| Objectives: In our previous studies,we have confirmed that Rosamultin,one of the effective components in the extract of Potentilla anserina L and its n-butanol fraction,which play an active role in antioxidant,anti-ischemia,anti-hypoxia and other widely bioactive.In addition,it has been proved that Rosamultin could prevent hypoxia-induced endothelial cells,neuron and myocardial cells injury,but the potential signaling pathway mechanism of it to exert anti-hypoxia is not clear.In the present study,we use Sprague Dawley rats to establish acute hypobaric hypoxia modle,and study the protective effects of Rosamultin on acute hypoxic rats.In addition,acute hypoxic cerebral edema is associated with dysfunction of vascular endothelial cells,we use vascular endothelial cells to establish hypxia-induced injury model,and analyze the expression of PI3K/Akt signaling pathway which regulated by Rosamultin,and explore the possible molecular mechanism of Rosamultin on preventing hypoxic cerebral injury.Methods: The research includes two parts:Part 1: Protective effects of Rosamultin on brain injury in rats with acute hypobaric hypoxia and regulation of pAkt protein expression1 The 50 SD rats were randomly divided into: normal control group,acute hypoxic model group,Rosamultin high concentration group,Rosamultin low concentration group and dexamethasone group.2 Except the control group,other groups should be establish a acute hypobaric hypoxia model using the low pressure oxygen cabin imitating a plateau of 7000 meters(temperature15±2 ℃,humidity 30%±5%)for 18 h.3 NO content in plasma were determined by using the biochemical method,as well as the content of MDA,GSH and LDH,SOD activity in brainhippocampus of different groups.4 Water concentration in brain were determinated by physical method.5 With H.E stain,we can study the morphological changes of brain tissues after hypobaric hypoxia injury in different groups.6 Immunohistochemical was used to investigate the expression of AQP1 and AQP4.7 Western Blot was used to investigate the effect on protein expression levels of pAkt.Part 2: Mechanism of PI3-K/Akt pathway mediated Rosamultin attenuates vascular endothelial cells injury induced by acute hypoxia1 The endothelial cells were randomly divided into different groups:control group,different concentrations of LY294002 intervention group(20μmol/L,10 μmol/L,5 μmol/L),hypoxia-induced injury model group,hypoxia-induced injury with different concentrations of LY294002 intervention group,hypoxia-induced injury with Rosamultin intervention group,hypoxia-induced injury with Rosamultin and different concentrations of LY294002 intervention group,hypoxia-induced injury with Rosamultin and2-Methoxyestradiol intervention group.2 To induce hypoxic condition in cell culture system,a tri-gas incubator is generally used(5% CO2,95% N2,37 ℃)for 2 h.3 Cell viability was determined using the conversion of MTT to formazan.LDH activity in culture medium were determined by using the biochemical method,as well as the content of MDA,GSH,CAT and SOD activity in cells under hypoxia condition.4 Trypan blue staining was used to detected the survival rate and cell membrane integrity in hypoxia environment.5 Hochest/PI and DAPI staining were used to observe cell apoptosis of different groups under hypoxia condition.6 Flow cytometry was used to detect cell apoptosis of different groups in hypoxia environment.7 Immunofluorescence microscope technology was used to observed theexpression of pAkt under hyoxia condition in different groups.8 Western Blot was used to investigate the expression levels of Akt,pAkt,HIF-1α,HIF-1β,Bax,Bcl-2,Cyt C,Caspase-9,pro-Caspase-3 and cleaved-Caspase-3 in different groups.Results:Part 1: Protective effects of Rosamultin on brain injury in rats with acute hypobaric hypoxia and regulation of pAkt protein expression1 Compared to control group,the activities of LDH and MDA contents in brain tissues were raised(P < 0.01),and the activities of SOD and GSH contents in brain tissues were decreased,as well as the NO contents in plasma(P <0.01,P <0.05).Rosamultin and dexamethasone could attenuate acute hypobaric hypoxia injury in rats brain in varying degrees(P <0.01,P <0.05).2 Compared to control group,the water concentration in rat brains was significantly increased after acute hypobaric hypoxia(P < 0.01),but treatment with different concentration of Rosamultin and dexamethasone could decrease the water concentration in rat brains(P <0.01,P <0.05).3 The morphological changes was observed by H.E staining,the piamater and the cerebral cortex of rats were seprated after hypoxia,and many red cells which were stained with eosin can be observed between them.The clearance around the capillaries in the cerebral cortex increased,and the main character of neurocytes in hippocampus was apoptosis or necrosis.Howere,treatment with different dose of Rosamultin and dexamethasone could attenuate the injury which mentioned above.4 Immunohistochemistry showed that the number of cells which expressed AQP1 and AQP4 was significantly increased in rat brain after hypoxia(P < 0.01),but when given different dose of Rosamultin and dexamethasone treatment were significantly reduced the number of AQP1 and AQP4 positive cells(P <0.01).5 The expression of pAkt were increased after hypoxia(P < 0.01);Compared to model group,the expression of pAkt was significantly increasedin high dose of Rosamultin group(P <0.01).Part 2: Mechanism of PI3-K/Akt pathway mediated Rosamultin attenuates vascular endothelial cells injury induced by acute hypoxia1 In hypoxia environment,LY294002 could significantly decrease cell viability compared with model group(P < 0.01).Rosamultin could significantly improve cell viability compared with model group(P <0.01),but it could be reversed by LY294002(P <0.01).2 Rosamultin could significantly decrease the release of LDH and MDA content(P <0.01),also improved the activities of SOD,CAT and GSH(P <0.01),but all of these effects could be reversed by LY294002(P <0.01,P <0.05).3 Trypan blue staining showed that the increased number of staining cells and the declined survival rate of cells induced by hypoxia could be reversed by Rosamultin(P < 0.01),but it could be abolished by LY294002(P <0.01).4 Hochest/PI staining,DAPI staining and flow cytometry results showed that Rosamultin could prevent hypoxia-induced endothelial cells apoptosis,but it also could be reversed by LY294002.5 Under normoxic conditions,the green fluorescence(pAkt)of FITC labeled cells was extremely weak.But the fluorescence was enhanced in hypoxic environment,and futher enhancement of fluorescence intensity after treatment with Rosamultin,but LY294002 could abolish it.6 Under hypoxic conditions,the protein expression of pAkt and HIF-1αexpressed a lot(P <0.01),both expression levels were further increased after treatment with Rosamultin(P < 0.01),and LY294002 could significantly reduce the expression level of them(P < 0.01).However,only HIF-1αexpression could be reduced by 2-Me(P < 0.01),it had no effect on the expression level of p Akt(P > 0.05).There was no difference in the expression of Akt and HIF-1β among the groups(P >0.05).7 The expression of Bax,Cyt C,Caspase-9 and cleaved-Caspase-3 were increased after hypoxia(P < 0.01),in contrast,Bcl-2 and pro-Caspase-3expression levels decreased(P < 0.01),and it could be reversed by Rosamultin(P <0.01).But at the same time to give the LY294002 or 2-Me treatment could abolish the protective effect of Rosamultin(P <0.01).Conclusions:1 Rosamultin can attenuate rat brain injury induced by acute hypobaric hypoxia through increasing the free radical scavenging ability of rats.2 Rosamultin can reduce the degree of cerebral edema in rats induced by acute hypobaric hypoxia by inhibiting the over expression of AQP1 and AQP4.3 Hypoxia can prevent acute hypoxic brain injury by up-regulating the expression of pAkt.4 Under hypoxic condition,Rosamultin can up-regulate the expression of HIF-1α through PI3K/Akt signaling pathway,and improve the functional damage of vascular endothelial cells induced by acute hypoxia. |
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