<i>EZH2</i>基因敲除对局灶节段性肾小球硬化小鼠足细胞损伤和JAK2/STAT3信号通路的影响

刘卫英; 隋晓露; 张燕子; 陈继红

doi:10.7619/jcmp.20220847

EZH2基因敲除对局灶节段性肾小球硬化小鼠足细胞损伤和JAK2/STAT3信号通路的影响

广东省深圳市宝安区人民医院肾内科, 广东深圳, 518000

基金项目: 广东省深圳市宝安区高层次人才创新项目

详细信息

通讯作者:
陈继红, E-mail: chenjihong0606@hotmail.com

中图分类号: R692.3;R363
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出版历程
- 收稿日期: 2022-03-16
- 网络出版日期: 2022-11-17

Effect of EZH2 knockout on podocyte injury and JAK2/STAT3 signaling pathway in mice with focal segmental glomerulosclerosis

Department of Nephrology, Bao'an District People's Hospital of Shenzhen of Guangdong Province, Shenzhen, Guangdong, 518000

摘要

摘要:
目的
探讨EZH2基因敲除对局灶节段性肾小球硬化（FSGS）模型小鼠肾脏足细胞损伤和JAK2/STAT3信号通路的影响。
方法
将60只Cas9小鼠随机分为EZH2敲除组和EZH2未敲除组，每组30只。EZH2敲除组小鼠肾静脉注射重组腺病毒（AAV9-sgRNA-EZH2），EZH2未敲除组小鼠肾静脉注射磷酸盐缓冲液（PBS），且2组小鼠均单次尾静脉注射阿霉素，分别建立EZH2敲除FSGS模型和EZH2未敲除FSGS模型。采用苏木素-伊红（HE）染色法观察2组小鼠肾组织病理变化；采用双重免疫荧光染色法观察2组小鼠足细胞nephrin、podocin表达情况；采用TUNEL法检测2组小鼠足细胞凋亡指数；采用免疫印迹（Western blot）法检测2组小鼠肾组织JAK2、STAT3蛋白表达水平。
结果
与EZH2未敲除组FSGS小鼠相比，EZH2敲除组FSGS小鼠肾小球病变更严重，足突广泛融合，肾小球基底膜明显增厚，系膜基质增多，毛细血管闭塞。与EZH2未敲除组FSGS小鼠相比，EZH2敲除组FSGS小鼠足细胞nephrin、podocin表达减少，差异有统计学意义（P < 0.01）。EZH2敲除组足细胞凋亡指数为（40.94±2.13）%，高于EZH2未敲除组的（21.23±3.30）%，差异有统计学意义（P < 0.01）。EZH2敲除组FSGS小鼠JAK2、STAT3蛋白表达水平依次为（2.67±0.41）、（2.37±0.53），分别高于EZH2未敲除组的（1.72±0.31）、（1.70±0.48），差异有统计学意义（P < 0.01）。
结论
EZH2基因敲除可能参与JAK2/STAT3信号通路的激活，并加重FSGS小鼠肾脏足细胞损伤。
- EZH2基因 /
- 局灶节段性肾小球硬化 /
- JAK2/STAT3信号通路 /
- 足细胞 /
- 基因敲除
Abstract:
Objective
To explore effect of EZH2 knockout on podocyte injury and JAK2/STAT3 signaling pathway in mice model with focal segmental glomerulosclerosis(FSGS).
Methods
Sixty Cas9 mice were randomly divided into EZH2 knockout group(n=30) and the EZH2 non-knockout group (n=30). Mice in EZH2 knockout group were intravenously injected with recombinant adenovirus (AAV9-sgRNA-EZH2), those in the EZH2 non-knockout group were intravenously injected with phosphate buffered solution (PBS). Mice in the two groups were injected with doxorubicin once through tail vein to establish EZH2 knockout FSGS model and EZH2 non-knockout FSGS model, respectively. The renal tissue pathological changes were observed by hematoxylin and eosin (HE) staining. The nephrin and podocin expressions were observed by double immunofluorescence. Podocyte apoptosis index was detected by TUNEL. The JAK2 and STAT3 protein expressions were detected by western blot method.
Results
Compared with mice in the EZH2 non-knockout group, the mice had more serious glomerular pathological changes, widely fused foot processes, significantly thickened glomerular basement membrane, increased mesangial matrix and blocked capillaries. Compared with FSGS mice in the EZH2 non-knockout group, the expressions of nephrin and podocin in podocytes of FSGS mice in the EZH2 knockout group were decreased (P < 0.01). The apoptosis index of podocytic cells in the EZH2 knockout group was (40.94±2.13)%, which was higher than (21.23±3.30)% in the EZH2 non-knockout group (P < 0.01). The protein expression levels of JAK2 and STAT3 in the EZH2 knockout group were (2.67±0.41) and (2.37±0.53) respectively, which were higher than (1.72±0.31) and (1.70±0.48) in the EZH2 non-knockout group (P < 0.01).
Conclusion
EZH2 gene knockout may be involved in the activation of JAK2/STAT3 signaling pathway and aggravate renal podocyte injury in FSGS mice.
- EZH2 gene /
- focal segmental glomerulosclerosis /
- JAK2/STAT3 signaling pathway /
- podocyte /
- gene knockout

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图 1 2组FSGS小鼠肾组织光镜表现

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图 2 2组FSGS小鼠肾组织电镜表现

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图 3 2组FSGS小鼠肾组织podocin、podocin表达情况(双重免疫荧光染色法，放大400倍)

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图 4 2组FSGS小鼠足细胞凋亡情况(光镜图，放大400倍)

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图 5 2组FSGS小鼠JAK2、STAT3蛋白免疫印迹图

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表 1 2组FSGS小鼠生化指标水平比较(x±s)

指标	EZH2未敲除组(n=30)	EZH2敲除组(n=30)
24 h尿蛋白定量/mg	241.87±31.21	386.65±42.30^**
BUN/(mmol/L)	13.08±3.08	19.54±3.22^**
Cr/(μmol/L)	73.50±8.96	96.48±9.32^**
ALT/(U/L)	41.10±3.91	42.59±7.07
AST/(U/L)	38.35±7.30	40.84±5.41
BUN: 尿素氮; Cr: 肌酐; ALT: 丙氨酸氨基转移酶; AST: 天门冬氨酸氨基转移酶。与EZH2未敲除组比较, **P < 0.01。

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表 2 2组FSGS小鼠足细胞JAK2、STAT3蛋白表达水平比较(x±s)

指标	EZH2未敲除组(n=30)	EZH2敲除组(n=30)
JAK2蛋白	1.72±0.31	2.67±0.41^**
STAT3蛋白	1.70±0.48	2.37±0.53^**
与EZH2未敲除组比较, **P < 0.01。

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