Discussion on Effect of Zuoguiwan on Skin Development of Kidney-deficient Rats Based on Autophagy

Ji-gang CAO; Ying LIU; Hong-ling SANG; Min ZHAO

doi:10.13422/j.cnki.syfjx.20210502

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Discussion on Effect of Zuoguiwan on Skin Development of Kidney-deficient Rats Based on Autophagy

Classic Prescriptions | 更新时间：2021-03-29

- Discussion on Effect of Zuoguiwan on Skin Development of Kidney-deficient Rats Based on Autophagy
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 8, Pages: 1-7(2021)
- 作者机构：
  
  湖北中医药大学，武汉 430065
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20210502
  CLC： R2-0;R22;R285.5;R289
- Received：27 November 2020，
  
  Published Online：26 February 2021，
  
  Published：20 April 2021
- 稿件说明：
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曹继刚,刘颖,桑红灵等.基于细胞自噬探讨左归丸对肾虚仔鼠皮肤发育的影响[J].中国实验方剂学杂志,2021,27(08):1-7.

CAO Ji-gang,LIU Ying,SANG Hong-ling,et al.Discussion on Effect of Zuoguiwan on Skin Development of Kidney-deficient Rats Based on Autophagy[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(08):1-7.
曹继刚,刘颖,桑红灵等.基于细胞自噬探讨左归丸对肾虚仔鼠皮肤发育的影响[J].中国实验方剂学杂志,2021,27(08):1-7. DOI： 10.13422/j.cnki.syfjx.20210502.

CAO Ji-gang,LIU Ying,SANG Hong-ling,et al.Discussion on Effect of Zuoguiwan on Skin Development of Kidney-deficient Rats Based on Autophagy[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(08):1-7. DOI： 10.13422/j.cnki.syfjx.20210502.

摘要

目的

研究左归丸对先天肾虚仔鼠发育过程中皮肤内细胞自噬的影响及其机制，探索补肾填精法在生长发育异常中的应用价值。

方法

36只雌雄比例为2∶1的大鼠进行配对，复合应激法刺激孕鼠构建先天肾虚仔鼠模型，根据将孕鼠处理方式不同，分为正常组，肾虚组，左归丸低、高剂量组（2，8 g·kg

-1

）。正常组不造模给予生理盐水灌胃，肾虚组造模给予生理盐水灌胃，左归丸低、高剂量组造模给予左归丸悬浊液灌胃。切取出生后21 d仔鼠的背部皮肤，苏木素-伊红（HE）染色观察仔鼠皮肤显微结构形态，测量真皮层和表皮层厚度，蛋白免疫印迹法（Western blot）和实时荧光定量聚合酶链式反应（Real-time PCR）分别检测仔鼠皮肤分泌型糖蛋白3a（Wnt3a）和

-连环蛋白（

-catenin） mRNA和蛋白表达量，免疫双荧光检测皮肤内自噬指标微管相关蛋白轻链3（LC3）和人死骨片1-泛素结合蛋白p62（SQSTM1-p62）的表达。

结果

正常组仔鼠皮肤层次清楚，结构正常，肾虚组真皮层胶原排列疏松，左归丸低、高剂量组皮肤组成基本正常；与正常组比较，肾虚组仔鼠在皮肤的表皮层增厚，真皮层变薄（

0.05）；与肾虚组比较，左归丸低、高剂量组皮肤的表皮层和真皮层厚度的改变明显恢复（

0.05）。与正常组比较，肾虚组仔鼠Wnt3a和

-catenin蛋白和mRNA表达明显下降（

0.05）；与肾虚组比较，左归丸低、高剂量组Wnt3a和

-catenin蛋白和mRNA表达升高（

0.05）。同时免疫荧光法检测发现与正常组比较，肾虚组的LC3的表达上调，SQSTM1-p62表达下调；与肾虚组比较，左归丸可以逆转SQSTM1-p62和LC3的异常表达（

0.05）。

结论

左归丸可以通过增加仔鼠皮肤内Wnt3a和

-catenin蛋白和mRNA的表达，下调皮肤细胞自噬水平，改善先天肾虚仔鼠皮肤异常发育。

Abstract

Objective

To study the effect and mechanism of Zuoguiwan on the autophagy of cells in the skin during the development of congenital kidney-deficient mice， and to explore the application value of kidney tonifying and essence filling method in abnormal growth and development.

Method

36 rats with a 2∶1 ratio of male to female were paired， and the pregnant rats were stimulated by combined stress method to build a model of congenital kidney-deficient rats. According to different treatment methods， the pregnant rats were divided into the control group， the kidney-deficient group， and the Zuoguiwan low and high dose groups （2， 8 g·kg

-1

）. The control group was given normal saline without modeling. Kidney deficiency group was modeled before intragastric administration of normal saline. Zuoguiwan small dose and high dose groups received intragastric administration of Zuoguiwan suspension. 21 days after the birth of mice， the back skin was taken to observe the skin microstructure and morphology of the mice by HE staining and detect the thickness of dermis and epidermis. The gene and protein expression levels of Wingless3a and

-catenin in the skin of neonatal rats were measured by Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Double immunofluorescence assay was used to detect the skin autophagy indicators Microtubule-associated protein light chain 3 （LC3）and Sequestosome 1 （SQSTM1-p62）.

Result

In the control group， the skin layers were clear and the structure was normal， in the kidney deficiency group， the collagen arrangement in the dermis was loose， the skin composition in Zuoguiwan low and high dose groups was basically normal. Compared with the control group， the epidermal layer of the kidney deficiency group was thickened and the dermis was thinned （

0.05）. Compared with the kidney deficiency group， the thickness changes of epidermal layer and dermis layer were significantly restored in Zuoguiwan low and high dose groups （

0.05）. Compared with the control group， the expression of Wnt3a and

-catenin protein and gene in the kidney deficiency group significantly decreased （

0.05）. Compared with the kidney deficiency group， Wnt3a and

-catenin protein and gene expression increased in Zuoguiwan low and high dose groups （

0.05）. At the same time， immunofluorescence assay showed that compared with the control group， the expression of LC3 was up-regulated and the expression of SQSTM1-p62 was down-regulated in the kidney deficiency group， while Zuoguiwan could reverse such abnormal expression （

0.05）.

Conclusion

Zuoguiwan can down-regulate the autophagy level of skin cells and improve the abnormal skin development of congenital renal deficiency by increasing the expression of Wnt3a and

-catenin protein and gene in the skin of neonatal rats.

关键词

Keywords

references

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