| Our laboratory screened out the Chinese herbal formula Xiang-Qi-Tang frommany drugs based on the traditional chinese veterinary medicine.Xiang-Qi-Tang ismade of andrographis, astragalus, cyperus rotundus by a certain percentage,which hasthe effect of heat-clearing and detoxifying,spleen-strengthening and liver-qiregulatingand Tonifying spleen. To feed chickens for the preparation three days before attackbacteria,the protection rate of artificial infection Pathogenic avian Escherichia coliO78up to90.0%; Treat with the preparation of the typical symptoms of infectedchickens,the cure rate is88.3%, whose effect is close to levofloxacin. But,the effect invitro antibacterial test is not clear compared with levofloxacin or some otherantibiotics, we analysis of the preparation in prevention and cure chickencolibacillosis may have other pharmacological mechanism.So,we intend to in-depthstudy from the anti-inflammatory effect and pharmacological mechanism ofXiang-Qi-Tang. With stimulating of LPS, the RAW264.7macrophages were be thebasis in this explore, we established models of inflammation and researched theanti-inflammatory activity and pharmacological mechanism of anti-inflammation ofXiang-Qi-Tang and its active ingredients by ELISA and Western blot in vitro.Macrophages involved in the inflammatory process indirectly or directly throughthe secretion of cytokines of different functions in the inflammatory responses.Macrophages in vivo could produce proinflammatory cytokines such as tumornecrosis factor α,interleukin1β, interleukin6and anti-inflammatory cytokines suchas interleukin10to mediate inflammatory disease process when stimulated bylipopolysaccharides.Accordingly,it is possible to reach a certain level of antagonisticrole of inflammation through the regulation of cytokines in macrophages.We exploredthe effects of Xiang-Qi-Tang and its active components on the secretion ofinflammatory cytokines through the inflammation model in vitro stimulated bylipopolysaccharides in RAW264.7macrophages in this study. Results shown that the production levels of TNF-α,IL-1β and IL-10were very significantly elevated thatwere induced by LPS after18hours compared with the normal control group inRAW264.7macrophages, different concentrations of Xiang-Qi-Tang(1mg·L-1and5mg·L-1) after3hous of pretreatment were able to significantly reduce the secretorylevel of TNF-α that was induced by LPS in RAW264.7macrophages （P＜0.01）,which indicated that Xiang-Qi-Tang of Chinese herbal formula could reduce theinflammatory response through inhibiting the secretion of TNF-α. We studied theeffects of its active components to further study the specific anti-inflammatoryactivity of Xiang-Qi-Tang, according to the results of giving Xiang-Qi-Tang of5mg·L-1, astragaloside of0.2mg·L-1, α-Cyperone of0.02mg·L-1and andrographalide of0.2mg·L-1which were pretreated macrophages3hours and then incubated with LPS18hours shown that each group was able to significantly reduce the secretory levelsof LPS-induced RAW264.7macrophage TNF-α, IL-1β and IL-6（P＜0.01） andsignificantly increase the secretory level of IL-10（P＜0.01） comparing with LPScontrol group. Experimental results indicated that Xiang-Qi-Tang might play ananti-inflammatory effect by inhibiting the generation of pro-inflammatory cytokinesand increasing the quantity of anti-inflammation cytokines.MAPK signal transduction pathway and NF-κB signaling pathway are twowidespread signaling cascades that mediate inflammatory response in Eukaryotic cells.The main members of MAPK family contain ERK1/2,JNK1/2and p38MAPK whichparticipate in the regulation of inflammation.At the resting state, NF-κB and IκBcould group to the trimers heterologous present in the cytoplasm to the non-activation,and then transferred to the nucleus,start gene transcription and translation of variousinflammatory mediators play to the level of transcription regulation role. Therefore, ascontrol inflammation blocking target, MAPK and NF-κB is considered an importantpart of the anti-inflammatory mechanism. This experiment based on the discussion ofthe RAW264.7macrophages, used of LPS stimulation, to establish in vitro model ofinflammation. The results shown that RAW264.7macrophages were treated by LPSof1mg·L-1after18hours,the phosphorylatory level of ERK1/2is extremelysignificant elevated; different concentrations of Xiang-Qi-Tang that pretreatedmacrophages by3hours Could significantly inhibit the expression of p-ERK1/2at1mg·L-1and5mg·L-1（P＜0.01）, point out that Xiang-Qi-Tang could achieve its anti-inflammatory effects by inhibiting the phosphorylation level of ERK1/2.Tofurther determine the specific anti-inflammatory mechanism of Xiang-Qi-Tang,westudied its active components. The results shown that Xiang-Qi-Tang and its activeingredient which pretreated macrophages3hours and incubated with LPS for18hours were able to significantly reduce the phosphorylation levels of ERK12, JNK1/2and p38MAPK（P＜0.01）, compared with LPS controlgroup.Meanwhile,Xiang-Qi-Tang and its active ingredient could extremely elevatedthe protein expression levels of p65and IκB-α and significantly reduced theexpression levels of p-p65and p-IκB-α in NF-κB signaling pathway. Theexperimental results show that Xiang-Qi-Tang of Chinese herbal formula might play arole of anti-inflammatory effect by blocking the activation of intracellulary MAPKsignaling pathway and NF-κB signaling pathwa.COX-2has a special role in arachidonic acid cleavage of an important class ofprostaglandins in the rate-limiting enzyme in the inflammation and pain. Theexperiment set up the inflammation model using LPS stimulate macrophages in vitroto research the impact of Xiang-Qi-Tang on the COX-2signaling pathway inmacrophages.The results show that Xiang-Qi-Tang and its active components couldsignificantly inhibite the secretory level of LPS-induced macrophage PGE2（P＜0.01）,the expression level of COX-2was significantly increased after LPS of1mg·L-1treated RAW264.7macrophages18hours,Xiang-Qi-Tang of1mg·L-1and5mg·L-1that pretreated macrophages for3hours could significantly inhibit the expression ofCOX-2（P＜0.01）,which reveals that Xiang-Qi-Tang can express its anti-inflammatoryeffects by inhibiting the expression of COX-2protein,at the same time,Xiang-Qi-Tang and its active ingredient that pretreated macrophages for3hours andthen co-incubated with LPS for18hours could significantly reduce the expressionlevel of COX-2as well（P＜0.01）.The experimental results show that Xiang-Qi-Tangof Chinese herbal formula can inhibit the activation of COX-2signaling pathways inmacrophages.In summary, we studied the anti-inflammatory mechanism of Xiang-Qi-Tangthrough the establishment of inflammation model of RAW264.7macrophages inducedby LPS via using ELISA and Western blot respectively in vitro. Results show thatXiang-Qi-Tang could achieve its anti-inflammatory effects by inhibiting the expression of pro-inflammatory cytokines and increasing the secretory level ofanti-inflammatory cytokines,which were regulated by MAPK signaling pathway andNF-κB signaling pathway.Furthermore, Xiang-Qi-Tang could restrain the activation ofMAPK and NF-κB signaling pathway.Meanwhile, Xiang-Qi-Tang could play ananti-inflammatory effect through inhibiting the activation of intracellular COX-2signaling pathway which is involved in the reduction of PEG2, sequentially interferewith the metabolism of arachidonic acid. |
PDF Full Text Request