KLF7对创伤性脑损伤海马神经元细胞模型凋亡和增殖的影响

Effect of KLF7 on the proliferation and apoptosis of hippocampal neurons induced by traumatic brain injury

目的 探讨KLF7对创伤性脑损伤诱导海马神经元细胞(HT22细胞)增殖、凋亡损伤作用及其机制。方法 HT22细胞培养和TBI体外模型(stretch联合OGD)制备,应用AAV-KLF7、AAV-NC腺病毒转染HT22,将细胞分为control组(未处理细胞)、SOAN组(stretch+OGD处理后应用AAV-NC转染)、SOAK组(stretch+OGD处理后应用AAV-KLF7转染、SOAK-AG490)(SOAK组加入AG490)和SOAN-AG490组(SOAN组加入AG490),应用Western blots和qRT-PCR检测各组KLF7及p-JAK2、t-JAK2、p-STAT3、t-STAT3、凋亡因子Bcl-2、Bax和C-caspase3表达水平;免疫荧光染色检测各组KLF7及βⅢ-tubulin表达,乳酸脱氢酶(LDH)活性试剂盒测定各组LDH表达,Caspase-3活性试剂盒检测各组Caspase-3活性;PI免疫荧光染色及CCK-8法检测各组细胞增殖和活性情况;ChIP检测转录因子KLF7与STAT3启动子的结合作用和结合位点。结果 与control组比较,stretch+OGD损伤后KLF7表达、PI阳性细胞百分比、LDH和caspase-3活性、p-JAK2/t-JAK2和p-STAT3/t-STAT3比值、Bax和C-caspase-3蛋白表达均显著增高;细胞活性、βⅢ-tubulin和Bcl-2表达均显著降低(P<0.05);与SOAN组比较,SOAK组KLF7表达、细胞活性、βⅢ-tubulin和Bcl-2表达、p-JAK2/t-JAK2和p-STAT3/t-STAT3比值均显著增高,PI阳性细胞百分比、LDH和caspase-3活性、Bax和C-caspase-3蛋白表达均显著降低(P<0.05);SOAK-AG490组PI阳性细胞百分率、LDH和caspase3活性增加,p-JAK2/t-JAK2和p-STAT3/t-STAT3相对表达及细胞活力降低(P<0.05)。SOAN、SOAN-AG490、SOAK-AG490三组间PI阳性细胞百分率、LDH和caspase3活性、Bcl-2、Bax和C-caspase-3蛋白表达和细胞活力无显著性差异(P>0.05)。ChIP结果表明KLF7与p-STAT3之间有直接相互作用。结论 KLF7能够抑制海马神经元TBI损伤细胞模型凋亡,机制可能与上调JAK2/STAT3信号通路表达有关。

Objective To investigate the effect of KLF7 on the proliferation and apoptosis of hippocampal neurons(HT22 cells) induced by traumatic brain injury(TBI) and its mechanism. Methods HT22 cells were cultured and the TBI model in vitro(stretch+OGD) was prepared. The cells were divided into 5 groups: control group, stretch+OGD+AAV-NC group(SOAN, AAV-NC transfection after stretch+OGD treatment), stretch+OGD+AAV-KLF7 group(SOAK, AAV-KLF7 transfection after stretch+OGD treatment), and stretch+OGD+AAV-KLF7+AG490 group(SOAK-AG490, AG490 pretreatment was performed prior to SOAK group) and stretch+OGD+AAV-NC+AG490 group(SOAN-AG490, AG490 pretreatment was performed prior to SOAN group). The expression of Bcl-2, Bax, C-caspase3, and KLF7, p-JAK2, t-JAK2, p-STAT3, t-STAT3 in groups were detected by Western Blots and RT-PCR. The expression of KLF7 and βⅢ-tubulin in each group was detected by immunofluorescence staining. Lactate dehydrogenase(LDH) and Caspase-3 activity was detected by LDH and caspase-3 activity kit. PI immunofluorescence staining and CCK-8 assay were used to detect cell proliferation and activity. ChIP detects the interaction between KLF7 and pSTAT3.Results Compared with the control group, the expression of KLF7, the ratio of LDH and caspase-3 active, PI positive cells, p-JAK2/t-JAK2 and p-STAT3/t-STAT3, Bax and C-Caspase-3 were significantly increased after stretch+OGD injury. The cell activity, the expression of βIII-tubulin and Bcl-2 were significantly decreased(P<0.05). Compared with SOAN group, the expression of KLF7, βIII-tubulin and Bcl-2, p-JAK2/t-JAK2 and p-STAT3/t-STAT3 were significantly increased(P<0.05), and the percentage of PI positive cells, LDH and caspase-3 activity, the expression of Bax and C-caspase-3 were significantly decreased in SOAK group(P<0.05). Compared with SOAK group, the percentage of PI positive cells, LDH and caspase3 activity increased, and the relative expression of p-JAK2/t-JAK2 and p-STAT3/t-STAT3 cells decreased in SOAK-AG490 group(P<0.05). There were no significant differences in the percentage of PI positive cells, LDH and Caspase-3 activity, the expression of Bcl-2, Bax and C-Caspase-3, cell viability in SOAN, SOAN-AG490 and SOAN-AG490 group(P>0.05). ChIP results indicate a direct interaction between KLF7 and P-STAT3.Conclusion KLF7 can inhibit the apoptosis of TBI damaged hippocampal neurons, and the mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.