Experimental Study Of ROS/NLRP3 Pathway Promoting The Secretion Of Inflammatory Cytokines In Pyroptosis Of Chondrocytes

Objective:1.To verify that chondrocyte pyroptosis is a mechanism leading to osteoarthritis;2.To verify the pathway by which increased ROS can activate the pyroptosis of chondrocytes;3.To verify that NLRP3 is involved in the pyroptosis of chondrocytes,which leads to the secretion of inflammatory cytokines;Methods:In this experiment,cell lines of C28 chondrocytes were subcultured in medium（1%fluid containing a mixture of penicillin and streptomycin;5-10%fetal bovine serum;90%to 95%DMEM without fetal bovine serum）.The cell lines were divided into 3 groups according to different experimental treatment conditions.Normal cell group:（Group A,saline was added with the same volume of inducer）;1μg/m L LPS+ATP-induced group（group B,1μg/m L LPS and 5mg/m L）;10μg/m L LPS+ATP-induced group（group C,10μg/m L LPS and 5mg/m L ATP）.Incubation was carried out in an incubator with a temperature of 37°C and a CO₂ volume ratio of 5%until the cells were spread to about75%-85%of the Petri dish or 6-well plate.The cells were induced with different concentrations of LPS for 12 hours,followed by the addition of 5mg/ml ATP for 3 hours.After drug intervention,ROS,LDH,NLRP3,Caspase-1,Caspase-4 and GSDMD were detected by Western Blot,and IL-1βand IL-18 were detected by Elise method.Results:1.CCK-8 cell proliferation-toxicity test:the size of cell survival.（1）The induction time of LPS was the same in each group.The results showed that the cell survival rate of group B（89.63%±0.67%）and group C（81.22%±2.79%）decreased significantly with the increase of inducer LPS concentration,which was lower than that of group A（100%±0%,P<0.05）.Moreover,the cell survival rate of group C was significantly lower than that of group B（P<0.05）.When the induction time of LPS was 12 h,the cell viability of group B（81.39%±1.98%）and group C（77.57%±1.87%）decreased significantly with the increase of inducer LPS concentration,which was lower than that of group A（100%±0%,P<0.05）.Moreover,there was no significant difference in cell survival rate between group C and group B（P>0.05）.When the induction time of LPS was 24 h,the results showed that the cell survival rate of group B（68.88%±3.57%）and group C（63.20%±1.97%）decreased significantly with the increase of inducer LPS concentration,which was lower than that of group A（100%±0%,P<0.05）.Moreover,there was no significant difference in cell survival rate between group C and group B（P>0.05）.（2）The induction was the same in each group:firstly,with the extension of induction time,the cell survival rate at 24h decreased significantly and was lower than that at 6h and12h（P<0.05）cell survival rate.Moreover,the survival rate of 6h cells was not significantly different from that of 12h cells（P>0.05）.With the extension of induction time,the cell viability at 24h decreased significantly and was lower than that at 6h and 12h（P<0.05）cell survival rate.Moreover,the survival rate of 6h cells was not significantly different from that of 12h cells（P>0.05）.2.ROS detection:The results showed that,compared with group A,C28 cells in groups B and C had significantly higher intracellular green fluorescence intensity after LPS induction.Meanwhile,the fluorescence intensity in C28 cells in group C was significantly higher than that in group B,indicating that LPS could make cells produce more reactive oxygen species.3.LDH activity:With the increase of inducer LPS concentration,LDH content in cell culture medium of group B（181.50%±7.78%）and group C（240.00%±21.21%）was significantly increased,and was higher than that of group A（100%±0%,P<0.05）.Moreover,the content of LDH in cell culture medium of group C was significantly higher than that of group B（P<0.05）.4.IL-1βand IL-18 were measured by ELISE:The results showed that the IL-1βcontent in cell culture medium of group B（1.6863±0.0416）and group C（2.0230±0.0783）was significantly higher than that of group A（1±0,P<0.05）with the increase of inducer LPS concentration.Moreover,the content in group C was significantly higher than that in group B（P<0.05）.The results showed that the IL-18 content in cell culture medium of group B（1.6913±0.1052）and group C（2.0533±0.1391）was significantly higher than that of group A（1±0,P<0.05）with the increase of inducer LPS concentration.Moreover,the content in group C was significantly higher than that in group B（P<0.05）.5.Western Blot analysis of the contents of NLRP3,GSDMD,Caspase-4 and Caspase-1:（1）Compared with group A（100%±0%）,the gray scale of NLRP3 in group B（152.00%±3.11%）and group C（184.35%±6.72%）was significantly higher than that in group A（P<0.05）.Moreover,the protein gray value of NLRP3 in group C was significantly higher than that in group B（P<0.05）.The results indicated that the expression of NLRP3was significantly increased with the increase of inducer LPS concentration.（2）Compared with group A（100%±0%）,the grayscale values of protein bands in group B（137.20%±2.97%）and group C（162.70%±3.04%）GSDMD were significantly higher than those in group A（P<0.05）.Moreover,the protein gray value of GSDMD in group C was significantly higher than that in group B（P<0.05）.The results indicated that the expression of GSDMD protein was significantly increased with the increase of inducer LPS concentration.（3）Compared with group A（100%±0%）,the gray values of caspase-4 protein bands in group B（130.60%±1.13%）and group C（161.10%±3.54%）were significantly higher than those in group A（P<0.05）.Moreover,the protein gray value of Caspase-4 in group C was significantly higher than that in group B（P<0.05）.The results indicated that the expression of Caspase-4 protein increased significantly with the increase of inducer LPS concentration.（4）Compared with group A（100%±0%）,the gray values of caspase-1 protein bands in group B（136.90%±1.98%）and group C(（152.35%±3.61%）were significantly higher than those in group A（P<0.05）.Moreover,the protein gray value of Caspase-1 in group C was significantly higher than that in group B（P<0.05）.The results indicated that the expression of caspase-1 protein increased significantly with the increase of inducer LPS concentration.Conclusion:1.Pyroptosis of chondrocytes is one of the pathogenesis of osteoarthritis;2.The increase of ROS can activate the pyroptosis pathway of chondrocytes;3.NLRP3 is involved in pyroptosis of chondrocytes and promotes the secretion of inflammatory cytokines.4.The increase of ROS in chondrocytes induced by LPS can promote the pyrolysis of chondrocytes by activating inflammasome NLRP3,and then lead to the secretion of inflammatory cytokines.