Exogenous H 2 S donor GYY4137 enabled SPC to attenuate myocardial IRI-induced cardiac infarction size and cardiac dysfunction in diabetic rats but was abolished by SIRT1 inhibition
The characteristics of rats acquired in the experimental animals are shown in Fig. 1b-e. Compared with age-matched control rats, the type 1 diabetic rats after 4 weeks of STZ-induced diabetes showed characteristic symptoms of diabetes including polyphagia, polydipsia and hyperglycemia, as evident by increased food consumption, water intake and blood glucose levels (Fig. 1b-d, P < 0.05). In addition to these results, the levels of H2S in plasma were shown in Fig. 1e. The levels of H2S in I/R group and SPC group were significantly more than those in the Sham group, and the myocardial levels of H2S in H group, SPC + H group and SPC + H + EX527 group were further enhanced when compared with I/R group and SPC group (Fig. 1e, P < 0.05).
As shown in Fig. 2, the marker enzymes associated with infarction size and myocardial injury were measured to confirm the cardioprotective effect of SPC on myocardial diabetic IRI. The results showed that the myocardial infarction area was significantly increased in other groups when compared to Sham group (Fig. 2a-b, P < 0.05). Compared with I/R group, myocardial infarction area in diabetic rats treated with SPC showed no significant changes (Fig. 2a-b, P > 0.05), while myocardial infarction area of diabetic rats treated with exogenous H2S donor GYY4137 showed significant reduction (Fig. 2a-b, P < 0.05). After administration of SPC and exogenous H2S donor GYY4137, myocardial infarction area of diabetic rats in SPC + H group was further decreased (Fig. 2a-b, P < 0.05). However, the hearts of diabetic rats were treated after SPC by combining GYY4137 and SIRT1 signal pathway inhibitor EX527 in SPC + H + EX527 group. The results revealed that the myocardial infarction area of diabetic rats was remarkably increased (Fig. 2a-b, P < 0.05). Meanwhile, the HW and BW of diabetic rats were estimated. The BW in all groups showed no significant differences (Fig. 2c, P > 0.05). The differences of HW and HW/BW in all groups showed a similar trend with that of myocardial infarction area. The HW and HW/BW showed significant elevation in other groups when compared to Sham group (Fig. 2d-e, P < 0.05). The HW and HW/BW showed no significant differences between I/R group and SPC group (Fig. 2d-e, P > 0.05). Compared with SPC group, the HW and HW/BW were found to be significantly reduced in H group, and further reduced in SPC + H group (Fig. 2d-e, P < 0.05). But the heart weight and HW/BW in SPC + H + EX527 group were obviously elevated as compared to SPC + H group (Fig. 2d-e, P < 0.05).
Simultaneously, the myocardial CK-MB, LDH and cTnl levels of diabetic rats were also detected. As shown in Fig. 2f-h, the CK-MB, LDH and cTnl levels of diabetic rats in other groups were obviously higher than those in Sham group (Fig. 2f-h, P < 0.05). Compared with I/R group, the CK-MB, LDH and cTnl levels of diabetic rats in SPC group showed no significant reduction (Fig. 2f-h, P > 0.05), while CK-MB, LDH and cTnl levels were significantly decreased in H group, and further inhibited in SPC + H group (Fig. 2f-h, P < 0.05). Nonetheless, after treatment with SPC, GYY4137 and EX527, the CK-MB, LDH and cTnl levels in SPC + H + EX527 group were significantly elevated when compared with SPC + H group (Fig. 2f-h, P < 0.05).
To further confirm the effects of GYY4137 on myocardial diabetic IRI and whether SIRT1 was involved in the cardioprotective effects of SPC and H2S, cardiac function of all groups was detected by echocardiography and the representative M-mode echocardiograms of all groups were presented in Fig. 3a. Left ventricular systolic function was reflected by EF%, FS%, SV, LVIDs and LVIDd, left ventricular diastolic function was reflected by LVPWs and LVPWd, and cardiac remodeling function was observed by IVSs and IVSd. As shown in Fig. 3b-j, the results showed that EF%, FS%, SV, LVPWs, LVPWd, IVSs, IVSd were significantly lowered, while LVIDs, LVIDd were significantly increased in other groups than in Sham group (Fig. 3b-j, P < 0.05). These cardiac function indexes showed no statistical differences between I/R group and SPC group (Fig. 3b-j, P > 0.05), while SPC treatment did not improve cardiac function of diabetic rats. Compared with SPC group, EF%, FS%, SV, LVPWs, LVPWd, IVSs, and IVSd were significantly increased in H group, and further elevated in SPC + H group, while LVIDs and LVIDd were significantly decreased in H group, and further declined in SPC + H group (Fig. 3b-j, P < 0.05). In contrast, EF%, FS%, SV, LVPWs, LVPWd, IVSs, and IVSd in SPC + H + EX527 group were obviously inhibited as compared with SPC + H group, and LVIDs and LVIDd in SPC + H + EX527 group showed significant improvement as compared with SPC + H group (Fig. 3b-j, P < 0.05).
Effects of SPC on myocardial damage and fibrosis in GYY4137 Treated Diabetic Rats with or without EX527 Treatment
As shown in Fig. 4, the area of fibrosis fraction and leucocyte infiltration were measured by histological analysis. HE and Masson staining showed that the structure of myocardial tissue in Sham group remained clear, the cardiomyocytes were arranged neatly, and no fibrosis fraction and leucocyte infiltration were observed. In I/R group, the cardiomyocytes were arranged in disordered manner, and enlarged. A mass of fibrosis fraction and leucocyte infiltration were observed in I/R group when compared to Sham group (Fig. 4, P < 0.05). After SPC administration, the area of fibrosis fraction and leucocyte infiltration of diabetic rats in the myocardium o SPC group showed no significant reduction when compared to I/R group (Fig. 4, P > 0.05), and also SPC did not improve myocardial histological changes in diabetic rats after I/R. Nonetheless, after treatment with exogenous H2S donor GYY4137, the myocardial tissue showed a clearer structure, lighter myocardial damage, and lower area of fibrosis fraction and leucocyte infiltration in H group when compared to I/R group (Fig. 4, P < 0.05). Also the area of fibrosis fraction and leucocyte infiltration in the myocardium of diabetic rats in SPC + H group were further reduced when compared to I/R group (Fig. 4, P < 0.05). However, the myocardial tissue structure in SPC + H + EX527 group was worsened after administration of SIRT1 signaling pathway inhibitor EX527, while the myocardial area of fibrosis fraction and leucocyte infiltration of diabetic rats in SPC + H + EX527 group was significantly increased when compared with SPC + H group (Fig. 4, P < 0.05).
Effects of SPC on mitochondrial structure and function in GYY4137 Treated Diabetic Rats with or without EX527 Treatment
SIRT1 is an important gene in mitochondrial biogenesis and function in the cardiomyocytes of rodents [24]. To further indicate that exogenous H2S restores SPC-induced cardioprotection via maintaining normal mitochondrial structure and function, myocardial ultrastructural structure, myocardial ATP content and mitochondrial respiratory chain complex I-IV in all groups were detected. As shown in Fig. 5a, cardiomyocytes in Sham group appeared in well-arranged sarcomeres and intercalated disc manner, as well as normal mitochondria with no swelling and intact cristae density. Consistent with the previous studies, the ultrastructural damages were worsened in I/R group and SPC group. After ischemic reperfusion, cardiac muscle tissue showed absence and edematous separation of sarcomeres, vacuolation of mitochondria with a more pronounced derangement and mitochondrial membrane and cristae disruption. Compared with I/R group, SPC treatment did not decrease myocardial ultrastructural damages in SPC group, while exogenous H2S donor GYY4137 inhibited myocardial ultrastructural damages in H group and combined application of SPC and GYY4137 further reduced the ultrastructural damages in SPC + H group, showing relatively parallel arrangement of sarcomeres and normal structure of the mitochondria. However, after SIRT1 signal pathway inhibitor EX527 treatment, myocardial ultrastructural was found to be obviously deteriorated in SPC + H + EX527 group (Fig. 5a).
Moreover, myocardial ATP content and mitochondrial respiratory chain complex I-IV in all groups demonstrated similar trend. The myocardial ATP content and complex I-IV activities were shown to be significantly decreased in other groups when compared to those in Sham group (Fig. 5b-f, P < 0.05). Compared with I/R group, myocardial ATP content and complex I-IV activities of diabetic rats in SPC group showed no significant increase (Fig. 5b-f, P > 0.05), while myocardial ATP content and complex I-IV activities were markedly increased in H group, and further elevated in SPC + H group (Fig. 5b-f, P < 0.05). Nonetheless, after combining the utilization of SPC, GYY4137 and EX527, myocardial ATP content and complex I-IV activities in SPC + H + EX527 group were markedly decreased as compared to SPC + H group (Fig. 5b-f, P < 0.05).
SIRT1 resisted IRI-induced oxidative stress participates in restoring the effect of H2S to SPC cardioprotection in diabetic rats
Abnormal mitochondrial structure and functioning along with oxidative damage were induced by overproduction of ROS in rodents with diabetic hearts [25]. Next, immunohistochemistry was employed in experimental groups to investigate the levels of MPO, MDA and SOD after IRI insult of SPC treatment with GYY4137 in diabetic rats, and whether these effects were affected by SIRT1 inhibitor EX527. As shown in Fig. 6a-b, the relative 4-HNE levels in diabetic rats in other groups were significantly higher than in Sham group (Fig. 6b, P < 0.05). Compared with I/R group, the relative 4-HNE levels in SPC group showed no significant changes (Fig. 6b, P > 0.05), while the relative 4-HNE levels were obviously reduced in H group, and further decreased in SPC + H group (Fig. 6b, P < 0.05). Nonetheless, after treatment with SPC, GYY4137 and EX527, the relative 4-HNE levels in SPC + H + EX527 group were significantly enhanced as compared to SPC + H group (Fig. 6b, P < 0.05).
In addition, the biochemical markers of oxidative stress in all groups were detected and showed in Fig. 6c-e. The levels of MPO and MDA were significantly higher and the levels of SOD were obviously lower in other groups when compared to Sham group (Fig. 6c-e, P < 0.05). The levels of MPO, MDA and SOD showed no significant differences between I/R group and SPC group (Fig. 6c-e, P > 0.05), but the levels of biochemical markers in oxidative stress showed no change after SPC treatment. Compared with I/R group, the levels of MPO and MDA were significantly inhibited and the levels of SOD were significantly enhanced in H group, and the levels of MPO and MDA were further inhibited and the levels of SOD were further enhanced in SPC + H group (Fig. 6c-e, P < 0.05). However, after SPC treatment, GYY4137 combined with SIRT1 signaling pathway inhibitor EX527 was used for treatment in SPC + H + EX527 group, and the levels of MPO and MDA were significantly enhanced and the levels of SOD were remarkably inhibited when compared to SPC + H group (Fig. 6c-e, P < 0.05).
Exogenous H2S restored SPC-induced cardioprotection through SIRT1-regulated Nrf2/HO-1/Nox-2 signaling pathway activation in diabetic heart IRI
To further determine the effects of exogenous H2S restoration in SPC-induced cardioprotection in diabetic hearts, the protein expression levels of SIRT1-regulated Nrf2/HO-1/Nox-2 signaling pathway were measured by western blotting. As shown in Fig. 7a-e, the expression levels of cytoplasmic fraction SIRT1, Nrf2 and HO-1 were obviously down-regulated and the expression levels of cytoplasmic fraction Nox-2 were significantly up-regulated in I/R group and SPC group when compared to Sham group (Fig. 7a-e, P < 0.05). In addition, compared to I/R group, the expression levels of cytoplasmic fraction SIRT1, Nrf2, HO-1 and Nox-2 in SPC group showed no significant change (Fig. 7, P > 0.05), while the expression levels of cytoplasmic fraction SIRT1, Nrf2, and HO-1 were obviously up-regulated and the expression levels of cytoplasmic fraction Nox-2 was significantly down-regulated in H group (Fig. 7a-e, P < 0.05), and the expression levels of cytoplasmic fraction SIRT1, Nrf2, and HO-1 were further up-regulated and the expression level of cytoplasmic fraction Nox-2 was further down-regulated in SPC + H group (Fig. 7a-e, P < 0.05). In contrast, diabetic rat hearts underwent treatment with GYY4137 combined and SIRT1 signaling pathway inhibitor EX527 after SPC treatment in SPC + H + EX527 group, and the expression levels of cytoplasmic fraction SIRT1, Nrf2, and HO-1 were significantly down-regulated and the expression levels of cytoplasmic fraction Nox-2 were significantly up-regulated (Fig. 7a-e, P < 0.05).
Simultaneously, the expression levels of nuclear fraction SIRT1 and Nrf2 were measured by western blotting and the results showed a similar trend as that of cytoplasmic fraction. As shown in Fig. 7f-h, compared with Sham group, the expression levels of nuclear fractions SIRT1 and Nrf2 were shown to be significantly down-regulated in I/R and SPC groups (Fig. 7f-h, P < 0.05). In addition, compared to I/R group, the expression levels of nuclear fraction SIRT1 and Nrf2 in SPC group showed significant up-regulation (Fig. 7f-h, P > 0.05), while the expression levels of nuclear fractions SIRT1 and Nrf2 were obviously up-regulated in H group (Fig. 7f-h, P < 0.05), and the expression levels of nuclear fractions SIRT1 and Nrf2 were further up-regulated in SPC + H group (Fig. 7f-h, P < 0.05). In contrary, diabetic rat hearts were treated with GYY4137 and SIRT1 signaling pathway inhibitor EX527 after SPC treatment in SPC + H + EX527 group, and the expression levels of nuclear fractions SIRT1 and Nrf2 were remarkably down-regulated (Fig. 7f-h, P < 0.05). Our study results showed that the expression and distribution of SIRT1and Nrf2 demonstrated apparent changes after SPC, GYY4137 and EX527 administration. In SPC + H + EX527 group, Nrf2 was promoted entry into the nucleus, resulting in the downstream expression of antioxidant HO-1/Nox-2 pathway changes, and SPC mediated cardioprotection in diabetic rats were finally restored.