The Effects And Mechanism Of Advanced Glycation End Products On Human Chondrocytes Pyroptosis

OBJECTIVE:In this study,by collecting clinical samples from patients with osteoarthritis(OA)and normal people,and performing comparison,analysis,and in vitro cytology experiments,we initially explored the relationship between chondrocyte pyroptosis and OA,and advanced glycation end products(AGEs)effect and mechanism of action on chondrocyte pyroptosis.MATERIALS AND METHODS:(1)RT q PCR method was used to detect the expression of NLRP3,Gasdermin D(GSDMD),IL-1β,IL-18 m RNA in cartilage tissue of OA and normal group;NLRP3,GSDMD protein expression of cartilage in two groups was detected by Western-blot.ELISA was used to detect the differences in IL-1β and IL-18 levels in joint fluid between the two groups;the transmission electron microscope was used to observe the differences in cartilage morphology between the two groups.(2)Alcian Blue Staining method and typeⅡ collagenase immunofluorescence method were used to identify the isolated chondrocytes.(3)Intervene human normal chondrocytes with AGEs at a concentration of 300 μg/m L at different times(0,6h,12 h,24h,48 h,72h).RT q PCR and Western blot methods were used to detect the expression levels of NLRP3,GSDMD m RNA and protein,and transmission The morphological changes of chondrocytes after AGEs treatment were observed under electron microscope.(4)RT q PCR and Western blot were used to detect the expression levels of RAGE and PPARγ in cartilage tissues of normal and OA group;AGEs with a concentration of 300 μg/m L were used to intervene in human normal chondrocytes at different times(0,6h,12 h,24h,48 h,72h),RT q PCR and Western blot to detect the m RNA and protein expression levels of PPARγ and RAGE after AGEs acted on chondrocytes at different times;the expression of PPARγ,RAGE,NLRP3 and GSDMD after chondrocytes were interfered by the RAGE-specific inhibitor FPS-ZM1.(5)After pretreatment of normal chondrocytes with PPARγ inhibitor T0070907 or PPARγ agonist pioglitazone,RT q PCR detected the m RNA expression of NLRP3,GSDMD,IL-1β and IL-18,Western blot detected the expression level of NLRP3,GSDMD protein,ELISA The content of IL-1β and IL-18 in cell culture supernatant was detected.Results:(1)Compared with normal group,the expression of NLRP3,GSDMD,IL-1β,IL-18 genes and protein in OA patients increased significantly,however the expression of PPARγ decreased cleavely;the differences between the groups were statistically significant.In the OA cartilage tissue,the chondrocytes showed swelling,a large number of die holes and other cell pyromorphosis,while the normal group maintain the characteristic of normal chondrocyte.(2)The chondrocytes have strong activity and high purity obtained by mechanical separation combined with pancreatin-collagen sequential digestion.(3)Compared with the normal chondrocyte,the m RNA and protein expression Levels of NLRP3,GSDMD in normal chondrocytes treated by AGEs were up-regulated,and were closely related to the time of AGEs treatment.The effect was most effective when AGEs at 300μg/m L was treated for 72 h.Obviously,the difference is statistically significant.After treatment with 300μg/m L AGEs for 72 h,the chondrocytes had obvious swollen cells and a large number of die holes.(4)Compared with the normal group,the expression of RAGE m RNA and protein in the cartilage tissue of OA patients was up-regulated,however the expression of PPARγ was significant Ly down-regu Lated,The difference was statistically significant;compared with the normal group,the AGEs treated normal chondrocytes induce RAGE expression up-regulated and PPARγ expression was down-regulated,while RAGE inhibitor FPS-ZM1 cou Ld inhibit the effect of AGEs on the expression of chondrocytes RAGE and PPARγ.The differences were statistically significant.(5)The PPARγ inhibitor T0070907 can promote the expression of NLRP3,GSDMD,IL-1β,IL-18 at the m RNA and protein Levels in AGEs-induced human chondrocytes,however the PPARγ agonist pioglitazone can weaken GSDMD,IL-1β,IL-18 expression Levels,and the comparison results have statistical significance.Conclusion:(1)Pyroptosis occurs in osteoarthritis chondrocytes.(2)AGEs can induce the occurrence of pyroptosis of human normal chondrocytes in vitro.(3)AGEs have an effect on human chondrocyte pyroptosis through the RAGE/PPARγ signaling pathway.