补肾通腑方调控肠道菌群介导的LPS/TLR4/NF-κB通路改善痴呆认知障碍作用机制

Dementia is a common disease in the elderly with cognitive impairment as its main manifestation. Inflammatory brain damage is the main cause of cognitive impairment. Gut flora plays an important role in the brain-gut axis pathway, which mediates brain inflammation through LPS/TLR4/NF-κB pathway. Constipation is a common symptom in patients with dementia.Previous studies have found that Bushen Tongfu Prescription is effective in the treatment of cognitive impairment，but its mechanism has not been clarified. In this study, we hypothesized that: Bushen Tongfu Prescription can regulate gut flora, alleviate brain inflammation and improve cognitive impairment. In this study, 16SrDNA high throughput sequencing will be used to analyze the gut flora abundance, diversity and plasma LPS difference between cognitive impairment patients and healthy people, and the effect of Bushen Tongfu Prescription on gut flora. In the experimental study,the rat model of cognitive impairment will be established by intraperitoneal injection of LPS,the expression of LPS/TLR4/NF-κB pathway-related proteins in brain and gut tissues will be observed by Western Blot method; the activation of microglia in brain will be observed by immunofluorescence method, to explore the effect of Bushen Tongfu Prescription on LPS/TLR4/NF-κB pathway.This study focused on gut flora and LPS/TLR4/NF-κB inflammatory pathway, to explore the mechanism of Bushen Tongfu Prescription in improving cognitive impairment, and provide basis for clinical application.

脑部炎症损伤是导致痴呆患者出现认知障碍的主要原因，肠道菌群失调激活LPS/TLR4/NF-κB通路是加重脑部炎症损伤的关键因素。便秘是痴呆患者常见症状，前期研究发现，具有补肾益髓、通腑醒神功效的补肾通腑方改善认知障碍疗效确切，但其作用机制尚未阐明。本研究提出假说：补肾通腑方通过调节肠道菌群，减轻其介导的炎症损伤从而发挥治疗作用。拟开展：1）以痴呆患者为研究对象，采用16SrDNA高通量测序技术分析其与健康者肠道菌群丰度、多样性及血浆LPS差异，及补肾通腑方对肠道菌群的影响；2）采用腹腔注射LPS法建立认知障碍大鼠模型，通过Western Blot方法观察脑、肠组织中LPS/TLR4/NF-κB通路相关蛋白的表达，免疫荧光法观察脑部小胶质细胞活化情况，探讨补肾通腑方对LPS/TLR4/NF-κB通路影响。围绕肠道菌群及其介导的炎症通路，探讨补肾通腑方改善认知障碍作用机制，为临床应用提供依据。

认知障碍是老年人群常见的疾病，脑部炎症损害是导致患者出现认知障碍的主要原因。研究发现，肠道菌群失调激活LPS/TLR4/NF-κB通路是加重脑部炎症损伤的重要因素。随着年龄增长，老年人群的肠道菌群会随之发生改变。老年认知障碍患者的肠道菌群特征及其与认知功能的相关性需进一步明确。前期研究发现，肾虚、腑实证是老年认知障碍患者常见的中医证素，具有补肾益髓、通腑益智功效的补肾益智方改善认知障碍疗效确切，但其作用机制尚未阐明。本研究通过开展临床研究，分析了认知障碍患者和非认知障碍的健康对照者的肠道菌群丰度和多样性，揭示了老年认知障碍患者肠道菌群特征。在实验研究中，采用腹腔注射LPS法建立了认知障碍大鼠模型，采用Morris水迷宫法检测实验大鼠的学习记忆能力，采用HE染色观察海马区脑组织细胞形态，采用免疫荧光法观察脑部小胶质细胞活化情况，采用Western Blot方法检测了脑组织中LPS/TLR4/NF-κB通路相关蛋白的表达。结果显示，与非认知障碍的健康对照者相比，认知障碍患者肠道菌群丰度和多样性显著下降，血液中LPS表达水平升高。补肾益智方能够降低认知障碍模型大鼠脑组织中TLR4、NF-κB、TNF-α、IL-6、IL-1β等蛋白的表达，促进小胶质细胞向抗炎表型转化，改善实验大鼠的学习记忆能力。通过本研究阐释了补肾益智方减轻脑组织炎症损伤，改善认知功能的作用机制，揭示了肠道菌群与认知障碍的相关性，将为中医药防治认知障碍类疾病提供理论依据。