Explore Mechanism of Hei Dihuangwan in Treating Renal Anemia Based on Autophagy and Paracrine Secretion

ZHAO Ping; LIU Wei-wei; ZHANG Liang; ZHANG Fa-rong

doi:10.13422/j.cnki.syfjx.2017020146

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Explore Mechanism of Hei Dihuangwan in Treating Renal Anemia Based on Autophagy and Paracrine Secretion

更新时间：2024-01-04

- Explore Mechanism of Hei Dihuangwan in Treating Renal Anemia Based on Autophagy and Paracrine Secretion
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 23, Issue 2, Pages: 146-152(2017)
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- DOI：10.13422/j.cnki.syfjx.2017020146
  CLC： R285.5
- Published：2017
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ZHAO Ping, LIU Wei-wei, ZHANG Liang, et al. Explore Mechanism of Hei Dihuangwan in Treating Renal Anemia Based on Autophagy and Paracrine Secretion[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(2): 146-152.
DOI：

ZHAO Ping, LIU Wei-wei, ZHANG Liang, et al. Explore Mechanism of Hei Dihuangwan in Treating Renal Anemia Based on Autophagy and Paracrine Secretion[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(2): 146-152. DOI： 10.13422/j.cnki.syfjx.2017020146.

摘要

目的：观察黑地黄丸含药血清（medicated serum of Hei Dihuangwan，MSHDW）对肾性贫血大鼠骨髓基质细胞（bone marrow stromal cells，BMSCs）自噬和旁分泌作用的影响，探讨黑地黄丸（Hei Dihuangwan，HDW）治疗肾性贫血的可能作用机制。方法：建立肾性贫血大鼠模型，梯度密度离心和贴壁培养法分离正常和肾性贫血大鼠BMSCs，取第3代细胞饥饿诱导1 h后予不同处理分为5组，分别为正常组，模型组，HDW高、中、低剂量组。MTT法检测细胞存活率，电子显微镜观察自噬小体，实时荧光定量聚合酶链式反应（Real-time PCR）法测定自噬相关基因LC3Ⅰ，LC3Ⅱ，Beclin-1 mRNA表达，ELISA法检测培养液上清干细胞因子（SCF），促红细胞生成素（EPO），胰岛素样生长因子（IGF-1）水平，典型相关分析研究自噬基因与造血因子之间的相关性。结果：与正常组比较，模型组BMSCs存活率明显降低（P<0.01），HDW高、中、低剂量组较模型组细胞存活率升高（P < 0.05，P < 0.01），剂量越高作用越强；电镜下观察，模型组自噬小体多于正常组，HDW高、中、低剂量组自噬小体均少于模型组；模型组自噬相关基因mRNA水平高于正常组（P<0.01），HDW高、中剂量组自噬相关基因mRNA水平低于模型组（P < 0.05，P < 0.01）；模型组BMSCs培养上清造血因子水平低于正常组（P<0.01），HDW高、中剂量组造血因子水平高于模型组（P < 0.05，P < 0.01），剂量越高作用越强；典型相关分析显示，在MSHDW作用下，BMSCs自噬基因水平与抑制旁分泌作用呈高度正相关。结论： HDW治疗肾性贫血的作用机制可能与下调BMSCs自噬水平和促进旁分泌造血因子有关。

Abstract

Objective: To investigate the effects of medicated serum of Hei Dihuangwan (MSHDW) on autophagy and paracrine secretion in bone marrow stromal cells (BMSCs) and explore the mechanism of Hei Dihuangwan (HDW) on renal anemia. Method: Rat models of renal anemia were established

and then normal and renal anemia BMSCs were separated by using gradient density centrifugation and adherent culture method. The third generation BMSCs were used and after starvation induction for 1 h

they were divided into normal group

model group

HDW high dose

middle dose and low dose groups. Cell survival rate was detected by MTT method; electron microscopy was used to observe the autophagosomes; the mRNA expression levels of LC3Ⅰ

LC3Ⅱ

and Becline-1 were determined by Real-time PCR. ELISA was used to detect stem cell factor (SCF)

erythropoietin (EPO)

and insulin-like growth factor (IGF)-1 in culture supernatant. Finally

the correlation between autophagy genes and hematopoietic factors were investigated by using canonical correlation analysis. Result: As compared with normal group

BMSCs survival rate was significantly decreased in model group (P<0.01); the cell survival rates in HDW high dose

middle dose and low dose groups were higher than that in model group (P < 0.05

P < 0.01)

and the effect was enhanced with the increase of dose. Under the electron microscope

the number of autophagosomes in model group was larger than that in normal group; and the number in HDW various groups was all smaller than that in model group; mRNA expression level of autophagy related genes in model group was higher than that in the normal group (P<0.01); mRNA expression levels of autophagy related genes in HDW high dose and middle dose groups were lower than those in model group (P < 0.05

P < 0.01). Hematopoietic factors in culture supernatant of model group BMSCs were less than those in normal group (P<0.01); the levels in HDW high dose and middle dose groups were higher than those in model group (P < 0.05

P < 0.01)

and the effect was enhanced with the increase of dose. Canonical correlation analysis showed that

autophagy genes of BMSCs were highly positively correlated to inhibition of paracrine secretion under MSHDW effect. Conclusion: The mechanism of HDW in treating renal anemia may be related to down-regulating the levels of autophagy in BMSCs and promoting paracrine secretion hematopoietic factors.

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