| Objective:With neuroblastoma cells of rats(B35)OGD model as the foundation,through the method of serum pharmacology,observe the party ultimately also five soup drug-containing serum on OGD injury caused by cell apoptosis and its influence on apoptosis related proteins expression,discusses Yang five soup also supplement in ischemic cerebral apoplexy after brain tissue ischemia anoxic injury caused by the protection mechanism of apoptosis.It provides a scientific theoretical basis for the clinical application of buyang huanwu decoction in the treatment of ischemic stroke.Methods: 1.The construct of B35 cells Oxygen glucose deprivation model-OGD model.Cobalt chloride was used to simulate hypoxic environment(The concentration of Cobalt chloride respectively are 0.7mmol/ml、0.8mmol/ml0.9mmol/ml、1.0mmol/ml)and 1/2 saccharide medium to simulate saccharide deficiency environment.B35 cells were treated for 12 h,and cell survival rate was detected by cck-8,so as to select the appropriate cobalt chloride concentration for modeling.2.The OGD model was constructed with the concentration of cobalt chloride selected in the first step(the concentration of cobalt chloride was 0.8mmol/ml,and the 1/2 sugar-containing medium was prepared with DMEM-F12 and sugar-free DMEM).Flow cytometry was used to analyze the apoptosis rate of OGD cells,and the apoptosis status and morphology of apoptotic cells after OGD were analyzed by transmission electron microscopy to preliminarily evaluate whether the model was successfully constructed.3.To investigate the effect of serum containing buyang huanwu decoction(BYHWD)on the survival rate and apoptosis rate of B35 cells.The OGD cells were treated with high and low dose of serum containing buyang huanwu decoction,and the survival rate and apoptosis rate of the treated cells were analyzed by cck-8 and flow cytometry to evaluate the effect of serum containing buyang huanwu decoction on the survival rate and apoptosis rate of B35 cells.4.The changes of apoptosis-related proteins Bax,bcl-2 and caspase-3 before and after serum containing BYHWD treatment were detected by Western blot.To evaluate the regulatory effects of serum containing BYHWD treatment on the apoptosis-related proteins Bax,bcl-2 and caspase-3.Results: 1.With the increase of cobalt chloride concentration,the cell survival rate showed a trend of gradual decline.The cell survival rates of cobalt chloride concentrations of 0.7mmol/ml,0.8mmol/ml,0.9mmol/ml and 1.0mmol/ml were(85.38±2.1)%,(78.25±2.0)%,(54.24±2.1)% and(46±2.2)%,respectively.The concentration of cobalt chloride(0.8mmol/ml)was selected for OGD modeling,and it was detected by flow cytometry and transmission electron microscopy.Compared with the blank control group,the concentration of cobalt chloride could significantly induce cell apoptosis,and the difference was statistically significant.2.The cell survival rates of the blank control group,OGD group,low dose group and high dose group were(95.09±0.73)%,(49.77±2.52)%,(60.00±2.65)% and(64.85±1.44)%,respectively.Compared with the blank control group,the cell survival rate of OGD group was significantly reduced(P < 0.01).Compared with the model group,the cell survival rate of the low dose and high dose groups of BYHWD containing serum increased significantly(P < 0.05).There was a significant difference in the improvement of cell survival between the high-dose and low-dose BYHWD groups(P < 0.05).Both the high-dose and low-dose groups of BYHWD drug-containing serum reduced the apoptosis rate,p < 0.01,the difference was statistically significant.There was a difference between high and low dose of BYHWD drug serum and low dose group in the reduction of apoptosis rate(P < 0.05),and the difference was statistically significant.3.Compared with the blank control group,the expression levels of caspase-3 and bax proteins in the model group were significantly increased(P < 0.01),and the expression levels of bcl-2 proteins were significantly decreased(P < 0.01).Compared with the model group,the expression level of caspase-3 in the low-dose serum of buyang huanwu decoction decreased(P < 0.05),the expression level of bax showed no significant difference,and the expression level of bcl-2 increased(P < 0.05).Compared with the model group,the expression levels of caspase-3 and bax in the high-dose serum of buyang huanwu decoction decreased(P < 0.01)and the expression levels of bcl-2 increased(P < 0.05).There was no significant difference between the high dose group and the low dose group(P > 0.05).Conclusions: 1.Combined treatment of B35 cells with cobalt chloride(concentration of 0.8mmol/ml)to simulate hypoxia environment and 1/2 saccharide medium to simulate saccharide deficiency environment for 12 h can induce the apoptosis of normal B35 cells,and the OGD model of B35 cells was successfully made.2.The treatment of apoptotic cells with buyang huanwu decoction containing drug serum can reduce the apoptosis rate and increase the cell survival rate.3.OGD treatment increased the expression of pro-apoptotic proteins caspase-3 and bax in B35 cells and decreased the expression of anti-apoptotic proteins bcl-2.However,in the high-dose group treated with buyang huanwu decoction containing drug serum,the expression of caspase-3 and bax proteins could be reduced,and the expression of bcl-2 protein could be increased,so as to inhibit the apoptosis of nerve cells and play a neuroprotective role.This may be one of the mechanisms of buyang huanwu decoction to reduce the apoptosis of nerve cells and treat ischemic stroke.In summary,combined treatment of B35 rat neuroblastoma cells with simulated hypoxic environment of cobalt chloride and simulated hypoglycemic environment of 1/2 saccharide medium for 12 h can significantly induce apoptosis and reduce cell survival rate,and establish cell OGD model.Moreover,OGD treatment can increase the expression of pro-apoptotic proteins caspase-3 and bax,decrease the expression of anti-apoptotic proteins bcl-2,and increase the ratio of bcl-2 to bax,which is one of the mechanisms of neuronal cell apoptosis.After the cells were treated with a high dose of buyanghuanwu decoction containing serum(15%),the expression of pro-apoptotic proteins caspase-3 and bax was decreased,the expression of anti-apoptotic proteins bcl-2 was increased,and the ratio of bcl-2 to bax was increased,so as to inhibit cell apoptosis,improve cell survival rate and play a neuroprotective role.This may be one of the mechanisms of buyang huanwu decoction in the treatment of ischemic stroke. |
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