Based on the theory of the “gut-osteoporosis” axis and 16S rDNA technology, to explore the effect and mechanism of Modified Huoluo Xiaoling Dan (加味活络效灵丹) on the repair of bone tissue in the rats with steroid-induced avascular necrosis of the femoral head (SANFH), as well as its influence on the structure of the gut microbiota.

Using the random number table method, 24 SPF-grade male SD rats were divided into a normal group (n=8), a model group (n=8) and a treatment group (n=8). In the model group and the treatment group, the method of alternately injecting methylprednisolone sodium succinate into the gluteal muscles was used to establish the model, and the treatment group was given intragastric administration of Modified Huoluo Xiaoling Dan (preparation liquid). For the femoral samples of the rats, HE staining was performed and the empty bone lacuna rate was calculated. Three-dimensional reconstruction by Micro-CT was used to analyze and measure the bone histological parameters such as the bone volume fraction (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp) in the measurement area. Immunohistochemistry method was used to detect the expressions of bone tissue repair markers BMP, OPG and Runx2. For the fecal samples, 16S rDNA technology was used for gut microbiota sequencing and ASV analysis. The α-diversity and β-diversity were used to analyze the species diversity of the gut microbiota, and LEfSe analysis was used to detect the changes in the species differences of the gut microbiota at the family level and genus level.

Modified Huoluo Xiaoling Dan could significantly improve the empty bone lacuna rate of the femoral head (P<0.05) and bone histological parameters such as BV/TV, Tb. N, Tb. Th and Tb. Sp (P<0.01) in the rats with SANFH, and significantly up-regulate the expression levels of bone tissue repair markers BMP, OPG and Runx2 (P<0.01). The results of gut microbiota sequencing showed that α-diversity analysis indicated that there were obvious differences in the species diversity and richness of the gut microbiota structures of the rats in the three groups; β-diversity analysis showed that the flora structure of the treatment group was significantly separated from that of the model group, and was similar to that of the blank group. The difference among groups was greater than that within groups, and the difference was obvious. LEfSe analysis has found that at the family level and genus level, the gut microbiota of the rats in the model group showed significant “pro-inflammatory” characteristics, while the gut microbiota of the rats in the treatment group showed “anti-inflammatory-promoting repair” characteristics.

The effect of Modified Huoluo Xiaoling Dan on the repair of bone tissue in the rats with SANFH is closely related to its regulation of the gut microbiota. The intervention drug effectively reshapes the intestinal microecology disturbed by hormones, further confirming that the reshaping of the flora has a bidirectional regulatory effect on the “gut-osteoporosis” axis, playing a role in preventing and treating SANFH.