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浙江大学学报（医学版）
J Zhejiang Univ (Med Sci)  2020, Vol. 49 Issue (6): 705-713    DOI: 10.3785/j.issn.1008-9292.2020.12.05
    
Protective effect of iridoid glycosides of radix scrophulariae on endoplasmic reticulum stress induced by oxygen-glucose deprivation and reperfusion in vitro model
YE Jiayi(),GONG Hengpei,WANG Lingfeng,HUANG Zhen,QIU Fengmei,ZHONG Xiaoming*()
College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 311402, China
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Abstract  

Objective: To investigate the regulatory effect of iridoid glycoside of radix scrophulariae (IGRS) on endoplasmic reticulum stress induced by oxygen-glucose deprivation and reperfusion in vitro model. Methods: Rat pheochromocytoma PC12 cells were pretreated with IGRS (50, 100, 200 μg/mL) for 24h, and the in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) was applied. The cell viability was determined by MTT and lactate dehydrogenase (LDH) assay. The apoptotic rate was detected by flow cytometry. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), C/EBP homologous protein (CHOP), caspase-12 protein, and glucose-regulated protein-78(GRP78)were detected by Western blotting. The mRNA expression levels of sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), 1, 4, 5-triphosphate inositol receptor 1 (IP3R1), and ryanodine receptor 2 (RyR2)were detected by real-time RT-PCR. Free Ca2+ concentration [Ca2+]i was determined by using laser scanning confocal microscopy. Results: The damage caused by OGD/R to PC12 cells was significantly reduced by IGRS, with significant effect on increasing survival rate and reducing LDH release (all P < 0.01). The expression of GRP78, CHOP, Bax, and caspase-12 were down-regulated (all P < 0.01), and the expression of Bcl-2 and Bcl-2/Bax ratio was up-regulated (all P < 0.01); IGRS increased the expression of SERCA2 mRNA in PC12 cells after OGD/R injury (P < 0.01), decreased [Ca2+]i and down-regulated the expression of RyR2 mRNA and IP3R1 mRNA. Conclusion: IGRS has neuroprotective effect, which may alleviate cerebral ischemia-reperfusion injury by regulating SERCA2, maintaining calcium balance, and inhibiting endoplasmic reticulum stress-mediated apoptosis.

Key words： Iridoid glycosides of radix scrophulariae      Oxygen-glucose deprivation-reperfusion      Endoplasmic reticulum stress      Calcium homeostasis      Apoptosis      Cells     
Received: 22 July 2020      Published: 14 January 2021
CLC:  R285.5  
Corresponding Authors: ZHONG Xiaoming     E-mail: yejiayi0109@yeah.net;zxm_k6@sina.com
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YE Jiayi
GONG Hengpei
WANG Lingfeng
HUANG Zhen
QIU Fengmei
ZHONG Xiaoming
Cite this article:

YE Jiayi,GONG Hengpei,WANG Lingfeng,HUANG Zhen,QIU Fengmei,ZHONG Xiaoming. Protective effect of iridoid glycosides of radix scrophulariae on endoplasmic reticulum stress induced by oxygen-glucose deprivation and reperfusion in vitro model. J Zhejiang Univ (Med Sci), 2020, 49(6): 705-713.

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http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2020.12.05     OR     http://www.zjujournals.com/med/Y2020/V49/I6/705


玄参环烯醚萜苷对氧糖剥夺再灌注细胞模型内质网钙稳态的调控作用

目的: 阐明玄参环烯醚萜苷（IGRS）基于调控内质网应激反应对抗脑缺血再灌注损伤的作用及机制。方法: 采用IGRS（50、100、200 μg/mL）预处理PC12细胞24 h，然后构建氧糖剥夺/再灌注（OGD/R）细胞模型。MTT法检测细胞存活率，细胞内乳酸脱氢酶（LDH）释放法检测细胞损伤程度；流式细胞术检测细胞凋亡率，蛋白质印迹法检测B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、C/EBP同源蛋白（CHOP）、半胱氨酸天冬氨酸蛋白酶12（caspase-12）、葡萄糖调节蛋白78（GRP78）水平；实时逆转录PCR法检测肌浆网钙泵2（SERCA2）、1，4，5-三磷酸肌醇受体1（IP3R1）、兰尼碱受体2（RyR2）mRNA表达；激光共聚焦显微镜观察细胞质中的游离钙离子浓度。结果: IGRS预处理可以提高OGD/R细胞存活率、减少LDH释放（均P < 0.01）；降低细胞凋亡率，抑制GRP78、CHOP，Bax和caspase-12蛋白表达（均P < 0.01），上调Bcl-2和Bcl-2/Bax（均P < 0.01），增加细胞SERCA2 mRNA表达（P < 0.01），降低游离钙离子浓度，下调RyR2和IP3R1 mRNA表达。结论: IGRS具有明确的神经保护作用，可能是通过调节SERCA2维持钙平衡，进而抑制内质网应激介导的细胞凋亡来减轻脑缺血再灌注损伤。


关键词： 玄参环烯醚萜苷,  氧糖剥夺/再灌注,  内质网应激,  钙稳态,  细胞凋亡,  细胞 
引物名称 引物序列(5′→3′)
SERCA2：肌浆网钙泵2；IP3R1：1，4，5-三磷酸肌醇受体1；RyR2：兰尼碱受体2；GAPDH：甘油醛-3-磷酸脱氢酶.
SERCA2 正向：GTGAAGTGCCATCAGTATGACGG
反向：GTGAGAGCAGTCTCGGTAGCTT
IP3R1 正向：GAGCATTGTCACCACCTTCTTCAG
反向：GGCTTTCTGGCTCGGCATCAAC
RyR2 正向：CACTCCTCTATGGACACGCC
反向：CAAAGGCCAGTTTGTCGGTG
GAPDH 正向：ACAGCAACAGGGTGGTGAC
反向：TTTGAGGGTGCAGCGAACTT
Tab 1 Primer sequences for real-time RT-PCR
组别 n 细胞存活率 乳酸脱氢酶释放量
与正常对照组比较，**P＜0.01；与模型对照组比较，#P＜0.05，##P＜0.01.IGRS：玄参环烯醚萜苷.
正常对照组 3 100.00 100.00
尼莫地平对照组 3 102.68±3.50 108.07±4.89
IGRS对照组
  50 μg/mL 3 99.60±3.51 107.20±8.34
  100 μg/mL 3 101.35±2.55 111.44±4.24
  200 μg/mL 3 101.63±0.71 109.23±8.37
模型对照组 3 56.83±0.98** 172.63±4.59**
尼莫地平实验组 3 72.94±0.28## 134.68±5.15##
IGRS实验组
  50 μg/mL 3 63.74±1.57 165.48±7.37
  100 μg/mL 3 72.68±0.84## 146.31±3.59##
  200 μg/mL 3 72.35±0.71## 158.15±9.46#
Tab 2 Cells injury induced by oxygen-glucose deprivation and reperfusion  (${\bar x}$±s, %)
组别 n 细胞凋亡率 游离钙离子浓度
与正常对照组比较，*P＜0.05，**P＜0.01；与模型对照组比较，#P＜0.05，##P＜0.01.4-PBA：4-苯基丁酸；IGRS：玄参环烯醚萜苷.
正常对照组 3 8.10±0.60 100.00±11.83
4-PBA对照组 3 6.37±0.35 90.47±3.03
IGRS对照组
  50 μg/mL 3 8.50±0.75 97.14±5.43
  100 μg/mL 3 8.33±0.32 95.74±15.95
  200 μg/mL 3 7.93±0.68 101.10±28.90
模型对照组 3 24.13±0.57** 519.74±30.25*
4-PBA实验组 3 13.77±0.74## 237.52±32.63#
IGRS实验组
  50 μg/mL 3 22.10±0.61 337.63±8.24
  100 μg/mL 3 14.80±0.44## 251.07±23.82#
  200 μg/mL 3 15.80±1.82 186.40±33.38##
Tab 3 Cells apoptosis and free Ca2+ concentration in each group  (${\bar x}$±s, %)
Fig 1 Flow cytometric results of cell apoptosis in each group
Fig 2 Fluorescence results of [Ca2+]i in cells in each group
组别 IP3R1 RyR2 SERCA2
与正常对照组比较，*P＜0.05，**P＜0.01；与模型对照组比较，#P＜0.05，##P＜0.01.IGRS：玄参环烯醚萜苷；Ip3R1：1, 4, 5-三磷酸肌醇受体1；RyR2：兰尼碱受体2；SERCA2：肌浆网钙泵2.
正常对照组 1.000 1.000 1.000
模型对照组 2.243±0.103* 2.555±0.058** 0.259±0.025**
IGRS实验组
  50 μg/mL 2.090±0.130 2.050±0.110 0.340±0.030#
  100 μg/mL 1.804±0.130 1.810±0.120# 0.481±0.030##
  200 μg/mL 1.906±0.040 1.742±0.100# 0.501±0.020##
Tab 4 IP3R1、RyR2、SERCA2 mRNA expression in each group  (${\bar x}$±s, n=3)
Fig 3 Electrophoretogram of endoplasmic reticulum stress related proteins expression in each group
组别 n Bax Bcl-2 Bcl-2/Bax CHOP caspase-12 GRP78
与正常对照组比较，**P＜0.01；与模型对照组比较，#P＜0.05，##P＜0.01.4-PBA：4-苯基丁酸；IGRS：玄参环烯醚萜苷；Bax：Bcl-2相关X蛋白；Bcl-2：B细胞淋巴瘤-2；CHOP：CCAAT/增强子结合蛋白同源蛋白；caspase-12：半胱氨酸天冬氨酸蛋白酶12；GRP78：葡萄糖调节蛋白78.
正常对照组 3 1.000±0.320 1.000±0.046 1.000 1.000±0.090 1.000±0.120 1.000±0.160
4-PBA对照组 3 1.130±0.240 0.969±0.060 0.971±0.080 1.110±0.120 0.980±0.120 0.950±0.100
IGRS对照组
  50 μg/mL 3 1.310±0.390 0.950±0.100 0.950±0.090 1.010±0.150 1.120±0.220 1.110±0.180
  100 μg/mL 3 1.140±0.390 0.964±0.120 0.967±0.150 1.030±0.080 0.990±0.160 1.240±0.130
  200 μg/mL 3 1.150±0.360 0.978±0.100 0.980±0.120 0.980±0.130 1.000±0.090 1.040±0.020
模型对照组 3 4.020±0.260** 0.546±0.010** 0.546±0.030** 6.900±0.240** 2.230±0.090** 3.460±0.070**
4-PBA实验组 3 2.880±0.550## 0.811±0.060## 0.811±0.050## 4.420±0.470## 1.530±0.100## 2.580±0.270##
IGRS实验组
  50 μg/mL 3 3.490±0.370 0.661±0.210 0.662±0.031 6.860±0.310 2.090±0.070 3.300±0.160
  100 μg/mL 3 3.080±0.200## 0.765±0.065## 0.765±0.047## 4.690±0.520## 1.560±0.140## 2.700±0.190##
  200 μg/mL 3 3.30±0.360# 0.760±0.079## 0.761±0.077## 5.550±0.400## 1.420±0.240## 2.870±0.290##
Tab 5 Endoplasmic reticulum stress related proteins expression in each group  (${\bar x}$±s)
[1]   梁刚, 牛育苗, 李一涵 et al. 雷帕霉素在大鼠局灶性脑缺血再灌注后24 h给药对脑损伤的保护作用[J]. 浙江大学学报(医学版), 2018, 47 (5): 443- 449
LIANG Gang , NIU Yumiao , LI Yihan . Rapamycin treatment starting at 24 h after cerebral ischemia/reperfusion exhibits protective effect on brain injury in rats[J]. Journal of Zhejiang University (Medical Sciences), 2018, 47 (5): 443- 449
doi: 10.3785/j.issn.1008-9292.2018.10.01
[2]   LIN L, WANG K, YU Z. Ischemia-reperfusion injury in the brain: mechanisms and potentialtherapeutic strategies[J/OL]. Biochem Pharmacol (Los Angel), 2016, 5(4). DOI: 10.4172/2167-0501.1000213.
[3]   ZHANG H Y , WANG Z G , LU X H et al. Endoplasmic reticulum stress: relevance and therapeutics in central nervous system diseases[J]. Mol Neurobiol, 2015, 51 (3): 1343- 1352
doi: 10.1007/s12035-014-8813-7
[4]   BODALIA A , LI H , JACKSON M F . Loss of endoplasmic reticulum Ca2+ homeostasis: contribution to neuronal cell death during cerebral ischemia[J]. Acta Pharmacol Sin, 2013, 34 (1): 49- 59
doi: 10.1038/aps.2012.139
[5]   OKUBO Y . Neuronal cell death induced by the disruption of endoplasmic reticulum-mediated Ca2+ signaling[J]. Nihon Yakurigaku Zasshi, 2019, 153 (4): 155- 160
doi: 10.1254/fpj.153.155
[6]   国家药典委员会.中华人民共和国药典(一部)[S].北京: 中国医药科技出版社, 2020: 124.
National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (Part 1) [S]. Beijing: China Medical Science Press, 2020: 124. (in Chinese)
[7]   GONG P Y, HE Y W, QI J, et al. Synergistic nourishing 'Yin' effect of iridoid and phenylpropanoid glycosides from radix scrophulariae in vivo and in vitro[J/OL]. J Ethnopharmacol, 2020, 246: 112209. DOI: 10.1016/j.jep.2019.112209.
[8]   CHEN Y Y , ZHANG L , GONG X Y et al. Iridoid glycosides from radix scrophulariae attenuates focal cerebral ischemia-reperfusion injury via inhibiting endoplasmic reticulum stress-mediated neuronal apoptosis in rats[J]. Mol Med Rep, 2020, 21 (1): 131- 140
doi: 10.3892/mmr.2019.10833
[9]   李祚丹, 季金苟, 楚莎莎 et al. 玄参中环烯醚萜类物质的分离纯化工艺[J]. 中成药, 2015, 37 (6): 1367- 1369
LI Zuodan , JI Jingou , CHU Shasha et al. Study on purification of iridoids from figwort[J]. Chinese Traditional Patent Medicine, 2015, 37 (6): 1367- 1369
doi: 10.3969/j.issn.1001-1528.2015.06.049
[10]   WANG K , CHEN M , GONG H P et al. Calcium homeostasis disruption and endoplasmic reticulum stress mediats ischemia/reperfusion-induced PC12 cells apoptosis[J]. Int J Clin Exp Med, 2017, 10 (9): 14121- 14129
[11]   CHO S K , YOON S Y , HUR C G et al. Acetylcholine rescues two-cell block through activation of IP3 receptors and Ca2+/calmodulin-dependent kinase Ⅱ in an ICR mouse strain[J]. Pflugers Arch, 2009, 458 (6): 1125- 1136
doi: 10.1007/s00424-009-0686-7
[12]   吴勉华, 王新月 . 中医内科学[M]. 北京: 中国中医药出版社, 2012: 288- 289
WU Mianhua , WANG Xinyue . Internal medicine of traditional Chinese medicine[M]. Beijing: China Press of Traditional Chinese Medicine, 2012: 288- 289
[13]   SU Y , LI F . Endoplasmic reticulum stress in brain ischemia[J]. Int J Neurosci, 2016, 126 (8): 681- 691
doi: 10.3109/00207454.2015.1059836
[14]   FONT-BELMONTE E , GONZáLEZ-RODRíGUEZ P , FERNáNDEZ-LóPEZ A . Necroptosis in global cerebral ischemia: a role for endoplasmic reticulum stress[J]. Neural Regen Res, 2020, 15 (3): 455- 456
doi: 10.4103/1673-5374.266054
[15]   WEI H F , INAN S . Dual effects of neuroprotection and neurotoxicity by general anesthetics: role of intracellular calcium homeostasis[J]. Prog Neuropsychopharmacol Biol Psychiatry, 2013, 47:156- 161
doi: 10.1016/j.pnpbp.2013.05.009
[16]   ANSARI N , HADI-ALIJANVAND H , SABBAGHIAN M et al. Interaction of 2-APB, dantrolene, and TDMT with IP3R and RyR modulates ER stress-induced programmed cell death Ⅰ and Ⅱ in neuron-like PC12 cells: an experimental and computational investigation[J]. J Biomol Struct Dyn, 2014, 32 (8): 1211- 1230
doi: 10.1080/07391102.2013.812520
[17]   ZHAO H , TONG G , LIU J et al. IP3R and RyR channels are involved in traffic-related PM2.5-induced disorders of calcium homeostasis[J]. Toxicol Ind Health, 2019, 35 (5): 339- 348
doi: 10.1177/0748233719843763
[18]   HAMMADI M , OULIDI A , GACKIèRE F et al. Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78[J]. FASEB J, 2013, 27 (4): 1600- 1609
doi: 10.1096/fj.12-218875
[19]   YE Z , WANG N , XIA P P et al. Parecoxib suppresses CHOP and Foxo1 nuclear translocation, but increases GRP78 levels in a rat model of focal ischemia[J]. Neurochem Res, 2013, 38 (4): 686- 693
doi: 10.1007/s11064-012-0953-4
[20]   LEI X , LEI L , ZHANG Z et al. Diazoxide inhibits of ER stress-mediated apoptosis during oxygen-glucose deprivation in vitro and cerebral ischemia-reperfusion in vivo[J]. Mol Med Rep, 2018, 17 (6): 8039- 8046
doi: 10.3892/mmr.2018.8925
[21]   SCH?NTHAL A H . Pharmacological targeting of endoplasmic reticulum stress signaling in cancer[J]. Biochem Pharmacol, 2013, 85 (5): 653- 666
doi: 10.1016/j.bcp.2012.09.012
[22]   ZHANG A , ZHANG J , SUN P et al. EIF2alpha and caspase-12 activation are involved in oxygen-glucose-serum deprivation/restoration-induced apoptosis of spinal cord astrocytes[J]. Neurosci Lett, 2010, 478 (1): 32- 36
doi: 10.1016/j.neulet.2010.04.062
[23]   ADAMS C M , KIM A S , MITRA R et al. BCL-W has a fundamental role in B cell survival and lymphomagenesis[J]. J Clin Invest, 2017, 127 (2): 635- 650
doi: 10.1172/JCI89486
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