The Effect Of Ass1 On TLR4-mediated Metaflammation Damage In ? Cells

Background:The metaflammation induced by an excessive increase of free fatty acid(FFA)plays an important role in islet ?-cells damage,activation of toll like receptor 4(TLR4)is one of the pathways by which FFA triggers metaflammation.Our previous study found inhibition of TLR4 activity or expression could antagonize insulin resistance caused by high-fat diet and alleviate ?-cells dysfunction caused by lipotoxicity,however,there are no targeted intervention for metaflammation,and the mechanism of TLR4-mediated metaflammation damage in ? cells has not been fully clarified.Therefore,this study will further investigate the effects of TLR4 knockout(TLR4KO)on islet function and pancreatic proteomics in obese rats induced by high-fat diet and the character of arginosuccinate synthetase 1(Ass1),the nodal protein based on proteomic results,in TLR4-mediated metaflammation damage in ? cells.Objectives:This study was divided into three parts:1 To investigate the effect of TLR4 KO on islet ?-cells dysfunction and pancreatic proteomics in obese rats induced by high fat diet.2 To investigate the effect of TLR4 on Ass1 and its downstream arginine in the process of lipotoxicity affecting ? cells.3 To investigate the effect of Ass1 and arginine on lipotoxicity-damaged ? cells.Methods:1 To investigate the effects of TLR4 KO on islet dysfunction and pancreatic proteomics in SD obese rats induced by high fat diet1.1 To study the effect of TLR4 KO on islet dysfunction in obese ratsCRISPR/Cas9 gene editing technique was used to knockout the TLR4 gene in sprague-dawley(SD)rats to construct a TLRKO rat model,high fat diet was fed to SD rats for 16 weeks to construct a high fat die-induced obesity rat model.Obesity models was evaluated by Lee's index.Islet function were evaluated by intraperitoneal glucose tolerance test(IPGTT),insulin tolerance test(ITT),and homeostasis model assessment beta(HOMA-?).Rat serum insulin and IL-1,IL-6were detected by ELISA assay.The inflammatory infiltration of pancreatic islets was observed by hematoxylin-eosin staining.Pancreatic islets structural changes and the ratio of ? and ? cells were analyzed by immunofluorescence(IF).TUNEL assay was used to detect pancreatic cells apoptosis.1.2 To study the effect of TLR4 KO on pancreatic proteomics in obese ratsConstruct TLR4 KO and obese rat models.i TRAQ was used to detect pancreatic proteomics.Nitro reductase method was used to detect nitric oxide level of blood serum.Ass1,phosphorylated Ass1,L-arginine and L-citrulline contents of rat pancreas were detected by ELISA assay.2 To investigate the effect of TLR4 on ASS1 and arginine in the process of lipotoxicity damaging ? cells2.1 Investigate the changes of ASS1 and arginine contents during the process of lipotoxicity damaging ?-cell function1mmol/L palmitic acid(PA)was used to intervene ?-tc6 cells for 24 h to establish lipotoxic ?-cell model.Ass1,phosphorylated Ass1,L-arginine and L-citrulline contents,basal insulin secretion(BIS)and glucose-stimulated insulin secretion(GSIS)of ? cells were detected by ELISA assay.The apoptosis of ? cells were detected by flow cytometry.2.2 The effect of TLR4 on Ass1 and arginine in lipotoxicity damaged ?-cellsTLR4 activity was regulated by 1ug/ m L LPS(TLR4 agonist)or 4umol/L CLI-095(TLR4 agonist)and TLR4 gene was knocked out or overexpressed by CRISPR-Cas9 technology in ? cells.Ass1,phosphorylated Ass1,L-arginine and L-citrulline contents of ? cells were detected by ELISA assay.2.3 To investigate the relationship between TLR4 and ASS1 in regulating arginine production in ? cells2.3.1 Investigate the effect of ASS1 on arginine production in lipotoxic ? cellsASS1 activity was regulated by 1umol/L Phorbol 12-myristate 13-acetate(PMA,Protein Kinase C alpha(PKC?)agonist,)or 1mmol/L DL-?-methyl aspartic acid(MDLA,a specific inhibitor of ASS1),or using CRISPR-Cas9 technique to overexpress ASS1 gene in lipotoxicity damaged ? cells.The content of arginine in each group was detected by ELISA.2.3.2 Investigate the effect of ASS1 on TLR4 regulating arginine production in? cellsActivate TLR4 activity of ? cells,and regulate ASS1 activity or overexpress ASS1.The content of arginine in ?-cells was detected by ELISA.2.4 Verify the interaction between TLR4 and Ass1The co-localization of TLR4 and ASS1 in ?-tc6 cells was observed by immunofluorescence co-localization.Co-immunoprecipitation was used to verify the interaction between TLR4 and Ass1.3 To investigate the effects of Ass1 and arginine on lipotoxicity damaged ? cells3.1 Investigate the effects of ASS1 on lipotoxic ? cellsRegulate Ass1 activity or expression in lipotoxicity damaged ? cells,the apoptosis of ?-cells were detected by flow cytometry,expressions of proteins were detected by western blot,ELISA assay was used to detected insulin.3.2 Investigate the effects of arginine on lipotoxic ? cells10mmol/L arginine was used to interfere lipotoxicity damaged ? cells,the apoptosis of ?-cells were detected by flow cytometry,expressions of proteins were detected by western blot,ELISA assay was used to detected insulin.Results:1 TLR4 KO improved islet function and rebalanced the disorder of pancreatic proteomics in obese rats induced by high fat dietTLR4KO reduced the body weight,fasting blood glucose and serum inflammatory cytokines(IL-1,IL-6),improved glucose tolerance and insulin tolerance,insulin secretion,reduced islet inflammatory infiltrates and apoptosis of pancreatic cells,improved the distribution of ? and ? cells in pancreas of obese rat induced by high fat diet.TLR4KO decreased the expressions of 11 up-regulated proteins and increased the expressions of 7 down-regulated proteins in the pancreas of obese rats;GO analysis showed that the main biological processes of differential proteins were cellular macromolecular metabolism,cellular component organization or biogenetic process,and cellular nitrogen metabolism.The pathways involved in differential proteins mainly included metabolic pathways,arachidonic acid metabolism,extracellular matrix receptor interaction,pancreatic secretion.PPI analysis showed that serine-threonine kinase 39(Stk39)and Ass1 were nodal proteins among differential proteins,and they were involved in inflammatory response,external stimulus response,defense response,carboxylic acid metabolism and small molecule metabolism.TLR4 KO restored the contents of pancreas ASS1 and arginine in obese rats induced by high fat diet,and reduced the serum NO level in normal rats and obese rats.2 TLR4 mediated the inhibitory effect of PA on ASS1 and arginine production in ?-cell,and promoted the phosphorylation of ASS1PA,activating or overexpressing TLR4 decreased the content of Ass1 and arginine in ?-tc6 cells,promoted the phosphorylation of ASS1.Silencing or inhibiting TLR4 reduced the decrease of Ass1 and arginine in ?-tc6 cells induced by PA.Overexpressing Ass1 reduced the decrease of arginine in ?-tc6 cells induced by PA and LPS.There exists co-localization and interaction between TLR4 and ASS1.3 ASS1 could alleviate lipotoxicity damage in ?-cellsInhibition of Ass1 promoted cell apoptosis and protein expression of TLR4 and NF-?B,impaired glucose-stimulated insulin secretion in ?-tc6 cells.While overexpression of Ass1 improved cell apoptosis,antagonized the protein expression of TLR4 and NF-?B,improved the insulin secretion dysfunction induced by PA in ?-cells.Arginine improved insulin secretion of lipotoxicity damaged ?-cells,antagonized PA-induced increases of TLR4 and NF-?B protein expression,inhibited the protein expression of Ass1.Conclusions:Ass1 could alleviate the metaflammation damage induced by lipotoxicity in pancreatic ? cells through antagonizing TLR4-NF-?B pathway,which is probably related to the regulation of arginine production.