RIP3介导的坏死性凋亡在HT-22细胞牵张损伤模型中的作用
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摘要
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 Ⅱ(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/m TOR/p-m TOR 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) μm2vs(175.91 ±15.00) μm2, 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/l 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-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] 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 [(0.88±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], 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.
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 Ⅱ(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/m TOR/p-m TOR 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) μm2vs(175.91 ±15.00) μm2, 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/l 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-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] 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 [(0.88±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], 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.
引文
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[17]Liu Q,Qiu J,Liang M,et al.Akt and m TOR mediate programmed necrosis in neurons[J].Cell Death Dis,2014,5:e1084.
[18]Park J,Zhang J,Qiu J,et al.Combination therapy targeting Akt and mammalian target of rapamycin improves functional outcome after controlled cortical impact in mice[J].J Cereb Blood Flow Metab,2012,32(2):330-340.
[19]Liu L,Yim H,Choi JH,et al.ATM kinase promotes both caspase-8 and caspase-9 activation during TNF-α-induced apoptosis of He La cells[J].FEBS Lett,2014,588(6):929-935.
[20]Kaufmann T,Strasser A,Jost PJ.Fas death receptor signalling:roles of Bid and XIAP[J].Cell Death Differ,2012,19(1):42-50.
[21]Kaiser WJ,Sridharan H,Huang C,et al.Toll-like receptor 3-mediated necrosis via TRIF,RIP3,and MLKL[J].J Biol Chem,2013,288(43):31268-31279.
[2]Xu J,Liao W,Gu D,et al.Neural ganglioside GD2 identifies a subpopulation of mesenchymal stem cells in umbilical cord[J].Cell Physiol Biochem,2009,23(4-6):415-424.
[3]Lim ST,Esfahani K,Avdoshina V,et al.Exogenous gangliosides increase the release of brain-derived neurotrophic factor[J].Neuropharmacology,2011,60(7-8):1160-1167.
[4]Bleriot C,Lecuit M.The interplay between regulated necrosis and bacterial infection[J].Cell Mol Life Sci,2016,73(11-12):2369-2378.
[5]Takemoto K,Hatano E,Iwaisako K,et al.Necrostatin-1 protects against reactive oxygen species(ROS)-induced hepatotoxicity in acetaminophen-induced acute liver failure[J].FEBS Open Bio,2014,4:777-787.
[6]Karunakaran D,Geoffrion M,Wei L,et al.Targeting macrophage necroptosis for therapeutic and diagnostic interventions in atherosclerosis[J].Sci Adv,2016,2(7):e1600224.
[7]Xu Y,Wang J,Song X,et al.RIP3 induces ischemic neuronal DNA degradation and programmed necrosis in rat via AIF[J].Sci Rep,2016,6:29362.
[8]Kitur K,Wachtel S,Brown A,et al.Necroptosis promotes staphylococcus aureus clearance by inhibiting excessive inflammatory signaling[J].Cell Rep,2016,16(8):2219-2230.
[9]Bozec D,Iuga AC,Roda G,et al.Critical function of the necroptosis adaptor RIPK3 in protecting from intestinal tumorigenesis[J].Oncotarget,2016,7(29):46384-46400.
[10]Xu B,Xu M,Tian Y,et al.Matrine induces RIP3-dependent necroptosis in cholangiocarcinoma cells[J].Cell Death Discov,2017,3:16096.
[11]Kim SK,Yun M,Seo G,et al.Palmitate induces RIP1/RIP3-dependent necrosis via MLKL-mediated pore formation in the plasma membrane of RAW 264.7 cells[J].Biochem Biophys Res Commun,2017,482(2):359-365.
[12]Hackenberg K,Unterberg A.Traumatic brain injury[J].Nervenarzt,2016,87(2):203-214;quiz 215-216.
[13]Liu T,Zhao DX,Cui H,et al.Therapeutic hypothermia attenuates tissue damage and cytokine expression after traumatic brain injury by inhibiting necroptosis in the rat[J].Sci Rep,2016,6:24547.
[14]Edinger AL,Thompson CB.Death by design:apoptosis,necrosis and autophagy[J].Curr Opin Cell Biol,2004,16(6):663-669.
[15]Chen W,Zhou Z,Li L,et al.Diverse sequence determinants control human and mouse receptor interacting protein 3(RIP3)and mixed lineage kinase domain-like(MLKL)interaction in necroptotic signaling[J].J Biol Chem,2013,288(23):16247-16261.
[16]Wang Z,Jiang H,Chen S,et al.The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways[J].Cell,2012,148(1-2):228-243.
[17]Liu Q,Qiu J,Liang M,et al.Akt and m TOR mediate programmed necrosis in neurons[J].Cell Death Dis,2014,5:e1084.
[18]Park J,Zhang J,Qiu J,et al.Combination therapy targeting Akt and mammalian target of rapamycin improves functional outcome after controlled cortical impact in mice[J].J Cereb Blood Flow Metab,2012,32(2):330-340.
[19]Liu L,Yim H,Choi JH,et al.ATM kinase promotes both caspase-8 and caspase-9 activation during TNF-α-induced apoptosis of He La cells[J].FEBS Lett,2014,588(6):929-935.
[20]Kaufmann T,Strasser A,Jost PJ.Fas death receptor signalling:roles of Bid and XIAP[J].Cell Death Differ,2012,19(1):42-50.
[21]Kaiser WJ,Sridharan H,Huang C,et al.Toll-like receptor 3-mediated necrosis via TRIF,RIP3,and MLKL[J].J Biol Chem,2013,288(43):31268-31279.