益气续骨方调节PI3K/Akt信号通路对激素性股骨头坏死大鼠骨代谢的影响

Effect of decoction for replenishing Qi and bonesetting on bone metabolism in rats with steroid-induced osteonecrosis of femoral head via regulating PI3K/Akt signaling pathway

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摘要目的探究益气续骨方调节磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路对激素性股骨头坏死大鼠骨代谢的影响。方法将50只SD雄性大鼠随机分为正常组、模型组、益气续骨方低剂量组、益气续骨方高剂量组、益气续骨方+LY294002组,每组10只。除正常组外,其余组大鼠右臀肌肉注射醋酸泼尼松龙注射液4周建立激素性股骨头坏死模型。造模结束后,益气续骨方低、高剂量组大鼠分别给予益气续骨方0.5 mL(含生药0.285 g/mL)和1 mL(含生药0.57 g/mL)灌胃,益气续骨方+LY294002组大鼠给予益气续骨方1 mL灌胃并腹腔注射6 mg/kg的LY294002,正常组和模型组给予等体积生理盐水灌胃,均1次/d,连续干预8周。ELISA检测大鼠血清白细胞介素-4(IL-4)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、血钙、血磷、骨保护素(OPG)、骨钙素(OCN)、碱性磷酸酶(ALP)水平,Micro-CT检测观察股骨头形态,HE染色观察股骨头组织病理形态,TRAP染色观察破骨细胞生成情况,Western blot法检测股骨头组织中骨特异性转录因子2(Runx2)、骨形态发生蛋白2(BMP-2)、裂解半胱天冬酶-3(Cleaved-Caspase-3)、PI3K、Akt蛋白表达情况。结果与正常组比较,模型组大鼠血清IL-4、IL-6、TNF-α、OPG、ALP水平和骨小梁分离度(Tb.Sp)、空骨陷窝率及股骨头组织中Cleaved-Caspase-3蛋白相对表达量均明显升高(P均<0.05),血钙、血磷、OCN水平和骨体积分数(BV/TV)、骨小梁数目(Tb.N)、骨小梁厚度(Tb.Th)及股骨头组织中Runx2、BMP-2、p-PI3K/PI3K、p-Akt/Akt蛋白相对表达量均明显降低(P均<0.05),破骨细胞数量增多,股骨头破坏、骨小梁空间结构紊乱;与模型组比较,益气续骨方低、高剂量组大鼠血清IL-4、IL-6、TNF-α、OPG、ALP水平和Tb.Sp、空骨陷窝率及股骨头组织中Cleaved-Caspase-3蛋白相对表达量均明显降低(P均<0.05),血钙、血磷、OCN水平和BV/TV、Tb.N、Tb.Th及股骨头组织中Runx2、BMP-2、p-PI3K/PI3K、p-Akt/Akt蛋白相对表达量均明显升高(P均<0.05),破骨细胞数量减少,股骨头破坏程度减轻,骨小梁空间结构形态改善;LY294002可减弱益气续骨方对上述指标的改善作用,各指标与益气续骨方高剂量组比较差异均有统计学意义(P均<0.05)。结论益气续骨方可改善激素性股骨头坏死大鼠骨代谢,降低炎症因子水平,减少破骨细胞数量,减轻大鼠股骨头坏死,机制可能与激活PI3K/Akt信号通路有关。 Objective It is to investigate the effect of decoction for replenishing Qi and bonesetting(DRQB)on bone metabolism in rats with steroid-induced osteonecrosis of femoral head(SONFH)via regulating the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.Methods Fifty SD male rats were randomly divided into normal group,model group,DRQB low-dose group,DRQB high-dose group and DRQB+LY294002 group,with 10 rats in each group.The rats of all group except for the normal group were injected with prednisolone acetate intramuscularly in the right buttock for 4 weeks to establish model of SONFH.At the end of modeling,the rats in the low and high dose groups of DRQB were gavaged with 0.5 mL and 1 mL of DRQB,respectively,and the rats in the DRQB+LY294002 group were gavaged with 1 mL of DRQB and intraperitoneally injected with 6 mg/kg of LY294002,while the rats in the normal group and model group were given the same volume of normal saline by gavage,all once daily,continuously treated for 8 weeks.The serum levels of interleukin-4(IL-4),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),calcium,phosphorus,osteoprotegerin(OPG),osteocalcin(OCN)and alkaline phosphatase(ALP)of the rats were determined by ELISA,the morphology of femoral head was detected and observed by Micro-CT,the pathologic morphology of femoral head tissues was observed by HE staining,the osteoclastogenesis was observed by TRAP staining,and the expressions of bone-specific transcription factor 2(Runx2),bone morphogenetic protein 2(BMP-2),Cleaved-Caspase-3,PI3K,and Akt proteins in the femoral head tissues were detected by Western blot.Results Compared with the normal group,the serum levels of IL-4,IL-6,TNF-α,OPG and ALP,the trabecular bone resorption(Tb.Sp),percentage of empty lacunae,number of osteoclasts and relative expressions of Cleaved-Caspase-3 protein in femoral head tissue were significantly increased in the rats of the model group(all P<0.05),while the serum levels of calcium,phosphorus and OCN,bone volume fraction(BV/TV),number of trabeculae(Tb.N),trabecular thickness(Tb.Th),relative expressions of Runx2,BMP-2,p-PI3K/PI3K and p-Akt/Akt proteins in femoral head tissue were significantly decreased(all P<0.05),the femoral head was greatly damaged,and the spatial structure of the bone trabeculae was disordered.Compared with the model group,the serum levels of IL-4,IL-6,TNF-α,OPG and ALP,Tb.Sp,percentage of empty lacunae,number of osteoclasts and relative expressions of Cleaved-Caspase-3 protein in femoral head tissue were significantly decreased in the rats of the low and high dose groups of DRQB(all P<0.05),while the serum levels of calcium,phosphorus and OCN,(BV/TV,Tb.N,Tb.Th,relative expressions of Runx2,BMP-2,p-PI3K/PI3K and p-Akt/Akt proteins in femoral head tissue were significantly increased(all P<0.05),the damage of the femoral head was alleviated,and the spatial structure of bone trabeculae was improved;LY294002 could attenuate the improvement effect of DRQB on the above indexes,and the differences in all indexes were statistically significant when comparing with those of the high-dose group of DRQB(all P<0.05).Conclusion Decoction for replenishing Qi and bonesetting can improve bone metabolism,reduce the levels of inflammatory factors,decrease the number of osteoclasts,and attenuate the necrosis of the femoral head in rats with hormonal necrosis of femoral head,and the mechanism may be related to the activation of the PI3K/Akt signaling pathway.

作者李皎许康周志能马桃红石小东戚琬若王琪沙正才董玮 LI Jiao;XU Kang;ZHOU Zhineng;MA Taohong;SHI Xiaodong;QI Wanruo;WANG Qi;SHA Zhengcai;DONG Wei(Kunming Hospital of Traditional Chinese Medicine,Kunming 650500,Yunnan,China;Yunnan University of Traditional Chinese Medicine,Kunming 650500,Yunnan,China)

机构地区昆明市中医医院云南中医药大学

出处《现代中西医结合杂志》 CAS 2024年第9期1181-1187,1196,共8页 Modern Journal of Integrated Traditional Chinese and Western Medicine

基金云南省科技厅科技计划项目(202301AZ070001-152)。

关键词激素性股骨头坏死益气续骨方磷脂酰肌醇3-激酶蛋白激酶B 骨代谢 hormone induced necrosis of femoral head decoction for replenishing Qi and bonesetting phosphoinositide 3-kinase protein kinase B bone metabolism

分类号 R-332 [医药卫生]