淫羊藿多糖对再生障碍性贫血骨髓造血功能与Th17/Treg平衡的影响及其相关机制

Effect and Mechanism of Epimedium Polysaccharide on Bone Marrow Hematopoietic Function and Th17/Treg Balance in Aplastic Anemia

目的:观察淫羊藿多糖(EPS)对再生障碍性贫血(AA)小鼠骨髓造血功能及Th17/Treg平衡的影响,并初步探讨其作用机制。方法:40只BALB/C小鼠随机分为4组:对照(control)、模型(model)、司坦唑醇(stanozolol)和EPS组,每组10只。除对照组外,其余各组采用乙酰苯肼、2.0 Gy照射、环磷酰胺三联应用的方法建立AA小鼠模型。造模后,司坦唑醇组以4 mg/kg司坦唑醇混悬液灌胃,EPS组以100 mg/kg EPS灌胃,对照组和模型组同时以等体积0.9%氯化钠溶液灌胃,1/d×14。全自动动物血液分析仪检测外周血血红蛋白(Hb)、红细胞(RBC)、白细胞(WBC)、血小板(PLT)的变化,流式细胞术检测Treg、Th17细胞比例,ELISA测定白介素2(IL-2)、白介素11(IL-11)、肿瘤坏死因子α(TNF-α)的水平,计数骨髓有核细胞数,HE染色和免疫组化染色检测骨髓细胞数量变化及骨髓细胞增生与凋亡情况。采用Western blot检测信号转导和转录激活子3(STAT3)、维甲酸受体相关孤儿受体γ(RORγt)、转导和转录激活子5(STAT5)、叉状头转录因子3(Foxp3)蛋白的表达变化。结果:与模型组比较,司坦唑醇组和EPS组小鼠外周血Hb水平及RBC、WBC、PLT数均明显升高(P<0.05),Th17细胞比例明显降低(P<0.05),Treg细胞比例明显升高(P<0.05),血清中IL-2、TNF-α水平均明显降低(P<0.05),IL-11水平明显升高(P<0.05),骨髓有核细胞数均明显增加,Ki-67阳性率明显增加,Caspase-3阳性率则明显减少,同时,STAT3、RORγt蛋白表达明显降低,STAT5、Foxp3蛋白表达升高,差异均具有统计学意义(P<0.05)。结论:EPS能够促进AA小鼠骨髓造血功能恢复,改善Th17/Treg比例失衡,其机制可能与抑制STAT3/RORγt途径和促进STAT5/Foxp3途径有关。

Objective:To observe the effects of Epimedium polysaccharides(EPS)on bone marrow hematopoietic function and Th17/Treg balance in aplastic anemia(AA)mice,and preliminarily explore its therapeutic mechanism.Methods:Forty BALB/C mice were randomly divided into control(control),model(model),stanozolol(stanozolol)and epimedium polysaccharide(EPS)group,with 10 mice in each group.Except for the control group,Acetophenazine,Gy irradiation and cyclophosphamide triple application were used to establish AA models for the other groups.After the model was established,the stanozolol group was intragastrically administered with 4 mg/kg stanozolol suspension,the EPS group was intragastrically administered with 100 mg/kg epimedium polysaccharide,while the control group and the model group were given an equal volume of 0.9%sodium chloride solution by gavage once a day,for 14 consecutive days.The automatic animal blood analyzer was used to detect the changes in peripheral blood hemoglobin(Hb),red blood cells(RBC),white blood cells(WBC)and platelets(PLT),flow cytometry was used to detect the proportion of Treg and Th17 cells,the levels of interleukin 2(IL-2),interleukin 11(IL-11)and tumor necrosis factorα(TNF-α)were detected by ELISA,the number of nucleated bone marrow cells was counted,HE staining and immunohistochemical staining were used to detect the number,the proliferation and apoptosis of bone marrow cells,Western blot was used to detect the expression of signal transducer and activator of transcription 3(STAT3),retinoic acid receptor-related orphan receptorγ(RORγt),transducer and activator of transcription 5(STAT5)and fork head transcription factor 3(Foxp3).Results:Compared with the model group,the levels of Hb,RBC,WBC and PLT in the peripheral blood of mice in stanozolol and EPS group significantly increased,the proportion of Th17 cells was significantly reduced,and the proportion of Treg cells significantly increased.The levels of IL-2 and TNF-αin serum were significantly reduced(P<0.05),the level of IL-11 significantly increased(P<0.05),the number of bone marrow nucleated cells significantly increased(P<0.05),the positive rate of Ki-67 significantly increased(P<0.05)and the positive rate of Caspase-3 was significantly reduced(P<0.05).At the same time,the protein expression of STAT3 and RORγt significantly decreased,and the protein expression of STAT5 and Foxp3 increased,the difference showed statistically significant(P<0.05).Conclusion:EPS can promote the recovery of bone marrow hematopoietic function in AA mice and improve Th17/Treg imbalance,the mechanism may be related to the inhibition of STAT3/RORγt expression and promotion of STAT5/Foxp3 expression.