HepG2 Cells Cellular Uptake And Insulin Resistance Improvement Mechanism Of Tartary Buckwheat Protein-derived Peptide AFYRW

Objective:To elucidate the mechanism of tartary buckwheat protein-derived peptide AFYRW into HepG2 cells and its targeting,and to predict the interaction between the AFYRW and its targeting protein.The HepG2 cell model of insulin resistance as well as the C57/BL6 mouse model of type 2 diabetes were also established to elucidate the mechanism,which AFYRW improveing insulin resistance based on the SIRT1/PGC-1αsignaling pathway.Methods:1.Taking HepG2 cells as experimental objects.HepG2 cells cytotoxicity assessment of AFYRW was detected by CCK8 and erythrocyte hemolysis experiments.The mechanistic pathway of AFYRW entry into HepG2 cells was found by observing the sublocalization of AFYRW and detecting the uptake of FITC-AFYRW by HepG2cells after treatment with multiple endocytosis inhibitors by flow cytometry.AFYRW coupled to biotin,through biotin-streptavidin action and protein profiling,selected potential targets of AFYRW,and predicted the interaction between the two using molecular docking technology.2.18 male C57/BL6 mice were randomly divided into three groups:normal diet group(ND group),high fat diet group(HFD group),and AFYRW treatment group(HFD+AFYRW group).The ND group was fed with standard rat feed;the HFD group was fed with high fat diet;and the HFD+AFYRW group was intraperitoneally injected with AFYRW(20 mg/kg/d)for one week before high fat feeding,followed by AFYRW intraperitoneal injection for 6 weeks while maintaining high fat feeding.Six weeks later,the C57/BL6 mouse model of diabetes was established by continuous intraperitoneal injection of 55 mg/kg streptozotocin(STZ)for 3 days.After establishing successful model,mice were maintained on high fat feeding and AFYRW intraperitoneal injection for 6 weeks.After 6 weeks glucose tolerance was measured by OGTT.The mice were sacrificed and serum samples were collected to detect various blood lipid levels and inflammatory factor contents.Liver tissues were also collected to detect the accumulation of lipids in liver tissues and HE staining was used to observe the morphology of liver tissues.3.0.25 mmol/L palmitic acid(PA)stimulated HepG2 cells.meanwhile,resveratrol was given as a positive drug,and AFYRW(5μg/m L at low concentration,20μg/m L at high concentration)was incubated for 24 h.IR-HepG2 model was established and treated with intervention.After drugs treatment,the uptake of glucose in each group was detected,and the contents of manganese superoxide dismutase(Mn SOD)and mitochondrial isocitrate dehydrogenase(ICDHm),mitochondrial ROS,mitochondrial membrane potential were detected.Western blot was used to detect the expression of SIRT1/PGC1αsignaling pathway-related proteins.Results:1.AFYRW was non-toxic by CCK-8 as well as erythrocyte hemolysis(P>0.05).Confocal laser scanning results indicated that AFYRW could be localized in the nucleus.After pretreatment with endocytosis inhibitors(NH4Cl,CQ),especially caveolin-mediated endocytosis pathway inhibitor(Nystatin),the uptake of AFYRW by HepG2 cells decreased significantly(P<0.01).Biotin-streptavidin,protein profiling,molecular docking results indicated that PARP-1 was a potential target protein of AFYRW.2.In C57/BL6 diabetic model mice,blood glucose and lipid levels were increased(P<0.01),HOMA-IR index was significantly enhanced(P<0.01),and there was a severe inflammatory response(P<0.01).The ectopic accumulation of triglyceride(P<0.01)and free fatty acid(P<0.05)in liver tissue was severe,and there was significant steatosis in hepatocytes.The NAD~+content in liver tissue decreased(P<0.01).After AFYRW treatment,the blood glucose and blood lipid levels were decreased(P<0.01),the inflammatory response was attenuated(P<0.05),the HOMA-IR index was decreased(P<0.01),the ectopic accumulation phenomenon was reduced(P<0.01),and the hepatocyte steatosis was alleviated.The content of NAD~+in liver tissue increased(P<0.01).3.After palmitic acid stimulation,the glucose uptake ability was decreased(P<0.01),the contents of Mn SOD and ICDHm were reduced(P<0.01),Mito ROS was increased P<0.01),the mitochondrial membrane potential was significantly reduced,and SIRT1,PGC-1α,and NRF-1 expression was down-regulated(P<0.01)in IR-HepG2.After treatment with different concentrations of AFYRW,various parameters were improved in IR-HepG2.Among them,cells in the high-dose group(20μg/m L)had enhanced glucose uptake ability(P<0.01),increased antioxidant substance(Mn SOD and ICDHm)content(P<0.01),improved mitochondrial oxidative stress(Mito ROS)(P<0.01),gradually increased mitochondrial membrane potential,and up-regulated SIRT1,PGC-1α,and NRF-1 expression(P<0.05)Conclusions:1.AFYRW can enter cells and localize to the nucleus through the caveolin-mediated endocytic pathway,and it is speculated that PARP-1 may be a potential target molecule of AFYRW in nuclear proteins.2.AFYRW can improve insulin resistance by improving the disorder of glucose and lipid metabolism and alleviating liver injury.The mechanism may be related to the activation of SIRT1/PGC-1αsignal pathway to improve mitochondrial function.