芍药苷在溃疡性结肠炎中通过靶向NF-κB信号通路调控Th17/Treg平衡

Paeoniflorin regulates Th17/Treg balance by targeting NF-κB signaling pathway in ulcerative colitis

目的探究芍药苷对大鼠溃疡性结肠炎(ulcerative colitis,UC)发生过程中体内Th17/Treg平衡和核因子-κB(nuclear factor kappa-B,NF-κB)信号通路的影响。方法将24只7~8周龄SPF级雄性SD大鼠随机分为4组:正常组、模型组、芍药苷低剂量组和高剂量组,每组6只大鼠。模型组、低剂量组和高剂量组大鼠推注5%TNBS进行UC大鼠造模,然后低剂量组和高剂量组大鼠灌胃相应浓度的芍药苷,连续治疗14 d。治疗结束后,对各组大鼠进行疾病活动指数(disease activity index,DAI)评分,并通过苏木精伊红(HE)染色进行组织病理学观察。分离大鼠脾脏T淋巴细胞并通过流式细胞仪分析Th17和Treg细胞比例。通过免疫组化染色检测结肠组织中p-P65、视黄酸相关孤儿受体γt(retinoic acid related orphan receptor-γt,ROR-γt)和叉头盒蛋白P3(forkhead box P3,FOXP3)的表达。通过qRT-PCR检测结肠组织中白细胞介素-6(interleukin-6,IL-6)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、IL-10、IL-17、FOXP3和ROR-γt的mRNA表达。通过Western blotting检测结肠组织中P65的磷酸化水平。通过免疫荧光分析结肠组织中p65的核转位。结果与模型组相比,低剂量组和高剂量组大鼠的结肠长度显著增加[(8.20±0.47)cm vs(9.66±0.44)cm,(10.72±0.39)cm,P<0.05]。与模型组相比,低剂量组和高剂量组大鼠的DAI评分显著降低(2.83±0.41 vs 2.33±0.52,1.67±0.82,P<0.05)。与模型组相比,低剂量组和高剂量组大鼠的结肠形态有所改善,结构破坏明显缓解,仅出现轻微的炎症细胞浸润。与模型组相比,低剂量组和高剂量组Th17细胞占比和Th17/Treg比率显著降低,而Treg细胞占比显著升高(均P<0.05)。与模型组相比,低剂量组和高剂量组IL-6、TNF-α、IL-17和ROR-γt的mRNA表达水平显著降低(P<0.05),而TGF-β1、IL-10和FOXP3的mRNA表达水平显著升高(P<0.05)。与模型组相比,低剂量组和高剂量组的P65磷酸化水平均显著降低(P<0.05)。与模型组相比,低剂量组和高剂量组的细胞核中P65蛋白表达水平明显降低(P<0.05)。结论芍药苷纠正了TNBS诱导的UC大鼠体内Th17/Treg平衡并抑制了NF-κB信号通路的活化,芍药苷可能通过靶向NF-κB信号通路来调控Th17/Treg平衡,从而发挥其对UC的治疗作用。

Objective To investigate the effect of paeoniflorin on Th17/Treg balance and nuclear factor kappa-B(NF-κB)signaling pathway in the process of ulcerative colitis(UC).Methods Twenty-four SPF male SD rats aged 7-8 weeks were randomly divided into four groups:normal group,model group,low-dose group and high-dose group,with 6 rats in each group.Rats in model group,low-dose group and high-dose group were injected with 5%TNBS to establish a UC rat model,and then the rats in low-dose group and high-dose group were intragastrically administered with corresponding concentrations of paeoniflorin for 14 d.After the treatment,the rats in each group were scored by disease activity index(DAI),and the histopathological changes were observed by hematoxylin-eosin(HE)staining.The rat spleen T lymphocytes were isolated,and the ratio of Th17 and Treg cells was analyzed by flow cytometry.The expression of p-P65,retinoic acid related orphan receptor-γt(ROR-γt)and forkhead box P3(FOXP3)in colon tissue was detected by immunohistochemical staining.The mRNA expression of interleukin-6(IL-6),transforming growth factor-β1(TGF-β1),tumor necrosis factor-α(TNF-α),IL-10,IL-17,FOXP3 and ROR-γt in colon tissue was detected by qRT-PCR.Western blotting was used to detect the phosphorylation level of p65 in colon tissue.The nuclear translocation of p65 in colon tissue was analyzed by immunofluore-scence.Results Compared with model group,the colon length of rats in low-dose group and high-dose group was increased significantly[(8.20±0.47)cm vs(9.66±0.44)cm,(10.72±0.39)cm,P<0.05].Compared with model group,the DAI scores in low-dose group and high-dose group were significantly decreased(2.83±0.41 vs 2.33±0.52,1.67±0.82,P<0.05).Compared with model group,the colon morphology were improved in low dose group and high dose group,the structural damages were significantly relieved,and only slight inflammatory cell infiltration appeared.Compared with model group,the proportion of Th17 cells and the ratio of Th17/Treg in low dose group and high dose group were significantly reduced,while the proportion of Treg cells was significantly increased(P<0.05).Compared with model group,the mRNA expression levels of IL-6,TNF-α,IL-17 and ROR-γt in low dose group and high dose group were significantly reduced(P<0.05),while the mRNA expression levels of TGF-β1,IL-10 and FOXP3 were significantly increased(P<0.05).Compared with model group,the phosphorylation level of P65 in low dose group and high dose group was significantly reduced(P<0.05).Compared with model group,the expression level of P65 protein in the nucleus in low dose group and high dose group was significantly reduced(P<0.05).Conclusion Paeoniflorin corrects the Th17/Treg balance in UC rats induced by TNBS and inhibits the activation of the NF-κB signaling pathway.Paeoniflorin may regulate Th17/Treg balance by targeting the NF-κB signaling pathway,thereby exerting its therapeutic effect on UC.