The Role And Mechanism Of Radix Astragali Aqueous Extract On Striated Muscle Myotility

[Objective] To investigate effects and signal transduction mechanism of radix astragali aqueous extract on antioxidant activity and striated muscle myotility.[Methods] Kunming mice, Wistar rats and cardiac H9c2 cells were used.1. Experiment of anti-hypoxia in common temperature and normal pressure: Forty mice were random divided into four groups, control group(0.9%NaCl), Radix astragali aqueous extract low dosegroup(6g/kg), middle dose group (12g/kg)and large dose group(24g/kg). It is ten in every group. The anti-hypoxia time was measured after intragastric administration three weeks.2. The loading swimming time:Thirty mice were random divided into three groups, control group(0.9%NaCl), model group(0.9%NaCl) and Radix astragali aqueous extract group (12g/kg). It is ten in every group. The loading swimming time will be determined when the limbs ischemic/ reperfusion model had been prepared after intragastric administrati three weeks.3. Experiment of musculi contraction in vivo:Eighteen Wistar rats were random divided into three groups, sham group(0.9%NaCl), model group(0.9%NaCl) and Radix astragali aqueous extract group (12g/kg). It is six in every group. The skeletal muscle myotility were determined when the limbs ischemic/ reperfusion model had been prepared after intragastric administrati three weeks.4. The detection of antioxidant activity:The reactive oxygen species, superoxide dismutase activity and malondialdehyde content were dertermined after electrophysiological experiment in blood serum, gastrocnemius and cardiac muscle.5. Experiment of musculi contraction ex vivo:Wistar rats were random divided into three groups, control group(0.9%NaCl), model group(0.9%NaCl) and astragalosideⅣgroup (50μM). It is six in every group. The skeletal muscle myotility was dertermined and ultrastructural changes of gastrocnemius were observed by electron microscope.6. Experiment of heart function ex vivo:Wistar rats were random divided into two groups, control group(0.9%NaCl), model group(0.9%NaCl) and astragalosideⅣ group (50μmol/L). It is six in every group. The left ventricular-developed pressure and coronary flow were dertermined and ultrastructural changes of cardiac muscle were observed by electron microscope.7. The potential signaling pathway underlying the antioxidative effect of astragaloside IV:①The oxidative damage model in H9c2 cells:H9c2 cells were exposed to 500μM H2O2 for 20 min to cause mitochondrial oxidant damage.②Effects of astragaloside IV on GSK-3βand Akt phosphorylation:H9c2 cells were exposed to astragaloside IV(50μM) for 20 min. GSK-3p(Ser9) and Akt (Ser473) activities were determined by measuring their phosphorylation statuses with Western blot. H9c2 cells were exposed to LY294002 (10μM), an inhibitor of PI3K, before exposed to astragaloside IV for 20 min. Then, GSK-3(3(Ser9) activities was determined by measuring its phosphorylation statuses with Western blot.③Effect of astragaloside IV on the mPTP opening:Fluorescence dyes tetramethylrhodamine ethyl ester (TMRE) was used to image mitochondrial membrane potential (ΔΨm). Fluorescence images were obtained with confocal microscopy. H9c2 cells were exposed to astragaloside IV with 30μmol/L,50μmol/L, 60μmol/L,80μmol/L and 100μmol/L for 20 min. H9c2 cells were exposed to LY294002 (10μM), an inhibitor of PI3K, before exposed to astragaloside IV for 20 min. Then, mitochondrial membrane potential (ΔΨm) was determined by measuring fluorescence dyes TMRE with confocal microscopy.④The potential mechanisms underlying the inhibitory effect of astragaloside IV on the mPTP opening:The effect of astragaloside IV on TMRE fluorescence were observed whether or not to prevent by ODQ (5μM), a potent selective inhibitor of NO-sensitive guanylyl cyclase, and KT5823 (1μM), a selective inhibitor of PKG. In addition, H9c2 cells were exposed to ODQ (5μM) and KT5823 (1μM) before exposed to astragalosideⅣ(50μM) for 20 min. Then, GSK-3β(Ser9) and vasodilator-stimulated phosphoprotein (VASP), a substrate of PKG, activities was determined by measuring theirs phosphorylation statuses with Western blot.⑤Effect of astragaloside IV on NO generation:Fluorescence dyes 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate was used to image NO. Fluorescence images were obtained with confocal microscopy. The effect of astragaloside IV on NO was observed by the NOS inhibitor L-NAME and the PI3K inhibitor LY294002[Results]1. Effects of radix astragali aqueous extract on anti-hypoxia time:Radix astragali aqueous extract with middle dose and large dose could increase anti-hypoxia time compared with control group in mice (P<0.05), but not in low dose group(P>0.05).2. Effects of radix astragali aqueous extract on loading swimming time:After intragastric administration three weeks with radix astragali aqueous extract in mice, the loading swimming time significantly prolongated compaired with model group (P<0.05).The loading swimming time significantly shorted in model group compaired with control group (P<0.05).3. Effects of radix astragali aqueous extract on skeletal muscle myotility:Radix Astragali aqueous extract can enhance Pt during the gastrocnemius muscle's twitch take place and tetanus take place compaired with model group in rats (P<0.05).4. Effects of radix astragali aqueous extract on ROS, SOD and MDA in blood serum, SOD and MDA in gastrocnemius and cardiac muscle:Radix astragali aqueous extract can increase SOD, degrade MDA and ROS, compaired with model group in blood serum(P<0.05); Aqueous extract with low dose and middle dose can significant compaired with control group (P<0.05); Radix astragali aqueous extract can significant increase SOD and degrade MDA compaired with model group in either gastrocnemius muscle or cardiac muscle (P<0.05).5. Effects of astragaloside IV on skeletal muscle myotility ex vivo:Astragaloside IV can enhance Pt during the gastrocnemius muscle's twitch take place and tetanus take place compaired with model group in rats (P<0.05). 6. Effects of astragaloside IV on heart function ex vivo:The left ventricular-developed pressure and coronary flow were significant enhanced compaired with control group(P<0.05).7. Ultrastructural changes of gastrocnemius muscle and cardiac muscle were observed by electron microscope:Cytochondriome injury induced by anoxemia and ischemic was reversed when astragaloside IV had dealed with gastrocnemius muscle and cardiac muscle.8. The potential signaling pathway underlying the antioxidative effect of astragaloside IV:①Effects of astragaloside IV on GSK-30 and Akt phosphorylation:Astragaloside IV significantly increased GSK-3βphosphorylation at Ser9 in a dose-dependent manner with the peak at 50μM, suggesting that astragaloside IV can inactivate GSK-3βin cardiac cells. The effect of astragalosideⅣ(50μM) on GSK-3βphosphorylation was reversed by LY294002 (10μM), an inhibitor of PI3K, indicating an involvement of the PI3K/Akt pathway in the action of astragaloside IV. Moreover, astragaloside IV (50μM) increased Akt phosphorylation at Ser473, an effect that was nullified by LY294002, confirming the role of the PI3K/Akt signaling pathway in the action of astragaloside IV on GSK-3β.②Effect of astragaloside IV on the mPTP opening:Treatment of cells with 500μM H2O2 induced a marked decrease in TMRE fluorescence (49.69±6.34% of baseline in the control group), implying that the oxidative stress caused loss ofΔΨm. In contrast, cells treated with 50,60, and 80μM astragaloside IV showed much less decrease in TMRE fluorescence, indicating that astragaloside IV modulates the mPTP opening. Further experiments showed that astragaloside IV was not able to prevent TMRE fluorescence loss in the presence of LY294002, implying that the PI3K/Akt pathway contributes to the protective effect of astragaloside IV. ③The potential mechanisms underlying the inhibitory effect of astragaloside IV on the mPTP opening:The effect of astragaloside IV on TMRE fluorescence were able to prevent by ODQ (5μM), a potent selective inhibitor of NO-sensitive guanylyl cyclase, and KT5823 (1μM), a selective inhibitor of PKG, implying that the cGMP/PKG pathway may play a role in the action of astragaloside IV. In addition, the effect of astragaloside IV on GSK-3βphosphorylation was reversed by ODQ and KT5823. Moreover, astragaloside IV significantly increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a substrate of PKG, and this effect was reversed by ODQ and KT5823, further confirming that the cGMP/PKG signaling pathway is required for the inhibitory action of astragaloside IV on GSK-3β.σEffect of astragaloside IV on NO generation:Confocal microscopy shows that astragaloside IV markedly enhanced DAF-FM fluorescence intensity 10 min after the treatment compared with the control, indicating that astragaloside IV can produce NO in H9c2 cells. This effect of astragaloside IV was reversed by the NOS inhibitor L-NAME and the PI3K inhibitor LY294002, indicating that astragaloside IV produces NO through NOS and the PI3K/Akt pathway.[Conclusion]1. It's maybe concerned with antioxidant activity that radix astragali aqueous extract can raise skeletal muscle's contractibility and enhance heart function;2. Astragaloside IV may serve as an essential component of radix astragali aqueous extract;3. Astragaloside IV prevents oxidative stress injury by targeting the mPTP via inactivating GSK-3β;4. Astragaloside IV produces NO and cGMP-PKG signaling pathway may mediates the effect of astragaloside IV on the mPTP opening; 5. The PI3K/Akt pathway is responsible for NO generation by astragaloside IV.