雷公藤多苷对高糖诱导人肾小管上皮细胞凋亡及CXCL10/CXCR3轴的影响

doi:10.11958/20221078

天津医药 ›› 2023, Vol. 51 ›› Issue (2): 139-143.doi: 10.11958/20221078

雷公藤多苷对高糖诱导人肾小管上皮细胞凋亡及CXCL10/CXCR3轴的影响

李明霞¹(), 乔海霞², 王晓玲¹, 贾丽媛¹, 胡利梅¹, 任卫东¹^,^△()

1 河北北方学院附属第一医院内分泌科（邮编075000）
2 河北北方学院临床医学院

收稿日期:2022-07-08 修回日期:2022-10-16 出版日期:2023-02-15 发布日期:2023-02-24
通讯作者: ^△E-mail：591i@163.com
作者简介:李明霞（1980），女，副主任医师，主要从事内分泌与代谢病方面研究。E-mail：lmx15530396931@163.com
基金资助:
河北省医学科学研究课题计划项目(20190871)

Effects of tripterygium glycosides on high glucose induced apoptosis of human renal tubular epithelial cells and CXCL10/CXCR3 axis

LI Mingxia¹(), QIAO Haixia², WANG Xiaoling¹, JIA Liyuan¹, HU Limei¹, REN Weidong¹^,^△()

1 Department of Endocrinology, the First Hospital Affiliated to Hebei North University, Zhangjiakou 075000, China
2 Clinical Medical College of Hebei North University

Received:2022-07-08 Revised:2022-10-16 Published:2023-02-15 Online:2023-02-24
Contact: ^△E-mail：591i@163.com

李明霞, 乔海霞, 王晓玲, 贾丽媛, 胡利梅, 任卫东. 雷公藤多苷对高糖诱导人肾小管上皮细胞凋亡及CXCL10/CXCR3轴的影响[J]. 天津医药, 2023, 51(2): 139-143.

LI Mingxia, QIAO Haixia, WANG Xiaoling, JIA Liyuan, HU Limei, REN Weidong. Effects of tripterygium glycosides on high glucose induced apoptosis of human renal tubular epithelial cells and CXCL10/CXCR3 axis[J]. Tianjin Medical Journal, 2023, 51(2): 139-143.

摘要/Abstract

摘要：

目的探讨雷公藤多苷（TG）对高糖诱导人肾小管上皮细胞HK-2凋亡及CXC趋化因子配体10（CXCL10）/CXC趋化因子受体3（CXCR3）轴的影响。方法体外培养人肾小管上皮细胞HK-2，并分为对照组（含5.5 mmol/L葡萄糖培养基）、高糖组（含25 mmol/L葡萄糖培养基）和12.5、25、50 mg/L TG组（分别用12.5、25、50 mg/L的TG和25 mmol/L葡萄糖培养基）。四甲基偶氮唑盐比色法检测HK-2细胞活力；流式细胞术检测HK-2细胞凋亡情况；2’,7’-二氯二氢荧光素二乙酸酯法检测HK-2细胞活性氧（ROS）水平；黄嘌呤氧化酶法检测HK-2细胞超氧化物歧化酶（SOD）水平；酶联免疫吸附试验检测HK-2细胞炎性因子肿瘤坏死因子-α（TNF-α）、转化生长因子-β1（TGF-β1）水平；蛋白免疫印迹法检测HK-2细胞凋亡蛋白[胱天蛋白酶（Caspase）-3、Caspase-9]以及CXCL10、CXCR3蛋白表达。结果与对照组比较，高糖组HK-2细胞活力、SOD水平显著降低，凋亡率、ROS、TNF-α、TGF-β1水平及Caspase-3、Caspase-9、CXCL10、CXCR3蛋白表达升高（P<0.05）；与高糖组比较，12.5、25、50 mg/L TG组HK-2细胞活力、SOD水平升高，凋亡率、ROS、TNF-α、TGF-β1水平及Caspase-3、Caspase-9、CXCL10、CXCR3蛋白表达降低，且高剂量效果更好（P<0.05）。结论 TG可抑制高糖诱导的HK-2细胞凋亡、氧化应激和炎症反应，其机制可能与抑制CXCL10/CXCR3轴有关。

关键词: 雷公藤, 趋化因子CXCL10, 受体, CXCR3, 细胞凋亡, 氧化性应激, 雷公藤多苷, 高糖, 人肾小管上皮细胞HK-2

Abstract:

Objective To investigate effects of tripterygium glycosides (TG) on the apoptosis of human renal tubular epithelial cells HK-2 induced by high glucose and the CXC chemokine ligand 10 (CXCL10)/CXC chemokine receptor 3 (CXCR3) axis. Methods Human renal tubular epithelial cells HK-2 were cultured in vitro and divided into the control group (culture medium containing 5.5 mmol/L glucose), the high glucose group (culture medium containing 25 mmol/L glucose) and the 12.5, 25 and 50 mg/L TG groups (culture medium containing 12.5, 25 and 50 mg/L TG and 25 mmol/L glucose, respectively). Methyl thiazolyl tetrazolium (MTT) method was used to detect the viability of HK-2 cells. Flow cytometry was used to detect the apoptosis of HK-2 cells. 2',7'-dichlorodi-hydrofluorescein diacetate method was used to detect reactive oxygen species (ROS) level of HK-2 cells. Xanthine oxidase method was used to detect superoxide dismutase (SOD) level of HK-2 cells. Enzyme-linked immunosorbent assay was used to detect levels of inflammatory factors of tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) in HK-2 cells. Western blot assay was used to detect the expression of apoptotic proteins (Caspase-3, Caspase-9), CXCL10 and CXCR3 proteins in HK-2 cells. Results Compared with the control group, the HK-2 cell viability and SOD level were significantly reduced in the high glucose group, and the apoptosis rate, ROS, TNF-α, TGF-β1 levels and Caspase-3, Caspase-9, CXCL10, CXCR3 protein expression levels were significantly increased (P<0.05). Compared with the high glucose group, the HK-2 cell viability and SOD level were significantly increased in the 12.5, 25 and 50 mg/L TG groups, and the apoptosis rate, ROS, TNF-α, TGF-β1 levels and Caspase-3, Caspase-9, CXCL10, CXCR3 protein expression levels were significantly reduced, and high dose TG has better effect (P<0.05). Conclusion TG can inhibit high glucose induced apoptosis, oxidative stress and inflammation of HK-2 cells, and its mechanism may be related to the inhibition of CXCL10/CXCR3 axis.

Key words: Tripterygium Wilfordii, chemokine CXCL10, receptors, CXCR3, apoptosis, oxidative stress, tripterygium glycosides, high glucose, human renal tubular epithelial cells HK-2

中图分类号:

R587.24

图/表 7

表1 Comparison of HK-2 cell viability and apoptosis rate between the five groups

Tab.1 各组HK-2细胞活力、凋亡率比较（n=6，%，$\bar{x}±s$）

组别	细胞活力	细胞凋亡率
对照组	100.00±0.00	8.34±1.84
高糖组	62.75±3.46^a	36.75±2.73^a
12.5 mg/L TG组	76.42±3.21^b	29.63±2.19^b
25 mg/L TG组	85.14±3.89^bc	21.56±1.95^bc
50 mg/L TG组	93.22±4.16^bcd	11.42±1.54^bcd
F	116.531^＊＊	197.208^＊＊

图1 流式细胞术检测各组HK-2细胞凋亡

Fig.1 The apoptosis of HK-2 cells in each group detected by flow cytometry

表2 Comparison of ROS and SOD levels of HK-2 cells between the five groups

Tab.2 各组HK-2细胞ROS、SOD水平比较（n=6，$\bar{x}±s$）

组别	ROS（荧光强度）	SOD（U/mL）
对照组	100.49±8.17	16.72±1.96
高糖组	254.39±14.54^a	7.41±0.77^a
12.5 mg/L TG组	207.58±13.47^b	9.12±0.98^b
25 mg/L TG组	162.71±10.32^bc	11.36±1.25^bc
50 mg/L TG组	112.88±9.47^bcd	14.58±1.47^bcd
F	190.452^＊＊	48.180^＊＊

图2 荧光显微镜观察HK-2细胞ROS荧光图像（×200）

Fig.2 ROS fluorescence image of HK-2 cells observed by fluorescence microscope (×200)

表3 Comparison of inflammatory factors TNF-α and TGF-β1 levels of HK-2 cells between five groups

Tab.3 各组HK-2细胞炎症因子TNF-α、TGF-β1水平比较（n=6，ng/L，$\bar{x}±s$）

组别	TNF-α	TGF-β1
对照组	21.44±3.56	25.39±4.37
高糖组	68.79±7.11^a	77.92±7.81^a
12.5 mg/L TG组	55.36±6.68^b	61.43±5.76^b
25 mg/L TG组	35.42±4.45^bc	45.72±5.11^bc
50 mg/L TG组	24.91±3.13^bcd	31.24±4.69^bcd
F	90.086^＊＊	87.014^＊＊

表4 Comparison of protein expressions of Caspase-3, Caspase-9, CXCL10 and CXCR3 in HK-2 cells between the five groups

Tab.4 各组HK-2细胞Caspase-3、Caspase-9、CXCL10、CXCR3蛋白表达比较（n=6，$\bar{x}±s$）

组别	Caspase-3	Caspase-9	CXCL10	CXCR3
对照组	0.21±0.03	0.31±0.04	0.26±0.03	0.19±0.03
高糖组	0.73±0.09^a	0.84±0.10^a	0.77±0.08^a	0.83±0.11^a
12.5 mg/L TG组	0.58±0.07^b	0.72±0.08^b	0.61±0.07^b	0.62±0.09^b
25 mg/L TG组	0.39±0.04^bc	0.56±0.06^bc	0.49±0.04^bc	0.41±0.07^bc
50 mg/L TG组	0.28±0.03^bcd	0.41±0.05^bcd	0.31±0.03^bcd	0.24±0.04^bcd
F	84.457^＊＊	58.718^＊＊	90.857^＊＊	77.794^＊＊

图3 各组HK-2细胞Caspase-3、Caspase-9、CXCL10、CXCR3蛋白印迹图 A：对照组；B：高糖组；C：12.5 mg/L TG组；D：25 mg/L TG组；E：50 mg/L TG组。

Fig.3 Western blot assay of Caspase-3, Caspase-9, CXCL10 and CXCR3 of HK-2 cells in each group

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雷公藤多苷对高糖诱导人肾小管上皮细胞凋亡及CXCL10/CXCR3轴的影响

Effects of tripterygium glycosides on high glucose induced apoptosis of human renal tubular epithelial cells and CXCL10/CXCR3 axis

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