primary HSCs isolated from CCl4‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. Knockdown of PLK1 inhibited α‐SMA and Col1α1 expression and reduced the activation of HSCs in CCl4‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. We further showed that inhibiting the expression of PLK1 reduced the proliferation of HSCs and promoted HSCs apoptosis in vivo and in vitro. Furthermore, we found that the Wnt/β‐catenin signalling pathway may be essential for PLK1‐mediated HSCs activation. Together, blocking PLK1 effectively suppressed liver fibrosis by inhibiting HSC activation, which may provide a new treatment strategy for liver fibrosis.

MicroRNA-155 Modulates Hepatic Stellate Cell Proliferation, Apoptosis, and Cell Cycle Progression in Rats With Alcoholic Hepatitis via the MAPK Signaling Pathway Through Targeting SOCS1.

The aim of this study was to investigate the regulatory function of the non-coding microRNA-155 (miR-155) and suppressor of cytokine signaling 1 (SOCS1) in alcoholic hepatitis (AH) and its potential mechanism associated with the mitogen-activated protein kinase (MAPK) signaling pathway. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total bilirubin (TBIL), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured in a rat model of AH. The biological prediction website microRNA.org and dual-luciferase reporter gene assay were used to identify whether SOCS1 was a direct target of miR-155, and the effects of miR-155 and SOCS1 on the viability, cycle progression, and apoptosis of hepatic stellate cells were assessed using RT-qPCR, Western blot assay, MTT assay, Annexin V/PI double staining, and PI single staining. The levels of ALT, AST, MDA, and TBIL and the liver cell morphology were all prominently changed in AH model rats. miR-155 suppressed SOCS1 by specifically binding to SOCS1-3’-UTR to activate the MAPK signaling pathway. SOCS1 had low expression while miR-155 was highly expressed in AH rats. miR-155 promoted hepatic stellate cell viability and cycle progression and reduced cell apoptosis by silencing SOCS1. Together, we find that silenced miR-155 could upregulate SOCS1 and inactivate the MAPK signaling pathway, thereby inhibiting the proliferation of alcoholic hepatic stellate cells and promoting cell apoptosis.

Oridonin ameliorates carbon tetrachloride-induced liver fibrosis in mice through inhibition of the NLRP3 inflammasome.

Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) and accumulation of the extracellular matrix. There are limitations in the current therapies for liver fibrosis. Recently, oridonin was shown to induce apoptosis in HSCs. Thus, we aimed to determine the roles of oridonin in chronic liver injury and fibrosis. Liver fibrosis was induced by CCl4 in mice injected intraperitoneally with oridonin for 6 weeks. The administration of oridonin significantly attenuated liver injury and reduced ALT levels. In addition, Sirius Red staining and the expression of α‐smooth muscle actin (α‐SMA) were significantly reduced by oridonin in murine livers with fibrosis. The expression of NLRP3, caspase‐1, and IL‐1β was downregulated with the oridonin treatment. Furthermore, the expression of F4/80 in liver tissues was also decreased by oridonin treatment. These results demonstrate that oridonin ameliorates chronic liver injury and fibrosis. Mechanically, oridonin may inhibit the activity of the NLRP3 inflammasome and inflammation in the liver. These results highlight the potential of oridonin as a therapeutic agent for liver fibrosis.