Macrophages are a type of innate immune cell that activates the NLRP3 inflammasome, causing the release of the cytokine IL-1β, which is a crucial mediator of the inflammatory response. NLRP3 activation that is dysregulated worsens a variety of inflammatory and autoimmune diseases, as well as neurodegenerative diseases. Oleamide is an endogenous fatty acid amide that was first determined as a sleep-inducing molecule and later shown to have wide-ranging beneficial effects on the central nervous system. How oleamide influences human macrophage polarization and NLRP3-inflammasome activation remains unclear. The effect of oleamide on macrophage polarization was explored using anin vitroculture of primary human monocyte-derived macrophages (MDMs) supplemented with human serum-containing media. Cellular and molecular mechanisms of oleamide-regulated MDMs polarization were also investigated. Results showed that oleamide promoted naïve macrophages (M0) toward the M1 phenotype by upregulating M1-associated genes (IL-1β,iNOS,CXCL10), along with downregulation of M2-associated genes (Arg-1,CD206,CCL22). Cell surface expression indicated that oleamide enhanced CD80 expression in M0 naïve macrophages and hider CD206 and CD163 expression in M2 macrophages. Higher production of IL-1β cytokine was observed but with no alteration in IL-6 and TNF-α levels by MDMs and differentiated THP-1 models. Whether oleamide functioned as a second signal that activated the NLRP3 inflammasome and mediated IL-1β production was further investigated using LPS-primed MDMs followed by oleamide treatment that induced activation of inflammasome-related proteins including NLRP3, ASC, cleaved casp-1, and cleaved IL-1β. These findings suggested that oleamide promoted M1 macrophage polarization and increased IL-1β production by activating the NLRP3 inflammasome in primary MDMs. This research reveals a new function for oleamide as well as prospective targets for treating NLRP3-related inflammatory disorders.

Moringa oleifera (MO) is an important plant for traditional medicine. The present study aimed to identify the MO active phytochemical compounds for their ability against inflamed macrophages. An ethyl acetate extract fraction of MO was fractionation by flash column chromatography. Human macrophages were stimulated by Lipopolysaccharide and then treated with fractions of MO to examine their anti-inflammatory activity and cellular mechanism. The active fractions were analyzed by liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometer (LC-ESI-QTOF-MS). MO treated cells showed a decreased production of pro-inflammatory mediator in response to lipopolysaccharide. This was evident at both mRNA and protein levels. The study revealed that MO suppressed mRNA expression of IL-1, IL-6, TNF-α, PTGS2, NF-κB (P50), and RelA. Furthermore, the extract effectively inhibited the expression of inflammatory mediators, including IL-6, TNF-α, and cyclooxygenase-2. Interestingly, the effect of MO inhibited phosphorylation of IκB-α and the ability to reduce expression of the nuclear factor (NF)-κB p65, suppressing its nuclear translocation. Moreover, LC-ESI-QTOF-MS analysis of the MO active fraction revealed seven compounds, namely 3,4-Methyleneazelaic acid, (2S)-2-phenylmethoxybutane-1,4-diol, (2R)-2-phenylmethoxybutane-1, 4-diol, γ-Diosphenol, 2,2,4,4-Tetramethyl-6-(1-oxobutyl)-1,3,5-cyclohexanetrione, 3-Hydroxy-β-ionone, and Tuberonic acid. Our findings highlight the ability of MO compounds to inhibit inflammation through regulation of the NF-κB pathway.

Triple negative breast cancer is one of the most aggressive breast cancer type with abilities of early metastasis and chemoresistance. The tropical plant Citrus hystrix DC. has been reported to promote many biological activities including anticancer. However, the effect of C. hystrix against triple negative breast cancer has not yet been identified. This study aimed to evaluate the anticancer properties of C. hystrix leaf extract and its bioactive constituents citronellol and citronellal against the triple negative breast cancer MDA-MB-231 cell line. C. hystrix leaves were powdered and sequentially macerated. The in vitro anticancer effects of C. hystrix leaf extracts, and its bioactive constituents (citronellol and citronellal) were evaluated against MDA-MB-231 cell line using cytotoxic MTT assay, cell proliferation, wound scratch migration, colony formation, cell cycle, apoptosis assay, Hoechst staining, RT-qPCR, and Western blot analysis. Results showed that crude hexane extract, citronellol, and citronellal significantly reduced cell proliferation, colony formation, and cell migration by inducing cell cycle arrest, while also inducing apoptosis in MDA-MB-231 cells through inhibition of anti-apoptotic Bcl-2 expression, leading to activation of the caspase-3-dependent pathway. This study is the first report to demonstrate the effect of C. hystrix, citronellol, and citronellal against triple negative breast cancer MDA-MB-231 cells.

Moringa oleifera Lam. (MO) is a medicinal plant distributed across the Middle East, Asia, and Africa. MO has been used in the traditional treatment of various diseases including cancer. This study aimed to perform bioassay-guided fractionation and identification of bioactive compounds from MO leaf against MDA-MB-231 breast cancer cells. MO leaf was sequentially extracted with hexane, ethyl acetate (EtOAc), and ethanol. The most effective extract was subjected to fractionation. MO extract and its derived fractions were continuously screened for anti-cancer activities. The strongest fraction was selected for re-fractionation and identification of bioactive compounds using LC-ESI-QTOF-MS/MS analysis. The best anticancer activities were related to the fraction no. 7-derived crude EtOAc extract. This fraction significantly reduced cell viability and clonogenic growth and increased cells apoptosis. Moreover, sub-fraction no. 7.7-derived fraction no. 7 was selected for the identification of bioactive compounds. There were 10 candidate compounds tentatively identified by LC-ESI-QTOF-MS. Three of identified compounds (7-octenoic acid, oleamide, and 1-phenyl-2-pentanol) showed anticancer activities by inducing cell cycle arrest and triggering apoptosis through suppressed Bcl-2 expression which subsequently promotes activation of caspase 3, indicators for the apoptosis pathway. This study identified 10 candidate compounds that may have potential in the field of anticancer substances.

Squamous cell carcinoma is the most common type of head and neck cancer worldwide. Radiation and chemotherapy are general treatments for patients; however, these remedies can have adverse side effects and tumours develop drug resistance. Effective treatments still require improvement for cancer patients. Here, we investigated the anti-cancer effect of Moringa oleifera (MO) Lam. leaf extracts and their fractions, 3-hydroxy-β-ionone on SCC15 cell line. SCC15 were treated with and without MO leaf extracts and their fractions. MTT assay was used to determine cell viability on SCC15. Cell cycle and apoptosis were evaluated by the Muse™ Cell Analyser. Colony formation and wound closure analysis of SCC15 were performed in 6-well plates. Apoptosis markers were evaluated by immunoblotting. We found that Moringa extracts and 3-HBI significantly inhibited proliferation of SCC15. Moreover, they induced apoptosis and cell cycle arrest at G2/M phase in SCC15 compared to the untreated control. MO extracts and 3-HBI also inhibited colony formation and cell migration of SCC15. Furthermore, we observed the upregulation of cleaved caspase-3 and Bax with downregulation of anti-apoptotic Bcl-2, indicating the induction of cancer cell apoptosis. Our results revealed that MO extracts and 3-HBI provided anti-cancer properties by inhibiting progression and inducing apoptosis of SCC15.

Citrus hystrix DC. (CH) is found in many countries in Southeast Asia. This plant has been reported for anti-microbial, anti-cancer and anti-inflammatory bioactivities. However, the anti-inflammatory and anti-inflammasome properties of the leaves remain poorly understood. This study aimed to investigate the effect of CH leaves on NLRP3 and NF-κB signaling pathways. CH leaves were sequentially extracted using hexane, ethyl acetate and 95% ethanol to give three crude extracts. An active compound, lupeol was fractionated from the ethanolic extract using chromatographic techniques, and its structure was identified and confirmed by spectroscopic methods. Anti-inflammatory activities were observed on both lipopolysaccharide-stimulated and NLRP3 adenosine triphosphate-induced macrophages. The release of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) was analyzed by Enzyme-Linked Immunosorbent Assay (ELISA). Real-time qRT-polymerase chain reaction (PCR) was used to measure inflammatory-associated gene expression. NF-κB protein expressions were investigated using the immunoblotting technique. The active fraction of ethanolic CH leaves and lupeol significantly reduced the release of pro-inflammatory cytokines and suppressed the expression of both inflammasome genes and NF-κB proteins. The ethanolic extract of CH leaves and lupeol showed potent anti-inflammatory activities by targeting NF-κB and NLRP3 signaling pathways.