摘要
目的研究磷脂酰肌醇3激酶(phosphatidylin-ositol-3-kinase,PI3K)抑制剂Wortmannin通过PI3K/蛋白激酶B(protein kinase B,Akt)信号通路对克罗恩病(Crohn disease,CD)炎症大鼠的作用与机理。方法选取10周龄无特定病原体级健康雄性Wistar大鼠60只,建立CD大鼠模型,采用随机数字表法分为C1、C2、W1、W2、W3组,每组12只,分别采用0.9%氯化钠溶液、5%二甲基亚砜(dimethyl surfoxide,DMSO)及0.25、0.5、1.0 mg/kg Wortmannin+5%DMSO溶液腹腔注射处理12、24、48、72 h;并选取60只体质量等一般状况匹配的健康正常大鼠作为对照,采用随机数字表法分为CON1、CON2、CON3、CON4和CON5组,每组12只,处理方法及时间同上。各组均于处理12、24、48、72 h后,进行疾病活动指数(disease activity index,DAI)评分、结肠总长度测量,苏木精-伊红染色观察饲养72 h后结肠组织病理学改变,采用酶联免疫吸附法检测血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1(interleukin-1,IL-1)、白细胞介素-6(interleukin-6,IL-6)含量,逆转录-聚合酶链式反应检测结肠组织PI3K mRNA表达量,蛋白质免疫印迹法检测PI3K、Akt、磷酸化Akt(phosphorylated Akt,p-Akt)、核因子-κB(nuclear factor-κB,NF-κB)、TNF-α、IL-1、IL-6蛋白表达量,采用钙卫蛋白检测试剂盒测定粪便钙卫蛋白含量。结果饲养12、24、48、72 h,CON1、CON2、CON3、CON4、CON5组间及C1、C2组间DAI评分、结肠总长度及血清TNF-α、IL-1、IL-6含量和粪便钙卫蛋白含量比较差异均无统计学意义;饲养24、48、72 h,W1、W2、W3组DAI评分均低于C2组,且W3组饲养24 h DAI评分低于W2组,W2组饲养48、72 h DAI评分均低于W1组(P<0.05);饲养48、72 h,W1、W2、W3组结肠总长度均长于C2组,且W2、W3组均长于W1组,W3组长于W2组,W3组饲养12 h及W2、W3组饲养24 h结肠总长度均长于C2组,W3组饲养24 h长于W2组,差异有统计学意义(P<0.05)。饲养72 h,与C2组比较,W1、W2、W3组结肠组织病理学炎症表现依次好转。饲养12、24、48、72 h,与C2组比较,血清中TNF-α、IL-1、IL-6含量依次降低(P<0.05)。饲养72 h,CON1、CON2、CON3、CON4、CON5组及C1、C2组结肠组织中PI3K mRNA和PI3K、p-Akt、NF-κB、TNF-α、IL-1、IL-6蛋白表达量比较差异无统计学意义;与C2组比较,W1、W2、W3组72 h结肠组织PI3K mRNA,PI3K、p-Akt、NF-κB、TNF-α、IL-1、IL-6蛋白表达量依次降低,Akt蛋白表达量依次升高(P<0.05)。与C2组比较,W1、W2、W3组饲养12、24、48、72 h大鼠粪便中钙卫蛋白含量降低(P<0.05)。结论PI3K抑制剂Wortmannin可改善CD大鼠炎症表现,具有一定治疗作用,其机理可能是通过阻断PI3K/Akt通路,降低下游炎症因子水平,从而发挥抗炎效果。
Objective To study the effect and mechanism of phosphatidylin-ositol-3-kinase(PI3K)inhibitor Wortmannin on Crohn disease(CD)inflammatory rats through PI3K/protein kinase B(Akt)signal pathway.Methods 60 healthy male Wistar rats aged 10 weeks without specific pathogen were selected to establish a CD rat model,they were randomly divided into the C1,C2,W1,W2,and W3 groups using a random number table method,with 12 rats in each group.The rats were treated with intraperitoneal injections of 0.9%sodium chloride solution,5%dimethyl surfoxide(DMSO),and 0.25,0.5,and 1.0 mg/kg Wortmannin+5%DMSO solutions for 12,24,48,and 72 h,respectively.60 healthy normal rats with general matching conditions were selected as controls and they were divided into the CON1,CON2,CON3,CON4,and CON5 groups according to the random number table method,with 12 rats in each group and the treatment method and time were the same as above.After 12,24,48,and 72 h of treatment,each group underwent disease activity index(DAI)scoring,total colon length measurement,and Hematoxylin and eosin staining was used to observe pathological changes in colon tissue,after 72 h of feeding,enzyme linked immunosorbent assay was used to detect serum tumor necrosis factor-α(TNF-α),interleukin-1(IL-1),and interleukin-6(IL-6)levels,reverse transcription polymerase chain reaction was used to detect PI3K mRNA expression in colon tissue,and protein immunoblotting was used to detect the expression levels of PI3K,Akt,phosphorylated Akt(p-Akt),nuclear factor-κB(NF-κB),TNF-α,IL-1,and IL-6 proteins,and the content of fecal calprotectin was determined by using a calprotectin detection kit.Results There were no significant differences in DAI score,total colon length and serum TNF-α,IL-1 and IL-6 levels and fecal calprotectin content between CON1,CON2,CON3,CON4,CON5 groups and between C1 and C2 groups at 12,24,48 and 72 h after feeding.At 24,48 and 72 h of feeding,the DAI scores in the W1,W2 and W3 groups were lower than those in the C2 group,and the DAI score in the W3 group at 24 h of feeding was lower than that in the W2 group,and the DAI scores in the W2 group at 48 and 72 h of feeding were lower than those in the W1 group(P<0.05).At 48 and 72 h of feeding,the total colon length in the W1,W2 and W3 groups were longer than the C2 group,and the W2 and W3 groups were longer than the W1 group,the W3 group was longer than the W2 group,the total colon length in W3 group for 12 h and W2 and W3 groups for 24 h was longer than that in C2 group,and the W3 group for 24 h was longer than that in the W2 group,and the differences were statistically significant(P<0.05).After 72 h of feeding,compared with group C2,the histopathological inflammation of colon in groups W1,W2 and W3 was improved in turn.Compared with group C2,the contents of TNF-α,IL-1 and IL-6 in serum decreased in turn at 12,24,48 and 72 h(P<0.05).After 72 h of feeding,there was no significant difference in the expression of PI3 K mRNA and PI3 K,p-Akt,NF-κB,TNF-α,IL-1,IL-6 protein in colon tissue of CON1,CON2,CON3,CON4,CON5 groups and C1,C2 group.Compared with group C2,the expression of PI3 K mRNA,PI3 K,p-Akt,NF-κB,TNF-α,IL-1 and IL-6 protein in colon tissue of 72 h in W1,W2 and W3 groups decreased in turn,and the expression of Akt protein increased in turn(P<0.05).Compared with group C2,the content of calprotectin in feces of rats in group W1,W2 and W3 decreased at 12,24,48 and 72 h of feeding(P<0.05).Conclusion The PI3K inhibitor Wortmannin can improve the inflammatory performance of CD rats and has a certain therapeutic effect,its mechanism may be to block the PI3K/Akt signal pathway and reduce the level of downstream inflammatory cytokines,so as to achieve anti-inflammatory effect.
作者
钟骏桥
陈恩
陈青
易建中
方传发
ZHONG Junqiao;CHEN En;CHEN Qing;YI Jianzhong;FANG Chuanfa(Department of Gastrointestinal and Hernia Surgery,Ganzhou People's Hospital,Ganzhou,Jiangxi,341000,China)
出处
《当代医学》
2025年第8期1-8,共8页
Contemporary Medicine
基金
江西省卫生健康委科技计划(202511050)。
