Study On The Protective Effect And Its Mechanism Of Zige Lyophilized Powder For Injection In Rats With The Disturbance Of The Cerebral Microcirculation

BackgroundOur research team researched a pharmaceutical preparation with the anti-ischemic effect of Zige lyophilized powder that contained catalpol and puerarin and had a patent. Zige lyophilized powder for injection had the effects of the neural functional recovery in permanent cerebral ischemia of rats, reducing cerebral infarction area, angiogenesis, improving blood stasis and blood brain barrier (BBB) permeability, and so on. But we had not research the effects of Zige lyophilized powder for injection in the celebral microcirculation.ObjectiveIt aims to research possible mechanism of Zige lyophilized powder for injection against cerebral ischemia-reperfusion injury in the celebral microcirculation through the pathological causes of contraction, permeability and blood rheology of microvessel in experiments of animal and cell.Methods1. The improving effects of Zige lyophilized powder for injection against focal cerebral ischemia-reperfusion injury in the disturbance of microcirculation80male sprague-dawley rats were randomly divided into sham group (10), ischemia1h group (16/20), ischemia2h group (16/20), ischemia4h group (16/20), nimodipine injection (ischemia1h and reperfusion,2.00mg·kg-1,8/10), and were administered intraperitoneally at a dose of10mL·kg-1·d-1on the basis of body weight for14days with drugs in reperfusion for0h, sham group and model group were administered intraperitoneally with an equal volume of saline solution. It made a cerebral ischemia-reperfusion (I/R) model using the line in the left middle cerebral artery occlusion (MCAO) of rats and test neuro-behavioral evaluation by a method of modified neurological severity scores (mNSS) in ischemia for1h,2h,4h and reperfusion for1d,4d,7d and14d.540male sprague-dawley rats were randomly divided into sham group (60), model group (61/80), puerarin injection group(30.00mg·kg-1,68/80), nimodipine injection (2.00mg·kg-1,69/80), Zige lyophilized powder groups(16.40mg·kg-1,65/80;32.70mg·kg-1,68/80;65.40mg·kg-1,70/80) in ischemia for2h and reperfusion for1d,4d,7d and14d, and were administered intraperitoneally at a dose of10mL·kg-1·d-1on the basis of body weight for14days with drugs in reperfusion for0h, sham group and model group were administered intraperitoneally with an equal volume of saline solution. Blood flow volume of pia mater in rats was observed by laser doppler flowmetry. Pathomorphism changes and damaged conditions in rat cortex were observed by HE staining. The volume of the cerebral infarction was estimated by2%TTC staining. The water content was calculated by dry and wet weight method. The disruption of micro vessel permeability was detected by the EB content. The expression of CD31was detected by immunohistochemical staining method.960male sprague-dawley rats were randomly divided into sham group (96), model group (109/144), puerarin injection group(30.00mg·kg-1,120/144), nimodipine injection (2.00mg·kg-1,123/144), Zige lyophilized powder groups(16.40mg·kg-1,117/144;32.70mg·kg-1,122/144;65.40mg·kg-1,126/144) in ischemia for2h and reperfusion for1d,4d,7d and14d, and were administered intraperitoneally at a dose of10mL·kg-1·d-1on the basis of body weight for14days with drugs in reperfusion for0h, sham group and model group were administered intraperitoneally with an equal volume of saline solution.Nitric oxide synthase (NOS), endothelin-1(ET-1),6-ketone-prostaglandin F1α (6-Ke-to-PG-F1α) and thromboxane B2(TXB2) content of the brain microvessel in the lateral ischemic cerebral cortex was detected by enzyme immunoassay kits. The expression of MMP-9、ZO-1、Occludin and Claudin-5protein and mRNA of the brain microvessel in the lateral ischemic cerebral cortex was detected by fluorescence quantitative PCR (qPCR) and Western blot (WB).2. The protective effect of Zige lyophilized powder for injection against the acute disturbance of cerebral microcirculation in rats by intravenous injection of high molecule dextran120male rats were divided into normal control group, model group, nimodipine injection (2.00mg·kg-1), Zige lyophilized powder groups(16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) with20for each goup, and made window craniotomy after the administration of drugs for14days (tail vein injection,1time per day), the model of rats with the disturbance of cerebral microcirculation was estabished by intravenous injection of high molecule dextran (10%T-500,10mL·kg-1) after the last administrat-ion of drug for30min. The effects of micro-vein diameter for5min in normal rats and1,5,10,20,40,60min in making model of rats were observed through the inverted microscope. Blood flow volume on brain meninx was observed through laser doppler flowmetry. HCT was measured by electric resistance method, hemorheological indexes were observed by auto-hemorheological instrument.3. The protein expression levels of mmp-9to Zige lyophilized powder for injection on brain microvascular endothelial cells after hypoxia-reoxygenation injury were detected in vitroIt made a hypoxia-reoxygenation injury model on cultured BMECs of rat (simulated cerebral ischemia-reperfusion injury model) in vitro by oxygen-glucose deprivation. The proliferation of endothelial cells was detected by MTT method. Endothelial cells were cultured by oxygen-glucose deprivation for2h and reoxygenation for24h, it determined to detect the protein expression levels of mmp-9by WB method.Results1. The improving effects of Zige lyophilized powder for injection against focal cerebral ischemia-reperfusion injury in the celebral microcirculation(1) The focal cerebral ischemia-reperfusion model of rats and neuro-behavioral test after I/R rats have significantly neurological deficit,1d after I/R, the mNSS score of1h,2h and4h groups were5.68±0.75,9.23±1.05,11.53±1.881, respectively. Percentages of the infarction area of three groups in14d were0.1026±0.0214,0.2245±0.0228,0.3828±0.0318, respectively. The main infarction area of1h group was cortex, and the main infarction area of2h and4h groups was cortex and basal ganglia. The best time of making the ischemia-reperfusion model is2h to ischemia for subsequent experiments.(2) The effect of Zige lyophilized powder for injection against the model rats of blood flow volume, infarction volume and brain edema in cerebral microcirculation.In this experiment, Zige lyophilized powder(16.40,32.70and65.40mg-kg-1) showed a trend of slow recovery during reperfusion. In TTC staining, sham group appeared in deep red and had not the brain infarcts, model group had a large area of infarcts and located in the left side of the cortex and basal ganglia. Compared with model group, Zige lyophilized powder groups (65.40mg-kg-1) decreased significantly infarction volume in14d(P<0.05).Compared with sham group, the water content and EB content increased significantly (P＜0.05, P<0.01) in model group,and it made a successful model. Compared with model group in reperfusion for7d and14d, Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could inhibit significantly the water content and EB content in lesion side of the rat brain tissue (P<0.05,P<0.01), these showed that Zige lyophilized powder protected against cerebral ischemic injury by improving microvessel permeability.In HE staining, cells of brain ischemia side in all groups after cerebral ischemia-reperfusion appeared necrosis, shrinking nucleus into irregular shape, edema, loosen and infiltration by inflammatory cells. Zige lyophilized powder groups(16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could inhibit infiltration by inflammatory cells, and reduce intercellular space and edema in the corresponding time. (3) The effect of Zige lyophilized powder for injection against focal cerebral ischemia-reperfusion injury in microvascular density of the celebral microcirculation.In immunohistochemical experiment, a few of CD31cells expressed in sham group. It showed that the expression of CD31after ischemia-reperfusion in lateral cortex of rats had a few in reperfusion for1d, and had higher in reperfusion for4d and7d, and increased slowly or no growth in reperfusion after7d. Compared with model group in reperfusion for1d,4d,7d and14d, the expression of CD31of puerarin injection group (30.00mg·kg-1), Nimodipine injection (2.00mg·kg-1), Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) had higher significantly in the corresponding time except reperfusion for1d (P<0.01). These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by promoting angiogenesis.(4) The effect of Zige lyophilized powder against the microvascular contraction function after cerebral ischemia-reperfusion injury of rats in the microcirculationIn this experiment, compared with sham group, it showed that TNOS and iNOS enzyme activity had increased and cNOS enzyme activity had decreased significantly after ischemia-reperfusion in lateral cortex of rats (P<0.01). Compared with model group in reperfusion for1d, puerarin injection group (30.00mg·kg-1), Nimodipine injection (2.00mg·kg-1), Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) had reduce significantly TNOS and iNOS enzyme activity (P<0.05, P<0.01). Compared with model group in reperfusion for4,7and14d, all drug groups had reduce significantly TNOS and iNOS enzyme activity and increased significantly cNOS enzyme activity. These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving the microvascular contraction function in the celebral microcirculation.Compared with sham group, it showed that the level of ET-1after ischemia-reperfusion in lateral cortex of rats had a rising trend and increased gradually with prolonged observation. In reperfusion for1d, compared with model group, Zige lyophilized powder group (32.70mg·kg-1) could reduce significantly the level of ET-1 (P<0.05). In reperfusion for4,7and14d, compared with model group, Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) had reduce significantly the level of ET-1in micro vessel of the lateral ischemic cerebral cortex (P<0.01). These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving the microvascular contraction function and endothelial cell damage.In reperfusion for1,4,7and14d, compared with sham group, it showed that the level of TXB2had increased and6-Ke-to-PGF1α had decreased significantly in model group. Zige lyophilized powder groups(16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could inhibit significantly the level of TXB2and6-Ke-to-PGF1α(P<0.05,P<0.01)and the ratio of TXB2/6-Ke-to-PGF1α(P<0.01). These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving microvascular contraction function in microcirculation.(5) The effect of Zige lyophilized powder for injection against the microvascular permeability after cerebral ischemia-reperfusion injury of rats in the celebral microcirculationIn real-time quantitative PCR, the cortex of sham group had a few of mmp-9mRNA levels. After focal cerebral ischemia-reperfusion, comparing with sham group in reperfusion for1,4,7and14d, it showed that mmp-9mRNA levels in lateral cortex of rats had increased significantly in model group. Zige lyophilized powder groups (16.40mg·kg-1,32.70mg·kg-1and65.40mg·kg-1) could reduce significantly mmp-9mRNA levels. Comparing with sham group, it showed that claudin-5mRNA、occludin mRNA、zo-1mRNA levels in lateral cortex of rats had reduced significantly in model group. Three groups of Zige lyophilized powder could increase significantly claudin-5mRNA、occludin mRNA、zo-1mRNA levels respectively (P<0.05,P<0.01).In the experiment of western blot, a change trend of protein expression and genetic level is basically identical. Compared with sham group in reperfusion for1,4,7and14d, it showed that mmp-9protein expression in lateral cortex of rats had increase significantly in model group and all drug groups could reduce mmp-9protein expression significantly. Comparing with sham group, it showed that the protein expressions of claudin-5、occludin、zo-1in lateral cortex of rats had reduced significantly in model group. Three groups of Zige lyophilized powder could increase significantly claudin-5、occludi、zo-1levels in the corresponding time, respectively (P <0.05,P<0.01).These show that Zige lyophilized powder protect against cerebral ischemia-reperfusion injury by improving microvascular permeability.2. The protective effect of Zige lyophilized powder for injection against the acute disturbance of cerebral microcirculation in rats by intravenous injection of high molecule dextranCompared with normal control group in the corresponding time, it showed that pia mater microvenule diameter and microartery diameter in rats had shrink significantly and blood flow volume had decrease significantly in model group. Compared with model group, Zige lyophilized powder for injection(16.40,32.70and65.40mg·kg-1) had significantly expended micro-vein diameter (P<0.01),and returned normal trend of blood flow volume after10min of making model. Compared with normal control group, it showed that blood viscosity, erythrocyte agglutination index, plasma viscosity and hematocrit in rats had increase significantly in model group. Compared with model group, Zige lyophilized powder (65.40mg·kg-1)could significantly reduce blood rheology indicators (P<0.01),and Zige lyophilized powder for injection(16.40,32.70mg·kg-1)had could significantly reduce other blood rheology indicators except hematocrit (P<0.05,P<0.01).3. The protein expression levels of mmp-9to Zige lyophilized powder on brain microvascular endothelial cells (BMECs) after hypoxia-reoxygenation injury were detected in vitroThe IC50value is860μg·ml-1by MTT method in cultured BMECs by Zige lyophilized powder for injection. Compared with normal control group in the reoxygenation time for24h, it showed that the level of mmp-9protein had increase significantly in model group. Zige lyophilized powder groups (24.50、49.00and98.00μg·ml-1)had inhibit significantly the increased levels of mmp-9protein in a hypoxia-reoxygenation injury.Conclusions1. The best time of making I/R model by Longa method is2h to ischemia, and it is stability, controllability and repeatability. Zige lyophilized powder for injection showed a trend of slow recovery during reperfusion, and could decrease infarction volume, and inhibit significantly the water content and EB content in lesion side of the rat brain tissue. These showed the fact that it could reduce the damage rate of the blood brain barrier and brain edema. Zige lyophilized powder for injection can promote CD31expression in lateral cortex of rats, it would be promote angiogenesis after ischemica-reperfusion injury, and improve the cerebral microcirculation, which is beneficial to the recovery of damaged nerve tissue. The possible mechanisms of Zige lyophilized powder for injection in the improving celebral microcirculation would adjust the microvascular contraction function, and inhibit the enzyme activity of iNOS, and reduce the generated TNOS and ET-1content, and inhibit the level of TXB2and6-Ke-to-PGF1a. Zige lyophilized powder for injection can protect the microvascular integrity, and reduce microvascular permeability, and improve cerebral microcirculation through it can inhibit the increasing trend of mmp-9mRNA and protein expression and the decreasing trend of claudin-5, occludin, zo-1mRNA and protein level.2. Zige lyophilized powder for injection against the acute disturbance of cerebral microcirculation in rats can change trend of pia mater microartery and microvenule diameter, and improve celebral blood flow and blood viscosity, these showed that it would impove the effect of the acute disturbance of cerebral microcirculation.3. Zige lyophilized powder for injection had inhibited the increased levels of mmp-9protein in a hypoxia-reoxygenation injury on cultured BMECs. It showed that the possible mechanism of Zige lyophilized powder for injection on protecting the cell integrity would be associated with the decreasing mmp-9protein expression.