| After Ischemic brain tissue restoring blood supply in certain conditions, there was a more serious brain dysfunction and structural damage, known as cerebral ischemia-repe-rfusion injury, which is Common in shock, DIC reperfusion of microcirculation and Cerebrovascular embolization through again, cardiopulmonary cerebral resuscitation etc. Ischemic cerebral vascular disease is a neurological disease, which has a high rates of disability and death. The treatment principle is to restore a blood supply, but which also brought a reperfusion injury. Inhibiting reperfusion injury have become a important part of treatment of ischemic cerebral vascular diseases. With modern medicine on cerebral ischemia-reperfusion injury in deepening understanding of pathophysiological mechanisms, in recent years, Chinese medicine in preventing and treating cerebral ischemia-reperfusion injury of rapid development, related to Pharmacology, pharmacology, mechanism of acupuncture, and other content. Many studies have confirmed that Chinese medicine has a protective effect on cerebral ischemia-reperfusion injury.TongLuoHuaTan capsule was invented by Professor Fan Jiping according to the Antihypertensive and Syndrome classification method of stroke disease determined by the book "Diagnosis and clinical efficacy evaluation standards of stroke", and selecting the most common stroke type of wind phlegm blood stasis bi resistance thread card, combinating mentor Wang yongyan fellow's academic thought "heat-toxic affecting the brain collaterals", and with himself s clinical treatment experience of stroke disease. The capsule is made by gall powder, Gastrodia elata, Sanqi, Salvia miltiorrhiza, Tianzhu yellow, and rhubarb, which has a effect of promoting blood circulation, removing obstruction in channels, peliminating phlegm, calming wind. Its major effects are treating stroke precursor caused by phlegm-stasis in channels, syndrome of intermingled phlegm and blood stasis,which show symptoms of hemiplegia, facial paralysis, stiff tongue and impeded speech, or aphasia, hemianesthesia, slobbering, lip color a dark, tongue thick and greasy, tongue qualitative dark, petechia or bruise, tongue base vein stasis and dark, pulse astringent or wiry slide.Early effect experimental research of TongLuoHuaTan capsule indicates that TongLuoHuaTan capsule can clearly improve hemiplegia signs and the nervous system symptoms of large rat due to brain deficiency blood, narrow brain infarction focal area, clearly reduce the permeability of blood vessel of brain when it is cerebral ischemia, and reduce the moisture of brain organization, reduce brain edema and secondary lesion of neurons, significantly reduce large rat Council focal sexual brain deficiency blood of pathology morphology change, reduced pathomorphology change in focal cerebral ischemia of rat, clearly reduce platelet aggregation rate in focal cerebral ischemia of rat, significantly delay rats blood clots forming time, suppression thrombosis formed, Also can enhance the ability of hypoxia in mice.This experiment tried to study the neuroprotective effects and mechanism of TongLuoHuaTan capsule and its effective components of bear gall, provide objective experiment basis of TongLuoHuaTan capsule in preventing and treating cerebral ischemia-reperfusion injury.Research objectives:1. Discuss on neuroprotective effect of TongLuoHuaTan capsule and provide objective experiment basis of TongLuoHuaTan capsule in preventing and treating cerebral ischemia-reperfusion injury.2. Discussion on neuro-protective effect and mechanism of the effective components of bear gall of TongLuoHuaTan capsule.Research methods:Using Suture-occluded method to simulation focal cerebral ischemia-reperfusion injury rat model, test experimental animals'neurological recovery degree According to the nerve functional score and behavioral testing methods, test brain's pathology change by using HE dyeing and Nepal's dyeing method, and using immunohistochemical staining to detect the expression of GFAP, NF-kB, TNF-a, ICAM-1, IL-1beta, IL-8in brain organization, using westernblot technology to detect the protein expression of GFAP, and NF-kB in the brain organization, using RT-PCR method to detect the gene expression of GFAP and NF-kB in the brain tissue.Research results:1. Nerve behavior results of Cylinder Test1day after surgery, asymmetric score of sham-operation group is increased compared with the blank Group (P<0.05), and then back to normal level (compared with the blank control group P>0.05).1day after surgery, asymmetric score of the model group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05).1day after surgery, asymmetric score of the positive drug group is significantly increased compared with the blank control group (P<0.05), which did not return to normallevels on the7th day (P<0.05), asymmetrical score of the7th day is reduced compared to the model group (P<0.05).1day after surgery, asymmetric score of the low dose group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), asymmetrical score of the7th day was no statistical difference compared to the model group (P>0.05).1day after surgery, asymmetric score of the medial-dose group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), asymmetrical score of the7th day is lower compared to the model group and low dose group (P<0.05), which has no statistically significant difference compared with positive Group (P>0.05).1day after surgery, asymmetric score of the high-dose group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), asymmetrical score of the7th day is lower compared to the model group and low dose group (P<0.05), which has no statistically significant difference compared with positive Group, middle-doze group(P>0.05).1day after surgery, asymmetric score of the bear bile powder group is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), asymmetrical score of the7th day is lower compared to the model group and low dose group (P<0.05), which has no statistically significant difference compared with positive Group, middle-doze group and high doze group (P>0.05).2. Nerve behavior results of Narrow-alley Corner TestSham-operation group:1day after surgery, turning score is increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05). Model group:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normallevels on the7th day (P<0.05). Positive groups:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05). Turning score of the7th day is lower compared to the model group (P<0.05). Low dose group:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), Turning score of the7th day is no statistical difference compared to the model group (P>0.05).Middle-dose group:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), Turning score of the7th day is reduced compared to the model group and low-doze group (P>0.05). High dose group:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), Turning score of the7th day is reduced compared to the model group and low-doze group (P<0.05), which has no statistically significant difference compared with positive Group, middle-doze group (P>0.05). Group of bear bile powder:1day after surgery, turning score is significantly increased compared with the blank control group (P<0.05), which did not return to normal levels on the7th day (P<0.05), Turning score of the7th day is reduced compared to the model group and low-doze group (P<0.05), which has no statistically significant difference compared with positive Group, middle-doze group and high doze group (P>0.05).3. Pathological morphological changes in brain tissues of ratsMacroscopic observation shows that blank group, Sham-operation group, positive drug group and the high-dose group, middle-dose groups and low dose groups and model group for a total of eight group no significant pathological changes of rat brain.Microscopically HE dyeing shows the blank set of brain tissue in rats, cerebellar neuronal has no pathological changes at all levels, no degeneration in cerebellar Purkinje cells, and structural is normal. Sham-operation set of brain tissue of rat has no deformation on cerebellar neurons at all levels in the brain,, small vessels have mild congestion, no significant disease. Model set of brain tissue in rats has ischemic change on cerebrum and cerebellum, cell's nissl bodies disappears, cell nuclear pyknosis, nucleoli disappear, some staining to be gray, degeneration; some boundaries around the cell membrane are clear, stained uniform. Purkinje cells of the cerebellum and hippocampal cells do not see obvious changes. Three dose groups, drug-positive Group and by the Group of bear bile powder in rat brain: compared with the model group, all have different degree change of nerve cells of the brain.Nissl staining microscopic observation shows in the blank control group Nissl's body shows more in brain cell of rat. Sham-operation group shows more Nissl's body in the brain cells of rats. Model groups of brain cells of rats have a marked decline in Nepal's small body. Three dose groups, drug-positive Group and bear bile powder group, compared to the model Group's smaller body, have increased in different levels.4.7days after Rats'ischemia-reperfusion, using immunohistochemistry to determine deficiency blood then perfusion using Immunohistochemical method to detect the expression of GFAP, and NF-k b, and TNF-a, and ICAM-1, and IL-1β in thel brain tissue of rats, the results display that the expression of GFAP in Hippocampus CA1area of high doze group, middle doze group, low-doze group, bear gall group are significantly lower than that of model group (P<0.05), the expression of GFAP in cortex area of positive drug group is lower than than of model group (P<0.05), that of high doze group, middle doze group, low-doze group, bear gall group and positive drug group have no significant differences compared to model group (P>0.05); The expression of NF-Kb of drug-positive Group, and the low-dose group are lower than that of the model group (P<0.05). The expression of TNF-a, ICAM-1, IL-1β and IL-8in hippocampus and cortex area of brain tissue of model group are higher than than of the blank controller group and sham-operation group (P<0.05); The expression of NF-Kb of high doze group, middle doze group and bear gall group are all significantly lower than that of positive drug group (P<0.05), compared with the model group, the expression of ICAM-1of doze groups, positive drug group and bear gall group are lower (P<0.05), the reduce level of the expression of ICAM-1of the high doze group and bear gall group are higher than that of positive drug group and low doze group (P<0.05).5.7days after Rats'ischemia-reperfusion, using westernblot technology to detect the protein expression of GFAP and NF-kB in the brain organization, results shows:the protein expression of GFAP of doze groups, bear gall group and positive drug group are all lower than that of model group (P<0.05), and the protein expression of GFAP of doze groups have no difference (P>0.05). The protein expression of NF-kb in doze groups, bear bile group, positive drug group are less than that of the model group (P<0.05). The protein expression of NF-Kb in high doze group, middle doze group and bear bile group are significantly less than that of the low dose group (P<0.05).6.7days after Rats,ischemia-reperfusion, to detect the mRNA expression of GFAP and NF-kB in the brain organization, results shows:the mRNA expression of NF-kB of doze groups, bear gall group and positive drug group are all lower than that of model group (P<0.05), the mRNA expression of NF-kB of doze groups have no difference (P>0.05); the mRNA expression of GFAP of doze groups, bear gall group and positive drug group are all lower than that of model group (P<0.05), the mRNA expression of GFAP of doze groups have no difference (P>0.05).Conclusions:TongLuoHuaTan capsule and its active ingredients bear bile powder have a Significant neural protection, can reduce cerebral ischemia-reperfusion injury in rats with Brain tissue damage, which may due to its reduce the expression of TNF-alpha, NF-k b, ICAM-1, IL-1, R, IL-8, of GFAP and other related factors after ischemia-reperfusion injury in cerebral organization.
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