| Linarin(LIN)and phenylethanoid glycoside-rich extract(Ph G-RE)are pharmaceutical effective ingredients extracted from wild chrysanthemum and Cistanche deserticola,respectively.Both of them have multiple pharmaceutical functions,but their cardiovascular protection and ischemia-reperfusion functions are seldom studied.In the present study,models of myocardium H9c2 cell line stress injuries by hypoxia/reoxygenation as well as isolated rat heart ischemia-reperfusion were established.To study the protections and mechanisms of the drugs against myocardium cell hypoxia/reoxygenation and isolated heart ischemia-reperfusion injuries,myocardium cell and rat heart were pretreated with Linarin,rat heart were pretreated with Ph G-RE.The experiments and results were as follows:1.Protection functions and mechanisms of Linarin against myocardium ischemia-reperfusion injury1.1 Protection and mechanism of Linarin to H9c2 rat myocardial cellMethods: Linarin were added to H9c2 cell at concentrations 0.4,1.1,3,10,30,50,75 and 100 ?M for 48 h,MTT method was used to determin the safety dosage range of the drug.Groups with 3,10,30 and 50?M Linarin treatments were treated with hypoxia for 4h and then reoxygenated for 1h.Effects of Linarin treatment to the myocardial cell apopsis were observed: survival rate,activities of LDH and caspase-3 with spectrophotometer,protein levels of Nrf-2,p-Nrf-2,Nu-p Nrf-2,Cyto-p Nrf-2,PI3 K,AKT,p-PI3 K and p-AKT with Western Blot.Results: The safety concentration of Linarin is ?30 ?M,it has no adverse effect on H9c2 cell survival rate.The survival rates of treated groups in these contractions were higher than those of Model groups.Lower LDH leakage,lowed Caspase-3 activity,reduced hypoxia/reoxygenation inducted cell injury and apopsis were also observed in these groups.In these low concentration groups,protein levels of p-Nrf-2,Nu-p Nrf-2,Cyto-p Nrf-2,p-PI3 K and p-AKT were elevated,while Nrf-2,PI3 K and AKT were not changed in Western blot.The rises of Nu-p Nrf-2/Cyto-p Nrf-2?p-PI3K/PI3 K and p-AKT/AKT suggested that myocardial protection of Linarin might be caused by the activation of PI3K/AKT/Nrf-2 signal pathway,and promoted Nrf-2 nucleus transportation in the process.1.2 Linarin affected PI3K/AKT/Nrf-2 signal pathway and protected myocardial cells apopsis from hypoxia/reoxygenation injuryMethods: The study model was setup as H92 c cells pretreated with 3,10 and 30 ?M Linarin for 48 h,and then subjected with hypoxia/reoxygenation for 4h/1h.The experimental groups were Vehicle-Control,Model,positive control(EDV,30 ?M),LIN-L(3?M),LIN-M(10?M),LIN-H(30 ?M),LIN-M+IC87114.Effect of Linarin to PI3K/AKT/Nrf-2 was observed by using PI3K? antagonist IC87114 to suppress PI3 K.MTT was used to detect H9c2 survival rates.Activities of LDH,Caspase-3,SOD,GSH-Px and MDA content were measured.Expression levels of inflammatory factors IL-1?,IL-6 and TNF-? m RNA were detected with q RT-PCR.Fluorescence Inversion Microscopy was used to observe the cellular ROS levels.Level of HO-1,NF-?B,Cyto C and Bcl-2 were detected with Western Blot.Results: Linarin protection was supressed by applying PI3 K repressor IC87114,the cell survival rates were lowed.When comparing LIN-M+IC87114 and LIN-M groups,LDH leakage,Caspase-3 activity and MDA content were elevated,SOD and GSH-Px activities were repressed.Lower ROS content was detected with DAPI stained fluorescence inversion microscopy.These results suggested that Linarin pretratment enhanced antioxidation of myocardium.q RT-PCR detected lower expression levels of IL-1?,IL-6 and TNF-? after Linarin treatment,IC87114 could block the Linarin effect,incidated Linarin could supressed myocardial inflammation.Western Blot results showed that Linarin upregulated protein levels of p-PI3 K,p-AKT,p-Nrf-2,HO-1,NF-?B and Bcl-2;Cyto C was downregulated,PI3 K,AKT and Nrf-2 levels were not changted.The protein levels were reversed after IC87114 supplement.These results indicated that the myocardial protection of Linarin could be caused by the activation of PI3K/AKT/Nrf-2 signal pathway.1.3 Linarin effects on rat isolated myocardial ischemia-reperfusion injuryMethods: The rats were randomly divided into 7 groups: Vehicle-Control,Model,positive control(EDV,30 ?M),LIN-L(3?M),LIN-M(10?M),LIN-H(30 ?M),LIN-M+IC87114.The rat heart ischemia-reperfusion(I/R)model was setup by hearts isolation,and perfused for 30 min,paused for 30 min and reperfused for 60 min.The following indices were measured: haemodynamics including LVSP,+dp/dtmax and CF;heart enzyme AST,CK-MB and LDH activities;oxidation and stress related SOD,GSH-Px activities and MDA content;measure myocardial infarction size(MIS);histopathology changes detected by optical microscopy with HE staining;myocardial apoptosis rate was detected with TUNEL method;Western Blot was used to determine protein levels of NF-?B,Cyto C,Bcl-2,Nrf-2 and p-Nrf-2.Results: Haemodynamics LVSP,+dp/dtmax and CF were elevated after 3,10,30 ?M Linarin pretreatment.Lower myocardial CK-MB and AST activities and reduced LDH leakage were also observed;oxidation stress related MDA content was lowed,while SOD and GSH-Px activities were enhanced.The structural damage of rat myocardium was mitigated,the myocardial infarction sizes were reduced.TUNEL staining results showed reduced positive expression,indicated improved I/R induced myocardial apoptosis after Linarin application.Western Blot results showed upregulation of NF-?B and Bcl-2 protein,raised p-Nrf-2/Nrf-2,downregulated Cyto C,suggested that Linarin could protect rat myocardium from I/R injury by activating Nrf-2 signal pathway.2.Function and mechanism of alleviation of ischemia-reperfusion injury phenylethanoid Glycoside-Rich Extract(Ph G-RE)from Cistanche deserticola2.1 Ph G-RE alleviated ischemia-reperfusion injury through PI3K/AKT/GSK-3? signal pathwayMethods: Experimental animal model of ischemia-reperfusion(I/R)injury was established by using isolated rat heart for 30 min perfusion,30 min pause and 60 min reperfusion.The rats were divided into 3 groups randomly,namely control(sham),model(I/R)and medication(Ph G-RE).The myocardial infarction sizes(MIS)were measured;LVSP,+dp/dtmax and CF values were tested;AST,CK-MB and LDH were measured;myocardial oxidation stress related SOD and GSH-Px activities,and MDA content were measured with automatic biochemistry analyzer.Myocardial pathologic examination was conducted by optical microscopy with HE staining.Myocardial cell apoptosis was tested by TUNEL method.Protein levels of Cyto C,Bcl-2,Bax,cleaved-caspase-3,Procaspase-3,AKT,GSK-3?,p-AKT and p-GSK-3? were determined with Western blot.Results: Activities of CK-MB,LDH and AST were reduced in Ph G-RE treated myocardial tissue,accompanied with reduced MDA,whereas SOD,GSH-Px activities and LVSP,+dp/dtmax and CF were increased.These results suggested that Ph G-RE post treatment could improve myocardial antioxidation and I/R induced myocardial injury.Less TUNEL staining was observed in Ph G-RE treated myocardial tissue,less myocardial damage and myocardial infarction size were also observed.These results suggested that Ph G-RE post treatment repressed I/R induced myocardial tissue apoptosis.Protein levels of p-AKT,p-GSK-3? and Bcl-2 were elevated,the Bcl-2/Bax was also rised;Cyto C,Bax and cleaved-caspase-3 were reduced,indicated that Ph G-RE could protected myocardial tissue through mitochondria pathway and activation of PI3K/AKT/GSK-3? pathway.3.Conclusion:The study demonstrated that Linarin could upregulate NF-?B and Bcl-2 expression levels,and reduced mitochondria Cyto C release,by means of Nrf-2 activation through PI3K/AKT/Nrf-2 pathway activation.The Ph G-RE might repress myocardial apoptosis through PI3K/AKT/GSK-3? signal pathway.Both Linarin and Ph G-RE could protect myocardial tissue from hypoxia-reperfusion induced apoptosis. |
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