Effect of Bushen Huoxue formula on lipopolysaccharide-induced chondrocyte inflammation injury
Objective To determine the mechanism of Bushen Huoxue formula(BSHXF)for osteoarthritis by observing the effect of BSHXF-containing serum on lipopolysaccharide(LPS)-induced chondrocyte inflammation and apoptosis.Methods BSHXF-containing serum and blank serum was prepared.C28/12 human normal chondrocytes were induced by LPS to establish the chondrocyte inflammation model.The cells were divided into a blank group,a LPS model group,and a LPS+BSHXF group.The blank group was cultured with medium containing 10％blank serum,the LPS model group was cultured with medium containing 10 μmol·L-1 LPS and 10％blank serum,and LPS+BSHXF group was cultured with 10μmol·L-1 LPS and 10％drug-containing serum medium.After 48 h of intervention,CCK-8 method was used to detect the chondrocyte activity,LDH assay to detect cytotoxicity,ELISA to detect IL-6,iNOS and COX2 levels in the supernatant of the cell culture,and TUNEL staining to detect chondrocyte apoptosis.The expressions of apoptosis-related protein Cleaved-Caspase-9,Bcl-2,Bax,and p-IκBα,IκBα,p-p65 and p65,the key proteins of NF-KB signaling pathway,were detected by Western blot.Results ① Compared with the blank group,the activity of chondrocytes in the LPS group was decreased,the level of LDH was increased,the levels of inflammatory cytokines IL-6,iNOS and COX2 were increased.The apoptosis rate of chondrocytes was increased,Cleaved-Caspase-9 and Bax protein expression levels were increased,and Bcl-2 protein was decreased.The ratios of p-IκBα/IκBα and p-p65/p65 were increased.(2)Compared with the LPS group,chondrocyte viability was increased in the LPS+BSHXF group,and the level of LDH was decreased,the levels of inflammatory cytokines IL-6,iNOS and COX2 were decreased.The apoptosis rate of chondrocytes was decreased,Cleaved-Caspase-9 and Bax protein expression levels were decreased,and Bcl-2 was increased.The ratios of p-IκBα/IκBα and p-p65/p65 were also decreased.Conclusion BSHXF can enhance the activity of chondrocytes with LPS induced injury,reduce the toxicity of LPS to chondrocytes,reduce the release of inflammatory factors and inhibit chondrocyte apoptosis,whose mechanism of action may be through inhibiting NF-κB signaling pathway to exert chondroprotective effect.
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