灰树花提取物对脾虚小鼠免疫功能的影响及作用机制研究
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摘要
目的:本研究以灰树花提取物作为一种天然免疫调节剂,以脾虚小鼠模型作为观察对象,探讨灰树花提取物对脾虚小鼠的细胞免疫功能的影响,并探讨其作用机制,从现代免疫学角度揭示脾虚证发生、发展的本质,为临床的脾虚证提供一种天然有效的免疫调节剂。
材料与方法:将健康清洁级昆明小鼠随机分为6组,即空白对照组、脾虚组、脾虚+阳性药物组(2mg/Kg,香菇多糖)、脾虚+灰树花提取物低剂量组(5mg/Kg)、脾虚+灰树花提取物中剂量组(10mg/Kg)、脾虚+灰树花提取物高剂量组(20mg/Kg)。采用番泻叶-劳倦过度复合因素造模法制做脾虚小鼠模型,之后腹腔注射灰树花提取物10天,第11天处死动物,完整摘取小鼠脾脏及胸腺,测定其重量,计算脾指数和胸腺指数。常规方法制备小鼠腹腔巨噬细胞和脾细胞,检测小鼠腹腔巨噬细胞吞噬中性红、产生一氧化氮(NO)能力(Griess法),MTT法检测T、B淋巴细胞转化率、自然杀伤细胞(NK细胞)杀伤活性。酶联免疫吸附法(ELISA法)检测小鼠血清IFN-γ和IL-4的含量,流式细胞仪检测各组小鼠外周血CD3~+T、CD4~+T、CD8+T含量。免疫组化法观察脾脏凋亡相关蛋白Bcl-2、Bax、Fas、FasL的表达,并用逆转录聚合酶链反应(RT-PCR)测定脾脏凋亡相关基因Bcl-2、Bax、Fas、FasL mRNA的表达。
结果:
1.灰树花提取物对脾虚小鼠体重和胸腺指数、脾指数的影响
造模14天后脾虚组小鼠体重明显低于空白组(P<0.01),脾虚组小鼠胸腺指数和脾指数较空白组降低,但差异无统计学意义(P>0.05),其中脾指数下降较为明显。
给予灰树花提取物10天后,灰树花提取物低、中、高剂量组小鼠体重明显高于脾虚组(分别为P<0.05,P<0.01,P<0.01)。同时低、中、高剂量组灰树花提取物的脾指数明显高于脾虚组(分别为P<0.05,P<0.05,P<0.01)。
2.灰树花提取物对脾虚小鼠腹腔巨噬细胞的影响
脾虚组小鼠腹腔巨噬细胞吞噬中性红能力和产生NO能力均降低,高剂量灰树花提取物可明显提高小鼠腹腔巨噬细胞吞噬中性红能力(P<0.05)和产生NO能力(P<0.01)。
3.灰树花提取物对脾虚小鼠脾淋巴细胞的影响
脾虚组小鼠脾脏B淋巴细胞增殖能力比空白组明显下降(P<0.01),高剂量灰树花提取物能明显提高小鼠脾脏B淋巴细胞增殖能力(P<0.01)。
脾虚组小鼠脾脏T淋巴细胞增殖能力比空白组降低,中、高剂量的灰树花提取物能明显提高小鼠脾脏T淋巴细胞的增殖能力(P<0.05和P<0.01);并且高剂量灰树花提取物对小鼠脾脏T淋巴细胞增殖能力也强于空白组(P<0.05)。
脾虚组小鼠脾脏NK细胞杀伤能力比空白组明显降低(P<0.01),灰树花提取物中、高剂量组小鼠脾脏NK细胞杀伤能力均明显高于脾虚组(P<0.01)。
4.灰树花提取物对脾虚小鼠外周血T亚群的影响
脾虚组CD4~+ T淋巴细胞百分率与空白组相比未见显著性差异,脾虚组CD8+ T淋巴细胞百分率比空白组明显升高(P<0.05),灰树花提取物高剂量组小鼠外周血CD4 +T淋巴细胞百分率增高有显著性差异(P<0.05);各组CD8+ T淋巴细胞百分率未见显著性差异(P>0.05)。
脾虚组CD4 /CD8比值比空白组明显降低(P<0.01),中、高剂量组灰树花提取物可以明显提高脾虚小鼠的CD4 /CD8比值(P<0.01)。
脾虚组的CD3~+T淋巴细胞百分率比空白组明显降低(P<0.01),中、高剂量组灰树花提取物可以明显提高脾虚小鼠的CD3~+T淋巴细胞百分率(P<0.01)。
5.灰树花提取物对脾虚小鼠血清IFN-γ和IL-4含量的影响
造模后,脾虚组小鼠血清IFN-γ含量比空白组明显降低(P<0.01),血清IL-4含量明显比空白组增高(P<0.05),中、高剂量组灰树花提取物可以明显提高脾虚小鼠血清IFN-γ含量(P<0.01),高剂量组灰树花提取物可以降低血清IL-4含量(P<0.01)。
6.灰树花提取物对脾虚小鼠Bcl-2、Bax、Fas、FasL蛋白表达的影响
脾虚组Bax、Fas、FasL阳性产物平均灰度值(平均灰度值与表达量成反比)明显低于空白组(P<0.01),给予阳性对照组药物和低、中、高剂量的灰树花提取物后,Bax、Fas、FasL阳性产物平均灰度值比脾虚组明显升高(P<0.01)。脾虚组Bcl-2阳性产物平均灰度值明显高于空白组(P<0.01),给予阳性对照组药物和低、中、高剂量的灰树花提取物后,Bcl-2阳性产物平均灰度值比脾虚组明显降低(P<0.01)。
7.灰树花提取物对脾虚小鼠Bcl-2、Bax、Fas、FasLmRNA表达的影响
与空白组比较,脾虚组小鼠脾脏Bax、Fas、FasL mRNA表达显著升高(P﹤0.05,P﹤0.01和P﹤0.01),Bcl-2 mRNA表达显著降低(P﹤0.01);与脾虚组相比较,脾虚小鼠给予阳性对照药物和低、中、高剂量灰树花提取物后, Bax、Fas、FasL mRNA表达显著降低(P﹤0.01),Bcl-2 mRNA表达显著升高(P﹤0.01)。
结论:
1.通过番泻叶-劳倦过度复合因素造模法制做脾虚小鼠模型,造模后脾虚小鼠体重明显下降,脾指数下降,灰树花提取物能够增加脾虚小鼠的体重、有效改善脾虚所致的脾指数下降。
2.灰树花提取物可以通过增强脾虚小鼠腹腔巨噬细胞吞噬中性红的能力;可以通过NO途径,增加NO的产生量来发挥巨噬细胞的生物学功能;灰树花提取物可以通过增强脾脏B淋巴细胞和T淋巴细胞的增殖能力来提高脾虚小鼠的免疫功能;灰树花提取物还可以通过提高NK细胞的杀伤活性来提高脾虚小鼠的免疫功能。
3.灰树花提取物可以改善脾虚时造成的小鼠CD4 /CD8比值降低,可能主要是通过作用于Th细胞来提高细胞免疫功能。灰树花提取物可以有效地升高脾虚小鼠血清中IFN-γ含量,下调IL-4的含量,从而平衡Th1/Th2亚群,使Th2细胞反应向Th1细胞转化,改善脾虚病理改变。
4.灰树花提取物可能通过促进Bc1-2蛋白与基因的表达、抑制Bax、Fas、FasL蛋白与基因的表达、提高Bcl-2/Bax的比值,通过影响线粒体、死亡受体依赖的凋亡途径来抑制脾淋巴细胞凋亡的发生。
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). Serumγ-interferon (IFN-γ) 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 macrophages
The 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 mice
Spleen 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 blood
The 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-γand IL-4 level in spleen deficiency mice serum
The IFN-γlevel 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-γlevel 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 mice
The 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 mice
The 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-γlevel 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.
材料与方法:将健康清洁级昆明小鼠随机分为6组,即空白对照组、脾虚组、脾虚+阳性药物组(2mg/Kg,香菇多糖)、脾虚+灰树花提取物低剂量组(5mg/Kg)、脾虚+灰树花提取物中剂量组(10mg/Kg)、脾虚+灰树花提取物高剂量组(20mg/Kg)。采用番泻叶-劳倦过度复合因素造模法制做脾虚小鼠模型,之后腹腔注射灰树花提取物10天,第11天处死动物,完整摘取小鼠脾脏及胸腺,测定其重量,计算脾指数和胸腺指数。常规方法制备小鼠腹腔巨噬细胞和脾细胞,检测小鼠腹腔巨噬细胞吞噬中性红、产生一氧化氮(NO)能力(Griess法),MTT法检测T、B淋巴细胞转化率、自然杀伤细胞(NK细胞)杀伤活性。酶联免疫吸附法(ELISA法)检测小鼠血清IFN-γ和IL-4的含量,流式细胞仪检测各组小鼠外周血CD3~+T、CD4~+T、CD8+T含量。免疫组化法观察脾脏凋亡相关蛋白Bcl-2、Bax、Fas、FasL的表达,并用逆转录聚合酶链反应(RT-PCR)测定脾脏凋亡相关基因Bcl-2、Bax、Fas、FasL mRNA的表达。
结果:
1.灰树花提取物对脾虚小鼠体重和胸腺指数、脾指数的影响
造模14天后脾虚组小鼠体重明显低于空白组(P<0.01),脾虚组小鼠胸腺指数和脾指数较空白组降低,但差异无统计学意义(P>0.05),其中脾指数下降较为明显。
给予灰树花提取物10天后,灰树花提取物低、中、高剂量组小鼠体重明显高于脾虚组(分别为P<0.05,P<0.01,P<0.01)。同时低、中、高剂量组灰树花提取物的脾指数明显高于脾虚组(分别为P<0.05,P<0.05,P<0.01)。
2.灰树花提取物对脾虚小鼠腹腔巨噬细胞的影响
脾虚组小鼠腹腔巨噬细胞吞噬中性红能力和产生NO能力均降低,高剂量灰树花提取物可明显提高小鼠腹腔巨噬细胞吞噬中性红能力(P<0.05)和产生NO能力(P<0.01)。
3.灰树花提取物对脾虚小鼠脾淋巴细胞的影响
脾虚组小鼠脾脏B淋巴细胞增殖能力比空白组明显下降(P<0.01),高剂量灰树花提取物能明显提高小鼠脾脏B淋巴细胞增殖能力(P<0.01)。
脾虚组小鼠脾脏T淋巴细胞增殖能力比空白组降低,中、高剂量的灰树花提取物能明显提高小鼠脾脏T淋巴细胞的增殖能力(P<0.05和P<0.01);并且高剂量灰树花提取物对小鼠脾脏T淋巴细胞增殖能力也强于空白组(P<0.05)。
脾虚组小鼠脾脏NK细胞杀伤能力比空白组明显降低(P<0.01),灰树花提取物中、高剂量组小鼠脾脏NK细胞杀伤能力均明显高于脾虚组(P<0.01)。
4.灰树花提取物对脾虚小鼠外周血T亚群的影响
脾虚组CD4~+ T淋巴细胞百分率与空白组相比未见显著性差异,脾虚组CD8+ T淋巴细胞百分率比空白组明显升高(P<0.05),灰树花提取物高剂量组小鼠外周血CD4 +T淋巴细胞百分率增高有显著性差异(P<0.05);各组CD8+ T淋巴细胞百分率未见显著性差异(P>0.05)。
脾虚组CD4 /CD8比值比空白组明显降低(P<0.01),中、高剂量组灰树花提取物可以明显提高脾虚小鼠的CD4 /CD8比值(P<0.01)。
脾虚组的CD3~+T淋巴细胞百分率比空白组明显降低(P<0.01),中、高剂量组灰树花提取物可以明显提高脾虚小鼠的CD3~+T淋巴细胞百分率(P<0.01)。
5.灰树花提取物对脾虚小鼠血清IFN-γ和IL-4含量的影响
造模后,脾虚组小鼠血清IFN-γ含量比空白组明显降低(P<0.01),血清IL-4含量明显比空白组增高(P<0.05),中、高剂量组灰树花提取物可以明显提高脾虚小鼠血清IFN-γ含量(P<0.01),高剂量组灰树花提取物可以降低血清IL-4含量(P<0.01)。
6.灰树花提取物对脾虚小鼠Bcl-2、Bax、Fas、FasL蛋白表达的影响
脾虚组Bax、Fas、FasL阳性产物平均灰度值(平均灰度值与表达量成反比)明显低于空白组(P<0.01),给予阳性对照组药物和低、中、高剂量的灰树花提取物后,Bax、Fas、FasL阳性产物平均灰度值比脾虚组明显升高(P<0.01)。脾虚组Bcl-2阳性产物平均灰度值明显高于空白组(P<0.01),给予阳性对照组药物和低、中、高剂量的灰树花提取物后,Bcl-2阳性产物平均灰度值比脾虚组明显降低(P<0.01)。
7.灰树花提取物对脾虚小鼠Bcl-2、Bax、Fas、FasLmRNA表达的影响
与空白组比较,脾虚组小鼠脾脏Bax、Fas、FasL mRNA表达显著升高(P﹤0.05,P﹤0.01和P﹤0.01),Bcl-2 mRNA表达显著降低(P﹤0.01);与脾虚组相比较,脾虚小鼠给予阳性对照药物和低、中、高剂量灰树花提取物后, Bax、Fas、FasL mRNA表达显著降低(P﹤0.01),Bcl-2 mRNA表达显著升高(P﹤0.01)。
结论:
1.通过番泻叶-劳倦过度复合因素造模法制做脾虚小鼠模型,造模后脾虚小鼠体重明显下降,脾指数下降,灰树花提取物能够增加脾虚小鼠的体重、有效改善脾虚所致的脾指数下降。
2.灰树花提取物可以通过增强脾虚小鼠腹腔巨噬细胞吞噬中性红的能力;可以通过NO途径,增加NO的产生量来发挥巨噬细胞的生物学功能;灰树花提取物可以通过增强脾脏B淋巴细胞和T淋巴细胞的增殖能力来提高脾虚小鼠的免疫功能;灰树花提取物还可以通过提高NK细胞的杀伤活性来提高脾虚小鼠的免疫功能。
3.灰树花提取物可以改善脾虚时造成的小鼠CD4 /CD8比值降低,可能主要是通过作用于Th细胞来提高细胞免疫功能。灰树花提取物可以有效地升高脾虚小鼠血清中IFN-γ含量,下调IL-4的含量,从而平衡Th1/Th2亚群,使Th2细胞反应向Th1细胞转化,改善脾虚病理改变。
4.灰树花提取物可能通过促进Bc1-2蛋白与基因的表达、抑制Bax、Fas、FasL蛋白与基因的表达、提高Bcl-2/Bax的比值,通过影响线粒体、死亡受体依赖的凋亡途径来抑制脾淋巴细胞凋亡的发生。
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). Serumγ-interferon (IFN-γ) 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 macrophages
The 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 mice
Spleen 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 blood
The 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-γand IL-4 level in spleen deficiency mice serum
The IFN-γlevel 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-γlevel 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 mice
The 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 mice
The 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-γlevel 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.
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