Study On The Relationship Between The Effect Of Organs On The MIRI Effect And The Mitochondrial Quality Control Of The Active Ingredients

Chapter One The Therapeutic Effect of Orientin on MIRIObjectivePrevious study proved that pharmacological preconditioning (PPC) with orientin (Ori) could enhance cardioprotection against myocardial ischemia/reperfusion injury (MIRI) mimicking is-chemic preconditioning (IPC). The aim of this study to clarify the relationship of cardioprotection against MIRI of orientin and mitophagy.Methods90 Sprague-Dawley male rats were equally and randomly divided into 6 different groups: Sham group (Sham, vehicle), Model group (Model, vehicle), Low-dose orientin preconditioning group (Ori lmg/kg), Mid-dose orientin preconditioning group (Ori 2mg/kg), High-dose orientin preconditioning group (Ori 4mg/kg). After 7-day adaptation, every rat was subjected to precondi-tioning with Ori or vehicle via intraperitoneal injection. All rats, but of Sham, were exposed to modeling of MIRI with ligating the left anterior descending coronary artery (LADCA) for 30 min, and then releasing for 120min. Following detections were performed:1) ECG; 2) infarction size; 3) the level of CK-MB\LDH\AST; 4) histomorphology; 5) myocardial ultrastructure.Results1. Model vs. Sham. Compared with Sham, QRS internal and QT internal increased, ST seg-ment elevated in infarction and reperfusion phase. Infarction area of Model significantly increased, 22.22%±3.96%(Model) vs.2.96%±2.83%(Sham), respectively, P<0.001. The level of CK-MB in Model was obviously different to Sham,1917.77±648.13U/L (Model) vs.756.16±200.84 U/L (Sham), P<0.001. LDH in Model increased from 448.93±199.81 U/L (Sham) to 1603.55±588.11 U/L, P<0.001. Additionally, the AST level in was statistically different to Model,197.05±54.72 U/L vs.129.35±40.07 U/L, P<0.01. Histopathological results revealed that myocardium structure in Model were characterized as myocardial fibrosis, necrosis, cell interstitial edema and angiote-lectasis. In addition, myocardial ultrastructure, in Model group, presented totally huddle, brimming with a large amount of broken myofilament, tons of dissociative and damaged mitochondria, and unconsolidated stroma.2. Orientin-preconditioning groups vs. Model. In infarction and reperfusion phase, Ori enhenced HR, decresed QRS internal and QT internal, and decreased ST segment. In comparison with Model, Low-dose orientin preconditioning had no evidently effect on infarction size (P>0.05 vs.Model). While, intraperitoneal injection with 2.0 mg/kg and 4 mg/kg orientin evidently decreased infarction area,10.71±2.79(Ori 2mg/kg),2.66±1.84%(Ori 4mg/kg) vs. 22.22%±3.96%(Model); P<0.01 or P<0.01. There were no obvious difference between Low and Model in leakage of CK-MB, LDH and AST (P>0.05vs.Model). Mid- and high-dose of orientin, compared with Model, dramatically reduced the level of CK-MB [977.27±404.30 U/L (Ori 2mg/kg),940.96±301.70 U/L(Ori 4mg/kg) vs.1917.77±648.13 U/L(Model), P<0.01 or P<0.01], the level of LDH [684.64±335.57 U/L (Ori 2mg/kg),676.37±280.99 U/L (Ori 4mg/kg) vs. 1603.55±588.11 (Model); P<0.01 or P<0.01] and the level of AST [132.40±24.83U/L (Ori 2mg/kg),122.66±21.89 U/L (Ori 4mg/kg) vs.197.05±54.72 (Model), P<0.01 or P<0.01]. Patho-logical section in Ori 2mg/kg and 4mg/kg distinctly alleviated the symptoms of MIRI, shown as myocardial fiber orderly arranging, few degeneration and necrosis. Preconditioning with mid- and high-dose orientin displayed as, in large part, intact and consolidated sarcomere, ascertained pro-file of mitochondria and compacted stroma.3、OW vs. Model. Compared with Model, HR, QRS and QT internal increased in OW, and ST segment elevated. Myocardial slices in OW clearly diminished in infarction size,2.39%±1.67% (OW), compared with Model (22.22%±3.96%), P<0.001. Intraperitoneal injection 15μg/kg wort-mannin, compared with Model, significantly decreased the level of CK-MB [825.37±302.67U/L (OW) vs.1917.77±648.13U/L (Model), P<0.001], the level of LDH [454.13±141.92U/L (OW) vs. 1603.55±588.11U/L (Model), P<0.001] and the level of AST [132.10±26.96U/L (OW) vs. 197.05±54.72U/L (Model), P<0.01]. Slightly pathological alteration manifested in OW histomor-phology, such as myocardial myofilament fracture, mild cell interstitial edema without other patho-logical abnormity. Ultrastructure in OW shown that myofilament was partly separated without losing integrality, and majority of mitochondria are not broken.4. OW vs. Others. Compared with Ori 2mg/kg, HR、QRS internal、QT internal in OW de-creased, but ST segment elevated. Infarction area, intracellular enzymes leakage in OW was not statistically different to Sham (P>0.05 vs.Sham).ConclusionMid- and high-dose orientin preconditioning evidently diminished the infarction size, the leak-age of myocardial enzmes, the pathological damage, the myofilament fracture and mitochondrial injury, protecting rats heart from MIRI via, maybe, inhibiting mitophagy.Chapter Two Relationship Between Cardioprotection induced by OrientinObjectiveTo clarify cardioprotection induced by orienti from the perspective of mitochondrial manip-ulation.Methods36 Sprague-Dawley male rats were grouped and operated following the methods mentioned in PART ONE. Measurement of following indexes was performed: 1) Mitochondrial membrane potential(MMP); 2) Opening of mitochondrial permeabiility trans on pore(MFTP);3) Activity of mitochondrial respiratory chain complexes I~V 4) Grading of mi chondrial injury; 5) Counting of autophagosome.Results1.Model Sham By compared wSham,the MMP of Model decreased prominently0.68±0.07(Model) 2.31±0.19(Sham),P<0.001.MIRI modeling significantly enhanced MPTP opening,comparedwiSham,13012.70±2101.60RFU/(mgprotein)(Modd)w.9244.08±692.69RFU/(mg protein)ham),P<0.01.comparisoSham,MIRI modelig dramatically de-creased the level of complex I[90.91±18.18 nmol NADH/(min-mg protein)(Model)vs.206.06±10,50 nmol NADH/(min.mg protei)(Sham),P<0.001],the level of complex II[396.64±18,40 nmol DCPIP/(min-mg protein)(Model)vs,632.88±21.68 nmol DCPIP/(minmg protein)(Sham),P<0.01,the level of complex I~II9.52±4.58 nmol CoQH2/(min-mg protein)(Model)w.177.013.99 nmol CoQH2/(min.mg protein)(Sham),P<0.01],the level of complexIV[663.92±292.06 nmol Cyt c/(tninmg protein)(Model)s.1873.47±183.63 nmol Cyt c/(miii.mg protein)(Sham),<0.00。ande level of complex V7.51±18.56 nmol NADH/(ininmg protein)(Model)vs.80.39±16.08 nmol NADH/(min-mg protein)(Sham),P<0.03.Compared with Sham,mitodiondrial injury obviously accentuated,3.40±0.6G(Model)vs.0.73±0.12(Sham),P<0.01.The umber of autophagosome also increased in Model。9.0±3,compared with Sham(7.3±2.1),P<0.01.2.Orientin-precond oning groups .Ori Img/kg had no evident effect on MMP{P>0.05 V5.Model),but mid-and high-dose orientin preconditioning evidently enhanced ftie MMP[1.12±0.13(Ori 2mg/kg),1.44±0.15(On4mg/kg)VS.0.68±0.07(Model),P<0.01 orP<0.01.Ori Img/kg and Ori 2mg/kg had no effect of statistical discrepancy on MPTP(P>0.05 Model),but Ori 4mg/kg inhibited MPTP opening,9302.24±1181.29 RFU/(mg.protein)、13012.70±2101.60FU/(mg.protein)(Model),P<0.01.Ori Img/kg did not affect 1:he level of complex I~IV as astatistically different way CP>0.05 vs.Model).Mid-and high-dose Orientin preconditioning sig-nificantly increased 1:he level of complex I[145.45±18.19 nmol NADH/(min.mg pro1:ein)(Ori2mg/kg),169.70±21.00 nmol NADH/(min.mg protein)(Ori 4mg/kg)vs.90.91±18.18 nmol NADH/(min.mg protein)(Model),P<0.01 or P<0.01],the level of complex II[576.30±24.67 nmol DCPIP/in.mg protein)(Ori 4mg/kg)w.396.618.40 nmol DCPIP/(min.mg protein)(Modd),P<0.05],the level of complex III[i00.73±12.11 nmol CoQ(min.mg protein)(Ori 2mg/kg),126.68±16.08 nmol CoQH2/(min.mg protein)(Ori 4mg/kg)V0.05±4.58 nmol CoQH2/(min‘mg protein)(Model),P<0.01 or P<0.01]and the level of complex IV[1549.15±240.4 nmol Cyt c/(min.mg protein)(Ori 4mg/kg)V663.92±292.06 nmol Cyt c/(min.mg protein)(Modd),P<0.01.There was no statistical difference in complex V between groups of Orienti-preconditioning and Model(P>0.05 VModel).了here were sigificatly difference amonoriciiti-preconditioning groups and Model in mitochondrial injury[2.53±0.12(Low),1.80.42(Ori 2mg/kg),1.40±0.20(Ori4mg/kg)v5.3.40:t0.60(Model),f<0.05orf<0.01orP<0.01].Thenumberofaul;ophagosomein Low had no shtistically difference to Model(P>0.05 Model,but among Ori 2mg/kg,Ori4mg/kand Model,8.7±6.0(Ori 2mg/kg),4.7±1.51(Ori 4mg/kg),respectively,V9.0±3.6(Model),P<0.05 or P<0.01.3.OW. Model. Compared Model, 1:he level of MMP in OW obviously enhanced [1.94=t0.29(OW) 0.68±0.07(Model),P<0.001],but MPTP opening in OW did not obviously en-hanced P>0.05 w. Model]. The level of complex I~V in OW had no notably discrepanc:y to Model CP>0.05 Model). While extent of mitochondrial injury in OW dramatically declined com-pared wi]Vlodel [1.87±0.50(OW) v0.05±3.40±0.60(Model),P<0.01]. Additionally, by comparisonwi Model, tile number of aophagosome in OW evidently diminished [1.7±0.6(OW)w.19.0±3.6(Model),P<0.05].4.OW vs. Ori 2mg/kg. There was significat difference between OW and Ori 2mg/kg in 1;helevel of MMP,[1.94=b 0.29(OW) 1.12±〇.13(Ori 2mg/kg),P<0.01],tmt no obvious differencein MPTP opening(P>0.05). In addition,ere was statistical discrepancy between OW and Ori2mg/kg in Complex I [109.09±18.15 nmolNADH/(min-mg protein)(OW) vs. 145.45±18.19 nmol NADH/(min.mg protein)(Ori 2mg/kg),P<0.05], but no evident difference in complex II~V(P>0.05). Besides,although there was no remarkable(iifferency between OW and Ori 2mg/kg in mkochondrial injury(P>0.05), number of autophagosome in OW dramatiacally diminished compared wi On 2mg/kg,1.0.6(OW) vs. 8.7±6.0(Ori 2mg/kg),P<0.01.ConclusionMid- and high-dose orientin-preconditioning ameliorates michondrial quality and quantity by enhancing the level of MMP and complex I-IV, and reducing the MPTP opening and mitochon?drial injury. Additionally, Mid- and high-dose orientin-preconditioning dramatically diminish the number of autophagosome,which tends to clarify that orientin-preconditioning protecting rats heart from MIRI is connected with inhibition of mitophagy.