| BACKGROUND:Rheumatoid arthritis(RA)is a long-term autoimmune disease that can lead to progressive joint damage,deformity,and dysfunction.At present,some clinical drugs can slow down the progression of RA to a certain extent,but they are often combined with many adverse reactions.Traditional Chinese medicine has a long history and experience in the treatment of RA.Traditional Chinese medicine has the advantages of multiple targets and high safety,from which we can develop supplementary and alternative medicines for RA treatment.Curcumin has anti-inflammatory,antioxidant properties,and a good safety profile.Many studies have shown that curcumin showed good efficacy in the treatment of RA.The previous study of the research group proved that curcumin can improve joint inflammation in rats with adjuvant arthritis,inhibit synovial angiogenesis.Part of the mechanism has been elucidated.OBJECTIVE:The purpose of this paper is to explore the effect and mechanism of curcumin in the treatment of RA by using the network pharmacology method and in vitro and in vivo experiments.METHODS:1.Network pharmacology methods were used to predict the possible targets and molecular mechanisms of curcumin in the treatment of RA and obtain the intersection targets between curcumin and RA.We constructed a"drug-target-disease" protein-protein interaction network,screened out the core targets of curcumin in the treatment of RA,and performed GO and KEGG enrichment analysis,then verified the core targets by molecular docking.2.15 patients with RA who underwent knee surgery were selected as the RA group,and 10 patients with joint injuries who underwent knee surgery during the same period were selected as the control group.Synovial histopathological specimens and clinical data of RA patients were collected.Synovial tissue characteristics are assessed from a pathological perspective.Immunohistochemical staining was used to detect the expression of p-AKT,Bcl-2,and MMP-9 proteins in the synovial tissue of RA patients.3.In this in vivo study,CIA mouse models of RA were established.DBA/1 mice were divided into four groups:Control group,CIA group,Curcumin group,and MTX group,and treated by grouping.An Arthritis index score was used to assess the degree of inflammation.HE staining was used to observe the pathological morphology of the synovium.ELISA was used to detect the expression of TNF-α,IL-6,IL-17 in serum.TUNEL staining was used to measure the apoptosis of synovial tissue.Western-Blot was used to detect the expression of PI3K/AKT signaling pathway and apoptosis-related proteins in synovial tissue.4.Part 1 study in vitro:TNF-α was used to induce the MH7A cell line in order to create an in vitro model of RA.The MMT method was used to assess MH7A cell viability,demonstrating the effects of TNF-α and Curcumin concentrations.The MH7A cells were divided into three groups:Control group,TNF-α group(10 ng/mL TNF-α),and TNF-α+Curcumin group(10 ng/mL TNF-α+50 μM Curcumin).Cell culture and dosing were performed according to groups.The proliferation ability of MH7A cells was detected by the EdU cell proliferation assay and plate colony formation assay.The migration ability of MH7A cells was detected by a cell scratch assay.The invasion ability of MH7A cells was detected by the Transwell assay.The apoptosis of MH7A cells was detected by the Annexin-V method.qRT-PCR was used to detect the expression of TNF-α,IL-6 and IL-17 in MH7A.Western Blot was used to detect the expression of MMP-2,MMP-9,apoptosis-related proteins and PI3K/AKT signaling pathway-related proteins in MH7A.Part 2 in vitro study:IGF-1,the PI3K/AKT signal stimulator,was used to activate the activity of PI3K/AKT signal transduction,and its effects on the apoptosis rate and apoptosis-related proteins of MH7A cells were reversedRESULTS:1.We obtained 108 targets for curcumin through the Swiss Target Prediction.We obtained 4465 disease targets for RA through the Genecards database.We obtained 71 potential targets for curcumin in the treatment of RA.We constructed a protein interaction network with AKT1 as the main core target.The core targets mainly include AKT1,TNF,EGFR,STAT3,MMP9,etc.Through GO and KEGG enrichment analysis,it is shown that the biological function of GO is dominated by protein serine/threonine kinase activity,and the signaling pathway is the PI3K/AKT signaling pathway.We performed molecular docking simulations between curcumin and AKT1,and the results showed that curcumin could stably dock into the active pocket of the AKT1 protein structure with an optimal affinity of-5.1 kcal/mol.2.We collected 15 cases in the RA group and 10 in the control group.There was no significant difference in age and gender between the two groups(P>0.05).WBC,MONO,NEUT,CPR,RF,and Anti-CCP in the RA group were significantly higher than those in the control group(P<0.05 or P<0.01).The synovial tissue of patients with RA conformed to the typical features of synovitis,with synovial lining hyperplasia and inflammatory cell infiltration.The Krenn’s pathological score of synovial tissue in the RA group was significantly higher than that in the control group(P<0.05 or P<0.01).Compared with the control group,the expressions of p-AKT,Bcl-2,and MMP-9 in the synovial tissue of the RA group were significantly higher than those of the control group(P<0.05 or P<0.01).The expression of p-AKT was positively correlated with ESR(r=0.680,P=0.005)and DAS28-3(r=0.634,P=0.011).The expression of Bcl-2 was positively correlated with ESR(r=0.640,P=0.010)and CRP(r=0.584,P=0.022).The expression of MMP-9 was positively correlated with ESR(r=0.809,P=0.000)and DAS28-3(r=0.590,P=0.021).The expression of Bcl-2 and MMP-9 was positively correlated(r=0.630,P=0.012).3.The arthritis index scores of the mice in the CIA group gradually increased.After we intervened with Curcumin,the arthritis index score in the Curcumin group was significantly lower than that in the CIA group after 35 days(P<0.01).Curcumin ameliorated pathological manifestations such as inflammatory cell infiltration,synovial lining hyperplasia,and pannus formation in the synovial tissue of CIA mice.TNF-α,IL-6,and IL-17 levels in the serum of mice in the CIA group were significantly higher than those in the Control group(P<0.01).TNF-α,IL-6,and IL-17 levels in serum of mice in the Curcumin and MTX groups were significantly lower than those in the CIA group(P<0.01).Compared with the CIA group,the synovial cell apoptosis index was significantly increased in the Curcumin group and MTX group(P<0.05 or P<0.01).The ratios of p-PI3K/PI3K and p-AKT1/AKT1 in the Curcumin group and MTX group were significantly lower than those in the CIA group(P<0.01).Compared with the CIA group,the ratio of Bcl-2/Bax was significant decreased in the Curcumin group(P<0.01).Compared with the MTX group,the expression of Bcl-2,Bax,Cleaved caspase-3 and the ratio of Bcl-2/Bax in the synovial tissue of the Curcumin group had no significant difference(P>0.05).4.Part 1 in vitro study:TNF-α can increase the viability of MH7A cells.The most significant increase in MH7A cell viability was observed at 10 ng/mL TNF-α concentration.Curcumin has an inhibitory effect on the viability of MH7A cells in a dose-dependent manner.The calculated median inhibitory concentration(IC50)is 50.68 μM,so we used 50 μM as the administration concentration of curcumin in the subsequent experiments.As compared with the Control group,the EdU positive rate,the number of colonies formed,the wound healing rate,and the number of invasive cells in the TNF-α group were significantly increased(P<0.01),while the apoptosis rate was significantly decreased(P<0.05 or P<0.01).When compared with the TNF-α group,the EdU positive rate,number of colonies formed,wound healing rate,and number of invasive cells in the TNF-α+curcumin group were significantly decreased(P<0.01),while the apoptosis rate was significantly increased(P<0.01).TNF-α,IL-6,and IL-17 mRNA levels in MH7A were significantly higher in the TNF-α group compared with the control group(P<0.01).TNF-α,IL-6 and IL-17 mRNA levels were significantly lower in the TNF-α+curcumin group compared with the control group(P<0.01).MMP-2 and MMP-9 protein expression lecels were significantly higher in the TNF-α+curcumin group compared with the TNF-α group(P<0.01).MMP-2 and MMP-9 protein expression lecels were significantly lower in the TNF-α+curcumin group compared with the TNF-α group(P<0.01).The ratio of Bcl-2/Bax was significantly higher in the TNF-group than in the control group(P<0.01).The protein expression of Cleaved caspase-3 in MH7A was upregulated in the TNF-α+curcumin group compared with the TNF-α group,while the Bcl-2/Bax ratio was significantly decreased(P<0.01).The ratios of p-PI3K/PI3K and p-AKT1/AKT1 in the TNF-α group were significantly higher when compared with the control group(P<0.01).The ratios of p-PI3K/PI3K and p-AKT1/AKT1 in the TNF-α+curcumin group were significantly lower when compared with the TNF-α group(P<0.01).Part 2 in vitro study:The apoptosis rate of MH7A cells in the TNF-α group was significantly lower when compared with the control group(P<0.01).The apoptosis rate of MH7A cells was significantly higher in the TNF-α+curcumin group than in the TNF-α group(P<0.01),and the apoptosis rate of the MH7A cells in the TNF-α+curcumin+IGF-1 group was higher than that in the TNF-α group,with no statistically significant difference.Compared with the TNF-α+curcumin group,the apoptosis rate of MH7A cells in the TNF-α+curcumin+IGF-1 group was significantly lower(P<0.01).After MH7A cells were induced by TNF-α,the ratios of p-PI3K/PI3K and p-AKT1/AKT1 were significantly increased(P<0.01).After adding Curcumin,the ratios of p-PI3K/PI3K and p-AKT1/AKT1 were significantly lower compared with the TNF-α group(P<0.01).After adding IGF-1,the ratio of p-PI3K/PI3K was significantly higher compared with the TNF-α+curcumin group(P<0.01),and the ratio of p-AKT1/AKT1 was slightly higher compared with the TNF-α+curcumin group,with no statistically significant difference(P>0.05).After inducted by TNF-α,the level of Cleaved caspase-3 protein was significantly decreased(P<0.01),and the ratio of Bcl-2/Bax was significantly increased(P<0.01).When curcumin was added,the level of Cleaved caspase-3 protein increased significantly compared with the TNF-α group(P<0.01),while the Bcl-2/Bax ratio decreased significantly(P<0.01).After adding IGF-1,compared with the TNF-α+curcumin group,the level of Cleaved caspase-3 protein decreased(P<0.05),while the ratio of Bcl-2/Bax increased significantly(P<0.01).CONCLUSION:1.The overexpression of p-AKT,Bcl-2,and MMP-9 in the synovial tissue of RA patients is positively correlated with RA disease activity-related indicators.2.Curcumin can ameliorate joint inflammation in CIA mice by inhibiting the activation of PI3K/AKT signaling in the synovium,reducing the ratio of Bcl-2/Bax,and increasing the activity of caspase-3.3.Curcumin induces apoptosis of FLS cells by inhibiting the PI3K/AKT signaling pathway. |
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