Inhibitory effect of thymoquinone on neuroinflammation in Parkinson's disease model by regulating NLRP3 inflammatory bodies

Objective:To explore the intervention mechanism of Thymoquinone in regulating NLR Family Pyrin Domain Containing 3(NLRP3)inflammasome mediated on neuroinflammatory injury of dopaminergic neurons in Parkinson's disease.Methods:After establishment of the MPTP-induced PD mouse model,which was randomly divided into control group,control+TQ group,model group and model+TQ group.The effects of TQ on the motor deficits in PD mice were evaluated by open-field test and rotarod test.The effects of TQ on the expression of tyrosine hydroxylase,α-Synuclein and NLRP3 inflammasomes in midbrain of PD mice were determined by Western blot.In vivo,BV-2 cells were induced by lipopolysaccharide+and MPP to establish neuroinflammation models of Parkinson's disease.The cell supernatant was collected as conditioned medium and acted on human neuroblastoma(SH-SY5Y)cells to construct PD inflammatory injury model.MTT method determined the optimal concentration+of TQ and MPP in vitro intervention and the survival rate of cells in each intervention group;SH-SY5Y cells apoptosis was detected by TUNEL.Western blot was used to analyze the expression of Bax,Bcl-2,Caspase-3 proteins in each experimental group.The effect of TQ on the proinflammatory factor interleukin 1βby ELISA.The expression of related proteins NLRP3,ASC,Caspase-1,IL-1βand Tubulin were detected by Western blot after TQ treatment.Results:Compared with normal group,the motor function of model group was significantly decreased(P<0.01),the motor function of TQ group was significantly increased(P<0.01).In Western blot experiment,compared with normal group,theα-Syn level in model group was significantly increased(P<0.01).Compared with model group,TQ significantly decreased theα-Syn level of MPTP mice(P<0.01);Compared with model group,TH protein expression was significantly increased after TQ administration(P<0.01).TQ further inhibited the up-regulation of NLRP3,Caspase-1,IL-1β,ASC protein expression in PD mice(P<0.01).MTT detection found that the survival rate of SH-SY5Y cells after-1+100μmol∙L MPP treatment was significantly decreased,SH-SY5Y cells damage can be+relieved after TQ treatment.TUNEL staining showed that SH-SY5Y cells treated with MPP conditioned medium could induce apoptosis,and TQ pretreatment could significantly(P<0.01)reduce the apoptosis rate.Western blotting showed that MPP+conditioned medium caused the down regulation of Bcl-2/Bax in SH-SY5Y cells,while TQ inhibited the down regulation+of Bcl-2/Bax.Meanwhile,MPP conditioned medium induced the activation of Caspase-3 in SH-SY5Y cells,and caspase-3 expression was decreased by TQ(P<0.01).ELISA kit showed+that TQ inhibited MPP induced IL-1βcontent increases(P<0.01).Western blotting showed that TQ inhibited the protein expression of NLRP3,Caspase-1,IL-1βand ASC of BV-2 cells+induced by MPP(P<0.01).Conclusion:Thymoquinone can reduce the inflammatory damage of PD model neurons by inhibiting the activation of NLRP3 inflammasome in PD model,and thus play a protective role in dopaminergic neurons.

Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK-mT OR-ULK1 signaling pathway

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-m TOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, m TOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin（50 or 100 mg/kg） reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and p S317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-m TOR and p S757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-m TOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.

Effect of flunarizine on HSP70 gene expression for following transient ischemia

Objective To investigate the effects of flunarizine on HSP70 gene expression for following transient ischemia.Methods Northern blot and immunohistochmistry (IHC) were used to determine the expression of the HSP70 during different periods after post ischemic reperfusion with or without Flunarizine treatment in the gerbil. Results The HSP70mRNA expression was increased in the forebrain,but the HSP70 protein only expressed at the 1st day of reperfusion(P<0.05);Flunarizine treatment could increase the expression of HSP70 protein, but couldn’t significantly increase the expression of HSP70 mRNA (P >0.05). Conclusion Flunarizine could protect neurons from ischemic damage through increasing the HSP70 expression.