摘要
蒺藜(tribulus terrestris)是蒺藜科植物蒺藜的干燥成熟果实。蒺藜皂苷(gross saponins of tribulus terrestris, GSTT)是蒺藜的有效成分之一,是以甾体皂苷为主的十多种皂苷的混合物。研究证实蒺藜皂苷具有降低血压、舒张血管、抑制血小板聚集、降低血糖、抑制肿瘤生长等作用。关于蒺藜皂苷对于脑血管的作用报道较少。PKCε是蛋白激酶C (protein kinase C, PKC)家族中的一个亚型。研究表明,活化后的PKCε可在缺血性脑损伤中对神经细胞起到保护作用。本实验通过建立大鼠脑缺血再灌注损伤模型及用H2O2损伤PC12细胞建立氧化应激损伤模型,阐明蒺藜皂苷对脑缺血再灌注损伤的保护作用并探讨其机制,为蒺藜皂苷在临床应用提供科学依据。
在整体水平上,采用线栓法建立大鼠脑缺血再灌注损伤模型。实验结果表明,蒺藜皂苷能够降低脑缺血再灌注损伤大鼠的神经功能评分;降低脑组织匀浆中MDA、NO的含量,并提高SOD的活性;减小脑梗死面积,抑制神经细胞凋亡;免疫组化结果表明,蒺藜皂苷可上调PKCε和ERK1/2蛋白的表达,Western blot结果显示蒺藜皂苷可促进PKCε、ERK1/2和Bcl-2蛋白的表达,可使PKCε发生膜转位。初步提示蒺藜皂苷的保护作用可能是通过激活PKCε与ERK1/2的信号转导通路实现的。
大鼠肾上腺嗜铬细胞瘤细胞(pheochromocytoma cells,PC12)具有神经元的基本生物学特性,而且容易培养可传代,有利于量化评价药物作用,是一个比较理想的细胞模型。H2O2是体内氧化代谢的中间产物之一,大量积聚时会对细胞产生毒性作用。细胞水平实验采用H2O2损伤PC12细胞来模拟体内氧化应激模型。结果表明:蒺藜皂苷可提高PC12细胞生存力;降低培养液中LDH、MDA的含量,并提高SOD的活性;降低PC12细胞凋亡百分率,抑制细胞内钙超载,稳定细胞线粒体膜电位,降低PC12细胞内ROS含量,促进PKCε、ERK1/2和Bcl-2蛋白的表达。使用PKCε抑制剂白屈菜赤碱(chelerythrine,简称Che),可阻断蒺藜皂苷的上述保护作用。结果进一步提示蒺藜皂苷的保护作用可能是通过激活PKCε-ERK1/2信号转导通路而抑制神经细胞凋亡,从而发挥其保护作用。
综上所述,蒺藜皂苷可通过激活PKCε,促使其发生膜转位,并且上调ERK1/2蛋白量的表达从而对抗氧化应激损伤,减轻细胞内钙超载、稳定线粒体膜电位,上调抗凋亡蛋白Bcl-2含量,抑制神经细胞的凋亡,从而发挥对脑缺血的保护作用。
Cerebrovascular disease is a common disease in clinic with high morbidity, disablement rate and mortality rate. It seriously influences people’s life and health. Recent epidemiology investigation shows that about one million people are affected by cerebrovascular disease. Cerebrovascular disease is the leading cause of death higher than heart disease and cancer in our country, accounting for sixty to eighty percent. Ischemic cerebrovascular disease (ICVD) refers to occurrences of stenosis and occlusion of main arteries which supply blood to brain. Local brain tissue is ischemic and necrotic, and appears corresponding clinical symptoms. Currently, thrombolysis is the main therapeutic method for the treatment of ICVD through regaining blood reperfusion to the ischemic area. Although some progress has been made but we are still disappointed about reperfussion injury only through anticoagulation, neuroprotection and increasing blood volume. Moreover most drugs may produce adverse reactions. Therefore, the investigation on the mechanism of the cerebral ischemia injury , research and development of protective drugs for the ischemia-reperfusion injury is very important both in theoretical sdudies and practical uses.
Tribulus terrestris refers to the dry, ripe fruit of the plant caltrop. It has efficacies of nature micro-warm and acrid-bitter flavor and calming liver wind and promoting circulation. The gross saponins of tribulus terrestris(GSTT)is one of the most effective ingredients which is extracted from the whole plant of caltrop. It is a mixture of about ten kinds of saponins. In recent years, some intensive studies on chemical constituent, pharmacological effects, clinical application and toxic effects of GSTT have been made and better results about them have been available. Researches have shown that GSTT have the effects of vasodilating, lowering blood pressure, inhibiting platelet aggregation, reducing blood sugar levels, suppressing tumor growth and enhancing sexual function. Our research team has done a lot of studies on GSTT. Our research has shown that GSTT has the effect of cerebral ischemia-reperfusion injury. But it is unclear about that protective effect and molecular mechanism of GSTT on cerebral ischemia-reperfusion injury. The aim of this research is to set up the model of cerebral ischemia-reperfusion injury in vivo and in vitro and investigate the protective effects and mechanisms of GSTT on the ischemic cerebral injury. It can provide a theoretical basis for safer and more reasonable uses of this therapy in clinic.
We set up the cerebral ischemia-reperfusion model by blocking middle cerebral artery in vivo and observed effects of GSTT on the neurologic deficit score, SOD, MDA, NO contents and infarction area in the brain tissue. At the same time, some samples were prepared to store in the formaldehyde solution for HE and toluidine blue staining and TUNEL immunohistochemical performance. Other samples were taken to store in the liquid nitrogen for the measure of Western blot. The protein expression of PKCε, ERK1/2 and Bcl-2, were analyzed by Western blot. PKCεtranslocation and change of ERK1/2 were detected by immunohistochemical and Western blot.
Experiments results in vivo showed that GSTT could significantly reduce the neurologic deficit score, contents of MDA and NO, and increased the activity of SOD in brain tissue, and decreased cerebral infarction area and neuronal cell apoptosis. GSTT could improve cerebral ischemia-reperfusion injury. The results of immunohistochemical and western blot analysis showed that GSTT could upregulate the protein expression of PKCε, ERK1/2 and Bcl-2. GSTT could promote PKCεtranslocation and upregulated the expression of ERK1/2. It was demonstrated that GSTT could protect cerebral ischemia-reperfusion injury through strengthening anti-oxidative enzyme activities, improving the ability of cleaning free radicals, alleviating the injury induced by free radicals, promoting PKCεtranslocation, upregulating the expression of ERK1/2 and inhibiting cell apoptosis.
In order to further investigate the protective effects and mechanisms of GSTT on cerebral ischemia-reperfusion injury, we used 0.1mmol·L-1 hydrogen peroxide (H2O2) to damage pheochromocytoma cell (PC12 cell). PC12 cell is an ideal model because it can be easily cultivated and has basal neuron biological characteristics. H2O2 is one of middle metabolizing products of the oxidative metabolism. Its accumulation in the body may lead to toxic effect on cells. The survival ability was detected by methyl thiazolyl tetrazolium (MTT) method. The contents of LDH, SOD and MDA in cultured conditions were measured by using the instruments. We determined the optimum dose and studied of the mechanism of GSTT on PC12 cell. The optimum dose of GSTT is 10mg·L-1. The percentage of apoptosis rate and the content of ROS were measured with flowcytometry. Mitochondrial memberane potential (△Ψm) and intracellular calcium concentration were observed with laser confocal microscopy system. The change of ERK1/2 was observed with immunofluorescence method. At the same time, the protein expressions of PKCε, ERK1/2 and Bcl-2 were analyzed with Western blot.
The results showed that GSTT could increase the survival rate of PC12 cells, reduce the release of LDH from PC12 cells, increase the activity of SOD ,reduce the concentration of MDA and PC12 cells apoptosis percentage and intracellular calcium concentration, stabilize the mitochondrial membrane potential, reduce the content of ROS, improve PKCεactivation and translocation,upregulate the protein expressions of ERK1/2 and Bcl-2. The inhibition of PKCεcould block the protective effects of GSTT.The results revealed that GSTT could produce the protective action through activating PKCε-ERK1/2 signaling pathway and inhibiting neurocyte apoptosis possibly.
From the above, we can make a conclusion that protective effects of GSTT on cerebral ischemia-reperfusion injury in rats and oxidative stress damaged by H2O2 of PC12 cells are probably through strengthening anti-oxidative enzyme activity, improving the ability of cleaning free radicals, inhibiting lipid peroxidation injury, stabilizing the mitochondrial membrane potential, protecting mitochondrial function, promoting PKCεtranslocation, upregulating expressions of ERK1/2 and Bcl-2 and inhibiting neuron apoptosis.
Innovative ideas:
1. GSTT has been applied for many years in clinic. But it is unclear about protective effects and mechanisms of GSTT on cerebral ischemia-reperfusion injury. We study the protective effects of GSTT on neuron injury by in vivo and in vitro experiments。
2. The study of the effects of GSTT on free radical,intracellular calcium overload, mitochondrial membrane potential,apoptosis and the protein expression of PKCε、ERK1/2 have not been reported up to now.
3. Our research will provide a new thought and theoretical basis for extracting new and effective monomers of GSTT through an intensive exploration in effects of GSTT on cerebral ischemia.
引文
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