RIP3介导的坏死性凋亡在HT-22细胞牵张损伤模型中的作用

Effects of necroptosis induced by RIP3 in stretch injury model of HT-22 cells

目的探讨受体相互作用蛋白3(RIP3)介导的坏死性凋亡在HT-22细胞牵张损伤模型中的作用及其机制。方法将HT-22细胞接种在Bioflex培养板,采用细胞损伤控制仪(CIC),设定损伤参数(阀门压力30 PSI、气体脉冲压力3.5~4.5 PSI、气体脉冲时间50 ms),建立HT-22细胞牵张损伤模型。分别采用数字全息显微镜(DHM)、乳酸脱氢酶(LDH)试剂盒、流式细胞术、western blot法检测牵张损伤后6 h Ctrl组、CIC组、GSK’872组间细胞形态差异,LDH浓度变化,细胞周期分布,RIP3/受体相互作用蛋白1(RIP1)/混合系列蛋白激酶样结构域(MLKL)、Akt/p-Akt/m TOR/p-m TOR、Caspase-8/X连锁凋亡抑制蛋白(XIAP)蛋白表达变化。结果与CIC组相比,应用GSK’872后细胞平均数量[(244.67±11.68)vs(190.67±15.28),t=4.865,P<0.01]、细胞平均面积[(260.14±16.81)μm2vs(175.91±15.00)μm2,t=6.476,P<0.01]有所增加,细胞平均厚度有所减小[(6.12±0.47)μm vs(8.04±0.48)μm,t=4.942,P<0.01];LDH浓度有所下降[(222.74±11.06)ng/l vs(275.93±12.26)ng/l,t=5.581,P<0.01];细胞周期有所恢复[Sub-G1:(0.33±0.15)%vs(6.51±0.63)%,t=16.530,P<0.01;G0/G1:(46.67±2.96)%vs(33.04±7.07)%,t=3.085,P<0.05];能够降低RIP3[(0.73±0.04)vs(1.09±0.09),t=6.239,P<0.01]、RIP1[(0.75±0.05)vs(0.91±0.05),t=4.211,P<0.05]、MLKL[(0.56±0.03)vs(0.70±0.04),t=4.785,P<0.01]、Akt[(0.49±0.05)vs(0.77±0.05),t=6.763,P<0.01]、p-Akt[(0.88±0.05)vs(1.06±0.05),t=4.509,P<0.05]、m TOR[(0.81±0.02)vs(0.90±0.05),t=2.813,P<0.05]、p-m TOR[(0.65±0.05)vs(1.00±0.05),t=8.413,P<0.01]、XIAP[(0.50±0.05)vs(0.73±0.05),t=5.814,P<0.01]蛋白表达,并可促进Caspase-8蛋白表达持续升高[(0.96±0.05)vs(0.75±0.05),t=5.351,P<0.01],差异具有统计学意义。结论 RIP3介导的坏死性凋亡在HT-22细胞牵张损伤模型中起到重要作用,应用GSK’872可减轻HT-22细胞牵张损伤的程度,提示RIP3有可能成为将来临床上治疗颅脑创伤新的靶点。

Objective To investigate the effects of necroptosis induced by receptor-interacting protein 3 （RIP3） on stretch injury model of HT-22 cells and its mechanisms. Methods HT-22 cell lines were seeded with Bioflex cell plate, and stretch injuries with 30 psi for the regulator with a 50 msec pulse resulting in 3.5-4.5 peak injury pressure by cell injury controller H （CIC） instrument. The morphological changes of HT-22 cells were assessed by digital holographic microscopy （DHM）, the degree of injury was detected by lactate dehydrogenase （LDH） assay, the cell cycle was detected with Flow cytometry and the expression of RIP3/RIP1/mixed lineage kinase domain like （MLKL）, Akt/p-Akt/ mTOR/p-mTOR and Caspase-8/X-linked inhibitor of apoptosis protein （XIAP） were detected by Western Blot assay at 6 h post-CCI among Ctrl, CIC and GSK＇872 groups. Results Compared with the CIC group, cells treated with GSK＇872 exhibited an increase in number [（244.67±11.68） vs （190.67±15.28）, t=4.865, P〈0.01] and area [（260.14±16.81） μm^2 vs （175.91±15.00）μm^2, t=6.476, P〈0.01], however, a reduction in thickness [（6.12±0.47）μm vs （8.04±0.48） μm, t=4.942, P〈0.01] with DHM. In the presence of GSK＇872, the leakage of LDH was sharply decreased compared with the CIC group [（222.74±11.06） ng/1 vs （275.93±12.26） ng/l, t=5.581, P〈0.01]. What＇s more, GSK＇ 872 could convert the alteration of cell cycle and cell death [Sub-Gl： （0.33±0.15）% vs （6.51±0.63）%, t=16.530, P〈0.01; G0/G1 （46.67±2.96）% vs （33.04±7.07） %, t=3.085, P〈0.05] compared to the CIC group. Furthermore, GSK＇872 treatment could significantly decrease the levels of RIP3 [（0.73±0.04） vs （1.09±0.09）, t=6.239, P〈0.01], RIP1 [（0.75±0.05） vs （0.91-＋0.05）, t=4.211, P〈0.05], MLKL [（0.56±0.03） vs （0.70±0.04）, t=4.785, P〈0.01], Akt [（0.49±0.05） vs （0.77-＋0.05）, t=6.763, P〈0.01], p-Akt [（0.85±0.05） vs （1.06±0.05）, t=4.509, P〈0.05], roTOR [（0.81±0.02） vs （0.90±0.05）, t=2.813, P〈0.05], p-roTOR [（0.65±0.05） vs （1.00±0.05）, t=8.413, P〈0.01], XIAP [（0.50±0.05） vs （0.73±0.05）, t=5.814, P〈0.01], but increase the level of Caspase-8 [（0.96±0.05） vs （0.75±0.05）, t= 5.351, P〈0.01] compared with CIC group. Conclusion Necroptosis induced by RIP3 may play an important role in stretch injury model of HT-22 cells, what＇s more, GSK＇872 could reduce the degree of injury after CIC, which reveals that RIP3 may be a new target for clinical treatment of TBI in the future.