基于LC-MS/MS探讨脑心通胶囊对脑缺血再灌注损伤小鼠短链脂肪酸的调控作用

Regulatory Effect of Naoxintong Capsules on Short-chain Fatty Acids in Mice with Cerebral Ischemia-reperfusion Injury Based on LC-MS/MS

摘要:

目的 构建小鼠脑缺血再灌注模型，以短链脂肪酸为切入点，探究脑心通胶囊干预脑缺血再灌注的作用机制。方法 将C57BL/6J雄性小鼠按照随机数字表法分为假手术组、模型组、脑心通胶囊组（158.9 mg?kg^-1）和金纳多组（12.1 mg?kg^-1）。通过线栓法制备小鼠脑缺血再灌注（MCAO/R）模型。利用神经行为学评分、脑梗死面积测定、尼式染色和免疫荧光染色评价脑心通胶囊对MCAO/R小鼠脑损伤的保护作用；采用苏木素-伊红（HE）染色和蛋白免疫印迹法（Western blot）评价脑心通胶囊对MCAO/R小鼠肠道屏障的改善作用；利用液相色谱-质谱联用（LC-MS/MS）检测小鼠粪便中短链脂肪酸的含量。结果 与假手术组比较，模型组小鼠脑梗死面积、神经行为学评分及细胞凋亡率显著升高（P<0.01）；尼式体数量显著下降（P<0.01）；紧密连接蛋白-1（Claudin-1）和闭合蛋白（Occludin）蛋白表达水平明显下降（P<0.05）。与模型组比较，脑心通胶囊干预后明显减少小鼠的脑梗死面积（P<0.05）；降低神经行为学评分（P<0.01）和细胞凋亡率（P<0.01），增加尼式体数量（P<0.01）及Claudin-1和Occludin蛋白表达水平（P<0.01）。LC-MS/MS结果显示，与假手术组比较，模型组小鼠粪便中乙酸、丙酸、丁酸含量显著降低（P<0.01），戊酸、异戊酸、异丁酸含量显著上升（P<0.01）；脑心通胶囊干预后显著降低戊酸、异戊酸、异丁酸含量（P<0.01）。结论 脑心通胶囊能够改善脑及肠道屏障损伤，并能调控短链脂肪酸的含量从而发挥脑缺血再灌注的保护作用。

关键词:脑缺血再灌注损伤;肠道屏障;脑心通胶囊;短链脂肪酸;液相色谱-质谱联用仪（LC-MS/MS）

Abstract:

Objective To explore the mechanism of Naoxintong capsules' intervention in cerebral ischemia-reperfusion by building a mouse cerebral ischemia-reperfusion model based on short-chain fatty acids.Method C57BL/6J male mice were randomly divided into the sham group， model group， Naoxintong group （158.9 mg?kg^-1）， and Ginaton group （12.1 mg?kg^-1） according to the random number table method. The model of cerebral ischemia-reperfusion （MCAO/R） was prepared via the filament occlusion method. The effect of Naoxintong capsules on brain injury in MCAO/R mice was evaluated by the neuroethological score， cerebral infarction area determination， Nissl staining， and immunofluorescence staining. Hematoxylin-eosin （HE） staining and Western blot were employed to evaluate the effect of Naoxintong capsules on the intestinal barrier in MCAO/R mice. The content of short-chain fatty acids in mouse feces was detected by LC-MS/MS.Result Compared to the sham group， the model group exhibited significant increases in the cerebral infarction area， neuroethological score， and cell apoptosis rate （P<0.01）， with a notable decrease in the number of Nissl bodies （P<0.01）. The protein expression levels of Claudin-1 and Occludin were significantly reduced （P<0.05）. Compared with the model group， the intervention of Naoxintong capsules significantly decreased the cerebral infarction area （P<0.05） and improved the neuroethological score （P<0.01） and cell apoptosis rate （P<0.01）， with the number of Nissl bodies （P<0.01） and expression levels of Claudin-1 and Occludin proteins （P<0.01） increased. LC-MS/MS results showed that compared to the sham group， the model group featured a significantly reduced content of acetic acid， propionic acid， and butyric acid in feces （P<0.01）， while valeric acid， isovaleric acid， and isobutyric acid levels were increased （P<0.01）. The intervention of Naoxintong capsules notably lowered the content of valeric acid， isovaleric acid， and isobutyric acid （P<0.01）.Conclusion Naoxintong capsules can improve brain and intestinal barrier damage and play a protective role in cerebral ischemia-reperfusion by regulating the content of short-chain fatty acids.

Key words:cerebral ischemia-reperfusion injury;intestinal barrier;Naoxintong capsules;short-chain fatty acids;LC-MS/MS