| The function of immune cell in the central nervous system and rise systemic immune responses can be changed by Mercury, but its toxic mechanism has not been elucidated. Microglia are the main immune cells in the central nervous system and react quickly to acute and chronic infestation, namely microglial activation, and release various of cytokines and inflammatory mediators, which plays a role in the process and the prognosis of various of diseases in central nervous system,which caused by chronic inflammation. α7n ACh R, as a key point in the cholinergic anti-inflammatory pathway, regulate activation of microglial and inhibit inflammation.Objective: To investigate the toxic effects and underlying mechanisms of neuroimmunology induced by methylmercury chloride(Me Hg Cl) and the potenyial roles α7n ACh R played in neuroinflammationMethods:Methods: Part I:To investigate the effects of nicotine(nicotine, Nic) and Me Hg Cl on serum levels of IL-1β in rats by ELISA. Part II:The survival rate of BV2 microglia cells with different concentrations of Me Hg Cl, as well as with pretreatment of nicotine(Nic) and methyllycaconitine(MLA) with the MTT assay. The levels of IL-6 at 4h, 12 h, 24 h, 48 h, which were stimulated by Nic, MLA and Me Hg Cl were invistigated by ELISA. On the basis of the establishment of inflammation model, The cells were divided into groups, Me Hg Cl 12 h group, Me Hg Cl 24 h group, Nic 30 min + Me Hg Cl 12 h group, MLA 30 min + Me Hg Cl 12 h group. The expression levels of α7n ACh R m RNA, NF-κB(p65) m RNA and COX-2 m RNA were measured with q RT-PCR. and the protein levels of α7n ACh R and p65 by immunofluorescence.Results:1, The effects of Nic on α7n ACh R and Iba-1 positive cells in the DG district of rats chronic were observed with immunofluorescence. The results showed that, the α7n ACh R positive cells significantly decreased in model group than that in control group.. The Iba-1-IR positive microglia expressed increased, the cell body showes amoeba-like form in the model group, N intervention group α7n ACh R positive cells and microglia significantly increased the number of cells, microglia clean cut, with projections.2, The effects of Nic on α7n ACh R m RNA expression levels in hippocampus of rats exposure to Me Hg Cl were observed with RT-PCR. The results showed that compared with the control group, the model group α7n ACh R m RNA was significantly lower(P <0.001); compared with the model group, N intervention group α7n ACh R m RNA levels were significantly increased(P <0.001).3, Me Hg expossed rat hippocampal IL-1β, IL-6 and IL-10 were tested by Dot-Blot Compared with the control group, model group, IL-1β, and IL-6 were significantly increased(P <0.001, P <0.001), IL-10 was no significant difference(P> 0.05).4, The effects of Nic on IL-1β in serum of rats exposured to chronic low-dose Me Hg Cl were detected with ELISA. The results showed that compared with the control group, the model group serum IL-1β was significantly higher(P <0.001); compared with the model group, IL-1β level in Nic intervention group were significantly decreased(P <0.001).5, The cells viability were investigated with MTT assay. The effects of Nic and MLA on cells viability. MTT results showed that, Me Hg Cl cell viability in a dose-dependent(P<0.05,P<0.01,P<0.001)and time-dependent(P<0.05,P<0.01,P<0.001)manner reduced treatment with Me Hg Cl. Compared with the model group at the corresponding time, the cell survival were significantly impreoved in Nic and MLA intervention(P <0.05, P <0.01).6, The secretion of inflammatory cytokines exposed to Me Hg Cl in BV2 cells. The results showed that,the secretion levels of IL-6 and IL-10 were increased, and peaked at 24 h, compared with the control group(P <0.05, P <0.001), Whereas,IL- 6 IL-10 levels were lower in N and M intervention group than that of model group,(P <0.05, P<0.001); IL-1β levels was not change significantly at any time(P> 0.05).7, The m RNA levels of factors in cholinergic anti-inflammatory pathway. ①Me Hg Cl group of COX-2 m RNA expression levels increase in 12h(P <0.05), with the exposure time, the expression level decreases rapidly decreased significantly(P <0.001) at 24 h and 48 h their level. Nic intervention group and intervention group MLA COX-2 m RNA expression levels in 12 h compared with the model group was significantly higher(P <0.001), 24 h and 48 h in any course remain high. ②Me Hg Cl group p65 m RNA expression was significantly increased(P <0.001) in 12 h, while Nic intervention group and intervention group MLA 12hp65 m RNA expression levels were significantly suppressed(P <0.001). ③ Different concentrations Me Hg Cl of PC12 cells α7 n Ach R m RNA levels were significantly inhibited(P <0. 001), Nic MLA intervention group and intervention group α7 n Ach R m RNA levels were significantly increased(P <0. 001).8, The protein levels of cytokines factors in cholinergic anti-inflammatory pathway were invistigated with cytoimmunochemistry. Compared with the control group, BV2 cells treated with Me Hg Cl 24 h α7n ACh R caused reduced expression(P <0.01) and EP3 expression increased(P <0.01), the expression of p65 protein in the nucleus increased; compared with the model group, N and M in the intervention group and EP3 α7n ACh R protein expression increased(P <0.05, P <0.001), decreased expression of p65 in the nucleus.Conclusion:1, Me Hg Cl have cytotoxic effects on BV2 cells and PC12 cells in a manner of time-dependent and dose-dependent way. The α7n ACh R played inhibit role on the apoptosis induced by Me Hg Cl.2, Me Hg Cl induced Neuroinflammation were associated with the structures and functions impairment related to the cholinergic anti-inflammatory infornation signaling pathways, which is mediated by α7n ACh R.3, The m RNA levels and protein expressions of α7n ACh R regulated by Nic and MLA affected the expression levels of NF-κB(p65) and COX-2, which plays an anti-inflammatory role through cholinergic signaling pathways. |
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