Network pharmacology mechanism of Rosmarinus officinalis L.(Rosemary) to improve cell viability and reduces apoptosis in treating Alzheimer’s disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by neurodegeneration, nerve loss, neurofibrillary tangles and Aβ plaques. Different process of the AD pathology present more opportunities for treatment. In addition, the holistic approaches involving network pharmacology with traditional Chinese medicine (TCM) may be viable options for AD treatment, and lead to an effective cure for AD in the future. Therefore, this study explored the therapeutic effect and mechanism of Rosmarinus officinalis L(rosemary) in the treatment of Alzheimer's disease on basis of cell experiments, network pharmacology and molecular docking. We performed cell experiments, to investigate the therapeutic effects of Rosmarinus officinalis L on AD in vitro using CCK8 assay, flow cytometry and TMRE Kit. In addition, carnosic acid is a major antioxidant diterpenoid in Rosmarinus officinalis L. We performed cell experiments, to investigate the neuroprotective effects of carnosic acid on AD in virto using CCK8 assay and flow cytometry. Furthermore, the main antioxidant compounds of rosemary ware collected through literature reviews, PharmMapper and Swiss Target prediction ware used to identify their potential targets. Targets of AD were obtained from Genecards and OMIM. The intersection targets of the main active components of rosemary and the therapeutic targets of Alzheimer's disease were subsequently obtained by using online Venn diagram. Protein–protein interaction, Cytoscape, Gene Ontology, and Kyoto Encyclopedia of Genes were used to analyze potential targets and key pathways of rosemary in AD. Besides, through molecular docking, the interactions of the main active components of rosemary, and the predicted candidate targets were verified. Finally, quantitative Real-Time PCR (RT-qPCR) was performed to confirm the effectiveness of the genes. It was found that rosemary could reversed Aβ25–35 induced damage to mouse hippocampal neuron HT22 cells, significantly improved the viability of damaged cells, and reduced apoptosis. The results of fluorescent staining with Hoechst 33,342 and TMRE suggested that rosemary inhibited the reduction of mitochondrial membrane potential levels induced by Aβ25–35, which had a specific protective effect on AD in vitro. Additionally, a main antioxidant compound in rosemary, carnosic acid, also has neuroprotective effects. Eight main antioxidant compounds of rosemary ware collected. Network pharmacology and molecular docking, revealed that rosemary plays a therapeutic role in the treatment of Alzheimer's disease through the main active carnosic acid, carnosol, rosmarinol, rosmadial, genkwanin, cirsimaritin, rosmarinic acid and caffeic acid in Rosmarinus officinalis L, which affects the gene regulation of HRAS, ESR1, RHOA, IGF1, SRC, ANXA5, MMP9, MAPK14, NOS3, and PIK3R1, and participates in the PI3K-Akt, Rap1, MAPK, and estrogen signaling pathways. RT-qPCR indicated that rosemary could elevated expression of IGF1, MMP9 and decreased mRNA levels of SRC, MAPK14, compared with the control group. Rosemary is an important economic plant with multi-component, multi-target and multi-pathway synergistic effects.The findings highlight the effectiveness of rosemary in helping to increase cell viability and reduce apoptosis when treating mouse hippocampal neuron HT22 cells, thereby supporting its therapeutic potential in treating Alzheimer's disease.