Juanbi decoction regulates iron death of chondrocytes to treat osteoarthritis
Objective The mechanism of action of osteoarthritis(OA)induced by Juanbi decoc-tion is investigated in this test using an in vitro cell model.Methods The ATDC5 cells were divided into four groups:the normal group,theinterleukin(IL)-1 β group,the IL-1β+Juanbi decoction group,and the IL-1 β+Fer-1 group.In the IL-1 β group,Dulbecco's modified Eagle's medium(DMEM)was used to induce inflammation with 10 ng/ml IL-1 β.The IL-1 β+Juanbi decoction group was treated with DMEM medium to induce inflammation.The IL-1 β+Fer-1 set was added to induce inflammation in the DMEM medium.The expression of tumor necrosis factor-α(TNF-α),inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2),Collagen Ⅱ,Aggrecan,matrix metalloproteinase-13(MMP-13),p53,ACSL4,SLC7A11 and GPX-4 in chondrocytes was assessed via Western blotting.GPX-4 expression was e-valuated through immunofluorescence staining.Chemiluminescence was utilized to measure the levels of malondialdehyde(MDA),glutathione peroxidase(GSH),Fe2+,and mitochondrial iron in chondrocytes.One-way analysis of variance was employed to compare multiple groups.Results The relative protein ex-pression levels of TNF-α(3.282±0.261,2.255±0.253),iNOS(5.046±0.478,3.443±0.039),and COX-2(3.484±0.333,2.450±0.467)in the IL-1 β+Fer-1 group and IL-1 β+Juanbi decoction group were significantly lower compared to the IL-1 β group(4.384±0.601,6.863±0.637,4.812±0.496).The differences were statistically significant with t-values of 3.848,7.432,5.579,10.5,4.289,and 7.629 respectively(P＜0.05).The relative protein expression levels of MMP-13(2.738±0.224,4.276±0.663),p53(3.364±0.898,4.289±0.936),and ACSL4(2.897±0.767,2.776±0.594)in the IL-1 β+Fer-1 group and IL-1 β+Juanbi decoction group were significantly lower compared to the IL-1 βgroup(5.779±0.018,7.413±1.635,6.077±0.847).The differences were statistically significant with t-values of 10.64,5.258,4.752,3.666,6.048,and 6.279 respectively(P＜0.05).The relative pro-tein expression levels of Collagen Ⅱ(0.752±0.143,0.609±0.073),Aggrecan(0.663±0.139,0.570±0.022),SLC7A11(0.623±0.037,0.400±0.034),and GPX-4(0.686±0.041,0.537±0.065)in the IL-1 β+Fer-1 group and IL-1 β+Juanbi decoction group were significantly higher compared to the IL-1β group(0.298±0.049,0.268±0.154,0.097±0.021,0.163±0.063).The differences were statistically significant with t-values of 4.661,3.895,4.631,3.538,23.7,13.66,12.89 and 9.226 respectively(P＜0.05).The fluorescence intensity of GPX-4(15.476±2.519,12.133±0.703)in the IL-1 β+Fer-1 group and IL-1 β+Juanbi decoction group exhibited a statistically significant increase compared to that in the IL-1 β group(5.141±0.153,t=8.117,5.491,P＜0.05).The fluorescence in-tensity of mitochondria iron(9.689±2.680,11.043±1.818)in the IL-1 β+Fer-1 group and IL-1 β+Juanbi decoction group exhibited a statistically significant decline compared to that in the IL-1 β group(16.604±3.262,t=3.675,2.955,P＜0.05).The levels of MDA[(8.755±0.390),(10.915±0.379)nmol/ml]and Fe2+[(4.200±0.041),(5.189±0.083)nmol]in the IL-1 β+Fer-1 group and IL-1 β+Juanbi decoction group were significantly lower compared to the IL-1 β group[(4.200±0.041),(5.189±0.083)nmol].The differences were statistically significant with t-values of 14.96,7.573,59.230 and 39.090 respectively(P＜0.05).The level of GSH[(7.215±0.382),(6.988±1.246)μmol/g prot]in the IL-1 β+Fer-1 group and IL-1 β+Juanbi decoction group were significantly higher com-pared to the IL-1 β group[(4.826±0.426)μmol/g prot].The differences were statistically significant with t-values of 4.24 and 3.838 respectively(P＜0.05).Conclusion The Juanbi decoction exerts inhib-itory effects on chondrocyte ferroptosis and attenuates inflammation and extracellular matrix degradation by modulating the SLC7A11-GSH-GPX4 axis.
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