Study The Effect Of Peony Seed Oil On Anti-inflammation And Its Molecular Mechanism

Peony seed oil(PSO)is a new food resource and has been rated as a high-quality edible oil by experts.It is a golden yellow transparent liquid oil extracted from peony seeds.Unsaturated fatty acids are the most important active ingredients in PSO and the content of linolenic acid is about 60%.Studies have been shown that PSO has many physiological functions.Recent studies indicate that PSO may have anti-inflammatory biological activity,but its molecular mechanism of anti-inflammatory function has not been reported in the literature.In this study,sodium dextran sulfate(DSS)was used to induce in vivo inflammation model in ICR mice and lipopolysaccharide(LPS)was used to stimulate RAW264.7 macrophages to establish an in vitro inflammation model.The molecular mechanism of the evaluation of anti-inflammatory function of PSO and anti-inflammatory effect was jointly explored.The experimental results provide reference for the deep processing of peony seeds and the development of peony seed oil health food.Experimental observations showed that the PSO intervention group showed a better improvement in mental state than the DSS group,and the number of diarrhea rare and hematochezia mice was decreased,meanwhile,the lesions degree of colorectal was mitigated,and the disease activity index(DAI)score was reduced significantly.Hematoxylineosin(H&E)staining was used to detect the colonic lesions in mice.The microscopic images were analyzed and the lesions in the DSS group included mucosal erosion and glandular recess structural integrity was seriously damaged,meanwhile,a large number of inflammatory cells such as lymphocyte and neutrophil was infiltrated into colorectal tissues,and goblet cells significantly reduced and appeared ulcerative colitis symptoms.The PSO intervention group mice just has fewer colon bleeding and only a small amount of inflammatory cells were infiltrated.Colonic mucosal erosion reduced and recess structure arranged more neatly and the shape is relatively complete.By measuring the tissue damage factor myeloperoxidase(MPO)and malondialdehyde(MDA)in mouse plasma,it was found that the MPO and MDA in the DSS group were 3.5 and 3.2 fold higher than those in the control group,respectively.At the same time,the content of inflammatory mediators nitric oxide(nitrite/nitrate,NO)in the colon tissue was increased by 4.2 fold.Compared with the DSS group,the contents of MPO,MDA and NO in the PSO group were decreased by 20%,26%and 22%,respectively.From the perspective of biochemical indicators,it was found that PSO could reduce DSS-induced mouse injury.Real time-quantitative polymerase chain reaction(RT-qPCR)and Western blotting(WB)assay showed that cyclooxygenase-2(COX-2),tumor necrosis factor-a(TNF-α),interleukin-1β(IL-1β)and inducible nitric oxide synthase(iNOS)were expressed very low in the colonic tissues of control group.Compared with control group,the mRNA expression levels of COX-2,TNF-α,IL-1β and iNOS were significantly increased in the colonic tissues.In the PSO group,the mRNA expression levels of COX-2,TNF-α,IL-1β and iNOS were decreased by 53.6%,41.0%,50.6%and 54.2%,respectively,compared with the DSS group.The WB experiment further confirmed that these inflammatory cytokine protein expression levels have similar results.Phosphorylation of key target genes in mitogen-activated protein kinases(MAPKs)signaling pathway was detected in colon tissues.Phosphorylation of extracellular regulated protein kinases 1/2(p-ERK1/2)and phosphorylation of c-Jun N-terminal kinase in the DSS group(phosphorylation of extracellular regulated protein kinases 1/2,p-ERK1/2)The expression levels of p-JNK and p-p38 protein were 1.48 ± 0.23,1.88±0.24 and 3.21 ± 0.45 fold of the control group.The PSO group was 1.23±0.15,1.45±0.25 and 1.48±0.34 fold of the control group,respectively.The results show that PSO can inhibit the activation of MAPK signaling pathway,which may be related to its inhibition of inflammatory cytokine expression.In vitro,the effect of PSO on RAW264.7 cytotoxicity was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe nyltetrazolium bromide(MTS)method.The results showed that there was no significant difference in OD values between the PSO groups and the control group.It indicated that 0-400μg/mL PSO solution had no effect on the growth,state and viability of RAW264.7 cells,which proved to be non-toxic to cells and could be used in subsequent experimental studies.The mRNA expression levels of COX-2,TNF-α,IL-1β and iNOS inflammatory factors in LPS group were 5.66,7.59,6.78 and 2.62 fold of the control group,while PSO 25 μg/mL,50 μg/mL and 100 μg/mL.The three groups showed a significant dose-dependent decrease compared with the LPS group.Protein expression levels have similar results.The luciferase reporter gene technology was used to detect the transcriptional activity of inflammation-associated nuclear transcription factors in inflammatory cells.Compared with the control group,the relative luciferase activity(RLU)of AP-1/c-Jun(activator protein-1)and NF-KB/p65(nuclear transcription factor-KB)in the LPS model group increased by 1.89 fold and 2.22 fold,the RLU values of AP-1 and NF-κB in the PSO groups decreased in a dose-dependent manner,which were reduced by 32.3%,39.2%,47.1%and 36.9%,43.7%and 55.4%,respectively.The changes of c-Jun and p65 proteins in the cytoplasm and nucleus were as follows:compared with the control group,the expression levels of c-Jun and p65 proteins in the nucleus and cytoplasm of the LPS group were opposite and increased in the nucleus.The expression of c-Jun protein in the nucleus and cytoplasm of PSO group increased gradually compared with LPS group,while the expression of p65 protein decreased significantly in a dose-dependent manner.Therefore,PSO can significantly inhibit the translocation of c-Jun and p65 in the cytoplasmic nucleus of inflammatory cells,and reduce the transcriptional activity of AP-1 and NF-κB.The detection of phosphorylation activation levels of key target genes in the MAPKs pathway in cells showed that the expression levels of p-ERK1/2,p-JNK and p-p38 in PSO group decreased compared with LPS group,indicating that PSO may regulate key targets of MAPKs.Activation of gene phosphorylation inhibits the production of inflammation.LPS-induced RAW264.7 cells were treated with inhibitors of the MAPKs pathway target gene,and the expression of pro-inflammatory factor IL-1β and the phosphorylation activation of key target genes of MAPKs were detected by WB technique.From the results,compared with the LPS-inducing group,the expression level of inflammatory factor IL-1β and the key target genes of MAPKs in p-ERK1/2,p-JNK,and p-p38 were detected by PSO and MAPKs inhibitors.Protein expression levels were significantly reduced.This indicates that in the case where MAPK phosphorylation is blocked,PSO can synergize with inhibitors to inhibit the expression of inflammatory factors and exert anti-inflammatory effects.In summary,in vivo experiments,PSO can effectively improve the macroscopic phenotype of DSS-induced colon inflammation in mice,reduce DAI,alleviate pathological damage of colon tissue,reduce MPO,MDA content in colon tissue and NO content in serum;inhibits the high expression of proinflammatory genes such as COX-2,TNF-α,IL-1β and iNOS in colon tissues;reduces the phosphorylation levels of p38,JNK and ERK1/2 in colon tissues.In vitro,PSO down-regulates LPS-induced overexpression of inflammatory factors COX-2,TNF-α,IL-1β and iNOS in RAW264.7 cells;inhibits inflammation-associated transcription factors such as NF-κB,AP-1)changes in cytoplasmic nucleus and transcriptional activity;reduced phosphorylation of p38,JNK and ERK1/2 in MAPKs.In addition,in the presence of PSO,the addition of MAPKs inhibitors further inhibited the phosphorylation of p38,JNK and ERK1/2 and the expression of the inflammatory factor IL-1β in MAPKs.It suggests that the molecular mechanism of the anti-inflammatory mechanism of PSO is related to the inhibition of the expression of inflammatory factors by inhibiting MAPKs signaling pathways in colon tissues and cells,as well as inhibiting nuclear transcription factors in cells.