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Duhuo Jisheng Decoction inhibits the activity of osteoclasts in osteonecrosis of the femoral head via regulation of the RELA/AKT1 axis.

OBJECTIVE
To investigate the effect of Duhuo Jisheng Decotion (DHJSD) on the activity of osteoclasts in osteonecrosis of the femoral head (ONFH) and its underlying mechanism relating to the RELA/AKT1 axis.


METHODS
The TCMSP database was used to search for the effective ingredients and the targets of various Chinese medicines in DHJSD. Its targets were intersected with ONFH risk genes in DisGeNET and Malacards databases to obtain the potential target genes. qRT-PCR was used to detect the expression of potential target genes in ONFH tissues, and the ChIP experiment was used to verify the relationship between RELA and AKT1 promoter. An ONFH rat model was established and DHJSD was used for the treatment. The expressions of RELA and AKT1 in rats were intervened, and rats were grouped. qRT-PCR was applied to detect the expression levels of osteoclast markers ACP5, CTSK, and RANK in the tissues to evaluate the regulation of DHJSD on target genes and the mechanism of osteoclast differentiation.


RESULTS
A total of 231 effective targets of DHJSD were screened out in the TCMSP database. Intersection with ONFH risk genes yielded a total of 20 candidate genes. Protein-protein interaction analysis showed that AKT1 regulated other genes. KEGG functional enrichment analysis revealed that STAT1, AKT1, PPARG, PPARG, TNF and RELA were enriched in osteoclast differentiation pathway. Compared with normal tissues, the expression of STAT1 was decreased in ONFH tissues, and the expressions of AKT1, PPARG, TNF, and RELA were increased, among which, RELA and AKT1 are the most significantly increased genes (all P<0.05). ChIP experiment found that RELA had a binding relationship with AKT1 promoter. DHJST had the inhibitory effect on the expression of RELA and AKT1 in ONFH tissues, as well as the levels of ACP5, CTSK, and RANK. However, overexpression of RELA or AKT1 attenuated the inhibitory effect of DHJSD on the levels of ACP5, CTSK and RANK. Meanwhile, knocking down RELA partially reversed the effect of AKT1 on the effect of DHJSD.


CONCLUSION
DHJSD inhibits the activity of osteoclasts in ONFH by inhibiting the RELA/AKT1 axis. This study further clarifies the potential specific mechanism of DHJSD to improve ONFH.