海藻多糖对类风湿关节炎外周血单个核细胞中IL-37表达的影响及其抗炎机制

Effects of seaweed polysaccharides on the expression of IL-37 in peripheral blood mononuclear cells of rheumatoid arthritis and its anti-inflammatory mechanism

目的探讨海藻多糖对体外培养的类风湿关节炎患者外周血单个核细胞(PBMC)中白细胞介素-37(IL-37)表达的影响及其抗炎机制。方法分别收集类风湿关节炎患者和健康体检者外周血各30份,分离PBMC。①取健康体检者外周血PBMC作为对照组,给予正常培养;取类风湿关节炎患者外周血PBMC,随机分为10 mg/mL、20 mg/mL、30 mg/mL、40 mg/mL海藻多糖组,实时荧光定量PCR检测各组PBMC中IL-37 mRNA表达水平。②取健康体检者外周血PBMC作为对照组,给予正常培养;取类风湿关节炎患者外周血PBMC,随机分为10 mg/mL、20 mg/mL、30 mg/mL、40 mg/mL、50 mg/mL海藻多糖组,实时荧光定量PCR检测各组PBMC中诱导型一氧化氮酶(iNOS)mRNA表达水平。③取健康体检者外周血PBMC作为对照组;取类风湿关节炎患者外周血PBMC,随机分为患者组及10 mg/mL、20 mg/mL、30 mg/mL、40 mg/mL海藻多糖组,Griess法检测各组PBMC中一氧化氮(NO)水平。④实验分为3组,健康体检者外周血PBMC作为对照组,类风湿关节炎患者外周血PBMC作为患者组,类风湿关节炎患者外周血PBMC给予10 mg/mL、20 mg/mL、30 mg/mL、40 mg/mL海藻多糖培养作为海藻多糖组,酶联免疫吸附法(ELISA)检测各组上清中IL-37、IL-10、IL-4水平,并分析IL-37与IL-10、IL-4相关性。结果20 mg/mL、30 mg/mL、40 mg/mL海藻多糖组PBMC中IL-37 mRNA表达水平均明显高于对照组(P均<0.05),30 mg/mL、40 mg/mL海藻多糖组均明显高于20 mg/mL海藻多糖组(P均<0.05),30 mg/mL海藻多糖组和40 mg/mL海藻多糖组比较差异无统计学意义(P>0.05)。20 mg/mL、30 mg/mL、40 mg/mL、50 mg/mL海藻多糖组PBMC中iNOS mRNA表达水平均明显高于对照组(P均<0.05),且50 mg/mL海藻多糖组明显高于其他组(P均<0.05)。患者组PBMC中NO水平明显高于对照组(P<0.05),海藻多糖各组均明显高于患者组(P均<0.05)。患者组PBMC中IL-4与IL-10水平显著低于对照组(P均<0.05),IL-37水平明显高于对照组(P<0.05);海藻多糖组IL-37水平明显高于患者组(P<0.05),IL-10及IL-4水平均明显低于患者组(P均<0.05),且IL-37的升高与IL-10、IL-4的降低呈负相关关系(r=0.889,0.572,P<0.05)。结论海藻多糖对类风湿关节炎患者具有免疫调节作用,可能与上调PBMC中IL-37 mRNA、iNOS mRNA及NO表达水平,进而抑制IL-10、IL-4分泌有关。

Objective It is to investigate the effects of seaweed polysaccharides on the expression of interleukin-37(IL-37)in peripheral blood mononuclear cells(PBMCs)of patients with rheumatoid arthritis and its anti-inflammatory mechanism.Methods 30 examples of peripheral blood were respectively collected from patients with rheumatoid arthritis and healthy subjects,and PBMCs were isolated.①The peripheral blood PBMCs from healthy people were taken as a control group and were cultured normally;the peripheral blood PBMCs from patients with rheumatoid arthritis were taken and randomly divide into 10 mg/mL,20 mg/mL,30 mg/mL,and 40 mg/mL seaweed polysaccharide groups.Real-time quantitative PCR was used to detect the expression of IL-37 mRNA in each group of PBMCs.②The peripheral blood PBMCs from healthy people were taken as a control group and were cultured normally;the peripheral blood PBMCs from patients with rheumatoid arthritis were taken and randomly divide into 10 mg/mL,20 mg/mL,30 mg/mL,40 mg/mL and 50 mg/mL seaweed polysaccharide groups.Real-time quantitative PCR was used to detect the expression level of inducible nitric oxide(iNOS)mRNA in PBMC of each group.③The peripheral blood PBMCs from healthy people were taken as a control group and were cultured normally;the peripheral blood PBMCs from patients with rheumatoid arthritis were taken and randomly divide into 10 mg/mL,20 mg/mL,30 mg/mL,and 40 mg/mL seaweed polysaccharide groups.Griess method was used to detect the level of nitric oxide(NO)in each group of PBMC.④The experiment was divided into 3 groups.Peripheral blood PBMC of healthy subjects was used as the control group.Peripheral blood PBMC of patients with rheumatoid arthritis was used as the patient group.PBMCs from patients with rheumatoid arthritis were given seaweed polysaccharide culture as a seaweed polysaccharide group.The levels of IL-37,IL-10,and IL-4 in the supernatant of each group were detected by Enzyme-linked immunosorbent assay(ELISA),and the correlation between IL-37 and IL-10 and IL-4 was analyzed.Results The expression levels of IL-37 mRNA in PBMC of the 20 mg/mL,30 mg/mL,and 40 mg/mL seaweed polysaccharide groups were significantly higher than those in the control group(all P<0.05),and the levels in the 30 mg/mL and 40 mg/mL seaweed polysaccharide groups were significantly higher than that in the 20 mg/mL seaweed polysaccharide group(all P<0.05),and there was no significant difference between the 30 mg/mL seaweed polysaccharide group and the 40 mg/mL seaweed polysaccharide group(P>0.05).The levels of iNOS mRNA expression in PBMCs of the 20 mg/mL,30 mg/mL,40 mg/mL,and 50 mg/mL seaweed polysaccharide groups were significantly higher than those in the control group(all P<0.05),and the levels in the 50mg/mL seaweed polysaccharide group was significantly higher than that in the other groups(all P<0.05).The levels of NO in PBMC of the patient groups were significantly higher than that of the control group(P<0.05),and the levels in the seaweed polysaccharide groups were significantly higher than those of the patient group(P<0.05).The levels of IL-4 and IL-10 in the PBMC of the patient group were significantly lower than those of the control group(both P<0.05),and the levels of IL-37 were significantly higher than those of the control group(P<0.05);the levels of IL-37 in the seaweed polysaccharide groups were significantly higher than the patient group(P<0.05),the levels of IL-10 and IL-4 were significantly lower than those in the patient group(P<0.05),and the increase of IL-37 was negatively correlated with the decrease of IL-10 and IL-4(r=0.889,0.572,P<0.05).Conclusion Seaweed polysaccharides have immunomodulatory effects on patients with rheumatoid arthritis,which may be related to up-regulating the expression levels of IL-37 mRNA,iNOS mRNA and NO in PBMC in order to inhibit the secretion of IL-10 and IL-4.