Bioactive Compound Of Licorice As Additive Of HTK Solution Fortifies Myocardial Preservation In Grafts Of Rats With Prolonged Cold Ischemia

BackgroundCurrently,heart transplantation is still considered the primary criterion for end-stage heart failure treatment.This method can reduce mortality rate and improve the quality of patients' lives.However,heart transplantation is only available for a minority of patients due to organ shortage.Thus,the major limitation of this technique is the increasing number of patients versus the few number of donor organs.Prolonging cold ischemic time is a possible method to increase the source of heart donors.Inadequate myocardial protection associated with prolonged cold ischemia results in postoperative myocardial dysfunction.Therefore,developing new types of preservation solutions causing minor cold storage injury is considerably important.Histidine-tryptophan-ketoglutarate(HTK)solution has been widely used to preserve various grafts,which can compensate myocardial acidosis and prolong anaerobic glycolysis.Although HTK solution possesses a strong histidine buffer system,it enhances the oxidative stress during cold ischemia storage and reperfusion.Nevertheless,HTK solution cannot prevent the inflammation or cell apoptosis induced by ischemia/reperfusion injury.Hence,a new additive of the HTK solution should be developed to increase its effectiveness and avoid ischemia/reperfusion injury.Considerable attention has been focused on traditional Chinese medicine due to its distinct efficacy against oxidative stress,limited adverse reaction,and myocardial preservation during ischemia reperfusion injury.With the development of network and pharmacology,drug targets can be possibly predicted.Our previous studies showed that the licorice extracts,such as licochalcone B(LCB),licochalcone C(LCC),kaempferol(Kae),and rosmarinic acid(RosA),may exhibit cardioprotective effects via their antioxidative,antiapoptotic,and anti-inflammatory activities.LCB,LCC,Kae,and RosA can attenuate ischemia/reperfusion injury.Our previous work and relevant reports also developed a series of modified HTK solutions with LCB(1 ?g/L),LCC(2 mmol/L),Kae(15 mmol/L),and RosA(15?mol/L)of different concentrations on the basis of the mechanistic insights of cold storage-associated injury and the recognition of the cytoprotective effect of LCB,LCC,Kae,and RosA due to their antioxidative,anti-inflammatory,and antiapoptotic properties.The present study aimed to establish cold ischemia storage model,heart transplant model,and ischemia reperfusion rat's heart model(1)to evaluate the hypothesis that the addition of licorice extract to HTK can reduce the ultrastructural changes of donor heart subjected to prolonged cold ischemia and select the most effective drug,(2)assess the improved graft-protective efficacy of HTK solution,and(3)determine whether the new HTK solution confers cardioprotective effect against cold ischemia-induced injury by interfering with the PPAR-?(peroxisome proliferator activated receptor gamma)/NF-kB pathway.Part? Effects of licorice extract in prolonging the cold ischemia preservation time of donor heartObjective:This study aimed to determine the effects of licorice extract on donor heart in different cold ischemia preservation times via establishing cold ischemia storage and a heart transplant model.Methods:Sprague-Dawley(SD)rats were randomly divided into four groups:4,6,8,and 10 h cold ischemia storage groups.Each group was divided into the following five groups:HTK group(HTK alone),RosA group(containing 15 pmol/L RosA),Kae(containing 15 mmol/L Kae),LCB group(containing 1 ?g/L LCB),and LCC group(containing 2 mmol/L LCC).Each group contained 20 rats.The receptor group was also randomly divided into four groups.Each group was again divided into five groups,and each group contained five rats.A total of 400 rats from the donor group were operated under general anesthesia.Five kinds of 4 ? HTK solutions were perfused retrograde through the aortic root.Afterward,the rats' hearts were cut and immersed in five kinds of 4 ? HTK solutions for 4,6,8,and 10 h.Cell morphology was observed with HE coloration,and mitochondrial damage was assessed by transmission electron microscopy.The levels of creatine kinase-MB(CK-MB),cardiac troponin I(CTnI),tumor necrosis factor-a(TNF-a),interleukin-6(IL-6),and C-reactive protein(CRP)in storage solution in different cold ischemia storage times were determined by myocardial infarct size(IS),which was estimated using 2,3,5-triphenyltetrazolium chloride(TTC)staining.Cardiac ejection fraction(EF%)was detected by a small animal ultrasound after the transplant.The survival rate of the host was also recorded.Results:1.Comparison of the CK-MB activities and CTnI concentrations between groups.With the extension of cold ischemia time,the levels of CTnI and CK-MB released in the heart of rats increased(P<0.01).No significant difference was observed between the HTK solutions when the ischemic time was less than 6 h(P>0.05).When the cold ischemic time was more than 6 h,the addition of RosA and Kae significantly reduced the CTnI and CK-MB release in the hearts of rats.This result was induced by the cold ischemia storage(P<0.05).2.Comparison of the morphological structures of cardiac cells.When the ischemic time was less than 6 h,muscle fibers were neatly arranged.In addition,interstitial substance contained no edema,and muscle membrane displayed no damaged.Muscle fibers also showed no fracture,degeneration,or necrosis.When the ischemic time was more than 6 h,muscle fibers were irregularly arranged,and interstitial substance exhibited edema.Moreover,muscle membrane was damaged,and muscle fibers showed fracture,degeneration,and necrosis.The group containing RosA and Kae showed significantly less myocardial structure injury compared with that of the HTK group.3.Comparison of cardiac structure under an electron microscope.When the ischemic time was less than 6 h,myocardial cells in all groups showed normal structures with no significant mitochondrial and muscular changes.When the ischemic time was more than 6 h,the mitochondria became swollen,deformed,and crest-broken.Approximately 15 ?mol/L RosA exhibited better protection effect than those of other treatments.4.IS.The myocardial IS after 8 h of preservation was used as an example.The HTK group showed significantly increased infarct area.The ratios decreased in the RosA,Kae,LCB,and LCC groups.5.Comparison of the survival rates of rats.No significant difference was observed between the HTK group and other groups when the cold ischemia storage time was less than 6 h.When the ischemic time was more than 6 h,the survival rate in the RosA group significantly increased compared with that in the HTK group.6.Licorice extract improved the recovery of cardiac function after transplant.Rosemary acid significantly improved the functional recovery(the heart ejection fraction)during early reperfusion compared with that in the HTK group(P<0.01).The Kae and LCB addition also enhanced the cardioprotective effects(P<0.05).Conclusion:1.Prolonged cold ischemic storage can cause severe damage to myocardial functions and myocardial tissues.The EF%and survival rates were reduced.2.RosA,as an additive of HTK solution,strengthened the preservation efficacy of HTK for cardiac grafts subjected to prolonged cold ischemia time,reduced the myocardial injury,and improved the EF%and survival rates.3.Among the licorice extracts,the RosA addition resulted in better myocardial protection than those of other extracts.Part? Mechanism of RosA in enhancing myocardial protectionObjective:This study aimed to determine the myocardial protection effect of RosA with HTK addition and its mechanism.Methods:Adult SD rats were randomly divided into six groups:1)control group,2)I/R group,3)RosA group,4)GW9662 group(a PPAR-? inhibitor),5)RosA+I/R group,and 6)RA+GW9662+I/R group.Grafts were arrested and stored in HTK with or without RosA for 4 h at 4 ? following baseline hemodynamic measurements.After cold storage,grafts were reperfused via Langendorff,and hemodynamic concerned parameters were examined.Myocardial IS was estimated macroscopically using TTC staining.Coronary effluent was analyzed for lactate dehydrogenase(LDH)and CK release to assess the degree of cardiac injury.The contents of malondialdehyde(MDA),superoxide dismutase(SOD),CRP,IL-6,and TNF-a and the ratio of glutathione/glutathione disulfide(GSH/GSSG)were determined by ELISA method.The protein expression degrees of PPARy and NF-?B were assayed by Western blot analysis.Cardiomyocyte apoptosis analysis was performed using an In Situ Cell Death Detection Kit,POD.Results:The RosA(15 ?mol/L)pretreatment improved the maximum up/down rate of the left ventricular pressure(±dp/dtmax),increased the left ventricular developed pressure(LVDP),and decreased the levels of CK and LDH in coronary flow compared with that of the I/R group.RosA reduced the contents of MDA,CRP,IL-6,and TNF-a;increased the SOD level,the GSH/GSSG ratio,and the PPARy protein expression;and decreased the protein expression degrees of NF-?B.The IS and cell apoptosis in the hearts from RosA-treated group were lower than those in the hearts from the I/R control group.However,these RosA effects were reversed after cotreatment with GW9662.Conclusion:1.The reperfusion of hearts that underwent 4 h of cold ischemia preservation time can cause severe damage to myocardial systolic and diastolic functions and myocardial tissues,oxidant stress,inflammation reaction,and cell apoptosis.2.RosA can protect the heart against myocardial injury induced by I/R via its antioxidant,antiapoptotic,and anti-inflammatory activities.3.RosA plays an important role in myocardial protection by upregulating PPARy expression and reducing the activity of NF-?B,thereby inhibiting the inflammatory response and prolonging the heart cold ischemia time.