基于“肾主骨”理论探讨金匮肾气丸调控AGEs/RANKL/NF-κB信号通路对糖尿病性骨质疏松症小鼠的影响

Effect of Jingui Shenqiwan on Diabetic Osteoporosis in Mice via AGEs/RANKL/NF-κBPathway Based on Theory of "Kidneys Governing Bones"

目的:基于“肾主骨”理论研究金匮肾气丸通过调控晚期糖基化终产物(AGEs)/核转录因子-κB(NF-κB)受体激活剂(RANKL)/NF-κB信号通路对糖尿病性骨质疏松症(DOP)小鼠的影响。方法:选取40只6周龄骨骼肌特异性胰岛素样生长因子-1受体功能缺失(MKR)小鼠,雌雄各半,高脂饲料喂养8周建立DOP模型,将造模成功的小鼠随机分成模型组、金匮肾气丸低、高剂量组(1.3、2.6 g·kg^(-1))和阿仑膦酸钠组(0.01 g·kg^(-1)),每组10只;另取10只同龄FVB/N小鼠为正常组。各给药组分别予以相应药物灌胃,每日1次,连续4周。给药结束后,各组小鼠行空腹血糖(FBG)检测和口服糖耐量实验(OGTT);检测小鼠肾功能和肾指数;采用苏木素-伊红(HE)和马松(Masson)染色观察肾脏组织病理学变化;免疫组化检测小鼠肾脏组织AGEs、磷酸化核转录因子-κB(p-NF-κB)和RANKL蛋白表达水平;采用蛋白免疫印迹法(Western blot)检测肾脏组织AGEs/RANKL/NF-κB信号通路相关蛋白和股骨组织中骨保护素(OPG)、Runt相关转录因子2(RUNX2)蛋白的表达。结果:与正常组比较,模型组小鼠FBG显著升高(P<0.01),骨小梁退变,骨形态参数指标异常,OGTT的曲线下面积(AUC)显著升高(P<0.01),肾脏体积增大,肾功能指标、肾指数显著升高(P<0.01),肾小球、肾小管结构紊乱,肾脏组织AGEs、RANKL表达和p-NF-κB/NF-κB值明显升高(P<0.05),股骨组织中OPG和RUNX2表达显著降低(P<0.01);与模型组比较,金匮肾气丸各剂量组小鼠OGTT的AUC显著降低(P<0.01),组织病理分析显示,肾小球和肾小管的结构病变缓解,肾脏组织AGEs、RANKL的表达和p-NF-κB/NF-κB值明显降低(P<0.05,P<0.01),股骨组织中RUNX2和OPG表达明显升高(P<0.05,P<0.01)。结论:金匮肾气丸可通过改善肾功能,下调AGEs/RANKL/NF-κB信号通路抑制炎性反应,进而缓解DOP小鼠骨质疏松症状,起到从肾论治DOP的作用。

Objective:To investigate the effect of Jingui Shenqiwan on diabetic osteoporosis(DOP)in mice by regulating the advanced glycation end products(AGEs)/receptor activator of nuclear factor-κB ligand(RANKL)/nuclear factor-κB(NF-κB)signaling pathway based on the theory of"kidneys governing bones".Method:Forty 6-week-old male and female skeletal-muscle-specific,dominant negative insulin-like growth factor-1 receptor(MKR)mice were selected and fed on a high-fat diet for eight weeks to establish the DOP model.The model mice were randomly divided into a model group,low-and high-dose Jingui Shenqiwan group(1.3,2.6 g·kg^(-1)),and an alendronate sodium group(0.01 g·kg^(-1)),with 10 mice in each group.Additionally,10 FVB/N mice of the same age were assigned to the normal group.The corresponding drugs were administered orally to each group once a day for four weeks.After the administration period,fasting blood glucose(FBG)measurement and oral glucose tolerance test(OGTT)were conducted.Kidney function and kidney index were measured.Renal tissue pathological changes were observed through hematoxylin-eosin(HE)and Masson staining.Immunohistochemistry was performed to assess the protein expression levels of AGEs,phosphorylated NF-κB(p-NF-κB),and RANKL in renal tissues.Western blot analysis was conducted to measure the expression of proteins related to the AGEs/RANKL/NF-κB signaling pathway,osteoprotegerin(OPG),and Runt-related transcription factor 2(RUNX2)proteins in femoral bone tissues.Result:Compared with the normal group,mice in the model group exhibited significantly increased FBG(P<0.01),trabecular bone degeneration,abnormal bone morphological parameters,significantly increased area under the curve(AUC)of OGTT(P<0.01),enlarged kidney volume,significantly increased kidney function indicators and kidney index(P<0.01),disrupted renal glomeruli and renal tubule structures,significantly increased expression of AGEs,RANKL,and p-NF-κB/NF-κB in renal tissues(P<0.05),and significantly decreased expression of OPG and RUNX2 in femoral bone tissues(P<0.01).Compared with the model group,mice in the Jingui Shenqiwan groups showed a significant decrease in OGTT AUC(P<0.01).Histopathological analysis revealed alleviated structural lesions in renal glomeruli and renal tubules.Furthermore,the expression of AGEs,RANKL,and p-NF-κB/NF-κB in renal tissues was significantly reduced(P<0.05,P<0.01),and the expression of RUNX2 and OPG in femoral bone tissues was significantly increased(P<0.05,P<0.01).Conclusion:Jingui Shenqiwan can improve kidney function and downregulate the AGEs/RANKL/NF-κB signaling pathway to inhibit inflammatory reactions,thereby alleviating the symptoms of DOP in mice,demonstrating a therapeutic effect on DOP from the perspective of the kidney.