延胡索总生物碱对溃疡性结肠炎小鼠的作用及机制研究

Study on the Pharmacodynamic Effect and Mechanism of Total Alkaloids of Rhizoma Corydalis on Mice with DSS-induced Ulcerative Colitis

目的观察延胡索总生物碱对溃疡性结肠炎(UC)小鼠的影响,并初步探讨其可能机制。方法①将30只雄性C57BL/6小鼠随机分为正常组、模型组、阳性药组(柳氮磺胺吡啶,300 mg·kg^(-1))、延胡索总生物碱低剂量组(50 mg·kg^(-1))和延胡索总生物碱高剂量组(100 mg·kg^(-1)),每组6只。采用2.5%葡聚糖硫酸钠(DSS)自由饮用7 d建立UC小鼠模型;造模同时连续灌胃给予延胡索总生物碱干预7 d。观测小鼠体质量及便血等疾病症状,计算粪便性状指数;采用ELISA法检测小鼠血清中白细胞介素6(IL-6)及肿瘤坏死因子α(TNF-α)含量;测量结肠长度,采用HE染色法观察结肠组织病理变化。②采用脂多糖(LPS)诱导RAW264.7小鼠巨噬细胞体外炎症模型。不同浓度延胡索总生物碱干预后,采用MTT法检测细胞活性。3.13、6.25、12.5μg·mL^(-1)延胡索总生物碱干预后,检测上清液中IL-6、TNF-α及一氧化氮(NO)含量;qPCR法检测细胞TNF-α、IL-6 mRNA表达;流式细胞术检测巨噬细胞M1表型比例;Western Blot法检测细胞丝裂原活化蛋白激酶(MAPK)通路相关蛋白的表达。结果①与模型组比较,延胡索总生物碱低、高剂量组小鼠的体质量减轻幅度及粪便性状评分均有所降低,血清中IL-6、TNF-α含量均显著降低(P<0.01);高剂量组小鼠结肠长度明显增加(P<0.05);低、高剂量组小鼠肠道上皮细胞破坏程度有所减轻,溃疡灶数量有所减少,溃疡面积缩小,中性粒细胞等免疫细胞浸润程度减轻。②3.13、6.25、12.5μg·mL^(-1)延胡索总生物碱组的细胞相对存活率均高于95%。与LPS组比较,3.13、6.25、12.5μg·mL^(-1)延胡索总生物碱组RAW264.7巨噬细胞的TNF-α、IL-6 mRNA表达显著下调(P<0.01),细胞上清液中的NO、TNF-α、IL-6含量均显著降低(P<0.05,P<0.01),巨噬细胞的CD86分子表达水平显著降低(P<0.01),细胞p38磷酸化水平显著降低(P<0.01),但ERK和JNK蛋白磷酸化水平未见明显改变(P>0.05)。结论延胡索总生物碱对UC小鼠具有治疗作用,可能与其调控MAPK通路抑制p38磷酸化发挥抗炎活性有关。

Objective To observe the pharmacodynamic effect of total alkaloids of Rhizoma Corydalis on mice with ulcerative colitis(UC)and to preliminary explore the possible mechanism.Methods①Thirty male C57BL/6 mice were randomly divided into the normal group,the model group,positive drug group(salicylazosulfapyridine,300 mg·kg^(-1)),the low dose group of Rhizoma Corydalis alkaloid(50 mg·kg^(-1))and the high dose group of Rhizoma Corydali alkaloid(100 mg·kg^(-1)),six mice in each group.The mice model of UC was established by free drinking of 2.5%dextran sodium sulfate(DSS)for 7 days;At the same time,the total alkaloid of Rhizoma Corydalis was administered by gavage for 7 continuous days.The body mass,blood in stool and other disease symptoms in mice were observed,and the faecal trait index was calculated;the interleukin 6(IL-6)and tumour necrosis factorα(TNF-α)in serum were detectd by ELISA method;the length of the colon was measured and the histopathological changes of the colon were observed by HE staining method.②Lipopolysaccharide(LPS)was used to induce inflammation in RAW264.7 mouse macrophages in vitro.The cellular activity was detected by MTT method after the intervention with different concentrations of total alkaloids of Rhizoma Corydalis.3.13,6.25 and 12.5μg·mL^(-1) total alkaloids of Rhizoma Corydalis were used to detect the contents of IL-6,TNF-αand nitric oxide(NO)in the supernatant;the qPCR method was used to detect the mRNA expression of TNF-αand IL-6;flow cytometry(FCM)was used to detect the phenotypic proportion of M1 in macrophages;the Western Blot method was used to detect the related protein expression of mitogen-activated protein kinase(MAPK)pathway.Results①Compared with the model group,the mice in the low-and high-dose of Rhizoma Corydalis groups showed a reduction in body mass and faecal trait score,and the contents of IL-6 and TNF-αin serum were significantly reduced(P<0.01);the colon length of mice in the high-dose groups was significantly increased(P<0.05);the damage degree of intestinal epithelial cells was alleviated,the number of ulcer foci was reduced,the ulcer area was narrowed,and the infiltration of immune cells such as neutrophils was reduced in the low-and high-dose groups.②The relative cell survival rates of the 3.13,6.25 and 12.5μg·mL^(-1) total alkaloids of Rhizoma Corydalis groups were all higher than 95%.Compared with the LPS group,the mRNA expressions of TNF-αand IL-6 of RAW264.7 macrophages in the 3.13,6.25 and 12.5μg·mL^(-1) total alkaloids of Rhizoma Corydalis groups were significantly reduced(P<0.01),the contents of NO,TNF-αand IL-6 in the cell supernatant were significantly decreased(P<0.05,P<0.01),the molecule expression of CD86 in macrophages was significantly decreased(P<0.01),and cellular p38 phosphorylation levels were significantly decreased(P<0.01),but protein phosphorylation levels of ERK and JNK were not significantly changed(P>0.05).Conclusion The total alkaloids of Rhizoma Corydalis has therapeutic effect on ulcerative colitis,and the mechanism may be related to its anti-inflammatory activity by inhibiting the phosphorylation level of MAPK pathway p38.