The Mechanism Of Paraquat Induce Microglia Activation Via TLR4/NF-?B Signaling Pathway Mediated Inflammatory Leading To Neuronal Injury

Objective In this study,microglia cells model(BV2 cells)and neurons model(PC12 cells and primary neurons)were established.Firstly,we evaluated whether low concentration paraquat can activate microglia cells.Secondly,we explore the mechanism of microglia cells mediated inflammatory response via TLR4/NF-?B signaling pathway.Finally,to elucidate the connection between microglia activation and neuron injury.Methods In vitro microglia cell model(BV2 cells)and neurons model(PC12cells and primary neurons)were established.1.To explore the dose-effect and time-effect relationships of BV2 cells activation by low concentration PQ.BV2 cells were treated with final concentrations of 0,0.015,0.03,0.06,0.12,0.24,0.48?mol/L PQ and 0.05 mol/L MPP~+for 24 h and0.03?mol/L PQ for 0,12,24,36,48h.The viability of cells was messured by CCK8 in order to determine the infection conditions for subsequent experiments.2.To evaluate the effective indicators of activation of BV2 cells by low concentration PQ.BV2 cells were treated with final concentrations of 0,0.015,0.03,0.06 and 0.12?mol/L PQ and 0.05?mol/L MPP~+for 24h.The contents of tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?)and interleukin-6(IL-6)in BV2cells were deteced by enzyme-linked immunosorbent assay(ELISA),cell phagocytosis was deteced by flow cytometry and immunofluorescence assay(IF),and cell migration ability was deteced by Transwell assay.The cells were treated with final concentration of 0,0.03,0.06,0.12?mol/L PQ and 0.05?mol/L MPP~+for 24 h,Western Blot method was used to determine the M1 type microglia markers TNF-?,IL-1?,IL-6,nitric oxide synthase(iNOS),CD86 and M2 type microglia markers arginase-1(Arg-1),mannose receptor(CD206)protein expression level.3.To explore the mechanism of TLR4/NF-?B signaling pathway activation by low concentration PQ.BV2 cells were treated with final concentrations of 0,0.03,0.06,0.12?mol/L PQ,0.05?mol/L MPP~+and 0.1 mg/mL LPS(positive control)for24h.Western Blot method was used to determine the key protein expression of TLR4,MyD88,IKK,I?B-?,p-I?B-?and NF-?B protein expression of whole cells,cytoplasm and nucleus.4.To explore the role of TLR4 inflammatory response and BV2 cells activation by PQ.The TLR4 gene was silenced by BV2 cells transfected with short hairpin RNA(shRNA)to established the new cells,including Control shRNA and TLR4 shRNA BV2 cells.The viability of cells was messured by CCK8,and the TLR4 protein expression were detcted by IF and Western Blot method after TLR4 silence.Then,the method of Western Blot was used to determine the protein expression level of MyD88,IKK,I?B-?,p-I?B-?,NF-?B,and p-NF-?B in the signaling pathway after silencing treated by 0.06?mol/L PQ.Finally,TLR4 shRNA BV2 cells phagocytosis ability was detcted by IF and flow cytometry,migration ability was detcted by Transwell method,and the proteins expression level of M1/M2 microglia markers TNF-?,IL-1?,IL-6,iNOS,CD86,Arg-1,CD206 were detectd by Western Blot.5.To explore the role of NF-?B in inflammatory response by PQ.Firstly,the PDTC that inhibitor of NF-?B used to treated BV2 cells with final concentration of 0,12.5,25,50,100 and 200?mol/L for 1h,and 25?mol/L PDTC used to treated BV2cells for 0,0.5,1,1.5 and 2h.The relative survival rate of cells was measured by CCK8method to determine the intervention conditions for subsequent experiments.Then,the protein expression level of NF-?B,TNF-?,IL-1?and IL-6 that downstream of the signaling pathway was detected by the method of Western Blot treated by 0.06?mol/L PQ.6.To investigate the damage effect on primary cortical neurons intervention by BV2 cells infected by PQ.Five adult C57BL/6 SPF male mice and 10 female mice were mated according to female:male of 2:1,the method to judged the pregnant of female mice were to observe the vaginal plug and weight.Primary cortical neurons were extracted and cultured within 24 hours after the birth.Then,the primary neurons were specifically labeled with MAP2,NeuN and?3-tublin around the 14th day of culture by IF.Then,neurons with good growth and uniform density were taken,and the cell supernatant was extracted after 24h with the final concentration of 0,0.12 and1?mol/L of PQ infected BV2 cells,then use the cell supernatant to intervene the primary neurons for 24h.Finally,the apoptosis of the primary neurons was detected by TUNEL method.7.To investigate the damage effect on PC12 cells by BV2 cells infected by PQ.The cell supernatant was extracted from BV2 cells infected with PQ at the final concentration of 0,0.12,1 and 10?mol/L to intervened the PC12 cells for 24 and 48h.Then,the relative survival rate of PC12 cells was determined by CCK8 assay,the proliferation of PC12 cells was detectd by EDU assay,and the apoptosis rate of PC12cells was detectd by Annexin V-FITC/PI double staining assay.Results 1.The influence of BV2 cells proliferation by low concentration PQ:CCK8 results showed that compared with 0?mol/L PQ,the proliferation of BV2 cells in the 0.03-0.12?mol/L PQ group were significantly increased,the proliferation of cells in the 0.24-0.48?mol/L PQ group were inhibited,and the proliferation in the0.03?mol/L PQ and 0.05?mol/L MPP~+groups were significantly increased after 24h treated,with statistically significant differences(P<0.05).Therefore,0.015,0.03,0.06,0.12?mol/L PQ treated BV2 cells for 24h as the infection conditons for the subsequent experiment.2.The influence of BV2 cells activity by low concentration PQ:ELISA results showed that the levels of TNF-?,IL-1?and IL-6 in the cell supernatant of the infected group were significantly increased compared with 0?mol/L PQ,especially in 0.03 and0.06?mol/L PQ groups,with statistically significant differences(P<0.05).Then,IF,flow cytometry and Transwell chamber results showed that the phagocytosis and migration capacity of BV2 cells in 0.03,0.06?mol/L PQ groups were significantly enhanced,compared with 0?mol/L PQ,with statistically significant differences(P<0.05).Finally,Western Blot results showed that compared with 0?mol/L PQ,the protein expression level of M1 markers TNF-?,IL-1?,IL-6,iNOS and CD86 in each treatment group were significantly increased,while the protein expression level of M2markers Arg-1 and CD206 were significantly decreased,with statistically significant differences(P<0.05).3.The influence of TLR4/NF-?B signaling pathway of BV2 cells by low concentration PQ:Compared with 0?mol/L PQ,the key protein expression level of TLR4,MyD88,IKK,p-I?B-?in TLR4/NF-?B signaling pathway were increased,especially in 0.06?mol/L PQ group,the difference was statistically significant(P<0.05).Then,the expression of NF-?B in the whole cells increased after PQ exposure,but there was no significant difference between the groups,while the protein expression in the cytoplasm decreased,and increased in the nucleus significally,the expression peaked at 0.06?mol/L PQ group,compared with 0?mol/L PQ,the difference was statistically significant(P<0.05).4.The influence of shRNA silencing TLR4 gene on the activity of BV2 cells:CCK8results showed that compared with the control group,the cell survival rate of TLR4shRNA group was 86.75%,with statistically significant difference(P<0.05),and the TLR4 protein expression showed significantly lower than Control shRNA group by IF and Western Blot results,with statistically significant differences(P<0.05),these results indicated that new cells construction successfully.According to compare with the PQ infected group.Western Blot results showed that the key protein expression level of TLR4,MyD88,IKK,I?B-?,p-I?B-?,NF-?B,p-NF-?B in TLR4/NF-?B signaling pathway were significantly decrease after 0.06?mol/L PQ infected TLR4shRNA BV2 cells,and the phagocytosis,migration ability were also significantly decrease,M1 type markers TNF-?,IL-1?,IL-6,iNOS and CD86 protein expression level were significantly decrease,and the expression level of M2 type markers Arg-1and CD206 were significantly increased,with statistically significant differences(P<0.05).5.The influence of PDTC inhibitor intervention on the TLR4/NF-?B signaling pathway:CCK8 results showed that compared with the control group,BV2 cells were treated with 50,100 and 200?mol/L PDTC for 1h and 25?mol/L PDTC for 1.5h and 2h,the proliferation of the BV2 cells decreased significantly(P<0.05).Therefore,the BV2 cells were intervene with 25?mol/L PDTC for 1 hour.Western Blot results showed that the protein expresstion level of NF-?B,TNF-?,IL-1?and IL-6 in the downstream signaling pathway were significantly decreased,compared with the PQ infected group(P<0.05).6.The influence of supernatant PQ explore BV2 cells on primary cortical neurons:After neuronal exteaction,the cells began to stick to the wall at 4h,and the cell body was clear.At culture of 6-7 days,neurons begin to gather and synapses begin to grow.When the culture at 12-14 days,the synapses grow longer and form many branches,synapses between adjacent neurons interweave into a network.Then,the MAP2,NeuN,and?3-tublin were able to mark the primary cortical neurons.Therefore,these results indicated that primary cortical neurons extracted successfully.TUNEL results showed that,compared with the control group,the number of the neuron apoptosis increased gradually after intervention of BV2 supernatant with the increase of PQ concentration.7.The influence of supernatant PQ explore BV2 cells on PC12 cells:CCK8 results showed that compared with the control group,the proliferation of 0.12,1 and 10?mol/L PQ infected BV2 cells was not significantly decreased after intervention for24h,while the proliferation activity was significantly decreased after intervention for48h,with statistically significant differences(P<0.05).The results of EDU staining results was basically consistent with CCK8,showing that the proliferation of PC12cells was significantly inhibited after intervention for 48h in the 1 and 10?mol/L PQ infected supernatant groups.The result of apoptosis showed that after 24h of intervention,compared with the control group,the total apoptosis rate of the 1 and 10?mol/L PQ supernatant groups were increased,with statistically significant difference(P<0.05).However,the total apoptosis rate of 0.12,1 and 10?mol/L PQ supernatant group was 9.31%,16.25%and 22.11%after intervention for 48h,which were significantly higher than control group,with statistically significant differences(P<0.05).Conclusion PQ can abnormally activate the microglia and induce cell phenotype from M1 to M2 type,and TLR4/NF-?B signaling pathway plays an important role in microglia mediated inflammatory response.Meanwhile,abnormally activated of microglia can damage neurons by decreasing proliferation and increasing apoptosis rate.