The Enhancement Of Immune Function And Regulation Of JNK/NF-κB Signal Pathway Of Spleen Lymphocyte By Resveratrol-Treatment In Immunosuppressive Mice Induced By Cyclophosphamide

As a kind of wide variety of sources of natural polyphenol,Resveratrol(Res)exhibits antitumor and antioxidant activities with important physiological functions as well as biological activities.Researches on alteration of white blood cells,peritoneal macrophages and lymphocytes after treatment by Res have been used for investigating the immunity enhancement of Res in recent years.A lot of immunological studies are complicated networks involving many pathways,which are relative with immunity.Recent studies showed that Res could play different roles in cell lysis and treatment of inflammation through regulating Nuclear factor-kappa B(NF-κB)and Stress-activated protein kinase/c-Jun N-terminal kinase(SAPK/JNK)signal pathways in different tissues or organs,but the mechanism of Res in immune regulatory is still unknown.This research,based on immunosuppressed mice model induced by CY,is to study the effects of Res on immune function in mice and the underlying molecular mechanisms through evaluating the approximately horizontal(organ index),cellular level(proliferation of splenic cells,T-cell subpopulations and peritoneal macrophage phagocytosis),molecular level(cytokines)and signal pathway level(JNK and NF-κB signal pathway).The details show as following:1.The effects of Res on immune function in immunosuppressed mice.The establishrment of cyclophosphanide(CY)induced immunosuppressive mice model and groups with administration:Young adult male and female mice totaled 120 were divided into six groups and each group included 20 mice.The experimental groups were given 200μL CY at a dosage of 60mg/kg bw via intraperitoneal injection,once a day for 3 days,the normal control group received 200μL of physiological saline.After CY-treatment for 3 days,the mice in the resveratrol-treated groups(CY+H,CY+M and CY+ L)were treated with resveratrol at dosages of 60,30,and 15 mg/kg bw respectively in the positive group with levamisole at a dosage of 30 mg/kg bw in the normal control group and Modle(CY)group with physiological saline.The drugs were given by gavage(200μL),once daily for 7days.The weight of the mice were measured every day.The spleen,thymus and liver index were counted after the spleen,thymus and liver were weight.The splenocyte proliferation was determined by Methyl thiazolyl tetrazolium(MTT).T lymphocyte subsets were examined by flow cytometry.The phagocytic index,K,was calculated by the equation with the macrophage phagocytosis assay.The concentrations of IL-1α/β、IL-2、TNF-α and NF-κB were analyzed using commercially available ELISA kits.Results:①The spleen index was significantly increased in the resveratrol-treated groups(15,30 and 60 mg/kg bw)when compared with the CY-treated group(P<0.05),the treatment of 30 mg/kg group is the best.No significant differences were observed in thymus and liver index between the resveratrol-treated groups and CY-treated group although the index increased(P>0.05).②Lymphocyte proliferation in the normal control group,resveratrol-treated groups(15,30 and 60 mg/kg bw)and levamisole-treated group were significantly higher than that in the CY-treated group(P<0.05).③Compared with the CY group,treatament with levamisole and resveratrol(15,30 and 60 mg/kg bw)significantly increased the percentage of CD4+(P<0.05).The percentages of CD3+ and CD4+/CD8+ gradually increased by resveratrol-treatment,but there were no significant differences between the resveratrol-treated groups and the CY-treated group(P>0.05).The phagocytic index K of the mice treated with levamisole and resveratrol(15,30 and 60 mg/kg bw)were significantly higher compared with the CY-treated group(P<0.05),however,phagocytic percentage was significantly increased in the resveratrol-treated group(15mg/kg bw)only when compared with the CY group(P<0.05).④The phagocytic index K of the mice treated with levamisole and resveratrol(15,30 and 60 mg/kg bw)were significantly higher compared with the CY-treated group(P<0.05),however,phagocytic percentage was significantly increased in the resveratrol-treated group(15mg/kg bw)only when compared with the CY group(P<0.05),and was decreased with increasing dosage ⑤The IL-1α/β、IL-2、TNF-α and NF-κB concentrations in the resveratrol-treated groups were signifcantly lower than those in the CY-treated group(P<0.05).All these results showed that Res could upregulate many immune indices and showed significant stimulating effects on spleen.2.The effects of resveratrol on NF-κB signal pathway in immunosuppressed mice:In order to investigate the mechanism of regualation of Res with NF-kB signal pathway,Western blot analysis was uesed to detect the expressions of NF-κB in nucleus and IKK、IκB in cytoplasm.The expressions of NF-κB in spleen were detected by Immunohistochemistry.Results:① We found that resveratrol at doses of 15 mg/kg,30 mg/kg and 60 mg/kg could significantly up-regulate the relative densities of NF-kB and the nucleus and IKK in the cytoplasm compared with the CY group(P<0.05).IκB protein level in the cytoplasm was increased after CY treatment,but it showed no significant difference(P>0.05).② NF-κB and c-jun were mainly expressed in the nucleus.when compared to the CY group,NF-κB nuclear expression was positive in the resveratrol-treated groups(15,30 and 60 mg/kg).All results showed thay Res could effectively regulate the NF-κB signal pathway to regulate nuclear transcriptional and IL-2 cytokine secretion.3.The effects of resveratrol on JNK signal pathway in immunosuppressed mice:In order to investigate the mechanism of regualation of Res with upstream pathway of NF-κB,Western blot analysis was uesed to detect the expressions of c-jun in nucleus and SAPK/JNK in cytoplasm.The expressions of c-jun in spleen were detected by Immunohistochemistry.Results:① We found that resveratrol at doses of 15 mg/kg,30 mg/kg and 60 mg/kg could significantly up-regulate the relative densities c-jun in the nucleus and SAPK/JNK in the cytoplasm compared with the CY group(P<0.05)..②-jun were mainly expressed in the nucleus,when compared to the CY group,c-jun nuclear expression was positive in the 60mg/kg resveratrol-treated group(P<0.05),other resveratrol-treated groups showed an increased level with no significant difference(P>0.05).The immunohistochemistry results were consistent with the western blot results.The results indicated that Res could effectively regulate the JNK pathway and activate JNK/NF-κB pathway.In conclusion,Res can regulate the immune functiong in immunosuppressive mice by effecting immune organ index,spleen lymphocyte proliferation,T-cell subpopulations,peritoneal macrophage phagocytosis and serum cytokine with a great effect on the immune function of spleen.And on this basis we are supposed to evaluate the effects of Res on NF-κB and JNK pathway by Western blot analysis and Immunohistochemistry.The promising immunomodulatory mechanism of resveratrol in immunosuppressive mice is associated with activating JNK/NF-κB pathway to activation IKK.