Investigations On The Anti-inflammatory Substances And Mechanisms Of Polygonum Capitatum By Serum Chemistry And Serum Pharmacology

Objective: To understand the anti-inflammatory effect and mechanism as well as the anti-inflammatory substances of Polygonum capitatum through pharmacological and chemical investigations on its drug-containing serum. Methods: Serum pharmacology and serum chemistry are the two basic methods employed. Rats were used for the preparation of drug-containing serums. The inflammatory cell model was reproduced by LPS(10 ng·m L-1)-treated murine mononuclear macrophage RAW264.7 cells. Releases of NO and TNF-α were adopted as the activity indexes to screen for the anti-inflammatory extract with the drug-containing serums of the water extract(PCWES), alcohol-precipitated water extract(PCAWES), and alcohol precipitation of water extract(PCAPWES) of P. capitatum. Nitric oxide(NO) production was detected by the Griess assay, inflammatory factors(TNF-α 、 IL-6) productions were quantitated by the enzyme-linked immunosorbent assay(ELISA), the expression levels of their related inflammatory cytokine genes(i NOS、TNF-α and IL-6 m RNA) were determined by the real-time polymerase chain reaction(Real Time-PCR), and the expressions of regulatory proteins in NF-κB and MAPKs pathway(IκB-α, p65 and p38) were measured by Western blot technology. The metabolites in the drug-containing serums of P. capitatum were analyzed by UHPLC-Q-TOF. Random forest regression analysis with inhibition ratio of TNF-α production being the dependent variable and the peak intensity of each m/z being the independent variable was used to screen for the very important variables responsible for the anti-inflammatory substances present in the drug-containing serum. Results: All the drug-containing serums significantly inhibited, to different degrees, the release of NO and TNF-α in LPS-induced RAW264.7 cells. Whereas, PCAWES showed the strongest effects(P<0.05 as compared to other groups) with the inhibitory rate of NO and TNF-α production being 17.60% and 29.90%, respectively. Besides, the drug-containing serums of PCAWE inhibited the releases of NO, TNF-α and IL-6 in a dose dependent manner(P<0.01 or P<0.001). In addition, PCAWES significantly inhibited the expressions of i NOS, TNF-α and IL-6 m RNAs(P<0.001), reduced the expression level of p-IκBα protein in the NF-κB pathway, and inhibited the translocation of the p65 from the cytoplasm to the nucleus(P<0.001). PCAWES had no significant effect on the expression of p-p38 protein in the MAPKs pathway. The major drug-related compounds in the drug-containing serum of P. capitatum were the metabolites of gallic acid, but the compounds directly responsible for the anti-inflammatory activity were endogenous metabolites. Conclusion: PCAWES was the main anti-inflammatory extract of P. capitatum. The anti-inflammatory effect may involve stimulation of the anti-inflammatory endogenous molecules by the compounds of P. capitatum. Down-regulations of the expressions of NO, TNF-α and IL-6 m RNA and the expression of NF-κB pathway-related p I-κBα protein in the NF-κB pathway may be involved in the anti-inflammatory activity of P. capitatum.