The Anti-Inflammatory Effect Of Neochlorogenic Acid From Lonicerae Japonicae Flos On LPS-induced RAW264.7 Cells And Its Mechanism

Honeysuckle as blindly traditional Chinese herbal medicine, first recorded in the "Shen Nong’s Herbal Classic". Its early opening or dry buds can be used as medicine, with detoxification, expelling the effect of heat removal. The study found honeysuckle contains large amounts of flavonoids, terpenoids, organic substances, it has anti-inflammatory and antipyretic, lowering blood pressure, anti-virus, gallbladder, bleeding antioxidant role. The chlorogenic acid of coffee acid derivatives has been fully studied, but the research for isomers of nechlorogenic acid was rarely reported. The purpose of this paper is to study the anti-inflammatory effects of neochlorogenic acid on RAW264.7 cells induced by lipopolysaccharide (LPS) and the underlying mechanism of action.The main contents of this paper include:First, the use of different concentrations of the drug in the cell, to observe the growth state of the cells; the use of CCK-8 reagent to assess drug toxicity, and explore the optimum concentration; Griess Reagent was employed to detect the impact of neochlorogenic acid on NO; Enzyme-linked immunosorbent assay (ELISA) was utilized to detect the effects of neochlorogenic acid on the generation of PGE2; Real-time fluorescent quantitative method (RT-qPCR) was used to analyze the expression of iNOS, COX-2, TNF-α, IL-1β, IL-6 at the mRNA level; and Western blot was used to detect the expression of iNOS and COX-2 at the protein level, as well as the changes on NF-Kb and MAPKs pathways.Experimental results showed that:there is non-toxic for cells when the 50-200 μg· mL-1 drug concentration effect. Compared with the model group, neochlorogenic acid not only significantly inhibits the production of NO and PGE2 in RAW264.7 induced by LPS under the concentration of 200 μg · mL-1 (P< 0.01), but also significantly down-regulates the expressions of iNOS, COX-2, TNF-α, IL-1β, IL-6 at the mRNA and protein level (P< 0.01). Meanwhile, it exhibits an obvious inhibitory effect on P-p65 in NF-Kb pathway under the concentration of 200 μg·mL-1.Experimental results show that the nechlorogenic acid can inhibit the expression of NO and PGE2, also iNOS and COX-2 significantly were inhibited at protein and nucleic acid level. TNF-α, IL-1β, IL-6 were significantly reduced at nucleic acid level too. Neochlorogenic acid show powerful anti-inflammatory effects on LPS-induced RAW264.7, and the action mechanism may be related to the down-regulation of P-NF-κbp65.