Abstract: BACKGROUND: Osteonecrosis of the femoral head is a common and disabling disease, which is mainly characterized by microcirculation disorders and bone cell metabolism disorders. Luzhongjiangu decoction was developed by Shandong Academy of Chinese Medicine and used in the form of soup in the clinic, which has good efficacy in the treatment of osteonecrosis of the femoral head. However, its mechanism of action has not been clarified. 
OBJECTIVE: To study the effect mechanism of Luzhongjiangu decoction on intestinal flora in rats with osteonecrosis of the femoral head based on 16S rDNA sequencing technique.
METHODS: The model of osteonecrosis of the femoral head was established in Wistar rats by intragastric administration of retinoic acid. The therapeutic effect of Luzhongjiangu decoction was evaluated by serum hormone, bone histopathology and serum hormone levels. 16s rDNA sequencing technique was used to detect the intestinal flora of rats in the blank control group, model group and middle-dose Luzhongjiangu decoction group. The corresponding library was constructed and OTU clustering and microbial community diversity and abundance analysis were carried out to determine the composition of intestinal flora and the changes of species and diversity among groups.
RESULTS AND CONCLUSION: Luzhongjiangu decoction could significantly increase the expression of osteocalcin, osteopontin and other osteogenic related factors, alleviate the destruction of bone trabeculae, increase bone mineral density, and had a significant therapeutic effect on osteonecrosis of the femoral head, of which the middle dose group showed the most significant effect. The results of intestinal flora sequencing showed that Luzhongjiangu decoction improved the flora disorder of rats with osteonecrosis of the femoral head to some extent, and screened out different colonies such as Bacillus, Desulfurizans, Desulfurization, Isobacteria, Bifidobacterium and so on; it could up-regulate the abundance of beneficial bacteria such as Bifidobacterium, down-regulate the abundance of harmful bacteria such as Desulfovibrio, and improve the structure of intestinal flora. Functional prediction analysis indicated that Luzhongjiangu decoction could mainly affect amino acid metabolism and energy metabolism. Correlation analysis showed that the differential bacteria of Bifidobacterium and Intestinimonas in the middle dose group of Luzhongjiangu decoction were positively correlated with vitamin D3, estradiol and calcitonin, and negatively correlated with prostaglandin E2. In the model group, Escherichia-Shigella, Desulfovibrio, Globicatella and Streptococcus were positively correlated with prostaglandin E2 and negatively correlated with vitamin D3, estradiol and calcitonin. To conclude, Luzhongjiangu decoction may play a role in the treatment of osteonecrosis of the femoral head by regulating the structure of intestinal flora, up-regulating the abundance of beneficial bacteria and affecting the secretion of vitamin D3, estradiol, calcitonin and prostaglandin E2.

Key words: Luzhongjiangu decoction, osteonecrosis of the femoral head, 16S rDNA, intestinal flora, hormone, vitamin D3, estradiol, calcitonin