The Role Of FAK/Pyk2 In High Glucose-induced Pyroptosis Of Rat Renal Tubular Epithelial Cells

Objietive:To understand the expression changes of FAK,p-FAK,Pyk2,p-Pyk2 and pyroptosis-related proteins in rat renal tubular epithelial cells induced by high glucose at different time points;To investigate the role of FAK/Pyk2 in high glucose-induced pyroptosis of renal tubular epithelial cells.Method:1.The cultured cells were identified by immunocytochemical staining to detect epithelial cell marker proteins keratin CK-18,E-cadherin and mesenchymal cell marker protein α-smooth muscle actin(α-SMA).Rat renal tubular epithelial cells(NRK52E).2.Grouping after cell passage: 1)normal glucose control group;2)high glucose group,in which three time points of 24 h,48h and 72 h were set in the high glucose stimulation group.After culturing for a corresponding time,the cells were collected and total RNA and protein were extracted for subsequent experiments: Real Time-PCR was used to detect the m RNA levels of NLRP3,ASC,caspase-1,GSDMD,FAK,and Pyk2;Western Blot was used to detect FAK,p-FAK,Pyk2,Protein levels of p-Pyk2 and pyroptosis-related proteins NLRP3,ASC,pro-caspase-1,caspase-1,GSDMD,GSDMD-N.ELISA was used to detect the release of IL-1β and LDH in cells and cell culture supernatants.The renal tubular epithelial cells cultured in high glucose for 48 hours were selected as the research object for further study.NRK-52 E cells cultured in vitro were divided into 4 groups: 1)normal glucose control group;2)mannitol hypertonic group;3)high glucose group;4)high glucose + PF-562271(FAK,Pyk2 Small molecule inhibitor)group;After 48 hours of cell culture,the cells were collected and the protein was extracted.Western Blot was used to detect the protein expression of NLRP3,ASC,pro-caspase-1,caspase-1,GSDMD,GSDMD-N,FAK,p-FAK,Pyk2 and p-Pyk2.Result:1.Real Time-PCR results showed that compared with the normal glucose control group,the m RNA expressions of NLRP3,GSDMD,ASC,caspase-1,FAK and Pyk2 in each high glucose group were all increased(P<0.05);Western blot The results showed that compared with the normal glucose control group,the protein expressions of NLRP3,GSDMD-N,ASC,pro-caspase-1,caspase-1,p-FAK/FAK,p-Pyk2/Pyk2 were all increased in the high glucose groups.The highest expression at 48 hours(P<0.05);ELISA results showed that compared with the normal glucose control group,the intracellular levels of IL-1β and LDH in the high glucose groups increased,and the levels were higher at 48 hours of culture,and the levels in the culture supernatant increased.The release amount increased gradually(P<0.05).2.Western blot results showed that compared with the normal glucose group,the related proteins in the mannitol hyperosmolar group did not change significantly(P>0.05),the protein expression of NLRP3,GSDMD-N,ASC,pro-caspase-1,caspase-1,p-FAK/FAK,p-Pyk2/Pyk2 in the high glucose group was increased(P<0.05);Compared with the high glucose group: after intervention with PF-562271,the protein expression of NLRP3,GSDMD,GSDMD-N,ASC,pro-caspase-1,caspase-1,p-FAK/FAK,p-Pyk2/Pyk2 was decreased(P<0.05).Conclusion:1.The expression levels of pyroptosis-related proteins in rat renal tubular epithelial cells induced by high glucose increased,accompanied by the release of active inflammatory factors and the activation of FAK and Pyk2.2.After inhibiting the activation of FAK and Pyk2 with PF-562271,the expression of pyroptosis-related proteins decreased,suggesting that FAK/Pyk2 may be involved in the pyroptosis of renal tubular epithelial cells induced by high glucose.