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

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

LI Weiyi²，JIANGLu，ZHANG Zongxing¹²，CHENDan，BAOZhuoma¹²，HUANGLi，YUANLin12 1Hubeirocialybtofaticecedertolceeeubeini Enshi44500ohina;DepartmentofeneraledicinuCountyspitalofaditioalinseedicinoni China;DeprtmntofeumtologyhiHuiyiHialofumticseses，hi4in

Abstract：ObjectiveToinvestigatetheeffectofQiangguKangshuFormula（QGKSF）foraleviatingosteoclastdiferentiationin rheumatoidarthritisand theunderlying mechanism.MethodsRAW264.7cels cultured under hypoxicconditions were treatedwithRANKLtoinduceosteoclastdiferentiationandincubatedwithnormalratserumorserafromratsmedicated with methotrexate （MTX）or QGKSF atlowand high doses.Cellviability， TRAP-positive multinucleated cels andF-actinring formation inthe treated celswere assessed with CCK-8 assayTRAPstaining and Pallidin staining，respectively. Autophagy and autophagosomes in the cels were observed with MDC staining and transmission electron microscopy. ELISA was used to measureIL-6and TNF- α levels inteurepatatteexpressiosofH-1，c-2li，--， P62 and TRAP mRNAs and proteins were analyzed using RT-qPCR and Western bloting. Results In hypoxia- and RANKLinduced RAW264.7cels treated with normal ratserum，significant incrementsof TRAP-positivecellsandF-actin ring formationwereobservedwithanenhancedautophagicfluorescence intensityandincreasedautophagosomes.Treatmentof theinducedcelswithratseramedicatedwithMTXandlow-andhigh-doseQGKSFobviouslyreducedtheTRAP-positive cels，F-actinringsandautophagosomesaswellastheautophagicfluorescenceintensityRANKLtreatmentsignificantly increasedIL-6andTNF- α levelsinRAW264.7cels，whichwereobviouslydecreasedby treatmentwithMTX-and QGKSFmedicatedsaoitlysddotesesofF， LC3and TRAPand lowered P62 expresionsand these changes were effectively reversedbytreatment with MTX-and QGKSF-medicated sera.Conclusion QGKSF atenuatesRANKL-induced osteoclast diferentiation inhypoxicRAW264.7cels by inhibitingthe HIF-1α/BNIP3autophagy signaling pathwaysuggesting its potential for treatmentof bone destruction in rheumatoid arthritis.

Keywords： Qianggu Kangshu Formula;rheumatoidarthritisbone destruction;autophagy;HIF-1α/BNP3signalingpathway; osteoclasts

类风湿关节炎（RA）是一种以关节炎症、增生肿胀、软骨和骨破坏为特征的慢性炎症性关节疾病[12]。（剩余13226字）