| [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. |
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