鬼箭羽通过抑制AGEs-RAGE信号转导通路改善晚期糖基化终末产物诱导的小鼠肾足细胞损伤

doi:10.12122/j.issn.1673-4254.2025.09.13

摘要/Abstract

摘要：

目的观察鬼箭羽对晚期糖基化终末产物（AGEs）刺激的小鼠肾足细胞（MPC-5）AGEs-晚期糖基化终末产物受体（RAGE）信号转导通路及下游靶点的影响，并阐明鬼箭羽改善糖尿病肾脏疾病（DKD）足细胞损伤的机制。方法鬼箭羽干预糖尿病肾脏疾病动物模型，利用透射电镜观察肾脏形态与结构；实时荧光定量-聚合酶链反应（RT-qPCR）、免疫组化检测相关基因与核心蛋白的表达情况。体外培养MPC-5，采用浓度为50 mg/L糖基化终末期产物-牛血清白蛋白（AGEs-BSA）培养液孵育24 h，诱导建立糖尿病细胞模型。利用CCK-8法检测技术，评价造模后的细胞成活率。将对数生长期的MPC-5随机分为正常组、模型组、鬼箭羽组、RAGE激动剂（D-Ribose）组、鬼箭羽+RAGE激动剂（D-Ribose）组，利用CCK-8法筛选不同浓度鬼箭羽对MPC-5细胞活力的影响。蛋白免疫印迹法检测各组细胞RAGE、VEGFA、TNF-α、NF-κB（p65）、IL-6、Caspase-3蛋白表达；RT-qPCR检测RAGE、NF-κB（p65）、VEGFA、IL-6mRNA表达。结果体内结果表明，鬼箭羽干预后，与模型组相比，干预组动物模型肾脏组织足细胞病理变化有所改善。免疫组化结果显示，与模型组比较，鬼箭羽高剂量组RAGE、VEGFA、NF-κB（p65）、IL-6蛋白表达降低（P<0.05）；RT-qPCR结果显示，与模型组比较，鬼箭羽高剂量组RAGE、NF-κB、IL-6mRNA表达降低（P<0.05）。体外结果表明，与正常组相比，模型组细胞活力显著降低（P<0.05），RAGE、NF-κB（p65）、VEGFA、TNF-α、IL-6和Caspase-3蛋白表达升高（P<0.05），RAGE、NF-κB（p65）、VEGFA和IL-6 mRNA表达升高（P<0.05）；与模型组比较，浓度为550 mg/L的鬼箭羽干预成模后的MPC-5，24 h提高细胞活力最为显著（P<0.05），降低RAGE、NF-κB（p65）、VEGFA、TNF-α、IL-6和Caspase-3蛋白表达（P<0.05）；而鬼箭羽+RAGE激动剂可逆转RAGE激动剂对MPC-5模型中RAGE、VEGFA、TNF-α、IL-6和Caspase-3蛋白表达的改变（P<0.05），同时与RAGE激动剂组比较，鬼箭羽+RAGE激动剂组RAGE、NF-κB（p65）、IL-6、VEGFAmRNA表达显著降低（P<0.01）。结论鬼箭羽可以通过抑制AGEs-RAGE信号转导通路的激活，减少下游促炎细胞因子和血管内皮生长因子的表达，进而发挥改善AGEs诱导糖尿病肾脏疾病MPC-5损伤的作用。

关键词: 鬼箭羽, 糖尿病肾脏疾病, 足细胞, AGEs-RAGE信号通路, 晚期糖基化终末产物

Abstract:

Objective To investigate the mechanism by which Guijianyu ameliorates podocyte injury in a mouse model of diabetic kidney disease (DKD) induced by advanced glycation endproducts (AGEs). Methods Sixty db/db mouse models of DKD were randomized equally into 5 groups for treatment with saline, Guijianyu extract at 3 doses or irbesartan for 12 weeks, and the changes in renal pathology and structure were observed using transmission electron microscopy, and the expressions of related genes and key proteins were detected using RT-qPCR and immunohistochemistry. In cultured MPC-5 cells incubated with 50 mg/L AGEs-BSA for 24 h, the effect of different concentrations of Guijianyu extract on cell viability was examined with CCK-8 assay; Western blotting was performed to detect the protein expressions of RAGE, VEGFA, TNF-α, NF-κB(p65), IL-6 and caspase-3, and the mRNA expressions of RAGE, NF-κB (p65), VEGFA and IL-6 were detected with RT-qPCR. Results In mouse models of DKD, treatment with high-dose Guijianyu extract significantly reduced renal expressions of RAGE, VEGFA, NF-κB(p65), and IL-6 proteins and the mRNA expressions of RAGE, NF-κB, and IL-6. In MPC-5 cells, exposure to AGEs significantly reduced cell viability and increased the protein expressions of RAGE, NF‑κB (p65), VEGFA, TNF-α, IL-6 and caspase-3 (P<0.05) and mRNA expressions of RAGE, NF-κB (p65), VEGFA, and IL-6. Treatment with Guijianyu extract obviously improved cell viability and reduced the expressions of RAGE, NF-κB(p65), VEGFA, TNF-α, IL-6, and caspase-3. Furthermore, Guijianyu extract effectively reversed RAGE agonist-induced elevation of protein expressions of RAGE, VEGFA, TNF-α, IL-6, and caspase-3 and mRNA expressions of RAGE, NF-κB (p65), IL-6, and VEGFA in MPC-5 cells. Conclusion Guijianyu extract ameliorates AGEs-induced mouse renal podocyte injury in DKD by inhibiting the activation of AGEs-RAGE signaling pathway and reducing the expressions of pro-inflammatory cytokines and vascular endothelial growth factors.

Key words: Guijianyu, diabetic kidney disease, podocytes, AGEs-RAGE signaling pathway, advanced glycation endproducts

马倩倩, 牛钰琪, 左铭钰, 李鑫, 符竣轲, 王瑾瑾. 鬼箭羽通过抑制AGEs-RAGE信号转导通路改善晚期糖基化终末产物诱导的小鼠肾足细胞损伤[J]. 南方医科大学学报, 2025, 45(9): 1938-1945.

Qianqian MA, Yuqi NIU, Mingyu ZUO, Xin LI, Junke FU, Jinjin WANG. Guijianyu alleviates advanced glycation endproducts-induced mouse renal podocyte injury by inhibiting the AGEs-RAGE signaling pathway[J]. Journal of Southern Medical University, 2025, 45(9): 1938-1945.

图/表 10

表1 RT-qPCR引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Forward 5'-3'	Reverse 5'-3'
RAGE	CCACTGGAATTGTCGATGAGG	CTCGGACTCGGTAGTTGGACT
NF-κB	GCAGAAAGAAGACATTGAGGTGTAT	GCGATCATCTGTGTCTGGCA
IL-6	CCCCAATTTCCAATGCTCTCC	CGCACTAGGTTTGCCGAGTA
VEGFA	TAACGATGAAGCCCTGGAGTG	CACAGTGAACGCTCCAGGATTTA
β-actin	GTGACGTTGACATCCGTAAAGA	GTAACAGTCCGCCTAGAAGCAC

图1 电镜下db/db小鼠足细胞超微结构

Fig.1 Ultrastructure of podocytes of db/db mice under electron microscope (Original magnification: ×15 000). A: Normal group. B: Model group. C: Guijianyu low-dose group. D: Guijianyu medium-dose group. E: Guijianyu high-dose group. F: Irbesartan group.

图2 各组肾脏NF-κB、IL-6、VEGFA、TNF-α的免疫组化结果

Fig.2 Immunohistochemistry for NF-κB, IL-6, VEGFA, and TNF-α in mouse renal tissues in each group (×400). A: Normal group. B: Model group. C: Guijianyu low-dose group. D: Guijianyu medium-dose group. E: Guijianyu high-dose group. F: Irbesartan group.

表2 DKD模型小鼠肾脏NF-κB（p65）、VEGFA、TNF-α、IL-6阳性表达情况

Tab.2 Expression levels of NF-κB (p65), VEGFA, TNF-α and IL-6 proteins in the kidney of DKD mouse models in each group (Mean±SD, n=3)

Group	NF-κB (p65)	VEGFA	TNF-α	IL-6
A	0.120±0.036	0.067±0.015	0.085±0.017	0.102±0.011
B	0.210±0.010	0.123±0.005*	0.121±0.007*	0.143±0.015*
C	0.173±0.023*	0.097±0.015	0.112±0.001	0.123±0.012
D	0.146±0.015	0.087±0.012^#	0.108±0.004	0.117±0.015^#
E F	0.130±0.010^# 0.137±0.006^#	0.067±0.006^# 0.063±0.015^#	0.102±0.002^# 0.099±0.006^#	0.103±0.015^# 0.103±0.006^#
P	<0.001	0.013	0.003	0.012

表3 DKD模型小鼠肾脏RAGE、NF-κB（p65）、IL-6mRNA表达的影响

Tab.3 Expression levels of RAGE, NF-κB (p65) and IL-6 mRNA in the kidney of DKD mouse models in each group (Mean±SD, n=3)

Group	RAGE	NF-κB(p65)	IL-6
A	1.00±0.09	1.00±0.03	1.00±0.14
B	1.83±0.15*	1.71±0.16*	1.42±0.08*
C	1.25±0.11^#	1.22±0.26^#	1.21±0.07^#
D	1.13±0.06^#	1.10±0.18^#	1.18±0.05^#
E F	1.10±0.38^# 1.12±0.02^#	1.05±0.34^# 1.09±0.16^#	1.06±0.04^# 1.04±0.14^#
P	<0.001	0.013	0.002

表4 鬼箭羽对AGEs诱导的MPC-5细胞活性的影响

Tab.4 Effect of Guijianyu on AGEs-induced MPC-5 cell viability (%, Mean±SD, n=3)

Group	Cell viability
Normal	100.00±3.45
Model	67.86±0.93*
150 mg/L Guijianyu	84.33±8.45^#
350 mg/L Guijianyu	88.00±4.84^#
550 mg/L Guijianyu	96.28±2.94^#
750 mg/L Guijianyu	86.01±4.89^#
P	<0.001

表5 各给药组对AGEs诱导的MPC-5细胞活性的影响

Tab.5 Effect of Guijianyu on AGes-induced MPC-5 cell viability (%, Mean±SD, n=3)

Group	Cell viability
Normal	100±1.32
Model	67.78±5.06*
Guijianyu	88.66±2.39^#
RAGE agonist	48.38±7.00^##
Guijianyu+RAGE agonist	76.40±2.85**
P	<0.001

图3 各组细胞RAGE、NF-κB(p65)、VEGFA、TNF-α、IL-6、Caspase-3蛋白表达情况

Fig.3 Protein expressions of RAGE, NF-κB(p65), VEGFA, TNF-α, IL-6, and caspase-3 in MPC-5 cells in each group. 1: Normal group; 2: Model group; 3: Guijianyu group; 4: RAGE agonist group; 5: Guijianyu+RAGE agonist group.

表6 鬼箭羽对DKD模型MPC-5细胞中RAGE、VEGFA、TNF-α、NF-κB（p65）、IL-和Caspase-3蛋白表达的影响

Tab.6 Effects of Guijianyu on the protein expressions of RAGE, VEGFA, TNF-α, NF-κB (p65), IL-6 and Caspase-3 in MPC-5 cells in each group (Mean±SD, n=3)

Group	RAGE	NF-κB(p65)	VEGFA	TNF-α	IL-6	Caspase-3
1	1.00±0.05	1.00±0.05	1.00±0.05	1.00±0.05	1.00±0.05	1.00±0.05
2	2.01±0.17*	1.41±0.19*	1.55±0.27*	1.51±0.12**	1.53±0.14*	1.64±0.24*
3	1.14±0.31^#	0.91±0.14^#	1.15±0.16^#	1.06±0.14^#	1.16±0.14^#	1.20±0.25^#
4	2.11±0.29	1.17±0.13	1.57±0.29	1.37±0.23	1.37±0.07	1.81±0.13
5	0.96±0.56^##	0.99±0.24	1.11±0.12^##	0.96±0.39^##	1.01±0.20^##	1.13±0.14^##
P	0.002	0.023	0.015	0.039	0.002	0.001

表7 鬼箭羽对DKD模型MPC-5细胞中RAGE、NF-κB（p65）、IL-6、VEGFA mRNA表达的影响

Tab.7 Effect of Guijianyu on mRNA expressions of RAGE, NF-κB (p65), IL-6 and VEGFA in MPC-5 cells in each group (Mean±SD, n=3)

Group	RAGE	NF-κB (p65)	VEGFA	IL-6
1	1.00±0.07	1.00±0.05	1.00±0.11	1.00±0.11
2	4.44±0.40*	2.49±0.21*	1.68±0.11*	2.09±0.12*
3	1.30±0.20^#	1.15±0.07^#	1.13±0.30^#	1.11±0.05^#
4	5.04±0.88	2.64±0.15	2.77±0.08	3.62±1.19
5	1.48±0.40^##	1.36±0.24^##	1.31±0.27^##	1.34±0.25^##
P	<0.001	<0.001	<0.001	0.001

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