基于多重免疫组化技术探讨股骨头坏死愈胶囊治疗激素性股骨头坏死的作用机制

The mechanism of Gugutou Huaisiyu Jiaonang(股骨头坏死愈胶囊)in treatment of steroid-induced osteonecrosis of femoral head:a multiplex immunohistochemistry technique-based experimental study

目的:观察股骨头坏死愈胶囊治疗激素性股骨头坏死(steroid-induced osteonecrosis of femoral head, SONFH)的效果,基于多重免疫组化(multiplex immunohistochemistry, mIHC)技术探讨其治疗SONFH的作用机制。方法:将100只SD大鼠随机分为正常对照组、模型组、低剂量药物治疗组、高剂量药物治疗组。将模型组、低剂量药物治疗组、高剂量药物治疗组大鼠采用改良的脂多糖联合甲泼尼龙法建立SONFH模型。造模结束后,低剂量药物治疗组按照0.33 g·kg^(-1)的剂量给予股骨头坏死愈胶囊溶液(将股骨头坏死愈胶囊粉剂溶于水中)灌胃,高剂量药物治疗组按照0.67 g·kg^(-1)的剂量给予股骨头坏死愈胶囊溶液灌胃,正常对照组和模型组给予等量生理盐水灌胃。每日灌胃2次,连续治疗4周。治疗结束后,采用Micro-CT扫描分析大鼠股骨头松质骨微结构,组织切片染色观察大鼠股骨头组织病理学改变,并采用mIHC分析巨噬细胞极化及Toll样受体4(Toll-like receptor 4,TLR4)/髓样分化因子初次应答基因88(myeloid differentiation primary response gene 88,MyD88)/核因子-κB(nuclear factor-κB,NF-κB)信号通路相关蛋白表达。结果:(1)一般结果。模型组1只大鼠于造模后死亡,低剂量药物治疗组和高剂量药物治疗组分别有3只和2只大鼠在治疗过程中死亡。正常对照组大鼠精神状态良好、进食正常、毛发光泽无脱落,模型组、低剂量药物治疗组、高剂量药物治疗组大鼠在造模开始后第3天出现神情不振、进食减少、少量脱毛,1周后精神、进食等逐渐恢复。(2)大鼠股骨头松质骨微结构Micro-CT扫描分析结果。模型组大鼠股骨头松质骨骨体积分数、骨小梁厚度、骨小梁数量小于正常对照组(P=0.000,P=0.000,P=0.000),骨小梁分离度大于正常对照组(P=0.000);低剂量药物治疗组大鼠股骨头松质骨骨体积分数、骨小梁厚度与模型组的差异均无统计学意义(P=0.052,P=0.071),骨小梁数量大于模型组(P=0.012),骨小梁离散度小于模型组(P=0.001);高剂量药物治疗组大鼠股骨头松质骨骨体积分数、骨小梁厚度、骨小梁数量大于模型组(P=0.001,P=0.011,P=0.000),骨小梁离散度小于模型组(P=0.001),骨体积分数、骨小梁厚度、骨小梁数量、骨小梁离散度与低剂量药物治疗组的差异均无统计学意义(P=0.146,P=0.414,P=0.086,P=0.146)。(3)大鼠股骨头组织病理学观察结果。模型组大鼠股骨头空骨陷窝率、骨坏死发生率均高于正常对照组(P=0.000,P=0.000);低剂量药物治疗组大鼠股骨头空骨陷窝率低于模型组(P=0.000),骨坏死发生率与模型组的差异无统计学意义(P=0.054);高剂量药物治疗组大鼠股骨头空骨陷窝率和骨坏死发生率均低于模型组(P=0.000,P=0.000),空骨陷窝率低于低剂量药物治疗组(P=0.049),骨坏死发生率与低剂量药物治疗组的差异无统计学意义(P=0.556)。(4)巨噬细胞极化mIHC分析结果。模型组大鼠股骨头M1型巨噬细胞、M2型巨噬细胞占比均高于正常对照组(P=0.000,P=0.000);低剂量药物治疗组大鼠股骨头M1型巨噬细胞、M2型巨噬细胞占比与模型组的差异均无统计学意义(P=0.270,P=0.533);高剂量药物治疗组大鼠股骨头M1型巨噬细胞占比低于模型组(P=0.009),与低剂量药物治疗组的差异无统计学意义(P=0.131),M2型巨噬细胞占比高于模型组和低剂量药物治疗组(P=0.006,P=0.038)。(5)TLR4/MyD88/NF-κB信号通路蛋白表达mIHC分析结果。模型组大鼠股骨头TLR4、MyD88、NF-κB p65蛋白相对表达量均高于正常对照组(P=0.000,P=0.000,P=0.000);低剂量药物治疗组大鼠股骨头TLR4、MyD88蛋白相对表达量与模型组的差异均无统计学意义(P=0.268,P=0.280),NF-κB p65蛋白相对表达量低于模型组(P=0.034);高剂量药物治疗组TLR4、MyD88、NF-κB p65蛋白相对表达量均低于模型组和低剂量药物治疗组(TLR4:P=0.002,P=0.040;MyD8:P=0.000,P=0.013;NF-κB p65:P=0.000,P=0.039)。结论:股骨头坏死愈胶囊治疗SONFH,能够显著改善股骨头骨微结构、抑制骨坏死,其作用具有一定的剂量依赖性;其作用机制与调控TLR4/MyD88/NF-κB信号通路及巨噬细胞极化有关。

Objective:To observe the outcome of Gugutou Huaisiyu Jiaonang(股骨头坏死愈胶囊,GGTHSYJN)in treatment of steroid-induced osteonecrosis of femoral head(SONFH),and to explore its underlying mechanism via multiplex immunohistochemistry(mIHC)technique.Methods:One hundred Sprague-Dawley(SD)rats were randomly assigned into normal control group,model group,low-dose drug treatment group and high-dose drug treatment group.After adaptive feeding for one week,the rats in model group,low-dose drug treatment group and high-dose drug treatment group were subjected to intramuscular injection of lipopolysaccharide and methylprednisolone in turn for inducing SONFH.After the end of modeling,the rats in low-dose drug treatment group were intragastric administrated with 0.33 g/kg GGTHSYJN solution(GGTHSYJN powders were dissolved into water),the ones in high-dose drug treatment group with 0.67 g/kg GGTHSYJN solution,while the ones in normal control group and model group with the same dose of normal saline.All rats in the 4 groups were intragastric administrated twice a day for consecutive 4 weeks.After the end of treatment,the femur heads were harvested from the rats,and the cancellous bone microstructure of femur head was observed and analyzed by using Micro-CT scanning;meanwhile,the tissue sections of femur head were stained with hematoxylin-eosin(HE)for observing the histopathological changes.Furthermore,the macrophage polarization and the expressions of Toll-like receptor 4(TLR4)/myeloid differentiation primary response gene 88(MyD88)/nuclear factor-κB(NF-κB)signaling pathway-related protein were analyzed by using mIHC technique.Results:①One rat in model group died after the modeling,and 3 rats in low-dose drug treatment group and 2 ones in high-dose drug treatment group died during the treatment.Rats with good mental state,normal eating and drinking as well as healthy fur without shedding were observed in normal group.On day 3 after the beginning of the modeling,the rats in model group,low-dose drug treatment group,and high-dose drug treatment group gradually exhibited the symptoms as dispiritedness,reduced eating and drinking,as well as slight hair removal,and the signs gradually recovered after one week.②The normal control group exhibited higher bone volume fraction(BVF),thicker trabeculae,more trabeculae,and lower trabecular separation(Tb.Sp)in cancellous bone compared with that of model group(P=0.000,P=0.000,P=0.000,P=0.000).The differences in BVF and trabecular thickness were not statistically significant between low-dose drug treatment group and model group(P=0.052,P=0.071),while,the trabeculae was more and the Tb.Sp was lower in low-dose drug treatment group compared to model group(P=0.012;P=0.001).The high-dose drug treatment group displayed higher BVF,thicker trabeculae,more trabeculae,and lower Tb.Sp in cancellous bone compared with that of model group(P=0.001,P=0.011,P=0.000,P=0.001),while,in contrast to that of low-dose drug treatment group,the results revealed no significant differences(P=0.146,P=0.414,P=0.086,P=0.146).③The percentage of empty lacunae and the incidence rate of osteonecrosis in femur heads were higher in model group compared to normal control group(P=0.000,P=0.000).The percentage of empty lacunae in femur head was lower in low-dose drug treatment group compared to model group(P=0.000),while,the comparison of osteonecrosis incidence rate between the 2 groups revealed no significant difference(P=0.054).The percentage of empty lacunae and the incidence rate of osteonecrosis in femur heads were lower in high-dose drug treatment group compared to model group(P=0.000,P=0.000);furthermore,the percentage of empty lacunae was lower in high-dose drug treatment group compared to low-dose drug treatment group(P=0.049),while,the comparison of osteonecrosis incidence rate between the 2 groups revealed no significant difference(P=0.556).④The M1 macrophages and M2 macrophages accounted for a higher proportion in femur heads of rats in model group compared to normal control group(P=0.000,P=0.000).There was no statistical difference in the proportions of M1 macrophages and M2 macrophages between low-dose drug treatment group and model group(P=0.270,P=0.533).The M1 macrophages accounted for a lower proportion in high-dose drug treatment group compared to model group(P=0.009),while the M2 macrophages accounted for a higher proportion in high-dose drug treatment group compared to model group and low-dose drug treatment group(P=0.006,P=0.038);furthermore,the comparison of the proportion of M1 macrophages between high-dose drug treatment group and low-dose drug treatment group revealed no significant difference(P=0.131).⑤The relative expression levels of TLR4,MyD88 and NF-κB p65 protein in femur heads were higher in model group compared to normal control group(P=0.000,P=0.000,P=0.000).There was no statistical difference in the relative expression levels of TLR4 and MyD88 protein between low-dose drug treatment group and model group(P=0.268,P=0.280),while the relative expression level of NF-κB p65 protein was lower in low-dose drug treatment group compared to model group(P=0.034).The relative expression levels of TLR4,MyD88 and NF-κB p65 protein were lower in high-dose drug treatment group compared to model group and low-dose drug treatment group(TLR4:P=0.002,P=0.040;MyD88:P=0.000,P=0.013;NF-κB p65:P=0.000,P=0.039).Conclusion:GGTHSYJN can significantly improve the bone microstructure of femur head and inhibit osteonecrosis in treatment of SONFH,and it exhibits dose-dependence in the efficacy.It may work by regulating TLR4/MyD88/NF-κB signaling pathway and macrophage polarization.