Mechanism Of TRPV5 Channel In Pyroptosis Of Chondrocytes

Objective:To elucidate the role and mechanism of the TRPV5 calcium channel during pyroptosis of chondrocytes.To provide experimental basis and new therapeutic strategies for clinical studies of OA.Methods:In this experiment,C28 human normal chondrocyte cell line was selected and the cells were plastered with high sugar DMEM medium containing a mixture of penicillin and streptomycin（1%）and fetal bovine serum（10%）.The C28 chondrocyte cell line was transfected with lentivirus,and when the C28 chondrocytes were plastered and the cell growth covered 70%-80%of the culture plate area,the cells were transfected according to the transfection procedure provided by PPL（Plasmid and Protein Sharing Library）.Transfection experiments were divided into 3 groups:a.Normal group:C28cells（PBS solution）;b.Knockdown group:C28 cells（carrying TRPV5 knockdown target gene lentivirus）;c.NC group:C28 cells（lentiviral empty vector）,culture for 72h and replace with DMEM complete medium with 2.5ug/ml of puromycin,When the cells were filled with petri dish,the cells were subcultured with 0.1ug/ml purinomycin complete culture medium to screen out stable transfected cells.The formal experiment was then divided into 4 groups,as follows:（1）control group（above medium+saline in equal amount with reagent）;（2）NC group（above medium+saline in equal amount with reagent）;（3）LPS（1ug/ml）+ATP（5mM）group;（4）TRPV5 knockdown+LPS（1ug/ml）+ATP（5mM）group;after incubation in the same environment for 14 hours,the proliferation of chondrocytes was detected by CCK-8 method.The expression of IL-1βand IL-18 in the supernatant of chondrocyte culture was detected by ELISA.The release of Cell pyroptosis specific enzyme LDH in the supernatant of chondrocyte culture was detected by microplate method.The expression of proteins（Caspase-1,Caspase-4,NLRP3,GSDMD）associated with the process of chondrocyte heat apoptosis was detected using Western blot.The content of calcium ions in chondrocytes was detected by fluorescence microscope with calcium probe.Results:1.CCk-8 test cell survival rate:We evaluated the survival rate of chondrocytes in each group by inducing cells with1ug/ml LPS+5mM ATP and 10ug/ml LPS+5mM ATP and measuring the survival rate of cells at 14 h.The results were as follows:compared with the control group（97.47%±5.32%）,the proliferation rate of LPS（1ug/ml）+ATP（5mM）group（84.40%±3.98%）was significantly decreased（P<0.01）.Compared with the proliferation rate of LPS（1ug/ml）+ATP（5mM）group,the survival rate of TRPV5 knockdown+LPS（1ug/ml）+ATP（5mM）group was significantly increased（91.82%±1.30%）（P<0.01）.Compared with LPS（1ug/m L）+ATP（5mM）group,the proliferation rate of LPS（10ug/m L）+ATP（5mM）group was significantly decreased（76.46%±5.35%）（P<0.05）.Compared with TRPV5 knockdown+LPS（1ug/ml）+ATP（5mM）group,the proliferation rate of TRPV5 knockdown+LPS（10ug/ml）+ATP（5mM）group（83.04%±1.98%）was significantly decreased（P<0.0001）.There was no significant difference in survival rate between control group and NC group（P>0.05）.2.Determination of IL-1βand IL-18 by ELISA:（1）IL-1β:Compared with the control group（184.30±7.72）,the release of IL-1βin LPS（1ug/ml）+ATP（5mM）group（307.59±11.05）was significantly increased（P<0.0001）.Compared with LPS（1ug/ml）+ATP（5mM）group,IL-1βrelease in TRPV5knockdown+LPS（1ug/ml）+ATP（5mM）group（271.13±10.30）was significantly decreased（P<0.001）.There was no significant difference in IL-1βrelease between control group and NC group.（P>0.05）（2）IL-18:Compared with the control group（97.17±8.05）,the release of IL-18 in LPS（1ug/ml）+ATP（5mM）group（183.66±14.58）was significantly increased（P<0.0001）.Compared with LPS（1ug/ml）+ATP（5mM）group,The IL-18 release of TRPV5knockdown+LPS（1ug/ml）+ATP（5mM）group（146.85±7.34）was significantly decreased（P<0.001）.There was no significant difference in IL-18 release between control group and NC group（P>0.05）.3.LDH lactate dehydrogenase determination:In the LDH lactate dehydrogenase assay,Compared with the control group（181.89±10.48）,LPS（1ug/ml）+ATP（5mM）group（252.77±7.02）significantly increased LDH LDH dehydrogenase release（P<0.0001）.Compared with LPS（1ug/ml）+ATP（5mM）group,the release of LDH lactate dehydrogenase in TRPV5 knockdown+LPS（1μg/ml）+ATP（5mM）group（231.17±8.59）was significantly decreased（P<0.01）.There was no significant difference in LDH dehydrogenase release between control group and NC group（P>0.05）.4.Caspase-1,Caspase-4,NLRP3 and GSDMD were detected by Western Blot.（1）Caspase-1:Compared with the control group（0.76±0.02）,the protein gray level of caspase-1 in LPS（1ug/ml）+ATP（5mM）group（1.57±0.03）was significantly higher（P<0.001）.Compared with LPS（1ug/ml）+ATP（5mM）group,the protein band gray value of TRPV5 knockdown+LPS（1ug/ml）+ATP（5mM）group（0.94±0.06）was significantly decreased（P<0.001）.There was no significant difference in the gray values of protein bands between the control group and NC group.（P>0.05）.（2）Caspase-4:Gray value of Caspase-4 protein relative to control group（0.78±0.03）,the gray level of protein bands in LPS（1ug/ml）+ATP（5mM）group（1.43±0.03）was significantly higher（P<0.001）.Compared with LPS（1ug/ml）+ATP（5mM）group,the protein band gray value of TRPV5 knockdown+LPS（1ug/ml）+ATP（5mM）group（1.19±0.06）was significantly decreased（P<0.01）.There was no remarkable difference in the gray values of protein bands between the control group and NC group（P>0.05）.（3）NLRP3:Relative to the control group,NLRP3 protein gray value（0.89±0.04）,the gray level of protein bands in LPS（1ug/ml）+ATP（5mM）group（1.41±0.02）was significantly higher（P<0.001）.Compared with LPS（1ug/ml）+ATP（5mM）group,the protein band gray value of TRPV5 knockdown+LPS（1ug/ml）+ATP（5mM）group（1.16±0.03）was significantly decreased（P<0.01）.There was no meaningful difference in the gray values of protein bands between the control group and NC group（P>0.05）.（4）GSDMD:Compared with the GSDMD gray value of the control group,the gray level of protein bands in LPS（1ug/ml）+ATP（5mM）group（1.46±0.07）was significantly higher（P<0.001）.Compared with LPS（1ug/ml）+ATP（5mM）group,the protein band gray value of TRPV5 knockdown+LPS（1ug/ml）+ATP（5mM）group（1.20±0.06）was significantly decreased（P<0.01）.There was no significant difference in the gray values of protein bands between the control group and NC group（P>0.05）.5.Calcium overload measured by fluorescence microscope:Compared with the normal control group,the fluorescence intensity of calcium ions in chondrocytes（149.61±5.97）,intracellular calcium fluorescence intensity in LPS（1ug/ml）+ATP（5mM）group（188.24±7.25）was significantly increased（P<0.01）.Compared with LPS（1ug/ml）+ATP（5mM）group,the intracellular calcium fluorescence intensity of TRPV5 knockdown+LPS（1ug/ml）+ATP（5mM）group（165.59±1.94）was significantly decreased（P<0.05）.There was no significant difference in intracellular calcium fluorescence intensity between control group and NC group（P>0.05）.Conclusion:1.The release of inflammatory factors facilitated by pyroptosis of chondrocytes is one of the factors of the damage of articular cartilage destruction.2.The inward flow of Ca²⁺mediated by TRPV5 channel and activation of NLRP3may be an important mechanism of pyroptosis in chondrocytes.