Liver X Receptor Β Controls Hepatic Stellate Cell Activation Via Hedgehog Signaling

Fibrosis of the liver,triggered by chronic liver injury,which may progress to liver cirrhosis or even liver cancer without control.At present,studies have confirmed that liver fibrosis occurs primarily due to the activation of hepatic stellate cells（HSCs）.Under conditions of liver injury,HSCs are activated and undergo a transformation giving rise to a fibrogenic myofibroblastic phenotype.Therefore,it is of great clinical significance to explore the mechanism of HSC activation and its role in liver fibrosis progression.LXRs,occurring as two isoforms（αandβ）,function as sensors of cholesterol levels,prompting reverse cholesterol transport and its excretion into bile.Previous studies have revealed that LXRs play an important role in HSC activation,but the specificity of the two isoforms and their molecular mechanisms remain unresolved.Nevertheless,activation of LXRs leads to enhanced hepatic triglyceride synthesis and may give rise to liver steatosis,hampering therapeutic development based on LXR agonists.However,certain studies have indicated that it is LXRα,predominantly expressed in hepatocytes,that specifically mediates the increase of triglyceride synthesis.The aims of this research,therefore,were to investigate the antifibrogenic role of LXRβin the process of HSC activation in vitro and in vivo.If confirmed,LXRβ-selective agonists may be a potential therapeutic target to avoid HSC activation-associated fibrosis,simultaneously avoid undesirable LXRα-associated side-effects.Hedgehog（Hh）signaling has been revealed to be important in the promotion of HSC activation and conversion to the myofibroblastic phenotype.Previous studies have shown that LXRs can inhibit Hh signaling pathway in a variety of tumors,but the specific mechanism is unclear.Therefore,LXRs may regulate HSC activation by inhibiting the Hh signaling pathway in HSC.In this study,we first explored the role of LXRβin maintaining HSC quiet.Then,we observed the pathological changes in CCl₄-affected liver and under conditions of LXRβoverexpression.Finally,the Hh signaling pathway involved in LXRβregulation of HSC activation and preliminarily studied its potential mechanism were studied.Method:I.Role of LXRβin maintaining HSC quiet.1.Exression of LXRs and its downstream molecules were observed during mice primary HSCs activation.After mice primary HSCs extraction for 0,24,72,120,168 h were performed by qRT-PCR,and for 24,72,120,168 h were performed by western blot to analyse the expression of alpha smooth muscle protein（Acta2）,Collagen I,LXRαandβand their downstream molecular sterol response original binding protein 1c（Srebp-1c）and adenosine triphosphate combined box transporter A1（Abca1）.2.The influence of LXRβto mice primary HSCs activation was detected.After 72 h of isolation,primary HSCs were treated with LXR agonist T0901317 or antagonist SR9238,then determinated the expression of Srebp-1c,Abca1,Acta2,Collagen I.Subsequently,the endogenous expression levels of LXRαand LXRβwere measured in mice primary HCs and HSCs after 24 h of culture in vitro.Finally,after 72 h of of isolation,siRNA was used to silence LXRαor LXRβin mouse primary HSCs,and the expression of Srebp-1c,Abca1,Acta2,Collagen I were analysed.3.The influence of LXRβto CCl₄-induced liver fibrosis.Mice were divided into blank control group（control velicle）,control adenovirus group（Ad-control+CCl₄）,LXRs agonist group（T0901317+CCl₄）and LXR adenovirus overexpression group（Ad-LXRβ+CCl₄）.Mice were intraperitoneally injected with CCl₄ to construct chronic injury and liver fibrosis model.Hepatic histopathology was evaluated by H&E,Sirius Red and Masson staining to determinate the influence of LXRβ.4.The influence of LXRβto HSC activation in CCl₄-induced liver fibrosisThe effect of LXRβoverexpression on the number of activated HSCs was observed by Acta2 immunohistochemical staining5.The influence of LXRβto LXRs target genes in liver.Protein expression of LXR and its target genes were investigated by western blotting.II.The influence of LXRβto Hh signaling1.The influence of LXRβto Hh signaling in CCl₄-induced liver fibrosisThe expression of Hh signaling pathway in control group,Ad-control+CCl₄ group and Ad-LXRβ+CCl₄ group was analysed.2.The influence of Hh signaling to mice primary HSCs activation in vitroAfter mice primary HSCs extraction for 0,24,72,120,168 h,the expressions of Patch and Gli2 were detected by qRT-PCR,and for 24,72,120,168 h were detected by western blot.HSCs were treated with Hh signaling agonist Purmorphamine or antagonist GDC0449,then the expressions of Acta2 and Collagen I were analysed by qRT-PCR.3.The influence of LXRβto Hh signaling in mice primary HSCsHSCs were transfected siRNA or adenovirus-mediated vectors targeting LXRβ,the expression of Patch,Gli2,Acta2 and Collagen I were detected by western blot.HSCs were intervened with Purmorphamine after transfected Ad-LXRβor GDC0449 after transfected siRNA targeting LXRβ.The expression of Patch,Gli2,Shh,Acta2 and Collagen I were detected by western blot.Results:I.Role of LXRβin maintaining HSC quiet in vitro.1.Exression of LXRs and its downstream molecules were observed during mice primary HSCs activation.The expression of LXRαand LXRβ,and their target genes Srebp-1c and Abca1 significantly decreased during HSC activation.2.The influence of LXRβto mice primary HSCs activation.Firstly,T0901317 led to a significant increase in the mRNA levels of Abca1 and Srebp-1c,and suppressed the activation of the mouse primary HSCs.In addition,SR9238 decreased the expressions of LXRs target genes and notably promoted the activation of HSCs.LXRαwas mainly expressed in HCs,while,HSCs expressed high levels of LXRβ.Furthermore,no obvious effect by the LXRαknockdown was observed on Abca1 or Srebp-1c on the transcription level,and no significant activation of HSCs was noted.In contrast,LXRβsilencing led to a notable decrease in the expression levels of LXR target genes,and promoted HSCs activation,as measured by Acta2and collagen I mRNA expression.3.The influence of LXRβto CCl₄-induced liver fibrosis.After C57/BL6 mice received IP injections of CCl₄ for 5 weeks,hepatic histopathology was evaluated by H&E,Sirius Red and Masson staining.The Ad-control mice displayed extensive structural disorganization.The livers in T0901317 treatment group displayed less septa formation,but were characterized by diffuse distribution of microvesicular steatosis.Furthermore,overexpression of LXRβin CCl₄-induced mice resulted in a significantly lower degree of septa formation,but did not induce the pathological features of liver steatosis.4.The influence of LXRβto HSC activation in CCl₄-induced liver fibrosis.The number of Acta2⁺cells was markedly lower in the Ad-LXRβmice compared with that in the Ad-control mice.5.The influence of LXRβto LXRs target genes in liver.Protein expression of LXRs target genes were investigated by western blotting.Compared with the control groups,the T0901317-treated group displayed a significant increase in Abca1 and Srebp-1c expression levels.However,overexpression of LXRβin mice resulted in just a moderate increase of the expression levels of Abca1 and Srebp-1c compared with the T0901317-treated group.II.The influence of LXRβto Hh signaling1.The influence of LXRβto Hh signaling in CCl₄-induced liver fibrosisThe Gli2 nuclear staining exhibited markedly increased in the Ad-control group.However,the overexpression of LXRβsignificantly suppressed the number of Acta2⁺cells as well as the nuclear expression of Gli2.Compared with the control vehicle group,the upregulation of Gli2protein levels in CCl₄-induced fibrotic liver was accompanied by an increase in Collagen I and Acta2 protein expression levels.While the overexpression of LXRβin CCl₄-treated mice led to decrease in the expressions of Gli2,Collagen I and Acta2.2.The influence of Hh signaling to mice primary HSCs activation in vitroThe HSC activation process was accompanied by the activation of Hh signaling.The mRNA expression levels of Collagen I and Acta2 in HSCs were upregulated by Purmorphamine but decreased by GDC0449.3.The influence of LXRβto Hh signaling in mice primary HSCsOverexpression of LXRβdid not block purmorphamine-stimulated induction of Ptch and Gli2 but partially inhibited the upregulation of HSC activation markers.In contrast,GDC0449 significantly inhibited the protein levels of Ptch,Gli2,Collagen I and Acta2 in HSCs,while silencing LXRβpartially rescued the inhibiting effects of GDC0449.Additionally,Hh ligands Shh was drastically upregulated by purmorphamine treatment,but completely abolished while overexpression of LXRβin HSCsConclusion:The results of the present study demonstrate that LXRβregulates the activation of HSCs and prevents fibrosis,simultaneously avoiding undesirable liver steatosis side-effects.LXRβinhibits HSC activation via Hh signaling,and may by inhibiting Hh ligands Shh production.