Obestatin Plays Beneficial Role in Cardiomyocyte Injury Induced by Ischemia-Reperfusion In Vivo and In Vitro

Abstract

BACKGROUND Obestatin, primarily recognized as a peptide within the gastrointestinal system, has been shown to benefit the cardiovascular system. We designed this experiment to study the protective role and underlying mechanism of obestatin against ischemia-reperfusion(I/R) injury in myocardial cells. MATERIAL AND METHODS In an In vivo experiment, LAD was ligated for 0.5 h and then opened for reperfusion with obestatin for 24 h. Then, the infarction area was shown with TTC staining, and inflammation factors in serum were analyzed by qRT-PCR. In primary cultured cardiomyocytes, we measured the level of LDH, MDA, GSH, and SOD. Finally, we assessed cells apoptosis using flow cytometry and detected the concentrations of caspase-3, Bax, and Bcl-2 using Western blot analysis. RESULTS TTC staining showed that in the 3 obestatin groups, the infarct area became smaller with the increase of obestatin concentration. Obestatin also inhibited LDH expression in rat serum and decreased mRNA levels of TNF-α, IL-6, ICAM-1, and iNOS in rat cardiomyocytes after reperfusion. In primary cultured cardiomyocytes, obestatin decreased LDH content and increased GSH level after I/R injury. Obestatin was also found to antagonize the apoptosis of cardiomyocytes in a dose-dependent manner. Western blot analysis showed that obestatin downregulated the expression of caspase-3 and Bax and upregulated the expression of Bcl-2. CONCLUSIONS Obestatin can protect cardiomyocyte from I/R-induced injury in vitro and in vivo. This beneficial effect is closely related with its properties of anti-inflammation, anti-cytotoxicity, and anti-apoptosis. The protective effect of obestatin might be associated with activation of Bcl-2 and inhibition of caspase-3 and Bax.

Figure 1

(A) The difference in survival between model group and obestatin groups. We used log rank analysis to compare the survival difference between the various groups, especially the model group and the 3 obestatin groups. Combined with the data in Table 2, we found survival time was significantly shorter in the model group than in the control group. Obestatin treatment prolonged the survival time and decreased mortality rate, and this improvement was significant in the obestatin 50 nM/kg group. (B) Infarct area of rat heart was reduced by treatment with obestatin. In the model group, infarction size increased significantly compared to the control group, but in the obestatin reperfusion groups, infarction size gradually decreased compared to the model group and this reduction was most obvious when obestatin reached 50 nM/kg. * p<0.05 compared with model, ^### p<0.001 compared with control.

Figure 2

Obestatin lowered LDH concentration in rat serum after reperfusion. I/R injury induced a significant increase of LDH level, but the concentration of LDH decreased in the obestatin pretreatment groups, and when the obestatin dose reached 50 nM/Kg, it showed a significant decrease of LDH concentration. * p<0.05 compared with model, ^## p<0.01 compared with control.

Figure 3

Obestatin lowered mRNA expression of inflammation factors caused by I/R injury. mRNA levels of IL-6, IL-1β, TNF-α, ICAM-1, and iNOS were all elevated in the model group. Obestatin decreased the concentration of IL-6, TNF-α, ICAM-1, and iNOS, and this decrease was generally dose-dependent. In IL-6 and ICAM-1, 25 nM/Kg of obestatin significantly decreased the mRNA level, and in TNF-α and iNOS, 50 nM/Kg of obestatin reached statistical significance. * p<0.05 compared with model, ^## p<0.01 compared with control, ^### p<0.001 compared with control.

Figure 4

Determination of working concentration of obestatin. Cultured cardiomyocytes were treated with various concentrations of obestatin, from 10⁻¹⁰ M to 10⁻⁵ M. At the concentration of 10⁻⁵ M, obestatin significantly damaged cells. According to the results from the histogram, the maximum concentration of obestatin we chose for the following experiments was 10⁻⁸ M. ** P<0.01 compared with PBS group.

Figure 5

Obestatin changed the level of some cytokines related with oxidative stress in cultured cardiomyocytes. I/R injury significantly increased the level of LDH and MDA and decreased the concentration of GSH and SOD. Obestatin (10 nM) pretreatment exerted its anti-oxidative stress effect by promoting the expression of GSH and inhibiting the expression of LDH. In MDA and SOD, obestatin showed the tendency of decreasing the MDA level and increasing the SOD level, although without statistical significance. * p<0.05 compared with model, ^# p<0.05 compared with control, ^## p<0.01 compared with control, ^### p<0.001 compared with control.

Figure 6

Obestatin blocked apoptosis of cardiomyocytes induced by I/R. Ischemia-reperfusion injury promoted the apoptosis of cells, while obestatin blocked this phenomenon. This blockage emerged with a dose-dependent tendency, especially in the obestatin 10 nM group. A) Blank control group; B) Cardiomyocytes with I/R injury alone; C–E) Cardiomyocytes pretreated with obestatin (1, 3, and 10 nM, respectively) followed by the I/R injury. * p<0.05 compared with model, ^### p<0.001 compared with control.

Figure 7

Proteins levels of active caspase-3, Bcl-2, and Bax in treated cardiomyocytes were quantified via normalization to GAPDH. Obestatin decreased the level of active caspase-3 and Bax, and increased the level of Bcl-2. According to the results of densitometric analysis, this change showed a positive correlation with obestatin concentration. When obestatin concentration reached 10 nM, the inhibition or promotion had statistical significance. * p<0.05 compared with model, ^# p<0.05 compared with control, ^## p<0.01 compared with control.