基于“成骨-成血管”理论探讨全身振动疗法治疗激素性股骨头坏死的效果及作用机制

Efficacy and mechanism of whole body vibration therapy in treatment of steroid-induced osteonecrosis of the femoral head:a osteogenesis-angiogenesis theory-based experimental study

目的:观察全身振动疗法(whole body vibration therapy,WBVT)治疗激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)的效果,并探讨其治疗SONFH的作用机制。方法:将50只SD大鼠随机分为空白组、模型组、WBVT组、Yoda1组、WBVT联合蜘蛛毒液肽(Grammostola spatulata mechanotoxin 4,GsMTx4)组。模型组、WBVT组、Yoda1组、WBVT联合GsMTx4组大鼠采用脂多糖联合甲泼尼龙琥珀酸钠构建SONFH模型。造模后,WBVT组使用WBVT干预,Yoda1组使用Piezo1蛋白激动剂Yoda1干预,WBVT联合GsMTx4组使用WBVT和Piezo1蛋白抑制剂GsMTx4干预。干预结束后,进行大鼠股骨头组织病理学观察(计算股骨头空骨陷窝率)、骨微结构观察,以及股骨头内Piezo1、骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)、Runt相关转录因子2(Runt-related transcription factor 2,Runx2)、低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、分化簇31(cluster of differentiation 31,CD31)/内皮粘蛋白(endomucin,EMCN)蛋白表达量检测。结果:(1)大鼠股骨头组织病理学观察结果。空白组大鼠的股骨头内骨小梁致密且排列整齐。与空白组相比,模型组大鼠股骨头内的骨小梁较为稀疏,骨小梁细小、不连续,且排列紊乱。与模型组相比,WBVT组和Yoda1组大鼠的股骨头内骨小梁数量增多,排列较为整齐。与WBVT组相比,WBVT联合GsMTx4组大鼠的股骨头内骨小梁排列则较为紊乱。模型组、WBVT组、Yoda1组、WBVT联合GsMTx4组大鼠的股骨头空骨陷窝率均高于空白组(P=0.000,P=0.000,P=0.000,P=0.000),WBVT组、Yoda1组大鼠的股骨头空骨陷窝率均低于模型组(P=0.000,P=0.000),WBVT联合GsMTx4组大鼠的股骨头空骨陷窝率高于WBVT组(P=0.000)。(2)大鼠股骨头骨微结构观察结果。WBVT组和Yoda1组大鼠的股骨头骨体积分数、骨小梁厚度、骨小梁数量、骨小梁分离度与空白组的差异均无统计学意义(P=0.213,P=0.081,P=0.384,P=0.471;P=0.435,P=0.131,P=0.104,P=0.126)。模型组和WBVT联合GsMTx4组大鼠的股骨头骨体积分数、骨小梁厚度、骨小梁数量均低于空白组(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000),骨小梁分离度均高于空白组(P=0.000,P=0.000)。WBVT组和Yoda1组大鼠的股骨头骨体积分数、骨小梁厚度、骨小梁数量均高于模型组(P=0.000,P=0.002,P=0.000;P=0.000,P=0.007,P=0.014),骨小梁分离度均低于模型组(P=0.000,P=0.000)。WBVT组大鼠的股骨头骨体积分数、骨小梁厚度、骨小梁数量、骨小梁分离度与Yoda1组的差异均无统计学意义(P=0.194,P=0.223,P=0.332,P=0.071)。WBVT联合GsMTx4组大鼠的股骨头骨体积分数、骨小梁厚度、骨小梁数量均低于WBVT组(P=0.002,P=0.021,P=0.000),骨小梁分离度高于WBVT组(P=0.000)。(3)大鼠股骨头内Piezo1、BMP2、Runx2、HIF-1α、VEGF蛋白表达量检测结果。WBVT组和Yoda1组大鼠股骨头内Piezo1、BMP2、Runx2、HIF-1α、VEGF蛋白表达量与空白组的差异均无统计学意义(P=0.061,P=0.122,P=0.773,P=0.814,P=0.991;P=0.112,P=0.071,P=0.955,P=0.749,P=0.915)。模型组和WBVT联合GsMTx4组大鼠股骨头内Piezo1、BMP2、Runx2、HIF-1α、VEGF蛋白表达量均低于空白组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000,P=0.000,P=0.000)。WBVT组和Yoda1组大鼠股骨头内Piezo1、BMP2、Runx2、HIF-1α、VEGF蛋白表达量均高于模型组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000,P=0.000,P=0.000)。WBVT组大鼠股骨头内Piezo1、BMP2、Runx2、HIF-1α、VEGF蛋白表达量与Yoda1组的差异均无统计学意义(P=0.962,P=0.179,P=0.214,P=0.990,P=0.975)。WBVT联合GsMTx4组大鼠股骨头内Piezo1、BMP2、Runx2、HIF-1α、VEGF蛋白表达量均低于WBVT组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000)。(4)大鼠股骨头内CD31/EMCN蛋白表达量检测结果。模型组和WBVT联合GsMTx4组大鼠股骨头内CD31/EMCN表达量均低于空白组(P=0.000,P=0.000)。WBVT组和Yoda1组大鼠股骨头内CD31/EMCN表达量与空白组的差异均无统计学意义(P=0.412,P=0.991)。WBVT组和Yoda1组大鼠股骨头内CD31/EMCN表达量均高于模型组(P=0.000,P=0.000)。WBVT联合GsMTx4组大鼠股骨头内CD31/EMCN表达量低于WBVT组(P=0.000)。结论:WBVT可以促进股骨头坏死组织修复,其作用机制可能与上调Piezo1蛋白的表达影响HIF-1α/VEGF轴,进而促进股骨头内H型血管的生成、改善股骨头血供有关。

Objective:To observe the outcomes of whole body vibration therapy(WBVT)in treatment of steroid-induced osteonecrosis of the femoral head(SONFH),and to explore its underlying mechanism.Methods:Fifty Sprague-Dawley(SD)rats were randomized into blank group,model group,WBVT group,Yoda1 group,WBVT combined Grammostola spatulata mechanotoxin 4(GsMTx4)group.The rats in model group,WBVT group,Yoda1 group,WBVT combined GsMTx4 group were intervened by lipopolysaccharide and methylprednisolone sodium succinate for inducing SONFH.After successful modeling,the rats in WBVT group,Yoda1 group and WBVT combined GsMTx4 group were further intervened with WBVT,Yoda1(a Piezo1 agonist),as well as WBVT and GsMTx4(a Piezo1 inhibitor),respectively.After the end of intervention,the histopathological changes of the femur head were observed and the percentage of empty lacunae in femur head were calculated;meanwhile,the bone microstructure of the femur head was observed and analyzed;furthermore,the protein expression levels of Piezo1,bone morphogenetic protein 2(BMP2),Runt-related transcription factor 2(Runx2),hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),and cluster of differentiation 31(CD31)/endomucin(EMCN)in femur head were detected.Results:①The results of observation on histopathological changes in femur head tissues of rats.In rats from blank group,the dense and well-arranged trabeculae were found within the femur heads;compared with that of blank group,the sparse,small,discontinuous and disordered arranged trabeculae were presented within the femur heads in rats of model group;compared with that of model group,the trabeculae increased and relatively neatly arranged within the femur heads in rats of WBVT group and Yoda1 group;whereas,compared with that of WBVT group,the trabeculae disorderedly arranged within the femur heads in rats of WBVT combined GsMTx4 group.Besides,the percentage of empty lacunae in femur head was higher in model group,WBVT group,Yoda1 group,WBVT combined GsMTx4 group compared to blank group(P=0.000,P=0.000,P=0.000,P=0.000),and was higher in model group compared to WBVT group and Yoda1 group(P=0.000,P=0.000),and was higher in WBVT combined GsMTx4 group compared to WBVT group(P=0.000).②The results of observation on bone microstructure of femur head in rats.The differences were not statistically significant in bone volume fraction(BVF),trabecular thickness(Tb.Th),trabecular number(Tb.N),and trabecular separation(Tb.Sp)of femur head between WBVT group and blank group,as well as between Yoda1 group and blank group(P=0.213,P=0.081,P=0.384,P=0.471;P=0.435,P=0.131,P=0.104,P=0.126).The BVF and Tb.Th were smaller,the Tb.N was fewer,while,the Tb.Sp was greater in model group and WBVT combined GsMTx4 group compared to blank group(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000).The BVF and Tb.Th were greater,the Tb.N was more,while,the Tb.Sp was smaller in WBVT group and Yoda1 group compared to model group(P=0.000,P=0.002,P=0.000;P=0.000,P=0.007,P=0.014;P=0.000,P=0.000);while,the comparisons between WBVT group and Yoda1 group revealed no significant differences(P=0.194,P=0.223,P=0.332,P=0.071).Additionally,the BVF and Tb.Th were smaller,the Tb.N was fewer,while,the Tb.Sp was greater in WBVT combined GsMTx4 group compared to WBVT group(P=0.002,P=0.021,P=0.000,P=0.000).③The results of detection on the protein expression levels of Piezo1,BMP2,Runx2,HIF-1αand VEGF in femur head of rats.The differences were not statistically significant in the protein expression levels of Piezo1,BMP2,Runx2,HIF-1αand VEGF in femur head of rats between WBVT group and Yoda1 group(P=0.061,P=0.122,P=0.773,P=0.814,P=0.991;P=0.112,P=0.071,P=0.955,P=0.749,P=0.915);while,the protein expression levels of Piezo1,BMP2,Runx2,HIF-1αand VEGF in femur head of rats were lower in model group and WBVT combined GsMTx4 group compared to blank group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000,P=0.000,P=0.000).The protein expression levels of Piezo1,BMP2,Runx2,HIF-1αand VEGF in femur head of rats were higher in WBVT group and Yoda1 group compared to model group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000,P=0.000,P=0.000),while,the comparison between WBVT group and Yoda1 group revealed no significant differences(P=0.962,P=0.179,P=0.214,P=0.990,P=0.975).Additionally,the protein expression levels of Piezo1,BMP2,Runx2,HIF-1αand VEGF in femur head of rats were lower in WBVT combined GsMTx4 group compared to WBVT group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000).④The results of detection on the protein expression level of CD31/EMCN in femur head of rats.The protein expression level of CD31/EMCN in femur head of rats was lower in model group and WBVT combined GsMTx4 group compared to blank group(P=0.000,P=0.000),while,the comparisons between WBVT group and blank group,as well as between Yoda1 group and blank group revealed no significant differences(P=0.412,P=0.991).The protein expression level of CD31/EMCN in femur head of rats was higher in WBVT group and Yoda1 group compared to model group(P=0.000,P=0.000),while,was lower in WBVT combined GsMTx4 group compared to WBVT group(P=0.000).Conclusion:WBVT can promote the repair of necrotic tissues in femur head.It may work by affecting the HIF-1α/VEGF axis through up-regulating the expression of Piezo1 protein,thereby promoting the formation of type H blood vessels in femur head and improving the blood supply to the femur head.