Mechanism Of Morroniside Inhibiting The Activation Of Microglia In Parkinson’s Disease CircHIPK3/miR-124 Through Regulating STAT3/NLRP3 Pyroptosis Signaling Pathway

Objective1.To explore the correlation between the expressions of serum CircHIPK3 as well as miR-124 with the clinical severity of Parkinson’s disease.2.To explore the mechanism of CircHIPK3/miR-124 regulating STAT3/NLRP3 pyroptosis signaling pathway to promote microglia activation under LPS stimulation.3.To explore the mechanism of morroniside inhibiting microglia activation by regulating CircHIPK3/miR-124/STAT3/NLRP3 pyroptosis signaling pathway.Methods1.The expressions of CircHIPK3 and miR-124 in blood samples from 40 PD patients and 40 controls in our hospital were investigated.The Hoehn&Yahr grading system,Unified Parkinson’s Disease Rating Scale(UPDRS)version 3.0 and mini-mental state examination(MMSE)scale were used for the assessment of PD severity.Blood was drawn for serological examination.The expressions of CircHIPK3 and miR-124 in blood samples were detected by qRT-PCR.Receiver operating characteristic(ROC)curve was used to evaluate the expressions of CircHIPK3 and miR-124 in PD progression.2.qRT-PCR was used to detect the expression of CircHIPK3 and miR-124 in LPS treated and untreated BV2 cells(microglia).qRT-PCR was used to detect the expression of CircHIPK3 and miR-124 in SH-SY5Y cell line without any treatment or treated with BV2 cell medium treated with LPS.To study the role and mechanism of CircHIPK3 in PD neuroinflammation by gene gain and losing functions in vitro.DCFH-DA was used to detect the production of intracellular reactive oxygen species(ROS).Detection of TNF-α,IL-1β and IL-6 in BV2 cell supernatant was conducted by ELISA.The protein expressions of microglial markers CDllb and Iba-1,pyroptosis related factors NLRP3,caspase-1 and ASC,STAT3 and p-STAT3 were detected by Western blot analysis.In addition,the interaction between CircHIPK3,miR-124 and STAT3 was detected using bioinformatics,FISH,RIP,RNA pull down and luciferase reporter assays.3.BV2 cells were incubated with low and high concentrations of morroniside(10 and 100 μmol/L)for 2 h and then stimulated with LPS(1μg/ml)for 48 h.DCFH-DA was used to detect the production of intracellular reactive oxygen species(ROS).Detection of TNF-α,IL-β and IL-6 in BV2 cell supernatant was conducted by ELISA.The protein expressions of microglial markers CD11b and Iba-1,pyroptosis related factors NLRP3,caspase-1 and ASC,STAT3 and p-STAT3 were detected by Western blot analysis.Results1.The expression of CircHIPK3 in serum of PD patients was significantly higher than that in the control group,and the expression of miR-124 was significantly lower than that in the control group.There was a significant negative correlation between CircHIPK3 expression in serum with miR-124 expression.The expressions of CircHIPK3 and miR-124 in serum of patients with Hoehn&Yahr grade 4/5 were significantly higher and lower than that in patients with Hoehn&Yahr grade 2/3,respectively.The expressions of CircHIPK3 and miR-124 in serum of patients with Hoehn&Yahr grade 2/3 was significantly higher and lower than that in patients with Hoehn&Yahr grade 1.ROC curve analysis showed that both CircHIPK3 as well as miR-124 in serum could be used as diagnostic indicators of PD progression.CircHIPK3 expression in serum was positively correlated with UPDRS Ⅲ score,and miR-124 expression in serum was negatively correlated with UPDRS Ⅲ score.On the other hand,CircHIPK3 expression in serum was negatively correlated with MMSE score,and miR-124 expression in serum was positively correlated with MMSE score.2.LPS-treated BV2 cells exhibited higher CircHIPK3 expressions and lower miR-124 expressions.SH-SY5Y exhibited significantly impaired viability and elevated apoptotic rate,along with upregulated CircHIPK3 and reduced miR-124 expression,after treatment with the supernatants collected from LPS-treated BV2 cells.Upregulation of CircHIPK3 increased the IL-6,IL-1β and TNF-α secretion of BV2 cells.The protein expressions of microglial markers CD11b and Iba-1,as well as pyroptosis related factors NLRP3,Caspase-1 and ASC were also increased following CircHIPK3 expression.All these effects were reversed by miR-124 addition.CircHIPK3 can target miR-124 to promote STAT3 expression.Fish,RIP,RNA pull Down and luciferase reporter assays showed that CircHIPK3,miR-124 and STAT3 could interact with each other.3.The expression of CircHIPK3 in both low and high concentration of morroniside group was significantly lower than that in LPS stimulated group,while the expression of miR-124 was significantly higher than that in LPS stimulated group.Both low and high concentration of morroniside can inhibit LPS induced increased levels TNF-α,IL-1β and IL-6 in the supernatant of BV2 cell culture and production of reactive oxygen species.In addition,the expressions of microglial marker proteins CD11b and Iba-1,cell apoptosis factors NLRP3,caspase-1 and ASC,and STAT3 and p-STAT3 in the low and high concentration morroniside groups were significantly lower than that in LPS stimulated group.Conclusion1.The expressions of CircHIPK3 and miR-124 in serum were significantly positively and negatively correlated with PD progression,respectively.CircHIPK3 and miR-124 in serum may be used as markers for diagnosing PD progression.2.CircHIPK3/miR-124 promotes neuroinflammatory response in PD through STAT3/NLRP3 signaling pathway.3.Morroniside inhibits microglia activation by regulating CircHIPK3/miR-124/STAT3/NLRP3 pyroptosis signaling pathway.