The Role And Mechanism Of TGR5 In Alleviating Hepatic Ischemia-reperfusion Injury

Objective:Hepatic ischemia-reperfusion injury(HIRI)is an inevitable problem in hepatectomy and liver transplantation.Macrophage-mediated inflammatory response plays an important role in its pathogenesis.Plasma membrane-bound G-protein-coupled bile acid receptor 5(TGR5)is one of a family of G-protein-coupled receptors that have been shown to play a protective role in a variety of liver diseases.However,the protective mechanism of TGR5 in HIRI remains unclear.Therefore,we attempted to determine whether TGR5 improves HIRI by reducing inflammatory response or inhibiting apoptosis,and to clarify whether TGR5 plays a protective role in HIRI by activating the KEAP1-NRF2 signaling pathway.Methods:We injected a small interfering RNA of TGR5(si-TGR5)or TGR5 agonist(INT-777)into wild mice and established a model of liver partial warm ischemia/reperfusion.Depending on the intervention,they were divided into sham operation group(Sham),sham operation+si-TGR5 pretreatment group(Sham+si-TGR5),sham operation+INT-777 pretreatment group(Sham+INT-777),6-hour hepatic ischemia reperfusion group(IR6),IR6+si-TGR5 pretreatment group(1R6+si-TGR5)and 1R6+INT-777 pretreatment group(IR6+INT-777).Murine bone marrow-derived macrophages(BMDMs)were isolated from mice bone marrow for cell experiments.Depending on the intervention,they were divided into control group(Control),control+si-TGR5 pretreatment group(Control+Si-TGR5),control+INT-777 pretreatment group(Control+INT-777),hypoxia and reoxygenation group(H/R),and H/R+si-TGR5 pretreatment group(H/R+s1-TGR5)and H/R+INT-777 pretreatment group(H/R+INT-777).The degree of hepatic damage was assessed by the serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)using an automated chemical analyzer.Quantitative real-time PCR(qRT-PCR)analyses were used to detect the expression of Ribonucleic Acid(RNA).Western blot analysis was used to measure protein levels.Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)staining was used to evaluate apoptosis.The levels of cytokines were assessed using enzyme-linked immunosorbent assay(ELISA).Immunohistochemical and immunofluorescence were used to identify tissue intracellular antigens(peptides and proteins).Results:In vivo experiments,we found that(1)TGR5 can protect liver tissue against hepatic IRI;(2)TGR5 can reduce cell apoptosis in hepatic IRI;(3)TGR5 can inhibit hepatic inflammation induced by ischemia-reperfusion injury;(4)TGR5 can alleviate hepatic IRI through Keap1-Nrf2 signaling pathway.In vitro experiments showed that(1)TGR5 inhibited inflammatory response and apoptosis of bone marrow derived macrophages in H/R model;(2)TGR5 can alleviate the inflammatory response in macrophage H/R model through keap 1-Nrf2 pathway.Conclusions:Our results suggested that activation of the Keap1-Nrf2 signaling pathway is important for TGR5 protecting against HIRI.TGR5 could not only reduce hepatocellular apoptosis but also inhibit inflammatory response by regulating Keap1-Nrf2 signaling pathways.These findings might provide a new and potential therapeutic approach for preventing inflammatory response and hepatic I/R injury.Therefore,targeting the TGR5 molecule may benefit the outcome of liver surgery.