Anti-inflammatory Effects Of Wild Bitter Gourd Extracts : Through The Transcription Factor PPAR Activation And Regulate The Nitric Oxide Production

[[abstract]]Wild bitter gourd (Momordica charantia L. var. Abbreviata Seringe, or WBG) is a wild variety of bitter melon, which is native to tropical areas of Asia. Inflammation is a complicated immune process that can regulate the expression of various inducible enzymes, such as inducible nitric oxide synthase (iNOS) and Cyclooxygenase-2 (COX-2). Nitric oxide (NO) is produced at inflammatory sites by iNOS, and COX-2 will lead to produce the pro-inflammatory production: prostaglandin E2 (PGE2), and anti-inflammatory production: prostaglandin D2 (PGD2), and 15-deoxy-△12, 14-prostaglandin J2 (15d-PGJ2) is generated as a consequence of dehydration of PGD2, those are associated with the progress of a variety of inflammation-related diseases and tissue injury. Heme oxygenase-1 (HO-1), an inducible enzyme, has been recognized to have major antioxidant, immunomodulatory and anti-inflammatory properties. Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcriptional factors that belong to the steroid hormone nuclear receptor superfamily, which play important roles in the regulation of metabolic pathways, including those of lipid of biosynthesis and glucose metabolism, as well as in anti-inflammatory. Some studies shows that WBG has anti-inflammatory activity and also can up-regulates the expression of PPAR, thus, the aim of this study is to clarify the effective mechanisms of anti-inflammatory by fractions of WBG extract in LPS-stimulated RAW264.7 macrophages.The 50% EtOH / 50% H2O extract from whole fruit of WBG was partitioned by n-hexane (Partition n-hexane extract, PHE), ethyl acetate (Partition ethyl acetate extract, PEAE), n-butanol (Partition n-butanol extract, PBE) and H2O (Partition water extract, PWE) subsequently. The highest inhibition effect on NO2- production by fraction partitioned was separated by silica gel chromatography: 90% n-hexane / 10% ethyl acetate (Hex : EA=9/1), 50% hexane / 50% ethyl acetate (Hex : EA=1/1), 100% ethyl acetate (EA) and 100% methanol (MeOH), then analyzed of NO2-, PGE2 and ROS production, western blot and transient transfection transactivation assay. The result shows that PEAE could significantly inhibit the NO2- production, and could also activated PPARα and PPARγ, and therefore this study selected PEAE to separated from silica gel chromatography. All of fractions eluted from silica gel chromatography could significantly inhibit COX-2 protein expression and NO2-, PGE2 and ROS production in LPS-stimulated RAW264.7 cell. The Hex : EA=1/1 and EA fractions could significantly increased the expression of HO-1 protein. The EA fractions could significantly activated both PPARα and PPARγ, and the MeOH fractions could significantly activated PPARγ. Interesting, the fraction of Hex : EA=9/1 could significantly decreased the expression of COX-2 protein, but the fractions of Hex : EA=1/1, EA and MeOH could significantly increased the expression of COX-2 protein. In conclusion, the working mechanism of the anti-inflammatory feature by fractions eluted from silica gel chromatography were through the specific effects regulated, and which may associated with the COX-2 pathway