小檗碱通过靶向AKT1/HSP90AA1信号通路缓解低压低氧诱导的睡眠障碍OA

OBJECTIVE To explore the mechanism by which berberine alleviates sleep disorders caused by hypobaric hypoxia. METHODS The compound coptis gel patch (CCGP) was prepared. The contents of its active ingredients, viscoelasticity and transdermal release behavior were analyzed by thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), rheology and trans⁃ dermal diffusion techniques. Network pharmacology was used to predict the active ingredients of the compound and the core targets of the disease. Male C57BL/6 mice were exposed to a hypobaric hypoxia chamber simulating an altitude of 7 000 meters for 3 days to establish a low-pressure and low-oxygen model. Before entering the chamber, the mice were randomly divided into the model group, the model + acetazolamide group (50 mg·kg-1, intragastric administration, once daily), and the model + topical administration CCGP group (topical administration with a patch area of 1.2 cm × 1.2 cm, dose approximately 54 mg, once daily). The treatment lasted for 7 days, and normal C57BL / 6 mice were used as the control group. After the mice exited the cabin, such experiments as the righting reflex disappearance test, open field test, and new object recognition test were conducted to measure the sleep latency, sleep duration, spontaneous activity, anxiety-like behavior, and learning and memory. An automatic blood cell analyzer was used to detect white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and hematocrit (HCT) of the mice. The spleen was taken to calculate the spleen weight index. ELISA was used to detect tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and molecular docking were performed to identify the binding sites and affinities. RT-qP⁃ CR was employed to detect the mRNA expression levels of AKT serine/threonine kinase 1 (Akt1) and heat shock protein 90 α family class A member 1 (HSP90AA1). Western blotting was used to detect the protein expressions of phosphorylated AKT1 (p-AKT1) and HSP90AA1. The localization of p-AKT1 and HSP90AA1 in the hippocampus of the mice was detected using the immunofluorescence method. RESULTS TLC and HPLC analyses revealed that effective components such as berberine and cinna⁃ maldehyde could be detected in the CCGP. The rheological results indicated that the storage modulus (G′) of the sample was higher than the loss modulus (G″) within the tested range, pointing to appropriate viscoelastic properties. The in vitro transdermal diffusion experiment showed that berberine could be released through the CCGP for transdermal administration. Network pharmacology screening identified 72 common targets and 14 core targets. The GO and KEGG enrichment analysis revealed that the active components were mainly enriched in hypoxia-inducible factor 1 (HIF-1), phosphatidylinositol 3 - kinase (PI3K)/AKT, and Toll-like receptor and through other low-oxygen inflammatory-related path⁃ ways. Molecular docking analysis found that the core component berberine had a strong binding ability with targets such as AKT1 and HSP90AA1, and the binding energy was the strongest at -10.5 kcal·mol-1 (< -10.0 kcal·mol-1) with AKT1. Compared with the control group, the sleep latency of mice in the model group was significantly prolonged, the sleep duration was significantly shortened, the number of entries into the central area of the open field and the distance of movement were significantly reduced, the new object recognition index was significantly decreased, WBC, RBC, HGB, HCT, TNF - α, and IL-6 were all significantly increased, and the spleen weight index was also elevated. Compared with the model group, the above indicators in the model + CCGP group were significantly improved. Compared with the control group, hippocampal expressions of Akt1 and HSP90AA1 mRNA and p-AKT1 (Ser473) protein were significantly increased in the model group. Compared with the model group, the expres⁃ sions of these indicators in the CCGP-treated group were significantly decreased. The protein expres⁃ sions of HSP90AA1 did not differ significantly between the groups. The immunofluorescence results for p-AKT1 and HSP90AA1 were consistent with the Western blotting results. CONCLUSION The CCGP, with berberine as the marker constituent, can significantly improve sleep disturbance, anxiety-like behaviors,and cognitive impairment while reducing the increased peripheral blood cell counts and sple-nomegaly in a mouse model of hypobaric hypoxia.These effects are associated with inhibition of aber-rant AKT1/HSP90AA1 signaling and attenuation of the inflammatory response.