The Cardioprotection Effects Of GSK-3β Inhibitor On Myocardial Ischemia/reperfusion Injury And Its Underlying Mechanism In Rats

BackgroundGrowing evidence from both animal experiments and clinical observations indicates that inflammatory response and cytokine production are particularly active after myocardial infarction and contribute to cardiac function and eventual host outcome. Cytokines such as TNF-αreleased after myocardial ischemic injury can acutely regulate myocyte survival or deaths and trigger subsequent cellular inflammatory response. Many studies indicated that p38MAPK and NF-κB are more important translation signaling, they induced the inflammation cytokines. So the drugs which decrease the active of p38MAPK and NF-κB signaling can protects the heart from acute ischemia-reperfusion injury via inhibition of inflammation and apoptosis.Recently, the acute phase of I-R has been viewed as part of the innate immune response, with a lack of vascular perfusion and oxygenation. These non-infectious stimuli contribute to the inflammatory response, in part by signaling via Toll-like receptors (TLRs)-mediated pathways. The identification of TLRs on cardiomyocytes not only has brought new insights on the inflammatory response initiated by cardiomyocytes themselves, but also offered potential targets to reduce I-R injury. Till date, there are no other studies on the role of TLR2 in MI-R injury documented. It has not been reported until now whether drug administration could modulate TLR2 /NF-κB signaling following MI-R injury in rats.Introduction of reperfusion therapy has markedly improved prognosis of patients with acute myocardial infarction (AMI). However, current reperfusion therapy cannot afford sufficient myocardial salvage in approximately 25% of patients, who subsequently develop severe heart failure. Thus, there is a clinical need for novel therapy to protect cardiomyocytes from ischemia/ reperfusion-induced necrosis. Many drugs which lead to myocardial protection from infarction induce Ser 9 -phosphorylation of GSK-3βand regulate of mPTP opening. Several reports have shown that chemical inhibitors of GSK-3βcan reduce the levels of pro-inflammatory cytokines in acute systemic inflammation. However, it is not known whether the GSK-3βinhibitor TDZD-8-elicited anti-inflammatory property contributes to the cardioprotective and prosurvival effects, if so, what the molecular mechanism is.Objectives1. To determine whether treatment with TDZD-8 may inhibit inflammation cytokines induction and reduce acute inflammatory response in MI/R.2. If so, to investigate the possible mechanism(s) involved in TDZD-8-induced inflammation cytokines induction in MI/R.3. To determine the modulatory role of TDZD-8 on TLR2/NF-κB signaling following MI-R injury in rats.4. To elucidate whether the anti-inflammatory and anti-apoptosis effect afforded by TDZD-8 may contribute to its cardio-protection in MI/R.Methods1. Rats were anesthetized and myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 6-0 silk and making a slipknot around the left anterior descending coronary artery. After 30 minutes of ischemia, the slipknot was released and the myocardium was reperfused for 6 hours. The administration of different concentration TDZD-8 (from 0.1 mg/kg to 1 mg/kg intravenous bolus) was gave 5 min prior to surgical procedures. Hemodynamic data were continuously monitored on a polygraph (RM-6200C) and simultaneously digitized by using a computer interfaced with an analog-to-digital converter. Blood samples were drawn from caudal vein before ischemia, 30 minutes after ischemia and 2/6 hours after reperfusion respectively to measure blood glucose levels. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured spectrophotometrically (Beckman DU 640, USA) at 6 hours after reperfusion.2. The animal model was the same as that in the first part. Rats were received DMSO or TDZD-8 (1 mg/kg intravenous bolus) 5 min prior to surgical procedures. After 6h reperfusion, the myocardial infarct size was determined by means of a double-staining technique and was analyzed by digital imaging systems. Evan's blue stained area, TTC stained area and TTC stained negative area were measured digitally using Image Pro Plus software. Myocardial apoptotic index was analyzed by TUNEL assay. A double-staining technique was used, i.e., TUNEL staining for apoptotic cell nuclei and DAPI staining for all myocardial cell nuclei. Myocardial TNF-αand IL-6 were detected using the enzyme-linked immunosorbent assay (ELISA) kits. Myocardial myeloperoxidase (MPO) was also deteced at the end of reperfusion. NF-κBp65, p38MAPK and GSK-3βSer9 phosphorylation were determined by western blot at the end of 30 min reperfusion.3. The animal model was the same as that in the first part. Real-time reverse transcriptase- polymerase chain reaction (RT-PCR), and immunohistochemist -ry (IHC) were used to analyze the presence and quantity of TLR2 mRNA and protein. Rats were received TDZD-8 (1 mg/kg) 5 min prior to surgical procedures. TNF-αmRNA and IL-6 mRNA were analyzed by RT-PCR. The activation of NF-κB was detected by western blot. The relation of TLR2 with NF-κB and its downstream cytokines was analysised. Myocardial infarct size was assessed by Evans blue-TTC staining.Results1. Low and high concentration of TDZD-8 did not change the level of blood glucose and heart rate, blood pressure before and after I-R in all study groups.±dp/dtmax decreased in MI/R group and Low concentration of TDZD-8 after 2h and 6h of reperfusion compared with sham group (P<0.05). High concentration of TDZD-8 increased±dp/dtmax compared with MI/R group (P<0.05). 2. MI/R resulted in a significant increase in TUNEL-positive nuclei (AI: 22±2% vs. sham groups, p <0.05) Administration of TDZD-8 caused a significant reduction in AI compared with the MI-R group (12±1%, p < 0.05). The infarct size (IS)/AAR ratio was about 58.64±9.45% in MI-R group. However, the IS/AAR ratio was reduced in TDZD-8 animals (34.42±8.65% of AAR) by nearly 43% compared with MI-R animals (p < 0.05). Administration of TDZD-8 alone did not affect the apoptosis and infarct size.3. Following 6h reperfusion, the activity of MPO was significantly elevated after MI-R when compared to the sham groups (p < 0.05). In the MI-R group, reperfusion resulted in a noticeable increase in cardiac TNF-αcontent compared with the sham groups (165.5±9.6 pg/mg protein, p < 0.05). The administration of TDZD-8 caused a significant attenuation of cardiac TNF-αcontent (107.4±7.3pg/mg protein vs. MI-R group, p <0.05). The same trend was observed in the levels of IL-6. Compared with the sham groups, MI-R-mediated injury significantly increased IL-6 levels (44.7±7.5 pg/mg protein, p <0.05), while TDZD-8 administration significantly decreased (29.28±6.3 pg/mg protein vs. MI-R group, p <0.05).4. Consistent with the levels of p-NF-κB p65 and p-p38MAPK protein resulted in 3.4-fold and 4-fold increase, but decrease in GSK-3βSer9 phosphorylation in myocardium in I/R rats. Treatment with TDZD-8 resulted in the decrease of p-NF-κB p65 and p-p38MAPK protein, and increased the p-GSK-3βSer9.5. After reperfusion, TLR2 mRNA expression was greater and increased over time. At 1, 6, 12 and 24h reperfusion, expression of the TLR2 gene was 2.1-, 2.9- , 4.7- and 3.5-fold higher compared to the sham group (p<0.001), respectively. IHC staining confirmed the expression of TLR2 occurred mainly in cardiac myocytes. In cardiac sections adjacent to the site of ischemic injury, enhanced and predominantly sarcolemmal staining of TLR2 was observed. Scattered foci of intense TLR2 staining involving 2 or more contiguous myocytes were noted in the I-R tissue.6. To investigate whether the functional effects of TLR2 are associated with pro-inflammatory cytokine production in MI-R, the levels of tissue TNF-αand IL-6 mRNA were measured after 1 h reperfusion. These mRNA levels were significantly increased after 1h reperfusion (TLR2: 2.1±0.1-fold higher than sham; TNF-α: 4.5±0.4-fold higher than sham, and IL-6: 4.3±0.4-fold higher than sham, p<0.05). The mRNA levels of TNF-αand IL-6 were positively correlated with TLR2 mRNA levels after 1 h reperfusion. (TLR2 vs. TNF-αmRNA: r=0.65, p<0.05; TLR2 vs. IL-6 mRNA: r=0.68, p<0.05). Treatment with TDZD-8 had a significant effect on the mRNA levels of TLR2, TNF-αand IL-6 compared with MI-R (2.1±0.1 to 1.5±0.1; 4.5±0.4 to 3.0±0.5; 4.3±0.4 to 2.7±0.3; respectively, p<0.05). There was a significant correlation between TLR2 mRNA levels and the levels of phospho- NF-κB p65 protein after 1h reperfusion. (TLR2 mRNA vs. p- NF-κB p65: r=0.89, p<0.001).The levels of phospho-NF-κB p65 protein were reduced in the nuclear fractions of the hearts of animals that received TDZD-8 (p < 0.05 vs MI-R group). The ratio of the infarct size (IS)/AAR was reduced in the animals treated with TDZD-8 by nearly 33% compared with the MI-R animals (p<0.05). Conclusions1. We have demonstrated that GSK-3βinhibitor TDZD-8 reduced myocardial infarct size, decreased neutrophil infiltration and suppressed NF-κB and p38 MAPK activation and associated proinflammatory cytokine expression, and reduced myocardial apoptosis in a rat model of MI-R through increasing the phospho- GSK-3βSer9 in MI/R.2. The functional effects of TLR2 are associated with pro-inflammatory cytokine production in MI-R and GSK-3βinhibitor reduced TLR2/ NF-κB signaling following MI/R.3. The anti-inflammatory property elicited by GSK-3βinhibitor may contribute to its cardioprotective effects in I/R injury and inflammatory related illness.