RIP3介导的坏死性凋亡在C57BL/6小鼠颅脑创伤模型中的作用

Effects of necroptosis induced by receptor interacting-protein 3 in traumatic brain injury model of C57/BL mice

目的探讨受体相互作用蛋白3(RIP3)介导的坏死性凋亡在C57BL/6小鼠颅脑创伤(TBI)模型中的作用及其机制。方法采用电子控制性皮质撞击仪(CCI),设定打击参数(速度5m/s、时间120 ms、深度1 mm),对小鼠顶叶大脑皮质进行精确撞击,建立颅脑创伤模型。将80只雄性C57BL/6小鼠随机分为4组,每组20只:Ctrl组不予处理,Sham组仅开骨窗,TBI组CCI打击后原位注射4μl的DMSO,GSK’872组CCI打击后原位注射4μl的GSK’872。再采用改良型神经功能缺损评分(mNSS)量表评估小鼠颅脑创伤后损伤程度,脑干湿比重法检测颅脑创伤后脑水肿程度,HE染色法检测小鼠脑皮层及海马区损伤程度,以创伤后觉醒时间评价损伤后恢复情况,Western blot法检测RIP3/RIP1/混合连接激酶结构域样蛋白(MLKL)、Akt/p-Akt/哺乳动物雷帕霉素靶蛋白(mTOR)/p-m TOR、含半胱氨酸的天冬氨酸蛋白水解酶8(Caspase-8)/X连锁凋亡抑制蛋白(XIAP)、Caspase-1/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)蛋白表达变化。结果与TBI组相比,GSK’872可明显降低m NSS评分[1 d:(11.95±1.50)vs(13.05±1.82),t=2.084,P<0.05;3 d:(8.95±1.39)vs(11.00±2.10),t=3.634,P<0.01;5 d:(6.75±1.33)vs(8.90±1.52),t=4.759,P<0.01;7 d:(4.00±1.08)vs(7.15±1.09),t=9.200,P<0.01],同时可明显减轻脑水肿程度[(77.91±0.84)%vs(80.34±0.94)%,t=8.692,P<0.01],减轻皮层及海马区损伤程度,并促进创伤后觉醒[(4.08±0.63)h vs(5.11±0.74)h,t=4.717,P<0.01]。Western blot结果显示,与TBI组相比,应用GSK’872后能降低RIP3[(0.70±0.03)vs(1.04±0.04),t=13.051,P<0.01]、RIP1[(0.93±0.02)vs(1.16±0.03),t=11.203,P<0.01]、MLKL[(0.75±0.04)vs(1.03±0.03),t=9.873,P<0.01]、Akt[(0.55±0.04)vs(0.77±0.05),t=6.278,P<0.01]、p-Akt[(0.80±0.04)vs(0.99±0.04),t=6.217,P<0.01]、m TOR[(0.48±0.05)vs(0.90±0.05),t=10.608,P<0.05]、p-m TOR[(0.59±0.06)vs(1.00±0.05),t=9.144,P<0.01]、Caspase-1[(0.80±0.04)vs(0.98±0.05),t=5.226,P<0.01]、NLRP3[(0.51±0.03)vs(0.89±0.03),t=15.590,P<0.01]、XIAP[(0.50±0.03)vs(0.83±0.03),t=13.340,P<0.01]蛋白表达,并可促进Caspase-8持续升高[(0.83±0.03)vs(0.71±0.03),t=5.044,P<0.01],差异具有统计学意义。结论 RIP3介导的坏死性凋亡在小鼠颅脑创伤中起重要作用,应用GSK’872可减轻小鼠颅脑创伤后的损伤程度,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 traumatic brain injury（TBI） model of C57BL/6 mice and its mechanisms.Methods C57 BL/6 mice were positioned beneath the controlled cortical impactor device（CCI） and subjected to impact injury at 1 mm depth of penetration,for a sustained depression of 120 msec and a velocity of 5 m/s.Ctrl group was not treated.Sham group was received craniotomy,without CCI injury.TBI group was received CCI injury and 4 μl DMSO injected.GSK＇872 group was received CCI injury and 4 μl GSK＇872 injected.Then,the degree of injury was detected by modified neurological severity scores（m NSS）,the degree of brain edema was measured with drying wet method,the injure degree of cortex and hippocampus was assessed by HE staining,the recovery after CCI was measured by awakening time and the expressions of RIP3/RIP1/MLKL,Akt/p-Akt/m TOR/p-m TOR,Caspase-8/XIAP,Caspase-1/NLRP3 were detected by Western blot assay.Results Compared with TBI group,GSK＇872 could significantly decrease the score of m NSS [1 d：（11.95 ±1.50） vs（13.05 ±1.82）,t =2.084,P〈0.05; 3 d：（8.95 ±1.39） vs（11.00±2.10）,t=3.634,P〈0.01：5 d：（6.75±1.33） vs（8.90±1.52）,t=4.759,P〈0.01; 7 d：（4.00±1.08） vs（7.15±1.09）,t=9.200,P〈0.01],decrease the degree of brain edema [（77.91 ±0.84） % vs（80.34±0.94） %,t =8.692,P〈0.01],reduce the degree of injury in cortex and hippocampus,and promote the awaking of mice after CCI [（4.08±0.63） h vs（5.11±0.74） h,t=4.717,P〈0.01].Furthermore,Western blotting assay reveled that GSK＇872 could significantly decrease the levels of RIP3[（0.70±0.03） vs（1.04±0.04）,t=13.051,P〈0.01],RIP1[（0.93±0.02） vs（1.16±0.03）,t=11.203,P〈0.01],MLKL[（0.75±0.04） vs（1.03±0.03）,t=9.873,P〈0.01],Akt[（0.55±0.04） vs（0.77±0.05）,t=6.278,P〈0.01],p-Akt[（0.80±0.04） vs（0.99±0.04）,t=6.217,P〈0.01],m TOR [（0.48±0.05） vs（0.90±0.05）,t=10.608,P〈0.05],p-m TOR [（0.59±0.06） vs（1.00±0.05）,t=9.144,P〈0.01],Caspase-1 [（0.80±0.04） vs（0.98 ±0.05）,t=5.226,P〈0.01],NLRP3 [（0.51 ±0.03） vs（0.89 ±0.03）,t=15.590,P〈0.01],XIAP [（0.50±0.03） vs（0.83±0.03）,t=13.340,P〈0.01],but increase the level of Caspase-8 compared with TBI group [（0.83±0.03） vs（0.71±0.03）,t=5.044,P〈0.01].Conclusion Necroptosis induced by RIP3 may play an important role in TBI,however,GSK＇872 could reduce the degree of injury after TBI,which reveals that RIP3 may be a new target for clinical treatment of TBI in the future.