Mechanism Of CaSR Mediated Activation Of NLRP3 Inflammasome In PD Inflammatory Cell Model

Objective To establish a model of MPP⁺（1-Methyl-4-phenylpyridinium）inducing Parkinson’s disease inflammatory cell model on human neuroblastoma cells（SH-SY5Y cells）,to investigate the effect of Calcium sensing receptor（CaSR）on the activation of NLR family pyrin domain containing3（NLRP3）inflammatorome in Parkinson disease（PD）inflammatory cell model.To investigate the role of CaSR/NLRP3 pathway on inflammatory cell models of Parkinson’s disease.Methods 1、SH-SY5Y cells were selected as experimental cells,and then the experiment was divided into 6 groups,which were blank control group,MPP⁺model group,CaSR inhibitor Calhex231 group,CaSR agonist Gd Cl₃ group,Calhex231+MPP⁺group,Gd Cl₃+MPP⁺group.2、Concentration selection of MPP⁺,CaSR inhibitor and CaSR agonist.SH-SY5Y cells were treated with different concentrations of MPP⁺（3.9μmol/L、7.8μmol/L、15.6μmol/L、31.3μmol/L、62.5μmol/L和125μmol/L、250μmol/L、500μmol/L、1mmol/L）,CCK8 method was used for detection,and the appropriate concentrations of MPP⁺were selected to construct PD inflammatory cell model.Different concentrations of Calhex231（0.39μmol/L,0.78μmol/L,1.56μmol/L,3.13μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L,100μmol/L）and Gd Cl₃（0.39μmol/L,0.78μmol/L,1.56μmol/L,3.13μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L,100μmol/L）were given to SH-SY5Y cells for intervention,and CCK8 method was used to detect and select the appropriate concentration for the experiment.3、The expression of Tyrosine hydroxylase（TH）in cells of each group was detected by Western blot.Western blot and cell immunofluorescence were used to detect the protein expression levels of CaSR in each cell line.Real-time quantitative PCR was used to detect m RNA expression levels of CaSR and NLRP3 inflammasome in each cell line,and the proliferation of each cell line was detected by CCK8 method.Results1、MPP⁺can inhibit the proliferation of SH-SY5Y cells,and is correlated with its concentration.The higher the concentration of MPP⁺,the more obvious the proliferation inhibition of SH-SY5Y cells.By checking the drug concentration used in similar experiments,MPP⁺of 250μmol/L was selected for PD modeling（P<0.05）.Compared with the blank control group,Calhex231 can promote cell proliferation,when the concentration of Calhex231 is 3.13μmol/L,it has a significant effect on cell proliferation（P<0.05）.Compared with the blank control group,Gd Cl3can inhibit the proliferation of SH-SY5Y cells,when the concentration of Gd Cl3 is25μmol/L,the cell proliferation was inhibited significantly（P<0.05）.Calhex231 of3.13μmol/L and Gd Cl₃of 25μmol/L was selected for the drug intervention concentration.2、Compared with blank control group,TH protein expression was decreased in MPP⁺model group（P<0.05）,the inflammatory cell model of PD was successfully established,protein and m RNA expression of CaSR and m RNA expression of NLRP3were increased in MPP⁺model group（P<0.05）.3、Compared with blank control group,cell proliferation was promoted in Calhex231 group,TH protein expression was increased in Calhex231 group,protein and m RNA expression of CaSR and m RNA expression of NLRP3 were decreased in Calhex231 group（P<0.05）.Compared with MPP⁺group,cell proliferation was promoted in Calhex231+MPP⁺group,TH protein expression was increased in Calhex231+MPP⁺group,protein and m RNA expression of CaSR and m RNA expression of NLRP3 were decreased in Calhex231+MPP⁺group（P<0.05）.4、Compared with blank control group,the cell proliferation of the Gd Cl₃ group was inhibited,TH protein expression was decreased in Gd Cl₃ group,protein and m RNA expression of CaSR and m RNA expression of NLRP3 were increased in Gd Cl₃ group（P<0.05）.Compared with MPP⁺group,the cell proliferation of the Gd Cl₃+MPP⁺group was inhibited,TH protein expression was decreased in Gd Cl₃+MPP⁺group,protein and m RNA expression of CaSR and m RNA expression of NLRP3 were increased in Gd Cl₃+MPP⁺group（P<0.05）.Conclusion In the Parkinson’s inflammatory cell model,CaSR inhibitors can reduce m RNA expression of NLRP3 inflammasome,reduce the inflammatory response of cells,and inhibit the proliferation of MPP⁺induced cell damage in Parkinson’s disease inflammatory cell model.These results suggest that CaSR/NLRP3 may be an important pathway of inflammatory response in PD inflammatory cell model.It may be involved in the occurrence and development of Parkinson’s disease.