Effects Of Acanthopanax Senticosus Polysaccharide Pretreatment On PC12 Cell Exposed To Oxygen-glucose Deprivation/reperfusion

Chapter 1 Protective effects of Acanthopanax senticosuspolysaccharide pretreatment on PC12 cell exposed tooxygen-glucose deprivation/reperfusionObjective:To study the protective effects of Acanthopanax senticosus polysaccharide(ASPS)pretreatment on PC12 cell exposed to oxygen-glucose deprivation/reperfusion.Methods:The oxygen-glucose deprivation/reperfusion model(OGD/R)of PC12 cell was established by anaerobic bag combined with oxygen-glucose deprivation to simulate cerebral ischemia-reperfusion injury.The impacts of ASPS pretreatment on the viability,apoptosis and cell morphology of PC12 cell exposed to oxygen-glucose deprivation/reperfusion were analyzed.The experiment was divided into five groups:control group,OGD/R model group,OGD/R+ ASPS(2.5,5 and 10μg/m L)groups.The ASPS-pretreated groups were pretreated with 2.5,5 and 10 μg/m L ASPS for 24 hours(5holes/group)before the beginning of OGD.After OGD for 12 h,similar concentrations of ASPS were given when PC12 cells were re-supplied oxygen and sugar and then continue to cultivate for 12h;The OGD/R model group only applied with the same volume of culture medium without ASPS;While control group was not treated by OGD or ASPS.For all groups,the viability of cells was observed by laser scanning confocal microscope,the level of apoptosis was measured by flow cytometry;and the morphology of cells was observed by transmission electron microscope.Results:Laser scanning confocal microscope showed that the cell viability of the OGD/R model group was significantly lower than that of the control group(79.93±2.73%vs 100%,P<0.01).Application of ASPS(2.5、5、10μg/m L)increased the cell viability(84.82±1.88、94.37±2.19、96.40±2.16%)and has a dose-effect relationship.The cell viability between 5 and 10 ug/m L ASPS-pretreated groups were significantlydifferent to that of OGD/R model group(P<0.01).Dose-dependent increasement of cell viability in ASPS-pretreated groups were observed.Flow cytometry showed that OGD/R increased the apoptotic rate(44.83±8.87%)significantly(P<0.001).ASPS pretreatment reduced apoptotic rates at a dose-dependent manner(41.37±5.61 、40.40±4.42 、 27.83±3.82% for 2.5,5 and 10 ug/m L ASPS pretreatment)(P<0.05).Transmission electron microscope showed that compared with the control group,the OGD/R model group showed irregular protuberances in the cell membrane,most of the nuclear membrane was intact,liquid droplets appeared in the cytoplasm.In ASPS-pretreated groups,the cell membrane was intact with no significant damage on the mitochondria and microtubule.Transmission electron microscope confirmed the protective effect of ASPS pretreatment on PC12 cell exposed to oxygen-glucose deprivation/reperfusion.Conclusions:ASPS pretreatment can reduce the morphological damage of PC12 cells and greatly attenuated oxygen-glucose deprivation/reperfusion(OGD/R)injury in PC12 cells by increasing cell viability and reducing apoptosis dose-dependently.Chapter 2 Protective Mechanism of Acanthopanax senticosusPolysaccharide pretreatment on PC12 cell exposed tooxygen-glucose deprivation/reperfusionObjective:To study the possible protective mechanism of Acanthopanax senticosus polysaccharide pretreatment on PC12 cell injury exposed to oxygen-glucose deprivation/reperfusion from the aspects of oxidative stress,inflammatory reaction and apoptosis.Methods:Indicators were studied.For oxidative stress,lipid peroxidation indexes including Superoxide dismutase(SOD),Glutathione(GSH),Ascorbic acid(As A),Total antioxidant capacity(T-AOC);8-oxoguanine DNA glycosylase(OGG1);Nitric oxide(NO)and Inducible nitric oxide synthase(i NOS);Cholinesterase,including acetylcholinesterase(ACh E)and butyrylcholinesterase(Bu Ch E)were detected.Inflammatory indicators,including inflammatory factors Interleukin-1(IL-1),Tumor necrosis factor-alpha(TNF-α)were detected.Apoptosis-related indicators,including caspase protease and Nuclear factor kappa-B(NF-κB)signaling pathway protein were detected.Results:In terms of lipid peroxidation,compared with the control group,OGD/R reduced the content of GSH(19.23±0.64mg/gprot),As A(0.28±0.02ug/mgprot),the activity of SOD(21.71±0.95 U/mgprot),and the value of T-AOC(0.43±0.01 U/mgprot)significantly(P<0.05).Application of ASPS(2.5、5、10μg/m L)increased the activity of11SOD(26.49±0.69、26.79±0.49、28.59±0.62U/mgprot),the content of GSH(21.49±1.82、24.77±2.29 、 25.28±2.88mg/gprot)and As A(0.53±0.02 、 0.54±0.01 、0.60±0.02ug/mgprot)and the value of T-AOC(0.44±0.02 、 0.45±0.01 、 0.46±0.02U/mgprot).The values of SOD and As A in all ASPS-pretreated groups,GSH between 5and 10 ug/m L ASPS-pretreated groups and T-AOC in 10 ug/m L ASPS-pretreated group were significantly different to that of OGD/R model group(P<0.05).Dose-dependent increasement of all indexes in ASPS-pretreated groups were observed.The results showed that ASPS pretreatment protected PC12 cell by increasing the activity of SOD,the content of GSH and As A,and finally increasing the value of T-AOC,alleviating the level of oxidative stress caused by oxygen-glucose deprivation/reperfusion.OGG1 is one of the key enzymes to repair oxidative damage of DNA.Compared with the control group,OGD/R increased the expression of OGG1(30.92±0.27ng/m L)significantly(P<0.001).ASPS pretreatment reduced the expression of OGG1 at a dose-dependent manner(25.92±0.16、23.98±0.09、23.63±0.19ng/m L for 2.5,5 and 10ug/m L ASPS treatment)(P<0.001).The results showed that ASPS pretreatment could reduce the oxidative damage of DNA in PC12 cells caused by oxygen-glucose deprivation/reperfusion,thus reducing the expression of OGG1.Cerebral ischemia will stimulate the release of NO and i NOS,in which i NOS will synthesize lots of NO,to aggravate nerve cell injury.Compared with the control group,OGD/R increased the content of NO(3.38±0.21umol/mgprot)and activity of i NOS(221.57±10.65U/mgprot)significantly(P<0.001).Application of ASPS(2.5、5、10μg/m L)reduced the content of NO(3.10±0.04、2.31±0.15、2.29±0.27umol/mgprot)and activity of i NOS(135.87±5.46、126.51±9.65、83.18±15.58 U/mgprot).The values of NO and i NOS in all ASPS-pretreated groups were significantly different to that of OGD/R model group(P<0.05).Dose-dependent reduction of all indexes in ASPS-pretreated groups were observed.The results showed that ASPS pretreatment could reduce the injury of PC12 cells caused by oxygen-glucose deprivation/reperfusion by reducing NO and i NOS.The activity of cholinesterase increases during oxidative stress and inhibits the release of neurotransmitters,which is related to the poor prognosis of ischemic stroke.Compared with the control group,OGD/R reduced the activity of ACh E(0.59±0.08U/mgprot)and Bu Ch E(0.87±0.20U/mgprot)(P<0.01).Application of ASPS(2.5、5、10μg/m L)increased the activity of ACh E(0.81±0.11、1.06±0.06、1.17±0.06U/mgprot)and Bu Ch E(0.90±0.23 、 0.96±0.14 、 1.18±0.13U/mgprot).The values of ACh E in all ASPS-pretreated groups and Bu Ch E in 10 ug/m LASPS-pretreated group were significantly different to that of OGD/R model group(P<0.05).Dose-dependent increasement of all indexes in ASPS-pretreated groups were observed.The results showed that ASPS pretreatment could reduce the injury of PC12 cells caused by oxygen-glucose deprivation/reperfusion by increasing the the activity of ACh E and Bu Ch E.During cerebral ischemia,ROS can stimulate microglia to release pro-inflammatory cytokines,including TNF-α,IL-1 and so on.Compared with the control group,OGD/R increased the expression of IL-1(43.04±3.44pg/m L)and TNF-α(175.39±8.67pg/m L)(P<0.001).Application of ASPS(2.5、5、10μg/m L)reduced the expression of IL-1(8.16±3.37、23.71±2.01、22.76±2.00pg/m L)and TNF-α(92.46±13.29、89.12±3.00、82.58±7.87pg/m L).The values of IL-1 and TNF-α in all ASPS-pretreated groups were significantly different to that of OGD/R model group(P < 0.001).Dose-dependent reduction of all indexes in ASPS-pretreated groups were observed.The results showed that ASPS pretreatment could reduce the injury of PC12 cells caused by oxygen-glucose deprivation/reperfusion by reducing the expression of inflammatory factors IL-1 and TNF-α.Apoptosis is the main form of cell injury during cerebral ischemia/reperfusion,and caspase protease is the key protein of apoptosis.Compared with the control group,OGD/R increased the expression of caspase-3(33.70±1.89μmol/L),caspase-8(30.00±0.87μmol/L)and caspase-9(30.85±0.72μmol/L)(P<0.001).Application of ASPS(2.5 、 5 、 10μg/m L)reduced the expression of caspase-3(19.55±0.70 、14.03±0.64、9.87±0.65μmol/L),caspase-8(19.54±0.59、13.96±1.28、10.63±0.86μmol/L)and caspase-9(21.52±0.87、19.37±0.68、13.04±1.78μmol/L).The values of all indexes in all ASPS-pretreated groups were significantly different to that of OGD/R model group(P<0.001).Dose-dependent reduction of all indexes in ASPS-pretreated groups were observed.ASPS pretreatment could reduce the injury of PC12 cells caused by oxygen-glucose deprivation/reperfusion by reducing the expression of caspase protease.Further analysis of apoptosis showed that compared with the control group,OGD/R increased the expression of NF-κB(67.97±1.69PU)significantly(P<0.001).ASPS pretreatment reduced the expression of NF-κB at a dose-dependent manner(65.98±2.12 、 63.90±1.67 、 61.06±2.20 PU for 2.5,5 and 10 ug/m L ASPS pretreatment)(P<0.01).The results showed that ASPS pretreatment could reduce the injury of PC12 cells caused by oxygen-glucose deprivation/reperfusion by reducing the expression of NF-κB.Conclusions:Acanthopanax senticosus polysaccharide pretreatment can alleviate the injury of PC12 cells caused by oxygen-glucose deprivation/reperfusion by reducing the level of oxidative stress,reducing DNA oxidative damage,reducing the expression of inflammatory factors IL-1 and TNF-α and reducing apoptosis.