| BackgroudIn recent years,with the changes in people’s lifestyles and diet,as well as the development of aging population,cardiovascular diseases have become the overriding global threat of human health.Meanwhile,myocardial infarction(MI)remains the major cause of death and disability in the world.Irreversible death of cardiomyocytes post-MI,result in the formation of reparative fibrotic and non-contractile scar in infarct area and degradation of original extracellular matrix(ECM),which impair cardiac geometry,structure and function,called ventricular remodeling.Ultimately,it would increase hospitalization and mortality related with heart failure(HF)after MI.Over the past few decades,cardiac tissue engineering and regenerative medicine have developed as a promising post-MI treatments.Direct intramyocardial injection of biomaterials,including injectable hydrogel,have shown promising benefit in preventence of cardiac maladaptive repair and cardiac dysfunction.Curcumin(Cur),a polyphenol compound extracted from Curcuma longa,is known to exhibit diverse pharmacologic protection properties,such as anti-inflammation,anti-oxidation,anti-apoptosis.Lots of researchs shown that curcumin could reduce ventricular remodeling caused by pressure overload or by MI and could improve heart function.However,the clinical application of Cur is limited because of its low bioavailability and poor chemical stability.Nitric oxide(NO),as a gas signal molecule in the body,has a variety of cardiovascular protection function,such as dilation of blood vessels,increase of coronary blood flow,prevention of platelet aggregation and promotion of angiogenesis.However,the short half-life within a few seconds of NO molecular restrict its utilization in vivo.Therefore,many biomaterials with efficient production and release of NO had been developed,which could be more effective,such as in healing skin wound healing,improving lower limb ischemia injury and promoting vascular regeneration post-MI.Recently,studies have shown that it were more effective in the combination therapy of different drugs than single drug for ischemic heart diseases treatment.Therefore,the small molecular hydrogel that we had prepared for the treatment of MI,could not only provide structural support for the weakened myocardial wall and enhance the compliance of ventricular wall motion,also provide platforms for drugs controlled deliveries of curcumin and NO.Finally,we speculate that combined curcumin and NO in mice MI treatment may have synergistic effect.ObjectiveWe developed and prepared a curcumin-NOsmall molecular hydrogel(Gel)which could controlled release nitric oxide(NO)in the addition of givenβ-galactosidase(β-gal).We also investigated the effects of hydrogel by intramyocardial injection on cardiac function,ventricular remodeling and neovascularization post-MI and explored the underlying molecule mechanism.MethodsWe previously synthesized a curcumin hydrogelator of Cur-FFE-ss-ERGD(named Comp I)and a nitric oxide hydrogelator of Nap-FFGGG-NO(named Comp II).In this study,Comp I and Comp II were added together at a mole ratio 4:1 in phosphate buffer saline(PBS)to form mixed suspension.Then the suspension was heated to form clear solutions and glutathione(GSH)was added.When the mixed solutions was cool down,it form a co-assembled small molecular curcumin-NOsmall molecular hydrogel(Gel).Several characteristics of hydrogel were performed,including the gel formation assay,mechanical property detected by rheometer,microstructure measured by high-resolution transmission electron microscopy(TEM),curcumin release assayed by liquid chromatography-mass spectrometry system(LC-MS)and NO release in response to 200 U/L β-galactosidase analyzed by nitric oxide assay reagent kit.Acute myocardial infarction model(AMI)was established by ligating the left anterior coronary artery(LAD)of male C57/BL6 mice and were randomly divided into three AMI groups,including saline group,Gel group and Gel+β-gal group.In saline group and Gel group,mice were respectively injected 20 μL saline or Gel into positions surrounding the infarct region,and then both group were injected 50 μ L saline by tail vein.Gel+β-gal group were given Gel injection as the Gel group and 50 μ Lβ-gal(0.4 mg/ml)by tail vein injection as well.Additionally,mice in sham group were operated as the same as AMI model but without ligating LAD or treatment.Four weeks later,the cardiac function was measured by echocardiography,including left ventricular(LV)internal diameter at end-diastole(LVIDd),LV internal diameter at end-systole(LVIDs),fractional shortening(FS)and ejection fraction(EF).Electrocardiograph(ECG)was also recorded by echocardiography examination.Then the mice were humanely euthanized and the heart were explanted,fixed and cut in 5μm thick sections.Masson staining were used to observe the heart structure and fibrosis.Myocardiocytes apoptosis were detected by Tunel staining.Neovascularization were analyzed by vWF immunofluorescence.The protein expression of SIRT1,TGFβ 1 and MMP-2 in heart tissue were tested by immunofluorescence staining.Furthermore,three fresh heart tissues of each group were taken out and grinded for protein quantitative analysis of SIRT1,TGFβ 1 and MMP-2 by Western Blot.Results1.Characterization of hydrogelThe appearance of hydrogel is like a yellow jelly,and it contained 0.5wt%of Compound I and 0.17wt%of Compound Ⅱ.The gel was stable without change of its appearance for at least one month at 37℃.We observed entangled long nanofibers with diameters of 20-80 nm and three dimensional(3D)networks structure within lots of water molecules in the gel tested by TEM.Both in dynamic frequency sweep test and dynamic strain sweep test,the value of elasticity(G’)of the Gel was about an order of magnitude greater its corresponding viscosity(G")value,indicating the formation of a true gel with strong properties of elasticity and compression resistance.The Gel could release curcumin monomer by the hydrolysis of the ester bond.Curcumin could be sustainably released from hydrogel at 24 h time point,and the release rate of Cur was about 2.43μg/mL at 37℃with about 0.84%of the total amount of Cur.It indicated that the Gel could controllably and constantly release NO over the 48 hours of experimental time in presence of β-galactosidase.About 26.14%of the total amount of NO was released from the gel at 48 hours.While,there was no NO release at the absence of β-galactosidase to the Gel.2.Results of echocardiographyECG showed that there was pathological Q wave formation in MI group,while ECG was normal in sham group,indicating that myocardial infarction model was successfully established.Compared with sham group,the cardiac function indexes of FS,EF values in saline group,Gel group and Gel+β-gal group were significantly decreased(P<0.01),while LVIDd,LVIDs values were significantly increased(P<0.05).The FS,EF values in Gel group as well as Gel+β-gal group were higher,while LVIDd,LVIDs values were lower than saline group(P<0.05).Compared with Gel group,the FS,EF values in Gel+β-gal group were significantly increased,while LVIDd,LVIDs values were significantly decreased(P<0.05).3.Results of Masson stainingLV volume and morphology were normal and with negligible amount of fibrosis collagen in sham group.While enlargement of LV chamber,irregular shape of heart,thinner thickness of LV wall as well as increased amount of fibrosis area were observed in saline group,and the difference was statistically significant compared to sham group(P<0.01).Compared with saline group,enlargement of LV chamber was alleviated and amount of fibrosis area was decreased in Gel group and Gel+ β-gal group(P<0.01).Compared with Gel group,heart morphology and structure were closer to normal heart shape and amount of fibrosis area decreased in Gel+β-gal group(P<0.05).4.Results of myocardiocytes apoptosisBy TUNEL assay,the percentage of apoptotic positive myocardiocytes was low in sham group,but was markedly increased in saline group(P<0.01).Compared with saline group,the Cur treatment in Gel group dramatically decreased cell apoptosis index(P<0.05).In Gel + β-gal group,the TUNEL-positive myocardial cell ratio was decreased,which was more significantly lower than that in Gel group(P<0.05).5.Angiogenesis in MI heartCompared with sham group,the number of angiogenesis in saline group and Gel group was increased,but the difference was not statistically significant(P>0.05).However,in Gel+ β-gal group,there was obviously increase of new vessel density about two-fold in the treated hearts,compared with other three groups(P<0.01).NO releasing from gel could effectively enhance angiogenesis in MI heart and reduce myocardial remodeling.6.Proteins expression of SIRT1,TGF-β1 and MMP-2 in MI heartThe protein expression of SIRT1 in saline group was lower than that in sham group(P<0.05),while the proteins expressions of TGFβ 1 and MMP-2 were increased(P<0.05).Compared with saline group,the protein expression of SIRT1 was increased in Gel group and Gel+β-gal group(P<0.05),while TGFβ 1 and MMP-2 decreased(P<0.05).Compared with Gel group,Gel+β-gal group more significantly increased the protein expression of SIRT1,and decreased the protein expression of TGFβ 1 and MMP-2(P<0.05).ConclusionWe firstly prepared a mixed curcumin-NO small molecule hydrogel,with good mechanical properties,which could slowly release curcumin under natural conditions and could controllably and sustainably release NO only in the condition of adding β-galactosidase.Curcumin-NO small molecule hydrogel could significantly improve cardiac function,reduce myocardial fibrosis,alleviate cell apoptosis and enhance angiogenesis post-MI in mice.The cardioprotection of hydrogel was more effective in the manner of simultaneous release both curcumin and NO than the release of curcumin alone,indicating synergistic effect of curcumin and NO in cardiac protection after MI in mice.The underlying molecular mechanism of synergistic effect may be related to the increase of SIRT1 expression and the decrease of TGF-β 1 and MMP-2 expression. |
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