| Objective:1.The effect of DAIa2 GIP on IL-1 β induced mitochondrial apoptosis was observed;2.To explore the molecular mechanism of DAIa2 GIP against chondrocyte injury;3.DAIa2 GIP can provide a new idea for the treatment of osteoarthritis,find a new breakthrough,and provide experimental basis for the research of osteoarthritis transformation medicine.Methods:In this experiment,the costal chondrocytes of SD rats born on the 7th day were carefully separated and digested,then cultured in DMEM medium containing 1% double antibody and 10% fetal bovine serum,and then made cell climbing tablets for cell identification by immunohistochemistry(IHC).The third generation cells were selected for this experiment.The cells were divided into 6 groups after different treatment on the basis of the above medium,including:(1)normal control group: no other drugs added to the original medium;(2)IL-1 β induction group : add IL-1 β(10 ng /m L);(3)IL-1β+DAla2GIP co incubation group: add IL-1 β(10 ng / m L),DAla2GIP(100p M);(4)IL-1β+DAla2GIP+pro3GIP(GIPR antagonists)co incubation group: add IL-1 β(10 ng / m L),DAla2GIP(100p M),pro3GIP(100p M);(5)DAla2GIP induction group: add DAla2GIP(100p M);(6)pro3GIP incubation group: add pro3GIP(100p M).After incubation in the same environment for 48 h,the cells in each group were tested for mitochondrial membrane potential,Annexin-V FITC Kit(phosphatidylserine eversion analysis)cell apoptosis,laser confocal microscopy cell calcium overload,ELISA method for cytochrome C content and Western blot method for p53 and Bcl-2 content.Results:1.The chondrocytes were identified as chondrocytes by SABC immunohistochemical method.The cells grew well.The cells in the dense area were mostly round,and the cells in the sparse area were mostly polygonal.The third generation cells were taken for follow-up experiment.2.Mitochondrial membrane potential and apoptosis of annexin-V FITC Kit:(1)Preservation of mitochondrial membrane potential in chondrocytes(Flow Cytometry): IL-1β+DAla2GIP co incubation group(93.15.10%±0.67%)is significantly higher than IL-1 β induction group(81.00%±1.48%)(P<0.01);There was no significant statistical difference between IL-1 β and IL-1β+DAla2GIP+pro3GIP co incubation group(81.65%±2.26%)(P>0.05),and both were lower than normal control group(95.10%±0.42%)(P<0.01);There was no significant statistical difference between normal control group,DAla2 GIP incubation group(94.95%±0.49%)and pro3 GIP incubation group(92.95%±0.35%)(P>0.05).(2)Preservation of mitochondrial membrane potential in chondrocytes(fluorescence microscope):IL-1β+DAla2GIP co incubation group(93.36%±2.34%)is significantly higher than IL-1 β induction group(89.86%±1.76)%(P<0.01);There was no significant statistical difference between IL-1 β and IL-1β+DAla2GIP+pro3GIP co incubation group(89.07%±1.40%)(P>0.05),and both were lower than normal control group(95.43%±1.31%)(P<0.01);There was no significant statistical difference between normal control group,DAla2 GIP incubation group(93.9%±2.28%)and pro3 GIP incubation group(93.5%±1.56%)(P>0.05).(3)Apoptosis rate of chondrocytes(Flow Cytometry):IL-1β+DAla2GIP co incubation group(1.05%±0.05%)is significantly lower than IL-1 β induction group(3.85%±0.55%)(P<0.01);There was no significant statistical difference between IL-1 β and IL-1β+DAla2GIP+pro3GIP co incubation group(3.25%±0.25%)(P>0.05),and both were higher than normal control group(0.95%±0.15%)(P<0.01);There was no significant statistical difference between normal control group,DAla2 GIP incubation group(1.7%±0.4%)and pro3 GIP incubation group(1.1%±0.4%)(P>0.05).(4)Apoptosis rate of chondrocytess(fluorescence microscope):IL-1β+DAla2GIP co incubation group(6.63%±2.34%)is significantly lower than IL-1 β induction group(10.13%±1.76%)(P<0.01);There was no significant statistical difference between IL-1 β and IL-1β+DAla2GIP+pro3GIP co incubation group(10.9%±1.40%)(P>0.05),and both were higher than normal control group(4.5%±0.75%)(P<0.01);There was no significant statistical difference between normal control group,DAla2 GIP incubation group(6.1%±2.29%)and pro3 GIP incubation group(6.5%±1.56%)(P>0.05).3.Calcium overload of cells measured by laser confocal microscopy(results expressed by fluorescence intensity):IL-1β+DAla2GIP co incubation group(52.59±3.00)is significantly lower than IL-1 β induction group(62.72±6.29)(P<0.01);There was no significant statistical difference between IL-1 β and IL-1β+DAla2GIP+pro3GIP co incubation group(62.87±3.75)(P>0.05),and both were higher than normal control group(51.79±3.76)(P<0.01);There was no significant statistical difference between normal control group,DAla2 GIP incubation group(51.18±2.61)and pro3 GIP incubation group(52.26±6.29)(P>0.05).4.Determination of cytochrome c by ELISA:IL-1β+DAla2GIP co incubation group(251.83nmol/L±19.16nmol/L)is significantly lower than IL-1 β induction group(189.85nmol/L±36.48nmol/L)(P<0.01);There was no significant statistical difference between IL-1 β and IL-1β+DAla2GIP+pro3GIP co incubation group(232.53nmol/L±15.38nmol/L)(P>0.05),and both were higher than normal control group(173.47nmol/L±35.16nmol/L)(P<0.01);There was no significant statistical difference between normal control group,DAla2 GIP incubation group(184.58nmol/L±33.748nmol/L)and pro3 GIP incubation group(187.41nmol/L±14.43nmol/L)(P>0.05).5.Determination of P53 by Western Blot(compare with relative gray value):IL-1β+DAla2GIP co incubation group(114.21%±6.62%)is significantly lower than IL-1 β induction group(141.37%±11.71%)(P<0.01);There was no significant difference between IL-1 β and IL-1β+DAla2GIP+pro3GIP co incubation group(139.39%±12.87)(P>0.05),and both were higher than normal control group(100.00%±14.47%)(P<0.01);There was no significant difference between normal control group,DAla2 GIP incubation group(98.94%±16.03%)and pro3 GIP incubation group(94.96%±16.65)(P>0.05).6.Determination of Bcl-2 by Western Blot(compare with relative gray value):IL-1β+DAla2GIP co incubation group(109.87%±14.64)is significantly higher than IL-1 β induction group(60.18%±6.74%)(P<0.01);There was no significant difference between IL-1 β and IL-1β+DAla2GIP+pro3GIP co incubation group(60.88%±8.80)(P>0.05),and both were lower than normal control group(100%±9.09%)(P<0.01);There was no significant difference between normal control group,DAla2 GIP incubation group(96.32%±8.35%)and pro3 GIP incubation group(97.82%±7.57%)(P>0.05).Conclusion:1Incubation with DAla2 GIP could alleviate the mitochondrial dysfunction induced by IL-1 β;2.DAla2 GIP antagonized the apoptosis of chondrocytes induced by IL-1 β;3.DAla2 GIP can promote the expression of Bcl-2 protein by inhibiting the intracellular calcium overload induced by IL-1 β and down regulating the expression of p53,so as to protect the stability of mitochondrial membrane potential,reduce the release of cytochrome c and inhibit the apoptosis of chondrocytes. |
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