The Anti-inflammatory Effects And Mechanism Of Parthenolide

[Objectives]1)To Test the anti-inflammatory effects of parthenolide on mouse macrophageRAW264.7 through measuring inflammatory related genes and cytokines,expression levels.2)To study the anti-inflammatory mechanism of parthenolide by testing the changes of protein phosphorylation level on NF-κB and MAPKs signal pathways in RAW264.7.3)To build the periodontitis model and periodontal regeneration model by ligature and surgical procedure for the further study about the anti-inflammatory effects of parthenolide.[Materials and Methods]1)The anti=inflammatory study of parthenolide monomer in vitroMouse macrophage RAW264.7 was used and three groups were included in the study.They were DMSO + LPS control group,Parthenolide lOμM + LPS group and BAY11-7082(NF-kB inhibitor)lOμM + LPS positive control group.Cells were treated with DMSO,parthenolide or Bay11-7082 for 1 hr prior to E.coli LPS stimulation.After LPS challenged for 4 hrs and 8 hrs,cells were collected and total mRNA were extracted.mRNA expression levels of IL-6,TNF-a,IL-1β,IL-10,Ptgs2,MMP13 and NOS2 were detected by qRT-PCR.After LPS challenged for 24 hrs and 48 hrs,supernatant were collected and cytokine expression levels of IL-6 and TNF-a were detected by ELISA.After LPS challenged for 0,15,30,60and 120 min,cells were collected and total protein was extracted.The protein phosphorylation level of Phospho-p65,Phospho-p38,Phospho-ERK,Phospho-JNK were detected by Western blot.2)The anti-inflammatory study of the fiber composite membrane loading parthenolide in vitroRAW 264.7 were used and three groups were included in the study.They were DMSO + LPS control group,Blank Membrane + LPS group and ParthenolideMembrane + LPS group.RAW 264.7 cells were treated with DMSO,blank membrane or parthenolide membrane for 7 days prior to E.coli LPS stimulation.After LPS challenged for 4 hrs and 8 hrs,cells were collected and total mRNA were extracted.mRNA expression levels of IL-6,TNF-a,IL-1β,IL-10,Ptgs2,MMP13 and NOS2 were detected by qRT-PCR.After LPS challenged for 24 hrs and 48 hrs,supernatant were collected and cytokine expression levels of IL-6 and TNF-a were detected by ELISA.After LPS challenged for 15,30,60 and 120 min,cells were collected and total protein was extracted.The protein phosphorylation level of Phospho-p65,Phospho-p38,Phospho-ERK,Phospho-JNK were detected by Western blot.3)The establishment and evaluation of rat experimental periodontitis model and periodontal regeneration modelSD rats were anesthetized with 10%chloral hydrate.The ligature was placed around the first molar on the right mandibular.One week later,the alveolar bone around the buccal root of the first and second molars was abraded by surgical procedure with size 3*2*1 mm3(length*width*depth).The exposed tooth roots were carefully denuded of periodontal ligament,cementum,and superficial dentin.Two weeks later,the rats were sacrificed and mandibular samples were collected.The experimental periodontitis model and periodontal regeneration model were evaluated by Micro-CT.[Results]1)Compared with DMSO + LPS control group,mRNA expression levels of IL-6,TNF-a,IL-1β,IL-10,Ptgs2,MMP13 and NOS2 in parthenolide lOμm + LPS group were significantly decreased at two different time points(P<0.01);cytokine expression levels of IL-6 and TNF-a were significantly decreased at two different time points(P<0.01);protein phosphorylation level of Phospho-p65,Phospho-p38,Phospho-ERK and Phospho-JNK were significantly decreased at different time points(P<0.05);2)Compared with DMSO + LPS control group and blank membrane + LPS group,mRNA expression levels of IL-6,TNF-a,IL-1μm,1L-10,Ptgs2,MMP13 and NOS2 in parthenolide membrane + LPS group were significantly decreased at two different time points(P<0.01);cytokine expression level of TNF-α was significantly decreased at two different time points(P<0.01);protein phosphorylation level of Phospho-p65,Phospho-ERK,Phospho-JNK were significantly decreased at different time points(P<0.05);however,the phosphorylation level of Phospho-p38 was increased at 15,30,60 and 120min after LPS challenged.3)Micro-CT scan showed that the mandibular first molar buccal root of rat was exposed to 1/3.There was a size 3*2*1(length*width*depth)mm3 bone defect between the root of first and second molar.[Conclusions]1)The anti-inflammatory mechanism of parthenolide and parthenolide membrane might be:a)inhibiting the mRNA expression of IL-6,TNF-α and IL-1β;b)inhibiting the mRNA expression of IL-10;c)downregulating Ptgs2 expression to reduce PGE2 production;d)reducing the mRNA expression of MMP13;e)inhibiting the mRNA expression of NOS to reduce NO secretion.2)The phosphorylation levels of proteins in NF-κB and MAPKs signal pathways could be downregulated by parthenolide.The blockade of these two signal pathways demonstrated the anti-inflammatory effect of parthenolide.3)The phosphorylation level of protein in NF-κB signal pathway could be downregulated by fiber composite membrane loading parthenolide.Some protein phosphorylation levels in MAPKs signal pathway could be reduced by fiber composite membrane loading parthenolide.4)The rat experimental periodontitis model and the periodontal regeneration model were successfully built.