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.