| ObjectiveBy observing the effect of Huangdi Anxiao capsule(HDAX)containing serum on the endoplasmic reticulum stress mediated apoptosis pathway of PC12 cells induced by high glucose,the mechanism of the Huangdi Anxiao capsule containing serum in the treatment of diabetic cognitive dysfunction(DCD)was discussed.Methods1 Establish the cell model of DCD in vitroPC12 cells were cultured in different concentrations of glucose(50,75,100,125,150 m M)for 24,48 and 72 hours,and the cell viability was detected by MTT assay.The mannitol balances osmotic pressure between different groups.To further determine the damage and apoptosis of PC12 cells induced by high glucose,the cell morphology was observed by inverted microscope,and the apoptosis was observed by Hoechst 33342 staining.2 Effect of Huangdi Anxiao capsule containing serum on cell damage in DCD cell modelThe experiment is divided into seven groups: The control group(Control),different concentration of Huangdi Anxiao capsule containing serum groups(5%,10%,15%,20%,25%,30%),each group respectively incubated 24 hours,MTT assays were used to measure the optimum concentration range of Huangdi Anxiao capsule containing serum.After PC12 cells were induced by high glucose to establish DCD cell model,different concentrations of Huangdi Anxiao capsule containing serum were given for further intervention,cell viability was detected by MTT assay.3 Effect of Huangdi Anxiao capsule containing serum on endoplasmic reticulum stress mediated apoptosis pathway in DCD cell modelThe experiment is divided into five groups: The control group(Control);model group(Model,glucose concentration 100 m M 48 h);Huangdi Anxiao capsule containing serum group(HDAX,HDAX concentration 15% after adding glucose concentration 100 m M 48 h);Inhibitor 4-phenylbutyric acid group(4-PBA,4-PBA concentration 5 μM after adding glucose concentration 100 m M 48 h);Huangdi Anxiao capsule containing serum+inhibitor 4-phenylbutyric acid group(HDAX+4-PBA,HDAX concentration 15% and4-PBA concentration 5 μM after adding glucose concentration 100 m M 48 h).After relevant treatment,cells were collected.The expression of GRP78,CHOP,Bax,Bcl-2,Caspase-12,Caspase-9 and Caspase-3 protein in PC12 cell were detected by Western blot,the expression of GRP78,CHOP,Bax,Bcl-2,procaspase-12,procaspase-9,procaspase-3m RNA in PC12 cell were detected by RT-q PCR.Results1 Establishment of PC12 cell injury model induced by high glucose in vitroAfter cultured PC12 cells with different concentrations of glucose(50,75,100,125,150 m M)for 24,48 and 72 hours,MTT showed that the cells treated with 100 m M glucose for 48 hours could significantly reduce cell viability,change cell morphology and promote cell apoptosis.2 Protective effect of Huangdi Anxiao capsule containing serum on cell injury in DCD cell modelCompared with the control group,there was no significant difference in cell viability when the concentration of Huangdi Anxiao capsule containing serum was 5%-15%(P<0.05),When the concentration of Huangdi Anxiao capsule containing serum was 20%,the cell viability began to decline;when the concentration was 30%,the cell viability decreased significantly(P<0.05),so 5%,10% and 15% were selected as the low,medium and high dose group of Huangdi Anxiao capsule containing serum.After treated with high glucose,the cell viability of the low,middle and high dose group of Huangdi Anxiao capsule containing serum was significantly higher(P<0.05,P<0.01),among which the high dose group(15%)had the best effect.3 Effect of Huangdi Anxiao capsule containing serum on endoplasmic reticulum stress mediated apoptosis in DCD cell model(1)Western blot results: Compared with the control group,GRP78,CHOP,Bax,Caspase-12,Caspase-9,and Caspase-3 protein expressions were significantly increased(P<0.01),the expression of Bcl-2 protein was significantly decreased in the model group(P<0.01).After treatment,protein expressions of GRP78,CHOP,Bax,Caspase-12,Caspase-9 and Caspase-3 were significantly decreased in HDAX group,4-PBA group and HDAX+4-PBA group(P<0.05,P<0.01),the expression of Bcl-2 protein was significantly increased,compared with the model group(P<0.05,P<0.01).(2)RT-q PCR results: Compared with the control group,the m RNA levels of GRP78,CHOP,Bax,procaspase-12,procaspae-9 and procaspase-3 in the model group were significantly increased(P<0.01),and the m RNA expression of Bcl-2 were significantly decreased(P<0.01).After treatment,the m RNA levels of GRP78,CHOP,Bax,procaspase-12,procaspase-9 and procaspase-3 in HDAX group,4-PBA group and HDAX+4-PBA group were significantly decreased(P<0.01),and m RNA levels of Bcl-2were significantly increased(P<0.01),compared with the model group.Conclusion1 High glucose can induce PC12 cell injury and increase apoptosis in a time and concentration dependent manner.Glucose concentration of 100 m M for 48 hours is the suitable experimental condition for the establishment of DCD cell model in PC12 cells in vitro.2 Huangdi Anxiao capsule containing serum can reduce the cell damage of DCD cell model.3 The neuroprotective mechanism of Huangdi Anxiao capsule containing serum on DCD cell model may be achieved by inhibiting the endoplasmic reticulum stress mediated apoptosis pathway. |
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