【作者】 张婷；

【导师】 俞捷； 顾雯；

【作者基本信息】 云南中医学院 ， 药物分析学， 2017， 硕士

【摘要】 目的:肠粘膜炎是化疗引起的临床常见不良反应,5-氟尿嘧啶(5-Fu)、伊立替康、甲氨蝶呤等主要抗肿瘤药物均易诱发肠粘膜炎。砂仁(Amomi Fructus)作为我国“四大南药”之一,是重要药食两用资源,也是《中华人民共和国药典》(2015年版)收录的重要品种,有着1300多年药用及食用历史,具有化湿开胃、温脾止泻和理气安胎的功效,是中医治疗胃肠疾病的常用药。本次研究关注砂仁挥发油(VOAV)提取物及其主要成分乙酸龙脑酯(BA)对5-FU诱导的大鼠肠粘膜损伤的保护作用,阐明其作用机理,为砂仁应用于化疗期间肠粘膜屏障受损的防治提供重要依据。方法:本课题研究将SPF级SD大鼠72只,随即分为9组,每组8只,分别为正常组(CON),5-FU+生理盐水组(MOD)、5-FU+金双歧(CBL,阳性药),5-FU+砂仁挥发油组(VOAV.L、VOAV.M、VOAV.H),5-FU+乙酸龙脑酯组(BA.L、BA.M、BA.H),化疗时间为5天,药物干预总共12天。实验期间记录大鼠每天的体重、食量,观察大鼠腹泻情况;实验结束后处死大鼠取小肠做病理切片;采用ELISA法检测测大鼠血浆中白介素-6(IL-6)、活性氧(ROS)的含量及小肠中ROS、核因子kappa B(NF-κB)、髓过氧化酶(MPO)、肿瘤坏死因子-α(TNF-α)的含量,评价砂仁挥发油对小肠炎症的调节;采用蛋白免疫印迹法检测VOAV及BA对大鼠肠道中p38 MAPK含量的影响。采用流式细胞术,检测VOAV及BA对肠道紧密连接蛋白occludin、ZO-1及对肠道细胞凋亡蛋白酶caspase-3含量的影响,结合voav及ba对血浆内毒素(lps)的调节,评价voav及ba对肠粘膜屏障功能的保护用;采用16srdnav4高变区测定技术,对肠道微生物的β多样性及out数目进行统计、物种注释及丰度分析,揭示voav及ba对肠道微生态的调节作用。结果:砂仁挥发油对5-fu所致肠粘膜炎大鼠炎症状态保护作用结果:voav及其主要成分ba可显著逆转由5-fu导致的肠粘膜炎大鼠体重、食量的降低及腹泻的发生,且高剂量效果最为显著。形态学上,5-fu可导致大鼠小肠粘膜发生明显损伤,突出表现为小肠绒毛变短及小肠隐窝结构破坏。而在给予voav或ba治疗后,绒毛结构基本恢复正常,voav有效减轻5-fu导致的小肠绒毛损伤,促进损伤修复,从而保护肠道上皮形态的稳定,并呈现一定剂量关系。voav及ba可显著降低5-fu导致的nf-κb表达量的升高,并显著降低外周血液及小肠中促炎因子(ros,il-6,tnf-α)含量的升高,效果与阳性药cbl相近且呈现一定剂量关系。此外小肠组织中mpo含量检测结果显示voav及ba亦可显著抑制由5-fu导致的大鼠小肠中mpo含量的升高,对5-fu导致的肠粘膜炎炎症状态有很好的改善作用。砂仁挥发油还可调节5-fu所致肠粘膜炎大鼠肠屏障功能损伤:voav及ba可显著逆转由5-fu导致的肠粘膜炎大鼠p38mapk及caspase-3表达量的升高,且呈一定剂量关系。同时,voav及ba可提高肠紧密连接蛋白zo-1、occludin的表达量,通过改善肠粘膜屏障功能,在一定程度上抑制肠源性内毒素lps易位,保护肠屏障功能的完整。砂仁挥发油对5-fu所致肠粘膜炎大鼠肠道微生物的调节:otu聚类分析显示,ba组与正常组分类距离接近,提示ba对这些otu的调节作用较为显著。pearson相关性分析结果显示,属于拟杆菌门、厚壁菌门及变形菌门中多数otus与炎症因子ros、mpo、lps及caspase-3呈正相关。门水平上,voav及ba可显著降低肠道菌群中变形菌门的相对丰度从而降低潜在致病菌。属水平上voav及ba可降低变形菌门中helicobacter、escherichia,拟杆菌门中bacteroides,及厚壁菌门中ruminococcus、parabacteroides、clostridum的相对丰度从而降低潜在致病菌来改善肠道微生态。同时VOAV及BA可升高厚壁菌门中Lactobacillus的相对丰度,BA可升高放线菌门中Bifidobacterium的比例,而VOAV可升高厚壁菌门中Roseburia和Coprococcus的相对丰度从而提升益生菌及相对丰度来改善肠道微生态。结论:砂仁挥发油及主要成分乙酸龙脑酯可抑制肠粘膜炎前体物质ROS的产生,从而抑制核因子NF-κB通路,降低炎症;抑制MPO、p38 MAPK的激活及Caspase-3表达,进而抑制细胞凋亡,维持细胞间紧密完整性,稳定肠粘膜机械屏障,进而抑制肠源性内毒素易位;同时通过调节肠道微生态平衡来减轻由5-FU导致的肠粘膜炎。更多还原

【Abstract】 Object:Intestinal mucositis is an major side effect caused by chemotherapy,such as 5-fluorouracil(5-FU)irinotecan,methotrexate and so on.Amomi Fructus,dry and mature fruit of Amomum villosum Lour.,A.villosum Lour.var.xanthioides T.L.Wu et Senjen and A.longiligulare T.L.Wu,is recorded and used in traditional Chinese medicine system as a excellent crude drug for the treatment of digestive system disorders and has more than 1300 years of medicinal and edible history.The objective of the study was to evaluate the effect of volatile oil of A.villosum Lour.(VOAV)and its main component,bornyl acetate(BA),on intestinal mucositis induced by 5-FU.In the meantime,the potential role of VOAV and BA on regulation of intestinal microflora,adjustment of inflammatory process and oxidative stress,maintaining intestinal permeability are investigated in order to provide more theoretical and scientific foundation for its clinic usage.Method:72 SD rats were randomly divided into normal group(CON),5-FU+saline group(MOD),5-FU+live combined Bifidobacterium and Lactobacillus tables(CBL),5-FU+VOAV(VOAV.H,VOAV.M,VOAV.L),5-FU+BA(BA.H,BA.M,BA.L),8 rats in each group.Chemotherapy lasted for 5 days,and drug intervention were continued for 12 days.Diarrhea occurrence,body weight and food intakes were daily recorded.Intestinal histology and inflammatory cytokines such as IL-6,ROS,NF-κB,TNF-α were tested to evaluate the effect of VOAV and BA on inflammation.Western blotting was used to detected the expression of p38 MAPK in intestinal tissue.The intestinal tight junction protein occludin and ZO-1 were measured by flow cytometry to future evaluate the improvement of VOAV and BA on intestinal barrier function.The levels of lipopolysaccharide was also tested.Based on determination of varied region V4 in 16 S rDNA,the regulation role of VOAV and BA in the intestinal microecology system was illustrated.Results:VOAV prevented inflammatory state in intestinal mucositis rats induced by 5-FU.Our study found that treatment with VOAV and its representative constituent,BA,could prevent diarrhea and reverse weight and food intake loss,which are important symptoms of intestinal mucositis induced by 5-FU.5-FU induced a significant decrease in villus height,an increase in the crypt depth,while treatment with VOAV and BA significantly relieved the histopathological changes.VOAV could effectively reduce the damage of small intestine caused by 5-FU and promote the repair of injury,so as to protect the stability of intestinal morphology in a dose dependent mannar.VOAV improved the injure of intestinal mucosal barrier in intestinal mucositis rats.VOAV and BA could significantly decrease the expression of NF-κB which was induced by 5-FU,and significantly reduce the content of inflammatory markers,such as ROS,IL-6 and TNF-α,in blood and small intestinal.The effect was similar with the positive drug and showed a certain dose relationship.VOAV and BA also could significantly inhibit the increase of MPO in rats induced by 5-FU,which representing an improvement of intestinal inflammation state.Both VOAV and BA could effectively reduce the expression of p38 MAPK and caspase-3 showed a certain dose relationship.Meanwhile,our study showed that VOAV and BA could increase the expression of intestinal tight junction protein ZO-1 and occludin,improve the intestinal mucosal barrier,and inhibit the translocation of LPS and protect the integrity of intestinal barrier function.VOA regulated intestinal microbial balance in intestinal mucositis rats induced by 5-FU.The OTUs cluster analysis showed that the group of BA was close the the CON,suggesting that it had a significant regulatory of these OTUs.Pearson correlation analysis showed that most of OTUs,which belong of Bacteroidetes,Frimicutes and Proteobacteri,was positively associated with inflammatory factors,such as ROS,MPO,LPS and Caspase-3.16 S rDNA sequencing results showed that there was no significant difference in the number of OUT among all groups.At the level of phylum,VOVA and BA could significantly reduce the relative abundance of Proteobacteria,which was usually considered as potential pathogen.VOAV and BA could reduce the relative abundance of some genera of Proteobacteria phylum,such as Helicobacter and Escherichia.They also reduced Bactetoides of Bacteroides phylum and Ruminococcus、Parabacteroides、Clostridum of Firmicutes phylum.Meanwhile,VOAV and BA effectively increased the abundance of Lactobacillus.BA could increase the abundance of Bifidobacterium and VOAV could increase the abundance of Roseburia,Coprococcus,which were considered as probiotics to improved intestinal microecological.VOAV and BA reduced the relative abundance of Helicobacter and Escherichia,the abundance of Bactetoides in Bacteroides phylum,and reduce the relative abundance of some genera of Firmicutes phylum,such as Ruminococcus、Parabacteroides、Clostridum,which are considered as potential pathogenic bacteria,to improve the intestinal microflora.At the same time,VOAV and BA both increased the relative abundance of Lactobacillus in Firmicutes phylum.In addition,BA could increase the relative abundance of Bifidobacterium in Actinomycete phylum and VOAV could increase the relative abundance of Roseburia,Coprococcus in Firmicutes phylum,which are considered as probiotics bacteria,to improve the intestinal microflora.Conclusion:VOAV and its main component BA could inhibit the production of ROS which was considered as precursor material of intestinal mucosa,and inhibit the NF-κB pathway to improve the state of inflammation.VOAV and BA could inhibit MAPK and caspase-3 signaling and suppressed the MPO activity,further decreased apoptosis,inhibit the translocation of LPS and protected the integrity of mucosal.At same time,VOAV and BA could regulate the intestinal microbial imbalance induced by 5-FU to stable intestinal mucosal mechanical barrier.更多还原