Mechanism Of Rosamultin On Inhabiting Ca²⁺ And Apoptosis Of EA.hy926 Endothelial Cells Induced By Hypoxia

Objectives: Studies have confirmed that Tibetan Potentilla anserina L, has significant effect on increasing immunity, anti-oxidation, anti-anoxia, anti-fatigue and other widely bioactive. It has been proved by the previous chemical constituents and pharmacological activity track that the active monomer of the n-butanol fraction(ND) of Potentilla anserina L which protecting myocardial from ischemia / reperfusion injury is Rosamultin. But the related signaling passway in hypoxia environment have not been established. Here we choose human EA.hy926 Endothelial cells as the research models to study the effect of Rosamultin on hypoxia- inducible factor-1 alpha(HIF-1α) signaling pathway 、 Ca2+ overloading and Mitochondrial apoptosis pathway, and also explore the possible molecular mechanism of Rosamultin.Methods: 1 Determination of drug concentration 1.1 Logarithmic growth phase EA.hy926 endothelial cells were randomly divided into control group, hypoxia model group, and Rosamultin(1×10-6 ~ 1×10-14 mol / L) treated group by MTT assay, LDH activity detect, investigate the protective effect Rosamultin on EA.hy926 hypoxic injury of endothelial cells, to determine the concentration of the drug experiments Rosamultin. 1.2 Logarithmic growth phase EA.hy926 endothelial cells, were randomly divided into normal control group, the hypoxia model group, and Verapamil(1×10-6 ~ 1×10-14 mol / L) treated group was observed by MTT Verapamil on Protective effects of hypoxic injury EA.hy926 endothelial cells to determine the concentration of the positive control drug administration. 2 Effects of Rosamultin protect human EA.hy926 endothelial from hypoxia induced oxidative damage 2.1 The ROS activity in hypoxia environment was detected by flow cytometry. 2.2 To observe the morphological by inverted phase contrast microscope and HE staining. 2.3 To investigate the effects of Rosamultin protect human EA.hy926 endothelial cell from hypoxia by trypan blue staining 3 Effects of Rosamultin protect human EA.hy926 endothelial from hypoxia induced intracellular calcium overload 3.1 The [Ca2+]i analysis of EA.hy926 in hypoxia environment was detected by Fluorescence microplate reader. 3.2 To observe the [Ca2+]i of EA.hy926 in hypoxia environment was detected by confocal microscopy. 3.3 The protein expression levels of Stim1、Orai1、Calpain1 were investigated by Western Blot. 4 Effects of Rosamultin protect human EA.hy926 endothelial from hypoxia induced apoptosis 4.1 Apoptosis of EA.hy926 was abserved by Hochest/PI and DAPI staining. 4.2 Apoptosis of EA.hy926 in hypoxia environment was detected by flow cytometry. 4.3 The protein expression levels of Bax/ Bcl-2、Cytochrome C、Caspase-9、Caspase-3 were investigated by Western Blot. 5 Immunofluorescence microscope technology was used to explore the effect of Rosamultin in hypoxia environment on nuclear translocation of HIF-1α. 6 Secretion capacity of i NOS and e NOS were examined by enzyme-linked immnosorbent assay(ELISA). Secretion capacity of NO was examined 7 The protein expression levels of p VHL、HIF-1α、HIF-1β、VEGH were investigated by Western Blot. 8 The effect of HIF-1αinhibitor 2- Methoxyestradiol on apoptosis of EA.hy926 in hypoxia environment.Results: 1 Determination of drug concentration 1.1 Determination of RosamultinCompared with the control group, cell viability of hypoxia model group decreased significantly, 1×10-6mol/L to 1×10-14 mol/L Rosamultin could significantly increased cell viability under hypoxic conditions(P<0.01). Wherein, protection effect of Rosamultin in 1×10-11 mol/L the most significant, and in the 1×10-11mol/L to 1×10-13 mol/L cell viability.The LDH analysis consistent with the cell viability analysis.so we choose 1×10-11mol/L to 1×10-13 Rosamultin. 1.2 Determination of VerapamilCompared with the control group, cell viability of hypoxia model group decreased significantly, 1×10-6mol/L to 1×10-14 mol/L Verapamil could significantly increased cell viability under hypoxic conditions(P<0.01). In order to be consistent with Rosamultin concentration, we chose 1×10-11 mol/L concentration of Verapamil. 2 Effects of Rosamultin protect human EA.hy926 endothelial from hypoxia induced oxidative damage 2.1 The measurement results of intracellular ROSRosamultin can reduce the accumulation of intracellular ROS induced by hypoxia. 2.2 Cell morphologyIn hypoxia environment Rosamultin can significantly improve proliferation activity of EA.hy926. 2.3 Trypan blue stainingCompared with normal control group, the hypoxia model group Aizen cells significantly increased,cell survival rate significant reduction. The cell viability of Rosamultin and Verapamil treated cells significantly increased 3 Effects of Rosamultin protect human EA.hy926 endothelial from hypoxia induced intracellular calcium overload 3.1 Fluorescence microplate reader testRosamultin can significantly improve [Ca2+]i overload of EA.hy926 endothelial cell induced by hypoxia by. 3.2 Confocal detectionIntracellular calcium ion concentration increased with prolonged hypoxia, Rosamultin treated cells was significantly decreased by fluorescence intensity. 3.3 Western BlotUnder normoxic conditions,The protein expression of Stim1、Orai1、Calpain1 expressed a lot. In hypoxic environment,it could up-regulate protein Stim1、Orai1、Calpain1 protein expression level. And compared with it, pretreatment with Rosamultin might markedly evaluate expression of Stim1、Orai1、Calpain1. 4 Effects of Rosamultin protect human EA.hy926 endothelial from hypoxia induced apoptosis a) Hochest / PI staining resultsCompared with control group, the hypoxia model group cell death increased significantly, the Vision is a large number of red fluorescent labeled cells, chromatin condensation increased, and after Rosamultin treatment decreased cell death, chromatin condensation weakened. b) DAPI stainingCompared with control group, the hypoxia model group cell nucleus shrinkage, color deepened, the rod can see the obvious apoptotic bodies, some cell nuclei swelling, broken, and after Rosamultin treatment, significantly improve the shrinking state of the nucleus. c) Flow cytometry ResultsCompared with the control group,In the hypoxia model group, cell apoptosis rate significantly increased(P<0.01), After treatment of Rosamultin(1×10-11,1×10-12,1×10-13mol/L) and verapamil(1×10-11mol/L) the apoptosis rate decreased significantly(P <0.01). Rosamultin can significantly inhibit apoptosis of EA.hy926 endothelial cell induced by hypoxia. d) Western BlotUnder normoxic conditions,The protein expression of Bax、Cytochrome C、Caspase-9、Caspase-3 were at a very low level and Bcl-2 expressed a lot. In hypoxic environment,it could up-regulate protein Bax、Cytochrome C、Caspase-9、Caspase-3 levels and down-regulate Bcl-2 protein expression level. And compared with it, pretreatment with Rosamultin might markedly evaluate expression of Bax 、Cytochrome C、Caspase-9、Caspase-3 and promote expression of Bcl-2. 5 Extracellular i NOS, e NOS, NO detection 5.1 Extracellular i NOS, e NOS content by ELISA assay.Rosamultin can significantly increase the release of e NOS and reduced the release of i NOS in hypoxia environment. And after each concentration Rosamultin and Verapamil treatment, the level of i NOS was significantly decrease(P <0.01), levels of e NOS significantly increased(P <0.01). 5.2 Extracellular NO contentCompared with control group, in the hypoxia model group, the NO level significantly decreased(P <0.01). And after each concentration of Rosamultin and Verapamil treatment, NO levels significantly increased(P <0.01). 6 Immunofluorescence was used to observe the results of the cell nucleus displacement of HIF-1αUnder normoxic conditions,little HIF-1α expressed in EA.hy926 cells. But in hypoxic environment,HIF-1α could be translocated into nuclear and expressed a lot. Compared with hypoxia control group,pretreatment with Rosamultin might promote HIF-1α into nuclear. 7 Western BlotUnder normoxic conditions,little HIF-1α and VEGF was expressed, p VHL expressed a lot.In hypoxic environment,it could up-regulate protein HIF-1αand VEGF levels and down-regulate p VHL protein expression level. And compared with it, pretreatment with Rosamultin might markedly promote expression of HIF-1α、VEGF and p VHL. Regardless of hypoxic or not HIF-1βexpressed the same level. 8 The inhibition of 2Methoxyestradiol 8.1 MTT, flow cytometry resultsOnce the express of HIF-1α was inhabited, the effect of Rosamultin on the apoptosis was suppression but not as low as merely hypoxia without any pretreatment. 8.2 Western BlotOnce the express of HIF-1α was inhabited, the effect of Rosamultin on the express of VEGF was suppression but not as low as merely hypoxia without any pretreatment.Conclusions:1 In hypoxic conditions, Rosamultin could promote endothelial cell proliferation, differentiation, and significantly inhibit apoptosis induced by hypoxia.2 Rosamultin can significantly reduced hypoxia-induced calcium overload and the protective effect may be actived by inhabiting store operated calcium channel.3 Rosamultin could protect EA.hy926 cells from hypoxia induced mitochondrial apoptosis.4 Rosamultin could protect EA.hy926 cells from hypoxia induced cell damage throμgh HIF-1αsignaling pathway...