The mechanism of Gugutou Huaisiyu Jiaonang(股骨头坏死愈胶囊)in treatment of steroid-induced osteone-crosis of femoral head:a multiplex immunohistochemistry technique-based experimental study
Objective:To observe the outcome of Gugutou Huaisiyu Jiaonang(股骨头坏死愈胶囊,GGTHSYJN)in treatment of ster-oid-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 GGTH-SYJN 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 in-tragastric 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-KB(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 trabe-culae 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 os-teonecrosis 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 osteone-crosis incidence rate between the 2 groups revealed no significant difference(P=0.054).The percentage of empty lacunae and the inci-dence 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 com-pared 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 treat-ment 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-KB signaling pathway and macrophage polarization.
femur head necrosishormonesGugutou Huaisiyu Jiaonangimmunohistochemistrymacrophagessignal transduction
万方数据
维普
