Protective Mechanism Of β-asarone On Beclin-1Dependent Autophagy In PC12Cells Following Oxygen-glucose Deprivation And Reoxygenation Induced Injury

ObjectiveFirst, study the relationship between autophagy and ATP during reoxyg enation.Second, study the role of autophagy in PC12cells subjected to2h of oxygen-glucose deprivation (OGD) with different time-points of reoxygenation.Third, study the effect of β-asarone on Beclin-1, intracellular free calcium concentration ([Ca²⁺] mitochondrial membrane potential (MMP), cell viability and cellular morphology in PC12cells treated with2h OGD followed by24h reoxygenation.MethodsFirst, establish an oxygen-glucose deprivation and reoxygenation induced injury model of PC12cells:The high glucose Dulbecco's modifed Eagle's medium (DMEM) was exchanged for Earle's balanced salt solution, and the plates were placed in a hypoxia chamber with1%O₂,94%N₂and5%CO₂for2h. Then the full culture medium replaced the Earle's balanced salt solution, and the plates were placed under an atmosphere of95%air and5%C02for different time-points of reoxygenation.Second, establish a method of high-performance liquid chromatography (HPLC) for detection of ATP. Study the specificity of the ATP in cell extracts by setting negative control group, standard control group, and sample control group.10mg ATP was dissolved with10mL double distilled water, the concentration of which was1. OOmg/mL.0.5mL β-asarone (1.00mg/mL) was diluted with9.5mL double distilled water to achieve the concentration of 50.0μg/mL16,32,48,64,80μLβ-asarone (50.0μg/mL) was diluted with appropriate double distilled water to achieve the concentration of0.080,0.160,0.240,0.320,0.400μg/mL.20μL of each concentration was injected for analysis. Investigate the linear relationship of the ATP concentration and peak area within0.080-0.400mg/mL ATP. For precision and accuracy, five replicate quality control samples at three concentrations (0.080,0.240and0.400(μg/20μL) were continuously assayed.600μL PBS was added to suspense the cells at three time-points of reoxygenation (0,10,24h).80μL of HClO₄(concentration50%, v/v) was added to the cell suspensions, then-80℃freezed and room temperature thawed for two times.0.1,0.2,0.4μg/mL ATP standard solution was added to the cell suspensions, respectively.300μL of2mol/L NaOH was added to adjust pH value to neutral. The neutral cell suspensions were then spun at12000rpm for15min. The upper] ayer was injected for analysis and for investigation of the recovery. For investigation of the stability of samples, ATP in cell extractions (reoxygenation time-point24h) were detected after storage at-80℃for0d,3d,7d.Third, establish a flow cytometric method for quantitative detection of Beclin-1expression:Investigate the influence of3-methyladenine (3-MA, the autophagic inhibitor),2%Bovine serum albumin/Phosphate buffer solution (BSA/PBS), the concentration of primary antibody (0.002,0.004,0.006μg/μL), incubation temperature (25,37℃), incubation time (15,30,60min), preserved time and methods (Sample preparation was determined immediately or determined after6h or24h of storage at4℃respectively. Sample preparation was determined after0,3or7d of storage fixing with1%paraformaldehyde at4℃, respectively.) on analysis of Beclin-1.Forth, study the role of autophagy and the relationship of autophagy with ATP. After growth of PC12cells for48h, the full culture medium wa s discarded, the cells was washed once with PBS and incubated with Earle' s balanced salt solution in a hypoxia chamber with a compact gas oxygen c ontroller to maintain oxygen concentration at1%by injecting a gas mixtu re of94%N2and5%CO2for2h. After OGD, the cells were transferred bac k to full culture medium with oxygen for0,4,10,24,48h. ATP (includi ng normal control group at24h reoxygenation) was measured using HPLC, c ell protein determined using the BCA assay, cell viability was measured b y3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assa y, cellular morphology was observed under inverted phase contrast microsc ope, and autophagosomes (model control group and normal control group at24h reoxygenation) were observed under transmission electron microscope. Cells grown in the same well were divided into two groups. One was used for ATP detection, and the other was used for Beclin-1analysis. Correlat ion of ATP and Beclin-1was analyzed.Fifth, Protective mechanism of β-asarone on Beclin-1in PC12cells following oxygen-glucose deprivation and reoxygenation induced injury. After growth for48h, PC12cells were incubated with full culture medium containing β-asarone (20,30or45μg/mL) or nimodipine (10μmol/L) under normoxic conditions for1h before OGD. The full culture medium containing drug was discarded. The cells were rinsed once with PBS, and incubated with Earle's balanced salt solution containing beta-asarone (20,30or45μg/mL) or nimodipine (10μmol/L) for2h OGD. The Earle's balanced salt solution was discarded, and then PC12cells were incubated with full culture medium free of drug under normoxic conditions for24h. After these treatments, Beclin-1,[Ca²⁺]i and MMP were analyzed by flow cytometry, cell viability was measured by MTT assay, cellular morphology was observed under inverted phase contrast microscope, and autophagosomes were observed under transmission electron microscope.Sixth, the methods of statistical analysis were used in this study. Measurement data were expressed as mean±standard deviation (Mean±SD). Statistical significance was determined by independent-samples t-test. Correlation analysis was determined by Pearson correlation analysis.ResultsFirst, a good linear relationship (r=0.9995) between ATP concentration and peak area in0.08-0.40mg/mL ATP. For precision and accuracy, the relative standard deviation (RSD) of the three concentrations (0.080,0.240and0.300μg/mL) were2.8%,2.1%and1.8%, respectively (five samples for each concentration level), indicating the precision and accuracy was good. The average recoveries of three concentrations (0.100,0.200and0.400μg/mL) were95.6±2.8%, respectively, and the RSD were2.9%.(five samples for each concentration level), indicating the method is accuracy. ATP concentration was0.331±0.010,0.312±0.012,0.303±0.008μg/mL, respectively, after0,3,7d of storage in-80℃refrigerator, indicating the stability of storage within7d is good.Second, Beclin-1(6.49±1.19%) expression was very low in normal control cells. However, after OGD/R treatment, Beclin-1expression (33.14±2.16%) was significantly upregulated. The autophagic inhibitor3-MA significantly reduced Beclin-1expression (15.27±1.51%) in OGD/R treated cells (P<0.001). Detected Beclin-1was35.40±2.13%or43.99±7.26%in blocked group or non-blocked group, respectively. These two groups were significant statistical difference (P<0.01). Detected Beclin-1was highest at0.004μg/μL of primary antibody (35.40±2.13%). Detected Beclin-1was decreased at0.002,0.006μg/μL of primary antibody (33.54±2.27%,29.95±1.76%). Detected Beclin-1was36.27±3.29%or36.70±2.84%in group incubated at room temperature (25℃) or at37℃, respectively. There was no significant statistical difference (P>0.05). Detected Beclin-1was36.38±3.51%or36.36±3.64%in sample incubated for30min or60min, respectively. The statistical difference was no significant (P>0.05). However, detected Beclin-1(31.67±3.76%) in sample incubated for15min was significantly decreased (P<0.05) compared to group incubated for30min or60min. Samples were stored at4℃and were not fixed with1%paraformaldehyde. There was no significant statistical difference (P>0.05) of detected Beclin-1(36.30±3.29%,34.93±3.38%, respectively) in group determined immediately or determined after6h of sample preparation. Compared to group determined immediately, detected Beclin-1(20.81±3.99%) was dramatically decreased in group determined after24h of sample preparation (P<0.001). Samples were all fixed with1%paraformaldehyde and stored at4℃. Detected Beclin-1was36.59±3.19%in sample after3d of storage and37.67±3.38%in sample in0d of storage. They had no significant statistical difference (P>0.05). Detected Beclin-1was significantly reduced in group after7d of storage (24.04±3.83%) compared to group in0d of storage (P<0.001).Third, during reoxygenation, the cell viability and ATP content decreased at first, and then increased, Beclin-1expression was upregulated at first, and then was downregulated, and cellular morphology was injury at first, and then was improved. Cell viability continued to decrease at0,4,10h reoxygenation (64.6±4.6%,51.8±1.6%,36.0±5.6%), and increase at24,48h reoxygenation (56.8±3.7%,73.4±4.3%). ATP continued to decrease at0,4h reoxygenation (3.4±0.5,3.2±0.4mg/g protein). It recovered at10h reoxygenation (4.1±0.7mg/g protein), but it was still lower than that before OGD (4.6±0.9mg/g protein). ATP continued to increase at24,48h reoxygenation (5.4±0.9,6.7±1.2mg/g protein). ATP at24h reoxygenation was lower than normal control (5.4±.09mg/g protein vs7.7±1.2mg/g protein, p<0.001). Beclin-1expression was upregulated at0,4,10,24,48h reoxygenation (10.5±1.4%,12.7±1.4%,16.1±1.8%,28.2±3.1%,20.0±3.2%). Beclin-1expression at48h reoxygenation was lower than that at24h reoxygenation. The numbers showed a trend of reduction at0h reoxygenation after2h OGD,, and continued to be reduced as well at4and10h reoxygenation, reaching its minimum at10h reoxygenation, while they were increased significantly at24and48h. Somas were smaller at0,4or10h reoxygenation than that before OGD. The cells exhibited round, gathered, slender and degenerated morphology at0,4and10h reoxygenation. Somas became satiation, and neurite became slender at24h reoxygenation. Somas was satiation, neurite interweave with each other to form a mesh at48h reoxygenation. Autophagosomes cound be hardly observed in the normal cultured cells, but some autophagosomes could be obviously observed in cells treated with OGD/R. At0h reoxygenation after2h OGD, a significant negative correlation was occurred between ATP and Beclin-1expression (r=-0.61, P<0.05), while there was not a significant correlation between ATP and Beclin-1expression at24h reoxygenation (r=0.24, P>0.05).Forth, After24h reoxygenation, the number of neurons showed a significant reduction, which was observed under inverted phase contrast microscope. The PC12cells exhibited round, slender and degenerated morphology. Some autophagosomes could observed under transmission electron microscopy. Beclin-1expression was increased (28.5±2.3%, vs normal control6.3±0.8%, p<0.001). Cell viability (OD value) was decreased (0.46±0.02, vs normal control0.79±0.04, p＜0.001). MMP was decreased (335.2±19.3, vs normal control (542.5±26.6), p<0.001).[Ca⁹2+)] was increased (294.9±42.7, vs normal control (124.7±14.9), p<0.001). Beta-asarone (20,30,45g/mL)μand nimodipine (a calcium antagonist,10(μmol/L) both significantly decreased Beclin-1expression (19.1±2.8%,17.6±2.6%,20.5±2.8%,15.6±2.5%, vs model control group, p<0.001) and [Ca²⁺](137.8±23.5,132.4±25.5,169.7±23.9,139.0±19.0,vs model control group, p<0.001), but increased MMP (422.4±27.5,478.7±21.6,414.1±25.1,483.5±28.2, vs model control group, p<0.01), cell viability (0.64±0.07,0.75±0.09,0.56±0.08,0.74±0.04, vs model control group, p<0.01) in PC12cells treated with OGD/R. They also increased the cell numbers and improved the cellular morphology in PC12cells subjected to OGD/R. For cell viability and [Ca²⁺]; there was no significant difference between beta-asarone (30μg/mL) group and normal control group normal control group (P>0.05).ConelusionsFirst, ATP can be well extracted from the cell using6%HClO₄(final concentration, v/v). HPLC can isolate complex components in the cells, and analyzed intracellular ATP content quickly, accurately and quantitatively.Second, the results got from flow cytometry and transmission electron microscopy indicates that OGD/R can generate autophagy. Flow cytometry c an easily, quickly, accurately and quantitatively determine Beclin-1. The results of screening analysis indicate that the optimization of multi-pa rameters for Beclin-1protein staining is as follows.2%BSA-PBS was used for sample block. Concentration of primary antibody was0.004μg/μL. Sam ples were incubated at room temperature(25℃) for30min. They were detec ted immediately or in6h storing at4℃or in3days fixing in1%paraf ormaldehyde and storing at4℃after sample preparation.Third, energy metabolism dysfunction can induce autophagy during OGD in PC12cells. Increased [Ca²⁺], and decreased MMP may induce autophagy during reoxygenation in PC12cells. Autophagy may be a protective effect on PC12cells treated with different time-points of reoxygenation after2h OGD.Forth, treatment with β-asarone (20,30or45μg/mL) for1h before OGD and2h during OGD can protect PC12cells against OGD/R induced injury, of which30μg/mL β-asarone is the most effective. It may reduce Beclin-1expression partly by decreasing [Ca²⁺],and increasing MMP in OGD/R treated PC12cells.β-asarone may improve cell function through calcium antagonism, and then reduces autophagy and improves cell viability.