Mechanism Of DAIa2GIP Inhibiting IL-1?-induced Endoplasmic Reticulum Stress And Apoptosis In Chondrocytes

Objective:1.To observe the protective effect of DAIa2 GIP on oxidative stress and apoptosis induced by IL-1? in vitro;2.To explore the molecular biological mechanism of DAIa2 GIP repairing chondrocyte injury.Methods:In this experiment,the SD rat costal chondrocytes were cultured on the 7th day after birth,and the third generation cells were used for the experiment.They were divided into normal control group,IL-1? induction group,IL-1?+DAla2GIP incubation group(drug treatment group),IL-1?+DAla2GIP+pro3GIP(GIPR antagonist)incubation.Group,DAla2 GIP incubation group,pro3 GIP incubation group.The normal control group was cultured in DMEM medium containing 1%double antibody and containing 10% serum.The IL-1? induction group was cultured in DMEM medium containing 10 ng/m L IL-1?,containing 1% double antibody and containing 10% serum.The IL-1?+DAla2GIP-incubation group(drug-treated group)was cultured in DMEM medium containing 10 ng/m L IL-1?,containing 1% double antibody,containing 100 p MDAla2 GIP,containing 10% serum;IL-1?+DAla2GIP+pro3 GIP The incubation group was cultured in DMEM medium containing 10 ng/m L IL-1?,1% double antibody,100 p M DAla2 GIP,100 p M pro3 GIP,10% serum;DAla2GIP incubation group containing 100 p M DAla2 GIP,containing 1% double antibody,The cells were cultured in DMEM medium containing 10% serum;the pro3 GIP incubation group was cultured in DMEM medium containing 1% double antibody,100 p M pro3 GIP,and 10% serum.After incubation for 48 h under the above conditions,TUNEL cell apoptosis,CCK-8 cell proliferation rate,intracellular reactive oxygen species detection and RT-PCR were used to detect the changes of calreticulin and caspase-12 m RNA expression in each group.Resules:1.CCK-8 method was used to detect the proliferation of chondrocytes in each group: 48 hours after drug treatment,the OD value of chondrocyte proliferation in IL-1?-induced group(1.27±0.01)and the proliferation of chondrocytes in IL-1?+DAla2GIP+pro3GIP group The values(1.34±0.01)were lower than the normal control group(1.78±0.03)(P<0.01).The OD value of chondrocyte proliferation in the IL-1?+DAla2GIP group was significantly higher than that in the IL-1? group(P<0.01);There was no significant difference in OD value(1.34±0.01)between chondrocytes in IL-1?-induced group and IL-1?+DAla2GIP+pro3GIP group(P>0.05);chondrocyte proliferation in normal control group and DAla2GIP-incubated group There was no significant difference in the OD value(1.70±0.01)between the OD value(1.72±0.02)and the pro3 GIP incubation group(P>0.05).2.TUNEL was used to detect the apoptosis rate of each group: compared with the normal control group(2.0±0.4),the apoptosis rate of IL-1? induced group was significantly increased(10.4±0.6)(P<0.01).The apoptosis rate of IL-1?+DAla2GIP group was compared.(1.8±0.4)was significantly lower than IL-1? induction group(P<0.01),and the apoptosis rate was significantly increased after adding GIP antagonist.The apoptosis rate of IL-1?+DAla2GIP+pro3GIP group was(9.0±0.1),significantly higher than IL-1?+DAla2GIP incubation group(P<0.01);DAla2GIP incubation group(2.1±0.1)and pro3 GIP incubation group(2.1±0.1)apoptosis rate and control group apoptosis rate no statistics Learning differences(P>0.05).3.Flow cytometry analysis of changes in reactive oxygen species in each group:Compared with the normal control group(1.7±0.6),the intracellular reactive oxygen species(6.4±0.8)in the IL-1?-induced group was significantly increased.High(P<0.01);intracellular reactive oxygen species(2.2±0.4)in IL-1?+DAla2GIP group was significantly lower than IL-1? induction group(P<0.01);IL-1?+DAla2GIP group was compared with IL-1?+DAla2GIP group.The intracellular reactive oxygen species(5.3±0.5)was significantly increased in the-1?+DAla2GIP+pro3GIP group(P<0.01).The active oxygen content(1.4±0.4)in the normal control group and DAla2 GIP group was compared with the GIPR antagonist group.There was no statistically significant difference between the active oxygen content(1.3 ± 0.2).4.Changes in the expression of calreticulin and caspase-12 m RNA in the RT-PCR assay: compared with the normal control calreticulin(1.65±0.58)and caspase-12(1.61±0.57),the IL-1? induction group The m RNA expression of calreticulin(9.42±1.11)and caspase-12(29.11±6.70)in chondrocytes was significantly increased,which was statistically significant(P<0.01).Compared with IL-1?-induced group,IL-1?+ The m RNA expression of calreticulin(4.10±1.23)and caspase-12(2.50±0.36)in the cartilage cells of DAla2 GIP group was significantly decreased(P<0.01).IL-1?+DAla2GIP+pro3 GIP incubation group The m RNA expression of calreticulin(7.11±1.57)and caspase-12(8.92±1.77)was significantly higher than that of IL-1?+DAla2GIP(P<0.01).Compared with the normal control group,DAla2 GIP was antagonized with GIPR.The m RNA expression of calreticulin and caspase-12 in the incubation group was not statistically significant(P>0.05).Conclusion:1.DAIa2 GIP effectively relieves IL-1?-induced chondrocyte endoplasmic reticulum stress;2.DAIa2 GIP inhibits IL-1?-induced chondrocyte apoptosis;3.DAIa2 GIP can alleviate the endoplasmic reticulum stress of chondrocytes by attenuating the active oxygen content in chondrocytes,and down-regulate the m RNA expression of calreticulin and caspase-12 to inhibit the apoptosis of chondrocytes.