短链脂肪酸对抑郁模型小鼠肠道菌群及海马TLR4/MyD88/NF-κB通路蛋白的影响

Effects of short-chain fatty acids on gut microbiota and hippocampal TLR4/MyD88/NF-κB pathway proteins in depression model mice

目的探讨短链脂肪酸(short chain fatty acid,SCFAs)是否通过改善抑郁模型小鼠肠道菌群失调及调控TLR4/MyD88/NF-κB炎性通路产生抗抑郁作用。方法60只SPF级6~8周龄C57BL/6J雄性小鼠根据随机数字表法分为对照组、抑郁模型组、SCFAs组,每组20只。抑郁模型组和SCFAs组小鼠采用慢性不可预知温和应激(chronic unpredictable mild stress,CUMS)方法建立抑郁模型,造模共进行8周,并从第6周开始,SCFAs组小鼠给予短链脂肪酸盐混合溶液饮用,模型组小鼠给予0.78%NaCl溶液饮用,直至造模完成。采用糖水偏爱实验(sucrose preference test,SPT)、强迫游泳实验(forced swimming test,FST)检测小鼠抑郁样行为,旷场实验(open field test,OFT)检测小鼠焦虑样行为。使用16S rRNA基因序列分析小鼠的肠道菌群;采用免疫荧光染色和Western blot方法测定海马组织中星形胶质细胞和TLR4/MyD88/NF-κB炎性通路的活化情况。采用SPSS 26.0进行统计分析,3组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果3组小鼠糖水偏爱率、强迫游泳不动时间和旷场中心区活动时间百分比均差异有统计学意义(F=10.554,10.912,12.599,均P<0.05)。抑郁模型组小鼠的糖水偏爱率和旷场中心区活动时间百分比均低于对照组(均P<0.05),强迫游泳不动时间高于对照组(P<0.05)。SCFAs组小鼠糖水偏爱率[(84.7±3.5)%,(75.3±6.0)%]和旷场中心区活动时间百分比[(7.4±1.4)%,(3.2±0.9)%]均高于抑郁模型组(均P<0.05),强迫游泳不动时间短于抑郁模型组[(110.5±21.5)s,(148.0±20.1)s;P<0.05]。3组小鼠肠道菌群的β多样性差异有统计学意义(P=0.001),与抑郁模型组相比,在科水平,SCFAs组理研菌科与拟杆菌科相对丰度增加,梭菌杆相对丰度降低;在属水平,梭菌属和普雷沃氏菌属相对丰度降低,另枝杆菌属相对丰度增加(均P<0.05)。免疫荧光结果显示,3组小鼠GFAP表达水平差异有统计学意义(F=16.565,P=0.004);抑郁模型组小鼠GFAP表达高于对照组和SCFAs组(均P<0.05)。Western blot结果显示,3组小鼠海马组织TLR4、MYD88、NF-κB表达水平均差异有统计学意义以(F=70.59,174.39,14.40,均P<0.05)。抑郁模型组小鼠TLR4、MyD88和NF-κB的蛋白表达水平均高于对照组和SCFAs组(均P<0.05)。结论SCFAs可以改善抑郁模型小鼠的抑郁样行为,降低星形胶质细胞的激活,这可能与改善肠道菌群失调及下调TLR4/MyD88/NF-κB通路蛋白的过表达有关。

Objective To investigate whether short chain fatty acid(SCFAs)intervention has an antidepressant effect by improving gut microbiota dysregulation and regulating the TLR4/MyD88/NF-κB inflammatory pathway in depression model mice.Methods Totally 60 SPF grade male C57BL/6 J mice aged 6-8 weeks were randomly divided into three groups:control group,depression model group,and SCFAs group,with 20 mice in each group.The mice in depression model group and SCFAs group were given the chronic unpredictable mild stress(CUMS)stimulations for 8 weeks to establish the depression model.From the 6th week,SCFAs group mice were given a mixed solution of short chain fatty acid salts for drinking,until modeling was completed,meanwhile mice in the model group were given 0.78%NaCl solution for drinking.The depression-like behavior was assessed using the sucrose preference test(SPT)and forced swimming test(FST)following modeling,and the open field test(OFT)was employed to evaluate the anxiety-like behavior of mice.16S rRNA gene sequence was used to analyze the gut microbiota of mice.The activation of astrocytes and TLR4/MyD88/NF-κB inflammatory pathway in hippocampus was determined by immunofluorescence staining and Western blot.SPSS 26.0 was used for statistical analysis,one-way ANOVA was used for comparison among the three groups,and LSD-t test was used for further pairwise comparisons.Results There were statistically significant differences in the sugar water preference rate,the immobility time in FST,and the percentage of activity time in OFT among the three groups(F=10.554,10.912,12.599,all P<0.05).The the sugar water preference rate and the percentage of activity time in OFT of the depression model group were both lower than those of the control group(both P<0.05),and the immobility time in FST was higher than that of the control group(P<0.05).The sugar water preference rate in SCFAs group((84.7±3.5)%,(75.3±6.0)%)and the percentage of activity time in OFT((7.4±1.4)%,(3.2±0.9)%)were both higher than those in the depression model group(both P<0.05),while the immobility time in FST was shorter than that in the depression model group((110.5±21.5)s,(148.0±20.1)s,P<0.05).There was a statistical difference in theβdiversity of gut microbiota among three groups(P=0.001).At the family level,compared with the depression model group,the relative abundance of Rikenellaceaee and Bacteroidaceae increased in the SCFAs group,while the relative abundance of Clostridia_UCG-014 decreased.At the genus level,the relative abundance of Clostridia_UCG-014 and Prevotella decreased,while the relative abundance of Alistipes increased(all P<0.05).The immunofluorescence results showed that there was a statistically significant difference in GFAP expression levels among the three groups of mice(F=16.565,P=0.004).The GFAP expression in the depression model group was higher than that in the control group and SCFAs group(both P<0.05).The Western blot results showed that there were statistically significant differences in the expression levels of TLR4,MYD88,and NF-κB ptoteins in the hippocampal tissue of the three groups(F=70.59,174.39,14.40,all P<0.05).The protein levels of TLR4,MyD88,and NF-κB in the depression model group were all higher than those in the control group and SCFAs group(all P<0.05).Conclusion SCFAs can ameliorate the depressive-like behavior in depression model mice and reduce the activation of astrocytes in the hippocampus,which may be associated with the improvement of dysregulated gut microbiota and down-regulation of the TLR4/MyD88/NF-κB pathway protein.