Effect Of Naoxintong Capsule On The Rats With Middle Cerebral Artery Occlusion

Objective: 1. Observe the cerebral tissue perfusion and edema zone changes in icerebral schemia rat models,using a method combined of MR diffusion weighted imaging(DWI) and perfusion weighted imaging(PWI). So that evaluating the effect of Naoxintong capsule on cerebral infarction volume. 2. Prepaire the middle cerebral artery occlusion rat model by zea longa method. Treat the models with naoxintong and explore the mechanism of the therapy effect. 3. The expression levels of protein and m RNA of NF-κB, MMP-9 and TNF-α were detected respectively by western blot and real time PCR after MCAO.Methods: 1. Group and administration: All animals are under " Guide for the care and use of Laboratory Animals ". Healthy SD male rats weighing between 200 to 220 g were fed in the manual control environment, drinking and eating free. The rats were randomly divided into(1) sham operated group: only neck surgery, no vascular ligation.(2) MCAO control group: normal saline administered after vascular ligation;(3) Naoxintong treatment group: Naoxintong administration 30 min after vascular ligation. Each group divided into 12 h, 1d, 3d, 5d, 7d subgroups, each subgroup has 6 rats. Administration: The Naoxintong powder dissolved in water with a density of 0.5mg/ml. Rats in Naoxintong treatment group were given Naoxintong in a dose of 0.45g/kg ? d. Sham operated group and MCAO control group were treated with equal volume of normal saline. 2. Model prepaire: The Middle Cerebral Artery Occlusion(MCAO) rats models were prepared by Zea Longa method. Bederson score were used to select the successful models. 3. MRI examination: The rats were examined by MRI respectively at 0.5d, 1 d, 3 d, 5 d, 7 d, using a 3.0T superconducting magnetic resonance imaging system. The rats were anesthetized by hydrated chloral(0.35g/kg) and examined the cranial coronal with a prone position. 4. Detection of AQP4, TNF-α, MMP-9 and NF-κB m RNA in rats brain:.Remove the brain and wash with saline. Taking 100 mg brain tissue from the area around infarction lesion. Extracte total RNA and synthesize c DNA under M-MLV reverse transcriptase. Then use the c DNA as a template to conduct the real time PCR and detect the relative expression. 5. The protein expression levels were detected by Western Blot: extracte the proteins using pre-cooled lysis buffer according to the istructions. Protein samples were separated on 10% SDS polyacrylamide gels, then transferred onto PVDF membranes, and blocked in 5% skimmed milk buffer for 1~2 h. The membranes were incubated over night at 4°C or 4~6h at 37°C. Wash the PVDF membrane in TBST 10 min for 3 times. Add the secondary antibody marked by horseradish peroxidase onto the PVDF membrane and incubate 1h at 37℃. Wash the PVDF membrane in TBST 10 min for 3 times. Protein expression was detected by an ECL detection system and exposed on an X-ray film. β-actin was used as a loading control. The optical densities of protein bands on the X-ray film were quantitatively analyzed by Quantity software. 6. Statistical Analysis: Statistical analysis was performed by the SPSS software 16.0. Statistical significant differences were evaluated by Student-t-test(two-tailed) or One-Way ANOVO. All data were expressed as mean ± SD. P<0.05 was considered statistically significant.Results: 1. The cerebral infarction area of naoxintong treatment group reduced compared with MCAO control group(P<0.05), indicating that Naoxintong mitigate cerebral infarction damage and protected the ischemia tissue. 2. The m RNAexpression level of TNF-α, MMP-9, NF-κB: The m RNA expression level of MCAO control group increased compared with sham operated group(P < 0.05). The m RNA expression level of naoxintong treatment group decreased compared with MCAO control group(P<0.05). 3. The protein expression level of TNF-α, MMP-9, NF-κB: The protein expression level of MCAO control group increased compared with sham operated group(P < 0.05). The protein expression level of naoxintong treatment group decreased compared with MCAO control group(P<0.05). 4. The brain edema of rat models: The expression level of AQP-4 increased in MCAO control group compared with sham operated group(P<0.05). The expression level of AQP-4 decreased in naoxintong treatment group compared with MCAO control group(P<0.05). Brain water content of naoxintong treatment group decreased compared with MCAO control group(P<0.05)..Conclusion: 1. Naoxintong.has a therapeutic effect on the cerebral infarction and decrease the infarction volume. 2. Naoxintong reduce both m RNA and protein expression levels of AQP4, TNF-α, MMP-9 and NF-κB. Thus reducing the inflammation in ischemic area and protect the brain tissue 3. Naoxintong attenuates ischemia-reperfusion injury, and significantly improved neurological function after ischemia. At the same time attenuates cerebral edema and cerebral infarct volume.