ROCK2 Promotes Sepsis-Triggered Liver Injury By Stabilizing HIF-1α

Background and ObjectiveSepsis is characterized by an aggressive inflammatory response to infection,and its adverse events,in particular,acute liver injury,can be deadly.Rho-associated coiled-coil-containing protein kinase 2(ROCK2)is a pivotal modulator of actin cytoskeleton dynamics,acting downstream of Rho GTPases involved in inflammation control.HIF-1α plays a central role in a variety of microenvironmental stresses,in addition to hypoxia,including exposure to infectious pathogens and inflammatory cytokines.Herein,we found that ROCK2 and HIF-1α were remarkably increased in liver injury triggered by sepsis in vitro,as well as in vivo.Knockdown of ROCK2 inhibited the production of inflammatory mediators,hepatocyte apoptosis,and the reduction of HIF-1α protein expression levels.Moreover,HIF-1α upregulation rescued such a decrease in the secretion of pro-inflammatory factors triggered by ROCK2 knockdown,whereas HIF-1α knockdown reduced the ROCK2-enhanced secretion of pro-inflammatory factors.The underlying mechanism involved the stabilization of HIF-1α by ROCK2 by preventing its ubiquitination and degradation.In conclusion,our study linked the two driving factors of sepsis,and we expect that it can lead to new treatment ideas and provide a new theoretical framework for sepsis-induced liver injury.Method1.The liver injury model of sepsis C57BL/6 mice was constructed by intraperitoneal injection of LPS(5 mg/kg).The liver tissue was taken at 0 hours and 6hours for HE staining,and enzyme-linked immunoassay was used to detect the inflammatory factor TNF at different time points.-α,IL-1β secretion,fluorescence quantitative PCR and western blotting to analyze the expression of ROCK2 in mouse liver tissues at different time periods.2.Down-regulate the expression of ROCK2 in mice by gene knockout technology,construct a mouse sepsis liver injury model,analyze the expression of ROCK2 in mouse liver tissue by fluorescent quantitative PCR and western blotting,and observe the effect of down-regulation of ROCK2 by gene knockout technology Expression changes,the secretion of inflammatory factors TNF-α,IL-1β,HE staining to observe the pathological changes of liver tissue.3.Screen the sh ROCK2 plasmid with the best down-regulation effect and down-regulate the expression of ROCK2 in human normal hepatocytes(HL7702),add cell culture medium containing LPS(10μg/ml)to construct the HL7702 inflammation environment,and detect the inflammatory factor TNF-α by ELISA,IL-1β secretion changes.At the same time,the p-ROCK2 plasmid with the best up-regulation effect was selected and the expression of ROCK2 in HL7702 was up-regulated.The expression of ROCK2 in HL7702 was analyzed by western blotting.Finally,the inflammatory factor TNF-α of cells in the inflammatory environment was detected by enzyme-linked immunoassay.,IL-1β secretion changes.4.After changing the expression of ROCK2,Western blotting was used to detect and analyze the expression of HIF-1α in mouse liver and HL7702,and observe the correlation between HIF-1α and ROCK2 expression.Finally,the expression of HIF-1α was up-regulated in the cells with stable low expression of ROCK2 and the expression of HIF-1α was decreased in the cells with over-expressing ROCK2,and the secretion of inflammatory factors TNF-α and IL-1β was detected by enzyme-linked immunoassay.5.Use Co-IP to detect whether ROCK2 and HIF-1α in HL7702 can directly bind to each other.On the basis of blocking cell protein degradation and synthesis,Western blotting was used to detect the effect of overexpression and silencing ROCK2 on the expression of HIF-1α.Co-IP detects the effect of overexpression and silencing of ROCK2 in HL7702 on the ubiquitination level of HIF-1α.Result1.The results of HE staining of mouse liver tissue showed that the degree of liver injury was significantly worsened after 6 hours of intraperitoneal injection of LPS.Similarly,we found that the concentration of TNF-α and IL-1β in mouse serum also increased 6 hours after intraperitoneal injection of LPS(p<0.01).Fluorescence quantitative PCR was used to detect the expression level of ROCK2 gene at different time points after LPS injection,and it was enhanced 6 hours after LPS injection(p<0.05),which was consistent with the results of western blot(p<0.05).It shows that the expression of ROCK2 in septic liver tissue is significantly up-regulated.2.Compared with wild-type(WT)mice,Western blot and real-time PCR showed that ROCK2 expression in heterozygous ROCK2-deficient mice(KD)was significantly down-regulated(p<0.05).6 hours after intraperitoneal injection of LPS(5 mg/kg)in KD and WT mice,ROCK2 gene and protein expression were significantly up-regulated(p<0.05).The results of enzyme-linked immunosorbent assay showed that in heterozygous ROCK2-deficient mice(KD),the release of pro-inflammatory cytokines TNF-α and IL-1β was significantly reduced(p<0.05).Finally,HE staining was used to observe the changes in liver tissue of ROCK2 heterozygous deficient mice(KD)after lipopolysaccharide treatment(p<0.05).These results indicate that loss of heterozygosity in mouse ROCK2 inhibits the development of liver injury induced by lipopolysaccharide.3.The results of western blotting and real-time PCR showed that after lipopolysaccharide treatment,the levels of ROCK2 protein and m RNA increased significantly(p<0.05).Down-regulation of ROCK2 can reduce the secretion of HL7702 inflammatory factors TNF-α and IL-1β(p<0.05).At the same time,after treatment with lipopolysaccharide transfected with p-ROCK2,the production of inflammatory cytokines was significantly increased(p<0.05),indicating that the expression of ROCK2 affected the secretion of inflammatory factors in HL7702 cells after treatment with lipopolysaccharide.4.Using the same liver injury model of sepsis in mice,a consistent result on the level of hypoxia-inducible factor-1α protein was obtained by Western blotting,which indicates that hypoxia-inducible factor-1α is overexpressed in the liver of mice triggered by lipopolysaccharide(P<0.05).In addition,our study found that up-regulating the expression of HIF-1α can reverse the decrease in the secretion of inflammatory factors caused by silencing ROCK2(p<0.05),while reducing the expression of HIF-1α can inhibit the increase in secretion of inflammatory factors caused by overexpression of ROCK2(p<0.05).5.Co-IP results confirmed that ROCK2 does not directly bind to HIF-1α.The protease inhibitor MG132 was further used to treat HL7702,and the expression of HIF-1α increased with the accumulation of MG132’s time of action(p<0.05).In the case of blocking proteasome degradation,after silencing and overexpression of ROCK2,HIF-1α protein expression did not change significantly and the degradation rate of HIF-1α in cells was significantly reduced(p<0.05).Our further study found that ROCK2 can inhibit the ubiquitination degradation process of HIF-1α in HL7702.ConclusionWe confirmed that ROCK2 stabilizes hypoxia-inducible factor-1α by inhibiting ubiquitin degradation.Our research results provide new ideas for the molecular mechanism of liver injury caused by sepsis,and provide a new target for the treatment of sepsis.