【摘要】 目的观察左归丸加减对雌激素缺乏骨丢失模型小鼠脾脏辅助性T细胞亚群17(Th17)/调节性T细胞亚群(Treg)亚群平衡的影响,并探讨其作用机制。方法 50只BALB/c小鼠按随机数字表法随机分为假手术组、卵巢切除模型组、左归丸加减(简称中药)小、中、大剂量组,共5组,每组10只。中药组分别给予中药小、中、大剂量(7.25、14.50、29.00 g/kg),1天1次,1次约0.5 m L,连续用药12周;同时假手术组及模型组给予等量的生理盐水。采用酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)法检测各组血清雌二醇(E2)水平,双能X线骨密度仪检测股骨骨密度(bone mineral density,BMD),流式细胞仪胞内外双染色法检测脾脏单个核细胞Th17/Treg亚群比例,RT-PCR法检测脾脏单个核细胞IL-17A、TGF-βmRNA表达。结果与假手术组比较,模型组E2、BMD均降低(P<0.05),Th17亚群比例、Th17/Treg比值均升高(P<0.05),Treg亚群比例明显降低(P<0.05),IL-17A mRNA表达明显升高(P<0.05),TGF-βmRNA表达明显降低(P<0.05);与模型组比较,中药中、大剂量组BMD值明显升高(P<0.05),Th17亚群比例、Th17/Treg比值降低(P<0.05)、Treg亚群比例升高(P<0.05),IL-17A mRNA表达降低(P<0.05)及TGF-βmRNA表达升高(P<0.05)。相关性分析显示BMD与血清E2水平、Treg亚群比例呈明显正相关(P<0.05),与Th17亚群比例呈明显负相关(P<0.05);血清E2水平与Treg亚群比例呈明显正相关(P<0.05),与Th17亚群比例呈明显负相关(P<0.05)。结论 Th17/Treg亚群平衡状态与雌激素缺乏骨丢失存在相关性,左归丸加减能降低雌激素缺乏骨丢失小鼠Th17亚群比例,升高雌激素缺乏骨丢失小鼠Treg亚群比例。左归丸加减可能通过调节雌激素缺乏骨丢失小鼠Th17/Treg亚群平衡达到治疗作用,且该方无明显雌激素样作用。更多还原

【Abstract】 Objective To observe the effect of Modified Zuoguiwan( MZ) on the balance between helper T cell subsets 17( Th17) and regulatory T cell subsets( Treg) in estrogen deficiency induced bone loss mice and to explore its mechanism. Methods Totally 50 BALB / c mice were divided into the sham-operation group,the ovariectomy model group,the low dose MZ group,the middle dose MZ group,and the high dose MZ group by random digit table,10 in each group. Mice in the low,middle,and high dose MZgroups were respectively administered with MZ at the daily dose of 7. 25,14. 50,and 29. 00 g / kg by gastrogavage,0. 5m L each time for 12 successive weeks. Meanwhile,mice in the sham-operation group and the ovariectomy model group were administered with equal volume by gastrogavage,0. 50 m L each time. The serum estradiol( E2) level was assessed by enzyme linked immunosorbent assay( ELISA). Bone mineral density( BMD) of thigh bone was measured with dual energy X ray absorptiometry. In addition,the population of Th17 / Treg subsets in spleen mononuclear cells was analyzed by extracellular and intracellular staining method using flow cytometry. Moreover,the mRNA expression of IL-17 A and TGF-β in the spleen mononuclear cells was detected by reverse transcription polymerase chain reaction( RT-PCR). Results Compared with the sham-operation group,both E2 and BMD significantly decreased,the percentage of Th17 subset and Th17 / Treg ratio both increased,the percentage of Treg subset obviously decreased,the expression of IL-17 A mRNA significantly increased,and the expression of TGF-β mRNA significantly decreased in the ovariectomy model group( all P <0. 05). Compared with the model group,BMD obviously increased,the percentage of Th17 subset and Th17 / Treg ratio both decreased,the percentage of Treg subset obviously increased,the expression of IL-17 A mRNA significantly decreased,and the expression of TGF-β mRNA significantly increased in the middle dose MZ group and the high dose MZ group( all P < 0. 05). Correlation analyses showed that BMD was positively related to both the serum E2 level and the percentage of Treg subset( P < 0. 05),but negatively related to the percentage of Th17 subset( P < 0. 05). In addition,the serum E2 level was positively related to the percentage of Treg subset,but obviously negatively related to that of Th17 subset( P < 0. 05). Conclusions There was correlation between Th17 / Treg imbalance and E2 deficient bone loss. MZ could decrease the proportion of Th17 subset,but elevate the proportion of Treg subset in E2 deficient bone loss mice. It could achieve therapeutic effect through adjusting the balance of Th17 / Treg in E2 deficient bone loss mice.更多还原