Study on the neuroprotective effect of Zhimu-Huangbo extract on mitochondrial dysfunction in HT22 cells induced by D-galactose by promoting mitochondrial autophagy

Zhimu-Huangbo (ZB) herb pair is a common prescription drug used by physicians of all dynasties, and has significant neuroprotective effect, such as the ZB can significantly promote neuronal cell regeneration, repair neuronal damage, and improve cognitive disorders. However, its ingredients are urgently needed to be identified and mechanisms is remained unclear. Using ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS), the study of neuroprotective mechanism of Zhimu-Huangbo extract (ZBE) is investigated, and the network pharmacology technology and experimental validation is also performed. Firstly, UPLC-Q-TOF-MS technology was used to characterize the chemical components contained in the ZBE. After that, the TCMSP database and the Swiss Target Prediction method were used to search for potential target genes for ZBE compounds. At the same time, the OMIM and GeneCards disease databases were used to search for Alzheimer's disease (AD) targets and expanded with the GEO database. Then, GO and KEGG enrichment analysis was performed using OECloud tools. Subsequently, the potential mechanism of ZBE therapeutic AD predicted by network pharmacological analysis was experimentally studied and verified in vitro. In the UPLC-Q-TOF-MS analysis of the ZBE, a total of 39 compounds were characterized including Neomangiferin, Oxyberberine, Timosaponin D, Berberine, Timosaponin A-III, Anemarsaponin E, Timosaponin A-I, Smilagenin and so on. A total of 831 potential targets and 13995 AD-related target genes were screened. A further analysis revealed the number of common targets between ZBE and AD is 698. Through GO and KEGG enrichment analysis, we found that ZBE's anti-AD targets were significantly enriched in autophagy and mitochondrial autophagy related pathways. The results of cell experiments also confirmed that ZBE can promote mitochondrial autophagy induced by D-galactose (D-gal) HT22 cells through the PTEN-induced kinase 1/Parkin (PINK1/Parkin) pathway. ZBE can promote autophagy of mitochondria and play a protective role on damaged neurons.