Effects Of Tetrahydrocurcumin On Myocardial Ischemia-Reperfusion Injury

ObjectiveRhizoma Curcumae Longae is a traditional Chinese medicine with a long history in China.It has the functions of promoting blood circulation and removing blood stasis,promoting Qi and relieving pain,and has always played an important role in the treatment of "xiongbi".Curcumin is the main active ingredient extracted from turmeric root of the ginger family plant,and has various pharmacological effects such as antioxidant,anti-inflammatory,hypolipidemic and hypoglycemic.However,due to the low intestinal absorption rate and rapid metabolism of curcumin,it is difficult to explain its remarkable pharmacological activity.Oral intake of curcumin is rapidly converted into various metabolites,such as dihydrocurcumin,tetrahydrocurcumin,octahydrocurcumin,etc.Among them,Tetrahydrocurcumin(THC)is the most important and most active natural metabolite.It has better biological activity than curcumin in antioxidant,hypoglycemic and hypolipidemic.Therefore,it is speculated that curcumin may be metabolized to more active THC in vivo to exert pharmacological effects.Myocardial ischemia-reperfusion belongs to the category of "xiongbi" in traditional Chinese medicine.Modern pharmacological studies have found that during myocardial ischemia-reperfusion,the production of a large amount of reactive oxygen species can induce mitochondrial damage,further causing apoptosis and excessive autophagy.Therefore,inhibiting apoptosis and excessive autophagy in the reperfusion stage has become an important target for the treatment of myocardial ischemia-reperfusion.Previous studies have found that THC can improve doxorubicin-induced cardiotoxicity,and can also inhibit myocardial inflammation and oxidative damage in mice with sepsis,reduce cardiomyocyte apoptosis,and protect the heart.Based on the above analysis,this study intends to observe the protective effect of THC on myocardial ischemia-reperfusion injury through in vivo and in vitro experiments,and to explore the role of PI3K/Akt/mTOR signaling pathway in apoptosis and autophagy in vitro.effect.This experiment is divided into three parts:Methods1.Effect of tetrahydrocurcumin on cardiac function in rats with myocardial ischemia-reperfusion injurySD rats with a body weight of about 200±20 g were selected,and the rat model of myocardial ischemia-reperfusion injury was established by ligating the left anterior descending coronary artery of the rat for 60 min and then restoring blood perfusion for 24 h.The Sham group and the THC group were only threaded without ligation.The electrocardiogram of the rats were measured 24 hours after operation.According to the electrocardiogram,the rats with successful modeling were randomly divided into myocardial ischemia-reperfusion group(I/R)and tetrahydrocurcumin treatment group(I/R+THC).The THC group and I/R+THC group were given intraperitoneal injection of tetrahydrocurcumin(50 mg/kg)for 4 consecutive weeks,and Sham group and I/R group were given the same dose of 0.2%Tween in normal saline solution for 4 consecutive weeks.After 4 weeks of administration,echocardiography was used to detect the changes of left ventricular structure and cardiac function;enzyme-linked immunosorbent assay was used to determine the myocardial enzymes in serum:creatine kinase(CK),lactate dehydrogenase(LDH),α-Hydroxybutyrate Dehydrogenase(α-HBDH)content changes;H&E staining was used to observe the pathological changes of the myocardial tissue of the rats in each group,and TUNEL staining was used to observe the apoptosis of myocardial cells in each group.Masson staining was used to observe the myocardial collagen fibrosis of rats in each group.2.Protective effect of tetrahydrocurcumin on hypoxia/reoxygenation injury of H9c2 cardiomyocytesCells were cultured in DMEM supplemented with 10%FBS.Cells were cultured at 37℃ in a humidified incubator with 5%CO2.For the establishment of the hypoxia-induced cardiomyocyte injury model,the cells were washed with PBS,and cultured in serum/glucose-free DMEM under hypoxic conditions(5%CO2,1%O2 and 94%N2)for 24 h.Subsequently,the cells were cultured under normoxic conditions(5%CO2)for 12 h to simulate reoxygenation.Cells under normoxic conditions served as control.To analyze cytotoxicity,two alternative methods were used.The viability of the cells was determined using a CCK-8 assay kit and cell injury was assessed using a LDH assay kit.For the CCK-8 assay,H9c2 cardiomyocytes were plated in 96-well plates.Briefly,after hypoxic treatment for 24 h,cells were incubated immediately with THC(0,0.5,1.0 and 2.0 μM)at the onset of reoxygenation,after which CCK-8 solution was added to the culture medium and cell viability was determined.Cellular injury was measured using a LDH assay kit.Briefly,the supernatant was used to detect LDH release using the LDH assay kit according to the manufacturer’s protocol after simulation of H/R and treatment with THC.H9c2 cardiomyocytes in logarithmic growth phase were randomly divided into 4 groups:Control group(blank control group),THC group(THC 2.0 μM drug control group),H/R group(hypoxia and reoxygenation group),H/R+THC group(THC 2.0 μM hypoxia-reoxygenation drug treatment group).After corresponding treatment of cells in each group,the activities of antioxidant enzymes SOD,CAT and MDA were detected by ELISA.The membrane potential was detected by JC-1 membrane potential detection kit.Cell apoptosis was detected by TUNEL in situ labeling and Annexin V-FITC/PI double staining.The ultrastructure of cardiomyocytes was observed by electron microscope,and the number of autophagosomes was counted.The mCherry-GFP-LC3B lentivirus was used to observe the flow of autophagy.Western blot was used to detect the expression levels of apoptosis-related proteins Bcl-2,Bax and caspase-3,and autophagy-related proteins LC3Ⅱ/Ⅰ,Beclin-1 and p62.3.THC mediated PI3K/AKT/mTOR signaling during H/R challenge.Western blot analysis of PI3K/Akt/mTOR signal transduction pathway-related proteins PI3K,p-PI3K,Akt,p-Akt,mTOR,p-mTOR and hypoxia-inducible factor HIF-la protein expression levels.H9c2 cardiomyocytes were randomly divided into normal control group(Control),normal tetrahydrocurcumin control group(THC),hypoxia/reoxygenation model group(H/R),tetrahydrocurcumin postconditioning group(H/R+THC).H9c2 cells in group Control were cultured normally,and H9c2 cells in the drug posttreatment group,cells were incubated immediately with THC(2.0 μmol/L)at the onset of reoxygenation.12 hours after the reperfusion,Western blot detection of apoptosis-related protein Bcl-2,Bax and cleaved caspase-3;autophagy-related protein LC3II/I,Beclin-1,p62 expression;hypoxia-inducible factor 1α(HIF-1α)and PI3K/Akt/mTOR pathway-related protein PI3K,Akt,p-Akt,mTOR and p-mTOR protein.Results1.Effect of tetrahydrocurcumin on cardiac function in rats with myocardial ischemia-reperfusion injury(1)The functional level of the left ventricle of the heart was examined by M-mode ultrasound,and results indicated that THC intervention for 28 days can significantly reduce the left ventricular end-systolic diameter(LVESD)and end-systolic volume(LVESV),and reduce the degree of ventricular dilatation after I/R.At the same time,THC treatments significantly increased ejection fraction(EF),short axis contraction rate(FS)and stroke volume(CO),and improved myocardial contractility and compliance.(2)Compared with the sham operation group,the three myocardial enzymes of MIRI rats were significantly increased at all time points after the operation.Treatment with THC partially inhibited the increase of serum myocardial enzymes caused by I/R,significantly reduce the levels of serum CK,LDH,and α-HBDH.(3)The results of HE staining showed that the myocardial tissue structure in the sham operation group was complete;rats in the I/R group showed severe necrosis of some myocardial cells,with a large number of neutrophils aggregated,and inflammatory cell infiltration was obvious.THC can significantly improve I/R rat inflammatory lesions,reduce cardiac tissue inflammatory cell infiltration,decrease the degree of myocardial fibrosis in I/R rats,and improve the pathology trend.(4)The apoptosis of rat myocardial cells was detected by TUNEL.Compared to the sham group,the apoptosis in myocardial tissue was increased significantly in the I/R operation group.While the rat myocardial cell apoptosis in I/R+THC groups was less than that of the I/R group.(5)The results of Masson staining showed that THC interventions effectively improved myocardial fibrosis in heart.2.Protective effect of tetrahydrocurcumin on hypoxia/reoxygenation injury of H9c2 cardiomyocytes(1)The viability of cells was evaluated using a CCK-8 assay and the LDH release was detected by LDH assay kit.Treatment of H/R-exposed H9c2 cells with THC significantly increased the cell viability.Additionally,THC significantly reduced LDH release in a dose-dependent manner.From the above results,we found that 2.0 μM THC led to the highest cell viability and the lowest LDH release.Therefore,2.0μM THC was employed for the subsequent experiments.(2)Compared with H/R group,THC effectively improved antioxidant activity by increasing SOD and CAT activities and decreasing MDA level.(3)The mitochondrial membrane potential of cardiomyocytes in the Control group and the THC group was higher,and the red fluorescence was stronger.Mitochondrial membrane potential decreased in H/R group,and the decrease in mitochondrial membrane potential in H/R group was reversed after THC intervention.(4)Compared with H/R group,THC significantly decreased the apoptotic ratio,diminished the ratio of Bax/Bcl-2 and cleaved caspase-3.(5)In addition,THC effectively decreased Beclinl expression and LC3Ⅱ/Ⅰ ratio,but increased p62 expression.Compared with the H/R model group,the number of autophagosome was significantly decreased and the autophagy was effectively inhibited by post-treatment with THC.3.THC mediated PI3K/Akt/mTOR signaling during H/R challenge.(1)Furthermore,THC promoted the phosphorylation of PI3K/Akt/mTOR and induced the expression of hypoxia-inducible factor 1α(HIF-1α)after H/R.(2)Compared with Control group,p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR expression in H/R group were significantly decrease,and HIF-1α expression was significantly increase;Compared with H/R group,p-PI3K,PI3K,p-Akt/Akt,p-mTOR/mTOR and HIF-1α expression in H/R+THC group were significantly increase;Compared with H/R+THC group,p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR and HIF-1αexpression in H/R+THC+LY and H/R+THC+Ra group were significantly decrease.(3)Compared with Control group,Bax/Bcl-2 and Cleaved caspase-3 expression in H/R group were significantly increase;Compared with H/R group,Bax/Bcl-2 and Cleaved caspase-3 expression in H/R+THC group were significantly decrease;Compared with H/R+THC group,Bax/Bcl-2 and Cleaved caspase-3 expression in H/R+THC+LY and H/R+THC+Ra group were significantly increase.(4)Compared with Control group,LC3Ⅱ/Ⅰ and Beclinl expression in H/R group were significantly increase,and p62 expression was significantly decrease;Compared with H/R group,LC3Ⅱ/Ⅰ and Beclinl expression in H/R+THC group were significantly decrease,and p62 expression was significantly increase;Compared with H/R+THC group,LC3Ⅱ/Ⅰ and Beclin1 expression in H/R+THC+LY and H/R+THC+Ra group were significantly increase,and p62 expression in H/R+THC+LY and H/R+THC+Ra group were significantly decrease.Conclusion(1)Study of the protective effect on the rat MIRI model,it was found that THC can improve the impaired heart function and serum myocardial enzyme performance,reduce ventricular enlargement,depress myocardial cell apoptosis and tissue fibrosis-like changes,and ameliorate MIRI damage and the pathological deterioration.(2)THC effectively protected cardiomyocytes against H/R-induced cellular injury,oxidative burst,and cardiomyocyte apoptosis and autophagy.(3)These effects were at least partially mediated by PI3K/Akt/mTOR signaling.These results suggest that THC might be further developed as a potential therapeutic candidate for protection against myocardial H/R injury.