摘要
目的通过网络药理学和动物实验探讨芩柏加减方通过核因子κB(nuclear factorκB,NF-κB)/肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)信号通路对溃疡性结肠炎(ulcerative colitis,UC)的保护机制。方法将48只SPF级SD大鼠利用2,4,6-三硝基苯磺酸(trinitrobenzene sulfonic acid,TNBS)建立UC模型,造模成功后随机分为模型组(以生理盐水灌肠)、奥沙拉嗪组(以200 mg/kg奥沙拉嗪灌肠)、芩柏加减方低剂量组(以1.43 g/kg芩柏加减方灌肠)和芩柏加减方高剂量组(以2.86 g/kg芩柏加减方灌肠),每组12只,每天灌肠2次,连续14 d,另取12只正常饲养的SD大鼠作为正常对照组。于给药第1、7、14天每组随机取4只大鼠进行麻醉采血并取结肠组织。采用免疫组织化学法检测结肠组织中核因子κB亚基p65亲和肽(nuclear factorκB p65,NF-κB p65)、TNF-α、白细胞介素-10(interleukin-10,IL-10)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的表达来评价芩柏加减方对UC大鼠的影响,同时采用TUNEL荧光法检测评价大鼠结肠组织细胞调亡情况;利用ELISA法评价血清中细胞因子NF-κB p65、TNF-α、IL-10、Caspase-3的水平差异变化情况。结果通过网络药理学富集分析发现芩柏加减方中5种主要活性成分与UC成明显相关性且与NF-κB p65/TNF-α信号通路密切相关。经UC造模发现:与正常对照组比较,模型组大鼠结肠组织中TNF-α、Caspase-3和NF-κB p65水平均升高(P<0.05),IL-10水平显著降低(P<0.05)。HE染色结果显示,正常对照组大鼠结肠腺体排列整齐,未见炎性浸润;模型组的结肠腺体排列紊乱,肌底膜断裂且伴有明显的炎性浸润,凋亡指数相比正常对照组均升高(P<0.05);奥沙拉嗪组及芩柏加减方低、高剂量组随着给药时间的延长,结肠病理损伤均得到不同程度的缓解。经检测给药14 d后,血清中TNF-α、Caspase-3、IL-10和NF-κB p65水平结果与组织病理检测结果高度一致性。相比模型组,奥沙拉嗪组、芩柏加减方高剂量组大鼠血清中TNF-α、Caspase-3和NF-κB p65含量明显降低(P<0.05),IL-10水平显著升高(P<0.05)。结论芩柏加减方对TNBS诱导的UC模型具有一定保护作用,能降低细胞凋亡指数,可能是通过调节NF-κB/TNF-α信号通路来降低炎症因子分泌、减轻机体炎症反应、提高抗炎因子表达、促进黏膜愈合来实现。
Objective To investigate the protective mechanism of modified Qinbo Formula on ulcerative colitis(UC)through nuclear factor kappa-B(NF-κB)p65/tumor necrosis factor-α(TNF-α)signaling pathway by network pharmacology and anima experiments.Methods Forty-eight SPF SD rats were used to establish UC model by 2,4,6-trinitrobenzene sulfonic acid(TNBS).After successful modeling,they were randomly divided into model group(enema with normal saline),olsalazine group(enema with 200 mg/kg olsalazine),low-dose modified Qinbo Formula group(enema with 1.43 g/kg modified Qinbo Formula),and high-dose modified Qinbo Formula group(enema with 2.86 g/kg modified Qinbo Formula),with 12 rats in each group,twice a day for 14 consecutive days.And another 12 SD rats were taken as normal control group(normal feeding).Then,4 rats in each group were randomly selected and anaesthetized for blood collection and colon tissue sampling on the 1st,7th,and 14th day after administration.The expressions of NF-κB p65,TNF-α,interleukin-10(IL-10),and Caspase-3 in colon tissue were determined by immunohistochemical(IHC)to evaluate the effects of modified Qinbo Formula on UC rats.Meanwhile,TUNEL fluorescence method was used to evaluate the apoptosis of colon tissue cells in rats,and ELISA was used to evaluate changes in level differences of NF-κB p65,TNF-α,IL-10,and Caspase-3 in serum.Results Based on network pharmacology enrichment analysis,it was found that the five major active ingredients in modified Qinbo Formula were significantly associated with UC and also closely related to the NF-κB p65/TNF-αsignaling pathway.Through UC modeling,compared with the normal control group,the levels of TNF-α,Caspase-3,and NF-κb p65 in colon tissue of rats in the model group significantly increased(P<0.05),while the level of IL-10 significantly decreased(P<0.05).HE staining showed that the colonic glands of the normal control group were arranged neatly and regularly without inflammatory infiltration,but the colonic glands of the model group were arranged in disorder,with ruptured muscle basement membrane and obvious inflammatory infiltration,and the apoptosis index was higher than that of the normal control group(P<0.05).The pathological damage of colon was alleviated to varying degrees in the olsalazine group and the low-and high-dose modified Qinbo Formula groups with the prolongation of administration time.After 14 days of administration,the levels of TNF-α,Caspase-3,and NF-κB p65 in serum were highly consistent with the results of histopathological examination.Compared with the model group,the levels of TNF-α,Caspase-3,and NF-κB p65 in serum of rats in the olsalazine group and the high-dose modified Qinbo Formula group were significantly lower(P<0.05),while the level of IL-10 was significantly higher(P<0.05).Conclusion Modified Qinbo Formula has certain protective effects on TNBS induced UC model and can reduce the apoptosis index,which may be achieved by regulating the NF-κB/TNF-αsignaling pathway to reduce inflammatory factors secretion and the body's inflammatory response,increase the expression of anti-inflammatory factors,and promote mucosal healing.
作者
陆文洪
王真权
熊家青
刘丽芳
李逵
LU Wenhong;WANG Zhenquan;XIONG Jiaqing;LIU Lifang;LI Kui(The Second Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410005,China;The First Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China)
出处
《湖南中医药大学学报》
CAS
2023年第10期1793-1802,共10页
Journal of Hunan University of Chinese Medicine
基金
湖南省教育厅科研课题(20C1422)
湖南省普通高等学校教学改革研究项目(HNJG-2021-0586)
湖南中医药大学教学改革立项项目(2020-JG042)
湖南中医药大学校院联合基金教学改革研究重点项目(2022-LHJG003)。
