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Aims: Prior to reperfusion, Calpains remain inactive due to the acidic pH and elevated ionic strength in the ischemic myocardium; but Calpain is activated during myocardial reperfusion. The underlying mechanism of Calpain activation in the ischemia-reperfusion (I/R) is yet to be determined. Therefore, the present study aims to investigate the mechanism of Calpain in I/R-induced mice., Main Methods: In order to detect the function of Calpain and the NLRP3/ASC/Caspase-1 axis in cardiomyocyte pyroptosis, endoplasmic reticulum (ER) stress and myocardial function, the cardiomyocytes were treated with hypoxia-reoxygenation (H/R), and NLRP3 were silenced, Calpain was overexpressed and Caspase-1 inhibitors were used to determine cardiomyocyte pyroptosis. The results obtained from the cell experiments were then verified with an animal experiment in I/R mice., Key Findings: There was an overexpression in Calpain, ASC, NLRP3, GRP78 and C/EBP homologous protein (CHOP) in cardiomyocytes following H/R. A significant increase was witnessed in lactic acid dehydrogenase (LDH) activity, cardiomyocyte pyroptosis rate, Calpain activity, reactive oxygen species (ROS) concentration, as well as activation of ER stress in cardiomyocytes after H/R. However, opposing results were observed in H/R cardiomyocytes that received siRNA Calpain, siRNA NLRP3 or Caspase-1 inhibitor treatment. Overall, the results obtained from the animal experiment were consistent with the results from the cell experiment., Significance: The silencing of Calpain suppresses the activation of the NLRP3/ASC/Caspase-1 axis, thus inhibiting ER stress in mice and improving myocardial dysfunction induced by I/R, providing a novel therapeutic pathway for I/R., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To investigate the effect of NACHT-LRR-PYD- containing proteins 3 (NLRP3) mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation (H/R) injury. Methods: In order to observe whether H/R-treatment could cause pyroptosis, H9c2 cells were divided into 2 groups randomly using the lottery method: control group(without H/R-treatment) and H/R group (in which the H9c2 cells were underwent H/R-treatment). In order to clarify the role of pyroptosis in H/R-injury, H9c2 cells were divided into 4 groups randomly using the lottery method: control group(in which the H9c2 cells were cultivated with normal medium); YVAD group(in which the H9c2 cells were pretreated with z-Val-Ala-Asp(Ome)-fluoromethylketone (Z-YVAD-FMK) 20 μm for 4 hours, then replaced with normal medium); H/R group(H9c2 cells underwent H/R-treatment); YVAD+H/R group (in which the H9c2 cells were pretreated with 20 μm Z-YVAD-FMK for 4 hours before H/R-treatment). To determine whether H/R-induced cell pyroptosis is associated with NLRP3, H9c2 cells were divided into 4 groups randomly using the lottery method: control group (in which cells were transfected with a control nonspecific siRNA); si-NLRP3 group (in which cells were transfected with NLRP3-targeting siRNA); H/R group(in which cells were transfected with a control nonspecific siRNA before H/R-treatment); si-NLRP3+H/R group(in which the H9c2 cells were transfected with NLRP3-targeting siRNA before H/R-treatment). Pore formation on cell membrane was detected by propidium iodide (PI) staining. Cell viability was detected by CCK8 reagent. The protein expression of Caspase-1, interleukin-1β (IL-1β) and NLRP3 was detected by Western blot. Results: (1) The positive rate of PI staining ((26.46±5.15)% vs. (1.69±0.73)%, P< 0.01), expression of NLRP3 (0.57±0.16 vs. 0.23±0.06, P< 0.01), expression of Caspase-1 (1.07±0.13 vs. 0.37±0.08, P< 0.01), and expression of IL-1β (0.38±0.08 vs. 0.16±0.05, P< 0.01) were significantly higher in H/R group than in control group. (2)The cell vitality was significantly higher in YVAD+H/R group than in H/R group ((87.31±9.05)% vs. (73.30±7.19)%, P< 0.05).The positive rate of PI staining was significantly decreased in YVAD+H/R group than in H/R group ((18.12±4.36)% vs. (26.45±4.60)%, P< 0.05). The expression of Caspase-1 (0.72±0.12 vs. 1.07±0.15, P< 0.05) and IL-1β(0.29±0.07 vs. 0.39±0.06, P< 0.05) were significantly lower in YVAD+H/R group than in H/R group. (3) The cell vitality was significantly increased in si-NLRP3+H/R group than in H/R group ((85.46±7.71)% vs. (72.41±5.53)%, P< 0.05). The positive rate of PI staining was significantly lower in si-NLRP3+H/R group than in H/R group ((18.22±4.20)% vs. (26.73±3.26)%, P< 0.05). The expression of Caspase-1(0.87±0.07 vs. 1.15±0.15, P< 0.05) and IL-1β(0.41±0.07 vs. 0.58±0.10, P< 0.05) were significantly decreased in si-NLRP3+H/R group than in H/R group. Conclusion: NLRP3 mediated pyroptosis is involved in H/R injury of myocardial cells.

Objective: To investigate the effect and related mechanism of salvianolate on myocardial ischemia-reperfusion (I/R) injury in rats. Methods: Thirty-six adult Sprague-Dawley rats were divided into three groups ( n= 12 each) using random number table method: control group (coronary artery was not ligated) , I/R group (myocardial I/R model was established by ligation and opening of left anterior descending coronary artery) , and salvianolate+I/R group (5 mg/kg of salvianolate was injected through the tail vein at the time of reperfusion) .Triphenyltetrazolium chloride (TTC) stain was utilized to measure the myocardial infarct size. The ELISA method was used to detect myocardial necrotic markers, including cardiac troponin T (TnT) , creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) . Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to analyses the levels of apoptosis. The levels of cleaved Caspase-3 protein were analyzed with Western blot.Cold luminescence method was used to detect the ATP level of myocardial tissue. The levels of 8-hydroxy-2 ' -deoxyguanosine (8-OHdG) in myocardial tissue were detected by immunofluorescence. Results: (1) The infarct size in control group, I/R group and salvianolate+I/R group were 0, (23.90±5.66) %, and (12.06±5.97) % , respectively ( P< 0.01 or 0.05) . (2) The TnT level was (0.04±0.03) , (16.96±2.80) , and (6.95±2.31) ng/ml, the CK-MB level was (43.6±23.5) , (1 135.8±180.6) ,and (390.3±121.5) U/L, the LDH level was (119.0±58.6) , (1 838.6±543.8) , and (631.6±228.3) U/L in control group, I/R group and salvianolate+I/R group, all significantly lower in salvianolate+I/R group than in I/R group (all P< 0.01) . (3) The rate of TUNEL positive myocardial cells were (1.07±1.16) % , (21.36±4.11) % ,and (13.30±3.67) % in control group, I/R group,and salvianolate+I/R group (all P< 0.01) . (4) The cleaved Caspase-3 expression was 0.11±0.05, 0.84±0.20,and 0.43±0.09 in control group, I/R group, and salvianolate+I/R group (all P< 0.01) . (5) The ATP level of myocardial tissue was (100.0±0.0) % , (34.2±9.2) % ,and (62.1±18.0) % respectively in control group, I/R group, and salvianolate+I/R group (all P< 0.01) . (6) There was almost no 8-OHdG expression in the myocardial tissue of control group. The expression of 8-OHdG in the myocardial tissue of I/R group was greater than that of the control group. The expression of 8-OHdG in the myocardial tissue of salvianolate+I/R group was less than that of the I/R group. Conclusion: Salvianolate may alleviate myocardial I/R injury of rat through reducing the mitochondrial DNA oxidative damage, protecting mitochondrial function and inhibiting the apoptosis of myocardial cells.

Objective: To investigate the possible mechanism related to the protective effects of salvianolate in H9c2 cells underwent hypoxia/reoxygenation (H/R)-injury. Methods: H9c2 cells were divided into four groups: control group, salvianolate group (S group), H/R group, and salvianolate+ H/R group(S+ H/R group), in which the H9c2 cells were pretreated with salvianolate before H/R-treatment.Apoptotic cells were detected by Tunel assays and AnnexinⅤ-FITC apoptosis detection kit.The intracellular ATP level, the change of mitochondrial membrane potential and the mitochondrial DNA oxidative damage were also determined in these groups. Results: (1) The apoptosis rate of H/R group(26.36±5.14)% was significantly higher compared to control group(2.71±1.66)%( P =0.000 4), which could be significantly reduced in S+ H/R group(17.28±4.75)%( P =0.012 8 vs. H/R group , P =0.003 9 vs. control group). The ratio of AnnexinⅤ and PI double positive cells in H/R group(28.23±6.73)% was significantly higher compared to control group(3.53±2.83)%( P =0.001 1), which was significantly reduced in S+ H/R group(18.10±4.56)%( P =0.037 2 vs. H/R group, P =0.038 3 vs. control group). (2)The ATP level of H9c2 cells in H/R group(49.05±10.12)% was significantly lower than in control group 100%( P =0.000 5), which was significantly increased in S+ H/R group(68.67±13.32)%( P =0.019 9 vs. H/R group). Confocal microscope showed that red fluorescence was dominant in the control group, red fluorescence was significantly reduced, while green fluorescence was significantly increased in H9c2 cells of H/R group and the fluorescence ratio of red to green in H/R group((37.13±8.47)%) was significantly decreased compared to control group (100%, P =0.000 1), fluorescence ratio of red to green was significantly increased in S+ H/R group((63.77±12.32)% vs. H/R group, P =0.007 3). (3)The mitochondrial DNA oxidative damage in different groups: there was only few 8-hydroxyguanine (8-OHdG) expression, which marked as green, in control group, and 8-OHdG expression was significantly upregulated in H/R group, moreover, the 8-OHdG was co-localized with mitochondria.The expression of 8-OHdG was significantly lower in S+ H/R group compared to H/R group. Conclusion: Salvianolate can reduce mitochondrial DNA oxidative damage, and protect mitochondrial function, thus inhibit myocardial cell apoptosis and eventually reduce the myocardial H/R-injury in H9c2 cells.

Objective: To investigate the effects of lycopene on primary cultured neonatal mouse cardiomyocytes with hypoxia/reoxgenation (H/R) injury and explore related mechanism., Methods: Primary cultured neonatal mouse cardiomyocytes were randomly divided to control group (control); lycopene group (5 μmol/L, lyc); H/R group (4 hours hypoxia followed by 6 hours reoxgenation); lycopene+ H/R group (lyc+ H/R, the cardiomyocytes were incubated with 5 μmol/L lycopene for 4 hours before H/R treatment). The cell viability of cardiomyocytes was assessed by CCK-8 assay. The apoptotic rate of cardiomyocytes was evaluated by flow cytometry using AnnexinV-PI double staining. Western blot was used to determine the GRP78, CHOP, Bax and Bcl-2 protein expression in cardiomyocytes. The mRNA expressions of ATF6、eIF2α and sXbp-1 were detected by real-time PCR. The fluorescence intensity for reactive oxygen species (ROS) in cardiomyocytes was measured with Olympus fluorescence microscope., Results: Compared to control group, the cell viability of cardiomyocytes was significantly reduced ((64.28±6.12)% vs. (100.00±4.98)%, P<0.01), the apoptotic rate ((24.42±1.76)% vs. (5.16±1.31)%, P<0.01) and ratio of Bax/Bcl-2 (2.33±0.20 vs. 1.00±0.09, P<0.01) significantly increased, the ATF6, eIF2α and sXbp-1 mRNA expression, the CHOP and GRP78 protein expression (1.98±0.15 vs. 1.00±0.12, 2.09±0.11 vs. 1.00±0.09) as well as fluorescence intensity for ROS ((262.13±22.03)% vs. (100.00±12.35)%) were markedly increased in H/R group (all P<0.01). Compared to the H/R group, pretreatment with lycopene markedly improved the cell viability of cardiomyocytes ((81.75±6.85)%, P<0.01), significantly decreased the apoptotic rate ((17.24±2.02)%, P<0.01) and ratio of Bax/Bcl-2(1.64±0.13, P<0.01), significantly down-regulated the mRNA expression levels of ATF6, eIF2α and sXbp-1, and the protein expression levels of CHOP (1.54±0.12) and GRP78 (1.53±0.12), significantly reduced the fluorescence intensity for ROS ((171.18±19.09)%, all P<0.01)., Conclusions: Lycopene could attenuate hypoxia/reoxygenation-injury in primary cultured neonatal mouse cardiomyocytes, possibly through inhibiting the ER stress and alleviating the ER stress-induced apoptosis.

Objective: To investigate the possible mechanism of lycopene on protecting against hypoxia/reoxygenation (H/R)-injury., Methods: Primary cultured cardiomyocytes, isolated from neonatal mouse, were divided into three groups randomly: control group (C) ; H/R group(4 h H followed by 8 h R); lycopene+H/R group(L+H/R), in which the cardiomyocytes were pretreated with lycopene for 4 h before H/R. The survival of cardiomyocytes was counted. Apoptotic cells were detected by TUNEL assays. The release of cytochrome c from mitochondrial matrix into the cytosol, the activity of caspase-3, intracellular ROS levels and the activity of calpain were also determined in these groups respectively at the same time., Results: The pretreatment of cardiomyocytes with lycopene significantly improved the survival of cardiomyocytes [C: (89.84 ± 5.15)%, H/R: (63.59 ± 5.11)%, L+H/R: (79.25 ± 1.48)%, P < 0.05] and reduced the extent of apoptosis [C: ( 10.37 ± 1.25)%, H/R: (32.03 ± 4.79)%, L+H/R: (22.57 ± 3.22)%, P < 0.05], significantly reduced caspase-3 activation [C: (2.61 ± 0.19), H/R: (5.82 ± 0.92), L+H/R: (3.74 ± 0.64) pNA pmol/µg protein, P < 0.05]. To further study the mechanism underlying the benefits of lycopene, interactions between lycopene and calpain activation were examined. Lycopene pretreatment of cardiomyocytes suppressed the activation of calpain(C:272.33 ± 300.46, H/R: 1156.00 ± 212.02, L+H/R: 607.33 ± 166.23, P < 0.05) by reducing the H/R induced increased intracellular ROS levels [C: 100%, H/R: (239.79 ± 27.27)%, L+H/R: (188.19 ± 17.63)%, P < 0.05]., Conclusion: Lycopene may protect against hypoxia/reoxygenation-induced injury by preventing calpain activation.