穗花杉双黄酮通过NF-κB通路在放射性肠炎中的消炎、抗纤维化和抗菌作用

The anti-inflammatory,antifibrotic and antibacterial function of Amentoflavone in radiation proctitis via NF-κB pathway

目的探讨穗花杉双黄酮(Amentoflavone,AF)在放射性肠炎中的消炎、抗纤维化和抗菌作用。方法选取雄性SD大鼠45只,随机分3组:空白组、辐射组(IR组)、辐射后AF干预组(IR+AF组)。采用高能X射线照射建立急性放射性肠炎大鼠模型,IR+AF组予60 mg·kg-1·d-1AF腹腔注射,空白组和IR组予生理盐水腹腔注射,分别于不同时段留取大鼠小肠组织、血清与粪便,观察肠黏膜组织病理学,采用ELISA法检测血清炎性因子TNF-α和IL-1β水平,qPCR检测组织NF-κB表达,应用16S rRNA测序方法对粪便样本进行测序及生物统计分析。另建立TNF-α诱导IEC6细胞损伤模型,采用qPCR法检测上皮细胞间质转化相关标志物:E-cadherin和Vimentin,采用划痕实验观察AF的修复作用。结果IR组大鼠肠上皮结构及黏膜屏障均被破坏,IR+AF组未见明显的肠道结构破坏,胶原纤维增生明显减少。与空白组、IR+AF组相比,IR组的菌群α-多样性更低,β-多样性更显著,而血清TNF-α和IL-1β的分泌增加(P<0.05)。AF干预后NF-κB表达下降,炎性因子明显减少(P<0.05),且大肠杆菌志贺菌、类杆菌的丰度下降,趋势一致,而普雷沃菌、乳酸杆菌属的丰度升高。TNF-α刺激后IEC6间质化,而AF可逆转这种变形,伴E-cadherin恢复、Vimentin表达下降。划痕实验提示AF促进上皮细胞修复。结论AF可抑制NF-κB信号通路,减少TNF-α等炎性因子刺激,抑制IEC6间质化,促进肠黏膜愈合,恢复肠道菌群多样性,起到消炎、抗纤维化和抗菌的多重作用。

Objective To explore the anti-inflammatory,antifibrotic and antibacterial function of Amentoflavone(AF)in radiation proctitis.Methods Forty-five male SD rats were randomly divided into 3 groups:blank group,radiation group(IR group)and AF intervention group(IR+AF group).The model of acute radiation proctitis rats were built by 14 high energy X rays radiation.Rats in IR+AF group were intraperitoneal injection 60 mg·kg-1·d-1 AF,in IR group and blank group did with saline.The feces,blood serum and intestinal tissue,the intestinal mucosa and the intestine barrier of rats were collected and observed.The expression level of TNF-αand IL-1βby ELISA and the relative expression of NF-κB by qPCR were measured.Then the DNA sequence and biometrical analysis of the feces were analyzed by 16S rRNA sequencing method.We built IEC6 cell damage model induced by TNF-α,then used qPCR to detect the relative expression level of the markers of epithelial-mesenchymal transition,E-cadherin and Vimentin and applied wounding healing assay to assess the repair effect.Results The intestinal mucosa and the intestine barrier of the IR group rats were both destroyed,while no significant change was observed in IR+AF group and collagen fibrous proliferation was inhibited.IR group had lowerα-diversity analysis while higherβ-diversity analysis.The level of TNF-αand IL-1βwere higher(P<0.05).After the treatment of AF,Prevotella and Lactobacillus were more various than before,however,Escherichia-shigella and Helicobacter were opposite and the level of inflammatory factor declined significantly(P<0.05).After inducing by TNF-α,IEC6 occurred epithelial-mesenchymal transition with the lower expression of E-cadherin and higher expression of Vimentin,while AF could inhibit these changes.Wounding healing assay indicated that AF promoted the repair process of the intestine epithelial cell.Conclusion AF,as an anti-inflammatory,antifibrotic and antibacterial drug,can inhibit the NF-κB pathway,reduce the inflammatory stimulation,such as TNF-α,inhibit IEC6 epithelial-mesenchymal transition,promote healing of the intestinal mucosa and recover the diversity of the intestinal bacterium.