强骨康疏方通过抑制HIF-1α/BNIP3自噬信号通路减少类风湿性关节炎大鼠的破骨细胞分化

doi:10.12122/j.issn.1673-4254.2025.07.05

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (7): 1389-1396.doi: 10.12122/j.issn.1673-4254.2025.07.05

强骨康疏方通过抑制HIF-1α/BNIP3自噬信号通路减少类风湿性关节炎大鼠的破骨细胞分化

李玮怡¹^,²(), 江露³, 张宗星¹^,², 陈丹¹, 包卓玛¹^,², 黄丽⁴, 袁林¹^,²()

^1.湖北民族大学风湿性疾病发生与干预湖北省重点实验室，湖北恩施 445000
^2.湖北民族大学医学部，湖北恩施 445000
^3.石柱县中医院全科医学科，重庆 409100
^4.恩施慧宜中西医结合风湿医院风湿病科，湖北恩施 445000

收稿日期:2024-09-26 出版日期:2025-07-20 发布日期:2025-07-17
通讯作者: 袁林 E-mail:1255688798@qq.com;171354806@ qq. com
作者简介:李玮怡，在读硕士研究生，E-mail: 1255688798@qq.com
基金资助:
国家自然科学基金(82460827);湖北省科技计划项目(2021CSA069);风湿性疾病发生与干预湖北省重点实验室2024年度开放基金(PT022406)

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

Weiyi LI¹^,²(), Lu JIANG³, Zongxing ZHANG¹^,², Dan CHEN¹, Zhuoma BAO¹^,², Li HUANG⁴, Lin YUAN¹^,²()

^1.Hubei Provincial Key Laboratory of Rheumatic Disease Occurrence and Intervention, Hubei Minzu University, Enshi 445000, China
^2.Health Science Center, Hubei Minzu University, Enshi 445000, China
^3.Department of General Medicine, Shizhu County Hospital of Traditional Chinese Medicine, Chongqing 409100, China
^4.Department of Rheumatology, Enshi Huiyi Hospital of Rheumatic Diseases, Enshi 445000, China

Received:2024-09-26 Online:2025-07-20 Published:2025-07-17
Contact: Lin YUAN E-mail:1255688798@qq.com;171354806@ qq. com
Supported by:
National Natural Science Foundation of China(82460827)

摘要/Abstract

摘要：

目的研究强骨康疏方（QGKSF）对HIF-1α/BNIP3自噬信号通路的调控作用及其改善类风湿关节炎的作用机制。方法制备含药血清，RAW264.7细胞体外培养，低氧+RANKL诱导破骨细胞。分别设置对照组、模型组、甲氨蝶呤组（MTX）、 QGKSF低剂量组（QGKSF-L）、QGKSF高剂量组（QGKSF-H）。CCK-8法检测细胞活力；TRAP染色观察各组TRAP阳性多核细胞的数量；鬼笔环肽染色检测F-肌动蛋白环形成情况；MDC染色观察自噬泡；透射电镜观察自噬小体；ELISA检测炎症细胞因子（IL-6、TNF-α）水平；Western blotting检测HIF-1α、BNIP3、Bcl-2、Beclin1、LC3-Ⅰ、LC3-Ⅱ、P62、TRAP的蛋白水平； RT-qPCR检测HIF-1α、BNIP3、Bcl-2、Beclin1、LC3、P62、TRAP的mRNA水平。结果与对照组比较，模型组可见大量TRAP阳性细胞，F-肌动蛋白环形成增多，自噬荧光强度增加，自噬小体增多（P<0.001）；与模型组比较，MTX组、QGKSF-L组和QGKSF-H组的TRAP阳性细胞数量和F-肌动蛋白环形成减少，自噬荧光强度减弱，自噬小体减少（P<0.01）。与对照组相比，模型组IL-6、TNF-α水平升高（P<0.001）；经过MTX和QGKSF治疗后IL-6、TNF-α水平有所降低（P<0.001）。与对照组相比，模型组HIF-1α、BNIP3、Bcl-2、Beclin1、LC3、TRAP蛋白及mRNA水平升高，P62蛋白及mRNA水平降低（P<0.01）；经MTX及QGKSF治疗后，HIF-1α、BNIP3、Bcl-2、Beclin1、LC3、TRAP蛋白及mRNA水平降低，P62蛋白及mRNA水平升高（P<0.05）。结论低氧条件下，QGKSF可能通过抑制HIF-1α/BNIP3自噬信号通路来减少RANKL诱导的RAW264.7向破骨细胞分化，从而发挥治疗RA骨破坏的作用。

关键词: 强骨康疏方, 类风湿关节炎骨破坏, 自噬, HIF-1α/BNIP3信号通路, 破骨细胞

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

李玮怡, 江露, 张宗星, 陈丹, 包卓玛, 黄丽, 袁林. 强骨康疏方通过抑制HIF-1α/BNIP3自噬信号通路减少类风湿性关节炎大鼠的破骨细胞分化[J]. 南方医科大学学报, 2025, 45(7): 1389-1396.

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.

图/表 9

表1 引物序列

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

图1 低氧状态下72 h及120 h QGKSF及MTX对细胞存活率的影响

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.

图2 RAW264.7向OC分化的TRAP染色结果

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).

图3 QGKSF对低氧+RANKL诱导的RAW264.7肌动蛋白环的影响

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.

图4 QGKSF对RAW264.7细胞自噬的影响

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.

图5 透射电子显微镜观察自噬小体和自噬溶酶体

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

图6 ELISA检测炎症因子IL-6、TNF-α水平

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.

图7 QGKSF对OC、自噬及信号通路相关蛋白的影响

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.

图8 QGKSF对OC、自噬及通路相关mRNA水平的影响

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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强骨康疏方通过抑制HIF-1α/BNIP3自噬信号通路减少类风湿性关节炎大鼠的破骨细胞分化

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

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