Qianggu Kangshu Formula attenuates osteoclast differentiation in rheumatoid arthritis by inhibiting the HIF-1α/BNIP3 autophagy signaling pathway

doi:10.12122/j.issn.1673-4254.2025.07.05

Abstract

Abstract:

Objective To investigate the effect of Qianggu Kangshu Formula (QGKSF) for alleviating osteoclast differentiation in rheumatoid arthritis and the underlying mechanism. Methods RAW264.7 cells cultured under hypoxic conditions were treated with RANKL to induce osteoclast differentiation and incubated with normal rat serum or sera from rats medicated with methotrexate (MTX) or QGKSF at low and high doses. Cell viability, TRAP-positive multinucleated cells and F-actin ring formation in the treated cells were assessed with CCK-8 assay, TRAP staining and Phalloidin staining, respectively. Autophagy and autophagosomes in the cells were observed with MDC staining and transmission electron microscopy. ELISA was used to measure IL-6 and TNF-α levels in the culture supernatant, and the expressions of HIF-1α, BNIP3, Bcl-2, Beclin1, LC3-I, LC3-II, P62 and TRAP mRNAs and proteins were analyzed using RT-qPCR and Western blotting. Results In hypoxia- and RANKL-induced RAW264.7 cells treated with normal rat serum, significant increments of TRAP-positive cells and F-actin ring formation were observed with an enhanced autophagic fluorescence intensity and increased autophagosomes. Treatment of the induced cells with rat sera medicated with MTX and low- and high-dose QGKSF obviously reduced the TRAP-positive cells, F-actin rings and autophagosomes as well as the autophagic fluorescence intensity. RANKL treatment significantly increased IL-6 and TNF-α levels in RAW264.7 cells, which were obviously decreased by treatment with MTX- and QGKSF-medicated sera. RANKL also significantly increased the mRNA and protein expression levels of HIF-1α, BNIP3, Bcl-2, Beclin1, LC3 and TRAP and lowered P62 expressions, and these changes were effectively reversed by treatment with MTX- and QGKSF-medicated sera. Conclusion QGKSF attenuates RANKL-induced osteoclast differentiation in hypoxic RAW264.7 cells by inhibiting the HIF-1α/BNIP3 autophagy signaling pathway, suggesting its potential for treatment of bone destruction in rheumatoid arthritis.

Key words: Qianggu Kangshu Formula, rheumatoid arthritis bone destruction, autophagy, HIF-1α/BNIP3 signaling pathway, osteoclasts

Weiyi LI, Lu JIANG, Zongxing ZHANG, Dan CHEN, Zhuoma BAO, Li HUANG, Lin YUAN. Qianggu Kangshu Formula attenuates osteoclast differentiation in rheumatoid arthritis by inhibiting the HIF-1α/BNIP3 autophagy signaling pathway[J]. Journal of Southern Medical University, 2025, 45(7): 1389-1396.

Figures/Tables 9

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequence 5'-3'
β-actin	F: GTGACGTTGACATCCGTAAAGA R: GTAACAGTCCGCCTAGAAGCAC
HIF-1α	F: TTGCTTTGATGTGGATAGCGATA R: CATACTTGGAGGGCTTGGAGAAT
BNIP3	F: GAAAAACAGCACTCTGTCTGAGGAA R: GACCAATCCCATATCCAATCTGA
BCL-2	F: TGACTTCTCTCGTCGCTACCGT R: CCTGAAGAGTTCCTCCACCACC
Beclin1	F: GCCAATAAGATGGGTCTGAAGTT R: CCACCTCTTCTTTGAACTGCTG
P62	F: CACTACCGCGATGAGGATGG R: CTGCACTTATAGCGAGTTCCCAC
LC3	F: TCCGACCGGCCTTTCAA R: TCACCCTTGTAGCGCTCGAT
TRAP	F: CACGATGCCAGCGACAAGA R: TGAAGCGCAAACGGTAGTAAGG

Fig.1 Effect of rat sera medicated with Qianggu Kangshu Formula (QGKSF) and methotrexate (MTX) on survival rate of RAW264.7 cells cultured in hypoxia for 72 h and 120 h (Mean±SD, n=5). *P<0.05, **P<0.01 vs Control group.

Fig.2 TRAP staining showing RAW264.7 cell differentiation into osteoclasts in different groups (Original magnification: ×200). ***P<0.001 vs Control group; ###P<0.001 vs Model group (Mean±SD, n=3).

Fig.3 Effect of QGKSF-medicated serum on actin ring formation in RAW264.7 cells induced by hypoxia +RANKL (×200). ***P<0.001 vs Control group; ###P<0.001 vs Model group.

Fig.4 Effect of QGKSF-medicated sera on autophagy in hypoxia- and RANKL-induced RAW264.7 cells (×200). ***P<0.001 vs Control group; ##P<0.01, ###P<0.001 vs Model group.

Fig.5 Observation of autophagosomes (red arrows) and autolysosomes (blue arrows) in RAW264.7 cells in different groups under transmission electron microscopy (×10 000).

Fig.6 Levels of inflammatory cytokines IL-6 and TNF‑α in RAW264.7 cell cultures determined with ELISA. ***P<0.001 vs Control group; ###P<0.001 vs Model group.

Fig.7 Effects of QGKSF-medicated sera on protein expressions of osteoclast markers, autophagy markers and the HIF-1α/BNIP3 signaling pathway in RAW264.7 cells. A: Protein expressions of osteoclast markers and the HIF-1α/BNIP3 signaling pathway. B: HIF-1α expression level. C: BNIP3 expression level. D: Protein expressions of autophagy markers. E: TRAP expression level. F: Bcl-2 expression level. G: Beclin1 expression level. H: P62 expression level. I: LC3 expression level. Data are presented as Mean±SD (n=3). **P<0.01, ***P<0.001 vs Control group; #P<0.05, ##P<0.01, ###P<0.001 vs Model group.

Fig.8 Effects of QGKSF-medicated sera on mRNA expressions of osteoclast markers, autophagy markers and the HIF-1α/BNIP3 signaling pathway in RAW264.7 cells. A: HIF-1α mRNA expression level. B: BNIP3 mRNA expression level. C: TRAP mRNA expression level. D: Bcl-2 mRNA expression level. E: Beclin1 mRNA expression level. F: P62 mRNA expression level. G: LC3 mRNA expression level. Data are presented as Mean±SD (n=3). ***P<0.001 vs Control group; #P<0.05, ##P<0.01, ###P<0.001 vs Model group.

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