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[1]簡羿,馬茂瀟,郭珈宜,等.基于多重免疫組化技術(shù)探討股骨頭壞死愈膠囊治療激素性股骨頭壞死的作用機制[J].中醫(yī)正骨,2024,36(02):23-31.
　JIAN Yi,MA Maoxiao,GUO Jiayi,et al.The mechanism of Gugutou Huaisiyu Jiaonang(股骨頭壞死愈膠囊)in treatment of steroid-induced osteonecrosis of femoral head:a multiplex immunohistochemistry technique-based experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(02):23-31.
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基于多重免疫組化技術(shù)探討股骨頭壞死愈膠囊治療激素性股骨頭壞死的作用機制()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期數(shù):
2024年02期
頁碼:
23-31
欄目:
基礎(chǔ)研究
出版日期:
2024-02-20

文章信息/Info

Title:
The mechanism of Gugutou Huaisiyu Jiaonang(股骨頭壞死愈膠囊)in treatment of steroid-induced osteonecrosis of femoral head:a multiplex immunohistochemistry technique-based experimental study
作者:
簡羿1; 馬茂瀟2; 郭珈宜2; 劉又文2; 岳辰2
(1.湖南中醫(yī)藥大學(xué)研究生院,湖南 長沙 410208; 2.河南省洛陽正骨醫(yī)院/河南省骨科醫(yī)院,河南 洛陽 471002)
Author(s):
JIAN Yi1; MA Maoxiao2; GUO Jiayi2; LIU Youwen2; YUE Chen2
1.Postgraduate College of Hunan University of Chinese Medicine,Changsha 410208,Hunan,China 2.Luoyang Orthopedic-Traumatological Hospital,Luoyang 471002,Henan,China
關(guān)鍵詞:
股骨頭壞死;  激素類;  股骨頭壞死愈膠囊;  免疫組織化學(xué);  巨噬細(xì)胞;  信號傳導(dǎo)
Keywords:
femur head necrosis;  hormones;  Gugutou Huaisiyu Jiaonang;  immunohistochemistry;  macrophages;  signal transduction
摘要:
目的:觀察股骨頭壞死愈膠囊治療激素性股骨頭壞死(steroid-induced osteonecrosis of femoral head,SONFH)的效果,基于多重免疫組化(multiplex immunohistochemistry,mIHC)技術(shù)探討其治療SONFH的作用機制。方法:將100只SD大鼠隨機分為正常對照組、模型組、低劑量藥物治療組、高劑量藥物治療組。將模型組、低劑量藥物治療組、高劑量藥物治療組大鼠采用改良的脂多糖聯(lián)合甲潑尼龍法建立SONFH模型。造模結(jié)束后,低劑量藥物治療組按照0.33 g·kg-1的劑量給予股骨頭壞死愈膠囊溶液(將股骨頭壞死愈膠囊粉劑溶于水中)灌胃,高劑量藥物治療組按照0.67 g·kg-1的劑量給予股骨頭壞死愈膠囊溶液灌胃,正常對照組和模型組給予等量生理鹽水灌胃。每日灌胃2次,連續(xù)治療4周。治療結(jié)束后,采用Micro-CT掃描分析大鼠股骨頭松質(zhì)骨微結(jié)構(gòu),組織切片染色觀察大鼠股骨頭組織病理學(xué)改變,并采用mIHC分析巨噬細(xì)胞極化及Toll樣受體4(Toll-like receptor 4,TLR4)/髓樣分化因子初次應(yīng)答基因88(myeloid differentiation primary response gene 88,MyD88)/核因子-κB(nuclear factor-κB,NF-κB)信號通路相關(guān)蛋白表達(dá)。結(jié)果:①一般結(jié)果。模型組1只大鼠于造模后死亡,低劑量藥物治療組和高劑量藥物治療組分別有3只和2只大鼠在治療過程中死亡。正常對照組大鼠精神狀態(tài)良好、進(jìn)食正常、毛發(fā)光澤無脫落,模型組、低劑量藥物治療組、高劑量藥物治療組大鼠在造模開始后第3天出現(xiàn)神情不振、進(jìn)食減少、少量脫毛,1周后精神、進(jìn)食等逐漸恢復(fù)。②大鼠股骨頭松質(zhì)骨微結(jié)構(gòu)Micro-CT掃描分析結(jié)果。模型組大鼠股骨頭松質(zhì)骨骨體積分?jǐn)?shù)、骨小梁厚度、骨小梁數(shù)量小于正常對照組(P=0.000,P=0.000,P=0.000),骨小梁分離度大于正常對照組(P=0.000); 低劑量藥物治療組大鼠股骨頭松質(zhì)骨骨體積分?jǐn)?shù)、骨小梁厚度與模型組的差異均無統(tǒng)計學(xué)意義(P=0.052,P=0.071),骨小梁數(shù)量大于模型組(P=0.012),骨小梁離散度小于模型組(P=0.001); 高劑量藥物治療組大鼠股骨頭松質(zhì)骨骨體積分?jǐn)?shù)、骨小梁厚度、骨小梁數(shù)量大于模型組(P=0.001,P=0.011,P=0.000),骨小梁離散度小于模型組(P=0.001),骨體積分?jǐn)?shù)、骨小梁厚度、骨小梁數(shù)量、骨小梁離散度與低劑量藥物治療組的差異均無統(tǒng)計學(xué)意義(P=0.146,P=0.414,P=0.086,P=0.146)。③大鼠股骨頭組織病理學(xué)觀察結(jié)果。模型組大鼠股骨頭空骨陷窩率、骨壞死發(fā)生率均高于正常對照組(P=0.000,P=0.000); 低劑量藥物治療組大鼠股骨頭空骨陷窩率低于模型組(P=0.000),骨壞死發(fā)生率與模型組的差異無統(tǒng)計學(xué)意義(P=0.054); 高劑量藥物治療組大鼠股骨頭空骨陷窩率和骨壞死發(fā)生率均低于模型組(P=0.000,P=0.000),空骨陷窩率低于低劑量藥物治療組(P=0.049),骨壞死發(fā)生率與低劑量藥物治療組的差異無統(tǒng)計學(xué)意義(P=0.556)。④巨噬細(xì)胞極化mIHC分析結(jié)果。模型組大鼠股骨頭M1型巨噬細(xì)胞、M2型巨噬細(xì)胞占比均高于正常對照組(P=0.000,P=0.000); 低劑量藥物治療組大鼠股骨頭M1型巨噬細(xì)胞、M2型巨噬細(xì)胞占比與模型組的差異均無統(tǒng)計學(xué)意義(P=0.270,P=0.533); 高劑量藥物治療組大鼠股骨頭M1型巨噬細(xì)胞占比低于模型組(P=0.009),與低劑量藥物治療組的差異無統(tǒng)計學(xué)意義(P=0.131),M2型巨噬細(xì)胞占比高于模型組和低劑量藥物治療組(P=0.006,P=0.038)。⑤TLR4/MyD88/NF-κB信號通路蛋白表達(dá)mIHC分析結(jié)果。模型組大鼠股骨頭TLR4、MyD88、NF-κB p65蛋白相對表達(dá)量均高于正常對照組(P=0.000,P=0.000,P=0.000); 低劑量藥物治療組大鼠股骨頭TLR4、MyD88蛋白相對表達(dá)量與模型組的差異均無統(tǒng)計學(xué)意義(P=0.268,P=0.280),NF-κB p65蛋白相對表達(dá)量低于模型組(P=0.034); 高劑量藥物治療組TLR4、MyD88、NF-κB p65蛋白相對表達(dá)量均低于模型組和低劑量藥物治療組(TLR4:P=0.002,P=0.040; MyD8:P=0.000,P=0.013; NF-κB p65:P=0.000,P=0.039)。結(jié)論:股骨頭壞死愈膠囊治療SONFH,能夠顯著改善股骨頭骨微結(jié)構(gòu)、抑制骨壞死,其作用具有一定的劑量依賴性; 其作用機制與調(diào)控TLR4/MyD88/NF-κB信號通路及巨噬細(xì)胞極化有關(guān)。
Abstract:
Objective:To observe the outcome of Gugutou Huaisiyu Jiaonang(股骨頭壞死愈膠囊,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.

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備注/Memo

備注/Memo:
基金項目:國家自然科學(xué)基金項目(82074472); 2022年度河洛青年人才托舉工程項目(2022HLTJ15)
通訊作者:岳辰 E-mail:[email protected]
更新日期/Last Update: 1900-01-01