Proteomic and metabolomic characterization of cardiac tissue in acute myocardial ischemia injury rats.

The pathological process and mechanism of myocardial ischemia (MI) is very complicated, and remains unclear. An integrated proteomic-metabolomics analysis was applied to comprehensively understand the pathological changes and mechanism of MI. Male Sprague-Dawley rats were randomly divided into a mock surgery (MS) group and an MI group. The MI model was made by ligating the left anterior descending coronary artery, twenty-four hours after which, echocardiography was employed to assess left ventricular (LV) function variables. Blood samples and left ventricular tissues were collected for ELISA, metabolomics and proteomics analysis. The results showed that LV function, including ejection fraction (EF) and fractional shortening (FS), was significantly reduced and the level of cTnT in the serum increased after MI. iTRAQ proteomics showed that a total of 169 proteins were altered including 52 and 117 proteins with increased and decreased expression, respectively, which were mainly involved in the following activities: complement and coagulation cascades, tight junction, regulation of actin cytoskeleton, MAPK signaling pathway, endocytosis, NOD-like receptor signaling pathway, as well as phagosome coupled with vitamin digestion and absorption. Altered metabolomic profiling of this transition was mostly enriched in pathways including ABC transporters, glycerophospholipid metabolism, protein digestion and absorption and aminoacyl-tRNA biosynthesis. The integrated metabolomics and proteomics analysis indicated that myocardial injury after MI is closely related to several metabolic pathways, especially energy metabolism, amino acid metabolism, vascular smooth muscle contraction, gap junction and neuroactive ligand-receptor interaction. These findings may contribute to understanding the mechanism of MI and have implication for new therapeutic targets.

Electroacupuncture preconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting mitophagy mediated by the mTORC1-ULK1-FUNDC1 pathway.

Myocardial ischemia/reperfusion (I/R) is an important complication of reperfusion therapy for myocardial infarction, and trimetazidine is used successfully for treatment of ischemic cardiomyopathy by regulating mitochondrial function. Moreover, electroacupuncture (EA) preconditioning was demonstrated to be cardioprotective in both in vivo rodent models and in patients undergoing heart valve replacement surgery. However, the mechanisms have not been well elucidated. Mitophagy, mediated by the mTORC1-ULK1-FUNDC1 (mTOR complex 1-unc-51-like autophagy-activating kinase 1-FUN14 domain-containing 1) pathway, can regulate mitochondrial mass and cell survival effectively to restrain the development of myocardial ischemia/reperfusion injury (MIRI). In this study, we hypothesized that EA preconditioning ameliorated MIRI via mitophagy. To test this, rapamycin, an mTOR inhibitor, was used. The results showed that EA preconditioning could reduce the infarct size and risk size, and decrease the ventricular arrhythmia score and serum creatine kinase-myocardial band isoenzyme (CK-MB), lactate dehydrogenase (LDH), and cardiac troponin T (cTnT) in MIRI rats. Moreover, it also attenuated MIRI-induced apoptosis and mitophagy accompanied by elevated mTORC1 level and decreased ULK1 and FUNDC1 levels. However, these effects of EA preconditioning were blocked by rapamycin, which aggravated MIRI, reduced adenosine triphosphate (ATP) production, and antagonized infarct size reduction. In conclusion, our results indicated that EA preconditioning protected the myocardium against I/R injury by inhibiting mitophagy mediated by the mTORC1-ULK1-FUNDC1 pathway.

Effect of moxibustion preconditioning on autophagy-related proteins in rats with myocardial ischemia reperfusion injury.

BACKGROUND: Autophagy has increasingly been recognized as playing an essential role in the pathogenesis of myocardial ischemia reperfusion injury (MIRI). Moxibustion, a form of heat therapy commonly used in traditional Chinese medicine (TCM), has been shown to exhibit cardioprotective effects. However, whether the cardioprotective effect of moxibustion is related to the regulation of autophagy remains unknown. This study aimed to investigate the possible mechanism underlying the cardioprotective effect of moxibustion preconditioning at PC6 on MIRI by measuring the expressions of proteins involved in the regulation of autophagy. METHODS: Male Sprague-Dawley rats were randomly divided to receive moxibustion preconditioning or autophagy inhibitor 3-Methyladenine (3-MA) intervention. Then the MIRI model was established by ligating the left anterior descending (LAD) coronary artery for 30 minutes followed by reperfusion for 4 hours. After 4 hours of reperfusion, the myocardial infarction area was assessed using Evans blue and TTC staining, and cTnT and lactate dehydrogenase (LDH) levels in the serum were determined by ELISA. Hematoxylin and eosin (H&E) staining was performed for morphological evaluation of ventricular tissues. Expressions of autophagy components Beclin 1, Bcl-2, and Akt were assessed using quantitative real-time PCR, immunohistochemistry (IHC) and western blot. RESULTS: Moxibustion preconditioning significantly reduced the necrotic area and the levels of cTnT and LDH were similar to the 3-MA intervention, also attenuated morphological alterations were induced by MIRI. Simultaneously, the mRNA and protein expressions of Beclin 1 and Akt were up-regulated, while those of Bcl-2 were down-regulated by MIRI. Moxibustion preconditioning and 3-MA intervention reversed MIRI-induced changes in Beclin 1, Akt, and Bcl-2 expressions. CONCLUSIONS: Moxibustion preconditioning at PC6 can attenuate myocardial injury for MIRI in a similar way to 3-MA intervention. This cardioprotective effect of moxibustion preconditioning may be mediated by modulating autophagy via regulation of Beclin 1, Bcl-2 and Akt.

Changes in the Serum Metabolome of Acute Myocardial Ischemia Rat Pretreatment with Electroacupuncture.

Myocardial infarction (MI), the most common symptom is chest pain, occurs when blood flow decreases or stops to a part of the heart, causing damage to the heart muscle. Electroacupuncture pretreatment (EP) is a recent observation which has been shown to induce ischemic tolerance like the ischemia preconditioning, suggesting that EP may be a promising preventive strategy for individual susceptibility to MI. This study investigated mechanisms that underlie the effect of EP on MI through the use of gas chromatography-mass spectrometry (GC-MS)-based metabolic profiling. Male Sprague-Dawley rats were randomly divided to receive or not receive three days of EP at PC6 (Neiguan). Then on the fourth day, each group was further divided to undergo mock surgery or MI, induced by ligation of the left anterior descending coronary artery. After 24h, the blood samples and hearts were collected for the follow-up research. The results showed that treatment by EP significantly reduced the levels of CK-MB, cTnT, AST, and MDH in serum and decreased myocardial infarction area. According to GC-MS-based serum metabolic profiling and analysis, a total of 636 characteristic peaks were identified, including 158 known and 478 unknown metabolites. MI caused comprehensive metabolic changes in glycolysis-related metabolites, malate-aspartate shuttle (MAS) metabolites, and purine metabolites with anti-oxidant functions, while EP reversed more than half of the differential metabolic changes, mainly affecting amino acid and energy metabolism, especially the glutamate metabolism and MAS. In a word, our findings suggest that EP exerts its cardioprotective effect on MI by regulating amino acid and energy metabolisms. Meanwhile, GC-MS-based metabolomics provided a powerful way to characterize the metabolic features of MI, with and without EP, and thereby improved our understanding of the effect and mechanisms of EP.

Hepatoprotective effect of silver nanoparticles synthesized using aqueous leaf extract of Rhizophora apiculata.

Background: Liver plays a vital role in the elimination of xenobiotics that can induce hepatotoxicity in living organisms.Silver nanoparticles have evolved recently as an alternative in various industries and are used for their biomedical applications.Rhizophora apiculata is a least studied mangrove-based plant that has been used in the traditional medicine of Southeast Asia for its healing properties. It is a well-known fact that the generation of free radicals has been associated with oxidative stress.  Methods: Hence, in this study we used carbon tetrachloride as a hepatotoxin to induce liver damage. The protective effects of silver nanoparticles synthesized using Rhizophora apiculata on hepatotoxin-induced liver damage in experimental mice were assessed.  Results: The results of the assessment indicate that silver nanoparticles were effective in protecting the liver from damages induced by carbon tetrachloride.  Conclusion: Among existing literature, this is the first ever approach for hepatoprotective effect of nanoparticles derived using plant extract from mangrove ecosystem.

[Time-dependent Protective Effect of Electroacupuncture on Ischemic Myocardium and Changes of Myocardial Autophagy and Apoptosis Related Protein Expression in Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at different time-points on the injured myocar-dium and expression of myocardial Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ proteins in acute myocardial ischemia-reperfusion injury (MIRI) rats so as to explore its mechanisms underlying myocardial protective effect via reducing cardiomyocyte autophagy and apoptosis. METHODS: A total of 66 adult SD rats were randomly divided into sham operation (sham), model, EA-R 0min(R＝ reperfusion), EA-R 30min, EA-R 60min, and EA-R 120min groups, with 6 rats being in the sham group and 12 rats being in each of the other 5 groups. The MIRI model was prepared by ligating the anterior descending branch (ADB) of the left coronary artery for 30 min followed by reperfusion for 4 h. In the sham group, the ADB was only threaded without ligation. EA was applied to bilateral "Neiguan" (PC 6) for 20 min at 0, 30, 60, and 120 min when reperfusion. Evans Blue-triphenyltetrazolium chloride (TTC) double staining was performed to determine the myocardial infarction area (MIA) and the ratio of the infarct size of the area at risk (IS/AAR). ELISA was performed to measure serum cardiac troponin 1 (cTn-Ⅰ) content, and Western blot was used to measure the expression of apoptosis-related proteins Bax and Bcl-2 and autophagy related proteins Lc 3 Ⅱ and Lc 3 Ⅰ in the left cardiac ventricle tissue. RESULTS: Compared with the model group, the percentages of MIA in the EA-R 30min, EA-R 60min, and EA-R 120min groups, and the IS/AAR in the EA-R 0min, EA-R 30min, EA-R 60min and EA-R 120min groups were significantly reduced (P<0.05, P<0.01). Comparison among the 4 EA groups showed that the percentages of MIA and the IS/AAR were considerably lower in the EA-R 30min, EA-R 60min, and EA-R 120min groups than in the EA-R 0min group (P<0.05, except IS/AAR in the EA-R 120min group), but significantly higher in the EA-R 60min and EA-R 120min groups than in the EA-R 30min group (P<0.05, P<0.01), suggesting a better therapeutic effect of EA intervention at 30 min of MIRI in improving MI. In comparison with the sham group, myocardial cTn-Ⅰ content and Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ levels in the model group were significantly increased (P<0.01). After EA intervention, the increased cTn-Ⅰ content and Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ levels in the EA-R 0min, EA-R 30min, EA-R 60min, and EA-R 120min groups were significantly reduced (P<0.05, P<0.01). The cTn-Ⅰ content was obviously lower at EA-R 30 min, but markedly increased at EA-R 120min than at EA-R 0min (P<0.05). The expression of Bcl-2 was obviously higher at EA-R 30min than at EA-R 0min (P<0.05). No significant differences were found among the 4 EA intervention time-points in the levels of Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ (P>0.05). CONCLUSION: EA intervention can reduce MIA in MIRI rats, which is possibly closely related to its effects in reducing apoptosis and autophagy. The best intervention time is at 30 min after MI reperfusion, but the difference of effects of EA at different time-points is independent of Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ expression.

[Effects of electroacupuncture at different time during reperfusion on the expression of Bcl-2 and Beclin1 in myocardial tissue in rats with myocardial ischemia reperfusion injury].

OBJECTIVE: To compare the effects of electroacupuncture (EA) at different time during reperfusion on the expression of autophagy-related protein Bcl-2 and Beclin1 in myocardial tissue in rats with myocardial ischemia reperfusion injury (MIRI), and to explore the autophagy-related mechanism of EA on protecting MIRI. METHODS: A total of 72 SD rats were randomly divided into a sham operation group, a model group, a RA group, a RB group, a RC group and a RD group, 12 rats in each group. Except the sham operation group, the rats in the remaining groups were treated with ligating the left anterior descending artery (LAD) for 30 minutes followed by reperfusion to establish the model of MIRI. The rats in the sham operation group were treated with crossing a line through the LAD. The rats in the model group did not receive treatment. The rats in the RA group, RB group, RC group and RD group were treated with EA at "Neiguan" (PC 6) for 20 min, starting at 0 h, 0.5 h, 1 h, 2 h after reperfusion. Evans Blue-TTC double-staining was employed to evaluate myocardial infarct size; the serum CK-MB was detected by ELISA and the expression of Bcl-2 and Beclin1 protein in myocardial tissue were detected by Western blot. RESULTS: Compared with the model group, the percentage of myocardial infarct size in the RB group, RC group and RD group was decreased significantly (all P<0.05), and the reduction in the RB group was more significant than that in the RC group and RD group (both P<0.05). Compared with the sham operation group, the expression of CK-MB and Beclin1 in the model group was significantly increased, but the expression of Bcl-2 was significantly decreased (all P<0.01). Compared with the model group, the expression of CK-MB and Beclin1 was decreased significantly in RA group, RB group, RC group and RD group (all P<0.05), but the expression of Bcl-2 was significantly increased (all P<0.05). Compared with the RA group, the expression of CK-MB was decreased in the RB group and RC group (both P<0.05) but the expression of Bcl-2 was increased (both P<0.01); the expression was not significantly different from that in the RD group (P>0.05); the increasing of Bcl-2 in the RB group was more significant than that in RC group (P<0.05). The expression of Beclin 1 in the RB group was significantly lower than that in the RA group (P<0.05), but there was no significant difference among other EA groups (all P>0.05). CONCLUSION: EA at different time during reperfusion could reduce myocardial infarct size in rats with MIRI, and EA at 0.5 h after reperfusion has best efficacy; this protective effect may be achieved by increasing Bcl-2 expression and reducing Beclin1 expression to inhibit overautophagy during reperfusion.

[An autophagic mechanism study on effect of electroacupuncture at different times pretreating myocardial ischemia-reperfusion injury].

OBJECTIVE: To investigate the difference of protective effect of electroacupuncture (EA) at different times pretreating myocardial ischemia-reperfusion injury (MIRI) and the protein expressions of ULK1 and Beclin-1 in myocardial tissue, so as to explore the relation of the difference and autophagic mechanism. METHODS: Sixty-three male SD rats were randomized into 7 groups, a sham group, a model group, an EA pretreating for 1 d (EA-1d) group, an EA pretreating for 2 d (EA-2d) group, an EA pretreating for 3 d (EA-3d) group, an EA pretreating for 4 d (EA-4d) group, an EA pretreating for 5 d (EA-5d) group, 9 rats in each group. All the rats in the pretreating groups were treated with EA 1-5 days before MIRI surgery. EA (2 Hz/100 Hz, 2 mA) was used at bilateral "Neiguan (PC 6)" for 20 min. All the rats except for those in the sham group was underwent left thoracotomy and ligation of the left anterior descending (LAD) coronary artery for 30 minutes followed by 4 hours of reperfusion to establish the MIRI model. The same operation was performed in the sham group except for the ligation of the LAD. Throughout the experiment, electrocardiogram was continuously monitored. The myocardial infarct sizes were assessed by Evans blue and triphenyltetrazolium chloride (TTC) staining. The serum concentrations of cardiac troponinⅠ(cTnⅠ) was detected by ELISA. The expressions of ULK1 and Beclin-1 in the heart tissues were analyze by Western blot. RESULTS: Compared with the sham group, the concentrations of cTnⅠ and the protein expressions of ULK1 and Beclin-1 increased in the model group (all P<0.05). Compared with the model group, the infarct sizes decreased in the EA-1d, EA-2d, EA-3d, EA-4d and EA-5d groups (all P<0.05), with less risk sizes in the EA-3d, EA-4d and EA-5d groups (all P<0.05). The levels of cTnⅠin the EA-4d and EA-5d groups decreased ( both P<0.05); the expressions of ULK1 protein decreased in the EA-1d, EA-2d, EA-3d, EA-4d and EA-5d groups (all P<0.05); the expressions of Beclin-1 protein decreased in the EA-2d, EA-3d, EA-4d and EA-5d groups (all P<0.05). The infarct sizes in the EA-4d and EA-5d groups were lower than that in the EA-1d group (both P<0.05). The cTnⅠconcentration in the EA-4d group was less than that in the EA-1d group (P<0.05). CONCLUSION: Pretreatment with EA for 1-5 days can improve the infarct size in MIRI, with better effect of the pretreatment for 4-5 days. The cardioprotective effect may be related to the inhibition of autophagy. But the difference of the protective effects is not related to the protein expressions of ULK1 and Beclin-1.

[Effect of Moxibustion Preconditioning with Seed-sized Moxa Cones on Myocardial Infarction Size and Beclin 1 Expression in Myocardial Ischemia-reperfusion Injury Rats].

OBJECTIVE: To observe the effect of moxibustion (Moxi) preconditioning with seed-sized moxa cones on myocardial ischemia-reperfusion injury (MI/RI) at different stages, and to analyze the correlation between this effect and the expression of autophagy related protein Beclin 1. METHODS: This study contains two parts: 1) changes of myocardial pathological injury and percentages of myocardial infarcted area at different time-points after modeling and Moxi intervention, and 2) effect of Moxi on contents of serum cardiac troponin T(cTnT) and expression of myocardial Bcl-2, Bax and Beclin 1 proteins. In the first part, 42 SD rats were randomly divided into model group, 1 day (d) Moxi group, 2 d Moxi group, 3 d Moxi group，4 d Moxi group, 5 d Moxi group and 7 d Moxi group. The model of MI/RI was established by ligating the left anterior descending coronary artery (LAD) for 30 min and reperfusion for 240 min. The electrocardiogram (ECG) of standard limb lead Ⅱ was monitored and the heart was taken 4 h after reperfusion for examining myocardial infarcted size with triphenyl tetrazolium chloride (TTC) staining. In the second part, 48 SD rats were randomized into sham-operation, model, moxibustion and autophagy inhibitor (3-MA) groups, with 12 rats in each group. The serum cTnT level was assayed and histopathological changes of the myocardial tissue below the ligation site were examined with HE staining, and the expression levels of Bcl-2, Bax and Beclin 1 proteins in the myocardial tissue below the LAD-ligated site were detected using Western blot. RESULTS: Compared with the model group, the percentages of myocardial infarcted area were significantly decreased in the 4 d, 5 d and 7 d Moxi groups (P<0.05), but without significant differences among the 3 Moxi groups (P>0.05). The state of MI-induced breakage and disordered arrangement of myocardial fibers with interstitial edema and inflammatory cell infiltration at the MI stage in the Moxi group and at the reperfusion stage in the autophagy inhibitor group was relatively lighter. The levels of serum cTnT content and Bax/Bcl-2 and Beclin 1 protein expression at the MI and reperfusion stages were significantly higher in the model group than in the sham-operation group (P<0.01), and considerably lower in the Moxi and autophagy groups than in the model group (P<0.01). The serum cTnT content, ratio of Bax/Bcl-2 expression and Beclin 1 expression levels at the MI and reperfusion stages were significantly lower in the autophagy inhibitor group than in the Moxi group (P<0.05). CONCLUSIONS: Moxibustion with seed-sized moxa cones at "Neiguan" (PC 6) can effectively alleviate myocardial ischemia in MI/RI rats, which is probably related to its effect in down-regulating Bax/Bcl-2 and Beclin 1 expression and in inhibiting autophagy.