四神丸对脾肾阳虚型溃疡性结肠炎小鼠炎性树突状细胞的调节作用

Effect of Sishen Pill(四神丸)on Inflammatory Dendritic Cells in Colitis Mice of Spleen-Kidney Yang Deficiency

目的探讨四神丸(SSP)对脾肾阳虚型溃疡性结肠炎(UC)小鼠炎性树突状细胞的调节作用。方法将BALB/c小鼠随机分为正常组(Normal)、四神丸对照组(Normal+SSP)、模型组(DSS)、四神丸组(DSS+SSP),每组10只。除Normal组和Normal+SSP组外,其余各组采用“大黄+氢化可的松+DSS”的复合造模法模拟脾肾阳虚型UC小鼠模型。造模第15天,Normal+SSP组和DSS+SSP组均灌胃给予四神丸混悬液,2.5 g/kg,连续7 d。每日观察各组小鼠一般情况,并记录体质量及疾病活动指数(DAI)评分;取小鼠外周血进行三碘甲状腺原氨酸(T3)、甲状腺素(T4)、睾酮(T)检测;取小鼠结肠组织和脾脏,观察大体形态及结肠损伤情况,进行结肠病理损伤评分,同时HE染色后观察结肠黏膜病理改变;采用流式细胞分析仪检测脾脏组织CD11c^(+)CD103^(+)TNF-α^(+)、CD11c^(+)CD103^(+)INOS^(+)细胞水平;采用ELISA测定结肠白细胞介素(IL)-12、IL-6、IL-1β、肿瘤坏死因子α(TNF-α)、IL-10、干扰素γ(IFN-γ)表达水平。结果与Normal组相比,DSS组小鼠出现精神萎靡、嗜倦懒动、弓背扎堆、纳差食少、毛发黯暗、大便稀软带血等体征,且体质量明显下降及DAI明显升高(P<0.05,P<0.01);血清T3、T4、T水平明显降低(P<0.05,P<0.01);结肠重量指数、肠重指数、脾重指数及结肠病理损伤评分明显升高(P<0.05,P<0.01),结肠长度减少(P<0.05);脾脏CD11c^(+)CD103^(+)TNF-α^(+)、CD11c^(+)CD103^(+)INOS^(+)细胞水平显著升高(P<0.01);结肠IL-1β、TNF-α、IL-6、IL-12水平明显升高(P<0.05,P<0.01),IL-10、IFN-γ水平显著降低(P<0.01)。与DSS组相比,DSS+SSP组小鼠上述体征明显改善,体质量明显提升及DAI明显降低(P<0.05,P<0.01);血清T3、T4、T水平明显升高(P<0.05,P<0.01);结肠重量指数、肠重指数、脾重指数及结肠病理损伤评分明显降低(P<0.05,P<0.01),结肠长度增加(P<0.05);脾脏CD11c^(+)CD103^(+)TNF-α^(+)、CD11c^(+)CD103^(+)INOS^(+)细胞水平显著降低(P<0.01);结肠IL-1β、TNF-α、IL-6、IL-12水平显著降低(P<0.05,P<0.01),IL-10、IFN-γ水平显著升高(P<0.01)。结论四神丸可通过降低CD11c^(+)CD103^(+)TNF-α^(+)、CD11c^(+)CD103^(+)INOS^(+)细胞水平以重塑肠道促炎因子IL-1β、TNF-α、IL-6、IL-12和抑炎因子IL-10、IFN-γ的平衡,进而有效治疗脾肾阳虚型小鼠UC。

Objective To investigate the regulatory effect of Sishen Pill(四神丸,SSP)on inflammatory dendritic cells in mice with ulcerative colitis(UC)of spleen-kidney Yang deficiency.Methods BALB/c mice were randomly divided into normal group(Normal),Sishen Pill control group(Normal^(+)SSP),model group(DSS)and Sishen Pill group(DSS^(+)SSP),with 10 mice in each group.Except for Normal group and Normal^(+)SSP group,the other two groups used the compound model of“Dahuang(rhubarb)^(+)hydrocortisone^(+)DSS”to simulate the UC mouse model of spleen-kidney Yang deficiency.On the 15 th day of the modeling,both the Normal^(+)SSP group and the DSS^(+)SSP group were given 2.5 g/kg SSP suspension by intragastric administration for 7 days.The general condition of mice in each group was observed every day,and the disease activity index(DAI)was scored by recording body mass,fecal traits and occult blood.The peripheral blood of mice was taken for three iodine thyroid(T3),thyroxin(T4)and testosterone(T).Colonic tissue and spleen were taken out to observe the general morphology and colonic injury score,and the pathological changes of colonic mucosa were observed after HE staining.Flow cytometry was used to detect the numbers of CD11 c^(+)CD103^(+)TNF-α^(+)and CD11 c^(+)CD103^(+)INOS^(+)cells in spleen,and the expression levels of interleukin-12(IL-12),interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),interleukin-10(IL-10)and interferon-γ(IFN-γ)in colon were measured by ELISA.Results Compared with those of the Normal group,the mice in the DSS group showed signs such as lethargy,arched back,less poor food,dark hair,soft stool and blood in stool,and the body mass was decreased significantly and the DAI increased significantly(P<0.05).The levels of serum T3,T4 and T were significantly decreased(P<0.05,P<0.05).The colon weight index,intestine weight index,spleen weight index and colon pathological injury score were significantly increased,the length of colon was decreased,the levels of CD11 c^(+)CD103^(+)TNF-α^(+)and CD11 c^(+)CD103^(+)INOS^(+)cells in spleen were significantly increased,the expression levels of IL-1β,TNF-α,IL-6 and IL-12 in colon were significantly increased,and the expression levels of IL-10 and IFN-γ were significantly decreased.Compared with those of the DSS group,the above signs were significantly changed,the body weight was significantly increased and the DAI was significantly decreased in the DSS^(+)SSP group(P<0.05).The levels of serum T3,T4 and T were significantly increased(P<0.05),and the colon weight index,intestinal weight index,spleen weight index and colon pathological injury score were significantly decreased(P<0.05),and the colon length was increased(P<0.05).The levels of CD11 c^(+)CD103^(+)TNF-α^(+)and CD11 c^(+)CD103^(+)INOS^(+)cells in spleen and the expression levels of IL-1β,TNF-α,IL-6 and IL-12 in colon were significantly decreased(P<0.05),while the expression levels of IL-10 and IFN-γ were significantly increased(P<0.05).Conclusion SSP can reshape the balance of intestinal pro-inflammatory factors IL-1β,TNF-α,IL-6,IL-12 and anti-inflammatory factors IL-10 and IFN-γ by reducing the numbers of CD11 c^(+)CD103^(+)TNF-α^(+)and CD11 c^(+)CD103^(+)INOS^(+)cells,and then effectively treat mice with UC of spleen-kidney Yang deficiency.