Buyanghuanwu Decoction Protects RBMECs Againstinjury Caused By Oxygen-glucose Deprivation/Reperfusion

ObjectiveThis study was to confirm the protect function of Buyanghuanwu decoction against oxygen glucose deprivation and reperfusion injury, and will be the foundation to study mechanisms of Buyanghuanwu decoction on Cerebral ischemia-reperfusion injury. OGD reperfusion model was established to imitate the process of Ischemic reperfusion after cerebral stroke based on Buyanghuanwu soup rat brain ischemia Pharmacodynamics-Pharmacokinetics in vivo study. The changes in OD value, bovine serum albumin transmittance, expression of tight junction proteins and cell proliferation of primary rBMECs were observed and analyzed.Methods1 Isolation and cultivation of primary rBMECs1.1 Separate and extract the original rBMECsThe 10-day-old rats were disinfected and head cut, cut along the sagittal line sequentially and peeled skull, take out brains and removed the olfactory bulb, cerebellum and brain stem and other parts. The pia mater and large vessels at the surface of brain tissues was removed by rolling brain on the dry sterile filter paper, and then the left and right hemispheres were separated, the part of the hippocampus and white matter were removed and gray matter was reserved. Gray matter was shredded and centrifugated and the centrifugation was obtained. The concentration of 25% BSA was added into the centrifugation and be centrifugated by freezing centrifuge, and then rat brain microvessel fragments was obtained at the bottom of centrifuge tube. The precipitate layer was transferred to another centrifuge tube, adding 0.1% collagenase II and digested in 37℃ incubator. Type II collagenase was removed by centrifugation for 5 min after the digestion, the precipitate layer of single brain microvascular endothelial cells endothelial cells and beads were obtained. DMEM/F12 complete medium was added into the precipitate layer and mixed, the mixture was transferred to flasks and incubated in incubator with 37℃ and 5% C02 change the broth for 2-3days. The state of growth and converge of brain endothelial cells were observed under the inverted microscope.1.2 Identification of primary rBMECsThe climbing film covered with cells was took out from six-well plates and fixed with 4% poly formaldehyde. The identification of rBMECs was conducted via Ⅷ factor immuneofluorescence staining. The staining process was as follows:goat serum blocking 30min, rabbit anti-rat Ⅷ factor antibody incubation overnight at 4℃, adding fluorescent secondary antibody to the mixture and incubated for 1h in room temperature, and DAPI stained nuclear once again. After staining process, the climbing film was sealed. Cell morphology were photographed and positive cell rate was calculated under fluorescence microscopy.1.3 Determination of rBMECs growth curve using CCK-8 methodRBMECs were inoculated in 96 orifice and one plate was collected a day. The OD values at 450 nm wavelength was measured using microplate reader after the addition of CCK-8 solution. 2 Establishment of oxygen glucose deprivation reperfusion model2.1 Oxygen glucose deprivation and reperfusion treatmentThe oxygen glucose deprivation model was established by removing RBMECs out of incubator, replacing its medium to DMEM sugar-free medium and then incubated in incubator in which contained oxygen concentration of 1%and provide a circumstance of hypoxia. Based on this model, the oxygen glucose deprivation and reperfusion model was established through replacing suger-free medium to complete medium and providing enough oxygen in normoxic incubator.2.2 Evaluation of oxygen glucose deprivation and reperfusion modelThe morphological changes of rBMECs were observed under an inverted microscope after oxygen glucose deprivation and reperfusion treatment. The absorbance of cells from each group were detected by CCK-8 method and the mortality rate of cells from each group were calculated by Trypan blue exclusion method.3 Determination of safe concentration of Buyanghuanwu decoctionRBMECs were divided into control group, model group and Buyanghuanwu decoction treatment group in which 4 different doses (1μg·mL-1,10μg·mL-1, 100μg·mL-1,1000μg·mL-1) were set and contained. The effective concentration and safe concentration of Buyanghuanwu decoction to rBMECs were detected by CCK-8 method.4 Blood brain barrier permeability detectionRBMECs were divided into normal control group, oxygen glucose deprivation and reperfusion treatment group, Buyanghuanwu decoction of low dose (10 μg·mL-1) treatment group and high dose (100μg·mL-1) treatment group according to the screen of safe concentration. The liquid in Transwell chambers on both sides was collected after different treatment. Absorbance of the liquid was measured by fluorescence spectrophotometer and BSA transmittance was calculated to confirm the damage degree of oxygen glucose deprivation reperfusion treatment to BBB model and the protect function of Buyanghuanwu decoction to it.5 Detection of Occludin and ZO-1 protein levels by Western blotRBMECs were divided into normal control group, oxygen glucose deprivation and reperfusion treatment group, Buyanghuanwu decoction of low dose treatment group and high dose treatment group. The total protein of rBMECs from each group was extracted, content of Occludin and Z0-1 in protein was detected by Western blot to discuss the influence to claudin expression of in vitro BBB model using oxygen glucose deprivation and reperfusion treatment and Buyanghuanwu decoction.6 Detection of the expression level of Occludin and 20-1 protein mRNA by fluorescence quantitative RT-PCRRBMECs were divided into normal control group, oxygen glucose deprivation and reperfusion treatment group, Buyanghuanwu decoction of low dose treatment group and high dose treatment group. Total RNA of rBMECs from each group was extracted and the expression level of Occludin and Z0-1 protein mRNA was detected by fluorescence quantitative RT-PCR.7 Measurement of cell proliferation by CCK-8 methodRBMECs were divided into normal control group, oxygen glucose deprivation and reperfusion treatment group, Buyanghuanwu decoction of low dose treatment group and high dose treatment group. The absorbance values was readed by CCK-8 method to sdudy the promotion proliferation function of Buyanghuanwu decoction for oxygen glucose deprivation and reperfusion cells.8 Detection of cell proliferation by BrdUThe climbing film of cells were inoculated and divided into normal group, model group, Buyanghuanwu decoction of low dose treatment group and high dose treatment group. Each group was treated with oxygen glucose deprivation. The medium which contained BrdU and Buyanghuanwu decoction was replaced at the time of reoxygenation sugar by BrdU Incorporation test. The climbing film were placed into incubator for 24 h and then stained with BrdU immunofluorescence. The proliferation states of rBMECs was observed and labeling index of BrdU was calculated.9 Detection of cell cycle by flow cytometryThe RBMECs were inoculated in the culture medium and be divided into normal group, oxygen glucose deprivation reperfusion treatment group, Buyanghuanwu decoction of high and low dose group. The RBMECs were placed into normoxic incubator for 24h and stained with propidium iodide. Cell cycle distribution and proliferation index were detected and calculated.Results:1 Isolation and cultivation of primary rBMECs1.1 Separate and extract the original rBMECsThe rBMECs which extracted from rats showed the shape of branch and bead. rBMECs were adherent growth and endothelial cells were spread gradually from the shape of bead when cultured for 1 day; rBMECs were astral radial and shape of cells were changed to polygonal or fusiform after 2-3 day’s culture; The cells grow rapidly to reach confluence gradually when cultured up to 4-5 days; They merged into a tightly integrated dense monolayer and unique morphological characteristics was observed.1.2 Identification of primary rBMECsRBMECs Cytoplasm were stained by factor Ⅶ and showed green fluorescence under fluorescence microscop. The number of rBMECs accounts for more than 96% of total number of cells.1.3 Determination of rBMECs growth curve using CCK-8 methodThe growth curve of rBMECs was measured by CCK-8 method and the result showed that rBMECs proliferation got into logarithmic growth phase in 3rd and the proliferation peak appeared from 3-6 d. The growth of endothelial cell began to enter plateau and proliferation rate was slow down.2 Establishment of oxygen glucose deprivation and reperfusion modelThe morphology of rBMECs cells was like long spindle shape and clear boundary was observed before oxygen glucose deprivation. The adherent ability of rBMECs was decreased, the cells started to shrink and the gap between the cells was increased, the cell viability values was decreased significantly and the mortality rate of cells was elevated. The results indicated that damage was appeared obviously to rBMECs with treatment of oxygen glucose deprivation and reperfusion.3 Determination of safe concentration of Buyanghuanwu decoctionThe experimental results showed that the concentration of Buyanghuanwu decoction which will be the most effective cell protective function was in the range of 10 μg·mL-1-100μg·mL-1. And then the treatment of Buyanghuanwu decoction using low dose (10μg·mL-1) and high dose (100 μg·mL-1) were chosed for the subsequent trials.4 Blood brain barrier permeability detectionResults showed that BSA transmittance treated by oxygen glucose deprivation and reperfusion up to 25.13% compared to the control group. BSA transmittance treated by Buyanghuanwu decoction of low dose and high dose were 19.38% and 16.53% respectively. BSA transmittance was reduced treated by both different methods.5 Detection of Occludin and Z0-1 protein levels by Western blotThe result of Western blot was analyzed and showed that expression of claudin Occludin and Z0-1 were decreaced and permeability of BBB model in vitro was improved after the treatment of oxygen glucose deprivation and reperfusion. The down regulation of Occludin and Z0-1 was inhibited by the treatment of Buyanghuanwu decoction of low dose and high dose.6 Detection of the expression level of Occludin and Z0-1 protein mRNA by fluorescence quantitative RT-PCRThe result of Ct values was analyzed and showed that the expression level of Occludin and Z0-1 protein mRNA was decreased from both groups of oxygen glucose deprivation and reperfusion and Buyanghuanwu decoction of low dose and high dose compared to control group. And the values from Buyanghuanwu decoction of low dose and high dose was higher than the values from oxygen glucose deprivation reperfusion group.7 Effect of Buyanghuanwu decoction on the cell proliferation of rBMECs7.1 Detection of cell proliferation by CCK-8 methodThe OD value of rBMECs treated by oxygen glucose deprivation and reperfusion was decreased obviously 24 h later compared to control group, while the OD value was improved obviously treated by Buyanghuanwu decoction. It indicated that Buyanghuanwu decoction can promote the cell proliferation which treated by oxygen glucose deprivation and reperfusion.7.2 Detection of cell proliferation by BrdURBMECs proliferation after the treatment of oxygen glucose deprivation and reperfusion was promoted obviously with Buyanghuanwu decoction. It proved that Buyanghuanwu decoction can promote rBMECs proliferation and reduce the damage caused by the process of oxygen glucose deprivation and reperfusion.7.3 Detection of cell cycle by flow cytometryThe proportion of rBMECs treated with oxygen glucose deprivation and reperfusion at G1 period was increased and relatively decreased at S and G2 stage in which state proliferation index of rBMECs was decreased obviously. The proportion of rBMECs treated with Buyanghuanwu decoction at Gl stage decreased and increased at S and G2 stage in which stage proliferation index of rBMECs was increased.Conclusion:1 Isolation and culture of primary rBMECs establishement of oxygen glucose deprivation and perfusion modelThe cells isolated and cultured was stained with factor VIII antibody immunofluorescence and proved to be rBMECs and high in purity. The detection results of cell viability and mortality rate showed that the celection of oxygen glucose deprivation and reperfusion model was successful.2 Effect on in vitro blood-brain barrier with Buyanghuanwu decoctionThe trial result of bovine serum albumin permeability test showed that transmittance of bovine serum albumin was decreased when the BBB model which treated with oxygen glucose deprivation and reperfusion was treated with Buyanghuanwu decoction. The result of Western blot and fluorescence quantitative PCR experiments discovered that the expression of Occludin and ZO-1 protein were relatively increased when interpreted with Buyanghuanwu decoction. The study indicated that Buyanghuanwu decoction can improve the expression of Occludin and ZO-1 protein and reduce the permeability of blood-brain barrier.3 Effect of Buyanghuanwu decoction on proliferation of rBMECsThe cell OD value, BrdU labeling index and cell cycle were detected by CCK-8 assay, the result showed that Buyanghuanwu decoction, on the one hand, can promote rBMECs proliferation at the damage stage of oxygen glucose deprivation and reperfusion, on the other hand, can restore damaged blood-brain barrier by promoting the proliferation of endothelial cells and eventually against the damage of oxygen glucose deprivation and reperfusion.