Effect of microRNAs and microvesicles containing miRNAs from Moringa oleifera Lam. on human health

Traditional medicine is often chosen due to its affordability, familiarity with patient’s cultural practices, and wider access to the local community. Moringa oleifera Lam. (MO) is one of the most well known and widely distributed species of the Moringaceae family. Moreover, this plant is widely used in African communities by traditional medicine, and various preparations of MO are used for the treatment of several diseases. Plants play an important role in providing essential nutrients. What is interesting is that in the last decade the presence of plant microRNAs (miRNAs) that can regulate human gene expression in a Cross-kingdom manner is emerging. Moreover, these plant miRNAs can be transported by microvesicles, key mediators of cell-cell communication. Plant microvesicles (MVs) display similar features to the mammalian counterpart and might enhance plant miRNA delivery in mammalian cells. Firstly, considering the effects of plant secondary metabolites on human cells, their presence in different MO aqueous extracts from leaves and seeds, were characterised. Then the anti proliferative, cytotoxic, and pro apoptotic activities of the different MO aqueous extracts, in lymphoid and monocytoid cells, were investigated. The results demonstrated the anti proliferative and pro apoptotic effects on tumour cells of MO extracts. In contrast, the same effects were not seen on peripheral blood mononuclear cells (PBMCs) from healthy donors. Studying the pro apoptotic effect of MO seed aqueous extract (MOES) in more detail, this appears to be correlated with a decreased expression of B Cell Lymphoma 2 (BCL2) and Sirtuin 1 (SIRT1) proteins, which are deeply involved in apoptosis. Thus, MOES appeared to be able to differentially regulate proliferation and apoptosis in healthy cells and cancer cells. Interestingly this ability could be attributed to the presence of miRNAs in the extracts. Considering the potential role of plant miRNAs in Cross Kingdom interactions, their presence in the MOES was characterized. Moreover, it seems that plant miRNAs are transported by extracellular vesicles, that may naturally vehiculate them in mammals. Thus, the presence of miRNAs in MOES MVs was also characterized. Through a bioinformatic analysis were identified specific human apoptosis-related genes, potentially targeted by MO miRNAs (p-miRs). Therefore, we characterized the MVs (MOES MVs) purified from MOES and used flow cytometry analysis to quantify the ability to deliver their content to host cells. In tumour cell lines, MOES MVs treatment reduced viability, increased apoptosis levels associated with a decrease in BCL2 protein expression and reduced mitochondrial membrane potential. Interestingly, the effects observed with MOES MVs treatment were comparable to those observed with MOES and transfection with the pool of miRNAs isolated from MOES, used as a control. These results highlight the role of miRNAs transported by MOES MVs as natural bioactive plant compounds that counteract tumorigenesis. The potential role of plant miRNAs as potential treatment in human pathologies, prompted us to evaluate the effects of plant-enriched purified extract miRNAs from MOES on the immune response and on HIV infection. Bioinformatic analysis shows that the p-miRs, including p-miR160h, p-miR166, p-miR482b, p-miR159c, p-miR395d, p-miR2118a, pmiR393a, p-miR167f-3p, and p-miR858b are predicted to target with high affinity BCL2, IL2RA, TNF, and VAV1. All these genes have been proven to be involved in the cell cycle, apoptosis, immune response and in the regulation of HIV pathogenesis. The analysis of the effects of MO p-miRs transfected into HIV+ PBMCs revealed a decrease in viability associated with an increase of apoptosis. In particular, an increase of T helper cells expressing Fas Cell Surface Death Receptor (FAS) and a decrease of intracellular BCL2 protein expression was detected. Meanwhile it was confirmed that no effects were detected in PBMCs from healthy donors. In CD4+ T cells, transfection of p-miRs significantly reduced cell activation and modified the T cell differentiation, thereby decreasing both central and effector memory cells while increasing terminal effector memory cells. Interestingly, the p-miRs transfection induces a reduction of intracellular HIV p24 protein and a reduction of viral DNA integration. Finally, we evaluated the effect of synthetic (mimic) p-miR858b whose sequence is present in the MO p-miR pool and predicted to target VAV1, a protein involved in HIV-Nef binding. This protein plays a pivotal role in T cell antigen receptor (TCR) signalling, thus triggering the activation of several pathways. The transfection of HIV+ PBMCs with the synthetic p-miR858b resulted in a reduced expression of the proteins VAV1 and HIV p24. Overall, our evidence defines putative mechanisms underlying a supplementary benefit of traditional medicine, alongside current antiretroviral therapy, in managing HIV infection in resource-limited settings where MO remains widely available. All these findings can provide a valuable contribution to the process of regularisation of traditional medicine remedies and open the possibility to develop safe and sustainable nutraceutical products.