Procyanidins Alleviate Cadmium Induced Chicken Chondrocytes ECM Damage

Cadmium(Cd)is a toxic non-essential metal that enters the body mainly through the digestive and respiratory systems,and can cause damage to multiple organs and tissues of the body.Cd accumulates in cartilage and causes damage to cartilage cells,so it is important to study the pathogenesis of Cd-induced cartilage diseases.Matrix degrading enzymes play a key role in the catabolism of cartilage extracellular matrix(ECM),and the ERK1/2 signaling pathway in Mitogen-activated protein kinas(MAPK)can upregulate the expression of matrix degrading enzymes,thus promoting ECM damage.Procyanidins(PAs),also known as condensed tannins,are oligomers of flavan-3-ol units that are widely found in fruits,vegetables,nuts,bark,seeds,and other plants and have a variety of activities such as anticancer,anti-oxidative stress,and anti-inflammatory.It was found that PA can alleviate the damage to liver,kidney and reproductive system caused by Cd by scavenging free radicals,chelating metals and antioxidant.However,whether PA can alleviate the damage of Cd on chicken chondrocytes and its mechanism of action need to be further explored.In this study,we aimed to investigate the mechanism of ERK1/2 signaling pathway of Cd-induced cartilage ECM damage in chicken embryonic chondrocytes cultured in vitro,and to investigate whether PA could alleviate the effect of Cd on chicken chondrocyte ECM through ERK1/2 signaling pathway,in order to provide a theoretical basis for clinical studies on the use of PA for the prevention and treatment of Cd-induced cartilage diseases in poultry.1 Chicken chondrocytes ECM degradation induced by CdTo investigate the effect of different concentrations of Cd on ECM of chicken chondrocytes,chicken embryonic chondrocytes were used as the target of the experiment.After the cells grew to 80%-90%fusion,the cells were treated with 0,1,2.5,5 and 10μmol/L CdCl2 for 24 h.The cell viability was detected by CCK8 method,GAG by alcian blue staining,matrix degrading enzyme and ECM component transcript levels,Western blot for ECM component protein expression levels,and ELISA for ECM metabolite levels.The results showed that,compared with the control group,the chondrocyte survival rate was significantly reduced in the group treated with 5 μmol/L and above concentrations of Cd(p<0.05);the expression levels of matrix metalloproteinases(MMP1,MMP3,MMP13)and ADAMTS4 genes were significantly increased(p<0.05);the expression of ECM component GAG(p<0.05),ACAN,and COL2A1 expression levels at the protein and gene levels were significantly decreased(p<0.05);COMP levels in the culture medium supernatant were significantly increased(p<0.05).This indicates that Cd at concentrations of 5μmol/L and above inhibited protein synthesis in ECM components,promoted matrix degradation enzyme expression,and destabilized the ECM of chicken chondrocytes.2 Cd activates ECM degradation in chicken chondrocytes through ERK1/2 signal pathwayTo investigate the role of ERK1/2 signaling pathway in Cd-induced ECM disorder in chicken chondrocytes,the cells were treated with different concentrations of CdCl2(0,1,2.5,5,10 μmol/L)CdCl2 for 24 h.The expression levels of ERK1/2 phosphorylated protein were detected by Western blot;ERK1/2 specific inhibitor(U0126)was combined with Cd to treat chick embryo chondrocytes for 24 h.GAG was detected by alcian Blue staining,matrix degrading enzyme and ECM component gene transcript levels by qRT-PCR,ECM component as well as ERK1/2 signaling pathway protein levels by Western blot,and ECM metabolite detection by ELISA.The results showed that ERK1/2 phosphorylation levels were significantly increased in the Cd-treated group above 2.5 μmol/L compared with the control group(p<0.05).The gene level expression of MMP1,MMP3,MMP13,and ADAMTS4 was significantly decreased in the combined U0126 and Cd-treated group compared with the 5 μmol/L Cd-treated group.(p<0.05);GAG expression was significantly increased(p<0.05),none of the ACAN gene expression levels were significantly changed,COL2A1 expression levels were significantly increased(p<0.05);COL2A1 and ACAN protein expression levels were significantly increased(p<0.05);COMP levels in the culture medium supernatant were significantly decreased(p<0.05).It showed that the activation of ERK1/2 is involved in regulating the effect of Cd on the stability of ECM in chicken chondrocytes.3 The protective effect of Procyanidins on chicken chondrocytes ECMTo investigate the mitigating effect of PA on Cd-induced ECM degradation in chicken chondrocytes and the effect on ERK1/2 pathway,5μmol/L CdCl2 and 10 μmol/L PA were used to treat chicken chondrocytes alone or in combination for 24 h.CCK8 was used to detect cell viability,alcian blue staining to detect GAG,qRT-PCR to detect matrix degradation enzymes and ECM component genes.transcript levels,Western blot for ECM component protein levels,and ELISA for ECM metabolites.The results showed that,compared with the Cd-treated group,the chondrocyte viability of chick embryos in the combined Cd and PA-treated group increased significantly(p<0.05);the ERK1/2 phosphorylation level decreased significantly(p<0.05);the MMP1 expression level decreased significantly(p<0.05);the MMP3,MMP13 and ADAMTS4 expression levels decreased significantly(p<0.05);the GAG expression was highly significant(p<0.01);AC AN and COL2A1 gene and protein expression levels were significantly significantly increased(p<0.05);and COMP levels in cell supernatants were significantly reduced(p<0.05).It showed that PA inhibited the activation of ERK1/2 signaling pathway,promoted the expression of ECM components,decreased the expression of matrix degrading enzymes,and alleviated the damage of ECM in chicken chondrocytes by Cd.