扶芳藤含药血清对大鼠胰岛细胞的保护机制

蒋鹏; 高宏君; 尤剑鹏; 梁泰生; 顾新伟; 张建强; 梁方芳; 黄福; 武桢

doi:10.3969/j.issn.1674-7445.2018.04.009

扶芳藤含药血清对大鼠胰岛细胞的保护机制

doi: 10.3969/j.issn.1674-7445.2018.04.009

530011 南宁，广西中医药大学附属瑞康医院移植泌尿外科（蒋鹏、高宏君、梁泰生、顾新伟、张建强、梁方芳、黄福、武桢）；广西中医药大学（尤剑鹏）

基金项目:

广西自然科学基金 2017GXNSFAA198080

广西壮族自治区中青年教师基础能力提升项目 J17114

2017年研究生教育创新计划资助项目 YJSP201745

详细信息

作者简介:
蒋鹏，男，1988年生，硕士研究生，住院医师，研究方向为泌尿外科，Email：418894639@qq.com

通讯作者:
高宏君，男，1972年生，博士，主任医师，研究方向为器官移植，Email：gao4056@163.com

中图分类号: R617, R335+.6
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出版历程
- 收稿日期: 2018-03-10
- 网络出版日期: 2021-01-19
- 刊出日期: 2018-07-15

Protective effect and mechanism of serum containing Euonymus fortunei on rat pancreatic islet cells

Department of Transplantation and Urology Surgery, Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine, Nanning 530011, China

More Information

Corresponding author: Gao Hongjun, Email:gao4056@163.com

摘要

摘要: 目的探讨扶芳藤含药血清对大鼠胰岛细胞的保护作用及其机制。方法将40只雄性SD大鼠随机分为5组，每组8只，包括对照组（正常大鼠胰岛细胞予正常大鼠血清培养）、缺血预处理组（获取胰腺前阻断腹主动脉再开放，胰岛细胞予正常大鼠血清培养）、扶芳藤治疗组（正常大鼠胰岛细胞予大鼠扶芳藤含药血清培养）、扶芳藤组和空白组（正常大鼠分别予扶芳藤提取物或蒸馏水灌胃，用于制备大鼠血清）。采用双硫腙（DTZ）染色法观察并计算胰岛数量；采用吖啶橙（AO）/碘化丙啶（PI）染色法，计算胰岛细胞存活率；采用胰岛素释放实验计算刺激指数（SI）来评价胰岛细胞功能；采用谷胱甘肽（GSH）及一氧化氮（NO）试剂盒测定胰岛细胞内GSH、NO含量；采用逆转录聚合酶链反应（RT-PCR）法检测诱导型一氧化氮合酶（iNOS）信使核糖核酸（mRNA）的表达水平。结果经DTZ染色后胰岛细胞呈特异性猩红色，各组间胰岛细胞数量比较，差异无统计学意义（均为P > 0.05）。随培养时间延长，各组胰岛细胞活性均逐渐降低，分离培养72 h后，与对照组比较，扶芳藤治疗组细胞存活率更高，差异有统计学意义（P < 0.05）。胰岛素释放实验检测结果提示，与对照组相比，缺血预处理组及扶芳藤治疗组的SI均升高，差异均有统计学意义（均为P < 0.05）。与对照组比较，缺血预处理组及扶芳藤治疗组胰岛细胞的GSH含量均升高、NO含量均降低、iNOS mRNA表达水平均降低，差异均有统计学意义（均为P < 0.05）。结论扶芳藤能通过提高GSH、降低iNOS表达及NO产生，提高胰岛细胞存活率，增强胰岛功能。
- 胰岛细胞 /
- 扶芳藤 /
- 中药 /
- 缺血预处理 /
- 原代培养 /
- 胰岛细胞移植 /
- 糖尿病 /
- 壮医
Abstract: Objective To investigate the protective effect and mechanism of serum containing Euonymus fortunei on the rat pancreatic islet cells. Methods Forty male SD rats were randomly divided into 5 groups (n=8 in each group), including the control group (normal rat islet cells were cultured with normal rat serum), ischemic preconditioning group (abdominal aorta was blocked first and then re-opened before the pancreas was obtained, and the pancreatic islet cells were cultured with normal rat serum), Euonymus fortunei treatment group (normal rat islet cells were cultured with rat serum containing Euonymus fortunei), Euonymus fortunei group and blank group (normal rats were administered orally with Euonymus fortunei extract or distilled water for the preparation of rat serum). Diphenylthiocarbazone (DTZ) staining was utilized to observe and calculate the quantity of islets. Acridine orange (AO)/propidium iodide (PI) staining was adopted to calculate the survival rate of islet cells. The insulin release experiment was performed to calculate the stimulation index (SI) and evaluate islet cell function. The concentration of glutathione (GSH) and nitric oxide (NO) in islet cells was detected using GSH and NO kits. The expression level of inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) was quantitatively measured by reverse transcription polymerase chain reaction (RT-PCR). Results Islet cells were observed in specifically scarlet color after DTZ staining. The quantity of islet cells did not significantly differ among different groups (all P > 0.05). Along with the prolongation of culture time, the activity of islet cells in each group was gradually decreased. At 72 h after isolation and culture, compared with the control group, the survival rate of the cells was significantly higher in the Euonymus fortunei treatment group (P < 0.05). The insulin release test results demonstrated that compared with the control group, the SI of the ischemic preconditioning and Euonymus fortunei treatment groups was significantly increased (both P < 0.05). Compared with the control group, the GSH contents of pancreatic islet cells in the ischemic preconditioning and Euonymus fortunei treatment groups were considerably enhanced, the NO content was significantly decreased, and the expression level of iNOS mRNA was significantly down-regulated (all P < 0.05). Conclusions Euonymus fortunei can increase the survival rate of islet cells and enhance the function of pancreatic islets by increasing the level of GSH, down-regulating the expression of iNOS and decreasing the NO production.
- Pancreatic islet cell /
- Euonymus fortunei /
- Chinese medicine /
- Ischemic preconditioning /
- Primary culture /
- Islet cell transplantation /
- Diabetes mellitus /
- Zhuang medicine

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图 1 胰岛细胞分离培养后形态和细胞存活率观察（×200）

A、B、C图分别为对照组、缺血预处理组、扶芳藤治疗组胰岛细胞经DTZ特异性染色后呈猩红色；D、E、F图分别为对照组、缺血预处理组、扶芳藤治疗组胰岛细胞经AO/PI染色后检测细胞存活率

Figure 1. Observation of morphology and cell survival rate after isolation and culture of islet cells

下载: 全尺寸图片幻灯片

图 2 各组胰岛细胞iNOS mRNA表达水平

与对照组比较，^aP < 0.05

Figure 2. Expression of iNOS mRNA in islet cells of each group

下载: 全尺寸图片幻灯片

表 1 各组大鼠胰岛细胞分离培养不同时间后细胞存活率的比较

Table 1. Comparison of cell survival rate of rats islet cells among each group at different time after isolation and culture (x±s, %)

组别	n	细胞存活率
组别	n	24 h	48 h	72 h
对照组	8	82±5	75±4	63±7a
缺血预处理组	8	81±8	75±4	66±7a
扶芳藤治疗组	8	82±4	77±5	70±4^a
与对照组比较，^aP < 0.05

下载: 导出CSV

表 2 各组大鼠胰岛细胞分泌功能及GSH、NO含量

Table 2. Secretory function of islet cells and content of GSH and NO of rat islet cells in each group(x±s)

组别	n	胰岛素分泌量（μg/L）		SI	GSH含量（μmol/g prot）	NO含量（μmol/L）
组别	n	高糖	低糖	SI	GSH含量（μmol/g prot）	NO含量（μmol/L）
对照组	8	07.38±0.29	3.36±0.15	2.20±0.15^a	3.05±0.39^a	43±8^a
缺血预处理组	8	09.38±0.37	3.58±0.31	2.63±0.19^a	4.12±0.29^a	34±7^a
扶芳藤治疗组	8	11.44±0.39	3.89±0.23	2.95±0.23^a	4.07±0.52^a	32±3^a
与对照组比较，^aP < 0.05

下载: 导出CSV

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