The Protective Effect Of RGS14 On Hepatic Ischemia-reperfusion Injury And Its Mechanism

ForewordHepatic ischemia-reperfusion injury is a phenomenon in which liver cell dysfunction and structural damage aggravate with the restoration of blood flow after a period of liver ischemia.In clinical practice,liver ischemia-reperfusion injury not only occurs in liver transplantation and liver resection,but also in the case of liver trauma,shock,and gastric fundus esophageal vein rupture and bleeding caused by portal hypertension.After restoring normal blood perfusion,it can cause further damage to the liver.Liver ischemia-reperfusion injury can lead to elevated hepatic transaminase in mild cases,and severe liver function impairment or liver failure in severe cases,and even the outbreak of Systemic Inflammatory Response Syndrome(SIRS),which further causes multiple organ dysfunction(Multiple Organ Dysfunction Syndrome,MODS).Liver ischemia-reperfusion injury seriously affects patient prognosis.At present,the mechanism of hepatic ischemia-reperfusion injury is still not fully clarified.Therefore,it is necessary to further clarify the occurrence and development mechanism of hepatic ischemia-reperfusion injury,and to provide new targets and new ideas for its treatment.Hepatic ischemia-reperfusion injury is a complex network regulatory process involving multiple cells and multiple mechanisms.During the ischemia stage of the liver,with the continuous consumption of oxygen,hepatocytes,Kufer cells,hepatic sinusoidal endothelial cells,etc.change from aerobic metabolism to anaerobic metabolism,resulting in intracellular acidosis and changes in ion channels,which further lead to cellular changes.During the blood reperfusion stage,the massive infiltration of inflammatory cells and the massive release of inflammatory mediators further aggravate the apoptosis and necrosis of cells.Studies have shown that inflammation and apoptosis play an important role in the process of hepatic ischemia-reperfusion.Regulator of G-protein signaling 14(RGS14)is an important member of the RGS protein family.The proteins of this family share a conserved 120 amino acid RGS domain,and almost all RGS proteins have GTPase activating protein activity and negatively regulate G protein signaling.RGS 14 possesses multiple signaling regulatory elements,and in addition to the canonical RGS domain and GoLoco/GPR motif,RGS 14 also contains two tandem Ras/rap binding domains.RGS 14 is highly expressed in lymphoid tissues.Studies have shown that RGS 14 can affect the recruitment of chemokines to lymphocytes by regulating chemokine receptors,thereby affecting the adhesion and migration of lymphocytes;RGS 14 has also been shown to regulate the MAPK signaling pathway and inhibit pathological cardiac remodeling by regulating the MEK/ERK1/2 signaling pathway.However,whether and how RGS 14 can regulate hepatic ischemia-reperfusion injury remains unclear.Therefore,studying the molecular regulation mechanism of RGS 14 in hepatic ischemia-reperfusion injury is helpful for in-depth study of the mechanism of hepatic ischemia-reperfusion injury,and can provide new targets for improving ischemia-reperfusion injury and drug development.It has important social implications.In this study,we first started to study the correlation of RGS 14 expression in liver ischemia-reperfusion injury.Using RGS 14 knockout and hepatocyte-specific transgenic mice,combined with the corresponding RGS 14 knockdown and overexpression stable cell lines,to study the expression of RGS 14 in the liver.The role and regulation mechanism of RGS 14 in liver ischemia-reperfusion injury provide new ideas and potential therapeutic targets for the prevention and treatment of ischemia-reperfusion injury,and provide a new theoretical basis for clinical translation and application.Part 1:The expression of RGS14 in hepatic ischemia-reperfusion injuryPurpose:To investigate the relationship between RGS14 and hepatic ischemia-reperfusion injury.Method:1.A mouse model of liver ischemia-reperfusion injury was established.Real-Time PCR and Western-blot were used to detect the changes of RGS14 gene and protein in the ischemia-reperfusion liver of mice,and immunohistochemical analysis of the expression of RGS14 after liver ischemia-reperfusion was performed.2.A model of hepatocyte hypoxia/reoxygenation was established,and the changes of RGS14 gene and protein in hepatocytes after hypoxia/reoxygenation were detected by Real-Time PCR and Western-blot.3.The online gene database was mined to verify the changes of RGS14 gene after ischemia-reperfusion in the mouse liver.Results:In wild-type C57BL/6 mice,with the prolongation of ischemia-reperfusion time,the expression of RGS14 gene and protein in the liver was gradually and significantly up-regulated;the expression of RGS14 gene and protein in hepatocytes was significantly up-regulated after hypoxia/reoxygenation;Online gene database data showed that with the prolongation of ischemia-reperfusion time in mouse liver,the expression of RGS14 gene was gradually and significantly up-regulated.Conclusion:The expression of RGS14 was significantly up-regulated in hepatic ischemia-reperfusion injury,suggesting that RGS14 has a regulatory effect on hepatic ischemia-reperfusion injury.Part Ⅱ:Regulation of RGS14 on inflammatory response and apoptosis in liver ischemia-reperfusion injuryPurpose:1.To study the regulatory effect of RGS14 on liver ischemia-reperfusion injury.2.To study the regulatory effect of RGS14 on inflammatory response in liver ischemia-reperfusion injury.3.To study the regulatory effect of RGS14 on apoptosis in liver ischemia-reperfusion inj ury.Method:1.RGS14 knockout mice and hepatocyte-specific RGS14 transgene mice were constructed and identified.2.The effects of RGS14 deletion and hepatocyte-specific RGS14 transgene on liver ischemia-reperfusion injury in mice were detected.RGS14 deletion and hepatocyte-specific RGS14 transgene mice were used to establish liver ischemia-reperfusion injury model.Serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)values;pathological detection of liver pathological changes of mice in Sham group and I/R group and statistics of liver necrosis area.3.Detection of the regulatory effect of RGS14 deletion and hepatocyte-specific RGS14 gene overexpression on the inflammatory response in mouse liver ischemia-reperfusion injury.The liver ischemia-reperfusion injury model was established in mice with RGS14 deletion and hepatocyte-specific RGS14 transgene.The degree of infiltration of CD11b positive inflammatory cells in mouse liver tissue was determined by immunofluorescence.RT-PCR was used to determine the mRNA expression of inflammation-related factors in liver tissue.The protein expression levels of NF-κB signaling pathway-related molecules in liver tissues were determined by Western-blot(WB).4.Detection of Detect the regulatory effect of RGS14 deletion and hepatocyte-specific RGS14 gene overexpression on apoptosis in mice with liver ischemia-reperfusion injury.The liver ischemia-reperfusion injury model was established in mice with RGS14 deletion and hepatocyte-specific RGS14 transgene.Immunofluorescence and immunohistochemistry were used to determine the number of TUNEL and C-Caspase3 positive cells in mouse liver tissue.The expression of apoptosis-related factor mRNA in liver tissue was determined by RT-PCR.The expression levels of apoptosis-related proteins in liver tissue were determined by WB.5.Construction of RGS14 knockdown and overexpression stable cell lineL02 cells were infected with lentivirus,and stable transgenic cell lines with knockdown and overexpression of RGS14 gene were constructed respectively,and the expression level of RGS14 protein was determined by WB.6.Detection of the regulatory effect of RGS14 knockdown and overexpression on inflammatory response in hepatocyte hypoxia/reoxygenation assayHepatocytes were subjected to hypoxia/reoxygenation treatment for RGS 14 knockdown and overexpression.RT-PCR was used to detect the expression levels of inflammatory-related factors in cells,and WB to determine the protein expression levels of NF-κB signaling pathway-related molecules in cells.7.Detection of the regulatory effect of RGS 14 knockdown and overexpression on apoptosis in hepatocyte hypoxia/reoxygenation assayHepatocytes were subjected to hypoxia/reoxygenation treatment for RGS 14 knockdown and overexpression.RT-PCR was used to detect the expression levels of apoptosis-related factors in cells.The protein expression levels of apoptosis-related molecules in cells were detected by WB.Result:1.RGS 14 attenuates liver ischemia-reperfusion injury in miceCompared with the Sham group,the serum ALT and AST of the I/R group were significantly increased,and the serum ALT and AST of the RGS14-KO mice were significantly higher than those of the WT mice.Liver pathology showed that compared with the Sham group,the liver necrosis of the mice in the I/R group was more obvious,and the area of liver necrosis in the RGS14-KO mice was significantly larger than that in the WT mice.The increase of serum ALT and AST in RGS14-TG mice was significantly lower than that in NTG mice,and the area of liver necrosis in RGS14-TG mice was significantly lower than that in NTG mice.2.RGS14 attenuates the inflammatory response in mouse liver ischemia-reperfusion injuryCompared with the Sham group,the infiltration number of CD11b-positive inflammatory cells in the liver tissue of the I/R group mice was significantly increased,and the infiltration number of CD11b-positive inflammatory cells in the liver tissue of the RGS14-KO mice was significantly higher than that of the WT mice.The expression levels of inflammatory-related cellular molecules(Tnf,Il1b)and chemical factors(Ccl2)in liver tissue were significantly higher than those in WT mice.Western-blot showed that the activation level of NF-κB signaling pathway-related molecules in liver tissue was significantly higher in WT mice.In RGS14-TG mice,the inflammatory response showed the opposite trend.3.RGS14 attenuates apoptosis in mouse liver ischemia-reperfusion injuryCompared with the Sham group,the number of TUNEL-positive and C-Caspase3-positive cells in the liver tissue of the I/R group mice was significantly increased,and the number of TUNEL-positive and C-Caspase3-positive cells in the liver tissue of the RGS14-KO mice was significantly increased.more than in WT mice.The expression levels of apoptosis-related factors(Bax,Bcl2)in liver tissue were significantly higher than those in WT mice,and Western-blot showed that the activation levels of apoptosis-related molecules in liver tissue were significantly higher than those in WT mice.In DaRGS14-TG mice,the opposite trend was observed in apoptosis.4.RGS14 attenuates inflammatory response and apoptosis in hepatocyte hypoxia/reoxygenation inj uryKnockdown of RGS14 can significantly increase the expression levels of inflammatory factors and the activation levels of NF-κB signaling pathway-related molecules in hepatocytes after hypoxia/reoxygenation.Knockdown of RGS 14 can significantly increase the expression levels of apoptotic factors and the activation levels of apoptosis-related molecules in hepatocytes after hypoxia/reoxygenation.RGS14 overexpression significantly reduced intracellular inflammatory response and apoptosis after hepatocyte hypoxia/reoxygenation.Conclusion:RGS14 has a protective effect on hepatic ischemia-reperfusion injury by down-regulating the inflammatory response and apoptosis in ischemia-reperfusion injury.Part Ⅲ:Mechanism of RGS14 alleviating liver ischemia-reperfusion injury via TAK1-p38/JNK pathwayPurpose:To elucidate and verify the molecular mechanism of RGS14 regulating hepatic ischemia-reperfusion injury.Method:1.High-throughput omics screening of signaling pathways that RGS14 plays a regulatory roleRGS14-KO mice and WT mice were used to establish a model of liver ischemia-reperfusion injury.Liver tissue was collected,total RNA was extracted,and genes with significant differences in expression were screened.The KEGG signaling pathway was enriched,and bioinformatics analysis was used to find the signaling pathway significantly altered by the deletion of RGS14.2.In vivo validation of the screened signaling pathwaysRGB 14 deletion and overexpression mice were used to establish liver ischemia-reperfusion injury model,and the expression changes of MAPK signaling pathway and its upstream molecule TAK1 were detected.3.In vitro validation of the screened signaling pathwaysA stable cell line with RGS14 knockdown and overexpression was constructed.After undergoing hypoxia/reoxygenation,the expression changes of MAPK signaling pathway and its upstream molecule TAK1 were detected.4.Validation of the interaction between RGS14 and TAK1Laser confocal detection of RGS14 and TAK1 co-localization,co-immunopreci pitation(CO-IP)and GST-pulldown to verify the interaction between RGS14 and TAK15.Detection of specific domains of RGS14 interaction with TAK1According to the functional domains of TAK1 and RGS14,the functional truncations of TAK1 and RGS14 were constructed,and the specific domains of the interaction between RGS14 and TAK1 were determined by co-immunoprecipitation.6.1 In vitro reversal verification of TAK1-P38/JNK signaling pathway.Using RGS14 knockdown hepatocytes and normal control hepatocytes,TAK1-specific inhibitors 5Z-7-Ox and DMSO were added respectively.After hypoxia/reoxygenation,Western-blot was used to detect the level of TAK1-P38/JNK signaling pathway.6.2 In vitro reversal validation of the inflammatory response in the hepatocyte hypoxia/reoxygenation assay.RGS14 knockdown hepatocytes and normal control hepatocytes were used to add TAK1-specific inhibitor 5Z-7-Ox and DMSO,respectively.After hypoxia/reoxygenation,RT-PCR was used to detect inflammatory response-related factors(Tnf,Il1b,Ccl2)expression changes,Western-blot detection of NF-κB signaling pathway-related molecules levels.6.3 In vitro reversal validation of apoptosis in hepatocyte hypoxia/reoxygenation assay.RGS14 knockdown hepatocytes and normal control hepatocytes were used to add TAK1-specific inhibitor 5Z-7-Ox and DMSO,respectively.After hypoxia/reoxygenation,RT-PCR was used to detect apoptosis-related factors(Bax,Bcl2)expression changes,Western-blot detection of apoptosis signaling pathway-related molecules(C-Caspase3,Bax,Bcl2)levels.Result:1.High-throughput sequencing showed that after liver ischemia-reperfusion surgery in RGS14-KO and WT mice,there were a large number of significantly differentially expressed genes in liver tissue.GSEA analysis showed that inflammatory response and apoptosis-related genes were dominant in RGS14 deletion.KEGG signaling pathway analysis showed that MAPK signaling pathway was the most significant enriched signaling pathway.2.After RGS14-KO mice underwent liver ischemia-reperfusion surgery,the expressions of P-TAK1,P-P38,P-JNK and other proteins in liver tissue were significantly up-regulated.The corresponding RGS14-TG mice P-TAK1,P-P38,P-JNK and other protein expressions were significantly down-regulated.3.After RGS14 knockdown cell lines underwent hypoxia/reoxygenation,the expressions of P-TAK1,P-P38,P-JNK and other proteins were significantly upregulated.After RGS14 overexpressing cell lines underwent hypoxia/reoxygenation,P-TAK1,P-P38,P-JNK and other protein expressions were significantly downregulated.4.Laser confocal confirmed that RGS14 and TAK1 were co-localized in the cytoplasm;CO-IP experiment confirmed that RGS14 interacted with TAK1;GST-pulldown confirmed that RGS14 and TAK1 could interact directly.5.Mapping experiments confirmed that the amino acid sequence of 374-566 of the C-terminal of RGS14 and the amino acid sequence of 1-300 of the N-terminal of TAK1 were combined with each other.6.By specifically inhibiting the phosphorylation of TAK1,it can reverse the up-regulation of P-P38/P-JNK expression;it can counteract the up-regulation of inflammatory response-related factors(Tnf,Il1b,Ccl2)and the activation of NF-κB signaling pathway-related molecules;It can counteract the up-regulation of apoptosis-related factors(Bax,Bcl2)and the activation of apoptosis signaling pathway-related molecules(C-Caspase3,Bax,Bcl2).Conclusion:RGS14 regulates the P-38/JNK signaling pathway by inhibiting the phosphorylation of TAK1,reducing hepatic IRI.The regulatory effect of RGS14 on hepatic ischemia-reperfusion injury depends on TAK1 activation.