Anti- inflammatory effect of Mitragyna speciosa (Korth.) methanolic extract on macrophages and T cell-mediated delayed type hypersensitivity mouse model

Mitragyna speciosa (M. speciosa), a botanical member of the Rubiaceae plant family, possesses several pharmacological properties, including antioxidant, anti-bacterial, anti-diabetic, antiproliferative, and anti-inflammatory, among other biological activities. Despite considerable attention being given to exploring the medicinal benefits of M. speciosa, its effects on the mechanisms of immune responses are poorly understood. Thus, this study aimed to investigate the immunomodulatory activity of M. speciosa methanolic extract (MSME) in the innate and adaptive immune responses. The crude bioactive compound was extracted in methanol by Soxhlet, while UHPLC was used to screen the phytochemical content in MSME. The effects of MSME on innate immunity, including the release of immune mediators in lipopolysaccharide (LPS)-induced macrophage immune response such secreted nitric oxide (NO), reactive oxygen species (ROS) and cytokines were evaluated by Griess assay, 2’,7’- dichlorodihydrofluorescein diacetate (H2DCFDA), and a cytometric bead array (CBA), respectively. The expression of Toll-like receptor 4 (TLR4), cluster differentiation 14 (CD14), inducible nitric oxide synthase (iNOS), nuclear factor kappa-light-chain (NF-KB), and cytokine genes were assessed by a quantitative reverse transcription polymerase chain reaction (qRT-PCR). Moreover, the phagocytosis activity of LPS-activated macrophages in the presence of MSME was evaluated by a neutral red uptake and latex bead phagocytic assays. Additionally, the effect of MSME on adaptive immunity was investigated in lymphocyte proliferation and phenotype by using Jurkat T cell line and splenocytes via Cell-Quant assay and immunophenotyping, respectively. To further evaluate the effects of MSME on adaptive immune response, an animal model of Delayed-Type Hypersensitivity (DTH) response was developed through induction with sheep red blood cells (SRBC) in Balb/c mice. The complete blood count (CBC), foot paw thickness, spleen index, and antibody production from Balb/c mice were measured. Three classes of bioactive phytochemical compounds were found in MSME. They are Flavonoid, Saponin, and Polyphenol. Stimulation of macrophages with LPS triggers the activation of TLR4 and CD14 which enhances the phagocytic activity of the cells. Overexpression of NF-κB by LPS also activated the downstream signaling, NO, ROS, and cytokines production. MSME has shown an inhibitory effect on macrophage through a significant (p<0.05) inhibition of TLR4 and CD14, which also affect NF-κB, iNOS, and cytokine gene expression. In addition, MSME also attenuated the phagocytic activity of macrophages and reduced NO and ROS production. In adaptive immunity, MSME did not induce changes to the proliferation rate and phenotype of resting Jurkat T cell line and splenocytes. A significant (p<0.05) reduction of T cell and B cell proliferation was observed in Concanavalin A or LPS-stimulated splenocytes in vitro study. However, the proportion of CD4/CD8/CD19 T cell subsets are remained unchanged. In addition, Balb/c mice have developed foot paw thickness through SRBC-induced DTH reaction. Increased antibody production and exaggeration of paw thickness formation were noticeable when the mice were treated with drug inducer, Levamisole. On the other hand, oral administration of MSME exhibited a decrease in foot paw thickness formation and antibody production without affecting the spleen index and overall CBC. In conclusion, these findings suggested that the MSME modulates both innate and adaptive immunity by suppressing the inflammatory response. These findings suggest the potential for M. speciosa to be used as an alternative or adjunctive therapy to existing immunosuppressive drugs. Additionally, it may be effective in treating various inflammatory diseases, such as arthritis, allergies, inflammatory bowel disease, and other chronic inflammatory disorders.

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