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

doi:10.3877/cma.j.issn.2095-9141.2017.03.008

Abstract

Abstract:

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/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) μm² vs (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], mTOR [(0.81±0.02) vs (0.90±0.05), t=2.813, P<0.05], p-mTOR [(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.

Key words: Traumatic brain injury, Stretch injury, Necroptosis, Receptor-interacting protein 3, GSK’872, HT-22 cells

Zeqi Yu, Tailong Yi, Yue Tu, Xiaosa Yang, Jipeng Jiang, Xiaoyu Dong, Sai Zhang, Shixiang Cheng. Effects of necroptosis induced by RIP3 in stretch injury model of HT-22 cells[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2017, 03(03): 159-165.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhsjcswkdzzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-9141.2017.03.008

https://zhsjcswkdzzz.cma-cmc.com.cn/EN/Y2017/V03/I03/159

Figures/Tables 8

References 21

[1]	涂悦，杨细平，商崇智.醒脑静对颅脑创伤的保护作用[J].中国应用生理学杂志, 2014, 30(3): 230-232, 236.
[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 mTOR 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 HeLa 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.

组别	Sub-G1	G0/G1	S	G2/M
Ctrl	0.63±0.26	58.11±4.31	18.29±0.11	22.97±0.22
CIC	6.51±0.63^a	33.04±7.07^a	10.85±0.37^a	49.60±0.73^a
GSK ’872	0.33±0.15^c	46.67±2.96^ab	25.55±1.93^ac	27.44±3.82^c

组别	Sub-G1	G0/G1	S	G2/M
Ctrl	0.63±0.26	58.11±4.31	18.29±0.11	22.97±0.22
CIC	6.51±0.63^a	33.04±7.07^a	10.85±0.37^a	49.60±0.73^a
GSK ’872	0.33±0.15^c	46.67±2.96^ab	25.55±1.93^ac	27.44±3.82^c

Please choose a citation manager

Content to export