Study On Xanthan Gum Inhibiting Chondrocytes Apoptosis In Osteoarthritis And Its Action Mechanism

ObjectiveTo study the affection and mechanism of xanthan gum(XG)in the inhibiting of chondrocytes apoptosis in osteoarthritis(OA)and provide the basic data for the clinical application of XG.MethodsThe OA mode in vivo was induced by anterior cruciate ligament transaction(ACLT).After intervening with XG and sodium hyaluronate(SH),rabbit body temperature,the temperature of the chamber knee and knee width were record by thermometer and vernier caliper;the histological observation and scoring of the femoral condyle and tibial plateau cartilage were also evaluated to investigate the protective effect of XG in vivo.The protein expression of Caspase-3,Bax and Bcl-2 were detected to investigate the mechanism of XG in vivo.The OA mode in vitro was also induced by various concentration of sodium nitriprusside(SNP).Chondrocytes were induced by different concentrations of SNP(0.0625,0.125,0.25,0.5,1 and 2 mmol/L)for 12,24 and 48 h.These models were evaluated by the MTT assay and TUNEL staining assay.Rabbit articular chondrocytes were pre-incubated with various concentrations of XG for 24 h before 0.5 mmol/L SNP co-treatment for 24 h.The proliferation of chondrocytes was evaluated using MTT assay,Chondrocytes early apoptosis rates was evaluated using Annexin V-FITC/PI flow cytometry and morphology of apoptosis chondrocytes was observed by scanning electron microscopy(SEM);The loss/disruption of mitochondrial membrane potential was detected using Rhodamin 123 by confocal microscope and the concentration of prostaglandin E2(PGE2)in cell culture supernatants was evaluated using ELISA assay.And then the protein expression of Caspase-3,Bax and Bcl-2 were also detected by Western-blot to investigate the protective effect and the action mechanism of XG in vitro.ResultsThere is no different significant when XG in dose ranging from 1% to 2% were injected in articular in OA model compared with SH group in vivo,and the protein expression of Caspase-3 and Bax were down-regulated while the protein expression of Bcl-2 was up-regulated.The MTT results showed that chondrocytes apoptosis models induced by 0.25,0.5,1 and 2 mmol/L of SNP were significantly different(P<0.05)in apoptosis rate at 12,24 and 48 h.At 12,24 and 48 h,chondrocytes apoptosis models induced by 0.25,0.5,1 and 2 mmol/L of SNP were higher(P<0.05)than chondrocytes apoptosis models induced by 0 mmol/L of SNP in rate apoptosis.The pharmacodynamics results showed that additions of XG and SH alone did not adverse effects on chondrocytes proliferation.Treatment with SNP(0.5 mmol/L)alone for 24 h,the ratio of survival of chondrocytes was about 55.03%.pretreatment with XG(500,1 000,2 000 ?g/m L)and SH(1 000 ?g/m L),the percentages survival of chondrocytes were about 74.7%,82.25%,90.52% and 87.32% respectively.In addition,there was no significant difference of chondrocytes proliferation between pretreatment with XG(1 000 and 2 000 ?g/m L)and SH(1 000 ?g/m L)in SNP-induced chondrocytes(P>0.05);The apotosis rate of chondrocytes were declined in a dose-dependent manner(35.88%,28.26%,19.67%,12.78%,8.52% and 12.75%,respectively)when pretreatment with XG(10,100,500,1 000,2 000 ?g/m L)and SH(1 000 ?g/m L).In addition,the apoptosis of chondrocytes was no significantly difference between pretreatment XG(1 000 ?g/m L)and pretreatment SH(1 000 ?g/m L).The morphology of apoptosis chondrocytes was gradually ameliorated in a dose-dependent manner when pretreatment of chondrocytes with XG and SH respectively;the fluorescent population of chondrocytes was increased in a dose-dependent manner when pretreatment with XG;the ELISA assay showed that additions of XG and SH alone did not up-regulate the levels of PGE2,and the protein expression of Caspase-3 and Bax were down-regulated while the protein expression of Bcl-2 was up-regulated.ConclusionsIn this study,these results have showed that(1)articular injection XG could inhibit chondrocytes apoptosis,reduce inflammation,relieve pain,and anti-experimental OA process in OA model induced by ACLT and SNP;(2)different severity of apoptosis models were successfully established when the concentration of SNP ranging from 0.25 to 2 mmol/L and the time was 12,24 and 48 h.These models are of the characters of high success and a good reproducibility;(3)XG blocks SNP-induced apoptosis in rabbit articular chondrocytes by regulating the protein expression of Caspase-3,Bax and Bcl-2 in vitro and in vivo.