| ObjectiveThis study was aimed to discuss the pathogenesis and treatment of vascular dementia,and to explore the biological basis of the "kidney essence deficiency" in the process of vascular dementia development,and then to study modified shuyu pill’s effects of preventing vascular dementia and the mechanism. Method 1 Theoretical research:Access to relevant literature, starting from the basic theory of traditional Chinese medicine "kidney essence" theory, and combining with advances in the study of modern TCM syndrome differentiation and treatment of VD, we discuss the pathogenesis and treatment of vascular dementia. We traced back modified shuyu pill,and analysed the mechanism how it treated vascular dementia combining with the research achievements before. 2 Experimental study:120 SPF healthy adult male wistar rats having been raised adaptabilityly for 1 week,were divided into 4 groups by randomly:1.normal control group(referred to as the normal group hereinafter): 20 rats, without surgery, starting on the third day after modeling, with irrigation normal saline 10ml/kg/d*8 week.2.Sham operation group:20 rats were performed at neck skin partially,isolating carotid artery, neither ligation nor ligation.Then we sutured the skin directly after being exposured for 5 minutes.At the beginning of the third day after modeling, the rats were given normal saline 10ml/kg/d*8 weeks.3.The vascular dementia model group(hereinafter referred to as model group): The rats were conducted behavioral screening after modeling(modified 2-VO method),and then were randomly divided into two groups(model group and treatment group), with irrigation saline 10ml/kg/d*8 weeks.4.modified Dioscorea pill group(hereinafter referred to as the treatment group): grouping method was the same as that of the model group. On the third day after modeling, the rats were given irrigation stomach modified Dioscorea pill 10ml/kg/d*8 week.Record the general condition of rats,the recorded in different time points in rats death number to calculate the survival rate of rat. Morris water maze test in rats.HE staining under light microscope to observe the pathological sections of rat hippocampus.TUNEL fluorescence staining under optical microscope observation of cell apoptosis in hippocampus of rats. Using immunohistochemical method to observe the expression of ERK5 / Bm K1 related upstream signal factor(mekk2, mekk3, MEK5) and ERK5,and using the method of Western blot detection of mekk2, mekk3, MEK5, ERK5 protein expression. Result 1 Theoretical research:"Kidney essence deficiency" is the basic pathogenesis of VD: kidney essence deficiency is the occurrence of VD. Deficiency of kidney essence can be phlegm, blood stasis, phlegm and blood stasis in turn makes kidney worse, and ultimately the formation of "the true mingled with the false" vicious spiral. Kidney deficiency, phlegm and blood stasis is the premise of reciprocal causation influence each other, the occurrence of VD, and also the development and change of the inherent power of VD. So in the differentiation and treatment of VD, although the "deficiency of kidney essence" is the basis of VD, when reinforcing kidney fill fine puzzle and invigorating the spleen and eliminating phlegm for resuscitation, nourishing blood and promoting blood circulation Tongluo simultaneously to attack stubborn ills.The treatment of VD can be from kidney legislation: through the positive effect of Chinese medicine "Bushen" law treatment of VD, combining the research with modern technology of medical research results at home and abroad, indicating the close relationship between the function of brain and kidney and brain virtual lesions of routine treatment with "kidney".Analysis of the mechanism of modified Dioscorea pill in the treatment of VD: modified Dioscorea pill is by in Synopsis of prescriptions of the Golden Chamber "Shuyuwan" cut and, with "tonifying kidney and marrow educational" effect; tutor’s research team found in the previous study, modified Dioscorea pills in intervention of cognitive disorder made good clinical curative effect, and from the behavior, pathology and molecular biology and other aspects to some extent reveals the VD the pathological mechanism and process of development. 2 experimental result: 2.1 General state of rats in each groupNormal group: during the whole experiment, hair, nails, lips, eyes, spirit and appetite were generally in good condition.Model group: Before modeling, experimental rats hair, nails, lips, eyes, spirit and appetite were generally in good condition. After modeling, appear different degree of hair color has withered dark, Chun Jiazi dark, spirit is dispirited and activity becomes less, sleep increased and decreased appetite; some rats appeared different degree of ptosis, eye fissure decreases and eye mild depression; individual rats appear abdomen gradually fullness of spherical, with obvious eating and decreased defecation, coat color rough dull limbs gradually weight loss and death. After a period of time after feeding or drug administration, the general situation was improved after the model was made. Compared with the normal group and sham operation group, the general situation was poor.Sham operation group: before the operation, the rats of hair, nails, lips, eyes, spirit and appetite were generally in good condition. The emergence of varying degrees of hair color, dull listlessness and less activity within 3 days after the operation, the rest of the good general condition. With the normal feeding, the general situation of basic recovery. Compared with the normal group, the difference was not significant.Treatment group: rats, rats hair, nails, lips, eyes, spirit and appetite were generally in good condition. After modeling, appear different degree of hair color has withered dark, Chun Jiazi dark, spirit is dispirited and activity becomes less, sleep increased and decreased appetite; some rats appeared different degree of ptosis, eye fissure decreases and eye mild depression; individual rats appear abdomen gradually fullness of spherical, with obvious eating and decreased defecation, coat color rough dull limbs gradually weight loss and death. After a period of time after feeding or drug administration, the general situation was improved after the model was made. Compared with the sham operation group, the general situation is bad; compared with the model group, the general situation is good. 2.2 Survival of rats in each group:Animal survival in all groups were detected by Log-rank(Mantel-Cox).There was statistical significancebetween the model group and the normal group(P<0.05).The normal group, model group, sham operation group and drug group survival rate were 95%, 68.57%, 83.33% and 54.28%, and the final survival animals accounted for 71.3% of all animals. 2.3 The results of Morris water maze test:Navigation experiment results showed that at different time points escape latency compared 7 days after modeling(gavage before), model group(116.47± 6.99) and treatment group(116.89±5.44) average escape latency were higher than those of the sham operation group(94.85±13.47); rats after gavage for 4 weeks, treatment group(89.92±3.20) lower than that of model group(11.7±564); after modeling gavage for 8 weeks, treatment group(87.15±1.274) lower than that of model group(117.54±5.05); above data by statistical treatment, there were significant difference,P<0.05.Space exploration, the experimental results show that the different time averaged across the platform times compared after 7 days(before gavage), model group(3.15±0.12) and treatment group(2.89±0.09) average over platform times were lower than in the sham operation group(8.20±0.21); treatment group after modeling in gavage for 4 weeks(4.16±0.18) was significantly higher than that of 7 days after modeling Guan Weiqian. After modeling irrigation stomach for 8 weeks, the treatment group(6.21±0.25) was higher than that in the model group(352±0.23); above data by statistical processing, there are significant difference,P<0.01 or P<0.05. 2.4 Pathological observation of hippocampal nerve cellsNormal group: vertebral cells arranged neatly, the cell shape is complete, the outline is clear, the nucleus structure is clear, the kernel is clear.Model group: vertebral cells were sparse, irregular arrangement of loose, cell spacing widened, vertebral body cell volume became smaller, the nuclear volume became smaller, the staining deepened, the kernel is not clear, some of the cell necrosis.Sham operation group:the vertebral bodies were arranged neatly, the cell morphology was complete, the outline was clear, the nucleus structure was clear, and the nucleus was clear.Treatment group: compared with the model group, vertebral cells arranged more neatly, cell density increased, the cell morphology and integrity, the outline is clear, the nuclear volume increased, the kernel is more clear, compared with the model group. 2.5 Apoptosis of hippocampal neurons induced by fluorescence stainingTake TUNEL staining under light microscope observation, apoptotic cells were green.Cell apoptosis is as follows: 7 days after modeling(gavage): model group(120.35±27.77) than those in the sham operation group(23.22 ± 1.235) apoptosis digital with increased; rats after gavage for 4 weeks.Treatment group(85.74±25.49) than in the model group(123.59±27.24) the number of apoptotic cells was significantly lower;rats after intragastric administration for 8 weeks.Treatment group(5.486 ± 23.59) than the model group(13.076 ± 30.08) apoptosis display significantly reduced; above data by statistical processing.There are significant difference,P<0.01 or P<0.05. 2.6 Expression of MEKK2, MEKK3, MEK5 and ERK5 in rats(immunohistochemistry)Comparison of MEKK2 expression levels in each group: 4 weeks after modeling, the treatment group(2.1250±0.2830) was higher than the model group(0.4083±0.6000), the statistical analysis,P<0.01. Comparison of MEKK3 expression levels in each group: 8 weeks after the model, the model group(0.3542±0.3753) was lower than the sham operation group(0.8333 + 0.6986), the difference was significant, with statistical significance,P<0.05. Comparison of the expression level of each group of MEK5: after modeling gavage for 4 weeks, treatment group(180±0.1375) higher than that of model group(0.6000±0.2819). By statistical analysis,P<0.05. After modeling irrigation stomach for 8 weeks, the treatment group(1.2368±0.5482) was higher than that in the model group(0.5100±0.3389), the statistical analysis,P<0.05. 2.7 MEKK2, MEKK3 and MEK5, ERK5 protein expression(Western Blot)Comparison of the expression level of each group of mekk3: after modeling gavage for 8 weeks, the model group(0.3312±0.2790 lower than sham operation group 0.5721 + 0.4345, treatment group(0.5787±0.3650) higher than that of model group(0.3312±0.2790); the above data after treatment, there is significant difference,P<0.05).Comparison of the expression level of each group of MEK5: after modeling gavage for 4 weeks, the model group(0.1927±0.1100) lower than sham operation group(0.4521±0.2666) and treatment group(0.3928±0.2766) was higher than that in the model group(0.1927±0.1100), the above data, the statistical analysis, P<0.05). ConclusionThrough the theoretical research, in-depth study of the etiology and pathogenesis of VD, suggesting that "deficiency of kidney essence" is the basic pathogenesis of VD and "Bushen" can clinical symptoms effectively improve the VD, modified Dioscorea pills in VD foundation as "kidney essence deficit can be symptoms associated with the treatment of VD deficiency, phlegm and blood stasis in collaterals" under the guidance of the theory.The improved 2-VO model can improve the survival rate of the model animals, and the characteristics of the model are consistent with the characteristics of the VD model. Through this model, we can further explore the mechanism of VD.Occurred in the model group rats survival state and behavior changes,structural changes of the hippocampus and down-regulation of MEKK2,MEKK3,MEK5 and ERK5 protein expression,downregulated the ERK5/BMK1 signaling pathway,inhibit the regeneration of hippocampal neurons, which may is after cerebral hypoxia ischemia incidence of VD or with heavy VD process of one of the most important molecular mechanism.Modified Dioscorea pill improve the survival state of VD rats,can improve the learning and memory ability of VD rats,improve the structure of hippocampal tissue is disorder,and decrease the apoptosis of hippocampal neural cells,and compared with the model group were significantly up-regulated the expression of MEK5 ERK5 specific upstream protein to activate ERK5/BMK1 signaling pathway,promote the regeneration of hippocampal neurons,which may is one of the mechanisms of the effect of modified Dioscorea pill in the treatment of VD.Modified Dioscorea pills may play a biological basis of VD therapy by inhibition of ERK5/BMK1 signaling pathway down to promote the repair and regeneration of hippocampal neurons. |
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