Research For The Immunoregulatory Role And The Mechanism Of Lentinan In Burned Sepsis

Patients with burn sepsis have high frequency admitted to intensive care units(ICU) and has a high mortality. The host immune system has been proposed to play significant effect on the pathogenesis of burn sepsis, which include the homeostasis of the helper T cell, activation of T lymphocytes, CD4 + CD25 + regulatory T cell dysfunction and changes of cytokine profile. Excessive activated macrophages also plays an important role in sepsis and other related inflammatory diseases. Lentinan, extracted from mushrooms, has been served as immune-regulating and anti-cancer drugs. Studies have shown that lentinan can also strengthen the innate and acquired immune responses to enhance the anti-infection role of the host. Macrophages and T lymphocytes are the major effector cells of lentinan. However, the therapeutic effect and mechanism of lentinan has not been fully elucidated. In the present study, with burn sepsis mice model, we aim to investigate the therapeutic effect of lentinan on burn sepsis, including the expression of inflammatory factors, the function alterations of CD4+CD25+ regulatory T cell, macrophage macrophage cell proliferation and apoptosis, endocytosis and related pathways, which may clarify the effect of lentinan on the immune system and its regulation mechanism on the development of burn sepsis, thus providing more molecular basis for the treatment of burn sepsis.The main contents of this article are as follows: Part one Research for the Role of Lentinan in the survival rate andinflammatory factor in burned septic miceObjective: To establish a mouse model of burned sepsis, observe the mice’s change of biological condition, the liver in morphopathology, the survival rate of 5-day, the levels of IL-1、IL-10 and IFN-γ in the serum.Methods: 128 male mice were divided into model group, low(40mg/kg), medium(100 mg/kg) and high(250 mg/kg)concentrations of lentinan treatment group. Another 60 mice according to the drug concentration of Lentinan used were treated similarly as above and were randomly divided into four groups, which were to be slaughtered for observing the natural mortality. After the mice were anesthetized by pentobarbital sodium, scrape the fur on the back of the mouse, the bare skin of the mice were placed in boiling water to cause skin burns and formation of circular burn area with back diameter of about 1cm. The Pseudomonas aeruginosa(1×10~6 CFU/ml) was injected at the skin site. Model groups were injected with 40 ml/kg physiological saline, while the low, medium and high concentrations of lentinan treatment group were peritoneally injected into the mice burns with 40, 100 and 250 mg/kg lentinan. The mice were sacrificed, blood and liver samples were collected after the injection of lentinan for 1, 2, 3, 4 days. HE staining was performed to detect the liver pathological changes, And the expression levels of IL-1, IL-10 and IFN-γ in the peripheral blood was detected with ELLSA assay. Measurement data were presented as mean±SD.Statistical analysis was performed by the Student’s t test between two groups. Multiple groups were analyzed by ANOVA. numeration data were performed by chi square test. Survival analysis were performed by Kaplan-Meier analysis.A signifieant difference was presume data P value of <0.05.Data were analyzed using the statistical program SPSS version17.0.Results: After infection, mice were died witnin 24 h, and the survival rate was 10, the low, medium and high concentrations groups were died at 30,42,48 hours, and the survival rates were 33.33%, 60%, 86.67%, which was dose dependent. In the control group, the mice liver lobule with sepsis was changed; the liver cell were dissociated, large areas of cloudy swelling of liver cells can be seen with vacuolar degeneration, cell structural damage; periportal inflammatory cell infiltration, accumulation of red blood cells. After lentinan treatment, lobular structure of septic mice were become normal; some liver cell degeneration and necrosis accompanied by a small amount of inflammatory cell infiltration, andt the high concentration(250 mg/kg) had minimal damage of mice liver cells, suggesting its treatment effect on the septic mice.(3) The peripheral blood levels of IL-1 was comparatively higher in the lentinan low concentration group than the level of model group, however the difference was not statistically significant; the lentinan medium and high concentration group had comparatively higher expression of IL-1 than the level in model group, which had a statistically significant difference. The level of IL-10 in low, medium and high concentrations of lentinan group was lower than the model group at various time points; The level of IFN-γ in low concentrations of lentinan group was higher than the model group, buth with no statistical difference, The level of IFN-γ in medium and high concentration of lentinan group were higher than the model with statistically significant difference. Part two Investigation for the effects of Lentinan to CD4+T cell inburned septic miceObjective: To investigate the role of Lentinan to CD4+T cells and CD4+CD25+regulatory T cells,which in ERK1/2/Fox O1 signaling pathway stimulated by LPS.Methods: In vitro T cell experiments: An assay kit was used to isolate CD4+T cells and CD4+CD25+ regulatory T cells from mouse peripheral blood. Flow cytometry was performed to detect the proportions of CD4+ CD25+Fox P3+T cells and CD4+CD25+T cells; ELISA assay detected the level of IL-10 was by in serum of CD4+CD25+ regulatory T cells and the level of IL-2, IFN-γ and IL-4 in serum of CD4+T cells isolated from septic group and Lentinan interferenced groups. MLR assays were carried out by coculturing CD4+ T cells(isolated from sham mice) and CD4+ CD25+ Tregs(derived from mice with septic group on PBD 2, 3, and 4) at a ratio of 1:10 for 72 h with CD3 antibody. CD4+T cell proliferation and IL-4 and IFN-γ levels were determined by MTT or ELISA. OD values were determined at a wavelength of 540 nm.CD4+CD25+ regulatory T cells isolated from mice with burns plus P. aeruginosa infection on PBD 3 were treated as indicated. After 24 h, total RNA was extracted and subjected to q PCR analysis for IL-10 m RNA expression. CD4+CD25+ regulatory T cells obtained as in were incubated with lentinan(250 ng/ml) for 12h; then, LPS(1 mg/ml) were added to the cultures at the indicated time. Total and phospho-ERK1/2 were determined by Western blot, whereas GAPDH served as a loading control. Luciferase assay detected the effects of lentinan on Fox O1 activation induced by LPS(1μg/ml). Experiments were repeated at least thrice independently. Measurement data were presented as mean±SD.Statistical analysis was performed by the Student’s t test between two groups. Multiple groups were analyzed by ANOVA. numeration data were performed by chi square test. A signifieant difference was presume data P value of <0.05.Data were analyzed using the statistical program SPSS version17.0.Results: After scalding and injecting of Pseudomonas aeruginosa, the percentage of CD4+CD25+Fox P3+T cell in CD4+CD25+T cells was significantly increased. Low, medium and high doses of lentinan all reduced the lower the percentage of Fox P3+ cells. Meanwhile lentinan significantly suppressed CD4+CD25+ T cells secretion of IL 10, which was dose-dependent of lentinan.(2) ELISA assay showed that CD4+T cells secreted increased level of IL-2, IFN-γ and decreased level of IL-4. MTT assay showed that lentinan increased the viability of CD4+T cell with a statistically significant difference.(3) In vitro mixed lymphocyte reaction showed that lentinan played a suppressive role on CD3 antibody and IL-10 antibody induced CD4+T cell proliferation, meanwhile lentinan can decrease Th2 secreted IL-4 and elevated Th1 cells secrete IFN- γ.(4) On the third day of the treatment of burn sepsis model, the results showed that LPS significantly induced the level of IL-10 in the CD4+CD25+ regulatory T cells, while co-treatment with LPS and lentinan significantly reduced the level of IL-10, which was dose-dependent. Further studies showed that lentinan had a synergistic effect with p38 inhibitors, while lentinan had no effet on the expression of IL-10 when ERK inhibitor was added. The resulted suggested that lentinan regulated the expression of IL-10 is dependent on ERK pathway, but not depend on p38 pathway. Further study found that lentinan inhibited LPS-induced ERK1/2 phosphorylation, and it also suppressed Fox O1 phosphorylation levels and transcriptional activity. Part three Study for the actions of Lentinan to macrophagesRAW264.7 induced by LPSObjective: To discuss the effects of Lentinan in the activity, apoptosis, pinocytosis, NO and TNF-α secretion of RAW264.7 cells, as well as the phenotypic change of MHC.Methods: In vitro macrophages experiments: were seeded with 1×105 cells/well in the 96-well plate, three groups were divided: PBS, 10 μg/m L LPS and 10 μg/m L LPS plus 100 ng/ml Lentinan. MTT assay was performed to detect cell viability after stimulated by LPS 24 h,48h and 72 h in all groups, The rate of cell apoptosis and the expression of MHC I and MHC II was analyzed by flow cytometry. Cell apoptosis-related protein such as caspase 3, Bax and Bcl-2 in all groups was detected by Western blot. The amount of NO and TNF-α production was measured by Griess reaction assay and ELISA assay.Neutral red dye assay was performed to evaluate RAW264.7 cells endocytic activity. Statistical analysis was as same as part two.Results: MTT assay showed that lentinan significantly increased the viability of macrophages; Flow cytometry showed that lentinan signifycantly reduced the cells apoptosis rate, western blot showed that lentinan reduced the pro-apoptotic protein(Caspase3 and Bax) expression, and promote the expression of antiapoptotic proteins(Bcl-2); Furthermore, we showed that the level of NO and inflammatory cytokines TNF-α were significantly reduced by lentinan; neutral red experimental results showed that lentinan can significantly promote RAW264.7 cell endocytosis activity with a dose-dependent manner. Flow cytometry results showed that lentinan significantly upregulated RAW264.7 cell surface MHC II molecules, whereas it had no significant effect on the expression of MHC class I molecules. Part four Investigation for the mechanism of Lentinan to macrophagesRAW264.7 induced by LPSObjective: To observe whether the mechanism of effects of Lentinan to macrophages induced by LPS through inhibiting the ERK/NF-κB signaling pathway.Methods: RT-PCR and ELISA were performed to detect the m RNA and protein levels of TNF-α, RT-PCR and western blot assays were performed to determine i NOS m RNA and protein levels, western blot assay was performed to determine the expression of p ERK1/2, p JNK1/2 and nucleus NF-κB p65, and the promoter luciferase reporter gene assay was also performed for determination of NF-κB transcriptional activity.Results: We detected the upstream pathways of NO and TNF-α, which showed that lentinan significantly reduced LPS-induced levels of i NOS m RNA and protein; NF-κB promoter reporter experiments showed that lentinan can inhibit LPS-induced luciferase expression level; Compared with LPS group, the level of phosphorylated ERK1/2, JNK1/2 and NF-κB p65 were obviously lower with cells co-treated with lentinan and LPS.Conclusion:1. Injection of Lentinan into septic mice significantly enhanced the expression of pro-inflammatory cytokines IL-1 and IFN-γ, while reducing the expression of anti-inflammatory cytokine IL-10, which resulted partial recovery the immune system and reduced immnunoparalysis, enhanced immune cell function and reduced liver damage, leading to increase of the survival rate of the septic mice.2. Using CD4+CD25+Fox P3+ regulatory T cells, we confirmed that lentinan inhibited ERK-Fox O1 pathway, leading to reduced expression of IL-10, thus promoting the CD4+ T cell proliferation and Th1 cell polarization. Meanwhile, lentinan can inhibit the level of Th2-type cytokines IL-4 and enhance the level of Th1-type cytokines IL-2 and IFN-γ production, thereby enhancing the ability of microbial clearance and improving the symptoms of septic mice.3. Studies have shown that Th1 cells secrete IL-2 and IFN-γ and other Th1-type cytokines, which can mediate activation of macrophage. Our experiments further found that lentinan can enhance cell viability by reducing apoptosis in macrophages. Lentinan can also reduce the level of NO and TNF-α, promote macrophage endocytosis ability, increase the expression of MHC II, thereby increasing antigen presentation capabilities to enhance the immunity, leading to reduced symptoms of sepsis mice.4. To investigate the upstream pathways of NO and TNF-α, we found that lentinan can inhibit the expression of i NOS, the phosphorylation of ERK1/2 and JNK1/2, the transcriptional activity and nucleus expression of NF-κB p65. In summary, our study demonstrated that lentinan can suppress ERK-Fox O1 pathway of CD4+CD25+Fox P3+ regulatory T cells to promote Th1 cell polarization from CD4+ T cells; Meanwhile, lentinan can inhibit ERK1/2, JNK1/2, NF-κB pathways of macrophages to promote macrophage activation, thus enhancing immune cell function and improve the symptoms of septic mice.