牦牛热休克蛋白B11通过抗凋亡缓解热应激对肾细胞的体外损伤

孟德飞; 张康友; 颜勇; 魏玲; 泽仁翁姆; 丁则德米; 黄建钧; 梅群弟; 张楠驰; 魏勇; 王利

doi:10.16829/j.slxb.150842

兽类学报 >

2024 , Vol. 44 >Issue 4: 489 - 496

DOI: https://doi.org/10.16829/j.slxb.150842

研究论文

牦牛热休克蛋白B11通过抗凋亡缓解热应激对肾细胞的体外损伤

孟德飞 ,
张康友 ,
颜勇 ,
魏玲 ,
泽仁翁姆 ,
丁则德米 ,
黄建钧 ,
梅群弟 ,
张楠驰 ,
魏勇 ,
王利

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1 西南民族大学青藏高原动物遗传资源保护与利用教育部重点实验室, 成都 610041;
2 甘孜州雅江县农牧农村和科技局, 雅江 627450;
3 四川畜牧科学研究院, 成都 610066

孟德飞(1997-)，男，硕士研究生，主要从事动物免疫学研究.E-mail:defeimeng@126.com

收稿日期: 2023-08-21

修回日期: 2024-03-11

网络出版日期: 2024-08-03

基金资助

四川省科技厅科研项目(23QYCX0190)；西南民族大学中央高校基本科研业务费专项资金资助(ZYN2024163)

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Yak heat shock protein B11 alleviates the damage of kidney cells induced by heat stress in vitro through anti-apoptosis

MENG Defei ,
ZHANG Kangyou ,
YAN Yong ,
WEI Ling ,
Zerenwengmu ,
Dingzedemi ,
HUANG Jianjun ,
MEI Qundi ,
ZHANG Nanchi ,
WEI Yong ,
WANG Li

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1 Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education, Southwest Minzu University, Chengdu 610041, China;
2 Yajiang County Agriculture and Animal Husbandry Bureau of Ganzi Prefecture, Yajiang 627450, China;
3 Sichuan Institute of Animal Husbandry Science, Chengdu 610066, China

Received date: 2023-08-21

Revised date: 2024-03-11

Online published: 2024-08-03

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摘要

热休克蛋白B11(Heat shock protein beta-11,HSPB11)是小热休克蛋白家族的成员之一，具有抗凋亡、促进细胞存活和维持细胞形态等功能。在热应激条件下，自噬失调是急性肾损伤、肾损伤修复不全及慢性肾脏疾病的发病机制之一，而肾细胞的基础自噬是维持肾脏稳态、结构和功能的关键。为探讨牦牛HSPB11对热应激牛肾细胞(Madin Darby Bovine Kidney,MDBK)活性和凋亡的作用，本研究将MDBK细胞分为对照组、热应激组和处理组，采用PCR扩增、生物信息学分析、原核表达载体构建、CCK-8法、吖啶橙染色法、荧光酶标仪检测、荧光定量PCR等方法。扩增得到牦牛HSPB11目的片段757 bp；构建的系统进化树显示，牦牛与黄牛HSPB11的相似性高达99%；成功构建pET-32a-HSPB11重组质粒，表达获得约35 kDa的牦牛HSPB11；筛选得到牦牛HSPB11最佳浓度为10.0 μg/mL，作为处理组HSPB11浓度。研究结果显示，对照组细胞相对活性极显著高于热应激组(P=0.004)，处理组细胞相对活性极显著高于热应激组(P<0.001)。热应激组细胞出现收缩、细胞核裂解呈现碎片状，处理组少见细胞裂解。此外，热应激组MDBK细胞核酸荧光强度均极显著高于对照组和处理组(P<0.001)。热应激组caspase-3、caspase-9、Bcl-2的mRNA表达量极显著高于对照组和处理组(P<0.001)。综上，推测牦牛HSPB11通过凋亡途径缓解热应激对MDBK细胞损伤，为探究牦牛HSPB11的功能提供基础资料。

关键词： 热休克蛋白B11; 热应激; MDBK 细胞; 凋亡

本文引用格式

孟德飞 , 张康友 , 颜勇 , 魏玲 , 泽仁翁姆 , 丁则德米 , 黄建钧 , 梅群弟 , 张楠驰 , 魏勇 , 王利 . 牦牛热休克蛋白B11通过抗凋亡缓解热应激对肾细胞的体外损伤[J]. 兽类学报, 2024 , 44(4) : 489 -496 . DOI: 10.16829/j.slxb.150842

Abstract

Heat shock protein beta-11 (HSPB11) is a member of the small heat shock protein family, which has the functions of anti-apoptosis, promoting cell survival, and maintaining cell morphology. Under heat stress, dysautophagy is one of the pathogenesis of acute kidney injury, incomplete repair of kidney injury, and chronic kidney disease. The basic autophagy of kidney cells is key to maintaining kidney homeostasis, structure, and function. To study the role of yak HSPB11 on heat-stressed MDBK cell activity and apoptosis. Cells were divided into control group, heat stress group, and treatment group. PCR amplification, bioinformatics analysis, prokaryotic expression vector construction, CCK-8 method, acridine orange staining method, fluorescent enzyme labeling assay, fluorescence quantitative PCR, and other methods were used. A 757 bp amplified fragment of yak HSPB11 was obtained. Phylogenetic tree showed that the similarity between yak HSPB11 and Bos taurus HSPB11 was 99%. pET-32a-HSPB11 recombinant plasmid was successfully constructed and expression yielded about 35 kDa of yak HSPB11. The yak HSPB11 concentration of 10. 0 μg/mL was screened to be the optimal concentration as the treatment group. The results showed that the relative activity of cells was significantly different between the control group and the heat stress group (P = 0. 004), and the relative activity of cells was significantly different between the heat stress group and the treatment group (P<0. 001). In the heat stress group, the cells contracted and the nucleus lysed in fragments, while in the treatment group, cell lysed was rare. In addition, the nucleic acid fluorescence intensity of MDBK cells in the heat stress group was significantly higher than that in the control group and treatment group (P<0. 001). The mRNA expressions of caspase-3, caspase-9 and Bcl-2 in the heat stress group were significantly higher than those in the control group and treatment group (P<0. 001). In summary, it is hypothesized that yak HSPB11 alleviates MDBK cell damage by heat stress through the apoptotic pathway, which provides basic information for exploring the function of yak HSPB11.

Key words： HSPB11; Heat stress; MDBK cells; Apoptosis

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