| ObjectiveGout is an acute and chronic inflammation and tissue damage caused by the deposition of monosodium urate in joints,kidneys and subcutanea.Worldwide,the prevalence of gout is increasing year by year,placing a heavy burden on society and medical systems.The main pathogenesis of acute gout attack is blood stasis.The traditional Chinese medicine turmeric has the effect of breaking blood and qi,relieving pain through menstruation,and is commonly used in the treatment of gout.Bone marrow mesenchymal stem cells can migrate to organs or tissues that suffer from inflammation or injury,produce cytokines,and participate in tissue repair and regeneration,which has a potential therapeutic prospect for gout.Firstly,using the method of network pharmacology,the active ingredients,targets and pathways of turmeric in the treatment of gout were studied to provide theoretical basis for further experimental research Next,to study the mechanism of curcumin combined with bone marrow mesenchymal stem cells against acute gouty arthritis through in vivo and in vitro experiments,in order to explore more therapeutic strategies for clinical treatment of gout.Method:The active constituents and targets of turmeric were screened by TCMSP,ChEMBL,SwissTargetPrediction and STITCH database,and gout related proteins were screened by MeSH,GeneCards and UniProt.Cytoscape software was used to construct the "Active ingredient-target-gout" network diagram,and then the PPI diagram was constructed to conduct enrichment analysis of KEGG pathway,Reactome pathway,and WikiPathways using STRING database.Finally,molecular docking was used to verify the degree of binding between the key target and the chemical composition.J774A.1 murine macrophages with a cell density of 2x106 per well were seeded in 6well plates.After 24hrs incubation,macrophages were pre-treated with 10 or 20μM curcumin,or 10nM colchicine for 1hr.Macrophages were then primed with 1μg/mL LPS for 4hrs,and then co-treated with 250μg/mL MSU for another 3hrs or 100μg/mL MSU for another 24hrs.For time point experiment,macrophages were starved for 2hrs in serum-free medium(SFM).Cells were then pre-treated with 10 or 20μM curcumin,or 1 0nM colchicine(positive control)for 1hr,and co-treated with 1μg/mL LPS for 15mins.The cells were collected for western blotting analysis,while conditioned media was collected for ELISA,Griess assay or western blotting analysis.Western blotting detected the expression of iNOS,NLRP3,ASC,procaspase-1,pro-IL-1β,cleaved-caspase-1,cleaved-IL-1β,p-NFκB,NFκB.ELISA detected the secretion of IL-1β,TNF-α and IL-6.J774A.1 murine macrophages with a cell density of 2x106 per well were seeded in 6well plates.After 24hrs incubation,macrophages were pre-treated with 10μM curcumin,control-CM,curcumin-CM or 10nM colchicine for 1hr.Macrophages were then primed with 1μg/mL LPS for for 4hrs,and then co-treated with 250μg/mL MSU for another 3hrs.The conditioned media was collected for ELISA,while cells were collected for western blotting analysis.Western blotting detected the expression of iNOS,NLRP3,ASC,pro-IL-1β.ELISA detected the secretion of IL-1β.C57BL/6 mice were randomLy divided into six groups,including the groups of uninduced,vehicle,control-MSC(CTL-MSC),curcumin-treated MSC(curcumin-MSC),control-MSC-CM(CTL-MSC-CM),and curcumin-MSC-CM.2.75x104 MSCs resuspended in 15μL of SFM or 15μL of MSC-CM were injected intra-articularly.1hr after the pretreatments of MSCs or MSC-CM,all groups except uninduced group were injected with MSU crystals(lmg MSU crystals in 40μL of PBS)by intra-plantar injection,while uninduced group was injected with the same volume of PBS.Measurement on the thickness of foot paw was conducted by caliper after MSU injection for 24hrs,and mice were terminated.Western blotting detected the expression of NLRP3 and pro-IL-1 β.ELISA detected the secretion of IL-1β.Results:The following results were found through network pharmacology and molecular docking.The potential active ingredient of turmeric in the treatment of gout is curcumin,and the key targets include NLRP3,ASC(PYCARD),Caspase-1,IL-6,STAT3,TNF and other proteins.It may play anti-inflammatory,immune regulation and antioxidant stress functions by targeting NLRP3 inflammasome pathways.Anti-inflammatory effects of curcumin on activated macrophages:1.The IC50 of curcumin on J744A.1 macrophages was around 24μM,while the IC50 of colchicine on J744A.1 macrophages was around 70nM.2.The results indicated that the NO production and iNOS protein expression were suppressed by curcumin treatments on activated macrophages with a dose-dependent manner(P<0.05),which indicated the potential of curcumin on antiinflammation.3.From the results,the protein expression of iNOS,NLRP3,pro-caspase-1,pro-IL-1β,cleaved caspase-1,cleaved-IL-1β,p-NF-κB were downregulated in activated macrophages by curcumin(P<0.05).4.The level of IL-1β,TNF-α and IL-6 in activated macrophages were significantly suppressed by curcumin treatment compared to LPS-MSUinduced macrophages(P<0.001),with a dose-dependent effect.Anti-inflammatory effects of curcumin-educated MSCs on activated macrophages:1.there was no significant difference in cell morphology between the control and curcumintreated MSC.2.After priming the J774A.1 macrophages with 1 μg/mL LPS for and activated with 250μg/mL MSU,the macrophages became more spindle shaped compared to uninduced control.The J774A.1 macrophages by the treatment with curcumin-CM from set 1 showed less spindle while more ball shaped.3.The curcumin-CM from set 1 downregulated the protein expression of iNOS,NLRP3,pro-IL-1 β significantly in activated macrophages(P<0.01).4.Moreover,the ELISA results also indicated a significant downregulation in IL-1β from activated macrophages by curcumin-CM from set 1(P<0.05),which suggested a suppressive effect by curcumin-CM from MSC in set 1 on NLRP3 inflammasome activation in macrophages.Anti-inflammatory effects of curcumin-educated MSCs in mice with acute gouty arthritis:1.After the injection of MSU to the foot paw,the mice foot swelled,and the foot paw thickness increased significantly.Meanwhile,the treatment with MSC or MSC-CM suppressed the increase in foot paw thickness.However,only the curcumin-MSC-CM showed a stronger suppressive effect in reducing the foot paw thickness after MSU injection than the CTL-MSC-CM,while curcumin-MSC did not when compared to the CTL-MSC group.2.The injection of MSU increased the IL-1β level in foot paw,while the MSC or MSCCM treatment suppressed the increased level of IL-1β significantly(P<0.05).3.The treatment with MSC-CM downregulated the protein expression of NLRP3 and pro-IL-1βsignificantly(P<0.05).Conclusion:This study predicted the possible mechanism of curcumin in the treatment of gout,and its main component curcumin may play an anti-arthritis role through NLRP3 inflammasome pathway,reflecting the multi-target and multi-pathway action characteristics of traditional Chinese medicine.Curcumin treatment suppressed the NLRP3 inflammasome activation in macrophages and downregulated the pro-inflammatory cytokine(IL-1β,TNF-α and IL-6)production.In addition,Curcumin suppressed the NO production and iNOS protein expression.Curcumin-CM from set 1 exhibited a suppressive effect on NLRP3 inflammasome in activated macrophages.Moreover,the ELISA results also indicated a significant downregulation in IL-1β from activated macrophages by curcumin-CM from set 1.Curcumin-MSC-CM alleviated MSU-induced gouty arthritis in mice.Curcumin-MSCCM downregulated the IL-1β production in mice with MSU-induced gouty arthritis.Moreover,the treatment with Curcumin-MSC-CM suppressed the NLRP3 inflammasome activation.In conclusion,whether from the perspective of in vivo or in vitro studies,curcumin or curcumin combined with MSCs suppressed the secretion of inflammatory factors and the activation of NLRP3 inflammasome,so as to improve the symptoms of gouty arthritis and relieve the onset of acute gouty arthritis.In addition,curcumin combined with MSCs was more effective than curcumin alone.This study aimed to provide a new idea for clinical treatment of gouty arthritis. |
