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目的研究灵芝孢子对帕金森病(PD)模型鼠脑黑质酪氨酸羟化酶(TH)的影响,以探讨灵芝孢子对PD的脑保护作用。方法120只SD大鼠随机分为三组,PD组:立体定向注入6-羟多巴(6-OHDA),术后1周腹腔注入阿朴吗啡观察30min大鼠旋转的次数,连续至第四周每分钟大于6次者为成功的PD模型;灵芝孢子组:先用灵芝孢子粉灌胃3d,立体定向注入6-OHDA,继续灌胃4周,直至处死;正常对照组:立体定向注入脑黑质抗坏血酸生理盐水。处死后制作快速冰冻切片用免疫组化检测TH阳性细胞数,用原位杂交法检测THmRNA的阳性细胞数,用Westernblot检测TH的半定量变化,用高效液相色谱检测多巴胺水平的变化。结果免疫组化显示灵芝孢子组术侧鼠脑黑质TH阳性神经元数量为99·7±13·6,PD组为51·0±13·5,灵芝孢子组较PD组显著升高(P<0·05);原位杂交显示灵芝孢子组术侧鼠脑黑质THmRNA阳性神经元数量为180·3±24·3,PD组为72·7±15·0,灵芝孢子组较PD组显著升高(P<0·01);Westernblot检测显示TH蛋白灵芝孢子组较PD组显著增加;高效液相色谱检测术侧鼠脑黑质多巴胺(DA)水平灵芝孢子组为(0·7±0·3)ng/mg蛋白,PD组为(0·4±0·1)ng/mg蛋白,灵芝孢子组较PD组显著升高(P<0·05)。结论灵芝孢子能够提高TH的表达,并提高鼠脑黑质DA的水平,推测对6-OHDA诱导的PD可能有脑保护作用。
Objective To study the effect of ganoderma lucidum spores on tyrosine hydroxylase (TH) in substantia nigra of Parkinson’s disease (PD) model mice in order to investigate the protective effects of ganoderma lucidum spores on PD. Methods 120 Sprague-Dawley rats were randomly divided into three groups. The PD group was injected with 6-OHDA stereotactically. The rats were injected intraperitoneally with apomorphine for 1 week to observe the number of rat rotations for 30 minutes. More than 6 cycles per minute were successful PD models; Ganoderma lucidum spore group: first by gavage with ganoderma lucidum spore powder for 3 days, stereotactic injection of 6-OHDA, continuous gavage for 4 weeks until sacrifice; normal control group: stereotactic injection into the brain Sodium ascorbate normal saline. After being sacrificed, rapid frozen sections were prepared and the number of TH positive cells was detected by immunohistochemistry. The number of positive cells of TH mRNA was detected by in situ hybridization. The semiquantitative changes of TH were detected by Western blot and the changes of dopamine levels were detected by high performance liquid chromatography. Results Immunohistochemistry showed that the number of TH-positive neurons in the brain substantia nigra of the Ganoderma lucidum spore group was 99.7±13·6, and that of the PD group was 51.0±13·5. The Ganoderma lucidum spore group was significantly higher than the PD group (P <0·05); In situ hybridization showed that the number of TH mRNA-positive neurons in the substantia nigra of the Ganoderma lucidum spore group was 180·3±24·3, and that of the PD group was 72·7±15·0. The Ganoderma lucidum spore group was more prone than the PD group. Significantly increased (P<0.01); Western blot showed that the TH protein lucidum spore group significantly increased compared with the PD group; high-performance liquid chromatography detection of brain substantia nigra dopamine (DA) level Ganoderma spore group was (0 · 7 ± 0 · 3) ng / mg protein, PD group (0 · 4 ± 0 · 1) ng / mg protein, Ganoderma lucidum spore group was significantly higher than the PD group (P <0. 05). Conclusion Ganoderma lucidum spores can increase the expression of TH, and increase the level of DA in rat brain substantia nigra, suggesting that there may be brain protection against PD induced by 6-OHDA.