Gegen Qinlian tablets delay Alzheimer's disease progression via inhibiting glial neuroinflammation and remodeling gut microbiota homeostasis

Current therapeutic agents for AD have limited efficacy and often induce undesirable side effects. Gegen Qinlian tablets (GGQLT) are a well-known clearingheat formula used in clinical treatment of inflammatory diseases. Based on traditional Chinese medicine (TCM) theory, the strategy of clearing-heat is then compatible with the treatment of AD. However, it remains unknown whether GGQLT can exert neuroprotective effects and alleviate neuroinflammation in AD. This study aimed to evaluate the anti-AD effects of GGQLT and to decipher its intricate mechanism using integrative analyses of network pharmacology, transcriptomic RNA sequencing, and gut microbiota. The ingredients of GGQLT were analyzed using HPLC-ESI-Q/TOF-MS. The AD model was established by bilateral injection of Aβ1-42 into the intracerebroventricular space of rats. The Morris water maze was used to evaluate the cognitive function of the AD rats. The long-term toxicity of GGQLT in rats was assessed by monitoring their body weights and pathological alterations in the liver and kidney. Reactive astrocytes and microglia were assessed by immunohistochemistry by labeling GFAP and Iba-1. The levels of inflammatory cytokines in the hippocampus were evaluated using ELISA kits, RT-PCR, and Western blot, respectively. The potential anti-AD mechanism was predicted by analyses of RNA-sequencing and network pharmacology. Western blot and immunohistochemistry were utilized to detect the phosphorylation levels of IκB, NF-κB p65, p38, ERK and JNK. The richness and composition of gut bacterial and fungal microflora were investigated via 16S rRNA and ITS sequencing. Typical ingredients of GGQLT were identified using HPLC-ESI-Q/TOF-MS. GGQLT significantly improved the cognitive function of AD rats by suppressing the activation of microglia and astrocytes, improving glial morphology, and reducing the neuroinflammatory reactions in the hippocampus. RNA-sequencing, network and experimental pharmacological studies demonstrated that GGQLT inhibited the activation of NF-κB/MAPK signaling pathways in the hippocampus. GGQLT could also restore abnormal gut bacterial and fungal homeostasis and no longer-term toxicity of GGQLT was observed. Our findings, for the first time, demonstrate GGQLT exhibit anti-AD effects and is worthy of further exploration and development.