Effect Of ERK1/2Signalling Pathway On Cell Function And TNFR Expression Of Microglia Cells

Objective: To determine the effects of ERK1/2signaling pathway on the functionof microglia cell and the expression of TNFR.Methods: In this study, BV2(Mice microglia tumor cells) and PC12(Ratchromaffin tumor cells) cell lines were used to represent microglia and neuron,respectively. BV2cells were cultured with or without ERK1/2signaling pathwayblocker (PD98059) under varying degrees of hypoxia stimulation, whose conditionmedia were then used for PC12cells culture. CCK-8was performed to evaluate cellviability in PC12cells, in which way the effects of BV2cells in different conditionswere manifestated. Western blot was performed to determine the expression andphosphorylation of JNK, p38MAPK proteins and the expression of TNFR on membranesurface of BV2cells before or after the treatment of PD98059. ELISA was performed todetect the secretory expression of TNF-α, IL-1β and NGF in BV2cells.Results:1. Cell viability in normal control PC12group was100%, while cellviability was85.14%and40.97%in hypoxia (1h) group and hypoxia (12h) group,respectively;52.48%and27.10%in hypoxia (12h) group and hypoxia (12h)+PD98059group, respectively. Cell viability was obviously decreased after the blockage ofERK1/2signaling pathway（p<0.05）.2. Protein expression was shown with grey values. In hypoxia (1h) group, the greyvalue of intracellular p-JNK, p-p38MAPK,TNFR1,TNFR2of BV2cellswas0.0451±0.010,0.0549±0.015,0.0394±0.019,0.1281±0.002, respectively. In hypoxiahypoxia (1h)+PD98059group, the grey value of intracellular of BV2cells was0.1023±0.013,0.0815±0.012,0.0592±0.004,0.0533±0.001,respectively; In hypoxia(12h) group, the grey value of intracellular p-JNK, p-p38MAPK,TNFR1,TNFR2ofBV2cells was0.2866±0.013,0.1001±0.004,0.1261±0.018,0.0241±0.002，respectively.In hypoxia hypoxia (12h)+PD98059group, the grey value of intracellular of BV2cells was0.3845±0.017,0.1436±0.010,0.2882±0.016,0.0175±0.003， respectively. Theexpression of p-JNK,p-p38MAPK,TNFR1was increased after the blockage of ERK1/2signaling pathway, while TNFR2protein expression was decreased (p<0.05).3. TNF-α, IL-1β and NGF were secreted by BV2cells under hypoxia stimulation.In hypoxia (1h) group, the secretion level of TNF-α, IL-1β and NGF was808.97±1.35pg/ml,8.17±0.06pg/ml,209.53±2.74pg/ml, respetively; In hypoxia (1h)+PD98059group, that was1652.07±21.51,32.79±1.62,138.47±1.9, respectively; Inhypoxia (12h) group, the secretion level of TNF-α, IL-1β and NGF was1652.07±21.51pg/ml,32.79±1.62pg/ml,138.47±1.91pg/ml，respectively; In hypoxia(12h)+PD98059group, that was1864.12±17.33,56.33±1.12,82.15±0.87, respectively;.The expression of TNF-α and IL-1β was increased after the blockage of ERK1/2signaling pathway, while that of NGF was decreased (p<0.05).Conclusions:1. The ERK1/2signaling pathway in hypoxic condition involved inthe protective effect of microglia.2. The ERK1/2signal pathway in BV2cells under hypoxic condition influencesthe phosphorylation of JNK, p38MAPK.3. ERK1/2signaling pathway is involved in the regulation of microglial cellmembrane of TNFR and TNF-α, IL-1β, NGF secretion expression.