补肾化痰方对去势骨质疏松模型大鼠Th17/Treg平衡机制的影响

Mechanism of Bushen Huatan Prescription in Regulating Th17/Treg Balance in Ovariectomized Osteoporosis Rats

目的:研究补肾化痰方在防治绝经后骨质疏松症中对免疫T细胞亚群辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的影响。方法:6月龄SD雌性大鼠60只,按随机数字表分为假手术组,模型组,戊酸雌二醇组(0.184 mg·kg^(-1)),补肾化痰方低、中、高剂量组(4.7,9.4,18.8 g·kg^(-1)),除假手术组外,其他组均采用手术切除卵巢,制作绝经后骨质疏松症模型。术后1周灌胃给药,模型组和假手术组以等体积的生理盐水灌胃,1次/d,灌胃12周后取材。运用微计算机断层扫描技术(Micro CT)检测大鼠骨量及骨微结构;采用酶联免疫吸附测定(ELISA)检测血清中叉头框蛋白p3(Foxp3),维甲酸相关核孤受体γt(RORγt)含量;实时荧光定量聚合酶链式反应(Real-time PCR)检测骨组织中Foxp3,RORγt mRNA表达,蛋白免疫印迹法(Western blot)检测骨组织中Foxp3,RORγt蛋白表达量;运用流式细胞抗原检测,分析并比较各组Th17,Treg细胞的数量。结果:与假手术组比较,模型组大鼠骨量降低、骨小梁显著减少(P<0.01);骨微结构破坏,血清中Foxp3浓度显著降低,RORγt浓度显著升高(P<0.01),骨组织中Foxp3 mRNA及蛋白表达降低,RORγt mRNA及蛋白表达显著升高(P<0.01),骨组织中Treg细胞数量减少(P<0.01),Th17细胞数量增加,且Th17/Treg值显著升高(P<0.01);与模型组比较,各给药组骨量明显增加(P<0.05,P<0.01),血清中Foxp3浓度升高,RORγt浓度显著降低(P<0.01),骨组织中Foxp3 mRNA及蛋白表达明显升高(P<0.05,P<0.01),但低剂量组mRNA差异无统计学意义,RORγt mRNA及蛋白表达明显降低(P<0.05,P<0.01),骨组织中Treg细胞数明显升高,Th17细胞数量明显降低(P<0.05,P<0.01),且Th17/Treg显著降低(P<0.01)。结论:补肾化痰方可以提高去卵巢大鼠骨量,改善骨微结构,并使骨组织中Treg细胞数量增加而Th17细胞数量减少。进而推论补肾化痰方可能通过调节Th17/Treg平衡,而发挥防治绝经后骨质疏松症的作用。

Objective: To study the effect of Bushen Huatan prescription on helper T cell 17(Th17)/T regulatory cells(Treg)balance of immune T cell subsets in the prevention and treatment of postmenopausal osteoporosis. Method: Sixty 6-month-old female SD rats were randomly divided into sham operation group,model group,estradiol valerate group(0.184 mg·kg^(-1))and Bushen Huatan prescription low,medium and high groups(4.7,9.4,18.8 g·kg^(-1))according to the random number table. All the groups except the sham operation group received ovariectomy to make postmenopausal osteoporosis model. Intragastric administration was started 1 week after operation,and the rats in model group and sham operation group received equal volume of normal saline,once a day for 12 weeks. Microcomputed tomography(Micro CT)was then used to detect bone mass and microstructure of rats,the contents of Forkhead box protein(Foxp3)and retinoic acid related nuclear orphan receptor(RORγt)in serum were detected by enzyme-linked immunosorbent assay(ELISA),the mRNA expression levels of Foxp3 and RORγt in bone tissues were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot was used to detect the protein expression of Foxp3 and RORγt in bone tissues,the number of Th17 and Treg cells in each group was analyzed and compared by flow cytometry. Result: Compared with the sham operation group,the bone mass and trabeculae of the model group decreased(P<0.01),the bone microstructure was destroyed,the concentration of Foxp3 in serum decreased,the concentration of RORγt increased(P<0.01),the mRNA and protein expression levels of Foxp3 in bone tissues decreased,RORγt increased,the number of Treg cells in bone tissues decreased,number of Th17 cells increased(P<0.01),and Th17/Treg ratio increased(P<0.01)in model group. Compared with the model group,the bone mass in each treatment group increased(P<0.05,P<0.01),Foxp3 concentration in serum increased,RORγt concentration decreased(P<0.01),the mRNA and protein expression levels of Foxp3 in bone tissues increased significantly(P<0.05,P<0.01),but no statistical difference was shown in mRNA expression between low dose group and the model group. In addition,the mRNA and protein expression of RORγt decreased(P<0.05,P<0.01),number of Treg cells increased,number of Th17 cells decreased(P<0.05,P<0.01), and Th17/Treg ratio decreased in treatment groups(P<0.01). Conclusion: Bushen Huatan prescription can increase bone mass,improve bone microstructure,increase the number of Treg cells and decrease the number of Th17 cells in ovariectomized rats. It is concluded that Bushen Huatan prescription may play a role in preventing and treating postmenopausal osteoporosis by regulating Th17/Treg balance.