The Mechanism Of Lower Range Of Molecular Weight Of Xanthan Gum Inhibiting Apoptosis Of Chondrocytes Under Oxidative Stress

ObjectiveOsteoarthritis?OA?is a common disease characterized by progressive lesions of articular cartilage.Among the medicines for treating OA,sodium hyaluronate?SH?has been recognized as the effective drug in clinical practice for its significant curative effect along with limited side effects.However,patients need to receive frequent injections of SH,as it degrades quickly in the affected joint cavity,which increases the chance of infection and is inconvenient for patients.Xanthan gum,due to its unique properties,has the potential to replace SH as a candidate drug in the treatment of OA by making up for the shortcomings of degradability of SH and possessing the pharmacological effects of SH in the treatment of OA.We have previously reported that XG with molecular weight?Mw?in the range from 3×10 ⁶ Da to 5×10 ⁶ Da can inhibit articular cartilage degradation in a experimental OA model.The Mw of the polysaccharides drug is closely related to the efficacy of the disease.Therefore,we need to evaluate the therapeutic effects of xanthan gum with different ranges of Mw in experimental OA.Previously,we have proved that low range of molecular weight of xanthan gum?LRWXG??1.0×10⁶ Da to 1.5×10⁶ Da?has protective effect on chondrocyte under oxidative stress,but its mechanism is still unclear.Mitogen-activated protein kinase?MAPK?signaling pathways are closely related to chondrocyte apoptosis.The aim of this study was to elucidate the mechanism of LRWXG in the treatment of OA by measuring the expression of MAPKs signaling pathway related substances of rabbit chondrocytes.MethodThe molecular weight of the was identified by Multi-angle laser scattering method;The sample was identified by sophora bean gum experiment and infrared absorption spectroscopy according to the Chinese Pharmacopoeia of2015 edition.Cell Counting Kit-8?CCK-8?was used to detect the effect of LRWXG on the growth and proliferation of chondrocytes isolated from New Zealand white rabbits of 4 weeks old and cultured in vitro.Chondrocytes cultured in vitro were cultured with LRWXG concentration gradient?0,10,100,500,1 000,2 000?g/mL?.CCK-8 was used to detect the cell viability of the treated chondrocytes.Setting groups,including model group,LRWXG concentration gradient groups?0,10,100,500,1 000 ug/mL?,signaling pathway inhibitor groups?SP600125,SB203580?and signaling pathway inhibitor plus LRWXG groups?1000 ug/mL?.SP600125 and SB203580 are JNK and p-38 signaling pathway inhibitors.They can specifically block the phosphorylation of JNK and P-38proteins,thereby blocking the activation of corresponding signaling pathways.After treatment for 24 h,H₂O₂ solution was added to each group except for the control group with a final concentration of 0.5 mM and treated for 24 h before protein was harvested.CCK-8 was used to detect the survival rate of cultured cells;The levels of tumor necrosis factor-alpha?TNF-??,interleukin-1bet?IL-1??,cyclooxygenase-2?COX-2?and prostaglandin E2?PGE2?were measured by enzyme-linked immunosorbent assay?Elisa?.Western blot was used to detect the expression level of MAPKs signaling pathway-related proteins,including p-38,p-p38,JNK,p-JNK,Bcl-2,Bax,cleaved-caspase-3;The expression of matrix metalloproteinases?MMPs?family members MMP-1,MMP-13 and collagen type II?COLII?was detected by immunofluorescence.Results1.The molecular weight of the sample ranges from 1.0×10⁶ Da to 1.5×10⁶Da.After identification experiment and comparison of infrared spectras,we came to a conclusion that the purified samples were proved to be xanthan gum.2.CCK-8 cell viability test results showed that the cell viability of chondrocytes treated with LRWXG?10,100,500,1 000,2 000?g/mL?was not significantly different from that of the blank control group,indicating that LRWXG had no effect on normal growth of chondrocytes.3.The CCK-8 results indicated that,compared the model group against the control group,the viability of chondrocytes at 500?g/mLwas slightly increased?P<0.05?.LRWXG?1 000?g/mL?significantly increased the survival rate of chondrocytes?P<0.01?compared with the model group.Compared with the inhibitor groups?SP600125,SB203580,SP600125+SB203580?,the survival rates of chondrocytes of the groups containing LRWXG?1 000?g/mL?plus the inhibitor treatments were significantly improved?P<0.01?.The results indicated that the anti-apoptotic effect of LRWXG was significantly stronger than that of the tested signal pathway inhibitors in a dose-dependent manner.4.The elisa results demonstrated that compared with the model group,LRWXG inhibited the secretion of inflammatory-related factors in a dose-dependent manner.With the exception of PGE2,the content of the other three cytokines in the inhibitor groups were not significantly different from those of the model group?P>0.05?.The level of the cytokines in the groups consisting of LRWXG plus inhibitor were lower?P<0.05?or significantly lower?P<0.01?than the inhibitor group or the model group.These experimental results suggest that the inhibitory effect of LRWXG on inflammatory-related factors was stronger than the effects of the known MAPK signaling pathway inhibitors.5.The results of western blot showed that,compared with the model group,LRWXG effectively reduced the levels of p-p38,p-JNK,Bax and cleaved-caspase-3,while increased the expression of Bcl-2 in a dose-dependent manner.Among the signaling pathway inhibitors treatment groups,there were no significant difference in the expression of p-p38 and p-JNK proteins compared to the inhibitor plus LRWXG?P>0.05?.Conversely,the expressions of the the Bax and cleaved-caspase-3 proteins were significantly decreased,while the expression level of Bcl-2 was significantly increased?P<0.01?.These experimental results showed that the inhibitory effect of LRWXG on MAPK signaling pathways was similar to that of signal pathway inhibitors,and the inhibitory effect of LRWXG on apoptosis terminal-related proteins was stronger than that of the known MAPK signal pathway inhibitors.6.The results of immunofluorescent staining indicated that,compared with the model group,LRWXG inhibited the expression of MMP-1 and MMP-13,and increased the expression of COLII in a dose-dependent manner.Compared with model group,the expression levels of MMP-1 and MMP-13 decreased and COLII increased in inhibitor group,but the changes were not significant?P>0.05?.Compared with the inhibitor group,the expressions of MMP-1 and MMP-13 in the groups containing inhibitor plus LRWXG were significantly decreased,and the expression level of COLII was significantly increased?P<0.01?.These results suggested that the inhibitory effect of LRWXG together with treatment by the inhibitors displayed a protective effect greater than with the MAPK signaling pathways inhibitors considering MMPs and COLII expression.ConclusionLRWXG could play a role through the apoptosis-related signaling pathways of MAPKs,to play an anti-apoptotic and protective role in chondrocytes.