Effect Of Myeloid-Specific Knockout Of SNX16 On Hepatic Ischemia-Reperfusion Injury In Mice

Objective:Sorting Nexin 16(SNX16)is a member of the sorting nexin family that contains the conserved Phox-homology domain(PX domain)of phagocyte oxidase.SNX16 is mainly distributed in intracellular bodies.The role of SNX16 is to participate in the regulation of transporting intracellular proteins.Ischemia-reperfusion injury(I/R)refers to a syndrome in which tissue or organ is further damaged and deteriorates organ function by restoring the blood flow following a period of ischemia.Our previous experimental results have shown that SNX16 is involved in LPS-mediated inflammatory response in the body,but its role in sterile inflammation is still unclear.This study aims to investigate the effect of myeloid-specific knockout of SNX16 on liver ischemia-reperfusion injury in mice.Methods:1.SNX16flox/flox F/F(-)mice were bred with Lyz2-Cre(myeloid-specific expressing Cre)mice to generate myeloid-specific SNX16 knockout mice F/F(+);R/R F/F(+)mice were generated by crossing SNX16flox/flox Cre mice with Gt(ROSA)26mTmG(ACTB-td Tomato,-EGFP)Luo mice.2.The mice were divided into four groups: F/F(-)control group,F/F(-)experimental group,F/F(+)control group,and F/F(+)experimental group.3.The serum was extracted from mice,and the contents of ALT and AST were detected.The routine analysis of blood was performed for the number of white blood cells.The left/middle liver tissues were extracted,and the expression levels of MDA,SOD,LDH,and other oxidative stress indicators in the liver were detected.4.Mouse left/middle liver tissues were extracted,fixed with 4%PFA for 12 hours,dehydrated,and embedded in paraffin.HE staining was used to observe the size of the damaged area of the liver(to detect the degree of liver injury).Tunel(TdT-mediated d UTP Nick-End Labeling)staining was performed to measure the apoptosis in the liver in mice.5.Left/middle lobe liver tissues(about 3-4mm3)of mTmG Cre mice including R/R(+),R/R F/F(+)were extracted,fixed with 4%PFA,dehydrated in sucrose/embedded in OCT,and frozen at -80℃.8μm frozen sections were used to observe the distribution of myeloid-derived macrophages in liver tissue under the fluorescence microscope.6.Western Blot was used to detect the expression of TNF-α,MCP-1,IL-10,i NOS,CD206,BCL-2,BAX,and other inflammatory factors.Total RNA was extracted from the left/middle liver lobe of mice,and the expression of TNF-α,IL-6,and IL-1β m RNA was detected by RT-qPCR.Results:1.Myeloid-specific knockout SNX16 mice and myeloid-specific knockout SNX16 ROSA mTmG mice were successfully constructed.2.Myeloid-specific knockout of SNX16 significantly alleviated the degree of liver damage in IR mice and decreased the levels of ALT and AST in serum.3.Myeloid-specific knockout of SNX16 reduced the expression of inflammatory factors TNF-α,IL-6 and MCP-1 in the liver of IR mice,increased the expression of anti-inflammatory factor IL-10,maintained the stable number of white blood cells in IR mice,and reduced the content of oxidative stress indicators MDA,LDH and IL-1β,and increased the contents of antioxidant index SOD.The expressions of TNF-α,IL-6 and MCP-1 were decreased in the IR liver of FF(+)mice.4.The expression of M2 macrophage surface marker CD206 was increased,the expression of M1 macrophage surface marker i NOS was decreased in the IR livers in myeloid-specific SNX16 knockout mice.5.Myeloid-specific knockout of SNX16 increased The expression of BCL-2,an anti-apoptotic protein was increased,and the expression of apoptotic protein BAX was decreased,and the number of Tunel-positive cells was reduced in in the IR livers in myeloid-specific SNX16 knockout mice.Conclusion:In conclusion,myeloid-specific knockout of SNX16 significantly reduced the area of IR liver damage and significantly reduced the serum ALT and AST levels in IR mice;Myeloid-specific knockout of SNX16 reduced inflammation and oxidative stress in the liver tissue of IR mice,inhibited the infiltration of myeloid macrophages into the IR area,promoted M2 polarization,and significantly reduced apoptosis in the IR liver tissue in mice.Therefore,myeloid-specific knockout of SNX16 plays a protective effect on liver ischemia-reperfusion injury.