Objective To screen the optimized Buyang Huanwu Decoction (BYHWD);To verify it. Methods H2O2 was used to induce PC12 cell oxidative stress models. MTT method was used to determine the prevention effects of BYHWD at different concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 3.5 mg/mL) on in vitro oxidative stress cell models to define the optimized concentration. Orthogonal design was used to divide BYHWD single medicine into decomposed BYHWD groups, control group (only with DMEM), normal group (without H2O2 and medicine processing), and model group, to investigate the protective effects on PC12 cells. Optimized BYHWD was screened to decide the compatibility ratio of each medicine. MTT was used to detect the cell survival rate in each group. Middle cerebral artery occlusion was used to replicate MACO rat models. SD rats were randomly divided into sham-operation group, model group, BYHWD group and optimized BYHWD high-, medium-and low-dose groups. Each medication group was given relevant medicine for gavage. The screened results were verified. Results Compared with other decomposed BYHWD groups, the protective effects of the compatibility of Astragali Radix+Chuanxiong Rhizoma+Pheretima on PC12 cells was the best (P<0.05), which was nearly equaled to BYHWD. Compared with the model group, BYHWD and the optimized one could evidently reduce cerebral cortex infarction area and improve the impaired brain edema (P<0.05), and the medium-dose group was the best. Conclusion The optimized BYHWD ratio is:Astragali Radix:Chuanxiong Rhizoma:Pheretima=10:3:1.

Aim To evaluate the regulation of thin recipe of Buyang Huanwu decoction on cyclin-dependent kinase 5(Cdk5)expressions in hippocampus tissue of rats after cerebral ischemia.Methods Male SD rats were divided into sham-operation group,MCAO group,Buyang Huanwu decoction group(ig.3.15 g·kg-1)and its thin recipe composition group(ig.2.41 g·kg-1).Each group was then divided into five subgroups based on the time after administration for 1,3,7,14,28 d respectively.Cdk5 protein and mRNA levels in each group were examined by using immunohistochemistry,Western blot and real-time PCR respectively.Results The up-regulation of Cdk5 was observed in model rat hippocampus after cerebral ischemia 1 day,and kept increasing with the aggravation of ischemia injury,the peaked expression was observed after 7～14 d,while the downtrend was observed after 28 days compared with the corresponding sham-operation groups(P<0.01),suggesting that the Cdk5 signal pathway would be activated by cerebral ischemic injury.The expression of Cdk5 in thin recipe of Buyang Huanwu decoction group was significantly lower than that in model group at each time point(P<0.05),and there was also more obvious down-regulation with the extend of intervene time.The regulation effects of thin recipe of Buyang Huanwu decoction was up to the best after 28 days of administration,which indicated the thin recipe was positive to the abnormal expression of Cdk5,and there was no difference between Buyang Huanwu decoction and its thin recipe treated groups(P>0.05).Conclusion The thin recipe of Buyang Huanwu decoction could exert the protective effect by regulating Cdk5 after cerebral ischemia.

Objective To study the pharmacokinetic features of ferulaic acid, senkyunolide A and ligustilide in Buyang Huanwu associated prescriptions (Buyang HuanwuDecoction andNaojianTablets).MethodsHPLC-DAD was applied for simultaneous determination plasma concentration of three ingredients with jugular venous cannula rats after intragastric administration ofBuyang Huanwu associated prescriptions. The pharmacolinetic parameters of each ingredient was calculated by DAS2.0, and then the total quantum statistical moment (TQSM) standard similarity was used to measure the overall pharmacokinetics behaviors.Results There were great differences in the three ingredients after the administration of two prescriptions, while the total quantum statistical parameters were very closely. The TQSM pharmacokinetic parameters of the three components inBuyang HuanwuDecoction andNaojian Tablets showed that AUC, MRT, VRT were 240.6 and 133.0, 3.192 min and 3.259 min, 21.59 min2and 19.75 min2, respectively.The similarity was up to 0.977 8.Conclusion The metabolic processes in vivo ofBuyang Huanwu Decoction andNaojianTablets have similarities. The efficacy of Chinese herbal compounds mostly depends on the multi-components overall contributions.

Aim To observe the effects of thin recipe of Buyang Huanwu Decoction on angiogenesis and the signal pathway of Nrf2 / HO-1 after cerebral ischemic injury in rats. Methods Totally 120 SD rats were randomly divided into four groups: sham operation group,model group,Buyang Huanwu Decoction group and thin recipe of Buyang Huanwu Decoction group. The focal cerebral ischemia rat model was established by middle cerebral arterial occlusion. Each group was treated with corresponding treatment. Each group was detected after cerebral ischemia for day 1,day 3 and day 7, respectively. Immunohistochemical method was used to detect the plasma levels of factor VIII related antigen( vWF), determination of microvessel density (MVD). The expression of Nrf2,HO-1 gene and pro-tein in brain tissues was detected by Real-Time PCR (RT-PCR) and Western blot. Results ① Compared with sham-operation group, the expression of vWF in the model group was significantly increased on day 3(P< 0. 05). Compared with model group, the expression levels increased differently in each drug group on day 7 (P < 0. 05). ② The expression of Nrf2, HO-1 gene and protein in sham operation group showed a small a-mount of gamma expression. Compared with sham op-eration group at the same time point, the expression of Nrf2 protein was significantly increased on day 3(P <0. 01). Compared with model group at the same time point, the Nrf2mRNA and protein expression was up-regulated in each drug group. The Nrf2mRNA on day 1,the Nrf2 protein on day 1 and day 7 were significant-ly increased( P < 0. 01). Compared with sham opera-tion group at the same time point, the expression of HO-1mRNA and protein in the model group was signif-icantly increased on day 7(P < 0. 05). Compared with model group at the same time point, the HO-1mRNA on day 3, the HO-1 protein on day 3 and day 7 in each drug group were significantly increased (P < 0. 05,P <0. 01). Conclusions The thin recipe of Buyang Hua-nwu Decoction promotes brain angiogenesis after ische-mia. The effect may be related wih the expression of Nrf2 / HO-1 signal pathway.

Objective To observe the effects of thin recipe ofBuyang Huanwu Decoction on expressions of glutathione (GSH) antioxidant system including GSH, GSH-Px andγ-GCS in the brain of focal cerebral ischemia rats;To study its mechanism of antioxidant effect.Methods Totally 120 SD rats were randomly divided into four groups according to random number table method:sham-operation group, model group,Buyang Huanwu Decoction group and thin recipe ofBuyang Huanwu Decoction group. Except for the sham-operation group, the rest groups established focal cerebral ischemia rat model by middle cerebral arterial occlusion. The treatment groups were given corresponding medicine by gavage, while the sham-operation group and model group were given the same amount of normal saline 2 h after modeling. Each group was detected after cerebral ischemia for 1 day, 3 days, and 7 days respectively. The content of GSH and the activity of GSH-Px were detected. At the same time,γ-GCS mRNA and protein levels were determined by using real time-PCR and Western blot.Results The content of GSH and the activity of GSH-Px at each time point decreased in the model group (P<0.05), compared with the corresponding sham-operation group. But the expression ofγ-GCS mRNA and protein increased, with statistical significance on day 1 (P<0.05). Compared with model group, content of GSH and GSH-Px activity levels increased differently in each treatment group, whileγ-GCS mRNA and protein expression raised, with statistical significance on day 3 (P<0.05).Conclusion The thin recipe ofBuyang Huanwu Decoction plays antioxidant effect by regulating the expression of glutathione antioxidant system GSH, GSH-Px andγ-GCS after cerebral ischemia, which may be one of its therapeutic mechanisms for cerebral ischemia.

Objective To observe the effects of simplified recipe ofBuyang Huanwu Decoction on expression of APJ in the brain of local cerebral ischemia rats;To discuss its mechanism of action. MethodsFocal cerebral ischemia rat models were established by middle cerebral arterial occlusion. The adult rats were randomly divided into sham-operation group, model group,Buyang Huanwu Decoction group and simplified recipe ofBuyang Huanwu Decoction group. Administration groups were given relevant medicine for gavage. The expressions of APJ protein and APJ mRNA at different time points were detected by Western blot and RT-PCR.ResultsCompared with model group and sham-operation group, the expression of APJ protein and APJ mRNA at different time points in Buyang Huanwu Decoction group and simplified recipe ofBuyang Huanwu Decoction group significantly increased (P<0.05). The expression of APJ protein at different time points showed no difference between simplified recipe ofBuyang Huanwu Decoction group andBuyang Huanwu Decoction group;while the expression of APJ mRNA in simplified recipe ofBuyang Huanwu Decoction group was higher than Buyang Huanwu Decoction group (P<0.05).ConclusionSimplified recipe ofBuyang Huanwu Decoction plays a role in neural protection and restoration by promoting the expression of APJ in the brain of focal cerebral ischemia rats.

Objective To investigate the preventive effects of Buyang Huanwu Decoction (BYHWD) and its recipe composition (BYJJF) in focal ischemic brain injury condition in vivo/in vitro. Methods In vivo studies, SD rats were divided into sham-operation group, MCAO group, BYHWD group and BYJJF group based on rat weight, 10 rats in each group. The body weight, infarct area and brain water contents were determined. In vitro studies, H2O2 was used to damage PC12 cells, and the vitro oxidative stress cell model was established. PC12 cells were divided into normal group, blank control group, BYHWD and BYJJF groups with different concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 3.5 mg/mL). MTT method was employed to determine the protective effects of BYHWD and BYJJF on model cells.Results Vivo studies showed that after 7 days of treatment with BYHWD and BYJJF, those determinated quotas were all significantly improved compared with MCAO model rats (P<0.05), and there were no differences between BYHWD and BYJJF (P>0.05).Vtiro studies showed that the protective effects of BYHWD and BYJJF took place 2 hours later, and it was obvious in oxidative stress injury caused by H2O2, with statistical differences with model group (P<0.05). BYHWD and BYJJF could increase cell viability, and there was no difference between the groups with same concentration (P>0.05).Conclusion The research confirmed that BYJJF plays a significant role in improving the cerebral ischemia injury, which is the same performance as BYHWD, and BYJJF can save TCM resources under the precondition of TCM efficacy.

Objective To study the protective effect of the thin Recipe of Buyanghuanwu on cerebral ischemic injury of rats. Methods Block model in cerebral artery was established through using the suture method of middle cerebral artery. The rats were randomly divided into sham-operation group, model group, Buyanghuanwu group and thin recipe of Buyanghuanyin high, medium and low-dose groups. Buyanghuanwu group and thin recipe of Buyanghuanyin groups were given gavage with relevant medicine, and sham-operation group and model group were given gavage with the same amount of sterile distilled water for continuous 7 days. Matlab software programming was used to analyze data and observe the changes in neurological function, body weight, the area of cerebral infarction and wet/dry weight ratio of brain of the rats. Results Rats with cerebral ischemia suffered large area of infarction, brain edema and neurological dysfunction. The thin recipe of Buyanghuanwu of medium and low dose groups could effectively improve the indexes of model rats compared with the model group, the difference with statistical significance (P<0.05). Conclusion The thin recipe of Buyanghuanwu has protective effect on cerebral ischemia, and the medium dose is the optimal dose.

Objective To explore the effects of Buyang Huanwu decoction(BYHWD)on expressions of nuclear factor-κB p65(NF-κBp65)and its inhibitor( I-κB)in signal transduction of NF-κB in brain tissue of rats with focal cerebral ischemia injury. Methods 180 Sprague-Dawley(SD)rats were randomly divided into normal group,sham-operated group,model group,pynolidine dithiocarbamate(PDTC)group,minocycline(MC)group and BYHWD treatment group,each group 30 rats. The rats of PDTC group were given PDTC 100 mg?kg-1?d-1 by intra-peritoneal injection. In MC group,MC was given by filling the stomach,the dose was 2.35 g?kg-1?d-1,the drug solution was prepared by adding the distilled water,and the total volume of drug solution to fill the stomach was kept at the same volume in various groups,thus the concentration of the drug was different. In BYHWD group,BYHWD was given,the dose was reduced to 5 g?kg-1?d-1 according to the body surface area dose conversion formula about people and animals. In sham-operated group and model group,the distilled water was given in the same volume as other drug solution. The protein expression levels of NF-κBp65 and I-κB in ischemic tissues were examined by using immunohistochemical method on the time points 7,14 and 21 days after treatment in each group. Results Compared with model group, the cell numbers with expression of NF-κBp65 in PDTC group,MC group and BYHWD group were significantly decreased along with the prolongation of therapy time,the decrease in number was more and more,until 21 days,it reached the valley level(cell/400 times HP:44.00±6.91,45.33±6.55,18.67±2.14 vs. 126.00±5.78,all P<0.05);the number of cells with expression of I-κB was obviously increased,the differences being statistically significant(all P<0.05),but the differences in expression of NF-κBp65 among the treatment groups at the different time points were not statistically significant(all P>0.05). After treatment for 7 days,the number of cells with positive expression of I-κB protein in BYHWD group was less than that in MC group(cell/400 times HP:55.00±3.40 vs. 72.50±4.29,P<0.05);after treatment for 14 days,the number in BYHWD group was approximately the same as that in the MC group, the difference being not statistically significant(93.50±6.15 vs. 93.00±6.20,P>0.05),and after treatment for 21 days,the number in BYHWD group was significantly higher than that in MC group(88.83±4.95 vs. 71.17±7.16, P<0.05). Conclusion BYHWD can regulate the expressions of inflammatory cytokine I-κB and NF-κB in signal transduction of NF-κB in ischemic brain tissue to inhibit the inflammatory reaction,thus it has the protective effect on cerebral ischemia.

BACKGROUND:Caveolin-1 is expressed in mammalian brain and involved in the normal development of the brain, which can affect the proliferation of neural stem cells in the brain. OBJECTIVE:To acquire neural stem cells from caveolin-1 knockout embryonic mice in vitro and study their biological characteristics. METHODS:The whole brain was separated from C57BL/6 mice and caveolin-1 knockout C57BL/6 mice respectively at encyesis 14-16 days. Single cellsuspension was obtained by enzyme digestion, and cultured in the conditioned medium of neural stem cells. Fol owing 7 days of primary culture, the cells were induced in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10%fetal bovine serum for 7 days. RESULTS AND CONCLUSION:The major cells of the cellsuspensions from the fetal mouse brain were dead at 1 day after culture, and some single cells floated in the medium and their transmittance were better, and then they gradual y formed multicellular bal s after 3 days. A smal amount of cells were adhered at the bottom of culture plate after passage, and a great amount of cellbal s appeared after 7 days. The proliferation rate of neural stem cells from caveolin-1 knockout mice was higher than that from normal mice. The cellbal s were nestin-positive and their differentiated cells was positive for neurofilament 200, glial fibril ary acidic protein or O4, respectively. Al of the cells from normal mouse brain were positive for caveolin-1, but the cells from caveolin-1 knockout mice were negative for caveolin-1 by immunocytochemistry. Moreover, the speed of cellbal formation and the number of cellbal s in neural stem cells from caveolin-1 knockout mice were better than those from normal mice. Caveolin-1 negative neural stem cells were cultured successful y from caveolin-1 knockout mouse brain, and the results show that caveolin-1 can promote the proliferation of neural stem cells and inhibit their differentiation in vitro.