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

【作者】 李海燕；

【导师】 潘彦舒；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2017， 硕士

【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(P<0.05);与模型组相比,黄岑苷组大鼠的血清孕酮水平升高(P<0.05)。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(P<0.05);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(P<0.05)。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(P>0.05),黄芩苷组大鼠的血清孕酮水平升高(P<0.05);与模型组相比,黄芩苷组大鼠的血清孕酮水平明显升高(P<0.01);与黄芩苷组相比,抑制剂组大鼠的血清孕酮水平降低(p<0.05)。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(P>0.05或P<0.01);与模型组相比,黄芩苷组大鼠的血清ACTH水平有升高的趋势(P>0.05);与黄芩苷组相比,抑制剂组大鼠的血清ACTH水平降低(P<0.05)。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(P<0.05),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(P<0.05);与黄芩苷组相比,抑制剂组大鼠的神经功能评分明显升高(P<0.01)。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(P<0.05);与黄芩苷组相比,抑制剂组大鼠的抓力下降率明显增加(P<0.01)。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(P<0.05);与黄芩苷组相比,抑制剂组大鼠的脑梗死体积百分比增加(P<0.05)。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。更多还原

【Abstract】 Stroke is a group of limited or comprehensive brain dysfunction syndrome caused by acute cerebral circulation disorder,including ischemic and hemorrhagic strokes.It has the characteristics of high incidence,high morbidity,high mortality and high recurrence rate,and brings about a heavy burden to the family and society.while acute ischemic stroke is the most common type of stroke,accounting for about 60 percent to 80 percent.The study of the protective effect of progesterone on the brain began in the 80s of last century.At present,the treatment for acute traumatic brain injury with progesterone has entered the phase III clinical trials,however,its untoward reactions limits its application and promoting.A large number of clinical practice and research confirmed that,traditional Chinese medicine has played a certain role in the treatment of ischemic stroke in clinics.Through the literature research and the preliminary research work in our team,baicalin of scutellaria baicalensis georgi can play a better role in protecting brain against ischemic brain injury,and it can improve the level of serum progesterone in pregnant mice.Thus,we put forward the hypothesis that the protective effect of baicalin on brain is related to the increase of serum progesterone level,and we have confirmed that the protective effect of baicalin on female rats with ischemic brain injury was related to the improvement of serum progesterone level in our preliminary work.In this study,we need to further clarify whether the protective effect of baicalin on male rats with ischemic brain injury is related to the improvement of serum progesterone level,and need to investigate the possible mechanism of baicalin in improving the serum progesterone level in male rats with ischemic brain injury,in order to lay the foundation for the research to develop a natural substitute drug for the treatment for ischemic brain damage which has a similar effect on progesterone.Objective1.To investigate the effect of baicalin on the serum progesterone level and the expression of progesterone receptor in brain tissue of normal male rats and rats with ischemic brain injury.2.To investigate the possible mechanism of baicalin in improving the serum progesterone level in male rats with ischemic brain injury.3.To investigate whether it is one of the mechanisms of brain protection for baicalin by improving the level of serum progesterone in male rats.Method1.The left permanent middle cerebral artery occlusion model(pMCAO model)in male rats with suture method was created,the model rats were evenly divided into model group and baicalin group according to the neurological function score,and normal group and normal +baicalin group were set.The rats were administered after successful modeling in 24hours,once a day.Rats in baicalin group and normal+baicalin group were given baicalin solution for intraperitoneal injection,while rats in model group and normal group were given isometric saline for intraperitoneal injection.Abdominal aortic blood and brain were taken out after modeling 7 days,detecting the serum progesterone level by ELISA,the expression of progesterone receptor in brain tissue was detected by immunohistochemistry.2.The left permanent middle cerebral artery occlusion model(pMCAO model)in male rats with suture method was created,the model rats were evenly divided into model group,baicalin group and Inhibitor group according to the neurological function score,and the sham operation group without inserting the intraluminal thread.The rats were administered after successful modeling in 24 hours,once a day.Rats in baicalin group were given baicalin solution for intraperitoneal injection and distilled water for gavage,rats in model group and sham operation group were given isometric saline for intraperitoneal injection and distilled water for gavage,and rats in inhibitor group were given baicalin solution for intraperitoneal injection and bromocriptine for gavage(in order to inhibit the production of progesterone).Abdominal aortic blood and renicapsule were taken out after modeling 7 days,the serum progesterone level and ACTH level were detected by ELISA,and the expression of P450 cholesterol side chain cleavage enzyme(P450scc)mRNA and 3beta-Hydroxysteroid dehydrogenase(3β-HSD)mRNA in renicapsule which were related to progesterone synthesis were detected by fluorescent quantitative PCR.3.The left permanent middle cerebral artery occlusion model(pMCAO model)in male rats with suture method was created,the model rats were divided into model group,baicalin group and inhibitor group according to the neurological function score,and the sham operation group without inserting the intraluminal thread(method of administration with 2).The neurological function score of sham operation group,model group,baicalin group and inhibitor group 2-4 hours,1 days,3 days and 7 days later after successful modeling were measured,the grip strength of double foreleg was measured before modeling the night before jejunitas and 1 days,3 days and 7 days later after successful modeling were measured,and the reduction rate of grip strength was calculated.The brain were taken out after modeling in 7 days,TTC staining was used to observe cerebral infarction,calculating the infarct volume percentage with image analysis software,and morphological change of brain tissue were observed by HE staining.Result1.The effects of baicalin on the serum progesterone level and the expression of progesterone receptor in brain tissue of normal male rats and rats with ischemic brain injury1.1 The level of serum progesterone:Compared with normal group,the level of serum progesterone in model group decreased(P<0.05).Compared with model group,the level of serum progesterone in baicalin group increased(P<0.05).1.2 The expression of progesterone receptor in brain tissue:Progesterone receptors are mainly expressed in the cytoplasm and nucleus of neurons in the cerebral cortex.Compared with normal group,the expression level of progesterone receptor in normal+baicalin group increased in the parietal cortex(P<0.05).Compared with model group,the expression level of progesterone receptor in baicalin group increased in the ischemic penumbra(P<0.05).2.The possible mechanism of baicalin in improving serum progesterone level in male rats with ischemic brain injury2.1 The level of serum progesterone:Compared with sham operation group,the level of serum progesterone in model group has a trend in decreasing(P>0.05),the level of serum progesterone in baicalin group increased(P<0.05).Compared with model group,the level of serum progesterone in baicalin group significantly increased(P<0.01).Compared with baicalin group,the level of serum progesterone in inhibitor group decreased(P<0.05).2.2 The level of serum ACTH:Compared with sham operation group,the level of serum ACTH in model group and baicalin group increased in different degrees(P>0.05 or P<0.01).Compared with model group,the level of serum ACTH in baicalin group has a trend in increasing(P>0.05).Compared baicalin group,the level of serum ACTH in inhibitor group decreased(P<0.05).2.3 The expression levels of P450scc mRNA and 3β-HSD mRNA in renicapsule:Compared with sham operation group,the expression of p450scc mRNA and 3β-HSD mRNA in renicapsule increased in model group,baicalin group and inhibitor group(P<0.05),but the expression of baicalin group increased significantly.3.Baicalin can improve the level of serum progesterone in male rats with ischemic brain injury which may be one of the mechanisms of brain protection3.1 Neurological function score:Compared with sham operation group,the neurological function of model rats in each group was impaired significantly,and the neurological function score increased.The neurological function score in model group has a trend in decreasing,and the nerve function was gradually restored.The recovery of nerve function in baicalin group was much faster;The recovery of nerve function was inhibited in inhibitor group.On the 7th day,compared with model group,the neurological score of baicalin group decreased(P<0.05).Compared with baicalin group,the neurological function score of inhibitor group was significantly higher(P<0.01).3.2 The reduction rate of grip strength:Compared with sham operation group,the reduction rate of grip strength decreased significantly after successful modeling;In model group,the reduction rate of grip strength gradually decreased,and the grip strength gradually recovered;In baicalin group,the recovery of the grip strength was more obvious;The recovery of the grip strength was inhibited in inhibitor group.On the 7th day,compared with model group,the reduction rate of grip strength decreased in baicalin group(P<0.05).Compared with baicalin group,the reduction rate of grip strength significantly increased in inhibitor group(P<0.05).3.3 The volume percentage of cerebral infarction in rats:There was no white infarct in the brain of sham operated rats.Rats in model group,baicalin group and inhibitor group had different degrees of cerebral infarction.Calculating the percentage of cerebral infarction volume in rats,it can be seen that,compared with model group,the volume percentage of cerebral infarction in baicalin group decreased(P<0.05).Compared with baicalin group,the volume percentage of cerebral infarction in inhibitor group increased(P<0.05).3.4 The brain morphology observation:The edges of gray and white matter of brain in sham operation group were clear,the distribution of cells in parietal cortex was normal,and the morphological of nerve cells was diverse.The edges of gray and white matter of brain tissue in the model group was unclear,cortical parietal neurons were lost,and neurons underwent ischemic changes(shrinkage,and deep dyeing),gliocyte proliferation,small vessel occlusion,accumulation of interstitial fluid.The degree of damage reduced in baicalin group,and neuronal loss was reduced.Brain tissue injury was not improved in inhibitor group.Conclusion1.Baicalin can increase the serum progesterone level in male rats with ischemic brain injury,and promote the expression of progesterone receptors in the brain tissue in normal male rats and rats with ischemic brain injury.2.Baicalin enhances the serum progesterone levels in male rats with ischemic brain damage,which may be related to the promotion of ACTH production,thereby promote the synthesis of P450scc and 3p-HSD in the renicapsule which is related to progesterone synthesis.3.The protective effect of baicalin on male rats with ischemic brain injury may be related to increasing the level of serum progesterone.更多还原