| BackgroundThe report in Lancet points out that ischemic heart disease ranked in the first place in 235 kinds of disease all over the world, which result in death. In the past few decades, with the application of percutaneous coronary intervention, coronary artery bypass grafting, antiplatelet and anticoagulant drugs, ischemic cardiomyocytes can regain blood supply quickly, however, myocardial ischemia/reperfusion paradoxically reduces the beneficial effects, which lead to the deterioration of cardiac function and myocardial injury. This is called ischemia-reperfusion (I/R) injury. This is the main cause of heart failure and lead to increase in mortality of patients and decrease in quality of life. Cause of mechanism in myocardial I/R injury is more, including apoptosis, autophagy, oxidative stress, endoplasmic reticulum stress and so on, but this is still not very clear.Endoplasmic Reticulum(ER) stress is one of the major mechanisms following myocardial ischemia-reperfusion injury. ER stress was induced by dysfunction of ER because of ischemia and hypoxia. ER stress was involved in reactions of pathophysiological change of I/R. The degree of ERS influences the survival or death of cardiomyocytes. Elevated protein markers of were detected in myocardial I/R injury model in mice.Studies have been shown that apoptosis induced by ER stress is one of the important pathophysiological mechanism of myocardial ischemia-reperfusion injury. Weakening the degree of endoplasmic reticulum stress can protect myocardial ischemia-reperfusion injury. Therefore, it can be to intervent the apoptosis of myocardial I/R injury by regulating the ER stress. SIRT1 is one of NAD+dependent-acetylation enzyme in mammals, which participates in the regulation of metabolism, cell survival, life span and other important physiological process. Studies have shown that activation of SIRT1 can protect myocardial from injury. It can be seen as a new target for treatment of myocardial ischemia reperfusion. However, the role of SERT1 in the prevention and treatment for cardiovascular diseases is still poorly understood. Both ER stress and SIRT1 signaling pathways are considered as the important factors for deciding myocardial cell survival and death, the study found that there is a close connection between ER stress and SIRT1, However, the mechanism of myocardial ischemia-reperfusion mechanism influenced by them is unclear.Cardiomyocyte autophagy plays an important role in myocardial I/R. Previous studies of ischemia-reperfusion injury paid attention to myocardial necrosis and apoptosis. but recent studies have shown that autophagy disorders may make the heart occurre cardiac remodeling after myocardial infarction. Now the relationship between autophagy and apoptosis have been a hot research topic. Studies have shown that autophagy can inhibit apoptosis to protect myocardial cells. The regulation of autophagy is a complicated process, the mechanism of signal transduction molecules is very complex, AMPK/mTOR signaling pathway is a comparatively clear, mTOR is considered to be negative regulation of autophagy in mammalian. Hypoxia or ischemia can activate AMPK to induce autophagy and inhibit mTOR.Sulforaphane (SFN) is a kind of isothiocyanates from cruciferous plant. it is an indirect antioxidants which can induce Ⅱ detoxification enzymes and antioxidant enzyme gene. So it has the function for anti-oxidative stress, anti-inflammation, antitumor and cardioprotective role.Therefore, we propose a hypothesis:1.Sulforaphane prevents rat cardiomyocytes from hypoxia/reoxygenation injury in vitro via activating SIRT1 and subsequently inhibiting ER stress; 2. Induction of autophagy by sulforaphane pretreatment protect myocardial ischemia/reperfusion injury via AMPK/mTOR in mice. We utilized an in vivo mouse model of myocardial I/R injury and an in vitro neonatal rat cardiomyocytes(NRCs) model of hypoxia/reoxygenation(H/R) injury to investigate of sulforaphane on myocardial I/R injury and its underlying mechanisms.Objectives1.We intend to establish the model of H/R injury in NRCs and explore that Sulforaphane prevents rat cardiomyocytes from hypoxia/reoxygenation injury in vitro via activating SIRT1 and subsequently inhibiting ER stress;2. We establish H/R model in vitro and I/R model in vivo to confirme that induction of autophagy by sulforaphane pretreatment protect myocardial ischemia/reperfusion injury via AMPK/mTOR in mice.MethodsPart1Neonatal rat cardiomyocytess(NRCs) were isolated from newborn(1-2 day old) Sprague-Dawley rats. The experimental design was divided into four steps.Step 1, The aim of this step is to explore the optional reoxygenaiton time. After 72h, cultured cardiomyocytes were randomly divided into five groups:1)Control group:cardiomyocytes were maintained in normoxic condition without any treatment; 2)H3/R3 group:Hypoxia 3h, following Reoxygenation 3h.3)H3/R6h group:Hypoxia 3h, following Reoxygenation 6h.4)H3/R9h group:Hypoxia 3h, following Reoxygenation 9h; 5)H3/R12 group:Hypoxia 3h, following Reoxygenation 12h. Morphology of myocardiocytes was obserbed by inverted microscopy. Cell viability was detected by MTS. LDH activity was measured by LDH elisa Kit.Step 2, when the reoxygenation duration of experimental model was determined, cardiomyocytes were randomly divided into six groups to be exposed to the optional concentration:Control group, H/R group and SFN group(0.1,0.5,1.0,5μM). Cell viability was detected by MTS. LDH activity was measured by LDH elisa kit. MDA was measured by MDA kit. SOD was measured by SOD kit.Step 3, When ideal concentration of SFN was screened out, cardiomyocytes were randomly divided into four groups:1)Control group:Cardiomyocytes were treated with PBS and incubated in normoxic condition.2) SFN group: Cardiomyocytes were treated with 5μM sulforaphane and incubated in normoxic condition.3) H/R group:as above design, cardiomyocytes were subjected to hypoxia for 3h, followed by reoxygenation for 3h.4)SFN+H/R group:Cardiomyocytes pretreated with 5μM SFN were exposed to 3h of hypoxia followed by 3h reoxygenation. SFN was pretreated 1h befor hypoxia. Apoptotic index was measured by TUNEL. Caspase-3 activity was measured by Caspase-3 elisa kit. Proteins including Bcl-2, Bax, GRP78, CHOP, Caspase-12 were measured by Western blot.Step 4, To explore the cardioprotective role of SIRT1 activated by SFN pretreatment during H/R injury, cardiomyocytes were randomly divided into six group(as shown in figure 1.):1)Control group.2) EX-527 group(E).3)H/R group.4) EX-527(E)+H/R group.5) SFN+HR group.6) SFN(5μM)+EX-527(E)+HR group. EX-527 was pretreated 1h before hypoxia. The concentration of EX-527 is 1μM. Cell viability was detected by MTS. Mitochondrial membrane potential was measured by JC-1 probe. Proteins including Bcl-2, Bax, GRP78, CHOP, Caspase-12 were measured by Western blot.Part 2Wild-type male C57BL/6 mice (20～25 g) were used in this study. To simulate I/R model, the mice were underwent 45 min ischemia followed by 2 h reperfusion. This experiment was divided into 5 steps:Step 1, To explore whether H/R could induce autophagy and apoptosis in NRCs exposed to H/R, myocardial cells were divided into six groups:Con group; H3/R3 group; H3/R6 group; H3/R9 group; H3/R12 group; H3/R24 group. Apoptotic index was detected by TUNEL. The expression of LC3-Ⅱ was measured by Western blot.Step 2, To explore whether autophagy can inhibit apoptosis and protect myocardial cell under H/R injury. Myocardial cells were divided into four groups: Control group; Hypoxia/reoxygenation group(H/R); 3-MA group; Rapa groups; Through the double dye and LC3-Ⅱ protein, P62 protein expression detect autophagy flow; Autophagy flux was detected with Ad-mRFP-GFP-LC3. The apoptotis were measured by TUNEL and expression level of apoptotic protein including Bcl-2 and Bax.Step 3, To explore whether SFN could promote autophagy to inhibit apoptosis of cardiomyocytes. Myocardial cells were divided into 5 groups:Control group; H/R group; SFN group; 3-MA group; Rapa groups; Autophagy flux was detected with Ad-mRFP-GFP-LC3. The apoptotis were measured by TUNEL and expression level of apoptotic protein including Bcl-2 and Bax.Step 4, To explore whether SFN could induce autophagy to protect myocardial I/R injury in mice. Mice were divided into 7 groups:Sham group; I/R group; SFN group; Wor group; Rapa group; SFN+Wor group; SFN+Rapa group. Autophagy flux was detected by expression level of LC3-Ⅱ and P62. Autophaosomes were detected by electron microscopy.The apoptotis were measured by TUNEL and expression level of apoptotic protein including Bcl-2 and Bax.Step 5, To explore whether SFN could promote autophagy to protect cardiomyocytes from the apoptosis via AMPK/mTOR pathway. Myocardial cells were divided into 5 groups:Contro group, I/R group, SFN group, CC group, SFN+ CC group. Autophagy flux was detected by expression level of LC3-Δ and P62. The expression level of proteins including AMPK, p-AMPK, mTOR and p-mTOR were detectd by Western blot.All of the values were expressed as the Mean±SD and statistically ananlyzed with SPSS 19.0. One-way ANOVA was performed to test significane of biochemical data of different groups. A difference of P<0.05 was considered to be statistically significant. For each assessment, at least three independent experiments were performed.ResultsPart 13.1 The stress of Hypoxia/reoxygenation affected the morphology of myocardial cells.The morphology of cardiomyocytes was observed with inverted microscope. The cardiomyocytes in Control group grew and beat well, while cardiomyocytes in H3/R3 group shrinked significantly and beat frequency reduced. With the duration of reoxygenation, cell viability of myocardial cells decreased, the beat of cardiomyocytes was weak.In addition, some cardiomyocytes were floating on the culture medium. The cell vitality of cardiomyocytes in H3/R12 group was the worst and some cardiomyocytes were dead with no beat.3.2 SFN alleviated H/R injuryTo explore the optional reoxygenation time, cell viability of different time duration was measured by MTS. As shown in Fig 1-2.A. cell viability significantly declined in a time-dependent manner(P<0.05). The release of LDH was used as an index of cardiomyocyte injury. LDH activity increased with duration of reoxygenation prolonged. (Figure 1-2.B). Taken together, to conduct our experiment with a relatively ideal cell viability and to provide an accurate result, we decided the optional duration for mimicking H/R is hypoxia 3h and reoxygenation 3h.We next determine the effect of SFN on cradiomyocytes during H/R. The results demonstrated that H/R caused a significant decline in cell viability compared with the Control group (P<0.05). Pretreatment with SFN(0.1,0.5,1,5μM) was found to increase cell viability significantly in a dose-dependent manner. The cell viability and SOD activity went to the peak at a concentration of 5 μM, while the results MAD content declined on the contrary(P<0.05 vs H/R group, Figure 1-3,A-C). Therefore, 5μM SFN was used as treated group in subsequent experiments.3.3 SFN attenuated H/R-eliciated apoptotis in cultured neonatal rat cardiomyocytesAs shown in Figure 1-4, representative photomicrographs of TUNEL assay results depicted apoptotis of H/R-induced in cardiomyocytes. TUNEL staining showed that the apoptotic cells increased remarkedly in the I/R group compared with the Control group and SFN decreased apoptotic cells obviously(P<0.05).Caspase-3 has been known as a pivotal protein in the final pathway of apoptosis. To further characterize the inhibitory effect of SFN on myocardial cell apoptosis, we examined whether SFN could suppress caspase-3 activity. In our present study, the activity of caspase-3 was significantly greater in HR group than that in control group(P<0.05). however, SFN could decrease the activity of caspase-3 which induced by H/R injury(P<0.05). Therefore, SFN could attenuate H/R-induced apoptosis in cultured neonatal rat cardiomyocytes3.4 SFN preserved the cardiomyocytes against H/R injury partly through the attenuation of ER stress-induced apoptosis activationWe then moved to investigate the underlying mechanism of the protective effects offered by SFN. As shown in Figure 1-5, in accordance with ER stress-dependent apoptosis activation, H/R remarkedly suppressed Bcl-2/Bax ratio compared with control group(P<0.05). however, SFN pretrement could elevate the ratio of Bcl-2/Bax(P<0.05).As ER stress was often implicated in the activation of apoptosis during H/R injury, we next examined whether SFN preserved cardiomyocytes by modulating ER stress-dependent apoptosis. The levels of ER stress protein markers GRP78, CHOP and cleaved caspase-12 in H/R group were higher than those in H/R group(P<0.05), but lower in SFN and SFN+H/R groups compared with that in H/R group(P<0.05).3.5 SFN pretreatment increased the expression of SIRT1 protein and SIRT1 activation protected cardiomyocytes suffered H/RTo investigate whether SFN could activate SIRT1 signal pathway, we detected the protein expression of SIRT1 by western blotting. As shown in Fig 1-5, the expression of SIRT1 increased significantly in SFN pretreated group compared with the H/R group(P<0.05). Specific SIRT1 inhibitor Ex-527 could block the effect (P<0.05, Fig 1-7). However, no significance difference in Sirtl expression is detectable between control group and H/R group((P>0.05). To further verify our results, we detected the SIRT1 activity in different groups. we found that SFN could elevate SIRT1 activity and Ex-527 could decline SIRT1 activity to abrogate the effect of SFN.Moreover, H/R could decline the SIRT1 activity (P<0.05, Fig 1-8).As we know, mitochondria plays an essential function in cell apoptosis during H/R injury. The decline of mitochondrial membrane potential(△Ψm) was regarded as an early event in the apoptotic cascade. As shown in Fig 1-6, The red/green ratio was increased in SFN group, while the red/green ratio of fluorescence in H/R+SFN+EX-527 group significantly reduced respect to the SFN+H/R group. In accordance with the above results, decreased cell viability was detected by MTS assay in H/R+SFN+EX-527 group(P<0.05). Meanwhile, SIRT1 activation by SFN significantly elevated the ratio of Bcl-2/Bax in cardiomyocytes exposed to H/R(P<0.05). Compared with the H/R+SFN group, the expression of SIRT1 and the ratio of Bcl-2/Bax remarkedly decreased in H/R+SFN+Ex-527 group(P<0.05). Thus, SIRT1 pathway induced by SFN was involved in cardioprotection via antiapoptotic function.3.6 Effects of SIRT1 pathways in modulating ER stress-induced apoptosisTo explore whether the inhibiton of SIRT1 had an effect on cardioprotection against ER stress, we employed the inhibitor of SIRT1-specific inhibitor EX-527 to block SIRT1 pathway. EX-527 partially abolished the cardioprotection offered by SFN against H/R-induced ER stress-induced apoptosis signaling, which was assessed with upregulation of GRP78, CHOP and cleaved caspase-12 expression levels(P<0.05). SIRT1 specific inhibitor EX-527 significantly decreased cardioprotective effect of SFN(Fig 1-7).As shown in Fig 1-8, the role of SIRT1 signaling pathway in regulating ER stress-dependent apoptosis activation was also indicated by Bcl-2/Bax ratio. SIRT1 inhibitor blocked the cardioprotective effect of SFN against H/R injury. Therefore, SIRT1 activation could attenuate ER stress-induced cell apoptosis.Part 23.1 Autophagy and apoptosis were both induced in NRCs exposed to H/RTo explore whether H/R injury can induce autophagy in NRCs, we detected the expression level of LC3-Ⅱ by Western blot. Compared with the control group, H3/R3 autophagy level reached its peak, the differences were statistically significant (P<0.01);Compared with H3/R3 group, autophagy level in other H/R group declined gradually(P<0.01). Apoptosis index was detected by TUNEL. In order to understand the relationship between apoptosis and autophagy. Compared with the control group, apoptotic index in H/R group increased significantly(P<0.01).3.2 Autophagy could inhibited apoptosis in NRCs exposed to H/RIn order to study the relationship between autophagy and apoptosis, cardiomyocytes were exposed to H/R with or without autophagy inhibitor and promoter rapamycin. Compared with the control group, the expression of LC3-Ⅱ in H/R group were elevated significantly, the differences was statistically significant (P<0.05);Compared with H/R group, autophagy inhibitor 3-MA group inhibited the level of LC3-Ⅱ (P<0.01). The LC3-Ⅱ level in rapamycin group increased significantly, the difference was significant statistically (P<0.01).Compared with H/R group, the level of P62 in 3-MA group increased significantly, however, it decreased significantly in rapamycin group, the differences were statistically significant (P<0.05). Compared with control group, the level of the Bcl-2 in H/R group was obviously lower, the level of Bax was higher significantly, the differences were statistically significant (P<0.05); Compared with H/R group, the level of Bcl-2 in 3-MA group was decreased significantly(P<0.01). the level of Bcl-2 in rapamycin group was increased significantly(P<0.01).Compared with H/R group, the level of Bax in 3-MA group was increased significantly, while the level of Bax in rapamycin group was obviously reduced, the differences were statistically significant(P<0.05). Autophagy flux was detected by Ad-mRFP-GFP-LC3. Compared with control group, the level of GFP-mRFP in H/R group was obviously higher, mRFP level were increased significantly, the differences were statistically significant (P<0.05);Compared with H/R group, the level of GFP/mRFP in rapamycin group was increased significantly(P<0.01), while the level of GFP/mRFP in 3-MA group was declined(P>0.05). Compared with H/R group, the level of mRFP in 3-MA group was ); the level of mRFP in rapamycin group was obviously ). Compared with control group, the apoptosis index of H/R group was obviously higher(P<0.01); Compared with H/R group, apoptosis index in 3-MA group was lower obviously(P<0.01).3.3 SFN could induce autophagy to inhibit apoptosis in NRCs exposed to H/R.Compared with the control group, the level of LC3-Ⅱ and P62 in H/R group were elevated significantly, the differences were statistically significant (P<0.05); Compared with H/R group, the level of LC3-Ⅱ in SFN group was increased significantly, while the level of P62 was reduced obviously, the differences were statistically significant (P<0.05); Compared with SFN group, the level of LC3-Ⅱ in SFN+3-MA group was decreased, while the level of P62 was elevated, the difference were statistically significant (P<0.01). Compared with control group, the level of the Bcl-2 in H/R group was obviously lower, the level of Bax was increased significantly, the differences were statistically significant (P<0.05); Compared with H/R group, the level of Bcl-2 in SFN group was obviously higher, the difference is statistically significant (P<0.01). Compared with SFN group, SFN+3-MA group decreased obviously, while the level of Bax was rised apparently(P<0.01), while the level of Bcl-2 in SFN+ Rapa group increased slightly, while the level of Bax was lower slightly(P>0.05). Compared with H/R group, the levels of GFP/mRFP and mRFP in SFN group were obviously higher(P<0.01). Compared with SFN group, the levels of GFP/mRFP and mRFP in SFN+3-MA group were lower, the differences were statistically significant (P<0.05). Compared with SFN group, the levels of GFP/mRFP and mRFP in SFN+Rapa group were higher, the differences were not statistically significant (P>0.05). Compared with control group, the apoptotic index of H/R group was obviously higher(P<0.01). Compared with H/R group, the apoptotic index of SFN group was obviously reduced(P<0.01). Compared with SFN group, the apoptotic index in SFN+3-MA group was elevated (P<0.05), but there is no statistically significant difference in the SFN group+Rapa (P>0.05).3.4 SFN pretreatment could reduce the myocardial infarction area of ischemia-reperfusion injury in miceCompared with control group, the level of INF/AAR and INF/LV in I/R group were significantly elevated, the differences were statistically significant (P<0.05); Compared with I/R group, the level of INF/AAR and INF/LV in SFN group were significantly reduced(P<0.05); Compared with I/R group, the level of INF/AAR and INF/LV in Wor group were increased significantly(P<0.05), while the level of INF/AAR and INF/LV in Rapa group were obviously higher(P<0.05).3.5 SFN pretreatment could reduce serum CK and LDH activity.Serum CK and LDH activity were detected by elisa kit. Compared with control group, the level of CK and LDH activity in I/R group were significantly elevated, the differences were statistically significant (P<0.05); Compared with I/R group, the level of CK and LDH activity in SFN group were obviously reduced, the differences were statistically significant (P<0.05); Compared with I/R group, the level of CK and LDH activity in Wor group were obviously higher, the differences were statistically significant (P<0.05), the level of CK and LDH activity in Rapa group were significantly lower, and the differences were statistically significant (P<0.05); Compared with SFN group, the level of CK and LDH activity in SFN+Wor group were increased, the difference is statistically significant (P<0.01). the level of CK and LDH activity in SFN+Rapa group were reduced (P>0.05).3.6 SFN pretreatment induced autophagy to inhibit apoptosis and protect myocardial I/R injury mice.Compared with the control group, the level of LC3-Ⅱ in I/R group was significantly elevated, the differences were statistically significant (P<0.05); Compared with I/R group, the level of LC3-Ⅱ in SFN group was increased significantly, while the level of P62 was reduced obviously (P<0.05); Compared with I/R group, the level of LC3-Ⅱ in 3-MA group was decreased significantly, the level of P62 was increased obviously(P<0.05), while the level of LC3-Ⅱ and P62 in Rapa group was increased significantly, while the level of P62 was reduced obviously(P<0.05); Compared with SFN group, the level of LC3-Ⅱ in SFN+Wor group was decreased, while the level of P62 was elevated significantly(P<0.01).The level of LC3-Ⅱ in SFN+Rapa group was rised a little, while the level of P62 wa lower slightly, there is no statistically significant difference (P>0.05). Compared with I/R group, the level of the Bcl-2 in SFN group was obviously higher, while the level of Bax was reduced obviously (P<0.05); Compared with I/R group, the levels of the Bcl-2 in Wor group was obviously lower, while the level of Bax was increased obviously(P<0.05), while the level of Bcl-2 in Rapa group was obviously higher, while the level of Bax was reduced obviously(P<0.05); Compared with SFN group, the level of Bcl-2 in SFN+Wor group was lower, while the level of Bax was higher(.P<0.01).The level of Bcl-2 in SFN+Rapa group was increased a little, while the level of Bax was slightly lower, there is no statistically significant difference (P>0.05). Compared with I/R group, the levels of autophagosome in SFN group and Rapa were increased significantly, while the level of autophagosomes in Wor group were reduced significantly, (P<0.01). Compared with SFN group, the level of autophagosomes in SFN+Wor group were lower(P<0.05). Compared with I/R group, apoptotic index in SFN group and Rapa group were decreased significantly, while apoptotic index in Wor group was increased slightly(P<0.01). Compared with SFN group, apoptotic index in SFN+Wor group was elevated, but there is no statistically significant difference in the SFN+Rapa (P>0.05).3.7 SFN could induced autophagy to inhibit apoptosis and protected myocardial I/R injury via AMPK/mTOR pathway.Ccompared with control group, the level of LC3-Ⅱin H/R group was significantly elevated, the differences were statistically significant (P<0.05); Compared with H/R group, the level of LC3-Ⅱin SFN group was increased significantly, while the level of P62 was significantly reduced; CC group could inhibited the expression of LC3-Ⅱ significantly and elevated the expression level of P62 level(P<0.01). Compared with control group, the level of GFP-mRFP in H/R group was obviously higher, the level of mRFP level was increased significantly, the differences were statistically significant (P<0.05); Compared with H/R group, the level of GFP/mRFP and mRFP in SFN group and Rapa group were increased significantly(P<0.01). Compared with H/R group, the level of GFP/mRFP and mRFP in 3-MA group were reduced. Compared with Rapa and SFN group, the level of GFP/mRFP and mRFP in SFN+3-MA group was lower(P<0.05). Compared with SFN group, the level of GFP/mRFP and mRFP in SFN+Rapa group were higher, the differences were not statistically significant (P>0.05). Compared with control group, the expression level of p-AMPK/AMPK in H/R group was increased significantly, while the expression level of p-mTOR/mTOR was lower apparently(P<0.05); Compared with SFN group, the level of p-AMPK/AMPK in CC and SFN+CC group were decreased obviously, the differences were statistically significant (P<0.05).Conclusions1. Sulforaphane prevents rat cardiomyocytes from hypoxia/reoxygenation injury in vitro via activating SIRT1 and subsequently inhibiting ER stress;2. Induction of autophagy by sulforaphane pretreatment protect myocardial ischemia/reperfusion injury via AMPK/mTOR in mice. |
PDF Full Text Request