| 1.IntroductionIschemic heart disease,the major cause of mortality in current societies,is featured by restriction of blood supply to regions of the myocardium resulting in infarction and tissue necrosis.Nowadays,the treatment of this disease has entered into an era that mortality can be approximately halved by intervention procedures to recover blood flow rapidly,i.e.reperfusion,to the myocardial ischemia area.However,evidences have indicated that rapid coronary perfusion regaining may paradoxically impair cardiomyocytes,which is named ischemia/reperfusion(I/R)injury.The underlying signaling pathway in I/R injury is complicated and partially understood.Present concepts underline three major contributors to the formation of I/R injury:reactive oxygen species(ROS),Ca2+,and mitochondria damage.Although early and timely reperfusion therapy markedly decreased mortality in acute myocardial infarction,application of cardiac-protection strategy might further alleviate the injury during the therapy.Nitric oxide(NO)is a soluble gas molecule that exhibits various heart functions including antioxidant,vasodilator,antiplatelet aggregation,and antineutrophil actions.Usually,NO protects myocardium from I/R injury.However,when NO at excessive concentration,in the ?M scope,is cytotoxic due to its transformed to oxidized intermediates.During reperfusion stage,cardiomyocytes are not only accompanied by raised ROS levels,but also by declined NO production.Therefore,exogenous NO donors,such as organic compounds,metal complexes and micelles that can release NO in controllable manners may be a candidate drug to mitigate I/R injury.while it is not easy to always keeping the NO releasing in a beneficial low level within the complicated cells and organisms circumstances.On the other hand,it has been demonstrated that most of the toxic actions of NO are not directly attribute to NO itself but are mediated by its decomposed product of ONOO-,which is produced by a reaction between NO and a ROS of 02-.Hence,under ROS greatly produced reperfusion stage,NO donor coupled with another antioxidant will be a more promising strategy for I/R treatment because the antioxidants can avoid the detrimental effects of NO.Curcumin(Cur)is an active component of Curcumin longa,which shows pleiotropic biological effects such as cardio-protection,antioxidation,anti-inflammation and anti-tumorigenicity.Furthermore,researchers reveal that pretreatment of Cur relieves mitochondrial oxidative damage during I/R process.However,the poor solubility in aqueous solutions of Cur limits its further biological employment.The encapsulation of Cur in nanocarriers can solve this problem.Recently,a novel delivery strategy for hydrophobic therapeutics emerges as promising one,that is coupling hydrophobic drug molecules with peptides to make self-assembling drug-peptide amphiphiles.The resulting molecules can self-assemble into nanomaterials or hydrogels with high(up to 90 wt%),designable drug loadings(controlled by the variation of peptide length),and sustained or responsive drug release.Besides,supramolecular hydrogels formed by self-assembly of peptide derivatives are physical gels and are injectable,which can be directly injected into myocardial infarction region for topical release of bioactive components.We recently prepared two supramolecular hydrogels,one formed by a Cur-peptide conjugate and the other one by a peptide-NO donor conjugate.We demonstrated that a mixed component hydrogel based on these two gels could improve mice cardiac function and alleviate ventricular remodeling after acute myocardial infarction.However,the weight percentage of peptide-NO donor conjugate in the two components used for hydrogelations could not exceed 25%.In order to increase the amount of NO released from gels and make Cur and NO more closer,we optted to integrated Cur and NO in the same peptide to develop a single component hydrogel capable of releasing both Cur and NO simutaneously for the treatment of I/R injury.2.Experimental sectionGeneral methods1H NMR(Bruker AXR 400)was used to characterize the synthesized compounds.HR-MS were received from the VG ZAB-HS system(England).HPLC was conducted at LUMTECH HPLC(Germany)system using a C18RP column with MeOH(0.1%of TFA)as the eluents.TEM images were obtained at Tecnai G2 F20 system.LC-MS was conducted at the Shimadzu LCMS-20AD(Japan)system.The peptide derivative was prepared by solid phase peptide synthesis(SPSS)using 2-chlorotrityl chloride resin and the corresponding N-Fmoc protected amino acids with side chains properly protected.Rheology test was done on an AR 1500ex(TA instrument)system.25 mm parallel plates with a gap of 400 ?m were used in the experiment.Neonatal rat cardiomyocytes(NRCs)cultureAll animals' experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals,and protocols were approved by the Bioethics Committee of the Southern Medical University and the Institutional Animal Care and Use Committee in the College of life sciences,Nankai University.To induce hypoxia,cells were firstly incubated in a humidified environment at 37 ? in a tri-gas incubator equilibrated with 1%O2,5%CO2 and 94%N2 for 2-3 h.Post-hypoxic cardiomyocytes were incubated in the medium containing Curcumin,Nap-Cur and Nap-Cur-NO at series of concentrations(5?200?M).?-galactosidase solution at a dose of 0.2 U/mL was given in all re-oxygenation medium.Drug releasing in PBS100 ?L,1 wt%of the Nap-Cur-NO hydrogels were firstly prepared in each well of the 96 well plates at 37 ?.For detection and calculation curcumin releasing proportion,the fluorescence microplate and curcumin standard fluorescence intensity measurement and curve fitting method were applied.Determination of IC50 value of the compoundsThe IC50 values of curcumin,Nap-peptide,Nap-Cur,and Nap-Cur-NO were evaluated by the MTT assay.The cell viability percent was calculated according to the following Equation(1):the cell viability percent(%)= OD(sample)/OD(control)× 100%.The concentration of the compounds was recorded when 50%of cell viability.Intracellular ROS(reactive oxygen species)and TUNEL measurementsIntracellular ROS was quantified with 2,7-dichlorofluoresein diacetate(DCFH-DA)(Beyotime,Institute of Biotechnology,Jiangsu,China).After DAPI staining NRCs were visualized by fluorescent microscopy(?ex 495 nm,?em 519 nm).The percentage of apoptosis positive cells was calculated by image pro-plus 6.0.Apoptotic ratio= positive cell numbers/total cell numbers.I/R mouse modelAdult wildtype male C57BL/6 mice were provided by the Laboratory Animal Center of Guangdong Province and the animal center of Southern Medical University.Mice were anaesthetized with a mixture of 5 mg/kg xylazine and 100 mg/kg ketamine by intraperitoneal injection.Mice in all groups were received galactosidase(100 ?L,0.2 mg/ml)right after the start of reperfusion by tail vein injection.Myocardial infarct size(IS)measurement2,3,5-Triphenyltetrazolium chloride(TTC)and Evans blue dyestuff were used to detect myocardial IS as previously described.Western blottingSamples were obtained from NRCs.Image Pro-Plus 6.0 software was used to measure the OD of the bands.The relative expression level of protein was presented as an average ratio to GAPDH or its total-non-phosphorylated protein.Experiments were repeated three times for each experimental condition.Evaluation of LC3 puncta fluorescentNRCs were seeded in laser confocal petri dishes and transduced by an adenoviral vector with tandem fluorescent mRFP-GFP-LC3 at 20 MOI.Statistical analysisData are presented as the mean + standard error of the mean.Statistical comparisons of data were performed using one-way ANOVA with SPSS software(version 13.0,Chicago,IL,USA).A value of p<0.05 was identified statistically significant.3.ResultsDesign and synthesis of the gelator and Characteration of the hydrogelWe firstly prepared peptide derivative Nap-FFKEGG(compound 1)with side chain protected by solid phase peptide synthesis.The peptide derivative was used to react with propargylamine to afford compound 2 with an alkynyl group.After removing the side chain protecting groups by trifluoroacetic acid(TFA),compound 2 was coupled with curcumin glutaric acid N-hydroxysuccinimide(NHS)active ester to produce Nap-Cur(compound 3).Compound 3 with an alkynyl group could be used to couple with the NO donor with an azide group through the well-known click reaction to afford the title product(Nap-Cur-NO,compound 4).We used high performance liquid chromatography(HPLC)to purify compounds during reactions.After obtaining the title product,we tested the gelation ability of Nap-Cur-NO in saline solution.We first obtained a dark yellowish solution of it by adding Na2C03(2.5 equiv.)following by a heating-cooling process.Slowly adding 0.1 mM HCl saline solution to adjust the pH value back to 7.4 resulted in a hydrogel formation within 2 hours.The gel was stable for at least four weeks at room temperature(20-25 ?).We then used a rheometer to characterize the mechanical properties of the gel at 37 ?.The result obtained by dynamic frequency sweep showed that the elasticity(G')and viscosity(G")of the sample was about 50 and 8 Pa at the frequency value of 1 rad/s,respectively,suggesting the formation of a mechanically weak hydrogel.The transmission electron microscopy(TEM)image presented uniform and spiral nanofibers with diameter of about 80 nm in the hydrogel.These helical nanofibers formed larger bundles and entangled with each other to form three dimensional networks for hydrogelation.These results,in combine with other hydrogelators based on dipeptide FF,indicated that peptide derivatives based on FF were excellent hydrogelators.Release profile of NO and curcumin and the biocompatibility of hydrogelWe then monitored the release profile of NO and curcumin from the 1%wt gel.100 ?L PBS with or without 0.2 U/mL P-galactosidasein was placed on the top of 100 ?L of gel.The upper solution was removed at predetermined time point to measure the accumulated releasing amount of NO and curcumin.The releasing rate of NO and curcumin were constantly during the 12 hours experimental procedure while without burst release phenomenon.The IC50 values of curcumin,Nap-peptide,Nap-Cur,and Nap-Cur-NO were evaluated by the MTT assay.NRCs without the compounds were used as the control.The Nap-peptide without Cur showed no obvious toxicity to the cells at a concentration of 400?M,thus the group of Nap-peptide was removed in the subsequent experiment.The curcumin alone exhibited an IC50 value of 56.69 ?M against NRCs.The Nap-Cur and Nap-Cur-NO indicated a decreased inhibition capacity to the cells and the IC50 value of Nap-Cur was 243 ?M and the Nap-Cur-NO was 188.2 ?M respectively.These observations suggesting the cytotoxic effect of curcumin was lessened after binding to Nap-peptide and the well biocompatibility of the gel.Another MTT cell viability test was carried out to study the biocompatibility and evaluate the efficacy of synthesized compounds to reduce the damage during hypoxia/re-oxygenation to NRCs.Cells were exposed firstly to hypoxia for 2 or 3 h and then to re-oxygenation for 2 or 3 h to stimulate an ischemia-reperfusion injury.Post-hypoxic cardiomyocytes were then incubated in the culture medium containing Curcumin,Nap-Cur or Nap-Cur-NO at series of concentrations(5?200?M).?-galactosidase at a dose of 0.2 U/mL was given in all re-oxygenation medium.curcumin protected myocytes at low concentrations such as 5,50,and 100 ?M,while markedly decreased cardiomyocytes viability at the high concentration of 200?M during H/R.There were no cytotoxic effects of both Nap-Cur and Nap-Cur-NO to NRCs in H/R experiments at concentrations ranging from 5 to 200 ?M.These observations indicated better compatibility of Cur-peptide conjugates at high concentrations.Furthermore,Nap-Cur-NO significantly improved cardiomyocytes viability in a dose-dependent manner,and we recorded a 41.2%increase in cell viability at the highest concentration of 200?M.These results suggested the big potential of Nap-Cur-NO gel to alleviate H/R injury.We then choose 20 ?M as the dosing concentration for subsequent experiments which was the first concentration of the Nap-Cur-NO group showing a significantly statistical difference compared to the Control and Cur groups.The 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate(DAF-FM Diacetate)was a NO indicator that could be converted from a non-fluorescent molecule to a fluorescent one by reacting with NO molecules.NRCs in the control group barely exhibited fluorescene signals,suggesting the existence of little amount of NO in the cells.Compared with NRCs in normal condition,those with a H/R procedure showed stronger green fluorescence,suggesting the generation of more amounts of NO in cells under the H/R condition.The addition of Cur or Nap-Cur to cells with a H/R procedure could further increase green fluorescence(2 and 2.1-folds increase compared to that in the H/R group),probabbly due to the anti-oxidant property of Cur to stabilize NO molecules.Moreover,we observed even stronger fluorescence signals in cells in both Nap-Cur-NO group.Among cells in all groups,those in Nap-Cur-NO group exhibited the strongest fluorescence signal(1.6,1.7 and 3.3-folds contrast to that in Nap-Cur,Cur and H/R group,respectively).These observations further indicated the role of NO donor in NO liberation within cells.Hydrogel prevents hypoxia/re-oxygenation damage of neonatal rat cardiomyocytesIt is believed that the production of reactive oxygen species(ROS)during reperfusion was one of essential pathogenic factors leading to cardiomyocyte apoptosis and cardiac damage.As Nap-Cur-NO hydrogel was capable of releasing curcumin and NO,both served as ROS scavenger,we then measured the cellular ROS level and the extent of apoptosis in H/R cardiomyocytes being treated with different compounds.the ROS level in cardiomyocytes increased dramatically after H/R exposure.Treating the cells with 20?M of Cur,Nap-Cur,or Nap-Cur-NO during reperfusion procedure resulted in the inhibition of ROS generation.Compared with cells in Cur and Nap-Cur groups,those in Nap-Cur-NO group showed the greatest decrease in ROS level.The pattern of apoptosis in cardiomyocytes was consistent with that of ROS level in cell.About 26%of cells were apoptosis positive in H/R group,and the number was decreased to about 24,16 and 11%in which receiving treatment with Cur,Nap-Cur and Nap-Cur-NO,respectively.These results were also consistent with the MTT assay,indicating that Nap-Cur-NO hydrogel may protect cardiomyocytes from H/R-induced cell death by reducing cellular ROS level and apoptosis.Cardioprotective property of hydrogel during myocardial I/R injury in vivo.Hydrogels are promising biomaterials for tissue engineering,we next investigated the effect of Nap-NO-Cur hydrogel on cardioprotective property during myocardial I/R injury in vivo.C57BL/6 mice were randomly separated into five groups and one received sham surgery as the control group(sham group).The other four groups were first submitted to the ischemia for 30 min.and then received the following administrations for 5 min before 4 hours of reperfusion:intramyocardial injection of normal saline along the ischemia region(I/R+NS group),intramyocardial injection of Curcumin solution(I/R+Cur group),intramyocardial injection of Nap-Cur hydrogel(I/R+Nap-Cur group),and intramyocardial injection of Nap-Cur-NO hydrogel(I/R+Nap-Cur-NO group).Mice in all groups were received with galactosidase(100 ?L,0.2 mg/ml)right after the start of reperfusion through tail vein injection.The area at risk(AAR)and myocardial infarct proportion were determined by the triphenyltetrazolium chloride(TTC)-Evans blue method.The results indicated that the infarction size(IS,white color area among AAR)in mice administrated with both hydrogels was remarkably reduced,compared with that in mice in I/R+NS group.The IS in mice in I/R+Nap-Cur-NO group was the lowest,and the percentage of infarcted myocardium was 66,61,52 and 32%for mice in I/R+NS,I/R+Cur,I/R+Nap-Cur,and I/R+Nap-Cur-NO groups,respectively.The results further suggested the synergistic effect of Cur and NO for I/R treatment.We also used the TUNEL assay to calculate the percentage of apoptosis cardiomyocytes in I/R region.Cell apoptosis index(percentage of apoptosis-positive cells)of mice in Sham group was about 1.6%,and it markedly increased to about 57.2%in I/R+NS group.In the meantime,the percentage of apoptosis-positive cells decreased to about 43.0,36.2,and 23.8%in Cur,Nap-Cur,and Nap-Cur-NO group,respectively.These observations showed that Nap-Cur-NO hydrogel could greatly alleviate cardiomyocytes apoptosis.Both results obtained in vitro and in vivo clearly indicated that Nap-Cur-NO hydrogel was a promising biomaterial as an implant for the treatment of myocardial I/R injury.Hydrogel improved heart function and reduced collagen deposition after I/RIn order to evaluate the heart function of post-I/R mice,the echocardiography was also performed 2 weeks after the treatment.Injection of Nap-Cur and Nap-Cur-NO hydrogel could improve both fractional shortening(FS)and ejection fraction(EF)of hearts post-I/R compared with those in I/R + NS group.Among all treatments,the one with the Nap-Cur-NO hydrogel could elevate heart function most effectively due to the synergistic therapeutic effects of curcumin and NO.Besides,although the treatment with curcumin showed slightly effects in heat function,there were no statistically differences between Cur and I/R+NS groups.Masson's staining was applied to detect the collagen deposition in heart.After myocardial I/R injury,the emerging of fibrillation and formation of non-contractile functional cicatrix in infarct area resulted in reduction of myocardium compliance and deterioration cardiac function.Compared with the saline group,injection of curcumin solution,Nap-Cur and Nap-Cur-NO hydrogel could significantly decrease the proportion of collagen deposition,and the Nap-Cur-NO group showed minimum collagen accumulation in all I/R mice.These data revealed that synergistical release of curcumin and NO from Nap-Cur-NO hydrogel could effectively restore post-I/R heart function and mitigate collagen deposition as well as assist heart remodeling.we then examined the protein level of p-p38 MAPK,p-NF-?B and Bax/Bcl-2 ratio by western blotting.Compared with the control group,we found that the p-p38 MAPK expression increased,while p-NF-?B expression decreased in the H/R group.Furthermore,Nap-Cur-NO hydrogel given at the onset of reperfusion remarkably decreased the expressions of both p-p38 MAPK and p-NF-?B.These results suggested that combination of Cur and NO molecules offered an efficient protection for H/R-induced cardiomyocytes via anti-ROS associated effects.The Bax/Bcl-2 ratio was consistent with apoptotic index counted by TUNEL measurement,suggesting that treatment of Nap-Cur-NO hydrogel dramatically improved cardiomyocytes survival in H/R injury.Inhibition of autophagy by hydrogelAutophagy is a degradation pathway that digestive cell components into basic molecules through the lysosomal machinery.Generally speaking,autophagy is deemed a pro-survival protection mechanism to cells under stress or insufficient nutrient supply.However,research reported the notion that over stimulation of autophagy is responsible for mediating severe post-ischemia myocardial death and inhibiting autophagic pathways can attenuate I/R injury.Microtubule-associated protein light chain 3(LC3)is a major regulator of the formation of autophagosome which is the most widely used autophagy-related protein marker.Beclin-1 is an essential partner in the onset of autophagy,therefore this protein is regarded as another monitor of autophagy.The p62 protein serves as a link between LC3 and ubiquitinated substrates which finally degraded in autolysosomes,thus regarding as a negative index of autophagy.The mRFP-GFP-LC3 which is a tandem monomeric RFP-GFP-tagged LC3,another way to monitoring autophagy through directly fluorescence microscopy.ROS induces autophagy through several mechanisms.Thus,our Nap-Cur-NO hydrogel may inhibition autophagy through suppression ROS-associated signaling.To measure the autophagy level in H/R-induced primary cardiomyocytes,we assessed the expression of LC3,Beclin-1 and p62 at the protein level by western blotting and counted the yellow and red dots in NRCs transduced by an adenoviral vector with tandem fluorescent.The level of LC 3B ?/? and Beclin-1 expression were increased,while p62 expression was decreased in H/R group,as well as the formation of autophagosomes in cardiomyocytes markedly raised after H/R exposure.As for the Cur group,administration of curcumin solution displayed a further enhancement in autophagy level compared to H/R group.This may attribute to curcumin enable to induce autophagy through suppression PI3K/Akt/mTOR pathways.However,the expression of these autophagy biomarkers in Nap-Cur and Nap-Cur-NO groups was the complete opposite of the H/R group.Compared with the Nap-Cur group,treatment of Nap-Cur-NO hydrogel showed greater decrease not only in autophagy biomarkers but also in the numbers of autophagosomes.These results demonstrated that co-treatment of curcumin and NO molecules protected myocardial against H/R injury by inhibiting autophagy.4.ConclusionIn summary,we reported on a novel hydrogelator by bridging a NO donor and an antioxidant Cur through a self-assembling short peptide derivative.The resulting hydrogel could simultaneously release bioactive NO and Cur in vitro and in vivo.We demonstrated that the treatment of our hydrogel could significantly attenuate the I/R-induced injury in cell experiments and in the animal model.The underlined mechanism revealed that the hydrogel could reduce ROS level and thus inhibit the expression of ROS-associated p38 MAPK/NF-?B signaling pathway.Besides,the hydrogel also significantly suppressed over stimulated autophagy during I/R treatment which was responsible for mediating severe post-ischemia myocardial apoptosis and death.Our results demonstrated that self-assembling short peptides were powerful moieties to construct bioactive hydrogels and the hydrogel capable of releasing both NO and Cur might be an ideal candidate for the treatment of myocardial I/R injury. |
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