清热止血方联合雷公藤多苷对过敏性紫癜性肾炎血清源性多聚IgA诱导内皮损伤的保护作用及对NF-κB通路的影响

张霞; 徐闪闪; 王龙; 孙杨; 邸家琪; 代彦林; 丁樱

doi:10.3969/j.issn.1006-2157.2023.07.014

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清热止血方联合雷公藤多苷对过敏性紫癜性肾炎血清源性多聚IgA诱导内皮损伤的保护作用及对NF-κB通路的影响

实验研究 | 更新时间：2023-08-07

- 清热止血方联合雷公藤多苷对过敏性紫癜性肾炎血清源性多聚IgA诱导内皮损伤的保护作用及对NF-κB通路的影响
- Protective effect of Qingre Zhixue Formula combined with tripterygium wilfordii multiglucoside on endothelial injury induced by serum-derived poly IgA in Henoch-Schönlein purpura nephritis and effects on the NF-κB pathway
- 北京中医药大学学报 2023年46卷第7期页码：970-979
- 作者机构：
  
  1.河南中医药大学第一附属医院　郑州　450003
  2.河南中医药大学儿科医学院
  3.许昌市中医院
  4.杭州师范大学附属医院
- 作者简介：
  
  张霞，女，博士，主任医师
  # 丁樱，女，国医大师，教授，主任医师，博士生导师，主要研究方向：中医药防治小儿风湿免疫及肾脏疾病，E-mail：dingying3236@sina.com
- 基金信息：
  
  国家自然科学基金面上项目(81873343);河南省自然科学基金面上项目(222300420488);河南省科技研发计划联合基金项目(222301420078);河南省中医药科学研究专项(2019ZY1010)
- DOI：10.3969/j.issn.1006-2157.2023.07.014
  中图分类号： R285.5
- 纸质出版日期：2023-07-30，
  
  收稿日期：2022-12-31，
- 稿件说明：
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张霞, 徐闪闪, 王龙, 等. 清热止血方联合雷公藤多苷对过敏性紫癜性肾炎血清源性多聚IgA诱导内皮损伤的保护作用及对NF-κB通路的影响[J]. 北京中医药大学学报, 2023,46(7):970-979.

ZHANG Xia, XU Shanshan, WANG Long, et al. Protective effect of Qingre Zhixue Formula combined with tripterygium wilfordii multiglucoside on endothelial injury induced by serum-derived poly IgA in Henoch-Schönlein purpura nephritis and effects on the NF-κB pathway[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(7):970-979.
张霞, 徐闪闪, 王龙, 等. 清热止血方联合雷公藤多苷对过敏性紫癜性肾炎血清源性多聚IgA诱导内皮损伤的保护作用及对NF-κB通路的影响[J]. 北京中医药大学学报, 2023,46(7):970-979. DOI： 10.3969/j.issn.1006-2157.2023.07.014.

ZHANG Xia, XU Shanshan, WANG Long, et al. Protective effect of Qingre Zhixue Formula combined with tripterygium wilfordii multiglucoside on endothelial injury induced by serum-derived poly IgA in Henoch-Schönlein purpura nephritis and effects on the NF-κB pathway[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(7):970-979. DOI： 10.3969/j.issn.1006-2157.2023.07.014.

摘要

目的

探讨清热止血方联合雷公藤多苷对过敏性紫癜性肾炎患者血清源性多聚IgA诱导内皮损伤的保护作用及对NF-κB信号通路关键蛋白表达的影响。

方法

本研究共纳入就诊于河南中医药大学第一附属医院的80例过敏性紫癜性肾炎患者及38名健康对照者，分别收集血清，将血清IgA纯化，以制备血清源性多聚IgA。将60只SD大鼠按照随机数字表法分为清热止血方组(31.25 mg/kg)、雷公藤多苷组(46.88 mg/kg)、清热止血方联合雷公藤多苷组(31.25 mg/kg＋46.88 mg/kg)、醋酸泼尼松组(23.44 mg/kg)、生理盐水组，每组12只，灌胃2次/d，连续7 d。采用过敏性紫癜性肾炎患者血清源性多聚IgA(200 mg/L)刺激人脐静脉内皮细胞24 h，以构建人脐静脉内皮细胞损伤模型。将造模成功的一部分细胞分为模型组、清热止血方组、雷公藤多苷组、清热止血方联合雷公藤多苷组、醋酸泼尼松组及生理盐水组，另设空白组及健康组，并给予相应干预。另一部分造模成功的细胞采用siRNA转染抑制NF-κB信号通路，并将转染后的细胞分为模型＋转染组、清热止血方组、雷公藤多苷组、清热止血方联合雷公藤多苷组及醋酸泼尼松组，另设空白组、模型组、模型＋正常组，并给予相应干预。采用MTT法检测各组细胞活力，流式细胞术检测各组细胞凋亡率，蛋白质印迹法分别检测转染前后细胞磷酸化的核因子-P65(p-P65)、磷酸化的核因子-P50(p-P50)、核转录因子-κB激酶β(IKKβ)及核转录因子-κB抑制蛋白α(IκBα)的蛋白表达情况。

结果

与空白组比较，模型组细胞活力减弱(

0.05)；与模型组比较，各给药组细胞活力增强(

0.05)；与清热止血方联合雷公藤多苷组比较，清热止血方组、雷公藤多苷组、醋酸泼尼松组细胞活力减弱(

0.05)。与空白组比较，模型组细胞p-P65、p-P50及IKKβ蛋白的表达量升高(

0.05)，IκBα蛋白表达量降低(

0.05)。与模型组比较，各给药组细胞p-P65、p-P50及IKKβ蛋白表达量均降低(

0.05)；而IκBα的表达量升高(

0.05)。与清热止血方联合雷公藤多苷组比较，清热止血方组、雷公藤多苷组、醋酸泼尼松组p-P65、p-P50及IKKβ蛋白的表达量升高(

0.05)，而IκBα的表达量降低(

0.05)。siRNA转染后，与空白组比较，模型组、模型＋正常组细胞p-P65、p-P50及IKKβ蛋白的表达量升高(

0.05)，IκBα蛋白表达量降低(

0.05)。与模型组比较，模型＋转染组、清热止血方组、雷公藤多苷组、清热止血方联合雷公藤多苷组、醋酸泼尼松组细胞p-P65、p-P50及IKKβ蛋白表达量降低(

0.05)，IκBα蛋白表达量升高(

0.05)。

结论

清热止血方联合雷公藤多苷可能是通过抑制NF-κB通路的活化，进而提高过敏性紫癜性肾炎患者血清源性多聚IgA诱导的人脐静脉内皮细胞的活力，以发挥对内皮细胞的保护作用。

Abstract

Objective

We aimed to explore the protective effect of

Qingre Zhixue

Formula and tripterygium wilfordii multiglucoside on the endothelial damage induced by serum-derived poly IgA in patients with Henoch-Schönlein purpura nephritis and its effect on the expression of key proteins of the NF-κB signaling pathway.

Methods

A total of 80 patients with Henoch-Schönlein purpura nephritis from The First Affliated Hospital of Henan University of Chinese Medicine and 38 healthy controls were enrolled in this study. Serum IgA was purified from all participants in order to prepare serum-derived poly IgA. Moreover

60 Sprague-Dawley rats were divided into the following five groups (

=12 rats per group): the

Qingre Zhixue

Formula group (31.25 mg/kg)

the tripterygium wilfordii multiglucoside group (46.88 mg/kg)

the combined group (31.25 mg/kg + 46.88 mg/kg)

the prednisone acetate group (23.44 mg/kg) and the saline group. Rats in each group were gavaged twice a day for 7 days. Additionally

human umbilical vein endothelial cells (HUVECs) were stimulated with serum-derived poly IgA (200 μg/mL) derived from patients with Henoch-Schönlein purpura nephritis for 24 h to construct HUVECs injury model. The cells were divided into the following seven groups: the blank group

the healthy group

the model group

the

Qingre Zhixue

Formula group

the tripterygium wilfordii multiglucoside group

the combined group and the prednisone acetate group

and provide corresponding intervention. siRNA transfection was used to inhibit the NF-κB signaling pathway

and divide the transfected cells into the blank group

the model group

the model+ normal control group

the model+ transfection group

the

Qingre Zhixue

Formula group

the tripterygium wilfordii multiglucoside group

the combined group and the prednisone acetate group. Cell viability was detected by the MTT assay

the apoptosis rate was detected by flow cytometry

and the protein expression levels of p-P65

p-P50

IKKβ

and IκBα were detected by Western blotting before and after transfection.

Results

Compared with the blank group

the cell viability of the model group was decreased (

0.05). Compared with the model group

the cell viability of each treatment group was increased (

0.05). Compared with the combined group

the cell viability of the

Qingre Zhixue

Formula group

the tripterygium wilfordii multiglucoside group

and the prednisone acetate group was decreased (

0.05). Compared with the blank group

the protein expression levels of p-P65

p-P50 and IKKβ in the model group were increased (

0.05)

and the protein expression level of IκBα was decreased (

0.05). Compared with the model group

the protein expression levels of p-P65

p-P50 and IKKβ were decreased in each group (

0.05)

and the protein expression level of IκBα was increased (

0.05). Compared with the combined group

the protein expression levels of p-P65

p-P50

and IKKβ in the

Qingre Zhixue

Formula group

the tripterygium wilfordii multiglucoside group and the prednisone acetate group were increased (

0.05)

while the protein expression level of IκBα was decreased (

0.05). After siRNA transfection

compared with the blank group

the protein expression levels of p-P65

p-P50 and IKKβ in the model group and the model+ normal control group were increased (

0.05)

and the protein expression level of IκBα was decreased (

0.05). Compared with the model group

the protein expression levels of p-P65

p-P50 and IKKβ were decreased (

0.05)

and the protein expression level of IκBα was increased (

0.05) in the cells of the model+ transfection group

the

Qingre Zhixue

Formula group

the tripterygium wilfordii multiglucoside group

the combined group and the prednisone acetate group.

Conclusion

Qingre Zhixue

Formula combined with tripterygium wilfordii multiglucoside may inhibit the activation of the NF-κB pathway to improve the cell viability of HUVECs induced by serum-derived poly IgA in children with Henoch-Schönlein purpura nephritis

so it plays a role in protecting endothelial cells.

关键词

过敏性紫癜性肾炎清热止血方雷公藤多苷人脐静脉内皮细胞核转录因子－κB通路

Keywords

Henoch-Schönlein purpura nephritisQingre Zhixue Formulatripterygium wilfordii multiglucosidehuman umbilical vein endothelial cellsNF-κB pathway

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