| Purpose of researchThe inhibitory effect of Traditional Chinese Medicine on cerebralischemia-reperfusion injury is an important topic. With the development of the society, the incidence of the cerbralvascular diseases keeps increasing and become one of themajor diseases threatening the existing and health of the human kind.Ischemia-reperfusion is one of the important pathologic processes in the developmentprocess of Cerbralvascular Disease. It can induce further tissue injury and dysfunction.To control the ischemia-reperfusion injury has become the key factor in the treatmentof ischemic stroke in the present day. At present, the mechanism of the medicine forcerebral ischemia diseases is usually simple and it often has big side effects. On theother hand, Tradition Chinese Medicine has multi-level and multi-link treatmenteffects. So, research on the mechanism of the TCM treatment of cerebral ischemia isvery important in theory and clinic.The Cerbralvascular Disease is closely associated with Blood stasis syndrome(TCM). The TCM treatment of blood stasis syndrome always adopts the role ofblood-activating and stasis-resolving and use drags that regulated flow of qi incombination to invigorate qi and promote blood circulation. Chuanxiong is ablood-activating and stasis-resolving medicine which is often used in clinic. It has notonly the function of promoting blood, but also the effect of invigorating qi. It is one ofthe common TCM medicines for the treatment of ischemia cerbralvascular disease inclinic. To explain the mechanism of the inhibitory effects of Chuanxiong onischemia-reperfusion is very important for the research on the TCM treatment ofischemic encephalopathy.Based on the theory of Traditional Chinese Medicine and Modern Medicine about Cerebral ischemia disease, we will do the following research: (1)Prove that theeffective compound of Chuanxiong- Ligustrazine Hydrochloride can go through thenormal and Ischemia-Reperfusion Blood-Brain Barrier. Provide the evidence that ithas direct effect on brain tissues. (2) Do the multi-level research systemically on thefunctions of Ligustrazine Hydrochloride in the process of anti Ischemia-Reperfusion.Hope this research can provide the theory basis for using Chuangxiong orLigustrazine Hydrochloride in the treatment of cerbralvascular disease in clinic, andprovide a methodology platform of screening the TCM for treating cerebral ischemiadiseases.Research methodWe used SD rats as research objects. We prepared the model of Focal CerebralIschemia-reperfusion of Middle Cerebral Artery in rats, and studied the inhibitorymechanism of Ligustrazine Hydrochloride on ischemia-reperfusion injury.(1) To test if the Ligustrazine Hydrochloride can go through the Blood-BrainBarrier of the normal rats and the rats with cerebral ischemia-reperfusion injury andreach the brain tissue using High-performance Liquid Chromatography (HPLC); (2)Measure the weight of rat brain tissues of the normal group, ischemia-reperfusiongroup and other treatment groups. And the water contents in brain tissue weremeasured. (3) Stain by HE staining, and observe the shape change of the brain cell ofdifferent groups. (4) Observe the expression of GFAP using the method ofimmunohistochemistry stain. And show the change of the astrocyte; (5) Measure thechange of contents of NOS and iNOS in serum of rats in each group usingbiochemical method. (6) Measure the change of contents of SOD in serum and brainhomogenate of rats in each group using biochemical method; (7) Measure the changeof brain cell proliferation cycle and apoptosis of rats in each group using FCM; (8)Measure the expression change of hypoxia-inducible factor 1 alpha in the brain tissueof rats in each group using Real-Time PCR.ResultsLigustrazine Hydrochloride can be detected in the brain tissues of rats in eachgroup. That indicates it can go through the Blood-Brain Barrier, reach the brain and has effects on central nervous system. This result provides the premise conditions ofthe research of the effects and mechanisms of Ligustrazine Hydrochloride or evenChuanxiong on brain. (2) Water content of ischemia-reperfusion group is obviouslyhigher than sham-operation group (P＜0.01), and the water content of three medicinegroups is apparently reduced comparing with ischemia-reperfusion group (P＜0.05).The change is more apparent in the Ligustrazine Hydrochloride high-dose group andnimodipine group. It indicates that Ligustrazine Hydrochloride can apparently reducethe swelling dimension of contralateral hemisphere, and reduce the water content inthe lesion hemisphere. (3) The brain tissue of rats in ischemia-reperfusion groupswelled apparently and the appearance is dim. It can be seen under microscope thatthe pyramid cells are arranged disorderly, the cell space got widening and neurondegeneration is apparent. Some degenerated neuron appear apoptosis: The cells shrinkto rounded or ovoid shape. The cytoplastic shrinks and eosinophilic character increase.Homogeneous red-stained necrotic material, nucleus condensation and dissolution, and other necrotic features also can be seen. The degree of degeneration wassignificantly reduced in medicine group than in ischemia-reperfusion group. And it ismore apparent in the Ligustrazine Hydrochloride high-dose group and nimodipinegroup. Only a little of pyramid cells were deeply stained and had nucleuscondensation. (4) ischemia-reperfusion group’ s GFAP immunostaining was strongpositive. Cell body is mast, stain is deepening and the neurite is thickening and gettinglonger. But sham-operation group only had very few positive cell expressions. Theimmunostaining of medicine only had positive-feebly positive change. Thisdemonstrates that AST reactive hyperplasia is decreased and suggest that LigustrazineHydrochloride can inhibit the over-expression of AST with ischemia-reperfusion. Thecontents of NOS and iNOS in serum of rats in ischemia-reperfusion group issignificantly increased than sham-operated group. The contents of NOS and iNOS inserum of rats in medicine group decreased. And it is more apparent in the LigustrazineHydrochloride high-dose group and nimodipine group (P＜0.05). It suggested thatLigustrazine can decrease the content of NOS, especially iNOS, whenischemia-reperfusion occurs. Reduce the synthesis of NO. Reduce the nerve toxic reactions and the NO-induced neuronal apoptosis. (6) Activity of SOD in the serumand brain tissue of the ischemia-reperfusion group significantly decreased (P＜0.01), Activity of SOD in the serum and brain tissue of the three medicine group allincreased. And it is more apparent in the Ligustrazine Hydrochloride high-dose groupand nimodipine group (P＜0.05). It suggests that igustrazine can increase the activityof SOD in brain tissue, and help to eliminate free radicals, and reduce lipidperoxidation injury. (7) After ischemia-reperfusion, noticeable changes had takenplace in the cell cycle. The percentage of cells in G0/G1 phase decreased apparently.The cells entered S and G2/M phase increased a lot. It had a significantly differencecomparing with sham-operated group (P＜0.05). It shown that, afterischemia-reperfusion injury, cells were very active, and can reenter the cell cycle. Inthe rate of cell apoptsis, the brain cell of rats in the ischemia-reperfusion group is(21.61±4.4)%, a significantly increase comparing with sham-operated group(P＜0.05). The rate of cell apoptsis in the Ligustrazine Hydrochloride high-dose groupand nimodipine group is significantly lower than ischemia-reperfusion group (P＜0.05).The Ligustrazine Hydrochloride high-dose group had the biggest change. (8) Theexpression of HIF-1αin the ischemia-reperfusion group was much higher thansham-operated group measured by Real-Time PCR. It is lower in the LigustrazineHydrochloride low-dose group. But it significantly decreased in the LigustrazineHydrochloride high-dose group and nimodipine group. The nimodipine group isalmost same as sham-operated group.Conclusions and significanceThe mechanism of the inhibitory effect of Ligustrazine Hydrochloride onischemia-reperfusion is a hot topic in recent years and has got significant results. Butit still has some problems: (1) the researches are mostly focused on the mechanism ofusing Ligustrazine Hydrochloride to treat the ischemic heart disease. But the researchon the mechanism of the inhibitory effect of Ligustrazine Hydrochloride on cerebralischemia-reperfusion injury is much less. This is inconsistent with the widely using ofLigustrazine Hydrochloride in the treatment of Cerebrovascular Disease. (2) Most ofthe researches are focused the regulation of Ligustrazine Hydrochloride on the functional indices, and didn’ t prove it can pass through the BBB and has directeffects on central nervous system. Although some scholars prove that LigustrazineHydrochloride can go through the BBB, there isn’ t report about whether it still cango through the BBB, with ischemia-reperfusion and damaged BBB. Also there isn’ treport about the whether there is any difference in the going through level comparingwith the normal cases. (3) The research method is relatively simple. Most scholarsonly research on one aspect of the mechanism of the inhibitory effect of LigustrazineHydrochloride on ischemia-reperfusion. But the pathophysiological changes ofcerebral ischemia-reperfusion are in many ways and at different levels. So, it isobviously not comprehensive to study the effect of Ligustrazine Hydrochloride ononly one pathology link. In addition, because different researchers use differentischemia-reperfusion model, the various experimental results don’ t have thehorizontal comparison feasibility. These problems induced that the research onmechanism of the inhibitory effect of Ligustrazine Hydrochloride on cerebralischemia-reperfusion is not systemic and holistic.The innovation of this experiment proves is (1) Proveing LigustrazineHydrochloride can go through the normal and Ischemia-Reperfusion Blood-BrainBarrier, reach the brain tissue and has direct effect on brain tissues. And theincensement of the content of Ligustrazine Hydrochloride in the ischemia-reperfusionbrain tissue after medical treatment, indices that in this case, more medicine can reachthe brain tissue. This provides the preconditions for the research on how it reaches thebrain tissue and has direct effect on brain tissues. (2) This study descripts themechanism for the treatment of cerebral ischemia-reperfusion injury usingLigustrazine Hydrochloride at the overall level, and the level of cells, molecules andgenes according to a variety of pathological changes. (3)This experiment used a lot ofmeasure methods, among which the Real-time PCR is at the advanced technologicallevel. Comparing with traditional PCR technology, it realized not only the leap from aqualitative level to a quantitative level, but also has the characters of more specificity, effectively solving the pollution problems of PCR, and a higher degree ofautomation. (4)Experiment proved that Ligustrazine Hydrochloride has protection function on cerebral ischemia-reperfusion injury. The mechanism of effect is closelyrelated to decreasing brain edema, adjusting the reaction of AST to injury, anti-freeradical damage, reducing the toxicity of NO, preventing vasospasm, anti-apoptotic, reducing nerve cell degeneration and necrosis, Gene regulation and other aspects.(5)This experiment built a whole set of experimental methodology platform forfurther research on going through the BBB of TCM and anti Ischemia-Reperfusion, and also has great significance to promoting the modernization of traditional Chinesemedicine. |
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