Anti-inflammatory Activity And Modulatory Mechanism On MAPK And NF-?B Transduction Signal Pathways Of Gegen Decoction

Gegen Decoction as one of the representative decoction derived from"Treatise on Febrile Diseases"of Zhang Zhongjing in the Eastern Han Dynasty.Gegen Decoction has many products such as sweat solution,regulate the body fluid and dredge the meridians.Gegen Decoction is used in clinical practice with diseases such as frozen shoulder,viral enteritis,and cold.Gegen Decoction as anti-inflammatory medicine is used in clinically widespread,however the specific anti-inflammatory molecular mechanism of Gegen Decoction is still unclear.Therefore,the prescription of Gegen Decoction recorded in the2015 Chinese Pharmacopoeia was taken as an example to explore its anti-inflammatory activity.LPS-induced RAW 264.7 cells are used as an inflammatory model to explore the anti-inflammatory molecular mechanism.High performance liquid chromatography?HPLC?was used to determine the content of the effective components in the Traditional Chinese Medicines.The quality of the Chinese herbal medicines of Gegen Decoction was tested.Two batches of Gegen Decoction Chinese herbal medicines were selected randomly for content determination.The experimental results showed that the content of index components in the Chinese herbal medicines of Gegen Decoction were in compliance with the requirements of 2015Chinese Pharmacopoeia and the content of the index components was qualified.The Gegen Decoction was prepared according to the preparation method of the Geegen Decoction recorded in the 2015 Chinese Pharmacopoeia.The medicinal materials Pueraria lobata and ephedra were immersed in warm water for 30 min,and decocted with cassia twig,white peony root,licorice,ginger,jujube twice,30 min each time.The decoction was combined and concentrated to a certain relative density under reduced pressure.The Gegen Decoction solution was used in the subsequent experiments.The anti-inflammatory activity of Gegen Decoction was studied by exploring the effect of Gegen Decoction on carrageenan-induced paw swelling in rats and xylene-induced ear swelling in mice,which included these two acute inflammation models.The results of carrageenan-induced foot swelling in rats and xylene-induced ear swelling in mice showed that Gegen Decoction can inhibit carrageenan-induced foot swelling in rats and xylene-induced ear swelling in mice,compared with the model group.The degree of swelling of the Gegen Decoction group was significantly reduced,and the swelling inhibition rate was better than that of the commercially available Gegen Decoction Granules entirely.The anti-inflammatory mechanism of Gegen Decoction was explored via LPS-induced inflammatory reaction on mouse macrophages.LPS was used to stimulated mouse macrophage RAW 264.7 to induce inflammatory reaction,released inflammatory mediators such as NO and PGE₂,and inflammatory cytokines such as IL-6 and TNF-?,MAPKs and NF-?B singaling pathways were activated,investigated the Effects of Gegen Decoction on inflammatory cytokines,MAPK and NF-?B signaling pathways.The cytotoxicity of Gegen Decoction was evaluated by MTT assay.The results showed that Gegen Decoction had no obvious cytotoxicity on RAW 264.7 cells on the concentration of 10 mg/mL⁴0 mg/mL.The Griess method was used to detect the effect of Gegen Decoction on the release of inflammatory mediator NO from mouse macrophages.The result exhibited Gegen Decoction had a significant inhibitory effect on the release of inflammatory mediator NO from mouse macrophage RAW 264.7 induced by LPS.The effect of Gegen Decoction on the release of TNF-?,IL-6 and PGE₂ was detected by ELISA.The results showed that Gegen Decoction significantly inhibited the release of inflammatory mediator PGE₂ and inflammatory cytokines IL-6 and TNF-?on LPS-induced mouse macrophage cells RAW 264.7.Western blot was used to detect the the protein expression levels of Gegen Decoction on iNOS,COX-2,p-p38,p-ERK,p-JNK and I?B-?and p65 to explore the NF-?B and MAPK inflammatory signal pathway induced by LPS.The results showed that Gegen Decoction can significantly inhibit the high expression of iNOS and COX-2 on LPS-induced inflammatory proteins,and blocked the phosphorylation of ERK/JNK/MAPKs,inhibited the degradation of I?B-?protein and the translocation of NF-?B p65 protein into the nucleus.In summary,Gegen Decoction can down-regulate the protein expression of iNOS and COX-2 by blocking the phosphorylation of ERK/JNK/MAPKs and degradation of I?B-?/NF-?B and p65/NF-?B into nucleus,further more Gegen Decoction inhibited inflammatory mediators such as NO and PGE₂,as well as inflammatory cytokines TNF-?and IL-6 to exerted anti-inflammatory effects.The anti-inflammatory mechanism of Gegen Decoction on LPS-induced mouse macrophages has not been reported in detail.