Caffeic Acid 3,4-Dihydroxyphenyl Ester Contributes To The Amelioration Of Knee Osteoarthritis,a Mechanism Research In Mice Models

Objective:The aim of this study is to investigate the arthroprotective effects of Caffeic Acid3,4-Dihydroxyphenethyl Este(CADPE)and its effects on cartilage and subchondral bone degeneration,and to investigate the molecular mechanisms underlying the effects of CADPE.Methods:1.The effect and mechanism of CADPE on ATDC5 chondrocyte cell line.1)ATDC5 cells were induced to reach an inflammatory state with 10ng/m L of IL-1β.2)The cytotoxicity of different concentrations of CADPE on ATDC5 cells in normal and inflammatory states was assayed with CCK-8 reagent.3)High-density culture with ATDC5 cells was performed and IL-1β combination normal group cells were treated with CADPE(30μM,60μM,120μM),and the synthesis of extracellular matrix breakdown was assessed by Alisin blue staining after 7 days.4)Chondrocytes with or without IL-1β were treated with CADPE(30μM, 60μM,120μM)for Western blot,immunofluorescence,q RT-PCR experiments to detect ATDC5 cell-specific proteins(Sox9,Col2,MMP13) and genes(MMP13,ADAMTS4,Aggrecan,Sox9)expression.5)Western blot assays were performed in chondrocytes with or without IL-1β treated with CADPE(30μM)to detect the expression of NF-k B pathway(NF-κB-p65 、 p-NF-κB-p65)and MAPK pathway(ERK,p-ERK,p-P38, p-JNK)related proteins.6)Cartilage explants were cultured and treated with CADPE(30μM)with or without IL-1β for 24 h,48h,72 h,fixed,dehydrated,embedded,sectioned,and stained with red O-solid green and immunohistochemistry(MMP13,Col2)to assess cartilage tissue degeneration.2.The effect and mechanism of CADPE on osteoclasts1)Bone marrow mesenchymal cells were obtained from the femur and tibia of 4-6 week old mice and BMMs were induced to form with M-CSF(25ng/m L).2)CCK-8 assayed the toxic effects of different concentrations of CADPE on BMMs.3)BMMs were induced to differentiate into osteoblasts by adding M-CSF(25ng/m L)and RANKL(50 ng/m L)to the culture medium,while the cells were treated with CADPE(5μM,10μM)and TRAP staining was used to detect the formation of osteoblasts.4)After treatment with CADPE(5μM,10μM)containing M-CSF(25 ng/m L) and RANKL(50ng/m L)induced BMMs,osteoclast-specific proteins(c-Fos,CTSK,Integrin β3,NFATc1)and genes(CTSK,c-Fos)expression.5)CADPE(5μM)was pretreated for 2 hours,and proteins were extracted after stimulation with RANKL,and western blot was performed to detect the expression of NF-k B pathway and MAPK pathway(ERK、p-ERK、p-P38、NF-κB-p65、p-NF-κB-p65、p-JNK)related proteins.3.To investigate the role of CADPE in osteoarthritis in mice in vivo.1)Eighteen C57BL/6 male mice at 8 weeks were selected.12 mice were established as mouse OA models by medial meniscal instability(DMM),and after surgery,the mice were randomly divided into three groups: control group,DMM group,and DMM+10 mg.kg-1 CADPE group.CADPE was administered intraperitoneally to DMM mice for 8 consecutive weeks.8 weeks later,the mice were executed,and the tibia and femur were decalcified,dehydrated,and embedded,and the sections were used for subsequent experiments.2)The effect of CADPE treatment and retardation of osteoarthrosis was analyzed using H&E,staining with saffron O-solid green,TRAP staining,and immunohistochemistry(MMP13,Col2,p-NF-κB-p65).Results:1.The results of CCK-8 showed that CADPE at 30μM,60μM and 120μM concentrations had no significant toxic effects on ATDC5 cells;CADPE at 5μM and 10μM concentrations had no significant toxic effects on BMMs.2.The high density results showed that the color of IL-1β alone treated group became lighter compared to the control group;while the color deepened in a dose-dependent manner after co-treatment with IL-1β and different concentrations of CADPE compared to the IL-1β group.3.The results of osteoclast differentiation showed that a large number of positive TRAP-stained cells could be observed after the addition of RANKL(50ng/m L)to BMMs,while the number of positive cells decreased significantly after the addition of both RANKL and different concentrations of CADPE(5μM,10μM).4.The results of real-time fluorescence quantitative PCR and protein immunoblotting experiments showed that in ATDC5 cells,the expression of anabolic-related genes and proteins(Aggrecan,Sox9 and Col2)was upregulated in the CADPE-treated group compared with the IL-1β group, while the expression of catabolic-related genes and proteins(MMP13 and ADAMTS4)was downregulated;in osteoblasts In osteoblasts,the expression of c-Fos,NFACT1,Interrinβ3 and CTSK were down-regulated in the CADPE group compared to the RANKL group.5.The results of protein immunoblotting assay showed that in ATDC5 cells, the expression of phosphorylated NF-κB-p65 was down-regulated in the CADPE-plus group compared with the IL-1β group,while the expression of MAPK was not significantly changed;in osteoblasts,the phosphorylated NF-κB-p65 was not significantly changed in the CADPE-plus group compared with the RANKL group,and the phosphorylated ERK did not change much,but phosphorylated ERK was significantly decreased in the RANKL+5μM CADPE group.6.The results of immunofluorescence experiments showed that CADPE increased the expression of Sox9 and decreased the expression of MMP13.7.The results of human cartilage block showed that the staining of the CADPE group was enhanced compared with the IL-1β group,and the immunohistochemical results showed that the MMP13 positive cells were reduced and the Col2 positive cells were increased in the cartilage blocks of the CADPE group compared with the IL-1β group.8.The results of H&E and saffron O-solid green in mice showed that CADPE could rescue the loss of cartilage matrix due to inflammation.9.In vivo immunohistochemical results in mice showed that CADPE increased Col2 expression and decreased MMP13 and NF-k B pp65 expression compared to IL-1β group.Conclusion:Our study showed that CADPE has arthroprotective effects in vitro and in vivo,suggesting that CADPE may serve as a potential drug for the treatment of OA.