本文選題：促腎上腺皮質(zhì)激素 + 黃芩苷�。� 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：腦卒中,俗稱中風(fēng),是一組急性腦循環(huán)障礙所致的局限或全面性腦功能缺損綜合征,包括缺血性和出血性腦卒中兩大類,具有發(fā)病率高、致殘率高、死亡率高和復(fù)發(fā)率高的特點(diǎn),給家庭和社會帶來了沉重的負(fù)擔(dān)。而急性缺血性腦卒中是最常見的類型,約占全部腦卒中的60%-80%。孕激素腦保護(hù)作用的研究始于上個世紀(jì)八十年代,目前,孕激素對于急性創(chuàng)傷性腦損傷的治療已進(jìn)入Ⅲ期臨床試驗(yàn)階段,然而其不良反應(yīng)限制了其使用推廣。大量臨床實(shí)踐和研究證實(shí),中醫(yī)藥在臨床上對于缺血性腦卒中的治療也發(fā)揮了一定的作用。通過文獻(xiàn)調(diào)研及本團(tuán)隊(duì)的前期研究工作發(fā)現(xiàn),黃芩的有效成分黃芩苷對缺血性腦損傷可發(fā)揮較好的腦保護(hù)作用,并且它還能提高已孕小鼠的血清孕酮水平。由此,我們提出了黃芩苷發(fā)揮腦保護(hù)作用與其提高血清孕酮水平有關(guān)的假說,并在前期工作中證實(shí)了黃芩苷發(fā)揮對缺血性腦損傷雌性大鼠的腦保護(hù)作用與其提高大鼠的血清孕酮水平有關(guān)。本課題則需要進(jìn)一步明確黃芩苷對缺血性腦損傷雄性大鼠的腦保護(hù)作用是否與其提高大鼠的血清孕酮水平有關(guān),并探討黃芩苷提高缺血性腦損傷雄性大鼠血清孕酮水平的可能機(jī)制,為研發(fā)一種用于治療缺血性腦損傷的類孕激素樣作用的天然替代藥物奠定基礎(chǔ)。目的1.明確黃芩苷對正常及缺血性腦損傷雄性大鼠血清孕酮水平及腦組織中孕酮受體表達(dá)水平的影響;2.明確黃芩苷提高缺血性腦損傷雄性大鼠血清孕酮水平的可能機(jī)制;3.明確黃芩苷提高雄性大鼠血清孕酮水平是否是其發(fā)揮腦保護(hù)作用的機(jī)制之一。方法1.線栓法建立雄性大鼠左側(cè)永久性大腦中動脈梗阻模型(pMCAO模型),將造模成功的大鼠根據(jù)神經(jīng)功能評分均勻地分為模型組和黃芩苷組,另設(shè)正常組和正常加黃芩苷組。造模成功后24h給藥,每天1次。黃芩苷組和正常加黃芩苷組給予黃芩苷溶液腹腔注射,模型組和正常組給予等體積生理鹽水腹腔注射。于造模后第7d取材(腹主動脈血和大腦),應(yīng)用ELISA法檢測血清孕酮水平,免疫組織化學(xué)法檢測腦組織中孕酮受體的表達(dá)。2.線栓法建立雄性大鼠左側(cè)永久性大腦中動脈梗阻模型(pMCAO模型),將造模成功的大鼠按照神經(jīng)功能評分均勻地分為模型組、黃芩苷組和抑制劑組,假手術(shù)組不插入栓線。造模成功后24h給藥,每天1次。黃芩苷組給予黃芩苷溶液腹腔注射和蒸餾水灌胃,模型組和假手術(shù)組給予等體積生理鹽水腹腔注射和蒸餾水灌胃,抑制劑組給予黃芩苷溶液腹腔注射和溴隱亭灌胃(抑制孕酮的生成)。于造模后第7d取材(腹主動脈血和腎上腺),應(yīng)用ELISA法檢測血清孕酮和促腎上腺皮質(zhì)激素(ACTH)水平,應(yīng)用熒光定量PCR法檢測腎上腺組織中孕酮合成相關(guān)酶P450膽固醇側(cè)鏈裂解酶(P450scc)和3 β-輕基類固醇脫氫酶(3β-HSD)mRNA的表達(dá)。3.線栓法建立雄性大鼠左側(cè)永久性大腦中動脈梗阻模型(pMCAO模型),將造模成功的大鼠按照神經(jīng)功能評分均勻地分為模型組、黃芩苷組和抑制劑組,假手術(shù)組不插入栓線(給藥方法同2)。分別對假手術(shù)組、模型組、黃芩苷組和抑制劑組大鼠在造模成功后的2-4h、1d、3d和7d進(jìn)行神經(jīng)功能評分,分別于造模前一晚禁食前,以及造模成功后的1d、3d和7d進(jìn)行大鼠雙前肢抓力測定,計算抓力下降率。于造模后第7d取材(大腦),TTC染色法觀察腦梗死情況,圖像分析軟件計算腦梗死體積百分比,HE染色法觀察腦組織形態(tài)變化。結(jié)果1.黃芩苷對正常及缺血性腦損傷雄性大鼠血清孕酮水平及腦組織中孕酮受體表達(dá)的影響1.1血清孕酮水平:與正常組相比,模型組大鼠的血清孕酮水平降低(P0.05);與模型組相比,黃岑苷組大鼠的血清孕酮水平升高(P0.05)。1.2腦組織中孕酮受體的表達(dá):孕酮受體主要在大腦皮層神經(jīng)元的胞漿和胞核表達(dá),與正常組相比,正常加黃岑苷組的孕酮受體在大腦頂葉皮層的表達(dá)水平增加(P0.05);與模型組相比,黃芩苷組的孕酮受體在患側(cè)大腦頂葉皮層缺血半暗帶區(qū)的表達(dá)水平增加(P0.05)。2.黃芩苷提高缺血性腦損傷雄性大鼠血清孕酮水平的可能機(jī)制2.1血清孕酮水平:與假手術(shù)組相比,模型組大鼠的血清孕酮水平有降低的趨勢(P0.05),黃芩苷組大鼠的血清孕酮水平升高(P0.05);與模型組相比,黃芩苷組大鼠的血清孕酮水平明顯升高(P0.01);與黃芩苷組相比,抑制劑組大鼠的血清孕酮水平降低(p0.05)。2.2血清ACTH水平:與假手術(shù)組相比,模型組和黃芩苷組大鼠的血清ACTH水平都有一定程度的升高(P0.05或P0.01);與模型組相比,黃芩苷組大鼠的血清ACTH水平有升高的趨勢(P0.05);與黃芩苷組相比,抑制劑組大鼠的血清ACTH水平降低(P0.05)。2.3腎上腺組織中P450sccmRNA和3 β-HSDmRNA的表達(dá)水平:與假手術(shù)組相比,模型組、黃芩苷組和抑制劑組大鼠腎上腺組織中的P450sccmRNA和3β-HSDmRNA的表達(dá)均增加(P0.05),但是黃芩苷組的表達(dá)增加更加明顯。3.黃芩苷提高缺血性腦損傷雄性大鼠血清孕酮水平可能是其發(fā)揮腦保護(hù)作用的機(jī)制之一3.1神經(jīng)功能評分:造模成功后的各組大鼠與假手術(shù)組相比神經(jīng)功能明顯受損,神經(jīng)功能評分增加;模型組大鼠的神經(jīng)功能評分有逐漸減小的趨勢,神經(jīng)功能逐漸恢復(fù);黃芩苷組大鼠的神經(jīng)功能恢復(fù)加快;抑制劑組大鼠的神經(jīng)功能恢復(fù)受到抑制。在第7d時,與模型組相比,黃芩苷組大鼠的神經(jīng)功能評分降低(P0.05);與黃芩苷組相比,抑制劑組大鼠的神經(jīng)功能評分明顯升高(P0.01)。3.2抓力下降率:造模成功后的各組大鼠與假手術(shù)組相比抓力下降率明顯增加;模型組大鼠的抓力下降率有逐漸減小的趨勢,抓力逐漸恢復(fù);黃芩苷組大鼠的抓力恢復(fù)更為明顯;抑制劑組大鼠的抓力恢復(fù)受到抑制。在第7d時,與模型組相比,黃芩苷組大鼠抓力下降率減小(P0.05);與黃芩苷組相比,抑制劑組大鼠的抓力下降率明顯增加(P0.01)。3.3大鼠腦梗死體積百分比:假手術(shù)組大鼠的腦片未出現(xiàn)白色梗死灶,模型組、黃芩苷組和抑制劑組大鼠的腦片均出現(xiàn)不同程度的梗死。計算大鼠腦梗死體積百分比發(fā)現(xiàn),與模型組相比,黃芩苷組大鼠的腦梗死體積百分比減小(P0.05);與黃芩苷組相比,抑制劑組大鼠的腦梗死體積百分比增加(P0.05)。3.4腦組織形態(tài)觀察:假手術(shù)組腦組織灰質(zhì)和白質(zhì)的邊緣基本清楚,頂葉皮層區(qū)各層細(xì)胞的分布基本正常,神經(jīng)細(xì)胞形態(tài)多樣;模型組腦組織灰質(zhì)和白質(zhì)的邊緣不清,皮層頂葉神經(jīng)元大量丟失,神經(jīng)元出現(xiàn)缺血性改變(皺縮、深染),膠質(zhì)細(xì)胞增生,小血管閉塞,周圍有組織間液積聚;黃芩苷組神經(jīng)元的受損程度減輕,神經(jīng)元丟失減少;抑制劑組的腦組織損傷未得到改善。結(jié)論1.黃芩苷能夠提高缺血性腦損傷雄性大鼠的血清孕酮水平,促進(jìn)正常及缺血性腦損傷雄性大鼠腦組織中孕酮受體的表達(dá);2.黃芩苷提高缺血性腦損傷雄性大鼠的血清孕酮水平可能與其促進(jìn)ACTH的產(chǎn)生,進(jìn)而促進(jìn)腎上腺中孕酮合成相關(guān)酶P450scc和3 β-HSD的合成有關(guān);3.黃芩苷發(fā)揮對缺血性腦損傷雄性大鼠的腦保護(hù)作用可能與其提高大鼠的血清孕酮水平有關(guān)。
[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.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5

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