Molecular Mechanism Of TrkB-ASIC3 Signaling Involved In H₂O₂ Induced PC12 Cell Injury

Part one:ASIC3 is involved in H2O2-induced PC12 cell injuryObjective To explore whether acid-sensing ion channel 3(ASIC3)is involved in H2O2-induced PC 12 cell injury and its mechanism.Methods PC 12 cells were divided into 3 groups using a random number table method:the control group(Con group)was added with PBS buffer,the H2O2 group was added with 200?mol/L of H2O2,and the AMI+H2O2 group was pretreated with 100?mol/L of ASIC3 inhibitor amiloride(AMI)for 1 h before H2O2.After incubation for 24 h,cell viability,the release rate of lactate dehydrogenase(LDH),the apoptosis rate,the ASIC3,tropomyosin receptor kinase B(TrkB),Interleukin-1?(IL-1?),Interleukin-6(IL-6),Tumor Necrosis Factor-?(TNF-?)mRNA expression levels,the relative expression of ASIC3,TrkB,B-cell lymphoma-2 related X protein(Bax),B-cell lymphoma-2(Bcl-2),cleaved cysteinyl aspartate specific proteinase-3(Cleaved caspase-3)protein were detected.Results When compared with the Con group,H2O2 treatment reduced cell viability(P<0.01),condensed and fragmented the nucleus,increased the ratio of apoptosis and LDH release(P<0.01),increased ASIC3,IL-1?,IL-6,TNF-? mRNA expression(P<0.01),reduced TrkB mRNA expression(P<0.01),increased ASIC3,Bax,Cleaved caspase-3 protein expression(P<0.01),and reduced TrkB,Bcl-2 protein expression(P<0.01).AMI pretreatment increased cell viability(P<0.01),reduced densely stained cells,the ratio of apoptosis and LDH release(P<0.01),reduced IL-1?,IL-6,TNF-? mRNA expression(P<0.01),reduced Bax,Cleaved caspase-3 protein expression(P<0.05),increased Bcl-2 protein expression(P<0.01)and had no significant effect on TrkB protein expression.Conclusion ASIC3 and TrkB are involved in H2O2-induced PC 12 cell injury.Inhibition of ASIC3 can reduce inflammation and apoptosis,but does not affect TrkB expression.Part two:TrkB participates in H2O2-induced PC12 cell injury by regulating ASIC3 signalingObjective To elucidate the molecular mechanism of TrkB agonist 7,8-Dihydroxyflavone(7,8-DHF)attenuating H2O2-induced PC 12 cell injury by inhibiting ASIC3 signaling.Methods PC 12 cells were divided into 3 groups using a random number table method:the control group(Con group)was added with PBS buffer,the H2O2 group was added with 200 ?mol/L of H2O2,and the 7,8-DHF+H2O2 group was pretreated with 25 ?mol/L of 7,8-DHF before H2O2 for 1 h.Cells in each group were incubated for 24 h.Cell viability,the LDH release rate,the apoptosis rate,the expression levels of IL-1?,IL-6,TNF-? mRNA and ASIC3,Bax,Bcl-2,Cleaved caspase-3 protein were respectively detected.Results 7,8-DHF pretreatment obviously increased cell viability(P<0.01),reduced densely stained apoptotic cells,increased cell number,near-normal morphology,reduced LDH release,reduced IL-1?,IL-6,TNF-? mRNA expression(P<0.01),reduced ASIC3,Bax,Cleaved caspase-3 protein expression(P<0.01)and increased Bcl-2 protein expression(P<0.05).Conclusion Activation of TrkB can reduce PC 12 cell injury caused by H2O2,which might be associated with inhibiting ASIC3 signaling and reducing inflammation and apoptosis.