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浙江大学学报（医学版）
浙江大学学报（医学版）  2020, Vol. 49 Issue (6): 705-713    DOI: 10.3785/j.issn.1008-9292.2020.12.05
专题报道     
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
叶嘉仪(),龚恒佩,王凌峰,黄真,仇凤梅,钟晓明*()
浙江中医药大学药学院, 浙江 杭州 311402
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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摘要：

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    
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
收稿日期: 2020-07-22 出版日期: 2021-01-14
CLC:  R285.5  
基金资助: 国家自然科学基金(81573643);浙江省自然科学基金(LY20H280010)
通讯作者: 钟晓明     E-mail: yejiayi0109@yeah.net;zxm_k6@sina.com
作者简介: 叶嘉仪(1997-), 女, 硕士研究生, 主要从事防治脑神经损伤药物的开发研究; E-mail:yejiayi0109@yeah.net; https://orcid.org/0000-0001-7460-1237
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引用本文:

叶嘉仪,龚恒佩,王凌峰,黄真,仇凤梅,钟晓明. 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[J]. 浙江大学学报（医学版）, 2020, 49(6): 705-713.

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.

链接本文:

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

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
表 1  实时逆转录PCR引物序列
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#
表 2  各组细胞损伤情况比较
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##
表 3  各组细胞凋亡率和细胞质中游离钙离子浓度比较
图 1  流式细胞仪检测各组细胞凋亡结果
图 2  各组细胞质中游离钙离子荧光图
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##
表 4  各组IP3R1、RyR2、SERCA2 mRNA相对表达量比较
图 3  各组内质网应激相关蛋白表达电泳图
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##
表 5  各组内质网应激相关蛋白表达量比较
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