| Objective: The Protective function of zhongfengkang on blood brain barrier of focal cerebral ischemia rats was proved, and furthermore its function mechanism was probed into through the animal experiment. Methods: Part 1: The effect of zhongfengkang on BBB transparence, the brain edema and pathologic histology of focal cerebral ischemia rats 50 healthy rats in SD, the weight of 250-300 gs, were divided into five groups at random, that is, Sham operation group, model group, zhongfeng -kang high-dose group( call a high-dose group) , zhongfengkang low-dose group(call a low-dose group) and Buchangnaoxintong control group(name control group). each groups takes 3 rats as electric-microscope observation; each one takes 8 rats as examination of transparence of BBB and the brain edema.The rats of high-dose, low-dose group and control group were given medicine through watering stomach everyday from 5 days before operation to before death. The dose of zhongfengkang high-dose group is 18.8 g/ kg; The dose of zhongfengkang low-dose group is 9.4 gs/ kg; The dose of buchangnaoxintong group is 0.33 gs/ kg.The rats of sham operation group and control group were given 0.5% carboxymethylcellulose sodium liquid through watering stomach once a day. According to the report, To make the model of focal cerebral ischemia by inserting nylon thread, The rats belly were injected 10% chloral hydrate(the dose was 350 mgs/ Kg) ,after anaesthesia, it were laied on the back and fixed, the neck slices exact center, separating and exposing the right total carotid artery, outside carotid artery ,and inside carotid artery;in the outside and inside carotid artery crotch, outside carotid artery were ligated, the total carotid artery of the right side were sheared, inserting the nylon fish line head to burn a bluntness that diameter is the nylon fish line of the 0.25 mms, entering the line length about 18-19 mms. The middle carotid artery were blocked up in start of middle carotid artery,then the right total carotid artery and the nylon fish line were ligated together,sewed up the skin, put the rats back the cage inside, The rats keeped the single cage each one .The rats of sham operation group were set separate and exposed the vessel only, don't ligated the total carotid artery and outside carotid artery, don't inserted the nylon fish line.Choose the rat which wake up that left arms is crooked and runed about revolve toward left side or left limbs were paralysis as the successful model to block up to start experiment, otherwise the rats was seemed the failure, do not use them. The rat rectum temperature were measured in the surgical operation process; The rat rectum temperature were keeped in 36.5-37.5 ℃s by the electric blanket. After building the model rats the successfully in 6h,12h,24h,48h, the change of the rat BBB transparence and brain edema were examined, at the same time , the each rat BBB variety of the super microstructural were observed used by an electric-microscope in building the successful model of 48h . The examination of the transparence of BBB: Each animal were anaesthesia before 3h of ending experiment, after 2% EB NaCl(4ml/ Kg) were infused into vein, the head of rats were breaked to take the brain in each time, before taking out of the brain, in order to the clear dyestuff in blood, the NaCl were infused left ventricles to the clear liquid of right heart-auricle run off. Joinning 10% trichloroacetic acid to precipitate the protein after taking the brain, then using to have no water of ethanol to produce homogenate, centrifugating and washing for 3 times, The EB of brain tissue were extracted by thick ammonia of which's weight was 30 times of the brain tissue. It were centrifugated in the 3000 rs/ min for 15 mins, got out the above clear liquid, to measure content of EB by contrasting-color method ( λ=632 nms) in 722 light grid cent light photometers up the pure liquid, measuresing to the intensity of light absorberation, the blank comparison liquid were composed with the ammonia water, the ice acetic acid and ethanol. The contents ofEB( the μg/ ml wet weight) were got according to the standard curve. The examination of amount of water in brain tissue:After rat were sentenced to death, the right side brain of rats were taken out to measure wet weight, placed 90℃dry box of constant temperatures inside for drying 60 hs to measure dry weight, the amount of water in brain tissue were measured according to the Elltot formula calculation,that is, The amount of water in brain tissue = ( wet weight -dry weight)/ wet heavy×100% . The observation of the BBB super-microstructural:After rat were sentenced to death, a leaf brain tissue of the right side were taken out,the size of which is the 1 mm×1 mm×1 mm, putting it into 2.5% of glutaric dialdehyde-formaldehyde immediately to fixed for 24 hs, then fixing it again by 1% of OSO4, It were dehydrated by grain alcohol in ranks, and wraped to cover up by the Epon812 resin; The super thin leaf was sliced by sliced machine, dyed by sour of the acetic acid uranium-lemon acid lead, and it were observed by electric-microscope and photoed. The count data was expressed by means±standard deviation ( ±s), and it were statisticed using the SPSS for Windows 10.0 software processing, The single factor analysis of variance was adoptted when comparing among many groups, then Student-Newman-Keuls Test was adoptted when comparing with each other; The "t"examination was adoptted when comparing between two groups. Part 2: The effect of Zhongfengkang on the content of SOD,MDA,and NO in brain of focal cerebral ischemia rats The dividing groups,medication,the methed of model replications and statistics were same to above. Eight rats were taken Each group;The values change were measured in the 6h,12 h,24 h,48 h after building the model of focal cerebral ischemia successfully.After animal anaesthetizing, the heads were cut off to take out of the brain of right side, done away with part of the cerebellum and medulla, it was produced homogenate with homogenizer in environment of ice tray ,centrifugated with temperature 4 ℃(3500rs/ min for 5mins),and got out the above clear liquid to measure the contents of examineSOD,MDA in serum by contrasting-color method, and examine NO in brain issue by nitric acid deoxidize-enzyme method. the values were corrected by protein content of brain tissue. Part 3: The effect of Zhongfengkang on the content of TNF-α,IL-8 in brain of focal cerebral ischemia rats The dividing groups,medication,the methed of model replications and statistics were same to above. Eight rats were taken Each group;The values change were measured in the 6h,12h,24h,48h after building the model of focal cerebral ischemia successfully.After animal anaesthetizing, the heads were cut off to take out of the brain of right side, done away with part of the cerebellum and medulla, it was produced homogenate with homogenizer in environment of ice tray ,centrifugated with temperature 4 ℃(3500rs/ min for 5mins),and got out the above clear liquid to measure the contents of TNF-α,IL-8 in brain issue by radiation-immunization method. the values were corrected by protein content of brain tissue. Part 4: The effect of Zhongfengkang on the content of ICAM in brain of focal cerebral ischemia rats The dividing groups,medication,the methed of model replications and statistics were same to above. Eight rats were taken Each group;The values change were measured in the 6h,12h,24h,48h after building the model of focal cerebral ischemia successfully.After animal anaesthetizing, the heads were cut off to take out of the brain of right side, done away with part of the cerebellum and medulla, it was produced homogenate with homogenizer in environment of ice tray ,centrifugated with temperature 4 ℃(3500rs/ min for 5mins),and got out the above clear liquid to measure the contents of ICAM in brain issue by ABC-ELISA method. the values were corrected by protein content of brain tissue. Part 5: The effect of Zhongfengkang on the content of MMP-9 in brain of focal cerebral ischemia rats The dividing groups,medication,the methed of model replications and statistics were same to above. Eight rats were taken Each group;The valueschange were measured in the 6h,12h,24h,48h after building the model of focal cerebral ischemia successfully.After animal anaesthetizing, the heads were cut off to take out of the brain of right side, done away with part of the cerebellum and medulla, it was produced homogenate with homogenizer in environment of ice tray ,centrifugated with temperature 4 ℃(3500rs/ min for 5mins),and got out the above clear liquid to measure the contents of ICAM in brain issue by ABC-ELISA method. the values were expressed by OD values which measured by the enzyme-linked analyzer. Part 6: The effect of Zhongfengkang on the expression of AQP-4mRNA in brain of focal cerebral ischemia rats The dividing groups,medication,the methed of model replications and statistics were same to above. Eight rats were taken Each group;The expression of AQP-4mRNA of model group were measured in the 6h,12h,24h,48h by method of RT-PCR after building the model of focal cerebral ischemia successfully; The expression of AQP-4mRNA of each group were measured in the 48h by method of RT-PCR after building the model of focal cerebral ischemia successfully. Results: Part 1: The effect of zhongfengkang on BBB transparence, the brain edema and pathologic histology of focal cerebral ischemia rats 1. The effect of zhongfengkang on BBB transparence of focal cerebral ischemia rats Compare with the sham operation group,in 6h after cerebral ischemia, the content of EB in brain increased, in 12h(P<0.01), with the time of cerebral ischemia prolonging, the content of EB increased, and it reached top in 24h(P<0.01);Compared with model group of the same time, the content of EB in brain of each treatment groups were decreased in degrees(P<0.01), Table 1-2 2 The effect of zhongfengkang on the brain edema of focal cerebral ischemia rats Compared with sham operation group, in 6h after cerebral is chemia,brain edema were obvious in model group(P<0.01); With time of cerebra ischemia extending, the brain edema of focal cerebral ischemia rats devoloped, and it reach top in 24h(P<0.01); Compared with model group of the same time, the brain edema of each treatment groups were decreased,and the brain edema decrease of high dose group is most markedly (P<0.05orP<0.01), and in 6h after cerebral ischemia, the brain edema of high-dose group was lower than that of control group(P<0.05). Table 3-4 3. The effect of Zhongfengkang on the BBB super microstructural of local cerebral ischemia rats. The sham operation group: capillary vessel is normal, edema could not be seen the in endothelioid cells, chromatin's distributing was equable, chromosome is normal, karyotheca was integrated; granular body is abundant, girder was clear and integrated, the of rest organelle is normal. Fig. 1-2; The model group: high edema could be seen surrounding the capillary vessel, there are a little amount of thin long girder in tube antrum, the basic film increased slightly thick, nucleolus could be seen outside of basic film;The number of granular body was reduced in vessel endothelioid cells, the most of granular body's girder were inosculated and disappeared, partial karyotheca were inosculated. Fig. 3-4; Zhongfengkang low-dose group, the Buchangnaoxintong control group: the representation of two groups are similar, we could seen the edema of capillary vessel, the thin long prominency in tube antrum, the basic film is normal; many mitochondrion could be seen in endothelioid cells, partial mitochondrion's girder ruptured and inosculated, partial karyotheca inosculated. Fig. 7-10; Zhongfengkang high -dose group,the group of using dose of Zhongfengkang, we could see slight edema of capillary vessel, few of granular body ruptured and inosculated;The rest of representation are resemble with the normal group. Fig.5-6 Part 2: The effect of Zhongfengkang on the activity of SOD and content of MDA,NO in brain of focal cerebral ischemia rats1. The effect of Zhongfengkang on the activity of SOD in brain of focal cerebral ischemia rats. Compared with the group of sham operation , in 6h after cerebral ischemia, the activity of SOD of model group in brain was began to decrease, and reached the lowest in 48h(P<0.01);Compared with model group of the same time, the activity of SOD of each treatment groups were increased ,and the change of high does group were most remarkedly(P<0.05orP<0.01);. Table 1-2 2. The effect of Zhongfengkang on the content of MDA in brain of focal cerebral ischemia rats Compared with the group of sham operation, in 6h after cerebral ischemia, the content of MDA of model group in brain was began to increased;and reached top in 48h(P<0.01);Compared with model group of the same time, the content of MDA in brain of each treatment groups were decreased in some degrees, and the change of high does group were most remarkedly(P<0.05 or P<0.01). Table 3-4 3. The effect of Zhongfengkang on the content of NO in brain of focal cerebral ischemia rats Compared with the group of sham operation , in 6h after cerebral ischemia, the content of NO of model group in brain began to increase, and it reached top in 24h(P<0.01);Compared with model group of the same time, the content of NO in brain of Zhongfengkang high-dose and low-dose groups were decreased in 6h and 12h after cerebral ischemia,but it was no difference ramarkedly(P>0.05); the content of NO in brain of high-dose group were decreased in 12h and 24h after cerebral ischemia remarkedly(P<0.05 or P<0.01), and the content of NO in brain of Buchangnaoxintong group were decreased in 24h after cerebral ischemia remarkedly(P<0.05). Table 5-6. Part 3: The effect of Zhongfengkang on the content of TNF-α,IL-8 in brain of focal cerebral ischemia rats 1. The effect of Zhongfengkang on the content of TNF-αin brain of focal cerebral ischemia ratsCompared with the group of sham operation, in 6h after cerebral ischemia, the content of TNF-αof model group in brain began to increase, and it reach top in 12h(P<0.01);Compared with model group of the same time,, the content of TNF-αin brain of each treatment groups were decreased(P<0.05orP<0.01), and it were decreased most remarkedly in forepart of ischemia(P<0.05orP<0.01)Table 1-2. 2. The effect of Zhongfengkang on the content of IL-8 in brain of focal cerebral ischemia rats Compared with the group of sham operation, in 6h after cerebral ischemia, the content of interleukin-8 of model group in brain reduced(P<0.01),and it began to increase in 12h, reach top in 24h(P<0.01);Compared with model group of the same time,, the content of interleukin-8 in brain of each treatment groups were increased in 6h after cerebral ischemia(P<0.05),and the content of interleukin-8 of high-dose and low-dose group was decreased in 24h to 48h after cerebral ischemia. Table 3-4 Part 4: The effect of Zhongfengkang on the content of ICAM in brain of focal cerebral ischemia rats Compared with the group of sham operation , in 6h after cerebral ischemia, the content of ICAM-1 in brain of model group began to increase, and it reached top in 24h(P<0.01);Compared with model group of the same time, the content of ICAM-1 in brain of each treatment groups were decreased, and the high-does group were decreased most remarkedly(P<0.01); the content of ICAM-1 in brain of high-does group was lower than control group remarkedly(P<0.05). Table 1-2. Part 5: The effect of Zhongfengkang on the content of MMP-9 in brain of focal cerebral ischemia rats Compared with the group of sham operation , in 6h after cerebral ischemia, the content of MMP-9 in brain of model group began to increase, and it reached top in 24h(P<0.01); Compared with model group of the same time, the content of MMP-9 in brain of each treatment groups were decreased in some degrees(P<0.05orP<0.01). Table 1-2.Part 6: The effect of Zhongfengkang on the expression of AQP4mRNA in brain of focal cerebral ischemia rats Compared with the group of sham operation , in 12h after cerebral ischemia, the expression of AQP4mRNA of model groups in brain tissue began to increase ; With time of cerebra ischemia extending, the expression of AQP4mRNA of model group devoloped, and it reach top in 72h(P<0.01). Table 1. Fig. 1. Compared with the group of sham operation , in 48h after cerebral ischemia,the expression of AQP4mRNA of model groups in brain tissue increased (P<0.01). Compared with the model group, the expression of AQP4mRNA of high-dose and control groups was increased(P<0.05 or P<0.01). Table 2, Fig. 2. Conclusion: 1 Zhongfengkang could alleviate damage of vessel endothelioid cells after cerebral ischemia, improve super microstructure of BBB, keep integrality of BBB, and lighten edema of brain in effect. 2 Zhongfengkang could raise the activity of SOD and reduce content of MDA in the brain tissue of cerebral ischemia rats , which resisted the oxidation and reduce hurt of body, at the same time it could reduce content of NO in the brain tissue of cerebral ischemia rats in 24h and 48h, alleviate matrix-noxious of NO,and alleviate hurt of BBB . 3 Zhongfengkang could reduce content of TNF-αof model group in brain at forepart of cerebral ischemia, which could lighten damage to the vessel endothelioid and keep integrality of BBB, and reduce edema of brain in effect. 4 Zhongfengkang could reduce content of ICAM-1 in brain tissue, which could lighten conglutination between inflammation cell and vessel endothelioid cell, and alleviate damage of inflammation to protect BBB. 5 Zhongfengkang could reduce content of MMP-9 in brain tissue of cerebral ischemia, and reduce fundus membranous destroyed to keep integrality of BBB. 6 Zhongfengkang could reduce expression of AQP-4mRNA in brain...
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