Antioxidants Derived From Natural Ingredients Regulate Oxidative Stress In Osteoarthritis

Background:Osteoarthritis（OA）is the most common disabling joint disease and its pathological process has relation to oxidative stress.Furthermore,intra-articular injection of mesenchymal stem cells（MSCs）is a promising application for OA treatment due to its multi-directional differentiation and paracrine function.However,MSCs are susceptible to interference of the local micro-environment,which reduces their therapeutic effect.Recent studies have shown that antioxidants（allicin,sulforaphane,and lycopene）derived from natural ingredients（garlic,broccoli,and tomato）can reduce degree of oxidative s tress and content of cytokine and catabolic enzymes;poor therapeutic effect of MSCs in OA may be related to limited survival of MSCs in joint caused by excessive reactive oxygen,indicating that theses antioxidants might be beneficial for OA pathophysiology.In this study,we inquired into influences of biologically active substances（allicin,sulforaphane and lycopene）derived from these natural ingredients（garlic,broccoli,and tomato）to probe causes of these antioxidants regulating OA and MSCs-injection therapies.The widely studied antioxidant N-Acetyl-L-cysteine（NAC）is used as a classic control.PurposesThis study explored mechanisms and impacts of allicin,sulforaphane and lycopene on the phenotype of human or rat OA chondrocytes（OACs）,therapeutic effects of rat OA,phenotype of rat adipose stem cells（ADSCs）,and curative effect of rat ADSCs on rat OA.MethodPart 1:Effects of allicin,sulforaphane and lycopene on phenotype of human OA chondrocytes and osteochondral composites.1.Isolation and culture of OACs in vitroOA cartilages of knee-joint were acquired from TKA surgical OA-patients.OACs were isolated-cultured and its morphology were photographed in our studies.To identify primary chondrocytes,HE,alcian blue,safranine O,and COL 2 immunofluorescence staining were performed.2.Effect of allicin,sulforaphane,lycopene,and NAC on OACs survivalThe cytotoxicity of allicin,sulforaphane,lycopene,and NAC on OACs was measured by CCK-8 assay.For purpose of further proving the reliability of the concentrations of these drugs,OACs were cultured with the optimum concentrations of those drugs from results above.Cell viability was assessed by morphology of OACs and Calcein-AM/PI staining.3.Oxidative stress was induced by addition of H₂O₂ in vitroThe medium supplemented with 200μM H₂O₂ was used for stimulating to accomplish oxidative stress model in vitro.4.Acquisition process of human osteochondral tissuesFor isolation of human knee osteochondral tissues,the hollow medical electrical trepan-drill was used.The drill was perpendicular to surface of cartilage.Acquired specimen were used for follow-up experiments.Those samples were fixed-decalcified,embedded-sectioned,and then subjected to HE,safranin O and toluidine blue staining;5.Treatment of human OACsMedium containing H₂O₂ was added to OACs to complete induction of oxidative stress and then the antioxidant-containing medium was added for following treatment.After finishing anti-oxidative-stress processing,the following tests were prepared:The culture medium was collected and content of IL-6,TNF-α,and MMP-13 was examined;morphology and apoptosis ratio of OACs were detected by CCK-8,flow cytometry,and HE staining;the expression of i NOS,TNF-α,COL 2,IL-6,ACAN,SOX-9,COL 1,COL X,MMP-13 and Nrf2 in OACs was detected by immunofluorescence staining,western-blot and qPCR;expression of proteoglycans were detected by alcian blue and safranin O staining;qPCR was used to detect the levels of GPX1,GPX3,GPX4,SOD1,CAT,GST,NOX-4,IL-1β,ADAMTS-5 and COX-2;6.Research on the pathway of antioxidants regulating human OACsRetinoic acid was used to inhibit expression of Nrf2 in the OACs.The expression of Keap1,Nrf2,p-Nrf2,COL2,SOX-9,i NOS and IL-6 was detected by western-blot;Part 2:Therapeutic effects of intra-articular injection of allicin,sulforaphane and lycopene on rats OA.1.Isolation,extraction and culture of rat chondrocytesRat chondrocytes were extracted and isolated and P1 generation rat chondrocytes were selected for this part of the experiment.Taking morphology-pictures of rat chondrocytes under microscope for observation;2.Effects of allicin,sulforaphane and lycopene on cell viability of rat chondrocytesThe cytotoxicity of allicin,sulforaphane,lycopene,and NAC on rat chondrocytes was measured by CCK-8 assay to detect whether the concentration of antioxidants used in Part 1has biological toxicity on rat chondrocytes.The drug concentration was used in subsequen t intra-articular injection experiments;3.Establishment of rat OA model and intra-articular injectionThe rat OA model was established by ACLT surgery（OA model was completed at 4weeks after ACLT operation）.Antioxidants were injected into rat knee for 5 weeks（once a week）;4.Histological section staining of rat knee jointSamples were fixed-decalcified,embedded-sectioned,and subjected to HE,Safranin O-Fast Green and COL 2 staining;Part 3:Effects of allicin,sulforaphane and lycopene on phenotype of rat ADSCs.1.Culture of rat ADSCsRat ADSCs line was purchased,cultured and expanded in vitro.The P7 generation ADSCs were used in our following experiment;2.Effects of allicin,sulforaphane and lycopene on the cell viability of rat ADSC sThe cytotoxicity of allicin,sulforaphane,lycopene,and NAC on rat ADSCs was measured by CCK-8 assay according to the manufacturer’s instructions;3.Oxidative stress was induced by addition of H₂O₂ in vitroThe 200μM H₂O₂ were added into medium to induce oxidative stress for 8 h in rat ADSCs;4.In vitro treatment of rat ADSCsMedium containing H₂O₂ was added to rat ADSCs to complete induction of oxidative stress.After induction medium is aspirated,the antioxidant-containing medium was added for following treatment.After finishing anti-oxidative processing,the following tests were prepared:The expression of i NOS,p53,C-caspase3,SOX-9,COL 2,ACAN,RUNX2,IL-6,TNF-αand MMP-13 in rat ADSCs was detected;the culture medium of rat ADSCs was collected and expression of IL-6,TNF-α,and MMP-13 was detected by ELISA;the proliferation,migration and differentiation of rat ADSCs were detected;Reactive oxygen species levels in ADSCs were detected.Part 4:Study on effects of allicin,sulforaphane and lycopene on survival of rat ADSCs in OA knee joint and curative effect of rat ADSCs on rat OA.1.Labeling of rat ADSCsRat ADSCs were labeled with Di R-fluorochrome;2.Rat OA model and intra-articular injectionThe rat OA model was established by ACLT surgery（OA model was completed at 4weeks after ACLT operation）.Di R-labeled rat ADSCs+antioxidants were injected into the joint cavity of the rat.For the cell survival experiment,number of rats ADSCs in joint was detected on the 3rd day after the first injection.For experiment of lasting effects,OA rats have five intra-articular injection（once a week）;3.Histological section staining of rat knee jointRat samples were fixed-decalcified,embedded-sectioned,and subjected to HE,Safranin O-Fast Green and COL 2 staining;ResultPart 1:Effects of allicin,sulforaphane and lycopene on phenotype of human OA osteochondral composites and chondrocytes.1.The cultured cells displayed typical OACs morphology with paving-stone-like shape.Moreover,alcian blue staining,safranine O staining,and collagen II immunofluorescence staining revealed that the isolated cells highly expressed the glycosaminoglycans（GAGs）and collagen II,indicating the phenotype of OACs;2.Allicin at 31μM,sulforaphane at 7μM,lycopene at 1.2μM,and NAC at 31μM had no cytotoxic effect on OACs.Cell morphology and Calcein-AM/PI double stain results further confirmed that there was no cytotoxic effect;3.Articular cartilage matrix was severely degraded by H₂O₂ stimulation,but this degradation could be largely reversed by allicin,sulforaphane,lycopene,and NAC treatment;Damage of cartilage surface after H₂O₂ stimulation,but allicin,sulforaphane,lycopene,and NAC treatment largely protected structural integrity of cartilage.In statistical analysis of relative staining intensity,H₂O₂ group was weaker compared with control group,and allicin,sulforaphane,lycopene,and NAC group showed stronger intensity than H₂O₂group;4.OACs had size-reduced in morphology in H₂O₂ group compared with control group.Furthermore,the condensation of nuclear chromatin,less cellular matrix,and reduced connectivity between individual OACs was found in H₂O₂ group.With the supplement of allicin,sulforaphane,lycopene,and NAC,OACs showed slightly low signal of nucleus,more cell matrix and increased connectivity compared with H₂O₂ group.In addition,OACs viability was significantly decreased after exposure to H₂O₂,but was rescued with treatment of allicin,sulforaphane,lycopene,and NAC.OACs apoptosis in the H₂O₂ group（12.48±0.55%）was more remarkable than that in control group（0.76±0.09%）.The apoptotic proportion of OACs was 0.94±0.22%in allicin group,1.46±0.15%in sulforaphane group,1.21±0.16%in lycopene group,and 1.04±0.14%in NAC group,indicating that allicin,sulforaphane,lycopene,and NAC significantly inhibited H₂O₂-induced apoptosis of OACs;5.Intracellular i NOS in OACs was significantly up-regulated after stimulating with H₂O₂.However,treatment with allicin,sulforaphane,lycopene,and NAC decreased H₂O₂-induced i NOS elevation in immunofluorescence analysis.In addition,western blot and PCR results confirmed that allicin,sulforaphane,lycopene,and NAC manifestly inhibited the expression of i NOS;6.H₂O₂ decreased levels of antioxidant enzymes（GPX1,GPX3,GPX4,SOD1,CAT and GST）and Nrf2 in human OACs drastically decreased,and impro ved expression of NOX-4.The addition of allicin,sulforaphane,lycopene,and NAC to OACs incubated with H₂O₂ could significantly decrease expression of NOX-4 and increase majorities of antioxidant enzymes RNA expression levels,except for GPX1 in allicin group,GPX1 and GST in sulforaphane group,GPX3 in lycopene group,and GPX1 and SOD1 in NAC group;7.H₂O₂ significantly inhibited GAGs synthesis of OACs.In addition,H₂O₂-stimulated OACs in allicin,sulforaphane,lycopene,and NAC solution showed a better matrix deposition and the GAGs production compared to stimulated-OACs in DMEM/High glucose medium.The H₂O₂ group had fewer expressions in COL 2,aggrecan,and SOX-9compared to control group,whereas staining of the chondrogenic markers in allicin,sulforaphane,lycopene,and NAC group was much stronger than that in the H ₂O₂ group.Supplement of H₂O₂ significantly inhibited SOX-9,aggrecan,and COL 2 synthesis of OACs compared with control group,and replenishment with allicin,sulforaphane,lycope ne,and NAC improved secretion of chondrogenic marker at protein levels.Besides,PCR results almost showed similar trend;8.The addition of H₂O₂ greatly improved production of IL-6,MMP-13 and TNF-α.In addition,allicin,sulforaphane,lycopene,and NAC manifestly inhibited expression of IL-6,MMP-13.and TNF-α.Besides,H₂O₂ stimulation increased levels of IL-6,MMP-13,and TNF-αsecreted by OACs compared with control group,while treatment with allicin,sulforaphane,lycopene,and NAC decreased expression of IL-6,MMP-13,and TNF-αsecreted by OACs,except for IL-6 in lycopene group.H₂O₂ stimulation also remarkably improved protein expression of these bio-markers in comparison with the control group,while treatment with allicin,sulforaphane,lycopene,and NAC could significantly decrease levels of these cytokines.PCR results showed the same trend;9.At m RNA levels,the addition of H₂O₂ greatly improved the production of IL-1β,ADAMTS-5 and COX-2.In addition,allicin,sulforaphane,lycopene,and NAC manifestly inhibited expression of IL-1β,ADAMTS-5 and COX-2;10.Results of fibrotic markers（COL 1）suggested no prominent difference am ong each group.No prominent difference in expression of COL 1 among each group.In terms of hypertrophic phenotype（COL X）,H₂O₂ group improved expression of collagen X compared with control group,whereas treatment with allicin,sulforaphane,lycopene,a nd NAC decreased content of collagen X;11.The H₂O₂ treatment did not adjust levels of Keap1,Nrf2,and p-Nrf2 in OACs while addition of allicin,sulforaphane,lycopene,and NAC markedly increased expression of Nrf2 and p-Nrf2 and decreased the expression of Keap1.Furthermore,when total Nrf2protein levels were used as relative internal reference,treatment of allicin,sulforaphane,lycopene,and NAC significantly improved p-Nrf2 expression in H₂O₂-stimulated OACs.That is,activation ratio of p-Nrf2（p Nrf2/total Nrf2）in allicin,sulforaphane,lycopene,and NAC groups increased;12.Retinoic acid markedly decreased expression of Nrf2 and p-Nrf2 in H₂O₂-stimulated OACs in allicin,sulforaphane,lycopene,and NAC group.Besides,the expression of Keap1 reduced the rate of decline to a relatively small range compared with before.Activation ratio of p-Nrf2 was also decreased.Allicin,sulforaphane,lycopene,and NAC supplemented with retinoic acid did not remarkably decrease expression of i NOS and IL-6 compared with H₂O₂ group.Besides,with addition of retinoic acid,there was no notably increase in levels of SOX-9 and COL 2 in allicin,sulforaphane,lycopene,and NAC group compared with H₂O₂ group;Part 2:The alleviating effect of allicin,sulforaphane and lycopene on rat knee OA.1.The extracted rat chondrocytes are in good growth-condition,showing a typical polygonal pattern and uniform shape;2.Allicin at 31μM,sulforaphane at 7μM,lycopene at 1.2μM,and NAC at 31μM had no side effect on the viability of rat chondrocytes;3.Compared with normal rats,superficial structure of OA rats was unsmooth,and expression of proteoglycan and COL 2 was higher;compared with OA rats,the cartilage surface morphology of rats after the injection of antioxidants was knee was smoother,and expression of proteoglycan and COL 2 in superficial structure were relatively increased;Part 3:Effects of allicin,sulforaphane and lycopene on the phenotype of rat ADSCs.1.The purchased ADSCs displayed typical MSCs morphology with long-spindle-like shape in vitro;2.Allicin at 31μM,sulforaphane at 7μM,lycopene at 1.2μM,and NAC at 31μM had no cytotoxic effect on rat ADSCs viability;3.The H₂O₂ group had fewer expressions in COL 2,aggrecan,and SOX-9 and compared to control group,whereas expression of the chondrogenic markers in allicin,sulforaphane,lycopene,and NAC group was much stronger than that in the H ₂O₂ group.Besides,the data demonstrated that H₂O₂ improved levels of i NOS,p53,IL-6,RUNX2,TNF-α,C-caspase3,and MMP-13 in ADSCs.Addition of allicin,sulforaphane,lycopene,and NAC to ADSCs incubated with H₂O₂ could significantly decrease expression of i NOS,p53,C-caspase3,IL-6,TNF-α,MMP-13,and RUNX2;4.H₂O₂ stimulation increased levels of IL-6,MMP-13,and TNF-αsecreted by rat ADSCs compared with control group,while treatment with allicin,sulforaphane,lycopene,and NAC decreased expression of IL-6,MMP-13,and TNF-α;5.The osteogenic and chondrogenic differentiation in H₂O₂ group were weakened,and adipogenic differentiation was enhanced.Rat ADSCs in Allicin,sulforaphane,lycopene,and NAC group have more"calcium nodule"and proteoglycans and less fat vacuoles;the proliferation and migration of ADSCs in H₂O₂ group were weakened,and ADSCs in allicin,sulforaphane,lycopene and NAC groups have stronger proliferation and migration.Part 4:The study of allicin,sulforaphane and lycopene on survival of rat ADSCs in knee joint and curative effect of antioxidants-MSCs suspension on rat knee OA.1.After Di R-labeling,growth of rat ADSCs is active,and its membrane is well labeled when observed under the microscope;2.Compared with OA rats without synergistic injection of antioxidants,number of ADSCs labeled with Di R in joint has increased significantly in antioxidant-injection group;3.Compared with normal rats,OA rats and OA rats without synergistic injection of antioxidants have unsmooth superficial structure and expression of proteoglycan and COL 2is decreased;compared with OA rats without synergistic injection of antioxidants,after synergistic injection of antioxidants,superficial structure of rats is smoother and expression of proteoglycan and COL 2 is relatively increased;Conclusion1.Allicin,sulforaphane and lycopene reduce oxidative stress,increase expression of antioxidase,inhibit expression of inflammatory factors,improve chondrogenic phenotype and alleviate hypertrophy phenotype of human OACs by activating Keap1/Nrf2 pathway;2.Intra-articular injection of allicin,sulforaphane and lycopene can effectively alleviate the progress of OA in rats;3.Allicin,sulforaphane and lycopene reduce oxidative stress,inhibit expression of inflammatory factors,improve chondrogenic differentiation and alleviate apoptosis,senescence and hypertrophy phenotype in rat ADSCs;4.Intra-articular injection of allicin,sulforaphane and lycopene could protect survival of rat ADSCs in joint and may improve therapeutic effect of MSCs suspension on rat OA;...