骨碎补总黄酮经JAK2/STAT3/SOCS3通路抑制幼兔激素性股骨头缺血坏死骨组织中促凋亡因子表达的实验研究

Inhibitory effect of total flavonoids of rhizoma drynariae on the expression of pro-apoptotic factors in hormone-induced avascular necrosis of the femoral head in juvenile rabbits via JAK2/STAT3/SOCS3 pathway

目的:探讨骨碎补总黄酮(TFRD)对幼兔激素性股骨头缺血坏死病程及凋亡相关蛋白表达的影响及其可能机制。方法:动物实验研究。选用2月龄健康新西兰白兔110只,按随机数字表法分为对照组(10只)、模型组(50只)、TFRD组(50只)。模型组及TFRD组行双侧臀肌交替注射醋酸泼尼松龙注射液(7.5 mg/kg),每周2次,对照组同部位同频次注射等体积生理盐水(0.3 mL/kg);TFRD组于第1次注射醋酸泼尼松龙开始每天灌服TFRD溶液(35 mg/kg),对照组及模型组每日灌服等体积生理盐水6 mL,连续8周。根据是否发病将模型组分为模型发病组及模型未发病组;将TFRD组分为TFRD发病组及TFRD未发病组。造模结束后,取双侧股骨头行病理学检查;酶联免疫吸附实验(ELISA)测定各组股骨头组织中磷酸化蛋白酪氨酸激酶2(P-JAK2)、磷酸化信号转导与转录激活因子3(P-STAT3)、细胞因子信号传导抑制蛋白3(SOCS3)及凋亡相关因子表达情况;实时荧光定量聚合酶链反应(qPCR)检测蛋白酪氨酸激酶2(JAK2)、信号转导与转录激活因子3(STAT3)、SOCS3相对表达情况。结果:模型组坏死发生率22.86%(8/35);TFRD组坏死发生率15.79%(6/38)。微型计算机断层扫描技术示:与模型发病组比较,TFRD发病组骨体积分数和骨小梁厚度较高,骨小梁分离度较低,差异均有统计学意义(均P<0.05)。病理染色示:TFRD未发病组较其他实验组骨小梁形态更完整、骨小梁周围可见成骨细胞、空骨陷窝更少,病理变化得到明显改善。ELISA显示:与模型发病组比较,TFRD发病组P-JAK2、P-STAT3、B细胞淋巴瘤因子2(Bcl-2)相关X蛋白、半胱氨酸蛋白酶3表达均下降,Bcl-2表达增高,差异均有统计学意义(均P<0.05)。qPCR示:与模型发病组比较,TFRD发病组JAK2、STAT3、SOCS3表达下调,差异均有统计学意义(均P<0.05)。结论:TFRD可以经JAK2/STAT3/SOCS3通路抑制幼兔激素性股骨头缺血坏死骨组织中促凋亡因子表达,减缓幼兔激素性股骨头缺血坏死病程。

Objective To investigate the effects of total flavonoids of rhizoma drynariae(TFRD)on the progression of hormone-induced avascular necrosis of the femoral head and the expression of apoptosis-related proteins in juvenile rabbits and their possible mechanisms.Methods Animal experimental study.A total of 110 healthy 2-month-old New Zealand white rabbits were selected and randomly divided into the control group(10 rabbits),model group(50 rabbits),and TFRD group(50 rabbits).Prednisolone acetate(7.5 mg/kg)was injected into the bilateral gluteus alternately twice a week in the model and TFRD groups.The same volume of normal saline(0.3 mL/kg)was injected in the control group.Additionally,rabbits in the TFRD group were given the TFRD solution(35 mg/kg)daily from the first injection of prednisolone acetate,and rabbits in the control and model groups were given the same volume of normal saline 6 mL daily for 8 weeks.In the model and TFRD groups,those diseased rabbits were included in the diseased group,and those non-diseased rabbits were included in the non-diseased group.At the end of modeling,all rabbits were sacrificed,and their femoral heads were examined by histopathology.The expressions of phosphorylated protein tyrosine kinase 2(P-JAK2),phosphorylated signal transducer and activator of transcription 3(P-STAT3),suppressor of cytokine signaling 3(SOCS3),and apoptosis-related factors in the femoral head tissues of each group were determined by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative polymerase chain reaction(qPCR)was used to detect the relative expressions of protein tyrosine kinase 2(JAK2),signal transducer and activator of transcription 3(STAT3),and SOCS3.Results The incidence of necrosis in the model group was 22.86%(8/35),and the incidence of necrosis in the TFRD group was 15.79%(6/38).The micro computed tomography showed that the bone volume fraction and trabecular thickness were higher,while the trabecular separation was lower(all P<0.05)in the diseased rabbits in the TFRD group compared with those in the model group.Pathological staining showed that for non-diseased rabbits in the TFRD group,the shape of bone trabeculae was relatively complete,the osteoblasts around the bone trabeculae were visible,the empty bone lacunae were fewer,and the pathological changes were significantly improved.ELISA data showed that compared with the diseased rabbits in the model group,the expressions of P-JAK2,P-STAT3,B cell lymphoma 2(Bcl-2)-associated X protein,and cysteine protease-3 in the diseased rabbits in the TFRD group decreased significantly,while the expression of Bcl-2 increased remarkably(all P<0.05).qPCR data showed that compared with the diseased rabbits in the model group,the expressions of JAK2,STAT3,and SOCS3 in the diseased rabbits in the TFRD group were downregulated markedly(all P<0.05).Conclusions TFRD inhibits the expressions of pro-apoptotic factors in the bone tissue of the femoral head with hormone-induced avascular necrosis in juvenile rabbits via the JAK2/STAT3/SOCS3 pathway,thus slowing down the progression of the hormone-induced avascular necrosis.