Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells

BACKGROUND Liver fibrosis is the common pathological process associated with the occurrence and development of various chronic liver diseases.At present,there is still a lack of effective prevention and treatment methods in clinical practice.Hepatic stellate cell(HSC)plays a key role in liver fibrogenesis.In recent years,the study of liver fibrosis targeting HSC autophagy has become a hot spot in this research field.Angiotensin-converting enzyme 2(ACE2)is a key negative regulator of reninangiotensin system,and its specific molecular mechanism on autophagy and liver fibrosis needs to be further explored.AIM To investigate the effect of ACE2 on hepatic fibrosis in mice by regulating HSC autophagy through the Adenosine monophosphate activates protein kinases(AMPK)/mammalian target of rapamycin(mTOR)pathway.METHODS Overexpression of ACE2 in a mouse liver fibrosis model was induced by injection of liver-specific recombinant adeno-associated virus ACE2 vector(rAAV2/8-ACE2).The degree of liver fibrosis was assessed by histopathological staining and the biomarkers in mouse serum were measured by Luminex multifactor analysis.The number of apoptotic HSCs was assessed by terminal deoxynucleoitidyl transferase-mediated dUTP nick-end labeling(TUNEL)and immunofluorescence staining.Transmission electron microscopy was used to identify the changes in the number of HSC autophagosomes.The effect of ACE2 overexpression on Wu Y et al.ACE2 improves liver fibrosis through autophagy WJG https://www.wjgnet.com 4976 September 7,2023 Volume 29 Issue 33 autophagy-related proteins was evaluated by multicolor immunofluorescence staining.The expression of autophagy-related indicators and AMPK pathway-related proteins was measured by western blotting.RESULTS A mouse model of liver fibrosis was successfully established after 8 wk of intraperitoneal injection of carbon tetrachloride(CCl4).rAAV2/8-ACE2 administration reduced collagen deposition and alleviated the degree of liver fibrosis in mice.The serum levels of platelet-derived growth factor,angiopoietin-2,vascular endothelial growth factor and angiotensin II were decreased,while the levels of interleukin(IL)-10 and angiotensin-(1-7)were increased in the rAAV2/8-ACE2 group.In addition,the expression of alpha-smooth muscle actin,fibronectin,and CD31 was down-regulated in the rAAV2/8-ACE2 group.TUNEL and immunofluorescence staining showed that rAAV2/8-ACE2 injection increased HSC apoptosis.Moreover,rAAV2/8-ACE2 injection notably decreased the number of autophagosomes and the expression of autophagy-related proteins(LC3I,LC3II,Beclin-1),and affected the expression of AMPK pathway-related proteins(AMPK,p-AMPK,p-mTOR).CONCLUSION ACE2 overexpression can inhibit HSC activation and promote cell apoptosis by regulating HSC autophagy through the AMPK/mTOR pathway,thereby alleviating liver fibrosis and hepatic sinusoidal remodeling.

MicroRNA-194 inactivates hepatic stellate cells and alleviates liver fibrosis by inhibiting AKT2

BACKGROUND Activation of hepatic stellate cells(HSCs)is a pivotal event in the onset and progression of liver fibrosis.Loss of microRNA-194(miR-194)has been reported in activated HSCs,but the actual role of miR-194 in liver fibrosis remains uncertain.AIM To explore the role and potential mechanism of miR-194-mediated regulation of liver fibrosis in vitro and in vivo.METHODS The expression of miR-194 was examined in human fibrotic liver tissues,activated HSCs,and a carbon tetrachloride(CCl4)mouse model by qPCR.The effects of AKT2 regulation by miR-194 on the activation and proliferation of HSCs were assessed in vitro.For in vivo experiments,we reintroduced miR-194 in mice using a miR-194 agomir to investigate the functions of miR-194 in liver fibrosis.RESULTS MiR-194 expression was notably lacking in activated HSCs from both humans and mice.Overexpression of miR-194(OV-miR-194)inhibitedα-smooth muscle actin(α-SMA)and type I collagen(Col I)expression and suppressed cell proliferation in HSCs by causing cell cycle arrest in G0/G1 phase.AKT2 was predicted to be a target of miR-194.Notably,the effects of miR-194 knockdown in HSCs were almost blocked by AKT2 deletion,indicating that miR-194 plays a role in HSCs via regulation of AKT2.Finally,miR-194 agomir treatment dramatically ameliorated liver fibrosis in CCl4-treated mice.CONCLUSION We revealed that miR-194 plays a protective role by inhibiting the activation and proliferation of HSCs via AKT2 suppression.Our results further propose miR-194 as a potential therapeutic target for liver fibrosis.

Expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic stellate cells during rat hepatic fibrosis and its intervention by IL-10

AIM: To investigate the expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic fibrosis and the antifibrogenic role of exogenous interleukin-10 (IL-10).METHODS: Hepatic fibrosis was induced by CCl4administration and 60 male Sprague-Dawley rats were randomly divided into normal control group (group N, 8rats), CCl4-induced group (group C, 28 rats) and IL-10-treated group (group I, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type Ⅳ collagenase through portal vein catheter and the suspension was centrifuged by 11%Nycodenz density gradient to isolate hepatic stellate cells (HSCs). RT-PCR was used to analyze mRNA of MMP-2 and TIMP-1 from freshly isolated cells. Densitometric data were standardized with β-actin signals. Immunocytochemistry was performed to detect MMP-2 and TIMP-1 expression in HSC cultured for 72 h.RESULTS: Compared to group N in the 7th wk, MMP-2and TIMP-1 mRNA increased in group C (P= 0.001/0.001)and group I (P = 0.001/0.009). The level of MMP-2 and TIMP-1 mRNA in group I was significantly lower than that in group C (P= 0.001/0.001). In the 11th wk, MMP-2 mRNAin group I was still lower than that in group C (P = 0.005),but both dropped compared with that in the 7th week (P = 0.001/0.004). TIMP-1 mRNA in group I was still lower than that in group C (P= 0.001), and increased in group C (P = 0.001) while decreased in group I (P = 0.042)compared with that in the 7th wk. Same results were found by immunocytochemistry.CONCLUSION: Expression of MMP-2 and TIMP-1 is increased in hepatic fibrosis. IL-10 exhibits an antifibrogenic effect by suppressing MMP-2 and TIMP-1 expression.

Activated rat hepatic stellate cells influence Th1/Th2 profile in vitro

AIM: To investigate the effects of activated rat hepatic stellate cells(HSCs) on rat Th1/Th2 profile in vitro.METHODS: Growth and survival of activated HSCs and CD4+ T lymphocytes cultured alone or together was assessed after 24 or 48 h. CD4+ T lymphocytes were then cultured with or without activated HSCs for 24 or 48 h and the proportion of Th1 [interferon(IFN)-γ+] and Th2 [interleukin(IL)-4+] cells was assessed by flow cytometry. Th1 and Th2 cell apoptosis was assessed after 24 h of co-culture using a caspase-3 staining procedure. Differentiation rates of Th1 and Th2 cells from CD4+ T lymphocytes that were positive for CD25 but did not express IFN-γ or IL-4 were also assessed after 48 h of co-culture with activated HSCs. Galectin-9 expression in HSCs was determined by immunofluorescence and Western blotting. ELISA was performed to assess galectin-9 secretion from activated HSCs.RESULTS: Co-culture of CD4+ T lymphocytes with activated rat HSCs for 48 h significantly reduced the proportion of Th1 cells compared to culture-alone conditions(-1.73% ± 0.71%; P < 0.05), whereas the proportion of Th2 cells was not altered; the Th1/Th2 ratio was significantly decreased(-0.44 ± 0.13; P < 0.05). In addition, the level of IFN-γ in Th1 cells wasdecreased(-65.71 ± 9.67; P < 0.01), whereas the level of IL-4 in Th2 cells was increased(82.79 ± 25.12; P < 0.05) by co-culturing, as measured by mean fluorescence intensity by flow cytometry. Apoptosis rates in Th1(12.27% ± 0.99%; P < 0.01) and Th2(1.71% ± 0.185%; P < 0.01) cells were increased 24 h after co-culturing with activated HSCs; the Th1 cell apoptosis rate was significantly higher than in Th2 cells(P < 0.01). Galectin-9 protein expression was significantly decreased in HSCs only 24 h after coculturing(P < 0.05) but not after 48 h. Co-culture for 48 h significantly increased the differentiation of Th1 and Th2 cells; however, the increase in the proportion of Th2 cells was significantly higher than that of Th1 cells(1.85% ± 0.48%; P < 0.05).CONCLUSION: Activated rat HSCs lower the Th1/Th2 profile, inhibiting the Th1 response and enhancing the Th2 response, and this may be a novel pathway for liver fibrogenesis.

P2X7 receptor inhibitor suppressed extracellular ATP/LPS-primed human hepatic stellate cells activation via downregulating NLRP3 inflammasome

OBJECTIVE To investigate the effect of P2X7receptor(P2X7r)inhibition,using a specific inhibitor(A438079)to prevent the development of liver fibrosis on human hepatic stellate cells,LX-2.METHODS The supernatant from lipopolysaccharide(LPS)-stimulated RAW264.7 mouse macrophages was supplemented to LX-2 cells for 24 h.LX-2cells were primed with LPS for 4h and subsequently stimulated for 30 min with 3mmol·L-1 of adenosine 5′-triphosphate(ATP).A438079(10μmol·L-1)was supplemented to LX-2 cells 10 min prior to ATP.RESULTS Directly treated with LPS on LX-2 cells,mRNA expressions of IL-1β,IL-18 and IL-6 were increased,as well as P2X7 r.And caspase-1,ASC and NLRP3 mRNA expressions were increased with LPS stimulation.LPS stimulation also increasedα-SMA and collagenⅠ mRNA expressions.Interestingly treatment of LX-2cells with mediums from LPS-primed RAW264.7mouse macrophages exhibited greater increase of mRNA expressions of above genes than those in LX-2directly treated with LPS.Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1βmRNA expression.In addition treatment of LPS-primed LX-2 cells with 3mmol·L-1 ATP induced the significant increase of IL-1β,IL-6,caspase-1,pannexin-1,α-SMA and collagenⅠ mRNA expression,the increasing ofα-SMA protein expression and cleavage of IL-1β.These events were significantly suppressed by pretreatment with P2X7 rantagonist A438079.P2X7 rblockade also significantly reduced the protein expression ofα-SMA.CONCLUSION Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1βfrom extracellular ATP/LPS-stimulated human hepatic stellate cells.This study demonstrated that repression of the P2X7 rrepresents a novel potential therapeutic approach to control liver fibrosis.

P2X7r antagonist suppressed hepatic stellate cells activation through NLPR3 inflammasome signaling

P2X7 receptor （P2X7r） is important in inflammation and fibrosis. The aim of the present study was to investigate the effect of P2X7r inhibition, using a specific inhibitor （A438079） to prevent the development of liver fibrosis on human hepatic stellate cells, LX-2. The supernatant from lipopolysaccharide （LPS）-stimulated RAW 264.7 mouse macrophages was supplemented to LX-2 cells for 24 h. LX-2 cells were primed with LPS for 4 h and subsequently stimulated for 30 rain with 3 mmol · L^-1 of adenosine 5＇-triphosphate （ATP）. A438079 （ 10 μmol · L^-1） was supplemented to LX-2 cells 10 rain prior to ATP. Directly treated with LPS on LX-2 cells, mRNA ex- pressions of IL-1β, IL-18 and IL-6 were increased, as well as P2X7r. And caspase-1, ASC and NLRP3 mRNA ex- pressions were increased with LPS stimulation. LPS stimulation also increased oL-SMA and collagen I mRNA expres- sions. Interestingly treatment of LX-2 cells with mediums from LPS-primed RAW 264.7 mouse macrophages exhibi- ted greater increase of mRNA expressions of above genes than those in LX-2 directly treated with LPS. Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1β mRNA expression. In addi- tion treatment of LPS-primed LX-2 cells with 3 mM ATP induced the significant increase of IL-1β, IL-6, caspase- 1, pannexin-1, α-SMA and collagen I mRNA expression, the increasing of oL-SMA protein expression and cleavage of IL-1β. These events were significantly suppressed by pretreatment with P2X7r antagonist A438079. P2XTr blockade also significantly reduced the protein expression of oL-SMA. Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1β from extracellular ATP/LPS-stimulated human he- patic stellate cells. This study demonstrated that repression of the P2XTr represents a novel potential therapeutic ap- proach to control liver fibrosis.

Role of cyclooxygenase-2 in lipid metabolism in hepatic stellate cells

Hepatic stellate cells(HSCs) are a kind of adipocytes. In HSCs lipids mainly exist in the form of lipid droplets. They are abundantly found in the cytoplasm and their main constituents are triglycerides. Lipid metabolism in HSCs is closely related to its biological activity, however the mechanism of lipid droplets disappearance after HSC activation is not clearly established yet. Recent research shows that, cyclooxygenase-2 plays an important regulatory role in the lipid metabolism of HSCs. This paper seeks to review the subject based on studies that have been conducted so far to understand the role of cyclooxygenase-2 in the metabolism of lipids in HSCs.

Switching-on of serotonergic calcium signaling in activated hepatic stellate cells

AIM:To investigate serotonergic Ca 2+ signaling and the expression of 5-hydroxytryptamine(5-HT) receptors,as well as Ca 2+ transporting proteins,in hepatic stellate cells(HSCs) . METHODS:The intracellular Ca 2+ concentration([Ca 2+ ]i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system.Quantitative PCR was per-formed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum(ER) proteins involved in Ca 2+ storage and release in cultured rat HSCs. RESULTS:Distinct from quiescent cells,activated HSCs exhibited[Ca 2+ ]i transients following treatment with 5-HT,which was abolished by U-73122,a phospholipase C inhibitor.Upregulation of 5-HT2A and 5-HT2B receptors,but not 5-HT3,was prominent during trans-differentiation of HSCs.Pretreatment with ritanserin,a 5-HT2 antagonist,inhibited[Ca 2+ ]i changes upon application of 5-HT.Expression of type 1 inositol-5'-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca 2+ storage and release in activated HSCs.Ca 2+ binding chaperone proteins of the ER,including calreticulin,calnexin and calsequestrin,were up-regulated following activation of HSCs. CONCLUSION:The appearance of 5-HT-induced[Ca 2+ ]i response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca 2+ transporting/chaperone ER proteins may participate in the activating process of HSCs.

KN-93,a specific inhibitor of CaMKⅡ inhibits human hepatic stellate cell proliferation in vitro

AIM: To investigate the effects of KN-93, a CaMKⅡ selective inhibitor on cell proliferation and the expression of p53 or p21 protein in human hepatic stellate cells. METHODS: Human hepatic stellate cells (LX-2) were incubated with various concentrations (0-50 mmol/L) of KN-93 or its inactive derivative, KN-92. Cell proliferation was measured by CCK-8 assay, and the expression of two cell cycle regulators, p53 and p21, was determined by SDS-PAGE and Western blotting. RESULTS: KN-93 (5-50 mmol/L) decreased the proliferation of human hepatic stellate cells in a dose- dependent manner from 81.76% (81.76% ± 2.58% vs 96.63% ± 2.69%, P < 0.05) to 27.15% (27.15% ± 2.86% vs 96.59% ± 2.44%, P < 0.01) after 24 h treatment. Incubation of 10 mmol/L KN-93 induced the cell growth reduction in a time-dependent manner from 78.27% at 8 h to 11.48% at 48 h. However, KN-92, an inactive derivative of KN-93, did not inhibit cell proliferation effectively. Moreover, analysis of cell cycle regulator expression revealed that KN-93 rather than KN-92 reduced the expression of p53 and p21.

KLF4 Inhibits the Activation of Human Hepatic Stellate Cell In Vitro

Objective Hepatic stellate cells(HSCs)play a crucial role in liver fibrosis.Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs.Kruppel-like factor 4(KLF4)plays a pivotal role in a wide array of physiological and pathological processes.This study aimed to investigate the effect of KLF4 on the proliferation,apoptosis and phenotype of quiescent HSCs Methods We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector,to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection.Cell proliferation was assessed using the CCK-8 assay.Flow cytometry was used to detect the cell cycle distribution and apoptosis rate.Western blotting was used to determine the levels of some quiescence and activation markers of HSCs Results Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1,which are quiescent HSC markers,while significantly decreased the levels of N-cadherin and a-SMA,known activated HSC markers.In contrast,cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced Conclusion KLF4 inhibits the proliferation and activation of human LX-2 HSCs.It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.

肝星状细胞特异性Grk2基因敲除小鼠模型的制备及鉴定

目的利用Cre-loxP基因敲除技术建立肝星状细胞特异性G蛋白偶联受体激酶2(G protein-coupled receptor kinase 2,GRK2)基因敲除小鼠模型,为研究GRK2在肝星状细胞中的生物学功能提供动物模型基础。方法将loxP标记的Grk2基因小鼠(Grk2^(fl/fl))和Lrat-Cre工具鼠进行多次繁殖,建立肝星状细胞特异性Grk2基因敲除(Grk2^(ΔHSC))小鼠模型。观察和分析小鼠的生长繁殖情况;通过PCR反应鉴定flox和Cre基因型;免疫荧光双染检测肝星状细胞中GRK2表达;Western blot检测小鼠肝星状细胞及肺、脾、肾脏、心脏组织中GRK2蛋白表达;HE染色观察肝脏及肺、脾、心脏、肾脏组织学形态。结果成功鉴定Grk2^(ΔHSC)小鼠基因型;两组小鼠体质量、繁殖能力无明显差异;免疫荧光双染及Western blot结果表明,Grk2^(ΔHSC)小鼠的肝星状细胞中GRK2蛋白水平明显低于对照组小鼠,Grk2^(ΔHSC)小鼠肺、脾、肾脏和心脏组织中GRK2蛋白表达与对照组相比无明显变化;HE染色结果显示,Grk2^(ΔHSC)小鼠肝脏及主要组织结构与Grk2^(fl/fl)相比差异无显著性,可用于后续研究。结论本研究应用Cre-loxP技术成功构建了肝星状细胞特异性Grk2基因敲除小鼠,为进一步研究GRK2在肝脏中的作用提供了优良工具。

Angiotensin-converting enzyme 2 alleviates liver fibrosis through the renin-angiotensin system

The present letter to the editor is related to the study titled‘Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells’.Angiotensin-converting enzyme 2 can alleviate liver fibrosis by regulating autophagy of hepatic stellate cells and affecting the renin-angiotensin system.

Effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with IL-2 or TNF-α

Objective To study the effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with interleukin-2 (IL-2 ) or tumor necrosis factor-α (TNF-α).Methods Hepatic stellate cells were isolated from male Sprague-Dawley rats by using modified Friedman's method. Using the isolated cells cultured and treated with IL-2 or TNF-α, we studied the effects of vitamin E on their proliferation and collagen synthesis through an 3 H-thymidine and 3 H-proline incorporation assay, as well as through observation of these cells under a contrary phase microscope. Results Adding IL-2 increased the both proliferation and collagen synthesis of hepatic stellate cells. Their proliferation was also increased by the addition of TNF-α, although it decreased collagen synthesis. Vitamin E had marked inhibitory effects on the ability of cells treated with IL-2 or TNF-α to reproduce or synthesize collagen.Conclusion Vitamin E can inhibit the proliferation and collagen synthesis of hepatic stellate cells. It is possible that vitamin E affects liver fibrosis through these activities.

Effects of octreotide on expression of L-type voltage-operated calcium channels and on intracellular Ca^(2+) in activated hepatic stellate cells

Background The contractility of hepatic stellate cells (HSCs) may play an important role in the pathogenesis of cirrhosis with portal hypertension. The aim of this study was to research the effects of octreotide,an analogue of somatostatin,on intracellular Ca 2+ and on the expression of L-type voltage-operated calcium channels (L-VOCCs) in activated HSCs,and to try to survey the use of octreotide in treatment and prevention of cirrhosis with portal hypertension complications. Methods HSC-T_6,an activated HSCs line,was plated on small glass coverslips in 35-mm culture dishes at a density of 1×10 5/ml,and incubated in DMEM media for 24 hours. After the cells were loaded with Fluo-3/AM,intracellular Ca 2+ was measured by Laser Scanning Confocal Microscopy (LSCM). The dynamic changes in activated HSCs of intracellular Ca 2+ ,stimulated by octreotide,endothelin-1,and KCl, respectively,were also determined by LSCM. Each experiment was repeated six times. L-VOCC expression in HSCs was estimated by immunocytochemistry. Results After octreotide stimulation,a signifcant decrease in the intracellular Ca 2+ of activated HSCs was observed. However,octreotide did not inhibit the increases in intracellular Ca 2+ after stimulation by KCl and endothelin-1. Moreover,octreotide did not significantly affect L-VOCC expression. These results suggest that neither L-VOCC nor endothelin-1 receptors in activated HSCs are inhibited by octreotide. Conclusions Octreotide may decrease portal hypertension and intrahepatic vascular tension by inhibiting activated HSCs contractility through decreases in intracellular Ca 2+ . The somatostatin receptors in activated HSCs may be inhibited by octreotide.

Silencing ofα-complex protein-2 reverses alcohol-and cytokine-induced fibrogenesis in hepatic stellate cells

Background and aim:a-complex protein-2(aCP2)encoded by the poly(rC)binding protein 2(PCBP2)gene is responsible for the accumulation of type I collagen in fibrotic livers.In this study,we silenced the PCBP2 gene using a small interfering RNA(siRNA)to reverse alcohol-and cytokine-induced profibrogenic effects on hepatic stellate cells(HSCs).Methods:Primary rat HSCs and the HSC-T6 cell line were used as fibrogenic models to mimic the initiation and perpetuation stages of fibrogenesis,respectively.We previously found that a PCBP2 siRNA,which efficiently silences expression of aCP2,reduces the stability of type I collagen mRNA.We investigated the effects of the PCBP2 siRNA on cell proliferation and migration.Expression of type I collagen in HSCs was analyzed by quantitative real-time PCR and western blotting.In addition,we evaluated the effects of the PCBP2 siRNA on apoptosis and the cell cycle.Results:PCBP2 siRNA reversed multiple alcohol-and cytokine-induced profibrogenic effects on primary rat HSCs and HSC-T6 cells.The PCBP2 siRNA also reversed alcohol-and cytokine-induced accumulation of type I collagen as well as cell proliferation and migration.Moreover,the combination of LY2109761,a transforming growth factor-b1 inhibitor,and the PCBP2 siRNA exerted a synergistic inhibitive effect on the accumulation of type I collagen in HSCs.Conclusions:Silencing of PCBP2 using siRNA could be a potential therapeutic strategy for alcoholic liver fibrosis.

Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells

Liver fibrosis is a consequence of chronic liver disease,causing morbidity and mortality.Interleukin-33(IL-33)is a critical mediator of inflammation,which may be involved in the development of liver fibrosis.Here,we investigated the role of IL-33 in human patients and experimental bile-duct ligation(BDL)-induced fibrosis in mice.We report increased hepatic IL-33 expression in the murine BDL model of fibrosis and in surgical samples obtained from patients with liver fibrosis.Liver injury,inflammatory cell infiltration and fibrosis were reduced in the absence of the IL-33/ST2 receptor,and the activation of hepatic stellate cells(HSCs)was decreased in ST2-deficient mice.Recombinant IL-33 activated HSCs isolated from C57BL/6 mice,leading to the expression of IL-6,TGF-β,α-SMA and collagen,which was abrogated in the absence of ST2 or by pharmacological inhibition of MAPK signaling.Finally,administration of recombinant IL-33 significantly increased hepatic inflammation in sham-operated BL6 mice but did not enhance BDL-induced hepatic inflammation and fibrosis.In conclusion,BDL-induced liver inflammation and fibrosis are dependent on ST2 signaling in HSCs,and therefore,the IL-33/ST2 pathway may be a potential therapeutic target in human patients with chronic hepatitis and liver fibrosis.

Inhibition of ASCT2 induces hepatic stellate cell senescence with modified proinflammatory secretome through an IL-1α/NF-κB feedback pathway to inhibit liver fibrosis

Senescence of activated hepatic stellate cells(aHSCs) is a stable growth arrest that is implicated in liver fibrosis regression.Senescent cells often accompanied by a multi-faceted senescence-associated secretory phenotype(SASP).But little is known about how alanine-serine-cysteine transporter type-2(ASCT2),a high affinity glutamine transporter,affects HSC senescence and SASP during liver fibrosis.Here,we identified ASCT2 is mainly elevated in aHSCs and positively correlated with liver fibrosis in human and mouse fibrotic livers.We first discovered ASCT2 inhibition induced HSCs to senescence in vitro and in vivo.The proinflammatory SASP were restricted by ASCT2 inhibition at senescence initiation to prevent paracrine migration.Mechanically,ASCT2 was a direct target of glutaminolysisdependent proinflammatory SASP,interfering IL-1α/NF-κB feedback loop via interacting with precursor IL-1α at Lys82.From a translational perspective,atractylenolide Ⅲ is identified as ASCT2 inhibitor through directly bound to Asn230 of ASCT2.The presence of -OH group in atractylenolide Ⅲ is suggested to be favorable for the inhibition of ASCT2.Importantly,atractylenolide Ⅲ could be utilized to treat liver fibrosis mice.Taken together,ASCT2 controlled HSC senescence while modifying the proinflammatory SASP.Targeting ASCT2 by atractylenolide Ⅲ could be a therapeutic candidate for liver fibrosis.

敲低HSC-LX2人肝星状细胞钙网蛋白引起细胞内Ca^(2+)水平升高并促进细胞凋亡

目的探讨沉默钙网蛋白(CALR)基因对人肝星状细胞(HSC-LX2)凋亡及细胞内Ca^(2+)水平的影响.方法设计靶向CALR的小干扰RNA(siRNA),脂质体法转染至HSC-LX2细胞,筛选出敲低CALR的HSC-LX2细胞.采用异硫氰酸荧光素标记的膜联素V/碘化丙啶(annexinⅤ-FITC/PI)双标记结合流式细胞术检测细胞凋亡,Fluo 3-AM钙离子荧光探针负载结合激光共聚焦显微镜观察细胞内Ca^(2+)浓度变化,反转录PCR检测细胞CALR、B细胞淋巴瘤分子2(Bcl2)、Bcl2相关X蛋白(BAX)的mRNA水平,Western blot法检测CALR、BAX、Bcl2蛋白表达.结果敲低HSC-LX2细胞CALR后,细胞凋亡率显著增加,Ca^(2+)浓度也有明显升高,BAX的表达显著增加,Bcl2表达明显降低,且BAX/Bcl2比值显著升高.结论敲低HSC-LX2细胞CALR的表达会增加细胞Ca^(2+)水平,上调BAX/Bcl2的比值,促进细胞凋亡.

基于PI3K/AKT/Bcl-2信号通路探讨加味柴胡当归汤抑制肝星状细胞纤维化的机制

目的探究加味柴胡当归汤对肝星状细胞纤维化的抑制作用及其通过调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)/B细胞淋巴瘤(Bcl)-2信号通路抗肝纤维化的机制。方法培养永生化的大鼠肝星状细胞(HSC-T6),设置正常对照组(不作干预)、模型组[血小板衍生生长因子(PDGF)-BB干预],中药组(PDGF-BB+含药血清干预),激动剂+中药组(PDGF-BB+HY-101625+含药血清干预),抑制剂+中药组(PDGF-BB+LY294002+含药血清干预),采取不同干预方式进行实验。使用CCK-8试剂盒和细胞凋亡试剂盒检测细胞增殖活性和凋亡水平;采用酶联免疫吸附试验(ELISA)检测细胞α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(COL-Ⅰ)、基质金属蛋白酶(MMP)-2、MMP-9、MMP组织抑制剂(TIMP)-1表达水平;进行Western印迹实验分析细胞中Bcl-2、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶(Caspase)-3、Caspase-9、哺乳动物雷帕霉素靶蛋白(mTOR)表达;利用qRT-聚合酶链反应(PCR)法测定细胞中PI3K、p-PI3K、AKT、p-AKT mRNA表达。结果与正常对照组相比,模型组细胞增殖活性、α-SMA、COL-Ⅰ、TIMP-1、Bcl-2表达及PI3K、AKT、mTOR磷酸化水平显著升高,凋亡率、MMP-2、MMP-9、Bax、Caspase-3和Caspase-9表达显著降低(P<0.05)。与模型组相比,中药组、激动剂+中药组、抑制剂+中药组细胞增殖活性、Bcl-2、α-SMA、COL-Ⅰ、TIMP-1表达及PI3K、AKT、mTOR磷酸化水平明显降低,凋亡率、MMP-2、MMP-9、Bax、Caspase-3和Caspase-9表达水平明显升高(P<0.05)。结论加味柴胡当归汤可通过调控PI3K/AKT/Bcl-2信号通路,抑制肝星状细胞活化,逆转肝纤维化进程。

姜黄素通过EZH2/PPARγ途径抑制肝星状细胞活化

目的 研究姜黄素抑制肝星状细胞(hepatic stellate cells,HSCs)活化的分子机制。方法 大鼠肝星状细胞HSC-T6分为对照组、转化生长因子-β1(transforming growth factor-β1,TGF-β1)组、姜黄素组、EZH2过表达组、EZH2敲降组和EZH2抑制剂组。对照组正常培养,TGF-β1组用TGF-β1处理,姜黄素组用TGF-β1与姜黄素共处理,EZH2过表达组用TGF-β1处理同时转染EZH2真核表达载体,EZH2敲降组用TGF-β1处理同时转染EZH2干扰RNA,EZH2抑制剂组用TGF-β1与EZH2抑制剂DZNep共处理。提取RNA和蛋白质,采用real-time PCR和Western blot技术检测肝纤维化相关基因α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原蛋白a1(typeⅠcollagen a1,Col1a1)、组织金属蛋白酶抑制剂1(tissue inhibitor of metalloproteinase 1,Timp1)、组蛋白甲基转移酶EZH2和纤维化抑制因子PPARγ的表达,免疫荧光技术检测细胞内H3K27me3水平。结果 与对照组相比,TGF-β1组HSC-T6细胞中α-SMA、Col1a1、Timp1和EZH2的表达升高(P<0.01),H3K27me3水平升高,PPARγ的表达下降(P<0.01);而姜黄素组的结果与TGF-β1组相反(P<0.01或P<0.05)。与TGF-β1组相比,EZH2过表达组PPARγ的表达降低(P<0.05);而EZH2敲降组和EZH2抑制剂组PPARγ表达上调(P<0.05)。结论 姜黄素可通过抑制EZH2表达进而上调PPARγ表达水平,从而起到抑制HSCs活化的作用。