滋阴补肾方对糖尿病骨质疏松大鼠炎症反应和骨代谢的影响

Effect of Ziyin Bushen Prescription on inflammatory responses and bone metabolisms in diabetic osteoporosis rats

目的:探讨滋阴补肾方治疗糖尿病性骨质疏松症(diabetic osteoporosis,DOP)大鼠的效果及其机制。方法:将8周龄雄性SD大鼠按体质量排序法随机分为假手术组,模型组,滋阴补肾方低、中、高剂量组。统计治疗前后各组大鼠空腹血糖变化,评估疗效;HE染色观察大鼠股骨组织的病理变化,ELISA检测血清中炎症因子IL-1β、IL-6和TNF-α的水平,Western blot检测股骨组织中TLR4/NF-κB信号通路(TLR4、NF-κB p65、NF-κB p-p65)和骨代谢(OPG、RUNX2、RANKL)相关蛋白的表达水平。结果:与假手术组相比,模型组大鼠血糖水平为(19.42±1.08)mmol·L^(-1),显著升高(P<0.01);骨小梁间隔增大,骨小梁间的连接减少;血清中IL-1β(167.21±13.41)pg·mL^(-1)、IL-6(214.11±25.20)pg·mL^(-1)和TNF-α水平(282.41±16.60)pg·mL^(-1),均显著升高;股骨组织中TLR4(0.94±0.06)、NF-κB p-p65(0.91±0.12)和RANKL(1.29±0.09)蛋白表达水平均显著升高,OPG(0.22±0.09)和RUNX2(0.32±0.07)蛋白表达水平显著降低(P<0.01)。与模型组相比,滋阴补肾方高剂量组大鼠血糖水平(15.52±2.62)mmol·L^(-1),显著降低;骨小梁间隔变小,骨小梁间连接增多;血清IL-1β(84.68±9.28)pg·mL^(-1)、IL-6(88.83±7.01)pg·mL^(-1)和TNF-α水平(140.17±21.52)pg·mL^(-1),均显著降低;股骨组织中TLR4(0.31±0.07)、NF-κB p-p65(0.34±0.03)和RANKL(0.32±0.05)蛋白表达水平显著降低,OPG(0.71±0.06)和RUNX2(0.68±0.07)蛋白表达水平显著升高(P<0.05)。结论:滋阴补肾方可通过抑制TLR4/NF-κB通路激活抑制DOP大鼠炎症反应,并通过上调OPG/RANKL调节骨代谢,从而发挥降血糖和缓解骨质疏松的作用。

OBJECTIVE To explore the effect of Ziyin Bushen Prescription(ZBP)for diabetic osteoporosis(DOP)rats and elucidate its mechanism.METHODS Male Sprague-Dawley(SD)rats aged 8 weeks were randomized into five groups of sham operation,model,ZBP low/medium/high-dose according to weight ranking.The changes of fasting blood glucose before and after treatment were recorded for evaluating the efficacy.The pathological changes of femoral tissues were observed by hematoxylin-eosin(HE)stain.The serum levels of inflammatory factors interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of TLR4/NF-κB signaling pathway and bone metabolism(OPG,RUNX2&RANKL)related proteins in femoral tissue were detected by Western blot.RESULTS As compared with sham operation group,blood glucose level(19.42±1.08)mmol·L^(-1)dropped markedly in model group(P<0.01),space between bone trabeculae expanded,connections between bone trabeculae decreased,serum levels of IL-1β(167.21±13.41)pg·mL^(-1),IL-6(214.11±25.20)pg·mL^(-1)and TNF-α(282.41±16.60)pg·mL^(-1)spiked markedly,protein expressions of TLR4(0.94±0.06),NF-κB p-p65(0.91±0.12)and RANKL(1.29±0.09)rose sharply in femoral tissue while the expression levels of OPG(0.22±0.09)and RUNX2(0.32±0.07)proteins declined markedly(P<0.01).Compared with model group,blood glucose level(15.52±2.62)mmol·L^(-1)decreased obviously in ZBP high-dose group.Trabecular space shrank and connections between bone trabeculae increased,serum levels of IL-1β(84.68±9.28)pg·mL^(-1),IL-6(88.83±7.01)pg·mL^(-1)and TNF-α(140.17±21.52)pg·mL^(-1)decreased markedly.Potein expression levels of TLR4(0.31±0.07),NF-κB p-p65(0.34±0.03)and RANKL(0.32±0.05)declined obviously in femoral tissue and the expression levels of OPG(0.71±0.06)and RUNX2(0.68±0.07)proteins significantly increased(P<0.05).CONCLUSION ZBP may suppress inflammatory responses of DOP rats through arresting an activation of TLR4/NF-κB pathway and a up-regulation of OPG/RANKL,thus playing some role in lowering blood sugar and alleviating osteoporosis.