Antagonistic Effect of Salidroside on Podocyte Pyroptosis in Diabetic Kidney Disease Rats under Hypoxia Based on NLRP3/IL-1β/TGF-β1 Pathway

doi:10.12114/j.issn.1007-9572.2023.0678

Abstract

Abstract:

Background

Salidroside has been shown to protect diabetic kidney disease (DKD) rats, however, whether it is equally effective in a hypoxic environment and the specific mechanism of action remain unclear.

Objective

To observe the effects of salidroside on biochemical parameters, renal tissue pathological lesion, and the expression of cell pyroptosis-related proteins in a rat model of DKD under hypoxia, and explore its mechanisms of action.

Methods

From March 2022 to March 2023, forty 6-week-old SPF-grade SD male rats were used, with eight randomly selected as the control group, the remaining were modeled. Twenty-four DKD model rats were randomly divided into three groups of the model group, salidroside group, and salidroside+nod-like receptor protein 3 (NLRP3) activator group for intervention, with 8 in each group. After the intervention, blood was collected from the abdominal aorta for biochemical parameter testing, hematoxylin-eosin (HE) staining, and transmission electron microscopy were used to observe renal pathological changes. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of interleukin (IL) 1β and IL-18. Western blotting was used to measure the expression levels of Caspase-1, Gasdermin D (GSDMD), NLRP3, and transforming growth factor β1 (TGF-β1) in renal tissue.

Results

The body weight of the rats after modeling was significantly lower than that of the control group (P<0.05). Compared to the control group, the levels of triglyceride (TG), total cholesterol (TC), fasting blood glucose (FBG), urinary microalbumin (UMA), blood urea nitrogen (BUN), and serum creatinine (Scr) were significantly higher in the model group (P<0.05). Compared to the model group, the BUN, UMA, and Scr levels were significantly lower in the salidroside group (P<0.05). Compared to the salidroside group, the UMA, BUN, and Scr levels were significantly higher in the salidroside+NLRP3 activator group (P<0.05). HE staining and transmission electron microscopy revealed that renal tissue pathological changes in the salidroside group were significantly reduced than the model group, and aggravated in the salidroside+NLRP3 activator group. Compared to the control group, serum IL-1β and IL-18 levels were significantly higher in the model group (P<0.05) ; these levels were significantly lower in the salidroside group compared to the model group (P<0.05), and higher in the salidroside+NLRP3 activator group compared to the salidroside group (P<0.05). Compared to the control group, the expression of Caspase-1, GSDMD, NLRP3, and TGF-β1 proteins was significantly higher in the model group (P<0.05) ; it was significantly lower in the salidroside group compared to the model group (P<0.05), and higher in the salidroside+NLRP3 activator group compared to the salidroside group (P<0.05) .

Conclusion

Salidroside exerted therapeutic effects on DKD rats in a hypoxic environment without reducing blood glucose and lipid levels, this effect may be related to the inhibition of NLRP3, affecting the NLRP3/IL-1β/TGF-β1 signaling pathway, ultimately improving podocyte pyroptosis injury.

Key words: Diabetic kidney disease, Pyroptosis, Salidroside, Hypoxia, Rat, Podocytes

摘要：

背景

红景天苷对糖尿病肾病（DKD）大鼠具有保护作用，但在低氧环境下是否同样起效及具体作用机制尚不明确。

目的

观察低氧环境下红景天苷对DKD大鼠模型血生化指标、肾组织病理损伤、肾脏细胞焦亡相关蛋白表达情况，并探讨其作用机制。

方法

2022年3月—2023年3月40只6周龄SPF级SD雄性大鼠，随机抽取8只作为对照组，其余大鼠造模。将24只DKD成模大鼠随机分为模型组、红景天苷组、红景天苷+Nod样受体蛋白3（NLRP3）激活剂组进行干预，每组8只。干预结束后腹主动脉取血进行生化指标检测，苏木素-伊红（HE）染色及透射电镜观察大鼠肾脏病理改变情况，酶联免疫吸附试验（ELISA）检测大鼠血清白介素（IL）1β、IL-18水平。免疫印迹试验（Western bloting）检测肾组织半胱天冬酶1（Caspase-1）、消皮素D（GSDMD）、NLRP3和转化生长因子β1（TGF-β1）蛋白表达水平。

结果

造模后造模大鼠体质量低于对照大鼠，差异有统计学意义（P<0.05）。与对照组相比，模型组大鼠三酰甘油（TG）、总胆固醇（TC）、空腹血糖（FBG）、尿微量白蛋白（UMA）、尿素氮（BUN）、血肌酐（Scr）水平明显升高（P<0.05）；与模型组相比，红景天苷组大鼠BUN、UMA、Scr水平明显降低（P<0.05）；与红景天苷组相比，红景天苷+NLRP3激活剂组大鼠UMA、BUN、Scr水平明显升高（P<0.05）。HE染色及透射电镜结果可见红景天苷组大鼠肾脏组织病理改变较模型组明显减轻，红景天苷+NLRP3激活剂组较红景天苷组加重。与对照组相比，模型组血清IL-1β、IL-18水平明显升高（P<0.05）；与模型组相比，红景天苷组大鼠血清IL-1β、IL-18水平明显降低（P<0.05）；与红景天苷组相比，红景天苷+NLRP3激活剂组IL-1β、IL-18水平明显升高（P<0.05）。与对照组相对，模型组Caspase-1、GSDMD、NLRP3、TGF-β1蛋白表达明显升高（P<0.05）；与模型组相对，红景天苷组Caspase-1、GSDMD、NLRP3、TGF-β1蛋白表达明显降低（P<0.05）；与红景天苷组相对，红景天苷+NLRP3激活剂组Caspase-1、GSDMD、NLRP3、TGF-β1蛋白表达明显升高（P<0.05）。

结论

低氧环境下，红景天苷以不降低血糖和血脂为前提发挥了对DKD大鼠的治疗作用，考虑该作用可能与抑制NLRP3从而影响NLRP3/IL-1β/TGF-β1信号通路，最终改善足细胞焦亡损伤密切相关。

关键词: 糖尿病肾病, 细胞焦亡, 红景天苷, 低氧, 大鼠, 足细胞

LI Jiawu,QIN Feng,SONG Shengqin, et al. Antagonistic Effect of Salidroside on Podocyte Pyroptosis in Diabetic Kidney Disease Rats under Hypoxia Based on NLRP3/IL-1β/TGF-β1 Pathway[J]. Chinese General Practice, 2024, 27(21): 2617-2622. DOI: 10.12114/j.issn.1007-9572.2023.0678.
李佳武,秦凤,宋生琴等. 基于NLRP3/IL-1β/TGF-β1通路探讨低氧环境下红景天苷对糖尿病肾病大鼠足细胞焦亡损伤的拮抗效应[J]. 中国全科医学, 2024, 27(21): 2617-2622. DOI: 10.12114/j.issn.1007-9572.2023.0678.

Figures/Tables 7

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组别	只数	造模前	造模后	t_配对值	P值
对照大鼠	8	170.4±7.8	439.7±14.1	-53.99	<0.05
造模大鼠	24	173.0±6.6	328.7±17.7	-44.86	<0.05
t值		-0.95	16.08
P值		0.35	<0.05

组别	只数	造模前	造模后	t_配对值	P值
对照大鼠	8	170.4±7.8	439.7±14.1	-53.99	<0.05
造模大鼠	24	173.0±6.6	328.7±17.7	-44.86	<0.05
t值		-0.95	16.08
P值		0.35	<0.05

组别	只数	TC（mmol/L）	TG（mmol/L）	FBG（mmol/L）	UMA（mg/mmol）	BUN（mmol/L）	Scr（μmol/L）
对照组	8	4.44±0.63	2.61±0.48	4.88±0.61	1.67±0.41	3.55±1.11	63.38±14.69
模型组	8	9.81±1.54^a	9.81±1.65^a	22.29±2.64^a	5.64±0.58^a	11.60±2.24^a	291.00±29.52^a
红景天苷组	8	8.77±1.14	9.29±1.31	20.66±2.35	3.68±0.50^b	7.75±1.50^b	173.13±18.84^b
红景天苷+NLRP3激活剂组	8	8.14±0.59	9.80±1.60	21.18±1.81	5.30±0.64^c	11.96±2.10^c	272.75±24.81^c
F值		41.21	54.91	333.11	100.16	38.42	171.18
P值		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

组别	只数	TC（mmol/L）	TG（mmol/L）	FBG（mmol/L）	UMA（mg/mmol）	BUN（mmol/L）	Scr（μmol/L）
对照组	8	4.44±0.63	2.61±0.48	4.88±0.61	1.67±0.41	3.55±1.11	63.38±14.69
模型组	8	9.81±1.54^a	9.81±1.65^a	22.29±2.64^a	5.64±0.58^a	11.60±2.24^a	291.00±29.52^a
红景天苷组	8	8.77±1.14	9.29±1.31	20.66±2.35	3.68±0.50^b	7.75±1.50^b	173.13±18.84^b
红景天苷+NLRP3激活剂组	8	8.14±0.59	9.80±1.60	21.18±1.81	5.30±0.64^c	11.96±2.10^c	272.75±24.81^c
F值		41.21	54.91	333.11	100.16	38.42	171.18
P值		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

组别	只数	IL-1β	IL-18
对照组	8	10.18±1.90	20.85±2.70
模型组	8	40.57±4.01^a	87.79±4.24^a
红景天苷组	8	13.97±2.41^b	32.52±3.67^b
红景天苷+NLRP3激活剂组	8	33.08±5.19^c	73.89±7.30^c
F值		141.90	497.18
P值		<0.001	<0.001