| ObjectiveProanthocyanidins(PCs)were shown the inhibition of oxidative damages by improving Nrf-2 expression in many tissues.However,its protective effects against oxidative-stress-induced damages have not been clarified in the tendon tissues.The present study aimed to assess the protection of PCs against the oxidative cellular toxicity in tendon-derived stem cells(TDSCs)and the tendon tissues,elucidate whether PCs can ameliorate oxidative damage and its possible mechanism,and provide the evidences for the mechanism and repair of the tendon injuries.Methods1.Cell Experiments 1.1 The tendon-derived stem cells(TDSCs)were isolated from the patellar tendon of SD rats,and the cells were cultured to P3 for the subsequent experiments.1.2 The isolated cells were identified by flow cytometry assay with lineage-specific markers.1.3 Cytotoxicity assay for PCs at different concentrations was to rule out their cytotoxic effects on TDSCs.1.4 Cytotoxicity assay for H2O2 at different concentrations was to assess the half maximal inhibitory concentration(IC50)value for subsequent studies.1.5 The cytoprotective effects of PCs at different concentrations on H2O2-induced oxidative stress was determined by CCK-8 assay.1.6 Effects of PCs on H2O2-induced alteration of Nrf-2,GCLM,NQO-1 and HO-1 expression levels were determined by Real-Time PCR and Western Blot assay.2.Animal Experiments 2.1 A total of 15 healthy male SD rats were averagely divided into three groups:control,overuse,PCs + overuse groups.2.2 The PCs + overuse group was pre-treated with PC(100 mg/kg)in 0.9%saline by oral gavage.2.3 Rats in overuse groups underwent a 1-week treadmill training.Overuse activity consisted of treadmill running at 17 m/min on a 10° declination for 1 h per day,5 days per week.2.4 Rats were sacrificed 2 weeks after serum collection,and assessed for SOD,MDA,and anti-·OH and O2·-abilities.2.5 Tendon samples were obtained and fixed for histology.Results:1.At P3,a homogeneous population of fibroblast-like cells were obtained.In flow cytometric analysis,the results showed that 96.8%of the TDSCs were positive for the fibroblastic marker CD90 and negative for the endothelial cell marker CD31.According to cell morphology and flow cytometric analysis of MSC markers,we confirmed that the isolated cells are TDSCs,ruling out contamination by other cells.2.In CCK-8 assay,all PCs amounts showed no cytotoxicity on TDSCs,and the cell viability of TDSCs treated with H2O2 was obviously decreased,resulting from the H2O2-induced oxidative damage.However,the cell viability of TDSCs pre-treated with PCs was distinctly improved,even at a very low concentration,and this exhibited the efficient antioxidant effects of PCs on TDSCs.3.In Real-Time PCR,there was significant upregulation of Nrf-2,GCLM,NQO-1 and HO-1 mRNA expression levels after PCs and H2O2 treatment compared with the control group,and the alternations of PCs were higher than H2O2.PCs pre-treatment followed by H2O2 increased the expression levels of Nrf-2,GCLM,NQO-1 and HO-1 the most.4.Although there were no effects(P>0.05)on Nrf-2 amounts,a trend of increase was obtained for the PC + H2O2 group regarding protein expression Nrf-2 levels.The treatment resulted in increased GCLM and HO-1 protein amounts,and the differences were significant compared with the control group.5.In our overuse rats model,the production of MDA was highly increased in the overuse group,and the result indicated that our overuse activity rats model had successfully caused the oxidative stress to the rats.Also,the SOD activity,anti-· OH and O2·-capacity were improved a little.Rats treated with PCs showed significantly increased SOD activity,as well as anti-· OH and O2·-capacity,while MDA amounts were decreased.6.Tendon histology in the control group by H&E staining showed normal structure and staining.In addition,collagen fibers were parallel to each other,and packed closely.The cell nuclei were arranged in the middle of collagen fibers,with a fusiform or spindle shape.In the overuse group,the collagen fibers were fractured,and a more disordered structure was obtained,with more rounded nuclei observed.The histology of rats treated with PCs showed that collagen fibers were arranged disorderly,and light-colored or stained non-uniformly,with some nuclei becoming circular gradually.Conclusions:1.PCs had cytoprotective effects against oxidative-stress-induced cytotoxicity to TDSCs and tendon tissues;these effects were achieved via up-regulating Nrf-2 signaling pathway.2.Overuse activities indeed caused the oxidative damage to tendons,and the efficient antioxidant effects of PCs proteted tendon tissues by increasing antioxidant enzyme activities and oxygen free radicals scavenging ability of tendon tissue. |
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