Studies On Impact Of Grifola Frondosa Extract On The Cellular Immune Function In Spleen Deficiency Mouse And Its Mechanisms

Purpose:In order to investigate the impact of grifola frondosaextract on the cellular immune function in spleen deficiency mouse and its mechanisms, the spleen deficiency mouse model was established and the extract of grifola frondosa was used to be a natural immunomodulator, to reveal the nature of the occurrence and development of spleen deficiency syndrome, in order to provide a kind of natural and effective immunomodulator for clinical spleen deficiency syndrome.Material and method:The healthy and clean KM mice were randomly divided into six groups, that is, control group, spleen deficiency group, positive drug group (2mg/Kg, lentinan), low-dose of grifola frondosa extract group(5mg/Kg), middle-dose of grifola frondosa extract group (10mg/Kg), high-dose of grifola frondosaextract group (20mg/Kg). The spleen deficiency mice were prepared with Senna-feeding and overstrain method, then after intraperitoneal injection of grifola frondosa extract for ten days, animals were sacrificed on the eleventh day, the spleen and thymus weight were measured to calculate the spleen index and thymus index. Peritoneal macrophages and spleen cells were prepared by conventional methods, and the cells were detected by peritoneal macrophages phagocytosis of neutral red, NO-producing ability (Griess method), and lymphocyte transformation rate, NK cell killing activity(MTT method). Serum?-interferon (IFN-?) and interleukin-4 (IL-4) levels were detected by enzyme-linked immunosorbent assay (ELISA). The other remaining animals in each group were detected CD3~+ T, CD4~+ T, CD8+ T percent by flow cytometry. Four apoptosis-related protein such as Bcl-2, Bax, Fas, FasL were observed by immunohistochemistry in spleen, as well as the mRNA expression of Bcl-2, Bax, Fas, FasL were detected by reverse transcription polymerase chain reaction (RT-PCR).Results:1.Impact of grifola frondosa extract on body weights,thymus index and spleen index of spleen deficiency mice .After fourteen modeling days, the body weight of spleen deficiency group was significantly lower than control group (P <0.01),the thymus index and spleen index of spleen deficiency group were lowered than control group, but the difference was not statistically significant (P> 0.05).While the spleen index decreased more significantly.After given grifola frondosa extract for ten days, the body weights of low,middle and high dose of grifola frondosa extract group were significantly higher than that of spleen deficiency group (P <0.05, P <0.01, P <0.01). While the spleen index of low,middle and high dose of grifola frondosa extract group were significantly higher than that of spleen deficiency group (P <0.05, P <0.05, P<0.01)2. Grifola frondosa extract on mouse spleen cells of peritoneal macrophagesThe peritoneal macrophages phagocytosis of neutral red, NO-producing ability of Spleen deficiency group decreased, but the high dose of grifola frondosa extract could significantly improve the peritoneal macrophages phagocytosis of neutral red(P<0.05), as well as NO-producing ability of Spleen deficiency mice(P<0.01).3. Impact of grifola frondosa extract on spleen lymphocytes of spleen deficiency miceSpleen B lymphocyte proliferation of spleen deficiency group was significantly lower than the control group (P <0.01), high dose of grifola frondosa extract could significantly improve the ability of spleen B lymphocyte proliferation (P<0.01).Spleen T lymphocyte proliferation of spleen deficiency group was lower than the control group, middle and high doses of grifola frondosa extract could increase the T lymphocyte proliferation of spleen deficiency group(P <0.05 and P <0.01); and the T lymphocyte proliferation of high dose grifola frondosa extract group was higher than that of the control group (P <0.05).The NK cell killing activity of spleen deficiency group was lower than the control group significantly (P <0.01), while the middle and high dose of grifola frondosa extract could increase the capability of spleen NK cells significantly (P <0.01).4. Impact of grifola frondosa extract on T subsets in spleen deficiency mice peripheral bloodThe CD4~+ T lymphocyte percentage of spleen deficiency group had no significant difference compared with control group. While the CD8+ T lymphocyte percentage of spleen deficiency group was significantly higher than that of the control group (P<0.05).The high dose of grifola frondosa extract could significantly increase the CD4~+ T lymphocytes percentage in peripheral blood (P<0.05); the percentage of CD8+ T lymphocyte percentage in each group had no significant difference (P> 0.05).The CD4/CD8 ratio of spleen deficiency group was significantly lower than the control group (P <0.01), middle and high dose of grifola frondosa extract could significantly increase the CD4/CD8 ratio of spleen deficiency group (P<0.01).Spleen group the percentage of CD3~+ T lymphocytes was significantly lower than the control group (P<0.01), middle and high dose group grifola frondosa extract can significantly improve the mouse spleen CD3~+ T lymphocyte percentage (P<0.01).The CD3~+T lymphocyte percentage of spleen deficiency group was significantly lower than the control group (P<0.01), middle and high dose of grifola frondosa extract could increase the CD3~+ T lymphocyte percentage of spleen deficiency group significantly (P<0.01).5. Impact of grifola frondosa extract on IFN-?and IL-4 level in spleen deficiency mice serumThe IFN-?level in spleen deficiency mice serum was significantly lower than the control group (P <0.01),and the IL-4 level in spleen deficiency mice serum was significantly higher than the control group (P <0.05), middle and high dose of grifola frondosa extract could significantly increase the IFN-?level in spleen deficiency mice serum (P <0.01), high dose of grifola frondosa extract could decrease the IL-4 level in spleen deficiency mice serum (P<0.01).6. Impact of grifola frondosa extract on the expression of Bcl-2, Bax, Fas, FasL in spleen deficiency miceThe average gray values of the positive product of Bax, Fas, FasL in spleen deficiency mice (mean gray value was inversely proportional to the expression)were significantly lower than that of the control group. The average gray values of the positive product of Bax, Fas, FasL in drug group and three groups of grifola frondosa extract were significantly higher than the control group after given the positive drug and low, middle and high dose of grifola frondosa extract (P <0.01). The average gray values of the positive product of Bcl-2 in spleen deficiency mice was significantly higher than that of the control group. The average gray values of the positive product of Bcl-2 in drug group and three groups of grifola frondosa extract were significantly lower than the control group (P <0.01)after given the positive drug and low, middle and high dose of grifola frondosa extract .7. Impact of grifola frondosa extract on the expression of Bcl-2, Bax, Fas, FasL mRNA in spleen deficiency miceThe expression of Bax, Fas, FasL mRNA in spleen deficiency mice was significantly increased (P <0.05, P <0.01 and P <0.01) compared with the control group, while the expression of Bcl-2 mRNA was significantly decreased (P<0.01).The expression of Bax, Fas, FasL mRNA in positive drug group and three groups of grifola frondosa extract significantly decreased compared with the control group after given the positive drug and low, middle and high dose of grifola frondosa extract (P<0.01). while the expression of Bcl-2 mRNA was significantly increased (P<0.01)Conclusion:1.The body weights of spleen deficiency mice were significantly decreased after modeling by Senna-feeding and overstrain method, and the spleen index also decreased. Grifola frondosa extract could increase the body weights of spleen deficiency mice, and effectively increase the spleen index which decreased due to spleen deficiency.2. Grifola frondosa extract could enhance the phagocytosis of macrophages in spleen deficiency mice to increase the non-specific immune function; could increase the amount of NO production through the NO way to play a biological function of macrophages; grifola frondosa extract could improve the immune function of spleen deficiency mice through the enhancement of spleen B lymphocytes and T lymphocyte proliferation; grifola frondosa extract could also improve the immune function of spleen deficiency mice by improving the cytotoxic activity of NK cells.3.Grifola frondosa extract could improve the decreased CD4/CD8 ratio which caused by spleen deficiency, mainly probably by acting on the Th cells to enhance the cellular immune function of spleen deficiency mice. Grifola frondosa extract could effectively increase the IFN-?level and reduce the IL-4 level of spleen deficiency mice serum to balance the Th1/Th2 subsets, and to help the Th2 cells to transform to Th1 cells , and improve pathology of spleen deficiency.4. Grifola frondosa extract may be through the promotion of Bc1-2 protein and gene expression and inhibiting of Bax, Fas, FasL protein and gene expression, increasing the Bcl-2/Bax ratio, and through the mitochondria as well as death receptor dependent apoptotic pathway to suppress the spleen lymphocyte apoptosis.