黄芩苷调控雄性大鼠血清孕酮水平进而发挥脑保护作用的机制研究

发布时间：2018-07-07 16:58

本文选题：促肾上腺皮质激素 + 黄芩苷　；参考：《北京中医药大学》2017年硕士论文

【摘要】：脑卒中,俗称中风,是一组急性脑循环障碍所致的局限或全面性脑功能缺损综合征,包括缺血性和出血性脑卒中两大类,具有发病率高、致残率高、死亡率高和复发率高的特点,给家庭和社会带来了沉重的负担。而急性缺血性脑卒中是最常见的类型,约占全部脑卒中的60%-80%。孕激素脑保护作用的研究始于上个世纪八十年代,目前,孕激素对于急性创伤性脑损伤的治疗已进入Ⅲ期临床试验阶段,然而其不良反应限制了其使用推广。大量临床实践和研究证实,中医药在临床上对于缺血性脑卒中的治疗也发挥了一定的作用。通过文献调研及本团队的前期研究工作发现,黄芩的有效成分黄芩苷对缺血性脑损伤可发挥较好的脑保护作用,并且它还能提高已孕小鼠的血清孕酮水平。由此,我们提出了黄芩苷发挥脑保护作用与其提高血清孕酮水平有关的假说,并在前期工作中证实了黄芩苷发挥对缺血性脑损伤雌性大鼠的脑保护作用与其提高大鼠的血清孕酮水平有关。本课题则需要进一步明确黄芩苷对缺血性脑损伤雄性大鼠的脑保护作用是否与其提高大鼠的血清孕酮水平有关,并探讨黄芩苷提高缺血性脑损伤雄性大鼠血清孕酮水平的可能机制,为研发一种用于治疗缺血性脑损伤的类孕激素样作用的天然替代药物奠定基础。目的1.明确黄芩苷对正常及缺血性脑损伤雄性大鼠血清孕酮水平及脑组织中孕酮受体表达水平的影响;2.明确黄芩苷提高缺血性脑损伤雄性大鼠血清孕酮水平的可能机制;3.明确黄芩苷提高雄性大鼠血清孕酮水平是否是其发挥脑保护作用的机制之一。方法1.线栓法建立雄性大鼠左侧永久性大脑中动脉梗阻模型(pMCAO模型),将造模成功的大鼠根据神经功能评分均匀地分为模型组和黄芩苷组,另设正常组和正常加黄芩苷组。造模成功后24h给药,每天1次。黄芩苷组和正常加黄芩苷组给予黄芩苷溶液腹腔注射,模型组和正常组给予等体积生理盐水腹腔注射。于造模后第7d取材(腹主动脉血和大脑),应用ELISA法检测血清孕酮水平,免疫组织化学法检测脑组织中孕酮受体的表达。2.线栓法建立雄性大鼠左侧永久性大脑中动脉梗阻模型(pMCAO模型),将造模成功的大鼠按照神经功能评分均匀地分为模型组、黄芩苷组和抑制剂组,假手术组不插入栓线。造模成功后24h给药,每天1次。黄芩苷组给予黄芩苷溶液腹腔注射和蒸馏水灌胃,模型组和假手术组给予等体积生理盐水腹腔注射和蒸馏水灌胃,抑制剂组给予黄芩苷溶液腹腔注射和溴隐亭灌胃(抑制孕酮的生成)。于造模后第7d取材(腹主动脉血和肾上腺),应用ELISA法检测血清孕酮和促肾上腺皮质激素(ACTH)水平,应用荧光定量PCR法检测肾上腺组织中孕酮合成相关酶P450胆固醇侧链裂解酶(P450scc)和3 β-轻基类固醇脱氢酶(3β-HSD)mRNA的表达。3.线栓法建立雄性大鼠左侧永久性大脑中动脉梗阻模型(pMCAO模型),将造模成功的大鼠按照神经功能评分均匀地分为模型组、黄芩苷组和抑制剂组,假手术组不插入栓线(给药方法同2)。分别对假手术组、模型组、黄芩苷组和抑制剂组大鼠在造模成功后的2-4h、1d、3d和7d进行神经功能评分,分别于造模前一晚禁食前,以及造模成功后的1d、3d和7d进行大鼠双前肢抓力测定,计算抓力下降率。于造模后第7d取材(大脑),TTC染色法观察脑梗死情况,图像分析软件计算脑梗死体积百分比,HE染色法观察脑组织形态变化。结果1.黄芩苷对正常及缺血性脑损伤雄性大鼠血清孕酮水平及脑组织中孕酮受体表达的影响1.1血清孕酮水平:与正常组相比,模型组大鼠的血清孕酮水平降低(P0.05);与模型组相比,黄岑苷组大鼠的血清孕酮水平升高(P0.05)。1.2脑组织中孕酮受体的表达:孕酮受体主要在大脑皮层神经元的胞浆和胞核表达,与正常组相比,正常加黄岑苷组的孕酮受体在大脑顶叶皮层的表达水平增加(P0.05);与模型组相比,黄芩苷组的孕酮受体在患侧大脑顶叶皮层缺血半暗带区的表达水平增加(P0.05)。2.黄芩苷提高缺血性脑损伤雄性大鼠血清孕酮水平的可能机制2.1血清孕酮水平:与假手术组相比,模型组大鼠的血清孕酮水平有降低的趋势(P0.05),黄芩苷组大鼠的血清孕酮水平升高(P0.05);与模型组相比,黄芩苷组大鼠的血清孕酮水平明显升高(P0.01);与黄芩苷组相比,抑制剂组大鼠的血清孕酮水平降低(p0.05)。2.2血清ACTH水平:与假手术组相比,模型组和黄芩苷组大鼠的血清ACTH水平都有一定程度的升高(P0.05或P0.01);与模型组相比,黄芩苷组大鼠的血清ACTH水平有升高的趋势(P0.05);与黄芩苷组相比,抑制剂组大鼠的血清ACTH水平降低(P0.05)。2.3肾上腺组织中P450sccmRNA和3 β-HSDmRNA的表达水平:与假手术组相比,模型组、黄芩苷组和抑制剂组大鼠肾上腺组织中的P450sccmRNA和3β-HSDmRNA的表达均增加(P0.05),但是黄芩苷组的表达增加更加明显。3.黄芩苷提高缺血性脑损伤雄性大鼠血清孕酮水平可能是其发挥脑保护作用的机制之一3.1神经功能评分:造模成功后的各组大鼠与假手术组相比神经功能明显受损,神经功能评分增加;模型组大鼠的神经功能评分有逐渐减小的趋势,神经功能逐渐恢复;黄芩苷组大鼠的神经功能恢复加快;抑制剂组大鼠的神经功能恢复受到抑制。在第7d时,与模型组相比,黄芩苷组大鼠的神经功能评分降低(P0.05);与黄芩苷组相比,抑制剂组大鼠的神经功能评分明显升高(P0.01)。3.2抓力下降率:造模成功后的各组大鼠与假手术组相比抓力下降率明显增加;模型组大鼠的抓力下降率有逐渐减小的趋势,抓力逐渐恢复;黄芩苷组大鼠的抓力恢复更为明显;抑制剂组大鼠的抓力恢复受到抑制。在第7d时,与模型组相比,黄芩苷组大鼠抓力下降率减小(P0.05);与黄芩苷组相比,抑制剂组大鼠的抓力下降率明显增加(P0.01)。3.3大鼠脑梗死体积百分比:假手术组大鼠的脑片未出现白色梗死灶,模型组、黄芩苷组和抑制剂组大鼠的脑片均出现不同程度的梗死。计算大鼠脑梗死体积百分比发现,与模型组相比,黄芩苷组大鼠的脑梗死体积百分比减小(P0.05);与黄芩苷组相比,抑制剂组大鼠的脑梗死体积百分比增加(P0.05)。3.4脑组织形态观察:假手术组脑组织灰质和白质的边缘基本清楚,顶叶皮层区各层细胞的分布基本正常,神经细胞形态多样;模型组脑组织灰质和白质的边缘不清,皮层顶叶神经元大量丢失,神经元出现缺血性改变(皱缩、深染),胶质细胞增生,小血管闭塞,周围有组织间液积聚;黄芩苷组神经元的受损程度减轻,神经元丢失减少;抑制剂组的脑组织损伤未得到改善。结论1.黄芩苷能够提高缺血性脑损伤雄性大鼠的血清孕酮水平,促进正常及缺血性脑损伤雄性大鼠脑组织中孕酮受体的表达;2.黄芩苷提高缺血性脑损伤雄性大鼠的血清孕酮水平可能与其促进ACTH的产生,进而促进肾上腺中孕酮合成相关酶P450scc和3 β-HSD的合成有关;3.黄芩苷发挥对缺血性脑损伤雄性大鼠的脑保护作用可能与其提高大鼠的血清孕酮水平有关。
[Abstract]:Stroke, commonly known as stroke, is a group of limited or comprehensive brain function defect syndrome caused by acute cerebral circulation disorder, including two major categories of ischemic and hemorrhagic stroke. It has high incidence, high disability rate, high mortality and high recurrence rate, and has a heavy burden on family and society. Acute ischemic stroke is the most frequent. The study of the 60%-80%. progesterone protective effect of all cerebral apoplexy began in the 80s of the last century. At present, the treatment of progestin for acute traumatic brain injury has entered stage III clinical trials. However, its adverse reaction restricts its use. Large amount of clinical practice and research confirm that Chinese medicine is in clinical practice. It has also played a role in the treatment of ischemic stroke. Through literature research and the previous research work of this team, it is found that baicalin, the effective component of Scutellaria baicalensis, can play a better role in cerebral ischemic injury, and it can also improve the level of serum gestation in the pregnant mice. The hypothesis that the protective effect is related to the level of serum progesterone, and in the previous work proved that baicalin exerts its protective effect on the brain of the female rats with ischemic brain damage and the level of the serum progesterone in rats. To improve the level of serum progesterone in rats, and to explore the possible mechanism of baicalin to improve the level of serum progesterone in ischemic brain injury of male rats, and to lay the foundation for developing a natural substitute drug for the treatment of ischemic brain damage like progesterone like action. Objective 1. to make clear that baicalin is a big male to normal and ischemic brain damage. The effect of progesterone in rat serum and the expression of progesterone receptor in brain tissue; 2. it is clear that baicalin improves the possible mechanism of progesterone level in the serum of male rats with ischemic brain injury, and 3. it is one of the mechanisms of whether the level of progesterone in the serum of male rats is one of the mechanisms of the protective effect of the male rats. Method the method of 1. line embolus was established to establish the left male rat left The rat model of permanent middle cerebral artery obstruction (pMCAO model) was divided into the model group and baicalin group according to the neurological function score, and the normal group and the normal baicalin group were set up. The model group and the normal addition of baicalin group were injected with baicalin 1 times a day after the success of the model and the normal addition of baicalin group. The model group and the normal addition of baicalin group were injected with baicalin. The normal group was given intraperitoneal injection of equal volume of physiological saline. After 7d, the serum progesterone level was detected by ELISA method, and the expression of progesterone receptor in the brain tissue was detected by.2. thread thrombus method in the brain tissue to establish the permanent middle cerebral artery obstruction model (pMCAO model) on the left side of the male rats (pMCAO model), and the model was successful. The rats were divided into the model group, the baicalin group and the inhibitor group, and the sham operation group did not insert the thrombus line. After the success of the model, 24h was given 1 times a day. The baicalin group gave baicalin solution intraperitoneally and distilled water to stomach. The model group and sham operation group were given intraperitoneal injection of equal volume of saline and distilled water to stomach, and the rats in the sham operation group were intraperitoneally injected with distilled water. The preparation group was given baicalin solution intraperitoneally and bromocriptine gavage (inhibiting the formation of progesterone). After the model 7d was obtained (abdominal aorta blood and adrenal gland), serum progesterone and adrenocorticotropin (ACTH) levels were detected by ELISA method, and the P450 cholesterol side chain of progesterone synthesis related enzyme in adrenal tissue was detected by fluorescence quantitative PCR method. The expression of lysase (P450scc) and 3 beta light steroid dehydrogenase (3 beta -HSD) mRNA was used to establish a permanent middle cerebral artery obstruction model (pMCAO model) on the left side of the male rat (pMCAO model). The rat model was divided into the model group, the baicalin group and the inhibitor group, and the sham operation group did not insert the thrombus line (the method of administration). 2-4h, 1D, 3D and 7d of rats in the sham operation group, the model group, the baicalin group and the inhibitor group were divided into 2-4h, 1D, 3D and 7d, respectively, before the first night fasting, and 1D, 3D and 7d after the model success respectively. Observation of cerebral infarction, image analysis software to calculate the percentage of cerebral infarction volume, HE staining method to observe the change of brain tissue. Results 1. baicalin on the serum progesterone level and the expression of progesterone receptor in the brain tissue of male rats with normal and ischemic brain injury 1.1 serum progesterone level: compared with the normal group, the serum of model rats Progesterone level decreased (P0.05); compared with the model group, the serum progesterone level of the rats in the cen group increased (P0.05) the expression of progesterone receptor in the.1.2 brain tissue: progesterone receptor was mainly expressed in the cytoplasm and nucleus of the cerebral cortex neurons. Compared with the normal group, the progesterone receptor in the normal plus Cen glycoside group increased in the parietal cortex of the brain. (P0.05); compared with the model group, the progesterone receptor in the baicalin group increased in the ischemic penumbra region of the cerebral parietal cortex (P0.05).2. baicalin increased the possible mechanism of serum progesterone in the serum of the male rats with ischemic brain damage: 2.1 serum progesterone level: compared with the artificial hand group, the serum progesterone level of the model group was reduced. The trend (P0.05), the serum progesterone level of the baicalin group increased (P0.05); compared with the model group, the serum progesterone level of the baicalin group was significantly higher (P0.01). Compared with the baicalin group, the serum progesterone level of the inhibitor group was lower (P0.05).2.2 blood ACTH level: compared with the sham group, the model group and baicalin group were compared with the sham group. The serum level of ACTH increased to a certain extent (P0.05 or P0.01); compared with the model group, the serum level of ACTH in the baicalin group was higher (P0.05). Compared with the baicalin group, the serum ACTH level of the inhibitor group decreased (P0.05) the expression level of P450sccmRNA and 3 beta -HSDmRNA in the adrenal gland of.2.3: compared with the sham group, In the model group, the expression of P450sccmRNA and 3 beta -HSDmRNA in the adrenal gland of the baicalin group and the inhibitor group increased (P0.05), but the increase of the expression of baicalin group was more obvious that the level of the serum progesterone in the male rats with ischemic brain injury may be one of the mechanisms of the 3.1 neurologic function. Compared with the sham operation group, the nerve function score of the rats in the model group was gradually reduced and the nerve function was gradually restored, the nerve function recovery of the baicalin group was accelerated and the nerve function recovery of the inhibitor group rats was inhibited. At the time of 7D, the rats in the group of the inhibitor group were inhibited. Compared with the model group, the nerve function score of the baicalin group was lower (P0.05). Compared with the baicalin group, the neural function score of the inhibitor group was significantly increased (P0.01).3.2 grasping force decrease rate: the decrease rate of the grasping force in the rats of the model group was significantly increased compared with the sham operation group; the decrease rate of the grasping force in the model group was gradually reduced. The grasping force of the baicalin group was more obvious, and the arrest of the rats in the inhibitor group was inhibited. At 7d, the decrease rate of the grasping force decreased (P0.05) in the baicalin group compared with the model group, and the decrease rate of the grasping force in the inhibitor group was significantly increased (P0.01).3.3 rat cerebral infarction volume compared with the baicalin group. Percentage: there was no white infarct in the brain slices of the sham operation group. The brain slices of the model group, baicalin group and the inhibitor group were all infarcted in different degrees. The percentage of cerebral infarction volume in the rats was calculated and the percentage of cerebral infarction in the baicalin group decreased (P0.05) compared with the model group. Compared with the baicalin group, the inhibitor was compared with the group of baicalin. The percentage of cerebral infarction volume increased (P0.05).3.4 brain tissue morphology: the edge of gray matter and white matter in the brain tissue of the sham operation group was basically clear, the distribution of cells in the parietal cortex was basically normal, the morphology of the nerve cells varied, the gray and white matter of the brain tissue in the model group were not clear, the neurons of the cortex parietal lobe were lost and the neurons were lost. Ischemic change (crinkle, deep staining), glial cell proliferation, small vascular occlusion, and surrounding tissue fluid accumulation, the damage degree of the neurons in the baicalin group reduced, the neuron loss decreased, and the brain tissue damage in the inhibitor group was not improved. Conclusion 1. baicalin can raise the serum progesterone level of the male rats with ischemic brain injury and promote the level of serum progesterone. The expression of progesterone receptor in the brain tissue of male rats with normal and ischemic brain damage; 2. baicalin enhances the level of progesterone in the serum of the male rats with ischemic brain injury, which may promote the production of ACTH, and further promote the synthesis of the progesterone synthesis related enzyme P450scc and 3 beta -HSD in the adrenal gland; 3. baicalin plays a major role in the ischemic brain damage in male rats. The protective effect of rat brain may be related to the increase of serum progesterone level in rats.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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