The Protective Effects Of Cordycepin On DA Neurons Via Suppression Of TLR4/NF-?B/NLRP3-Mediated Pyroptosis In PD Models Induced By LPS

Objective:The present study was designed to explore the protective effects of cordycepin(Cor)on DA neurons and the underlying molecular mechanism against TLR4/NF-?B/NLRP3-mediated pyroptosis through establishing LPS-induced PD models both in vivo and vitro.Methods:1.In vivo:First of all,to select the optimum conditions for establishing a PD model,C57BL/6 mice were administered LPS by intranasal route.And then,the mice were randomly divided into five groups:the control group,the LPS group and Cor groups(2.5,5.0,10.0 mg/kg)to study the effects of Cor on them induced by LPS.Open-field,pole climbing and rotarod tests were applied to assess the motor function of mice.After that,the numbers of TH and Iba-1 positive cells in SN of mice were measured by immunohistochemistry.And the contents of DA and its metabolites in Str were determined by HPLC-ECD.TUNEL staining and ELISA were used to observe the numbers of apoptotic cells and levels of TNF-? and IL-1?,respectively.Western blotting was applied to detect the expressions of TH,iNOS,COX-2,TLR4,I?B,NF-?B p65,Caspase-1 p20,NLRP3,GSDMD and GSDMD-NT,investigating the neuroprotective effects of Cor and its preliminary mechanism.2.In vitro:Firstly,two kinds of conditioned medium(CM)were prepared,including CM1 and CM2.CM1 was the supernatant of BV-2 cells treated with LPS,and CM2 was that treated with LPS and Cor.In order to investigate the neuroprotective effect of Cor in vitro,CM1 was added into PC12 cells pretreated with Cor for 4 hours,and CM2 was done to normal PC12 cells.Then,the cell viability and apoptotic morphology of PC12 cells were measured by MTT assay and Hoechst 33258 nuclear staining,respectively.And the levels of apoptotic-related proteins,Bax,Bcl-2 and cleaved Caspase-3 were measured by Westem blotting.Secondly,we explored the anti-neuroinflammatory effects and its mechanism of Cor on BV-2 cells,induced by LPS in depth.Likewise,BV-2 cells were divided into the control group,the model group and Cor groups(0.5×10-6,2.0×10-6,8.0×10-6?M).The contents of NO and TNF-?,IL-1? and IL-18 of BV-2 cells were respectively tested by Griess method and ELISA after the cells were pretreated by Cor for 4h following by LPS(1 ?g/mL)treatment for 12 hours.The release of LDH was measured by colorimetric determination.And the ultrastructure of cell membrane and the levels of Iba-1 together with GSDMD-NT were imaged by scanning electron microscopy and immunofluorescence staining.Moreover,the mRNA expression of GSDMD-NT was evaluated by RT-qPCR.And Western blotting was used to detect the expressions of iNOS,COX-2,TLR4,I?B,NF-?B p65,Caspase-1,Caspase-1 p20,NLRP3,ASC,IL-1?,IL-18,GSDMD and GSDMD-NT,exploring the relevant mechanism of Cor against neuroinflammation.Lastly,in order to further explore the relationship between TLR4/NF-?B pathway and NLRP3 pathway,TLR4 inhibitors,CLi-095 and NLRP3 inhibitors,MCC950 were given to BV-2 cells,respectively,and then the expressions of related proteins described above were measured by Western blotting.Results:1.Establishment of a PD model of mice by intranasal administration with LPS successfully.It was found that the DA neurons of nigrostriatal system in mice were selectively damaged by LPS,which was added by intranasal administration for 30 days.Meanwhile,LPS also induced motor dysfunction and activated the glial cells in substantia nigra of mice,promoting the inflammatory response as a result.2.The protective effects and mechanism of Cor on DA neurons in LPS-PD mice.(1)The protective effects of Cor on DA neurons in PD mice:The results showed that the motor dysfunction and DA neurons loss induced by LPS exposure were apparently ameliorated by Cor-pretreatment,suggesting a beneficially neuroprotective effect.(2)Cor inhibited the neuroinflammation in SN of mice induced by LPS:Cor markedly suppressed the activation of microglia and reduced the releases of TNF-? and IL-1? as well.Meanwhile,the expressions of iNOS and COX-2 were obviously downregulated as expected.(3)Cor inhibited activation of TLR4/NF-KB/NLRP3-mediated pyroptosis:Cor pretreatment,downregulated significantly the levels of TLR4 and I?B of SN in mice and inhibited NF-?B p65 translocation from cytoplasm to nucleus consequently.Additionally,the expressions of NLRP3,Caspase-1 p20 and GSDMD-NT were declined obviously3.The protective effects of Cor on apoptosis of PC12 cells induced by CM.(1)Cor inhibited the apoptosis of PC12 cells induced by CM1:Here we observed that the injury of PC12 cells induced by CM1 was significantly improved after Cor-pretreatment.Additionally,Cor reduced apoptosis of PC 12 cells,and hence increased the cell viability More importantly,Cor significantly downregulated the expressions of Bax and cleaved Caspase-3,and increased the expressions of Bcl-2 and the ratio of Bcl-2/Bax as well,playing a direct neuroprotective role(2)The toxicity of CM2 to PC12 cells was decreased:We found that the injury effects induced by CM2,such as the decrease in cell viability and the increase in apoptosis rate of PC 12 cells,were much more less than those of CM1.Apart from this,the expressions of Bax and cleaved Caspase-3 were declined obviously,while the expression of Bcl-2 was increased The results indicated that Cor could inhibit the activation of BV-2 cells induced by LPS,and hence alleviate great damage of the supernatant against PC 12 cells as a result,suggesting the indirect protection of nerve cells.4.Anti-neuroinflammatory effects of Cor on BV-2 cells induced by LPS and its molecular mechanism.(1)Cor inhibited the neuroinflammation of BV-2 cells induced by LPS:It was observed that Cor effectively inhibited the excessive activation of BV-2 cells and the release or expression of inflammatory mediators such as NO,iNOS,COX-2,TNF-?,IL-1? and IL-18 in our study.(2)Cor suppressed pyroptosis by regulating TLR4/NF-?B/NLRP3 signaling pathways:The results showed that Cor inhibited LDH release and reduced membrane pore formation of BV-2 cells What's more,Cor significantly downregulated expressions of TLR4 and I?B and thus inhibited NF-?B p65 translocating from cytoplasm to nucleus,resulting in the down-expression of NLRP3,Caspase-1 p20,ASC,IL-1?,IL-18 and GSDMD-NT.Similar to the effects of Cor,the expressions of related proteins of TLR4 and NLRP3 pathways decreased significantly after inhibitors treatment,such as CLi-095 or MCC950.Conclusion:Cordycepin exerted neuroprotective effects on LPS-induced PD models partially through preventing TLR4/NF-?B/NLRP3-mediated neuroinflammation and pyroptosis.