Del-1促进SERCA2表达抑制ER应激、炎症和神经细胞凋亡保护小鼠SCI

doi:10.5246/jcps.2024.09.059

摘要/Abstract

摘要：

本文探索发育内皮细胞因子-1(Developmental endothelial locus-1, Del-1)对脊髓肌浆网内质网钙调蛋白2 (sarcoplasmic reticulum Ca²⁺ ATPase 2, SERCA2)的影响, 及其对脊髓损伤(spinal cord injury, SCI)的可能作用。48只小鼠随机均分为sham组、SCI组和SCI+CE组, 每组随机均分为两个亚组, Del处理亚组给予尾静脉注射Del-1 (1 μg/d)连续1周, 另一组注射等量生理盐水; 实验1周后处死小鼠, 取脊髓进行下一步处理。首先通过免疫印迹分析Del-1在脊髓中对SERCA2的作用; 然后通过免疫印迹和荧光TUNEL分析Del-1对小鼠SCI内质网应激、炎症和凋亡的作用; 最后通过免疫印迹和荧光TUNEL分析分析阻断SERCA2后Del-1对小鼠SCI内质网应激、炎症和凋亡的作用。结果表明Del-1处理增加了脊髓中SERCA2的表达(P < 0.01), 并且减弱了SCI诱导的ER应激、炎症和神经细胞凋亡(P < 0.01); 阻断SCI小鼠脊髓中SERCA2表达, 促进ER应激、炎症反应和神经细胞凋亡(P < 0.01), 但Del-1处理并不能减少阻断SERCA2对ER应激、炎症反应和神经细胞凋亡的影响(P > 0.05)。最终得出Del-1可以通过促进SERCA2表达降低脊髓损伤的ER应激、炎症反应和神经细胞凋亡。

关键词: 脊髓损伤, Del-1, 内质网钙调蛋白2, 炎症, 凋亡

Abstract:

This study aimed to investigate the impact of developmental endothelial locus-1 (Del-1) on sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) and its potential effects on spinal cord injury (SCI). A total of 48 mice were randomly assigned to the sham group, SCI group, and SCI + CE group. Each group was further divided into two subgroups. The Del treatment subgroup received tail-vein injections of Del-1 (1 μg/d) for a consecutive week, while the other group was injected with an equivalent volume of normal saline. After 1 week of experimentation, the mice were euthanized, and the spinal cord was extracted for further analysis. Initially, the impact of Del-1 on SERCA2 in the spinal cord was assessed using western blotting analysis. Subsequently, the effects of Del-1 on stress, inflammation, and apoptosis in the endoplasmic reticulum (ER) of SCI mice were analyzed through Western blotting analysis and fluorescent TUNEL. Finally, the study utilized Western blotting analysis and fluorescent TUNEL analysis to examine the consequences of Del-1 blocking SERCA2 on ER stress, inflammation, and apoptosis in mice. The results revealed that Del-1 treatment significantly increased the expression of SERCA2 in the spinal cord (P < 0.01) and mitigated SCI-induced ER stress, inflammation, and neuronal apoptosis (P < 0.01). Blocking SERCA2 expression in the spinal cord of SCI mice promoted ER stress, inflammatory response, and neuronal cell apoptosis (P < 0.01). However, Del-1 treatment did not alleviate the effects of blocking SERCA2 on ER stress, inflammatory response, and neuronal cell apoptosis (P > 0.05). In conclusion, this study proposed that Del-1 could reduce ER stress, inflammatory response, and nerve cell apoptosis in spinal cord injury by enhancing the expression of SERCA2.

Key words: SCI, Del-1, SERCA2, Inflammation, Apoptosis

Supporting:

王浩, 陈康雪, 张雨焱, 蒋奎军, 孙中磊. Del-1促进SERCA2表达抑制ER应激、炎症和神经细胞凋亡保护小鼠SCI[J]. 中国药学(英文版), 2024, 33(9): 795-804.

Hao Wang, Kangxue Chen, Yuyan Zhang, Kuijun Jiang, Zhonglei Sun. Developmental endothelial locus-1 enhances ER calmodulin expression, mitigates ER stress, suppresses inflammation, and attenuates neuronal apoptosis[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(9): 795-804.

图/表 6

Figure 1. Del-1 treatment increases SERCA2 expression levels in both normal and injured spinal cords. Sham group (normal control group without Del-1 treatment subgroup), sham + Del group (normal control Del-1 treatment subgroup), SCI group (SCI group without Del-1 treatment subgroup), SCI + Del group (SCI + Del-1 treatment subgroup), and the No. 1 mouse image of each group was taken as representative. Compared with the sham group normal saline subgroup **P < 0.01, compared with the SCI group normal saline subgroup #P < 0.05.

Table 1. The relative content of SERCA2 protein in the spinal cord of mice in each group (s, x ± s, n = 8).

Figure 2. (A) Immunoblotting analysis of CHOP, ELF2α, TNF-α, IL-6, Caspase-3, Bcl-2, and β-actin; (B) The relative content of CHOP protein with β-actin as the contrast band; (C) The relative ELF2α protein content with β-actin as the contrast band; (D) The relative content of TNF-α protein with β-actin as the contrast band; (E) The relative content of IL-6 protein with β-actin as the contrast band; (F) The relative content of Caspase-3 protein with β-actin as the contrast band; (G) The relative content of Bcl-2 protein with β-actin as the contrast band; (H) TUNEL immunofluorescence assessment, scale bar = 100 μm; (I) The percentage of TUNEL-positive cells; Values are expressed as mean ± SD. sham group (subgroup without Del-1 treatment), SCI group (subgroup without Del-1 treatment after SCI), SCI + Del (subgroup with Del-1 treatment after SCI), and the No. 1 mouse image of each group was taken as representative. Compared with the sham group, **P < 0.01, compared with the SCI group, ##P < 0.01.

Table 2. Relative contents of related proteins in each group of mice (s, x ± s, n = 8).

Figure 3. (A) Immunoblotting analysis of SERCA2, CHOP, ELF2α, TNF-α, IL-6, Caspase-3, Bcl-2 and β-actin; (B) The relative content of SERCA2 protein with β-actin as the contrast band; (C) The relative content of CHOP protein with β-actin as the contrast band; (D) Relative content of ELF2α protein with β-actin as the contrast band; (E) The relative content of TNF-α protein with β-actin as the contrast band; (F) The relative content of IL-6 protein with β-actin as the contrast band; (G) The relative content of Caspase-3 protein with β-actin as the contrast band; (H) The relative content of Bcl-2 protein with β-actin as the contrast band; (I) Immunofluorescence assessment of TUNEL, scale bar = 100 μm; (J) The percentage of TUNEL-positive cells; Values are expressed as mean ± SD. SCI group (without Del-1 treatment after SCI), SCI + CE group (SERCA2 blockade after SCI, without Del-1 treatment), SCI + Del group (SERCA2 blockade after SCI, Del-1 treatment), and the images of No. 1 mice in each group were taken as representative. Compared with the SCI group, **P < 0.01, compared with the SCI + CE group, NSP > 0.05.

Table 3. Relative contents of related proteins in each group of mice (s, x ± s, n = 8).

参考文献 28

[1]	Hurlbert, R.J.; Hadley, M.N.; Walters, B.C.; Aarabi, B.; Dhall, S.S.; Gelb, D.E.; Rozzelle, C.J.; Ryken, T.C.; Theodore, N. Pharmacological therapy for acute spinal cord injury. Neurosurgery. 2015, 76, S71–S83.
[2]	Khazaeipour, Z.; Taheri-Otaghsara, S.M.; Naghdi, M. Depression following spinal cord injury: its relationship to demographic and socioeconomic indicators. Top. Spinal Cord Inj. Rehabil. 2015, 21, 149–155.
[3]	Varma, A.K.; Das, A.; Wallace, G. 4th, Barry, J.; Vertegel, A.A.; Ray, S.K.; Banik, N.L. Spinal cord injury: a review of current therapy, future treatments, and basic science frontiers. Neurochem. Res. 2013, 38, 895–905.
[4]	Wolf, J.A.; Stys, P.K.; Lusardi, T.; Meaney, D.; Smith, D.H. Traumatic axonal injury induces calcium influx modulated by tetrodotoxin-sensitive sodium channels. J. Neurosci. 2001, 21, 1923–1930.
[5]	Kapoor, R.; Davies, M.; Blaker, P.A.; Hall, S.M.; Smith, K.J. Blockers of sodium and calcium entry protect axons from nitric oxide-mediated degeneration. Ann. Neurol. 2003, 53, 174–180.
[6]	Kapoor, R.; Davies, M.; Blaker, P.A.; Hall, S.M.; Smith, K.J. Blockers of sodium and calcium entry protect axons from nitric oxide-mediated degeneration. Ann. Neurol. 2003, 53, 174–180.
[7]	Sun, Z.L.; Liu, Y.F.; Kong, X.B.; Wang, R.J.; Xu, Y.Q.; Shang, C.Z.; Huo, J.R.; Huang, M.Q.; Zhao, F.; Bian, K.F.; Zhang, S.; Tu, Y.; Chen, X.Y. Exendin-4 plays a protective role in a rat model of spinal cord injury through SERCA2. Cell Physiol. Biochem. 2018, 47, 617–629.
[8]	Hajishengallis, G.; Chavakis, T. Endogenous modulators of inflammatory cell recruitment. Trends Immunol. 2013, 34, 1–6.
[9]	Choi, E.Y.; Chavakis, E.; Czabanka, M.A.; Langer, H.F.; Fraemohs, L.; Economopoulou, M.; Kundu, R.K.; Orlandi, A.; Zheng, Y.Y.; Prieto, D.A.; Ballantyne, C.M.; Constant, S.L.; Aird, W.C.; Papayannopoulou, T.; Gahmberg, C.G.; Udey, M.C.; Vajkoczy, P.; Quertermous, T.; Dimmeler, S.; Weber, C.; Chavakis, T. Del-1, an endogenous leukocyte-endothelial adhesion inhibitor, limits inflammatory cell recruitment. Science. 2008, 322, 1101–1104.
[10]	Eskan, M.A.; Jotwani, R.; Abe, T.; Chmelar, J.; Lim, J.H.; Liang, S.; Ciero, P.A.; Krauss, J.L.; Li, F.G.; Rauner, M.; Hofbauer, L.C.; Choi, E.Y.; Chung, K.J.; Hashim, A.; Curtis, M.A.; Chavakis, T.; Hajishengallis, G. The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss. Nat. Immunol. 2012, 13, 465–473.
[11]	Choi, E.Y.; Lim, J.H.; Neuwirth, A.; Economopoulou, M.; Chatzigeorgiou, A.; Chung, K.J.; Bittner, S.; Lee, S.H.; Langer, H.; Samus, M.; Kim, H.; Cho, G.S.; Ziemssen, T.; Bdeir, K.; Chavakis, E.; Koh, J.Y.; Boon, L.; Hosur, K.; Bornstein, S.R.; Meuth, S.G.; Hajishengallis, G.; Chavakis, T. Developmental endothelial locus-1 is a homeostatic factor in the central nervous system limiting neuroinflammation and demyelination. Mol. Psychiatry. 2015, 20, 880–888.
[12]	Kang, Y.Y.; Kim, D.Y.; Lee, S.H.; Choi, E.Y. Deficiency of developmental endothelial locus-1 (Del-1) aggravates bleomycin-induced pulmonary fibrosis in mice. Biochem. Biophys. Res. Commun. 2014, 445, 369–374.
[13]	Sun, J.L.; Park, J.; Lee, T.; Jeong, J.H.; Jung, T.W. DEL-1 ameliorates high-fat diet-induced insulin resistance in mouse skeletal muscle through SIRT1/SERCA2-mediated ER stress suppression. Biochem. Pharmacol. 2020, 171, 113730.
[14]	Hu, J.Z.; Long, H.; Wu, T.D.; Zhou, Y.; Lu, H.B. The effect of estrogen-related receptor α on the regulation of angiogenesis after spinal cord injury. Neuroscience. 2015, 290, 570–580.
[15]	do Couto Nicola, F.; Marques, M.R.; Odorcyk, F.; Arcego, D.M.; Petenuzzo, L.; Aristimunha, D.; Vizuete, A.; Sanches, E.F.; Pereira, D.P.; Maurmann, N.; Dalmaz, C.; Pranke, P.; Netto, C.A. Neuroprotector effect of stem cells from human exfoliated deciduous teeth transplanted after traumatic spinal cord injury involves inhibition of early neuronal apoptosis. Brain Res. 2017, 1663, 95–105.
[16]	Mekahli, D.; Bultynck, G.; Parys, J.B.; De Smedt, H.; Missiaen, L. Endoplasmic-reticulum calcium depletion and disease. Cold Spring Harb. Perspect. Biol. 2011, 3, a004317.
[17]	Kourtzelis, I.; Li, X.F.; Mitroulis, I.; Grosser, D.; Kajikawa, T.; Wang, B.M.; Grzybek, M.; von Renesse, J.; Czogalla, A.; Troullinaki, M.; Ferreira, A.; Doreth, C.; Ruppova, K.; Chen, L.S.; Hosur, K.; Lim, J.H.; Chung, K.J.; Grossklaus, S.; Tausche, A.K.; Joosten, L.A.B.; Moutsopoulos, N.M.; Wielockx, B.; Castrillo, A.; Korostoff, J.M.; Coskun, Ü.; Hajishengallis, G.; Chavakis, T. DEL-1 promotes macrophage efferocytosis and clearance of inflammation. Nat. Immunol. 2019, 20, 40–49.
[18]	Yan, S.; Chen, L.; Zhao, Q.; Liu, Y.N.; Hou, R.; Yu, J.; Zhang, H. Developmental endothelial locus-1 (Del-1) antagonizes Interleukin-17-mediated allergic asthma. Immunol. Cell Biol. 2018, 96, 526–535.
[19]	West, X.Z.; Malinin, N.L.; Merkulova, A.A.; Tischenko, M.; Kerr, B.A.; Borden, E.C.; Podrez, E.A.; Salomon, R.G.; Byzova, T.V. Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands. Nature. 2010, 467, 972–976.
[20]	Zhao, H.S.; Chen, S.R.; Gao, K.; Zhou, Z.P.; Wang, C.; Shen, Z.L.; Guo, Y.; Li, Z.; Wan, Z.H.; Liu, C.; Mei, X.F. Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway. Neuroscience. 2017, 348, 241–251.
[21]	Huang, J.H.; Yin, X.M.; Xu, Y.; Xu, C.C.; Lin, X.; Ye, F.B.; Cao, Y.; Lin, F.Y. Systemic administration of exosomes released from mesenchymal stromal cells attenuates apoptosis, inflammation, and promotes angiogenesis after spinal cord injury in rats. J. Neurotrauma. 2017, 34, 3388–3396.
[22]	Gu, S.X.; Xie, R.; Liu, X.D.; Shou, J.J.; Gu, W.T.; Che, X.M. Long coding RNA XIST contributes to neuronal apoptosis through the downregulation of AKT phosphorylation and is negatively regulated by miR-494 in rat spinal cord injury. Int. J. Mol. Sci. 2017, 18, 732.
[23]	Gao, K.; Shen, Z.L.; Yuan, Y.J.; Han, D.H.; Song, C.W.; Guo, Y.; Mei, X.F. Simvastatin inhibits neural cell apoptosis and promotes locomotor recovery via activation of Wnt/β-catenin signaling pathway after spinal cord injury. J. Neurochem. 2016, 138, 139–149.
[24]	Zhu, H.G.; Xie, R.; Liu, X.D.; Shou, J.J.; Gu, W.T.; Gu, S.X.; Che, X.M. MicroRNA-494 improves functional recovery and inhibits apoptosis by modulating PTEN/AKT/mTOR pathway in rats after spinal cord injury. Biomed. Pharmacother. 2017, 92, 879–887.
[25]	Kurnellas, M.P.; Nicot, A.; Shull, G.E.; Elkabes, S. Plasma membrane calcium ATPase deficiency causes neuronal pathology in the spinal cord: a potential mechanism for neurodegeneration in multiple sclerosis and spinal cord injury. FASEB J. 2005, 19, 298–300.
[26]	Dremina, E.S.; Sharov, V.S.; Kumar, K.; Zaidi, A.; Michaelis, E.K.; Schöneich, C. Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA). Biochem. J. 2004, 383, 361–370.
[27]	Kwon, C.H.; Sun, J.L.; Kim, M.J.; Abd El-Aty, A.M.; Jeong, J.H.; Jung, T.W. Clinically confirmed DEL-1 as a myokine attenuates lipid-induced inflammation and insulin resistance in 3T3-L1 adipocytes via AMPK/HO-1- pathway. Adipocyte. 2020, 9, 576–586.
[28]	Kumar, S.; Behera, S.; Basu, A.; Dey, S.; Ghosh-Roy, A. Swimming exercise promotes post-injury axon regeneration and functional restoration through AMPK. eNeuro. 2021, 8, ENEURO.0414–20.2021.