【作者】 莲花；

【导师】 麻春杰；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2014， 博士

【摘要】 目的：通过研究额尔敦-乌日勒对大脑中动脉阻塞／再灌注(MCAO/R)损伤大鼠海马及皮质神经营养因子的影响,明确传统蒙药额尔敦-乌日勒抗局灶性脑缺血再灌注的药理作用,并探讨其作用机制,证实额尔敦-乌日勒治疗缺血性脑卒中(ICS)之传统功效,为进一步研究额尔敦-乌日勒提供实验依据。方法：采用改良Zea-Longa线栓法构建MCAO/R模型。将MCAO/R模型成功的造模组大鼠再随机分为模型组、额尔敦-乌日勒组、银杏叶片组、尼莫地平片组4组,同时设假手术组,共5组。假手术组和模型组ig蒸馏水,1m1/100g,1次/d,连续14天；额尔敦-乌日勒组、尼莫地平片组、银杏叶片组分别以61.7mg/100g、6.17mg/100g、12.34mg/100g剂量ig给药,1次/d,连续给药14天。实验结束,对各组大鼠进行神经功能得分评价,行TTC染色,检测大鼠脑指数、脑含水率、脑梗率等指标；采用HE、 SP等免疫组化染色方法评价脑组织病理形态学的改变情况；采用ELISA及RT-PCR法检测各组大鼠海马及皮质相关神经营养因子(NTFs)的含量及基因表达。采用SPSS18.0统计软件进行数据分析。结果：1.制备MCAO/R损伤大鼠模型的评价：经过神经功能Bederson评分与TTC染色发现造模组大鼠左眼明显小于右眼,并且没有亮度；向右转圈、向右推抵抗力、右爪内收等症状明显；缺血局部有苍白色脑组织,主要位于左侧大脑皮质及海马区,梗死灶比较稳定。2.神经功能Bederson评分情况：假手术组大鼠无神经功能缺失症状；造模组大鼠神经功能缺损障碍明显(P<0.05)；治疗第15天,模型组大鼠精神状态、活动情况、饮食、二便及毛发光泽度等神经症状较差,体重下降,右眼比左眼明显小而没有亮度,向右转圈、向右推抵抗力及右爪内收等情况未见明显减轻；各治疗组大鼠神经功能Bederson得分明显低于模型组,神经功能明显好转(P<0.05),其中额尔敦-乌日勒组神经功能得分较低(P<0.05),并且与假手术组无统计学意义差异(P>0.05),神经功能恢复于临近正常,而尼莫地平片组与银杏叶片组无统计学意义差异(P<0.05)表明大鼠MCAO/R损伤后,在急性期大鼠神经行为缺失症状严重,通过不同的治疗方法可有效的改善神经功能的恢复,表明额尔敦-乌日勒能减轻大鼠神经功能损伤,具有改善神经功能恢复作用。3.各组大鼠海马及皮质TTC染色结果：假手术组各脑片中未见苍白色组织,模型组苍白色组织较其它组明显,各给药组苍白色组织明显少于模型组,其中额尔敦-乌日勒组苍白色组织少于其他治疗组,而银杏叶片组与尼莫地平片组没有明显区别。4.各组大鼠海马及皮质脑梗塞率、脑含水率、脑指数情况比较结果：额尔敦-乌日勒组、银杏叶片组脑梗塞率、脑指数明显低于模型组和尼莫地平片组(P<0.05),而额尔敦-乌日勒组与银杏叶片组脑梗塞率、脑指数组间比较无差异(P>0.05)；额尔敦-乌日勒组脑含水率明显低于模型组和银杏叶片组(P<0.05),而额尔敦-乌日勒组与尼莫地平片组间脑含水率无差异(P>0.05)。实验结果显示,大鼠MCAO/R损伤后,在急性期脑梗塞范围较大,尚伴有较为严重的脑水肿,给于药物干预可有效降低脑梗塞率、脑含水率及脑指数,其中额尔敦-乌日勒及银杏叶片能显著降低脑梗塞率、脑指数,作用优于尼莫地平片；而额尔敦-乌日勒及尼莫地平片显著降低脑含水率,作用优于银杏叶片组。5.各组大鼠海马及皮质病理形态学结果：假手术组海马及皮质各层细胞形态、结构、排列均正常,未见细胞黏附及炎性细胞嵌塞,也无梗死灶出现,细胞周围间隙无水肿；模型组大鼠海马及皮质色泽苍白,胞膜与周围分界明显,胞浆浓缩红染,细胞核固缩为三角形或溶解、碎裂,神经纤维呈空泡化或软化现象严重,有大量的炎性细胞或淋巴细胞浸润,神经细胞稀疏或变性坏死、萎缩、排列不规则、紊乱,水肿疏松；各给药组大鼠海马及皮质各层病理变化较模型组均有不同程度的减轻,其中额尔敦-乌日勒组和银杏叶片组神经细胞损伤密度显著下降(P<0.05),减轻了神经元缺失、变性坏死范围、变性坏死程度、间质水肿。免疫组织化学染色定位分析显示,阳性细胞为神经细胞和部分胶质细胞,胞浆、胞膜部位及突起呈深棕黄色。染色阳性细胞于缺血半暗区表达明显。并且各组大鼠皮质区BDNF、NGF、 PDGF阳性细胞数的表达较多于海马区,而海马区GDNF、bFGF、TGF-β阳性细胞数较多于皮质区；假手术组大鼠海马及皮质BDNF、NGF、 GDNF、bFGF、TGF-p、PDGF阳性细胞数较少(P<0.05)；各给药组大鼠海马及皮质BDNF、NGF、GDNF、bFGF、TGF-β阳性细胞数较多于模型组(P<0.05),而PDGF阳性细胞数少于模型组(P<0.05)；其中额尔敦-乌日勒组大鼠海马及皮质BDNF、NGF、 GDNF、bFGF、TGF-β阳性细胞数最多,而PDGF阳性细胞数最少(P<0.05)；银杏叶片组大鼠海马及皮质BDNF、NGF阳性细胞数较多于尼莫地平片组(P<0.05),而GDNF、bFGF、TGF-p. PDGF阳性细胞数与尼莫地平片组间无差异(P>0.05)6.采用双抗夹心ELISA法检测大鼠海马及皮质BDNF、NGF、GDNF、bFGF、TGF-β、 PDGF蛋白含量的结果：各组大鼠皮质区BDNF、bFGF、TGF-p、PDGF蛋白含量较高于海马区(P<0.05),而海马区PDGF蛋白含量高于皮质区；假手术组与模型组大鼠海马区GDNF蛋白含量明显高于皮质区(P<0.05),而各给药组大鼠皮质区GDNF蛋白含量高于海马区(P<0.05)。假手术组大鼠海马及皮质BDNF、NGF、GDNF、bFGF. TGF-p、PDGF蛋白含量较低；与模型组相比,各给药组大鼠海马及皮质BDNF、NGF、 GDNF、bFGF、TGF-p蛋白含量较高(P<0.05),PDGF蛋白含量较低(P<0.05)其中额尔敦-乌日勒组大鼠海马及皮质BDNF、NGF、GDNF、bFGF、TGF-β蛋白含量最高,PDGF蛋白含量最低(P<0.05)；与尼莫地平片组相比,银杏叶片组大鼠海马及皮质NGF蛋白含量较高(P<0.05),PDGF蛋白含量较低,而BDNF、GDNF、bFGF、TGF-β蛋白含量差异无统计学意义(P>0.05)7.采用RT-PCR法检测大鼠皮质BDNF、NGF、GDNF、bFGF、TGF-β、PDGF基因表达的结果：假手术组大鼠皮质BDNF mRNA、NGF mRNA、GDNF、mRNA、bFGF mRNA、TGF-βmRNA、PDGF mRNA相对表达量较低。与模型组相比,各给药组大鼠皮质BDNF mRNA、NGF mRNA、GDNF mRNA、bFGF mRNA、TGF-pmRNA相对表达量提高,PDGF mRNA相对表达量降低；其中尼莫地平片组大鼠皮质GDNF mRNA相对表达量及银杏叶片组BDNF mRNA、GDNF mRNA相对表达量较高(P<0.05)；额尔敦-乌日勒组大鼠皮质BDNF mRNA、NGF mRNA、GDNF mRNA、bFGF mRNA、 TGF-βmRNA相对表达量最高,而PDGF mRNA相对表达量最低(P<0.05)。与尼莫地平片组相比,银杏叶片组大鼠皮质bFGF mRNA相对表达量最高(P<0.05)；额尔敦-乌日勒组大鼠皮质BDNF mRNA、NGF mRNA、GDNF mRNA、bFGF mRNA、 TGF-βmRNA相对表达量最高,而PDGF mRNA相对表达量最低(P<0.05)。与银杏叶片组相比,额尔敦-乌日勒组大鼠皮质BDNF mRNA、NGF mRNA、GDNF mRNA、 TGF-βmRNA相对表达量增高,(P<0.05)结论：1.额尔敦-乌日勒对局灶性脑缺血再灌注损伤有较好的治疗作用。(1)通过神经功能Bederson评分额尔敦-乌日勒能显著减轻大鼠神经功能损伤,具有改善神经功能恢复作用。(2)额尔敦-乌日勒能显著减少脑梗塞苍白区面积。(3)额尔敦-乌日勒能显著降低脑梗塞率、脑含水率、脑指数。2.额尔敦-乌日勒能够通过诱导刺激内源性神经营养因子的分泌,加强神经营养因子的支持作用,促进了脑缺血损伤的恢复。(1)额尔敦-乌日勒可通过调节BDNF、NGF、GDNF、bFGF、TGF-β、PDGF等阳性细胞的内分泌,降低神经细胞损伤密度,并且减少神经元缺失或变性坏死(范围及程度),减轻细胞间质水肿,增多正常细胞数量、减轻阳性细胞反映强区域的病理变化,进而起到对受损神经元的保护或修复作用。(2)额尔敦-乌日勒可通过增加BDNF、NGF、bFGF、TGF-β蛋白含量,减少PDGF蛋白含量等途径发挥其靶源性营养作用和神经保护作用,进而达到保护或修复受损神经元、减轻脑缺血损伤的作用。(3)额尔敦-乌日勒可通过上调BDNF mRNA、NGF mRNA、GDNF、RNA、bFGF mRNA、TGF-βmRNA相对表达量,下调PDGF mRNA相对表达量等途径发挥其靶源性营养作用和神经保护作用,进而达到保护或修复受损神经元,抑制脑缺血损伤的作用。总之,额尔敦-乌日勒可能通过促进诱导MCAO/R损伤大鼠海马及皮质神经营养因子BDNF、NGF、GDNF、bFGF、TGF-p、PDGF等具有相互协同作用的NTFs的内分泌,发挥其靶源性营养作用和神经保护作用,进而达到保护或修复受损神经元,减轻脑缺血损伤的作用。其额尔敦-乌日勒的安神、舒筋活络功效可能与相互协同作用的NTFs的内分泌有关。该结果证实了额尔敦-乌日勒修复“白脉”损伤的传统功效。更多还原

【Abstract】 [Obejctive]:Our work is devoted to study the pharmacology effect of Eerdun-Wurile on the Neurotrophic factors (NTFs) in middle cerebral artery occlusion/reperfusion injured rats’hippocampus and cortex, which demonstrates the effect of Eerdun-Wurile on focal cerebral ischemia reperfusion and knows its mechanism. The result shows Eerdun-Wurile has therapeutic effects of ICS and provides data for for the further study.[Method]:Using modified Zea-Longa line plug method to build MCAO/R model. There are5groups in all. Successfully modeled rats were randomly divided into four groups:Model group, Nimodipine tablets group, Ginkgo Leaf tablets group, Eerdun-Wurile group plus the sham operation (SO) group. The SO group and model group with distilled water of ig for14consecutive days; Eerdun-Wurile group, Nimodipine tablets group and Ginkgo Leaf tablets group with dose administration61.7mg/100g,6.17mg/100g,12.34mg/100g,1times/d of ig for14consecutive days. Then carry out Bederson neurological score and evaluate TTC staining then measure the cerebral index, cerebral water ratio and cerebral infarction rate. Using HE, SP immunohistochemical staining methods such as ELISA and RT-PCR to detect the content of NTFs and gene expression in hippocampus and cortex in each group to evaluate the pathological changes in brain tissue. Using SPSS14.0statistical software analyze the data.[Result]:(1) Assessment of the establishment of MCRO/R injured rats model:After evaluate Bederson neurological score and TTC staining, the model manufacturing group:left eyes were much smaller than right eyes and dimmed; There were obvious symptoms like turning right, right pushing resistance, retracting right claws; And pale brain tissue in local ischemic region which mainly located in the left hippocampus and cortex. And the cerebral infarction was relatively stable(2) Bederson neurological score:SO group had no neurological deficit symptom; Model manufacturing group had significant neurological deficit(P<0.05); After15d treatment, the rats of model group had worsened spirit, activity, appetite, excretion and hair gloss. Other symptoms including losing weight and significantly smaller and dimmed left eyes. There were no significant relief of turning right, right pushing resistance and retracting right claws; The Bederson neurological score of drug groups were apparently lower than the model group which neurological function improved obviously (P<0.05). Eerdun-Wurile group had the lowest score (P<0.05) among them and no significant difference with So group (P>0.05). The nerve function deficient recovery was almost normal in Eerdun-Wurile group and had no significant difference with Nimodipine tablets group and Ginkgo Leaf tablets group (P< 0.05). These tests revealed that after the rats got MCAO/R injured, the neurological deficit become more serious in acute stage. Through different treatment the nerve function deficient recovery will be improved. The result shows that Eerdun-Wurile can reduce neurological deficit and improve neurological function recovery.(3) TTC staining results:There was no pale tissue in SO group. The model group had more clear pale tissue than drug groups. In drug groups, Eerdun-Wurile group was the least and the other two had no significant difference.(4) The comparison of the cerebral infarction rate, cerebral water ratio and cerebral index: The cerebral infarction rate and cerebral index of Eerdun-Wurile group were apparently lower than Nimodipine tablets group (P<0.05) and no significant difference between Ginkgo Leaf tablets group (P>0.05). Cerebral water ratio of Eerdun-Wurile group was significantly lower than the model group and Ginkgo Leaf tablets group (P<0.05) and no significant difference with Nimodipine tablets group (P>0.05). The results illustrated that when the rats get MCAO/R injured, the cerebral infraction region maximized along with serious cerebral oedema. Through various treatment the cerebral infarction rate, cerebral water ratio and cerebral index can be decreased. Among them, Eerdun-Wurile group and Ginkgo Leaf tablets group have a better effect of lowering cerebral infarction rate and cerebral index (P<0.05) than Nimodipine tablets group. On the other side, Eerdun-Wurile group and Nimodipine tablets group can remarkablely lower cerebral water ratio with high efficiency compared with Ginkgo Leaf tablets group.(5) The hippocampus and cortex pathomorphology result:The hippocampus and cortex cells’forms and structures were normal and arranged in order; There was no cell adhesion and embedded plug the gap without edema surrounding cells, no inflammatory cell infiltration and no infarction occurs. The hippocampus and cortex of model group turned pale and had clear outlines between surroundings and membrane; Cytoplasm was concentrated and red-stained. The nucleus pycnosised into triangle, dissolved or divided, the nerve fibers’vacuolated or softening was serious. There were a lot of inflammatory cells or lymphocyte infiltrations; And nerve cell became degenerated and necrosis thinning or atrophic, irregular disordered, oedematous osteoporosis. Each group’s hippocampal and cortical layers of varying degrees of pathological changes were reduced.The Eerdun-Wurile group and the Ginkgo Leaf tablets group’demaged nerve cells’density decreased significantly(P<0.05), neuronal loss reduced; Interstitial edema, degeneration and necrosis zones were narrowed and dropped. Immunohistochemical localization analysis showed:the positive cells were nerve cells, gliocytes, cytoplasm and cell membrane and the protuberances were dark nankeen. The positive cells were obvious in the ischemic penumbra. And the expression of BDNF, NGF、 PDGF positive cells in the cortex were more than the hippocampus area.Meanwhile, the GDNF, bFGF, TGF-β positive cells in the hippocampus had surpassed the cortex area. The SO group had the least BDNF, NGF, GDNF, bFGF, TGF-β, PDGF positive cells in its hippocampus and cortex area (P<0.05).Each drug group had more BDNF, NGF, GDNF, bFGF, TGF-β positive cells and less PDGF positive cells than the model group (P<0.05). Eerdun-Wurile group have the largest amount of BDNF、NGF、GDNF、bFGF、TGF-P positive cells in hippocampus and cortex and the least amount of PDGF positive cells (P<0.05). Ginkgo Leaf tablets group had more BDNF、NGF positive cells than Nimodipine tablets group, but GDNF、bFGF、TGF-β、PDGF positive cells had no significant difference between the two group.(P>005)(6) Measuring BDNF、NGF、GDNF、bFGF、TGF-β、PDGF protein content in the cortex and hippocampus by DAgS-ELISA ELISA:BDNF、bFGF、TGF-β、PDGF protein content in cortex of were higher than (P<0.05) hippocampus, while PDGFcontent in hippocampus was higher than cortex; Obviously the hippocampus area had much PDGF protein than the cortex area in So group and the model group (P<0.05). Each drug group had a higher PDGF protein content in cortex than in hippocampus(P<0.05). The So group had the lowest BDNF, NGF、GDNF、bFGF、TGF-β、PDGF protein content;The drug group had higher BDNF, NGF, GDNF bFGF, TGF-P protein content and lower PDGF protein content (P<0.05); Eerdun-Wurile group had the largest amount of BDNF、NGF、GDNF、bFGF、TGF-βand lowest amount of PDGF protein content in the hippocampus and cortex (P<0.05);Compared with Nimodipine tablets group, Ginkgo Leaf tablets group had a higher NGF protein content and lower PDGF protein content (P<0.05) and had no significant difference of BDNF、GDNF、bFGF、TGF-βprotein content (P>0.05)(7) Measuring BDNF、NGF、GDNF、bFGF、TGF-β、PDGF gene expression byRT-PCR:So group had lower relative quantitative expression of BDNF mRNA、NGF mRNA、GDNF mRNA、bFGF mRNA、TGF-βmRNA、PDGF mRNA.Compared with the model group, each drug group have higher BDNF mRNA、NGF mRNA、GDNF mRNA、bFGF mRNA、 bFGF mRNA、TGF-βmRNA relative quantitative expression and lower PDGF mRNA relative quantitative expression. Among them, GDNF mRNA relative quantitative expression in cortex of Nimodipine tablets group and BDNF mRNA、GDNF mRNA relative quantitative expression of Ginkgo Leaf tablets group were relatively high (P<0.05); Eerdun-Wurile group had highest BDNF mRNA、NGF mRNA、GDNF mRNA、bFGF mRNA、TGF-βmRNA and lowest PDGF mRNA relative quantitative expression (P<0.05). Compared with Nimodipine tablets group, Ginkgo Leaf tablets group had the highest bFGF mRNA relative quantitative expression (P<0.05). Eerdun-Wurile group had the highest BDNF mRNA、NGF mRNA、 GDNF mRNA、bFGF mRNA、TGF-βmRNA and lowest PDGF mRNA relative quantitative expression in cortex (P<0.05).Compared with Ginkgo Leaf tablets group, Eerdun-Wurile group had relative high BDNF mRNA、NGF mRNA、GDNF mRNA、TGF-βmRNA relative quantitative expression (P<0.05).[conclusion]:1.Eerdun-Wurile have curative effect on focal cerebral ischemia reperfusion.(1)Bederson neurological score shows Eerdun-Wurile can lessen neurological deficit and improve nerve function.(2)Eerdun-Wurile can decrease the pale infraction area.(3)Eerdun-Wurile can remarkably reduce cerebral infarction rate, cerebral water ratio and cerebral index.2. Eerdun-Wurile can stimulate the secretion of endogenous NGF, enhance NGF function and promote the recovery of ischemia injury.(1) Eerdun-Wurile can adjust the incretion of BDNF、NGF、GDNF、bFGF、TGF-β、 PDGF in the positive cell to reduce damaged nerve cells density, neuronal loss, necrosis narrow,the extent of interstitial edema and the histopathological changes of positive cells strongly reacted area. Meanwhile increase normal cells to repair injured neurons.(2) By increasing BDNF, NGF, GDNF,bFGF, TGF-β protein content and inhibiting the growth of PDGF protein, Eerdun-Wurile has pharmacology function of target-derived nutrition mechanism and neuroprotective effect to protect and repair the damage in cerebral ischemic in rats.(3) By increasingDNF mRNA、NGF mRNA、GDNF mRNA、bFGF mRNA、 TGF-βRNA relative quantitative expression and inhibiting the PDGF mRNA relative quantitative expression, Eerdun-Wurile has pharmacology function of target-derived nutrition mechanism and neuroprotective effect to protect and repair the damage in cerebral ischemic in rats.In general, Eerdun-Wurile can induce BDNF、NGF、GDNF、bFGF、TGF-p、 PDGF coordinative NTFs’ endocrine. Thus have the function of target-derived nutrition mechanism and neuroprotective effect to protective and repair the damage in cerebral ischemic. The result shows the traditional effect of Eerdun-Wurile on repairing nerve vascular.更多还原