GABRG2基因敲除小鼠和HT22细胞通过激活PKA/NF-κB信号通路影响MMP3的表达

GABRG2 Knockout Mice and HT22 Cells Affect MMP3 Expression by Activating the PKA/NF-κB Signaling Pathway

目的探究GABRG2基因敲除后小鼠海马和HT22细胞中基质金属蛋白酶(MMP)3的表达变化及潜在参与机制。方法动物实验:利用Cre/loxp重组酶系统所构建的GABAA受体γ2亚基(GABRG2)基因条件性敲除小鼠(GABRG2fl/wtCre+)模型为实验组,野生型小鼠作为对照组,用Western blot技术检测小鼠海马中GABRG2、PKA、p-PKA、NF-κB、MMP3蛋白的表达情况;免疫荧光技术观察海马中GABRG2、MMP3蛋白的表达情况;尼氏染色技术观察GABRG2基因缺失对小鼠海马神经元的影响。细胞实验:利用CRISPR/Cas9技术构建GABRG2基因敲除的HT22细胞为实验组,HT22细胞为对照组。Western blot技术检测各组细胞中GABRG2、PKA、pPKA、NF-κB、MMP3蛋白的表达情况;免疫荧光技术观察各组细胞中GABRG2、MMP3蛋白的表达情况。结果动物实验:Westernblot结果显示,与对照组小鼠相比,实验组小鼠海马组织中GABRG2蛋白表达降低(P<0.05),p-PKA、NF-κB、MMP3蛋白表达均升高(P均<0.05),PKA蛋白表达差异无统计学意义(P>0.05);免疫荧光结果显示,与对照组小鼠相比,实验组小鼠海马区GABRG2表达降低,MMP3表达升高,特别在海马CA3区表达明显升高。尼氏染色结果显示,与对照组小鼠相比,实验组小鼠海马神经元的结构松散,神经元离散程度提高。细胞实验:Western blot结果显示,与对照组细胞相比,实验组细胞GABRG2蛋白表达降低(P<0.05),pPKA、NF-κB、MMP3蛋白表达均升高(P均<0.05),PKA表达差异无统计学意义(P>0.05);细胞免疫荧光结果显示,与对照组细胞相比,实验组细胞GABRG2的表达降低,MMP3的表达升高。结论GABRG2基因缺失小鼠海马和HT22细胞均引起MMP3蛋白表达增高,其机制可能与上游PKA/NF-κB信号通路的调控有关。

Objective To investigate the expression changes and the potential mechanisms involved of matrix metalloproteinase(MMP)3 in mouse hippocampus and HT22 cells after GABRG2 knockdown.Methods Animal experiments:We constructed the GABAAreceptor γ2 subunit(GABRG2)gene conditional knockout mouse model(GABRG2fl/wtCre+)by Cre/loxp recombinase system as the experimental group and used the wild-type mice as the control group.We detected the expression of GABRG2、PKA、p-PKA、NF-κB、MMP3 protein in mouse hippocampus by Western blot technique;the expression of GABRG2 and MMP3protein in hippocampus was observed by immunofluorescence technique;the effect of GABRG2 gene deletion on mouse hippocampal neurons was observed by Nissl staining technique.Cell experiments:We constructed GABRG2 gene knockout HT22 cells by CRISPR/Cas9 technology as the experimental group and used the HT22 cells as the control group.We detected the expression of GABRG2,PKA,p-PKA,NF-κB,MMP3protein in each group of cells by Western blot technique;the expression of GABRG2 and MMP3 protein in each group of cells was observed by immunofluorescence technique.Results Animal experiments:Western blot resulted showed that,compared with the control mice,GABRG2 protein expression was decreased(P0.05)in hippocampal tissues of experimental mice.The immunofluorescence results showed that,compared with the control mice,the expression of GABRG2was decreased and the expression of MMP3 was increased in the experimental group,especially in the CA3region of the hippocampus.Nissl staining results showed that the neurons in the hippocampus of the experimental group were loosely structured and the neuronal discrete degree was increased compared with the control group.Cell experiments:Western blot results showed that the expression of GABRG2 protein was decreased(P0.05)in the experimental cells compared with the control cells.The results of cellular immunofluorescence showed that the expression of GABRG2 was decreased and the expression of MMP3 was increased in the experimental cells compared with the control cells.Conclusion Deletion of the GABRG2 gene will cause expression of MMP3 protein increasing in both the hippocampal of mice and HT22 cells by a mechanism that may be related to the regulation of the upstream PKA/NF-κB signaling pathway.