丁酸钠通过Toll样受体4/核转录因子-κB p65通路抑制海马体内小胶质细胞炎性活化的机制

Mechanisms of sodium butyrate inhibition of microglia inflammatory activation in hippocampus via Toll-like receptor 4/nuclear factor-κB p65 pathway

目的观察丁酸钠对脓毒症相关性脑病(SAE)小鼠远期焦虑样行为及大脑海马体内小胶质细胞炎性活化的影响。方法①动物实验:将50只6~8周龄C57BL/6小鼠按随机数字表法分为假手术组(Sham组,仅开腹找到盲肠,不穿孔也不结扎)、盲肠结扎穿孔术(CLP)所致SAE模型组(开腹找到盲肠,结扎后穿孔,旷场实验表明小鼠自主探索行为能力下降,有焦虑样行为表现,证明SAE模型复制成功)和丁酸钠预处理组(制模前以500 mg·kg^(-1)·d^(-1)剂量的丁酸钠灌胃,制模后以相同剂量继续灌胃,每日1次,均连续3 d)。术后7 d,采用旷场实验检测各组小鼠焦虑样行为;术后1 d、3 d,采用蛋白质免疫印迹试验(Western blotting)和酶联免疫吸附试验(ELISA)检测各组小鼠海马组织内白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)蛋白表达水平及含量的变化,采用免疫荧光染色观察小胶质细胞标记蛋白离子钙接头蛋白-1(Iba-1)和TNF-α蛋白的共定位情况。②细胞实验:将小鼠小胶质细胞株BV-2细胞分为空白对照组、脂多糖(LPS)模型组(用1 mg/L LPS处理细胞)、丁酸钠干预组(用1 mg/L LPS+5 mmol/L丁酸钠处理细胞)。采用Western blotting检测各组IL-1β、TNF-α、Toll样受体4(TLR4)、磷酸化核转录因子-κB p65(p-NF-κB p65)、核转录因子-κB p65(NF-κB p65)、NF-κB抑制蛋白-α(IκB-α)的蛋白表达情况;采用免疫荧光染色观察各组Iba-1、TNF-α的表达情况。结果①动物实验:与假手术组比较,SAE模型组制模后7 d小鼠中央区域活动路程、中央区域活动时间、总路程均明显下降〔中央区域活动路程(mm):13.45±3.97比161.44±27.00,中央区域活动时间(s):1.82±0.58比13.45±2.17,总路程(mm):835.01±669.67比2254.51±213.45,均P<0.05〕;小鼠海马组织大量神经细胞核固缩深染,神经细胞排列紊乱,海马体中小胶质细胞胞体明显增大,小胶质细胞标记的Iba-1/TNF-α阳性细胞(Iba-1^(+)/TNF-α^(+))数明显增多,海马组织匀浆上清液中促炎因子TNF-α、IL-1β含量和蛋白表达均于术后3 d明显升高〔TNF-α(ng/L):119.17±18.40比90.18±21.17,IL-1β(ng/L):407.89±70.64比313.69±34.63;TNF-α/GAPDH:1.42±0.50比0.80±0.08,IL-1β/GAPDH:1.27±0.22比0.85±0.25,均P<0.05〕;给予丁酸钠处理后,小鼠中央区域活动路程、中央区域活动时间均较SAE模型组明显增加〔中央区域活动路程(mm):47.39±15.63比13.45±3.97,中央区域活动时间(s):6.12±1.87比1.82±0.58,均P<0.05〕,但总路程无明显变化(mm:1550.59±1004.10比835.01±669.67,P>0.05),小鼠神经细胞核固缩深染情况较SAE模型组明显减轻,Iba-1^(+)/TNF-α^(+)阳性细胞数目明显减少,术后3 d海马组织匀浆上清液中促炎因子TNF-α、IL-1β含量和蛋白表达均较SAE模型组明显降低〔TNF-α(ng/L):64.95±9.10比119.17±18.40,IL-1β(ng/L):311.94±69.92比407.89±190.64;TNF-α/GAPDH:1.02±0.36比1.42±0.50,IL-1β/GAPDH:0.86±0.20比1.27±0.22,均P<0.05〕。②细胞实验:采用LPS干预后BV-2细胞中TNF-α的荧光强度明显增强,TNF-α、IL-1β、TLR4、p-NF-κB p65蛋白表达均升高(TNF-α/GAPDH:0.39±0.06比0.20±0.02,IL-1β/GAPDH:0.27±0.03比0.19±0.01,TLR4/GAPDH:0.55±0.12比0.33±0.09,p-NF-κB p65/NF-κB p65:0.55±0.05比0.29±0.04,均P<0.05),IκB-α的蛋白表达均较空白对照组明显降低(IκB-α/GAPDH:0.54±0.06比0.81±0.03,P<0.05);给予丁酸钠处理后TNF-α、IL-1β、TLR4、p-NF-κB p65的蛋白表达均较LPS模型组明显降低(TNF-α/GAPDH:0.26±0.02比0.39±0.06,IL-1β/GAPDH:0.11±0.04比0.27±0.03,TLR4/GAPDH:0.28±0.14比0.55±0.12,p-NF-κB p65/NF-κB p65:0.29±0.01比0.55±0.05,均P<0.05),IκB-α蛋白表达均较LPS模型组明显升高(IκB-α/GAPDH:0.75±0.01比0.54±0.06,P<0.05)。结论丁酸钠可通过拮抗LPS诱导的TLR4激活,从而抑制p-NF-κB p65的核移位及IκB-α降解,减少小胶质细胞活化及分泌炎性因子,最终改善脓毒症小鼠海马体内的炎症反应和远期焦虑样行为。

Objective To investigate the effects of sodium butyrate(NaB)on long-term anxiety like behavior and inflammatory activation of microglia in the hippocampus of sepsis-associated encephalopathy(SAE)mice.Methods①Animal experiment:fifty C57BL/6 mice aged 6-8 weeks were randomly divided into Sham group(only the cecum was found by laparotomy without perforation or ligation),and SAE model group caused by cecal ligation and puncture(CLP;SAE model group,the cecum was found by laparotomy and perforated after ligation.The open field test indicated that the ability of independent exploration decreased and showed anxiety like behavior,which proved that the SAE model was successfully replicated)and NaB pretreatment group was established(NaB was administered at a dose of 500 mg·kg^(-1)·d^(-1) for 3 days before modeling,and the same dose once a day for 3 days after modeling).Open field test was used to detect the anxiety like behavior of mice at 7 days.The protein expressions and content changes of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in hippocampus of mice at 1 day and 3 days after operation were detected by Western blotting and enzyme linked immunosorbent assay(ELISA).Immunofluorescence staining was used to observe microglia labeled protein ionized calcium bindingadaptor molecule-1(Iba-1)and TNF-αprotein co localization.②Cell experiment:mouse microglia cell line BV-2 microglia were divided into blank control group,lipopolysaccharide(LPS)group(cells were treated with 1 mg/L LPS),and NaB treatment group(cells were treated with 1 mg/L LPS+5 mmol/L NaB).The protein expressions of IL-1β,TNF-α,Toll-like receptor 4(TLR4),phosphorylated nuclear factor-κB p65(p-NF-κB p65),nuclear factor-κB p65(NF-κB p65)and NF-κB inhibitor protein-α(IκB-α)were detected by Western blotting.The expressions of Iba-1 and TNF-αin each group were observed by immunofluorescence.Results①Animal experiment:compared with the Sham group,the distance and duration of movement in the central area,the total distance moved of mice decreased 7 days after the establishment of SAE model group were decreased[distance of movement in the central area(mm):13.45±3.97 vs.161.44±27.00,duration of movement in the central area(s):1.82±0.58 vs.13.45±2.17,the total distance moved(mm):835.01±669.67 vs.2254.51±213.45,all P<0.05].In the hippocampus tissues of mice,a large number of nerve nuclei were pyknotic and deeply stained,and the arrangement of nerve cells was disordered.The cell bodies of microglia in mouse hippocampus increased significantly.The number of positive cells of Iba-1/TNF-α(Iba-1^(+)/TNF-α^(+))increased significantly.The contents and protein expression of proinflammatory factors TNF-α,IL-1βin hippocampal homogenate supernatant 3 days after operation in SAE model group were significantly higher than those in Sham group[TNF-α(ng/L):119.17±18.40 vs.90.18±21.17,IL-1β(ng/L):407.89±70.64 vs.313.69±34.63;TNF-α/GAPDH:1.42±0.50 vs.0.80±0.08,IL-1β/GAPDH:1.27±0.22 vs.0.85±0.25,all P<0.05].After intragastric administration of NaB,the distance and duration of movement in the central area of mice were significantly higher than those in SAE model group[distance of movement in the central area(mm):47.39±15.63 vs.13.45±3.97,duration of movement in the central area(s):6.12±1.87 vs.1.82±0.58,all P<0.05].There was no significant change in the total distance moved(mm:1550.59±1004.10 vs.835.01±669.67,P>0.05).The pyknosis and deep staining of nerve nuclei in mice were significantly less than those in SAE model group.The number of Iba-1^(+)/TNF-α^(+)positive cells decreased significantly.The contents and protein expression levels of proinflammatory factors TNF-α,IL-1βin hippocampal homogenate supernatant 3 days after operation were significantly lower than those in SAE model group[TNF-α(ng/L):64.95±9.10 vs.119.17±18.40,IL-1β(ng/L):311.94±69.92 vs.407.89±70.64;TNF-α/GAPDH:1.02±0.36 vs.1.42±0.50,IL-1β/GAPDH:0.86±0.20 vs.1.27±0.22,all P<0.05].②Cell experiment:after LPS intervention,the fluorescence intensity of TNF-αin BV-2 cells was significantly enhanced,the protein expression levels of TNF-α,IL-1β,TLR4 and p-NF-κB p65 protein increased(TNF-α/GAPDH:0.39±0.06 vs.0.20±0.02,IL-1β/GAPDH:0.27±0.03 vs.0.19±0.01,TLR4/GAPDH:0.55±0.12 vs.0.33±0.09,p-NF-κB p65/NF-κB p65:0.55±0.05 vs.0.29±0.04,all P<0.05),the expression level of IκB-αwas lower than that in the control group(IκB-α/GAPDH:0.54±0.06 vs.0.81±0.03,P<0.05).After NaB treatment,the fluorescence intensity of TNF-αin BV-2 cells was decreased.The protein expression levels of TNF-α,IL-1β,TLR4 and p-NF-κB p65 protein were significantly lower than that of LPS model group(TNF-α/GAPDH:0.26±0.02 vs.0.39±0.06,IL-1β/GAPDH:0.11±0.04 vs.0.27±0.03,TLR4/GAPDH:0.28±0.14 vs.0.55±0.12,p-NF-κB p65/NF-κB p65:0.29±0.01 vs.0.55±0.05,all P<0.05),the protein expression level of IκB-αwas significantly higher than that in the LPS group(IκB-α/GAPDH:0.75±0.01 vs.0.54±0.06,P<0.05).Conclusion NaB could antagonism the TLR4 activation induced by LPS,thus inhibiting p-NF-κB p65 nuclear transcription and IκB-αdegradation.It can reduce microglia activation and secretion of inflammatory factors,and finally improve the inflammation in the hippocampus of septic mice and long-term anxiety like behavior.