摘要
目的:观察补肾活血胶囊防治大鼠激素性股骨头坏死的效果,并基于微生物组学和代谢组学分析探究其作用机制。方法:将24只大鼠随机分为空白组、模型组和补肾活血胶囊组,每组8只。模型组、补肾活血胶囊组大鼠采用右侧臀肌注射甲泼尼龙琥珀酸钠建立激素性股骨头坏死模型,每周前3 d连续给药,每天一次,持续3周;在造模的同时,补肾活血胶囊组大鼠按照生药浓度5.4 g·kg^(-1)·d^(-1)的剂量给予补肾活血胶囊药液灌胃,每天一次,连续灌胃6周;空白组和模型组以等量纯净水灌胃。干预结束后,采用Micro-CT扫描仪扫描大鼠右侧股骨头,观察大鼠股骨头影像学变化,并分析股骨头骨小梁体积和骨体积;采用HE染色观察大鼠左侧股骨头骨小梁数量变化和脂肪细胞浸润情况;采用Western Blot法检测大鼠右侧股骨头骨形成关键蛋白Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)和Osterix的蛋白表达水平。采用16S核糖体DNA测序鉴定大鼠粪便中的菌群,并进行差异肠道菌群分析和功能预测。采用液相色谱法和质谱法检测各组大鼠粪便样本中的代谢物,并进行差异肠道代谢物分析和KEGG通路富集分析。结果:①股骨头Micro-CT扫描分析结果。空白组大鼠股骨头轮廓规则,软骨下骨骨小梁有序排列,无囊性变;模型组大鼠股骨头轮廓不规则,软骨下骨骨小梁缺失、紊乱,部分可见囊性变结构;补肾活血胶囊组大鼠股骨头损伤情况较模型组改善。模型组大鼠股骨头骨小梁体积和骨体积均小于空白组(P=0.002,P=0.000),补肾活血胶囊组大鼠股骨头骨小梁体积和骨体积均大于模型组(P=0.002,P=0.000),补肾活血胶囊组与空白组大鼠股骨头骨小梁体积和骨体积的组间差异均无统计学意义(P=0.994,P=0.186)。②股骨头病理学观察结果。空白组大鼠股骨头软骨下骨骨小梁分布均匀,无脂肪细胞浸润;模型组大鼠股骨头软骨下骨骨小梁稀疏或中断,有脂肪细胞浸润;补肾活血胶囊组大鼠股骨头软骨下骨骨小梁较模型组致密,脂肪细胞浸润较模型组减轻。③股骨头骨形成关键蛋白表达水平检测结果。模型组大鼠股骨头中RUNX2和Osterix的蛋白相对表达量均低于空白组(P=0.000,P=0.001),补肾活血胶囊组大鼠股骨头中RUNX2和Osterix的蛋白相对表达量均高于模型组(P=0.000,P=0.000),补肾活血胶囊组大鼠股骨头中RUNX2和Osterix的蛋白相对表达量与空白组比较,组间差异均无统计学意义(P=0.279,P=0.509)。④肠道菌群鉴定与分析结果。在门水平上,补肾活血胶囊组大鼠肠道菌群中帕特西菌门、疣微菌门和梭杆菌门的丰度较模型组下降;在属水平上,补肾活血胶囊组大鼠肠道菌群中嗜冷杆菌属的丰度较模型组升高,糖单胞菌属、苏黎世杆菌属和阿克曼菌属等的丰度较模型组降低。补肾活血胶囊组大鼠肠道菌群在类黄酮的生物合成、异黄酮的生物合成和肥厚型心肌病等方面的基因丰度显著低于模型组。⑤肠道代谢物检测与分析结果。补肾活血胶囊组大鼠与模型组的差异肠道代谢物,依据变量投影重要性由高至低排序,位于前20的依次为胆固醇硫酸盐、油酰胺、麻黄素、反式长春花酸、次黄嘌呤、刺桐素、N-乙酰神经氨酸、2-哌啶酮、左旋肉碱、4-吡哆酸、含有菲-6-酮结构的化合物、可的松、丁酸、脱氧腺苷、棕榈酰胺、原阿片碱、芥酰胺、雄烯二酮、反式脱氢雄甾酮以及N-甲基-1-谷氨酸。补肾活血胶囊组和模型组的差异肠道代谢物富集的通路主要有前列腺癌、类固醇激素的生物合成、α-亚麻酸代谢、维生素B6代谢、癌症中的信号通路等。结论:补肾活血胶囊能够改善股骨头骨微结构、促进骨形成、抑制骨坏死,其作用机制与调节肠道菌群及肠道代谢物存在密切关系。
Objective:To observe the outcomes of Bushen Huoxue Jiaonang(补肾活血胶囊,BSHXJN)against steroid-induced osteonecrosis of the femoral head(SONFH)in rats,and to analyze and explore its underlying mechanisms based on microbiomics and metabono-mics.Methods:Twenty-four rats were randomized into blank group,model group,and BSHXJN group,with 8 ones in each group.All rats but the ones in blank group were modeled by intramuscular injection of methylprednisolone sodium succinate into the right gluteal region for inducing SONFH on the first three days of each week,once a day for consecutive 3 weeks.During the SONFH modeling,the rats in BSHXJN group were intervened by intragastric administration with BSHXJN solution at a crude dose of 5.4 g/kg/day,while the ones in blank group and model group with the equivalent volume of purified water,once a day for consecutive 6 weeks.After the end of the last intervention,the rats were sacrificed and their bilateral femur heads and faeces were harvested.The right femur heads were scanned by a Micro-CT scanner to observe the imaging changes,and analyze the trabecular bone volume(TBV)and bone volume(BV).After that,the left femur heads were stained with HE to observe the changes in trabecular quantity and the degree of adipocyte infiltration.Furthermore,the protein expression levels of osteogenic markers including Runt-related transcription factor 2(RUNX2)and Osterix in the rat right femur heads were detected by using Western Blot,the microbial communities in the rat fecal samples were identified by 16S ribosomal DNA(rDNA)sequencing,followed by differential gut microbiota analysis and functional prediction.Moreover,the metabolites in the fecal samples of rats in each group were detected by liquid chromatography-mass spectrometry(LC-MS),and the differential gut metabolite analysis and KEGG pathway enrichment analysis were performed.Results:①Micro-CT analysis on femur heads.The femur heads in rats of control group exhibited regular contours with well-organized subchondral trabeculae,and no cystic changes,whereas the femur heads in model group displayed irregular contours,accompanied by missed and disordered subchondral trabeculae,and some cystic structures,while these deteriorations were mitigated in BSHXJN group compared to model group.Apart from that,the TBV and BV were smaller in model group compared to blank group,and were greater in BSHXJN group compared to model group(P=0.002,P=0.000;P=0.002,P=0.000),with no significant difference between BSHXJN group and blank group(P=0.994,P=0.186).②The pathological findings in femur heads.In blank group,the subchondral trabeculae of femur heads presented uniform distribution with no adipocyte infiltration;while that in the model group demonstrated sparse or discontinuous distribution,accompanied by adipocyte infiltration;whereas,the changes were ameliorated in BSHXJN group compared to model group,manifesting as denser subchondral trabeculae and significantly mitigated adipocyte infiltration in the femur heads.③The protein expression levels of osteogenic markers.The relative protein expression levels of RUNX2 and Osterix in the femur heads were lower in model group compared to blank group,and were higher in BSHXJN group compared to model group(P=0.000,P=0.001;P=0.000,P=0.000),with no significant difference between BSHXJN group and blank group(P=0.279,P=0.509).④The gut microbiota.At the phylum level,the abundance of Patescibacteria,Verrucomicrobiota,and Fusobacteriota in the gut microbiota decreased in BSHXJN group compared to model group;At the genus level,the abundance of Psychrobacter in the gut microbiota increased,while that of Candidatus Saccharimonas,Turicibacter and Akkermansia decreased in BSHXJN group compared to model group;Furthermore,the gene abundance related to flavonoid biosynthesis,isoflavonoid biosynthesis,and hypertrophic cardiomyopathy(HCM)in the gut microbiota was significantly lower in BSHXJN group compared to model group.⑤The gut metabolites.The differential gut metabolites between BSHXJN group and model group were sorted by the variable importance in projection(VIP)from high to low,and the top 20 ones were Cholesterol Sulfate,Oleamide,Ephedrine,trans-Vaccenic Acid,Hypoxanthine,Erythraline,N-Acetylneuraminic Acid(Neu5Ac),2-Piperidone,L-Carnitine,4-Pyridoxic Acid,Phenanthren-6-one-containing Compound,Cortisone,Butyric Acid,Deoxyadenosine,Palmitamide,Protopine,Erucamide,Androstenedione,trans-Dehydroandrosterone,and N-Methyl-1-glutamic Acid,and they were mainly enriched in Prostate Cancer pathways(KEGG:ko05215),Steroid hormone biosynthesis pathways(KEGG:ko00140),Alpha-linolenic acid metabolism pathways(KEGG:ko00592),Vitamin B6 metabolism pathways(KEGG:ko00750),and pathways in cancer(KEGG:ko05200).Conclusion:BSHXJN can ameliorate the bone microstructure of femur heads,promote osteogenesis,and inhibit osteonecrosis.It may work by modulating the gut microbiota and gut metabolite.
作者
李威
张博淳
周忠起
李广政
李刚
梁学振
LI Wei;ZHANG Bochun;ZHOU Zhongqi;LI Guangzheng;LI Gang;LIANG Xuezhen(The First Clinical Medical College of Shandong University of Traditional Chinese Medicine,Jinan 250014,Shandong,China;College of Traditional Chinese Medicine,Shandong University of Traditional Chinese Medicine,Jinan 250355,Shandong,China;The Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250014,Shandong,China)
出处
《中医正骨》
2025年第5期6-20,29,共16页
The Journal of Traditional Chinese Orthopedics and Traumatology
基金
国家自然科学基金项目(82205154,82074453)
山东省自然科学基金项目(ZR2024MH156)。
