| Background: Rheumatoid arthritis is an autoimmune disease characterized by chronic joint synovial inflammation.Glucocorticoid is a recommended drug for the "Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis",but long-term,high-dose use can cause serious side effects such as immunosuppression and osteoporosis.Our previous study showed that ESCIN can enhance the anti-arthritic effect of low-dose glucocorticoids,but the detailed mechanism of action is unclear and needs further clarification.Objective: We investigated the anti-RA effect of ESCIN combined with low dose of GCs(dexamethasone,Dex)and its underlying mechanism.Methods: I.In vivo experiment: Preparation of SD rats for adjuvant arthritis model,rats were randomly divided into model group(AIA),dexamethasone high dose(DEX,0.2 mg/kg)group,dexamethasone low dose(DEX,0.05 mg /kg)group,ESCIN 10 mg/kg group,DEX 0.05+ESCIN group,10 rats in each group,another 10 were used as normal control group(Control).The vehicle and the corresponding drug were administered intragastrically(ig.)daily for 14 days.1.Joint swelling and pathological examination: The swelling of the primary and secondary side joints of the hind limbs of rats was detected by the athlete’s foot swelling measuring instrument every 3 days.Hematoxylin and eosin(HE)staining was used to observe the pathology of the foot and ankle.2.Detection of inflammatory factors: Elisa method was used to detect serum TNF-α and IL-6 inflammatory factors.3,Immunological organ pathological examination: Spleen and thymus weighing,calculate the immune organ index;and observe the pathological changes by HE staining;4,GR and its downstream inflammatory pathway detection: gene chip detection of GR signaling pathway gene expression in rat’s secondary joint synovial tissue;Western blot was used to detect the expression of GR,nuclear p65(NF-κB p65),IκB-α,phosphorylated p65 and phosphorylated IκB-α protein in rat’s synovial membrane.2.In vitro experiment: LPS was used to stimulate RAW 264.7 macrophages for inflammatory models,which were divided into Control group,LPS group,DEX with high dose(50 n M)group,and DEX with low dose(12.5 n M)group.In the ESCIN 10 μM group and the DEX+ESCIN(12.5 n M+10 μM)group,the corresponding drugs were added to each well.After 2 h,LPS was added to induce inflammation.1.Detection of inflammatory factors: After 24 h,the Griess method was used to detect the expression of NO in the supernatant;after 6 h,the concentration of TNF-α and IL-6 inflammatory factors in the supernatant was determined by Elisa;2.The detection of GR and its downstream inflammatory pathway: After 1 h,RT-PCR was used to detect the expression of m RNA of GR.After 6 h,the expression of GR,p65,IκB-α,phosphorylated p65 and phosphorylated IκB-α protein were detected by Western blot.Results: In vivo experiment: 1.Effects on swelling of the joint: Compared with the Control group,the swelling rate and joint swelling index of the primary side and secondary side of the AIA group were significantly increased(P < 0.01),Joint synovial hyperplasia and cartilage destruction occurred;compared with AIA group,the joint swelling rate and joint swelling index of DEX with high dose group and DEX+ESCIN group were significantly decreased(P < 0.01),joint synovial hyperplasia and cartilage destruction was also significantly reduced;compared with the low-dose DEX group,the DEX+ESCIN group had better inhibition of joint swelling rate and joint swelling index,and joint damage was lighter.2.Effect on inflammatory factors: The levels of TNF-α and IL-6 in serum in the AIA group were significantly increased(P < 0.01),which compared with the control group;compared with the AIA group,the levels of TNF-α and IL-6 in serum in the DEX+ESCIN group were significantly lower(P < 0.01).Compared with the low-dose dexamethasone group,the TNF-α and IL-6 in serum in the DEX+ESCIN group were lower.3.Effect on immune organs: Compared with the control group,the thymus of the AIA group showed no obvious atrophy and the spleen increased slightly;the spleen and thymus of the high-dose DEX group were severely atrophied(P < 0.01),and the number of spleen lymph nodes significantly reduced,the cortex became thinner,the number of thymocytes decreased significantly,and vacuoles were formed in the cytoplasm of the cells.Compared with the high-dose DEX group,the spleen and thymus atrophy in the DEX+ESCIN group was significantly reduced.4.Effects on GR and its downstream inflammatory pathway: Compared with the control group,the expression of Nr3c1 gene in the synovial tissue of the AIA group was obviously down-regulated(P < 0.01),and the expression of tnf and Il-6 genes was up-regulated(P < 0.01);Compared with AIA group and DEX group,the expression of Nr3c1 gene in DEX+ESCIN group was significantly up-regulated(P < 0.01);compared with Control group,AIA group rats synovial tissue GR,IκB-α protein level was significantly decreased(P < 0.05),p65 and phosphorylated p65,phosphorylated IκB-α protein levels were significantly increased(P < 0.05);compared with AIA group,DEX+ESCIN group GR,IκB-α protein was significantly increased(P < 0.05),and the levels of p65 and phosphorylated p65 and phosphorylated IκB-α protein were significantly decreased(P < 0.05).In vitro experiment: In vitro experiment: 1.Effects on inflammatory factors: Compared with the Control group,LPS can stimulate the release of NO,TNF-α and IL-6 in RAW 264.7 macrophages(P < 0.01);Compared with AIA group,DEX+ESCIN group significantly inhibited the production of NO,TNF-α and IL-6(P < 0.01);compared with the low-dose DEX group,DEX+ESCIN group enhanced inhibition of TNF-α and IL-6.2.Effects on GR and its downstream inflammatory pathway: Compared with the Control group,the m RNA and protein expression of GR in RAW 264.7 macrophages was significantly decreased in LPS group(P < 0.05),phosphorylation of p65,phosphorylation.The level of IκB-α protein was significantly increased(P < 0.05).Compared with the LPS group,DEX+ESCIN group significantly increased the m RNA and protein expression levels of GR(P < 0.05).The expression of GR m RNA and protein in DEX+ESCIN group was increased,which compared with the high-dose DEX group.Compared with the Control group,the expression of GR and IκB-α protein in the LPS group were significantly decreased(P < 0.05),and the phosphorylated p65 and phosphorylated IκB-α protein levels were significantly increased(P < 0.05);compared with LPS group,the expression of GR and IκB-α protein in DEX+ESCIN group was significantly increased(P < 0.05),inhibiting phosphorylated p65,phosphorylated IκB-α protein.The level of expression was significantly decreased(P < 0.05);further blocking of GR blockade revealed that the DEX high,low dose and DEX +ESCIN combination inhibited the phosphorylation of p65 and phosphorylated IκB-α protein in the inflammatory pathway.Conclusion: ESCIN can enhance the anti-arthritic effect of low-dose glucocorticoids and reduce the side effects of glucocorticoids,and its mechanism may be related to the regulation of GR signaling pathway activation. |
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