Protective effects of Shenqi Granules and its component formulas on injured podocytes

WU Yansheng; GUO Xiaoping; WEI Lifeng; XU Rui; WANG Lin

doi:10.16305/j.1007-1334.2024.z20240610004

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Protective effects of Shenqi Granules and its component formulas on injured podocytes

更新时间：2024-12-23

- Protective effects of Shenqi Granules and its component formulas on injured podocytes
- Shanghai Journal of Traditional Chinese Medicine Vol. 58, Issue 12, Pages: 153-162(2024)
- 作者机构：
  
  上海中医药大学附属龙华医院肾病科（上海 200032）
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2024.z20240610004
  CLC：
- Published：10 December 2024，
  
  Received：10 June 2024，
- 稿件说明：
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吴燕升,郭小平,魏丽凤,等.参芪颗粒及其拆方对足细胞损伤的保护作用[J].上海中医药杂志,2024,58(12):153-162.

WU Yansheng,GUO Xiaoping,WEI Lifeng,et al.Protective effects of Shenqi Granules and its component formulas on injured podocytes[J].Shanghai Journal of Traditional Chinese Medicine,2024,58(12):153-162.
吴燕升,郭小平,魏丽凤,等.参芪颗粒及其拆方对足细胞损伤的保护作用[J].上海中医药杂志,2024,58(12):153-162. DOI： 10.16305/j.1007-1334.2024.z20240610004.

WU Yansheng,GUO Xiaoping,WEI Lifeng,et al.Protective effects of Shenqi Granules and its component formulas on injured podocytes[J].Shanghai Journal of Traditional Chinese Medicine,2024,58(12):153-162. DOI： 10.16305/j.1007-1334.2024.z20240610004.

摘要

目的

观察参芪颗粒及其拆方［益气方、活血方、清热化湿方（以下简称“化湿方”）］对足细胞损伤的保护作用，探寻各拆方的作用靶点，为参芪颗粒各成分的有效性提供现代药理学依据。

方法

采用细胞计数试剂盒（CCK-8）检测不同浓度嘌呤霉素氨基核苷（PAN）对小鼠肾足细胞MPC-5存活率的影响。选择合适浓度PAN刺激MPC-5的同时，用不同浓度参芪颗粒及益气方、活血方、化湿方，干预细胞24 h后，根据CCK-8检测得出各中药处理组药物浓度，将细胞分为10组，分别为：正常对照组、模型组、环孢素组、参芪颗粒组、益气方组、活血加化湿方组、活血方组、益气加化湿方组、化湿方组、益气加活血方组。干预24 h后，通过细胞划痕试验检测CCK-8检测获得的最佳浓度拆方对MPC-5细胞划痕愈合的作用。进一步以Western blot法检测足细胞黏附蛋白（nephrin）、p-nephrin（Tyr1217）的表达水平；免疫荧光检测足细胞nephrin及F-肌动蛋白（F-actin）的表达水平；透射电镜观察足细胞形态、细胞器、细胞骨架等超微结构变化，研究参芪颗粒对PAN诱导的损伤足细胞MPC-5的保护作用，以环孢素作为阳性对照，将不同治则进行组合，明确益气、活血、清热化湿与足细胞增殖、骨架蛋白表达、细胞骨架结构的关系。

结果

①CCK-8检测发现，PAN以时间和浓度依赖的方式降低足细胞的存活率，在所试验的浓度范围内，益气方和活血方对足细胞存活率没有影响，参芪颗粒的细胞毒性主要是来自化湿方。25 mg/L PAN为造模最佳浓度，0.5 g/L参芪颗粒、0.21 g/L 益气方、0.09 g/L 活血方、0.2 g/L化湿方为各药物干预最佳浓度。②划痕试验显示，与模型组比较，参芪颗粒及其拆方促进了足细胞的增殖和迁移（

0.05）。治疗组之间，益气方与活血加化湿方比较，化湿方组与益气加活血方组比较，伤口愈合面积无明显差异，但益气加化湿方在促进伤口愈合方面优于单独的活血方（

0.05）。③参芪颗粒及其拆方组可以提高nephrin、p-nephrin的表达水平。其中益气方组nephrin、p-nephrin表达高于活血加化湿方组（

0.05）；益气加化湿方组nephrin、p-nephrin的表达高于活血方（

0.05），化湿方组nephrin、p-nephrin表达与益气加活血方组比较差异无统计学意义（

0.05）。④参芪颗粒及其拆方可防止肌动蛋白细胞骨架的破坏；其中参芪颗粒组与拆方组之间的平均荧光强度差异无统计学意义（

0.05）。⑤参芪颗粒及其拆方可保持足细胞结构的完整性，减少足突消失；参芪颗粒及其拆方的应用使足细胞细胞质中足突、线粒体和溶酶体的数量增加，维持了细胞活力、细胞骨架和细胞超微结构稳定。

结论

参芪颗粒及其拆方可促进足细胞增殖和迁移，提高nephrin、p-nephrin的表达水平，保护激动蛋白细胞骨架，保持足细胞结构的完整性、增加足细胞线粒体和溶酶体的数量、减少足细胞足突消失。且全方组效果最佳，在拆方组中益气、化湿组的效果较好。

Abstract

Objective

To observe the protective effects of Shenqi Granules and its decomposed formulas［Yiqi Formula， Huoxue Formula， and Qingre Huashi Formula （Huashi Formula）］ on injured podocytes， explore their targets， and provide modern pharmacological evidence for the efficacy of each component of Shenqi Granules.

Methods

The Cell Counting Kit-8 （CCK-8） assay was used to evaluate the effects of different concentrations of puromycin aminonucleoside （PAN） on the viability of mouse podocyte clone 5 （MPC-5）. Following the determination of the optimal PAN concentration for stimulating MPC-5 cells， the cells were treated with varying concentrations of Shenqi Granules， Yiqi Formula， Huoxue Formula， and Huashi Formula for 24 h. Based on the CCK-8 assay results， the appropriate drug concentrations for each treatment group were determined， and the cells were divided into 10 groups： normal control group， model group， cyclosporine group， Shenqi Granules group， Yiqi Formula group， Huoxue plus Huashi Formula group， Huoxue Formula group， Yiqi plus Huashi Formula group， Huashi Formula group， and Yiqi plus Huoxue Formula group. After 24 hours of incubation， a scratch assay was performed to assess the effects of the optimal concentrations of the component formulas， as determined by the CCK-8 assay， on the scratch wound healing of MPC-5. Further analysis was conducted using Western blot to detect the expression levels of podocyte adhesion protein （nephrin） and phosphorylated nephrin （p-nephrin Tyr1217）. Immunofluorescence was used to assess the expression of nephrin and F-actin in podocytes. Transmission electron microscopy was used to observe ultrastructural changes in podocytes， including morphology， organelles， and cytoskeleton. These methods were used to investigate the protective effects of Shenqi Granules on PAN-induced podocyte MPC-5 injury， with cyclosporine serving as a positive control. Combinations of different therapeutic principles were tested to clarify the relationships between benefiting qi， activating blood circulation， clearing heat， resolving dampness， and their effects on podocyte proliferation， cytoskeletal protein expression， and cytoskeletal structure.

Results

①The CCK-8 assay revealed that PAN decreased podocyte viability in a time- and concentration-dependent manner. Within the tested concentration range， Yiqi Formula and Huoxue Formula had no significant effect on podocyte viability， while the cytotoxicity of Shenqi Granules was primarily attributed to its Huashi Formula component. The optimal concentration for modeling was determined to be 25 mg/L PAN， with optimal intervention concentrations of 0.5 g/L Shenqi Granules， 0.21 g/L Yiqi Formula， 0.09 g/L Huoxue Formula， and 0.2 g/L Huashi Formula. ②The scratch assay showed that， compared to the model group， Shenqi Granules and its component formulas promoted podocyte proliferation and migration （

0.05）. Among the treatment groups， there was no significant difference in wound healing area between the Yiqi Formula and the Huoxue plus Huashi Formula， or between the Huashi Formula and the Yiqi plus Huoxue Formula. However， the Yiqi plus Huashi Formula was more effective in promoting wound healing than the Huoxue Formula alone （

0.05）. ③Shenqi Granules and its component formulas could increase the expression levels of nephrin and p-nephrin. Notably， the Yiqi Formula group showed higher nephrin and p-nephrin expression than the Huoxue plus Huashi Formula group （

0.05）. Additionally， the Yiqi plus Huashi Formula group had higher nephrin and p-nephrin expression than the Huoxue Formula group （

0.05）. However， there was no statistically significant difference in nephrin and p-nephrin expression between the Huashi Formula group and the Yiqi plus Huoxue Formula group （

0.05）. ④Shenqi Granules and its component formulas were able to prevent the destruction of the actin cytoskeleton. There was no statisti

cally significant difference in the mean fluorescence intensity between the Shenqi Granules group and the component formula groups （

0.05）. ⑤Shenqi Granules and its component formulas maintained the structural integrity of podocytes and reduced foot process effacement. The application of Shenqi Granules and its component formulas resulted in an increase in the number of foot processes， mitochondria， and lysosomes in the podocyte cytoplasm， preserving cell viability， cytoskeletal integrity， and ultrastructural stability.

Conclusions

Shenqi Granules and its component formulas can promote podocyte proliferation and migration， increase the expression levels of nephrin and p-nephrin， protect the actin cytoskeleton， maintain the structural integrity of podocytes， increase the number of mitochondria and lysosomes， and reduce foot process effacement. Among the groups， the complete formula （i.e.， Shenqi Granules） showed the best effects， with the Yiqi and Huashi Formulas performing better among the component groups.

关键词

特发性膜性肾病足细胞损伤细胞骨架参芪颗粒中药研究

Keywords

idiopathic membranous nephropathypodocyte injurycytoskeletonShenqi Granulestraditional Chinese herbal medicine research

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