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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。
Objective: To observe the effect of medicated serum of Hei Dihuangwan (MSHDW) on autophagy and paracrine effect of bone marrow stromal cells (BMSCs) in renal anemia rats and to explore the effect of Hei Dihuangwan Dihuangwan, HDW) possible mechanism of treatment of renal anemia. Methods: BMSCs were isolated from normal and renal anemic rats by gradient density centrifugation and adherent culture. The third generation of cells were induced by starvation for 1 h, then divided into 5 groups according to different treatment: normal group , Model group, HDW high, medium and low dose groups. Cell viability was detected by MTT assay. Autophagosomes were observed by electron microscopy. The expression of LC3Ⅰ, LC3Ⅱ and Beclin-1 mRNA were detected by real-time PCR. (SCF), erythropoietin (EPO), and insulin-like growth factor (IGF-1) levels in canines. The correlation between autophagy genes and hematopoietic factors was investigated by canonical correlation analysis. Results: Compared with normal group, the survival rate of BMSCs in model group was significantly lower than that in normal group (P <0.01), while the survival rate of model group was higher (P <0.05, P <0.01) Under electron microscope, autophagosomes in model group were more than those in normal group. Autophagosomes in HDW medium, medium and low dose groups were less than those in model group. Autophagy-related gene mRNA level in model group was higher than that in normal group (P <0.01). The mRNA level of autophagy-related genes in high-dose and high-dose HDW groups was lower than that in model group (P <0.05, P <0.01). The level of hematopoietic factor in supernatant of BMSCs in model group was lower than that in normal group The levels of hematopoietic factors in the high and middle dose groups were higher than those in the model group (P <0.05, P <0.01), and the higher the dose, the stronger the effect. The typical correlation analysis showed that under MSHDW, the autophagy gene level and the inhibition of paracrine effect Highly relevant. Conclusion: The mechanism of HDW in the treatment of renal anemia may be related to the down-regulation of autophagy and the promotion of paracrine hematopoietic factors in BMSCs.