度洛西汀对肠易激综合征小鼠内脏高敏感及微生物群落的影响

Effects of duloxetine on visceral hypersensitivity and microbial community in mice with irritable bowel syndrome

目的 研究度洛西汀对避水应激导致的肠易激综合征(IBS)小鼠内脏敏感性的影响,并观察其对小鼠粪便微生物群及肠组织中肿瘤坏死因子α(TNF-α)及氧化应激相关生化指标的影响。方法 选取18只8周龄的C57BL/6小鼠,将其随机分为3组:对照组、避水应激+PBS组和避水应激+度洛西汀组,每组6只。对照组每日放置在没有注水的水箱中央平台上,持续1 h,每日每只灌胃200μL的PBS;避水应激+PBS组在避水应激结束后,灌胃200μL的PBS;避水应激+度洛西汀组在避水应激结束后,灌胃200μL含有3 mg/mL度洛西汀的PBS,避水应激造模持续10 d。造模结束后,采用腹壁撤退反射(AWR)评分评估内脏敏感性的变化,并通过16S rDNA和真菌核糖体DNA内部转录间隔区测序(ITS测序)分析各组小鼠粪便中的细菌及真菌在多样性、物种组成和差异物种的变化,同时检测十二指肠和升结肠组织中肿瘤坏死因子α(TNF-α)及氧化应激相关生化指标:总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)。结果 与对照组相比,避水应激+PBS组小鼠的内脏敏感性显著升高(P<0.05);与避水应激+PBS组相比,避水应激+度洛西汀组小鼠内脏敏感性显著降低(P<0.05)。在α多样性方面,相较于对照组,避水应激+度洛西汀组细菌群落的Chao指数显著降低(P<0.05),而其他指数无显著差异;对于真菌群落,与对照组相比,避水应激+PBS组及避水应激+度洛西汀组Chao指数和Sobs指数均显著降低(P<0.05),其他指标无显著变化。在β多样性方面,三组小鼠粪便细菌组成存在显著差异(P<0.05),避水应激+度洛西汀组与其他两组分离明显。LefSe的LDA分析揭示,在细菌方面,对照组主要包括丹毒丝菌科、另枝菌属、阿克曼氏菌属和疣微菌目,避水应激+PBS组为Eubacterium_siraeum_group,而避水应激+度洛西汀组为乳杆菌属和脱硫弧菌属;在真菌方面,对照组的生物标识涵盖硬壳囊菌科、折球菌科、弦霉属和假丝酵母属等,还包括一些未分类明确的属,避水应激+PBS组的生物标识为小囊菌科,避水应激+度洛西汀组的生物标识则为二孢菌科及其下属的一个未明确分类的属。生化检测结果表明,与对照组相比,避水应激+PBS组小鼠十二指肠和升结肠中TNF-α显著升高(P<0.05),避水应激+度洛西汀组TNF-α水平未显著升高(P>0.05),且三组间氧化应激相关生化指标(T-SOD、GSH-Px、MDA)也未见显著差异(P>0.05)。结论 度洛西汀可以降低避水应激所致的内脏高敏感,并对肠道微生物群产生一定影响,但对十二指肠和升结肠组织中TNF-α及氧化应激相关生化指标无显著影响。

Objective To investigate the effects of duloxetine on visceral sensitivity in mice with irritable bowel syndrome(IBS)in‐duced by water avoidance stress(WAS),and explore its effect on fecal microbiota and biochemical indicators related to tumor necrosis factor‐α(TNF‐α)and oxidative stress in intestinal tissues.Methods Eighteen 8‐week‐old C57BL/6 mice were randomly divided into three groups:control group,WAS+PBS group,and WAS+duloxetine group,with six mice in each group.The mice in control group were placed on the central platform of an empty water tank for 1 h daily and administered with 200μL PBS by gavage.The mice in WAS+PBS group received 200μL PBS by gavage after WAS.The mice in WAS+duloxetine group were administered with 200μL PBS containing 3 mg/mL duloxetine by gavage after WAS.The WAS model was established through continuous stimulation of water avoidance stress for 10 d.After modeling,the abdominal wall retraction reflex(AWR)score was used to assess the changes of visceral sensitivity.16S rDNA sequencing and fungal ribosomal DNA internal transcribed spacer(ITS)sequencing were employed to analyze the diversity,the species composition,and the differential species of bacteria and fungi in the feces of mice in each group.Simulta‐neously,TNF‐αand oxidative stress‐related biochemical indicators,including total superoxide dismutase(T‐SOD),glutathione peroxi‐dase(GSH‐Px),and malondialdehyde(MDA),were measured in the duodenal and ascending colonic tissues.Results Compared with control group,the visceral sensitivity of mice was significantly increased in WAS+PBS group(P<0.05);compared with WAS+PBS group,the visceral sensitivity of mice was significantly decreased in WAS+duloxetine group(P<0.05).In terms ofα‐diversity,Chao index of bacterial communities in WAS+duloxetine group was significantly lower than that in control group(P<0.05),while other indices showed no significant differences between two groups.For fungal communities,both Chao index and Sobs index were signifi‐cantly lower in WAS+PBS group and WAS+duloxetine group than in control group(P<0.05),while other indicators showed no signifi‐cant change.In terms ofβ‐diversity,there were significant differences in the fecal bacterial composition among the three groups(P<0.05),with a distinct separation between water avoidance stress+duloxetine group and the other two groups.LDA analysis using LefSe revealed that the bacteria mainly included Erysipelotrichaceae,Allobaculum,Akkermansia,and Verrucomicrobiales in control group,Eubacterium_siraeum_group in WAS+PBS group,and Lactobacillus and Desulfovibrio in WAS+duloxetine group.For fungi,the bio‐markers encompassed Ceratocystidaceae,Plectosphaerellaceae,Chordomycetes,Candida,and some unclassified genera in control group,Microascaceae in WAS+PBS group,and Dipodascaceae and an unclassified genus within this family in WAS+duloxetine group.Biochemical test results showed that compared with control group,TNF‐αlevel was significantly increased in the duodenal and ascending colonic tissues of mice in WAS+PBS group(P<0.05),while TNF‐αlevel did not increase significantly in WAS+duloxetine group(P>0.05).There were no significant differences in oxidative stress‐related biochemical indicators(T‐SOD,GSH‐Px,MDA)between the three groups(P>0.05).Conclusion Duloxetine can reduce the visceral hypersensitivity induced by WAS and exert the certain effect on intestinal microbiota,but it has no significant impacts on TNF‐αand oxidative stress‐related biochemical indicators in duodenal and ascending colonic tissues.