Effects Of Different Calcium Channel Blockers On Matrix Metabolism Of Chondrocyte In Osteoarthritis

Knee osteoarthritis(KOA)is a common and frequently-occurring disease in the middle-aged and elderly people.However,there are no effective treatment measures.So,what urgently needs to be solved is to investigate the pathogenesis of KOA and find drugs that can effectively treat or alleviate the progression of it.Voltage-dependent calcium channels are the main pathways for chondrocytes to regulate extracellular calcium entry into cells,and play crucial roles in maintaining the intracellular calcium homeostasis environment.Previous studies show that calcium channel blockers are widely used to lower blood pressure.Which drugs have the strongest protective effect remains unclear.Animal experiments often focus on single drug and the results are not consistent.In this study,in order to identify calcium channel blockers that have a protective effect on chondrocyte matrix metabolism,we used ATDC-5 cells to observe the effect of 10 commonly used calcium channel blockers for the treatment of hypertension on the anabolic and catabolic metabolic parameters of chondrocytes induced by IL-1β.Subsequently,q RT-PCR and Western blot methods were performed on the rat KOA model to verify the protective effect of the best drug.Methods:1 Effects of 10 calcium channel blockers including Nifedipine,Verapamil,Diltiazem,Felodipine,Amlodipine,Nicardipine,Lercanidipine,Nitrendipine,Nenidipine,and Lacidipine on ATDC-5 cells proliferation were measured by MTS assay.2 Induction of ATDC-5 cells: cells were induced by ITS for 7 days or 14 days.The expression of cartilage marker-related genes(Col2a1,Aggrecan-1,Col10a1 and Sox-9)was measured by qRT-PCR.IL-1β induced cells into chondrocytes with OA property,and q RT-PCR was used to test the expression of genes related to matrix metabolism.3 Effects of 10 calcium channel blockers on the relative genes expression of ATDC-5 cells cartilage matrix metabolism:10 calcium channel blockers including Nifedipine,Verapamil,Diltiazem,Felodipine,Amlodipine,Nicardipine,Lercanidipine,Nitrendipine,Nenidipine,and Lacidipine were studied by q RT-PCR for cartilage matrix anabolism(Col2a1,Aggrecan-1 and Sox-9)and catabolism(Mmp-3,Mmp-8 and Impact of Mmp-13)expression of chondrocytes.The effect of protective calcium channel blockers on the protein expression of type II collagen was verified by ELISA.4 Making and identifying KOA rat model: KOA rat model was made by Hulth method.4 weeks after the surgery,observing the major change of knee joint and taking pathological observation.5 Expression of chondrocytes matrix metabolism related genes in normal and KOA rats: Cultured normal and KOA chondrocytes from normal and KOA rats.Matrix anabolism(Col2a,Aggrecan-1,and Sox-9)and catabolism(Mmp-3,Mmp-13,Adamts-4,and Adamts-5)gene expression were measured by q RT-PCR.6 Effects of Nifedipine on cartilage matrix metabolism in KOA chondrocytes: The effects of Nifedipine on KOA chondrocyte matrix metabolism-related gene expression in rat were measured by qRT-PCR.The effect of Col II protein expression was measured by Western blot.Results:1.Effects of 10 calcium channel blockers on ATDC-5 cells proliferation::Nifedipine,Felodipine,Diltiazem,Amlodipine,Lercanidipine,Benidipine,and Lacidipine,the cell viability gradually decreased while concentration increasesing.Nicardipine and Nitrendipine showed cytotoxicity at 30 μM,and Verapamil did not show significant cytotoxicity in this experiment.2.Induction of ATDC-5 cells: After 14 days of ITS induction,the related genes of cartilage markers increased significantly.IL-1β induced anabolic and catabolism of cartilage extracellular matrix with OA property.3.Effects of 10 calcium channel blockers on the relative genes expression of ATDC-5 cells cartilage matrix metabolism: At low concentrations,Nifedipine reversed matrix anabolic(Col2a1 and Sox-9)and catabolic Mmp-3 which changed in chondrocyte with OA property in gene expression.Diltiazem,Felodipine and Lercanidipine increased the expression of Col2a1,and Verapamil,Amlodipine and Benidipine reduced the expression of Mmp-3,Mmp-8 or Mmp-13.Nitrendipine and Lacidipine increased the expression of Mmp-3,Mmp-8,and Mmp-13.4.Making and identifying KOA rat model: After the making KOA model by Hulth method for 4 weeks,the HE staining showed that the tide line was blurry,and toluidine blue and safranine O were lighter than normal and appeared to be unstained.5.Relative genes expression of cartilage matrix metabolism in articular chondrocytes isolated from normal and OA rat cartilage: Expression of matrix anabolic(Col2a1,Aggrecan-1 and Sox-9)in OA group was reduced significantly,and catabolism(Mmp-3,Mmp-13,and Adamts-5)gene expression was increased significantly.6.Nifedipine reversed the changes of gene expression of Col2a1,Aggrecan-1,Sox-9 and Adamts-5,and increased the protein expression of type II collagen in OA rat chondrocytes.Conclusion: At low concentrations,Nifedipine reversed anabolic and catabolic metabolism of osteoarthritic chondrocytes.The anabolism of chondrocyte matrix was increased by Felodipine,Diltiazem and Lercanidipine.Verapamil,Amlodipine,Nicardipine and Benidipine decreased the catabolism of chondrocyte matrix.The catabolism of chondrocytes was exacerbated by Nitrendipine and Lacidipine.Therefore,it is presumed that Nifedipine is best drug for protecting osteoarthritis cartilage among 10 calcium channel blockers.