基于IL-6/JAK2/STAT3通路探讨补肾化痰方改善去势大鼠骨丢失的作用机制

Mechanism of Bushen Huatan Recipe on improving bone loss of ovariectomized rats based on IL-6/JAK2/STAT3 pathway

目的探究补肾化痰方对去势大鼠骨丢失和IL-6/Janus激酶2(Janus kinase 2,JAK2)/信号转导与转录活化因子3(signal transducers and activators of transcription 3,STAT3)通路的影响。方法SD雌性大鼠随机分为对照组、模型组、戊酸雌二醇(0.184 mg/kg)组以及补肾化痰方低、中、高剂量(4.7、9.4、18.8 g/kg)组,除对照组外,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周给予戊酸雌二醇和补肾化痰方进行干预,给药12周后取材。采用Micro CT检测各组大鼠肱骨骨密度和骨矿物质含量;采用苏木素-伊红(HE)染色法观察各组大鼠股骨组织病理变化;采用碱性磷酸酶染色法考察各组大鼠骨髓成骨细胞数量;采用ELISA法检测各组大鼠血清中白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和IL-17水平;采用qRT-PCR法检测各组大鼠股骨组织IL-6、IL-6R、JAK2和STAT3mRNA表达情况;采用Western blotting法检测各组大鼠股骨组织IL-6、IL-6R、JAK2、p-JAK2、STAT3和p-STAT3蛋白表达情况。结果与对照组比较,模型组大鼠肱骨骨密度和骨矿物质含量显著降低(P<0.01);骨小梁减少、稀疏,脂肪细胞增多;血清中IL-6、TNF-α和IL-17水平显著升高(P<0.01);股骨组织IL-6、IL-6R、JAK2和STAT3 mRNA表达水平显著升高(P<0.01);IL-6、IL-6R、p-JAK2和p-STAT3蛋白表达水平显著升高(P<0.01)。与模型组比较,各给药组骨密度和骨矿物质含量显著增加(P<0.05、0.01);骨小梁增多,且结构较完整,脂肪细胞减少;补肾化痰方中、高剂量组大鼠骨髓成骨细胞数显著增多(P<0.05、0.01);各给药组大鼠血清中IL-6和TNF-α水平显著降低(P<0.01),戊酸雌二醇组以及补肾化痰方中、高剂量组大鼠血清中IL-17水平显著降低(P<0.01);股骨组织IL-6、IL-6R、JAK2和STAT3 mRNA表达水平显著降低(P<0.01),IL-6、IL-6R、p-JAK2和p-STAT3蛋白表达水平显著降低(P<0.01)。结论补肾化痰方能够改善去势大鼠骨丢失,其机制可能与抑制IL-6/JAK2/STAT3通路有关。

Objective To explore the effect of Bushen Huatan Recipe(补肾化痰方)on bone loss and IL-6/Janus kinase 2(JAK2)/signal transducers and activators of transcription 3(STAT3)pathway in ovariectomized rats.Methods SD female rats were randomly divided into control group,model group,estradiol valerate(0.184 mg/kg)group,low-,medium-and high-dose(4.7,9.4,18.8 g/kg)Bushen Huatan Recipe groups.Except for control group,the ovaries of all other groups were surgically removed to make a postmenopausal osteoporosis model.One week after operation,estradiol valerate and Bushen Huatan Recipe were given for intervention,and samples were obtained 12 weeks after administration.Micro CT was used to detect humerus bone mineral density and bone mineral content of rats in each group;Hematoxylin-eosin(HE)staining method was used to observe the pathological changes of femur of rats in each group;Alkaline phosphatase staining method was used to examine the number of osteoblasts in rats of each group;ELISA method was used to detect the levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and IL-17 in serum of rats in each group;q RT-PCR method was used to detect IL-6,IL-6 R,JAK2 and STAT3 m RNA expressions in femur of rats in each group;Western blotting method was used to detect IL-6,IL-6 R,JAK2,p-JAK2,STAT3 and p-STAT3 protein expressions in femur of rats in each group.Results Compared with control group,the bone mineral density and bone mineral content of humerus in model group were significantly reduced(P<0.01);Bone trabecula was reduced and sparse,and fat cells were increased;Levels of IL-6,TNF-αand IL-17 in serum were significantly increased(P<0.01);IL-6,IL-6 R,JAK2 and STAT3 m RNA expressions of femur were significantly increased(P<0.01);IL-6,IL-6 R,p-JAK2 and p-STAT3 protein expressions were significantly increased(P<0.01).Compared with model group,the bone mineral density and bone mineral content of rats in each treatment group were significantly increased(P<0.05,0.01);Bone trabecula were increased,structure was more complete,and fat cells were decreased;Number of bone marrow osteoblasts in rats of medium-and high-dose Bushen Huatan Recipe groups were significantly increased(P<0.05,0.01);Levels of IL-6 and TNF-αin serum of rats in each administration group were significantly reduced(P<0.01),IL-17 level in serum of rats in estradiol valerate group and medium-,high-dose Bushen Huatan Recipe groups were significantly reduced(P<0.01);IL-6,IL-6 R,JAK2 and STAT3 m RNA expressions of femur were significantly reduced(P<0.01),IL-6,IL-6 R,p-JAK2 and p-STAT3 protein expressions were significantly reduced(P<0.01).Conclusion Bushen Huatan Recipe can improve bone loss in ovariectomized rats,and its mechanism may be related to the inhibition of IL-6/JAK2/STAT3 pathway.