| Objective The aim of the present study is to explore the influence of ASIV intervention on the expression of AQP-4 and TNF-α in astrocytes after OGD/R.Methods 1. To dispense 100 ul of astrocytes suspension(20000 cells /well) in a 96-well plate. Astrocytes were incubated with various concentrations of TNF-α(0.01、0.1、1、10、100ng·mL-1) and various concentrations of astragaloside IV(0.001、0.01、0.1μmol·mL-1) separately, and astrocytes activity was measured by cell counting kit-8(CCK-8). 2. Astrocytes were incubated with various concentrations of TNF-α(0.5、5、10、30、50ng·mL-1), and AQP-4 gene level was assessed by RT-PCR. 3. Astrocytes were divided into five groups: sham group, TNF-α group, 0.001 μmol ·mL-1ASIV+TNF-α group, 0.01μmol·mL-1 ASIV+TNF-α group, 0.1μmol·mL-1 ASIV+TNF-α group, and AQP-4 gene level was assessed by RT-PCR. 4. Astrocytes were moved into an anaerobic 1%O2, 94%N2, 5%CO2 at 37(1℃ 、2、3、4h), then returned to the normal condition for 24 h. AQP-4 and TNF-α level was assessed by RT-PCR and WB. 5. Astrocytes were divided into four groups: control group, OGD/R group, OGD/R+0.001μmol·mL-1ASIV group, OGD/R+0.01μmol·mL-1ASIV group. AQP-4 and TNF-α level was assessed by RT-PCR and IF.Results 1. The effect of TNF-α on the activity of astrocytes:Compared with the control group, the activity of astrocytes was higher and had a tendency to rise, without statistical significance, when the concentration of TNF-α ranged from 0 to 10 ng·mL-1. When the concentration of TNF-α reached to 100 ng·mL-1, he activity of astrocytes declined significantly(P<0.05) compared with the control group. 2. The effect of ASIV on the activity of astrocytes:When the concentration of ASIV was 0.001、 0.01、 0.1μmol·mL-1, the activity of astrocytes was improved at a dose-dependent manner compared with the control group(P<0.05). 3. The effect of various concentrations of TNF-α on the AQP-4 gene expression in cultured astrocytes: RT-PCR analysis showed that compared with the control group the expression levels of AQP-4 in astrocytes treated with various concentrations of TNF-α were higher significantly and when the concentration of TNF-α was 10ng·mL-1, the role was the most obvious. 4. The effects of ASIV on AQP-4 gene expression induced by TNF-α in cultured astrocytes : The expression levels of AQP-4 in astrocytes treated with 0.001 、 0.01 、 0.1μmol ·mL-1ASIV + TNF-α were significantly lower than that of the TNF-α group, but still higher than that of the sham group. 5. The effect of different time of OGD/R on the AQP-4 gene expression: Compared with the control group, when the time of OGD were 1h and 2h, the AQP-4 level had no obvious difference, when the time of OGD were 3h and 4h, the AQP-4 level was higher(P<0.05) and the AQP-4 level of the former was higher than that of the latter(P<0.05). 6. The effect of different time of OGD/R on the AQP-4 expression: the AQP-4 levels of OGD/R groups were higer than the control group, and that of the OGD./R3 h group was the highest. 7. The effect of different time of OGD/R on the TNF-α gene expression: Compared with the control group, when the time of OGD were 1h and 2h, the TNF-α level was higher(P<0.05), and the TNF-α level of the former was higher than that of the latter(P<0.05), when the time of OGD were 3h and 4h, the TNF-α level had no obvious difference. 8. The effect of different time of OGD/R on the TNF-α expression: the TNF-α levels of OGD/R groups were higer than the control group, and that of the OGD./R1 h group was the highest. 9. The influence of ASIV intervention on the expression of AQP-4 gene in astrocytes after OGD/R: The AQP-4 gene level of the OGD3h+0.001μmol·mL-1ASIV was lower than that of the OGD3 h group and was still higher than that of the control group(P<0.05). The AQP-4 gene level of the OGD3h+0.01μmol·mL-1ASIV was lower than that of the OGD3 h group but was lower than that of the control group(P<0.05). 10. The influence of ASIV intervention on the expression of AQP-4 level in astrocytes after OGD/R : the AQP-4 level of OGD3h+0.001μmol·mL-1ASIV was lower than that of the OGD3 h group. 11. The influence of ASIV intervention on the expression of TNF-α gene in astrocytes after OGD/R: The TNF-α gene levels of the OGD1h+0.001μmol·mL-1ASIV and the OGD1h+0.01μmol·mL-1ASIV were lower than that of the OGD1 h group and were still higher than that of the control group(P<0.05). 12. The influence of ASIV intervention on the expression of TNF-α level in astrocytes after OGD/R: the TNF-α level of OGD1h+0.001μmol·mL-1ASIV was lower than that of the OGD1 h group.Conclusions 1. ASIV could inhibit AQP-4 gene expression induced by TNF-α; 2. OGD/R could improve AQP-4 and TNF-α expression and the expression of TNF-α in astrocytes after OGD/R was prior to that of the AQP-4; 3. ASIV could inhibit the expression of AQP-4 and TNF-α in astrocytes after OGD/R, and the inhibition of ASIV to AQP-4 expression may be realized by the inhibition of ASIV to TNF-α expression, and ASIV could have an effect on the edema by the chemism. |
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