| Background and objective As we known, myocardial reperfusion is the best effective in decreasing the infarct size and restoring cardiac function after acute ischemic events. However, ischemic tissue of the myocardium is susceptible to be further injury deriving from reperfusion. A variety of mechanisms of myocardial ischemia/reperfusion(I/R) injury, including cellular apoptosis, calcium overload, mitochondrial dysfunction and so on. Apoptosis participated in the enlargement of infarct size and heart failure. Apoptosis plays an important role in the pathogenesis of myocardial I/R injury. Previous studies have reported that endogenous substances soluble receptor for advanced glycation end products(s RAGE) can protect cardiomyocytes from apoptosis induced by I/R. However, the signaling mechanisms in cardioprotection by s RAGE are currently unknown. It has been reported that JAK2/STAT3 is related to I/R injury. The activated JAK2/STAT3 can protect against myocardial injury from I/R. Most important, the prior work suggested that s RAGE inhibits the reduction in protein level of phosphorylated STAT3. But the potential role of JAK2/STAT3 in the effect of s RAGE inhibition on I/R-induced myocardial apoptosis is unclear. The aims of the present study are to identify the effect of s RAGE on the I/R-induced myocardial apoptosis, detect the effect of s RAGE on JAK2 and STAT3 following I/R, and explore whether s RAGE inhibit apoptosis induced by I/R through JAK2/STAT3 activation.Methods we investigated the cardioprotective effect and potential molecularmechanisms of s RAGE inhibition on apoptosis in the mouse myocardial I/R as an in vivo model and neonatal rat cardiomyocyte subjected to ischemic buffer as an in vitro model. Cardiac function and myocardial infarct size following by I/R were evaluated with echocardiography and Evans blue/2, 3, 5-triphenyltetrazolium chloride. Apoptosis was detected by TUNEL staining and caspase-3 activity. Expression of the apoptosis-related proteins p53, Bax, Bcl-2, JAK2/p-JAK2, STAT3/p-STAT3, AKT/p-AKT, ERK/p-ERK, STAT5A/p-STAT5 A and STAT6/p-STAT6 were detected by western blot analysis. And then, to further test the concrete signaling pathways for inhibiting apoptosis, the TUNEL staining and caspase-3 activity were detected in in vitro with or without the JAK2 inhibitor AG 490, AKT inhibitor LY294002 and ERK inhibitor PD95089, respectively.Results 1. The effect of s RAGE on the cardiac function, myocardial infarct size and apoptosis following I/R in in vivo: Cardiac function: The EF and FS were significantly increased by 42% and 57% in I/R+s RAGE group compared to I/R group(p<0.05). Myocardial infarct size: The infarct size in mice were pretreated with s RAGE following I/R was significantly lowered compared to I/R group. This represents a significant 52% reduction of infarct size in IR+s RAGE group(p<0.05). Apoptosis: The number of TUNEL-positive cardiomyocytes、caspase-3 activity、p53 and the Bax/Bcl-2 ratio were significantly increased in I/R group compared to Sham group(p<0.05). However, the number of TUNEL-positive cardiomyocytes was markedly reduced by 66%、caspase-3 activity by 24%、p53 by 29% and ratio of Bax to Bcl-2 by 87% in I/R+s RAGE group compared with I/R group(p<0.05). 2. The effect of s RAGE on protein levels of apoptosis-related Compared with I/R group, the ratio of p-JAK2/JAK2 was significantly elevated by 92% in I/R+s RAGE group(p<0.05), the ratios of p-STAT3/STAT3 wassignificantly elevated by 281% and p-AKT/AKT was further increased by 31% in IR+s RAGE group(p<0.05). 3. The effect of s RAGE on I/R-induced myocardial apoptosis in the presence and absence of JAK2 inhibitor AG 490, AKT inhibitor LY294002 and ERK inhibitor PD95089 Compared with I/R+s RAGE group, the number of TUNEL-positive cardiomyocytes, caspase-3 activity, p53 protein level and the Bax/Bcl-2 ratio were markedly elevated in I/R+s RAGE+AG 490 group(p<0.05). The percentage of TUNEL-positive cardiomyocytes and the caspase-3 activity were significantly increased in I/R+s RAGE+LY294002 group compared with I/R+s RAGE group(p<0.05). But there was no difference in TUNEL-positive cardiomyocytes and caspase-3 activity between I/R+s RAGE+PD95089 group and I/R+s RAGE groups(p>0.05). 4.Test the concrete signaling pathways for s RAGE inhibits apoptosis: Compared with I/R group, p-JAK2 and p-STAT3 levels were significantly elevated by 26% and 156% in I/R+s RAGE group(p<0.05). Compared with I/R+s RAGE group, the levels of p-JAK2 and p-STAT3 were significantly decreased by 29% and 48% in I/R+s RAGE+AG 490 group(p<0.05). Compared with I/R group, the ratio of p-AKT/AKT was significantly increased by 38% in I/R+s RAGE group(p<0.05). Compared with I/R+s RAGE group, the ratio of p-AKT/AKT was reduced by 25% in I/R+s RAGE+AG 490 group(p<0.05). Compared with I/R group, the ratio of p-STAT3/STAT3 was significantly elevated by 150% in I/R+s RAGE group(p<0.05). There was no difference in the ratio of p-STAT3/STAT3 between I/R+s RAGE and I/R+s RAGE+LY294002 groups(p>0.05).Conclusion These results suggest that s RAGE protects cardiomyocytes fromapoptosis induced by I/R in vitro and in vivo by activating the JAK2/STAT3 signaling pathway.Background and objective As we known, myocardial reperfusion is the best effective in decreasing the infarct size and restoring cardiac function after acute ischemic events. However, ischemic tissue of the myocardium is susceptible to be further injuried deriving from reperfusion. The treatment and prognosis of coronary heart disease is restricted by ischemia/reperfusion(I/R) injury. It is no doubt that apoptosis plays an important role in the pathogenesis of myocardial I/R injury. Previous studies have reported that endogenous substances soluble receptor for advanced glycation end products(s RAGE) has the ability to protect against I/R injury through inhibiting myocardial apoptosis. But the mechanism of s RAGE-induced reduction in apoptosis caused by I/R is currently unknown. Prior work has demonstrated that the ubiquitin proteasome system(UPS) dysfunction is closely related to apoptosis. It has demonstrated that the protein expression of ubiquitin proteasome β-subunits were inhibited in response to STAT3 inhibitor. More interesting, s RAGE has the ability to inhibit the reduction on the protein expression of STAT3 induced by I/R. But the potential role of UPS in the effect of s RAGE inhibition on I/R-induced myocardial apoptosis is unclear. The aims of the present study are to identify the effect of s RAGE on the I/R-induced myocardial apoptosis, detect the effect of s RAGE on the activity and protein expression of UPS following I/R, and explore whethwe s RAGE inhibit apoptosis induced by I/R through UPS mediated STAT3.Methods Adult male C57 BL mice treated with or without s RAGE(100μg/day, i.p.) or saline were performed to ligate the left anterior descending coronary artery(LAD) as an in vivo model, and neonatal rat cardiomyocyte pretreated with or without s RAGE/s RAGE-containing adenovirus、UPS inhibitor Bortezimb and/or STAT3 inhibitor were subjected to ischemic buffer as an in vitro model. At the end of reperfusion, cardiac function and myocardial infarct size following by I/R were evaluated with echocardiography and Evans blue/2, 3, 5-triphenyltetrazolium chloride(TTC). The TUNEL and caspase-3 activity were detected to assess the myocardial apoptosis. In addition, the activity and expression of proteasome were detected with kit and western blot.Results 1.The effect of s RAGE on cardiac function, infarct size and myocardial apoptosis following I/R cardiac function:Compared with Sham group, EF and FS were significantly reduced in I/R group(p<0.05), but were significantly increased in I/R+s RAGE group compared to single I/R group(p<0.05). During the systolic period, compared with Sham group, the thickness of left ventricular anterior wall、left ventricular internal diameter、left ventricular volume were significantly increased in I/R and I/R+s RAGE group, and the thickness of left ventricular posterior wall in I/R group was elevated(p<0.05), but there were no difference between the two groups(p>0.05). During the diastolic period, there were no difference among the Sham、I/R+s RAGE and I/R groups(p>0.05). Infarct size: The infarct size in mice were pretreated with s RAGE following I/R was lowered compared to I/R alone group(p<0.05). Myocaidial apoptosis: Compared with I/R group, the number of TUNEL-positive cardiomyocytes and caspase-3 activity were significiantly reduced in I/R+s RAGEgroup(p<0.05). 2. The effect of s RAGE on the level of total ubiquitinated proteins, the activity and protein expression of UPS: The caspase-like, trypsin-like, chymotrypsin-like proteolytic activities of the proteasome were significiantly decreased in I/R group compared with Sham group, accompanied by ubiquitinated proteins level significiantly increased compared with Sham group(p<0.05). Howerer, the caspase-like, trypsin-like, chymotrypsin-like proteolytic activities of the proteasome were significiantly increased in I/R+s RAGE group compared with I/R alone group, and then the ubiquitinated proteins level was significiantly reduced in I/R+s RAGE group compared with I/R alone group(p<0.05). Compared with Sham group, the β1i and β5i protein levels were significiantly decreased in I/R alone group(p<0.05). Meanwhile, the β1 and β5 protein levels were significiantly reduced in I/R group compared with Sham group(p<0.05). However, β1i and β5i protein levels were significiantly increased in I/R+s RAGE group compared with I/R alone group(p<0.05). Otherwise, s RAGE-treated decreased the β5 protein level compared with I/R alone group(p<0.05). The protein level of constitutively and inducible β-subunits were detected in vivo, and the result indicated that s RAGE can significantly increase the expression of β1i and β5i, so we further test the protein expression of β1i and β5i in the presence and absence of s RAGE. Compared with the Con+GFP group, the β1i and β5i protein levels were significiantly reduced in I/R+GFP group(p<0.05). Compared with the I/R+GFP group, the β1i and β5i protein levels were significiantly increased in I/R+Ad-s RAGE group(p<0.05). 3. The effect of s RAGE on the UPS activity and myocardial apoptosis in the presence and absence of UPS inhibitor Bortezimb Compared with I/R+s RAGE group, the caspase-like, trypsin-like, chymotrypsin-like proteolytic activities of the proteasome were significiantlydecreased, the number of TUNEL-positive cardiomyocytes and caspase-3 activity were significiantly increased in I/R+s RAGE+BTZ group(p<0.05). 4. The effect of s RAGE on myocardial apoptosis 、β1i and β5i protein expression in the presence and absence of STAT3 inhibitor: Apoptosis: The number of TUNEL-positive cardiomyocytes and the caspase-3 activity were significiantly reduced in I/R+GFP group compared to I/R+Ad-s RAGE group(p<0.05), and then, both the number of TUNEL-positive cardiomyocytes and the caspase-3 activity were significiantly increased in I/R+Ad-s RAGE+S3I-201 compared to I/R+Ad-s RAGE group(p<0.05). In addition, the number of TUNEL-positive cardiomyocytes and the caspase-3 activity were also significiantly increased in I/R+Ad-s RAGE+AG 490 compared to I/R+Ad-s RAGE group(p<0.05). The protein levels of β1i and β5i were significiantly increased in I/R+s RAGE/Ad-s RAGE group compared to I/R+GFP group(p<0.05). Compared with I/R+s RAGE/Ad-s RAGE group, the protein levels of β1i and β5i were significiantly reduced in both I/R+s RAGE+AG 490 and I/R+Ad-s RAGE+S3I-201 groups(p<0.05).Conclusion Our data suggest that the effect of s RAGE inhibition on apoptosis induced by I/R apart from activating STAT3 to regulate the activity and protein expression of UPS. We predict that s RAGE is a novel intervention to target UPS activation for preventing and treating myocardial apoptosis. |
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