| Objective: To study the inhibitory effect of activated MC1 R on microglial BV2 inflammation induced by lipopolysaccharide(Lipopolysaccharide,LPS)and its related mechanism,and to further explore its neuroprotective effect.Methods:(1)the inflammation model of Parkinson’s disease(PD)was established by inducing microglia BV2 inflammation by LPS(100ng/ml).Cell grouping:(1)normal control group;(2)model group: LPS(100ng/ml);(3)MC1R receptor agonist BMS-470539(25umol/L)group;(4)BM S-470539(25umol/L)+LPS group.(2)BV2 cells were treated with MC1 R receptor agonist BMS-470539 for 24 hours,and then the effect of MC1 R receptor agonist on BV2 cell viability was analyzed by CCK-8 method.(3)the effect of MC1 R agonist on the production of NLRP3 inflammatory bodies and inflammatory factors in BV2 cells induced by LPS: BV2 cells were pretreated with MC1 R agonist BMS-470539 for 1 hour,and then BV2 cells were treated with LPS for24 hours.the protein expression levels of NLRP3 and IL-1 β induced by MC1 R agonists were detected by Western blotting,the levels of nitric oxide(NO)in the supernatant of BV2 cells were detected by griess,and the levels of TNF-α and IL-6 in the supernatants of BV2 cells were detected by ELISA.(4)Co-culture:BV2 cells were pre-incubated with MC1 R receptor agonist BMS-4705391 for 1hour,and then BV2 cells were cultured with LPS for 24 hours.The conditioned supernatant of BV2 cells was used to interfere with PC12 cells for 24 hours.The activity of PC12 cells was detected by CCK-8 method.(5)Mechanism study:BV2 cells were pretreated with MC1 R agonist BMS-470539 for 1 hour,and then BV2 cells were treated with LPS for 15 minutes.The effects of MC1 R agonists on NF-kappa B p65,Pmura NF-kappa B p65,Pmure ERK1 and 2 Magi Akt and P-AMPK were detected by Western blotting.The effect of MC1 R agonist on the entry of NF-κ B p65 into the nucleus was detected by cellular immunofluorescence.BV2 cells were pretreated with BMS-470539,a MC1 R agonist,for 1 hour,and then BV2 cells were treated with LPS for 24 hours.The protein levels of HIF-1 α,NR4A2 and NRF2 were detected by Western blotting to determine whether HIF-1 α agonist(DMOG)could reverse the anti-inflammatory effect of MC1 R agonists.Results:(1)BMS-470539(0-50 umol/L),an agonist of MC1 R,had no significant effect on the viability of BV2 cells,and DMOG(0-2 mmol/L),an agonist of HIF-1 α,had no significant effect on the viability of BV2 cells.(2)compared with the normal control group,LPS increased the protein expression of NLRP3,IL-1 β,TNF-α and IL-6.Compared with LPS intervention group,MC1 R receptor agonist BMS-470539 decreased the expression of NLRP3,IL-1 β and NO,but could not reduce the expression of TNF-α and IL-6.(3)BV2 cells were pretreated with BMS-470539,a MC1 R agonist,for 1hour,and then LPS was added to interfere with BV2 cells for 24 hours.The cell viability of PC12 cells in LPs group was lower than that in normal control group for 24 hours,while that in BMS group,BMS+LPS group and LPS group was higher than that in normal control group and LPS group.(4)compared with the normal control group,LPS intervention could promote the transcription of NF-κ B p65 into the nucleus,MC1 R agonists could partially inhibit the entry of NF-κ B p65 into the nucleus,LPS intervention could increase the protein levels of NF-NF-κ B p65,P-ERK1/2,P-Akt and HIF-1 α,but had no significant effect on the total protein expression of NR4A2 and NRF2.MC1 R agonists could inhibit the expression of p65,P-Akt,P-ERK1/2 and HIF-1 α of PMI NF-κ B,and could also promote the phosphorylation of AMPK,but had no significant effect on the expression of NR4A2 and NRF2.(DMOG),a HIF-1 α agonist,reversed the effect of MC1 R agonists on reducing NLRP3 and NO.Conclusion:(1)MC1R can inhibit neuroinflammation by reducing the release of NLRP3 and NO by inhibiting HIF-1 α.(2)The anti-inflammatory effect of 2MC1 R may also be related to the inhibition of classical pathways such as NF-κ B,MAPK,Akt and the promotion of AMPK. |
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