Investigation On Anti-inflammatory Activatities And Action Mechanism Of Terpenoids From Alisma Orientale And Radix Paeoniae Alba,and Novel Macrolactams

Inflammation is a complex defense reaction of body response to external stimulus and injury.On the other hand,the excessive inflammation will cause more severe tissue injury and induce a variety of inflammatory diseases,therefor affecting our health.In recent years,more and more studies show that natural products are the rich resource to discover new anti-inflammatory drugs with special molecular mechanisms.Some anti-inflammatory terpenoids including Alisol F and 25-anhydroalisol from Alisma orientale and paeonidanin(PD),albiflorin(AF),paeoniforin(PF),4-O-methylpaeonifl-orin(MPF),benzoylalbiflorin(BAF),galloylalbiflorin(GAF),debenzoylalbiflorin(DAF),4-O-methylbenzoylpaeoniflorin(MBPF)and paeonidanin A(PDA)from Radix Paeoniae Alba were isolated in our previous work.In the present research,we decipHered the anti-inflammatory activity and mechanism of these terpenoids in LPS-induced RAW 264.7 cells and LPS/D-gal-induced liver injury in mice.The effects of compounds on expression of inflammatory mediators and cytokines,as well as the activation of MAPK,NF-?B,JAK/STAT and PI3K/Akt signal pathway were examined.At the same time,we also investigate the effect and action machnism of five new macrolactams from the Streptomyces sp.(T101)derivertived from Vicugna pacos feces on antiinflammation.Part IThe anti-inflammatory activity and anti-inflammatory mechanism of alisol F and 25-anhydroalisol F were investigated in LPS-induced RAW 264.7 cells.The results demonstrated that alisol F and 25-anhydroalisol F suppressed the LPS-induced RAW 2647 cells production of nitric oxide(NO),interleukin-6(IL-6),tumor necrosis factor alpHa(TNF-?)and interleukin-1?(IL-1?),as well as inhibited the mRNA levels of TNF-?,IL-1? and IL-6.In addition,alisol F and 25-anhydroalisol F inhibited mRNA and protein expression of iNOS and COX-2,and attenuated the phosphorylation of ERK,JNK,p38 and stat3.Western blot results showed that alisol F and 25-anhydroalisol F significantly inhibited LPS-induced the activation of p-p65 and p-IK?B-?,IKB-? degradation,increased the expression of p65 in cytoplasm and reduced its expression in nucleus.Immunofluorescence results showed that alisol F and 25-anhydroalisol F inhibited the translocation of p65.In agreement with these in vitro observations,we further found that alisol F obviously improved liver pathological injury by inhibiting the production of TNF-?,IL-1? and IL-6,and significantly decreased the serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels in LPS/D-gal-induced mice.In addition,pHospHorylations of ERK,JNK and attenuation of total IKB-a were further confirmed to be reduced in alisol F-treated liver tissues.Thus,these in vivo results clearly confirmed the in vitro data suggesting that alisol F protect liver by inhibiting the inflammatory processes.In summary,alisol F and 25-anhydroalisol F induced the expression of various inflammatory mediators via inhibited the activation of MAPK,NF-?B and JAK/STAT signaling pathways.Part IIThe anti-inflammatory activity,structure-function relationship and anti-inflammatory mechanism of nine monoterpenoids were explored in LPS-induced RAW 264.7 cells.The results demonstrated that the most of monoterpenoids suppressed the LPS-induced production of NO,TNF-a and IL-6.The anti-inflammatory activities of these monoterpenoids were closely related with their structure characteristics.Paeoniflorins(PF,MPF and MBPF)and paeonidanins(PD and PDA)present stronger anti-inflammatory activities than those albiflorin derivatives(AF,BAF,GAF and DAF).Furthermore.The results RT-qPCR and Weston blot method indicated BMPF could down-regulate the mRNA and protein expression level of iNOS in LPS-stimulated RAW 264.7 cells.MBPF inhibited the expression of p-ERK,p-p38,p-JNK,p-c-Jun,p-p65,p-Akt and p-I?B-? protein,attenuated degradation of IKB-a protein in LPS-induced RAW 264.7 cells,as well as increased the expression of p65 in cytoplasm and induced its expression in nucleus.In summary,this is first report demonstrating that MAPK,NF-?B and PI3K/Akt signaling pathways are involved in mediating the roles of BMPF in suppressing the expression and production of pro-inflammatory cytokines in LPS-induced RAW 264.7 cells.Part IIIThe anti-inflammatory activity and anti-inflammatory mechanism of five new macrolactams were researched in LPS-induced RAW 264.7 cells.The results showed that the compounds had no cytotoxicity,and inhibited the production of NO.In addition,the compounds significantly inhibited production of TNF-?,IL-1? and IL-6 in LPS-induced RAW 264.7 cells,as well as inhibited their mRNA levels.It indicated the 497-1 shown strong anti-inflammatory activity,therefore,497-1 was selected for further mechanism research.The 497-1(5-40 ?M)significantly inhibited the release of inflammatory cytokines such as TNF-?,IL-1? and IL-6 by reducing the expression of mRNA levels.The results indicated that 497-1 not only inhibited the expression of mRNA and protein of iNOS and COX-2,but also lowered expression of p-ERK,p-JNK,p-p38 and p-Akt protein.Meanwhile,497-1 significantly inhibited LPS-induced the activation of p-p65 and p-IKB-?,I?B-? degradation,increased the expression of p65 in cytoplasm and reduced its expression in nucleus,as well as immunofluorescence results showed it inhibited the translocation of p65.All the evidences demonstrating that 497-1 suppressed the expression of pro-inflammatory cytokines via inhibiting the activation of MAPK,NF-?B and PI3K/Akt signaling pathways in LPS-induced RAW 264.7 cells.