The Role And Mechanism Of Lnc-MALAT1 In MPP+-Induced Astrocytes-mediated Inflammation

Objective:Neuroinflammation plays a key role in the occurrence and development of Parkinson’s disease,and various studies have shown that lnc-MALAT1 plays an important regulatory role in Parkinson’s disease models.However,the regulatory role and mechanism of lnc-MALAT1 in the neuroinflammation process of PD remain unclear.In this paper,the expression of lnc-MALAT1 in mouse primary astrocytes was inhibited by small interfering RNA,and the astrocytes were stimulated by MPP⁺.The expression levels of TNF-α,IL-6,IL-1β,Caspase-1 and NLRP3 were detected,and then the expression levels of the above inflammatory factors in the control group,MPP⁺group,MPP⁺+MALAT1-si RNA-NC group and MPP⁺+MALAT1-si RNA group were compared.To investigate the effect of down-regulation of lnc-MALAT1 on MPP⁺-induced inflammatory response in mouse astrocytes and further explore the effect of lnc-MALAT1 on Parkinson’s disease and its possible mechanism,so as to provide new research ideas for clarifying the pathogenesis of Parkinson’s disease and new research directions for finding the etiology and treatment of Parkinson’s disease.Methods:The primary astrocytes in the brain of C57BL/6 mice were extracted,and the purity of the extracted primary astrocytes was identified by GFAP staining using immunofluorescence assay.Astrocytes were treated with MPP⁺of six different concentrations（50μmol/L,100μmol/L,200μmol/L,250μmol/L,400μmol/L,800μmol/L）,and CCK8 kit was used to determine the cell viability of cells treated with different MPP⁺concentrations for 24h to explore the appropriate concentration of drugs.According to the results of CCK8 cell viability determination,the appropriate concentration of MPP⁺was selected to treat astrocytes to construct a model of astrocytes inflammation.The lnc-MALAT1 interference vector was constructed and transfected into cells.The background and post-interference expression levels of lnc-MALAT1 in astrocytes were detected by q PCR,and an effective interference vector was selected.Astrocytes were treated according to the above explored MPP⁺concentration.The experiment was divided into four groups:Control group,MPP⁺group,MPP⁺+MALAT1-si RNA-NC group,and MPP⁺+MALAT1-si RNA group.Immunofluorescence was used to detect the activation of NLRP3inflammatory bodies in different treatment groups;ELISA was used to detect the expression levels of inflammatory factors TNF-α,IL-6,Caspase-1 and IL-1βin the supernatant;NLRP3 protein expression was determined by Western Blot.Results:1.The purity of the extracted primary astrocytes was above 64.1%.2.The damage of MPP⁺to astrocytes increased with the increase of drug concentration.In this experiment,250μmol/L MPP⁺was selected for subsequent cell treatment,and the cell survival rate was 76%at this concentration.3.Three interference vectors（MALAT1-si RNA-286 group,MALAT1-si RNA-1763 group and MALAT1-si RNA-3875 group）were constructed.The interference efficiency of three different si-RNAs was detected by qPCR.Compared with the si-NC group,the silencing effect of MALAT1-si RNA-1763 group on lnc-MALAT1 was the most obvious,and the silencing efficiency was 52%（P<0.05）.4.By detecting the expression levels of TNF-α,IL-6,Caspase-1,IL-1β,NLRP3 inflammasome and NLRP3 protein in the control group,MPP⁺group,MPP⁺+MALAT1-si RNA-NC group and MPP⁺+MALAT1-si RNA group,it was found that the expression levels of the above indicators in astrocytes treated with MPP⁺were higher than those in the control group,and the difference was statistically significant（P<0.05）.However,the expression levels of TNF-α,IL-6,Caspase-1,NLRP3inflammasome and NLRP3 protein in MPP⁺+MALAT1-si RNA group were significantly lower than those in MPP⁺+MALAT1-si RNA-NC group（P<0.05）.Conclusion:Lnc-MALAT1 may be involved in the development of Parkinson’s disease by regulating the levels of TNF-α,IL-6,Caspase-1,IL-1β,NLRP3 inflammasome and other related inflammatory factors produced by astrocytes.