Immunoregulatory Effect And Mechanism Of Punicic Acid

Punicic acid(C18:3,PA)is a unique Omega-5 long-chain polyunsaturated fatty acid,concentrated mainly in pomegranate seed oil,with a content of 56% ～ 83%.Studies have shown that PA has physiological activities such as anti-inflammatory,antioxidant,anti-cancer,modulating lipid metabolism and anti-diabetic,however,the immunomodulatory effects of PA and its mechanism are still unclear.This study firstly enriched PA from pomegranate seed oil and elucidated its immunomodulatory activity and mechanism at the cellular level in vitro.Then we further evaluated the effect of PA on immune function of immunocompromised mice at animal level,and also analyzed its effect on intestinal flora composition and lipid metabolism in immunocompromised mice,and revealed the possible mechanism of immunomodulatory effect of PA through improving the composition of intestinal flora and regulating lipid metabolism.Firstly,PA in pomegranate seed oil was extracted by cold pressing method and enriched by molecular distillation,the results of gas chromatography showed that the content of PA increased from 59.28% to 87.45% after enrichment.Next,the in vitro immunomodulatory activity of PA was investigated using mouse mononuclear macrophages RAW264.7 as the study subject and Lipopolysaccharide(LPS)as a positive control.The effects of PA on the cell proliferation,morphology,phagocytosis,enzyme activity and cytokines of RAW264.7 cells were investigated,while the expression of proteins related to immune regulatory signaling pathways in the cells was detected by Western-blot.The results showed that low concentrations of PA promoted the proliferation of RAW264.7 cells,and the cell morphology was changed and phagocytosis was enhanced after incubation with PA,while the secretion of cytokines and cell enzyme activity were increased;and the Western-blot results indicated that PA activated NF-κB and MAPK signaling pathways in the cells.In vitro cellular experiments suggest that PA may enhance the immune activity of cells by activating NF-κB and MAPK pathways.Secondly,the in vivo immunomodulatory activity of PA was investigated using cyclophosphamide(CTX)-induced immunocompromised mouse model with levamisole hydrochloride(LH)as a positive control.The experiments mainly assessed the changes of body weight,immune organ index,histological morphology of spleen and thymus,proliferation capacity of splenic lymphocytes and serum levels of immunoglobulins and cytokines in mice,while the changes of NF-κB signaling pathway-related protein expression in spleen were detected by Western-blot.The results showed that after intraperitoneal injection of CTX,the mice showed a significant decrease in body weight,atrophy of immune organs and disorders of various immune indexes.After gavage of PA,all immune indexes were gradually restored,mainly manifested as weight gain,increase of immune organ index,improvement of spleen and thymus tissue morphology as observed by H&E staining,enhancement of splenic lymphocyte proliferation capacity,increase of immunoglobulin and cytokine content in serum,and Western-blot results showed that PA ameliorated the inhibition of NF-κB signaling pathway by CTX.In vivo animal experiments suggest that PA may exert immunomodulatory effects in mice by regulating the NF-κB signaling pathway.Finally,16 S r DNA sequencing technology and LC-MS/MS analysis were used to detect the intestinal flora and serum lipid composition of mice,and the association between the two was analyzed by Spearman statistical analysis method.The results showed that PA ameliorated the intestinal flora disorder caused by CTX and improved the community structure of the intestinal flora,with increased levels of probiotic bacteria(Bacteroidetes,Lactobacillaceae and Bacteroidaceae)and decreased levels of pathogenic bacteria(Helicobacteraceae,Helicobacter).Meanwhile,PA improved the serum lipid composition,with a decrease in Triglyceride(TG)content and an increase in the ratio of Phosphatidylcholine(PC)and Phosphatidylethanolamine(PE).The association analysis showed that the regulation of lipid disorders was associated with changes in the structure of the intestinal flora.In summary,PA has good immunomodulatory activity both in vivo and in vitro.This study provides a theoretical basis for its use as an immunomodulatory adjuvant or for functional product development,and also provides new ideas for the exploitation of pomegranate seeds.