Steroid-induced osteonecrosis of the femoral head (SONFH) is common and disabling. The efficacy of Huoxue Tongluo Capsule has been consistently demonstrated in the prevention and treatment of osteonecrosis of the femoral head, but there is no research on whether the treatment can be carried out by regulating the intestinal flora.

To establish SONFH rat model intervened by Huoxue Tongluo Capsule, analyze and compare the diversity of intestinal flora in each group of rats, screen out SONFH-specific altered bacterial genera and observe the effect of Huoxue Tongluo Capsule on intestinal flora.

24 male SD rats were selected from April to October 2022 and divided into the blank group (n=8), model group (n=8) and Huoxue Tongluo group (n=8) by random number table method. The SONFH model was prepared. The Huoxue Tongluo group was given 0.63 g·kg^-1·d^-1 Huoxue Tongluo Capsule by gavage on the 3rd to 28th day of modeling. The blank group and model group were given an equal volume of 0.9% sodium chloride solution by gavage. After drug intervention, rat femurs were collected for HE staining and Micro-CT analysis. Rat feces were collected for 16S rDNA sequencing. α diversity analysis was used to evaluate species diversity, β diversity analysis was used to explore the similarity or dissimilarity of sample community composition, and linear discriminant analysis (LEfSe) was used to compare the differences of intestinal flora among the three groups. COG function was used to predict the intestinal flora function of the rats in the three groups.

There were significant differences in the rate of empty bone lacunae among the three groups, and the rate of empty bone lacunae in the model group was higher than that in the blank group, while the rate of empty bone lacunae in the Huoxue Tongluo group was lower than that in the model group (P<0.05). There were significant differences in the bone volume (BV/TV), bone trabeculae number (Tb.N), bone trabeculae thickness (Tb.Th) and bone trabeculae separation (Tb.Sp) among the three groups, and the BV/TV, Tb.N and Tb.Th of the model group were lower than those of the blank group, while the Tb.Sp of the model group was higher than that of the blank group (P<0.05). The BV/TV, Tb.N, Tb.Th of the Huoxue Tongluo group were lower than those of the blank group, while the Tb.Sp was higher than that of the blank group (P<0.05). There were significant differences in the intestinal flora structure of rats among the three groups, the number of intestinal flora ASVs in the model group was higher than the blank group, the number of intestinal flora ASVs in the Huoxue Tongluo group was lower than the model group (P<0.05). There were significant differences in the Shannon index among the three groups, and the Shannon index in the model group was higher than the blank group (P<0.05). The PCoA plot of β-diversity analysis showed that there was partial overlap among the three groups, but the community structure of the three groups could be distinguished. The results of similarity analysis showed that the differences among the groups were greater than that within groups, and the differences were statistically significant (R=2 062, P=0.01). The results of LEfSe analysis showed that the abundances of Desulfovibrio, Hafnia-Obesumbacterium, Leuconostoc and Staphylococcus were increased, the abundances of Bacteroides and Prevotella _UCG-001 were decreased (P<0.05). The abundances of Oxalobacteraceae and Ruminococcus_torques_group were increased and the abundances of Rekenella and Kineothrix were decreased in the Huoxue Tongluo group compared with the model group (P<0.05). The prediction results of COG function in the three groups of rats showed that compared with the blank group, COG function of the model group mainly focused on carbohydrate transport and metabolism, inorganic ion transport and metabolism, amino acid transport and metabolism, lipid transport and metabolism, cell wall/membrane/envelope biogenesis. The function of the Huoxue Tongluo group focused on inorganic ion transport and metabolism, carbohydrate transport and metabolism, and the levels of replication, recombination and repair, transcription, cell cycle control, cell division, chromosome division, and cell motor function were increased compared with the model group.

The bacterial diversity of SONFH rats was higher than that of normal rats, and the increased bacteria genera was mainly pathogenic bacteria, with the decrease of corresponding beneficial bacteria genera. Huoxue Tongluo Capsule may achieve the therapeutic purpose of SONFH by changing the abundance and diversity of intestinal flora and regulating the abundance of Oxalobacteraceae, Ruminococcus_torques_group and other genera.

激素性股骨头坏死（SONFH）是一种常见且致残性高的疾病，活血通络胶囊已被多次证明在防治股骨头坏死上疗效显著，但是治疗是否通过调节肠道菌群目前少见研究。

建立SONFH大鼠模型并采用活血通络胶囊进行干预，分析比较各组大鼠肠道菌群的多样性，筛选出SONFH特异性改变的菌属并观察活血通络胶囊对肠道菌群的作用。

2022年4—10月选取雄性SD大鼠24只，采用随机数字表法分为空白组（n=8）、模型组（n=8）、活血通络组（n=8），建造SONFH模型，活血通络组造模第3~28天给予0.63 g·kg^-1·d^-1活血通络胶囊灌胃，空白组和模型组给予等体积0.9%氯化钠溶液灌胃。药物干预结束后收集大鼠股骨进行HE染色、Micro-CT分析。收集大鼠粪便进行16S rDNA测序。采用α多样性和β多样性分析评估物种组间和组内群落的多样性及差异性，采用线性判别分析（LEfSe）比较三组大鼠肠道菌群差异。采用COG功能预测三组大鼠肠道菌群功能。

模型组空骨陷窝率高于空白组、活血通络组（P<0.05）。模型组骨体积分数（BV/TV）、骨小梁数量（Tb.N）、骨小梁厚度（Tb.Th）低于空白组，骨小梁分离度（Tb.Sp）高于空白组（P<0.05）；活血通络组BV/TV、Tb.N、Tb.Th高于模型组，Tb.Sp低于模型组（P<0.05）。三组大鼠肠道菌群结构存在差异，与空白组相比，模型组肠道菌群ASV数目增加；与模型组比较，活血通络组肠道菌群ASV数目减少。模型组Shannon指数高于空白组（P<0.05）。β多样性分析主坐标分析（PCoA）图显示，三组样本存在部分重叠，但是能区分三组的群落结构。相似性分析结果显示，组间差异大于组内，差异有统计学意义（R=2 062，P=0.01）。LEfSe分析结果显示，与空白组比较，模型组脱硫弧菌（Desulfovibrio）、Hafnia-Obesumbacterium、明串珠菌属（Leuconostoc）、葡萄球菌属（Staphylococcus）丰度升高，拟杆菌属（Bacteroides）、普雷沃氏菌属_UCG-001丰度降低（P<0.05）；与模型组比较，活血通络组草酸杆菌属（Oxalobacteraceae）、扭链胃球菌属（Ruminococcus_torques_group）丰度升高，理研菌属（Rekenella）、Kineothrix菌属丰度降低（P<0.05）。三组大鼠COG功能预测结果显示，与空白组比较，模型组菌群COG功能主要集中于碳水化合物运输与代谢、无机离子转运和代谢、氨基酸转运和代谢、脂质转运和代谢、细胞壁/膜/包膜生物发生；与模型组比较，活血通络组功能集中于无机离子转运和代谢、碳水化合物运输与代谢，且复制、重组和修复、转录、细胞周期控制、细胞分裂和染色体分裂、细胞运动功能水平升高。

SONFH大鼠菌群多样性较正常大鼠增加，增加的菌属主要是致病菌属，而相应有益菌属则出现减少。活血通络胶囊可能通过改变肠道菌群的丰度和多样性以及调控Oxalobacteraceae、Ruminococcus_torques_group等菌属丰度而实现治疗SONFH的目的。