Weipan Xu

Beta-1-adrenergic receptors mediate Nrf2-HO-1-HMGB1 axis regulation to attenuate hypoxia/reoxygenation-induced cardiomyocytes injury in vitro.

Backgroud/Aims: The aim of the study was to evaluate the effects of beta1-adrenergic receptors (β1-ARs) -mediated nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1)-high mobility group box 1 protein (HMGB1) axis regulation in hypoxia/reoxygenation (H/R)-induced neonatal rat cardiomyocytes. Methods: The neonatal cultured cardiomyocytes were concentration-dependently pretreated by dobutamine (DOB), a selective β1-adrenergic receptor agonist, in the absence and/or presence of LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), SB203580 (a p38mitogen-activated-protein kinase (p38MAPK) inhibitor), Nrf2siRNA and HO-1siRNA, respectively, and then treated by H/R. The effects and mechanisms by which H/R-induced cardiomyocytes injury were evaluated. Results: Significant increase of HO-1 was found in neonatal cultured cardiomyocytes treated with DOB, when compared to the control group. Significant change for Nrf2 translocation was also revealed in neonatal cultured cardiomyocytes treated with DOB. Insignificant decreases of NF-kappaB p65 activation and HMGB1 release were observed in H/R-induced neonatal cultured cardiomyocytes treated with DOB, when compared to the control group. Importantly, DOB treatment significantly increased the cell viability and decreased the levels of LDH and MDA in H/R-induced cardiomyocytes injury. However, DOB failed to increase HO-1, inhibit NF-kappaB p65 activation, prevent HMGB1 release and attenuate H/R-induced cardiomyocytes injury when the cultured cardiomyocytes were pretreated by Nrf2siRNA, HO-1siRNA, PI3K inhibitor (LY294002) and p38MAPK inhibitor (SB203580), respectively. Conclusions: β1-ARs-mediated Nrf2-HO-1-HMGB1 axis regulation plays a critical protective role in H/R-induced neonatal rat cardiomyocytes injury in vitro via PI3K/p38MAPK signaling pathway.

Exogenous High-Mobility Group Box 1 Protein Injection Improves Cardiac Function after Myocardial Infarction: Involvement of Wnt Signaling Activation

Exogenous high-mobility group box 1 protein (HMGB1) injection could prevent left ventricular remodeling and enhance left ventricular function during myocardial infarction (MI). However, the mechanism remains unclear. This paper was to investigate in the mechanism of cardioprotection of HMGB1 during MI in rats. Anesthetized male rats were treated once with HMGB1 (200 ng) 4 h after MI and then executed after 7 and 28 days, respectively. Cardiac function, collagen deposition, and dishevelled-1 and β-catenin protein expression were measured. After MI 7 days or 28 days, the left ventricular ejection fraction (LVEF) was significantly decreased compared to that of sham-operated control group (P < 0.05). However, the LVEF HMGB1-treated groups were significantly higher compared to those of the MI group in both 7 days and 28 days (P < 0.05). The collagen volume fraction was significantly reduced in the HMGB1-treated group in infarcted border zone. HMGB1 could activate the expression of dishevelled-1 and β-catenin proteins (P < 0.05). Our study suggested that exogenous high-mobility group box 1 protein injection improves cardiac function after MI, which may be involved in Wnt/β-catenin signaling activation.

Short-Term Hesperidin Pretreatment Attenuates Rat Myocardial Ischemia/Reperfusion Injury by Inhibiting High Mobility Group Box 1 Protein Expression via the PI3K/Akt Pathway.

Background/Aims: Hesperidin pretreatment has been shown to protect against myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism is poorly understood. This study aimed to investigate the cardioprotective effects of a 3-day hesperidin pretreatment on I/R injury and to further explore whether its mechanism of action was associated with the inhibition of high mobility group box 1 protein (HMGB1) expression via the PI3K/Akt pathway. Methods: In a fixed-dose study, hematoxylin and eosin staining and myocardial enzyme measurements were used to determine the optimal dose of hesperidin that elicited the best cardioprotective effects against I/R injury. Furthermore, rats were pretreated with 200 mg/kg hesperidin, and infarct size and the levels of myocardial enzymes, apoptosis, inflammatory and oxidative indices, and HMGB1 and p-Akt expression were measured. Results: Our results indicated that while different 3-day hesperidin pretreatment doses promoted histopathological changes and reduced myocardial enzymes induced by I/R the optimal dose was 200 mg/kg. Moreover, the 200 mg/kg hesperidin pretreatment not only significantly decreased the infarct size as well as myocardial enzyme levels but also inhibited myocardial apoptosis, the inflammatory response and oxidative stress. Additionally, hesperidin downregulated HMGB1 expression and upregulated p-Akt expression in the myocardium. LY294002, a specific PI3K inhibitor, partially reversed the decreased HMGB1 expression, increased p-Akt expression induced by hesperidin and abolished the anti-apoptotic, anti-inflammatory and anti-oxidative effects of hesperidin. Conclusion: These findings suggest that short-term pretreatment with hesperidin protects against myocardial I/R injury by suppressing myocardial apoptosis, the inflammatory response and oxidative stress via PI3K/Akt pathway activation and HMGB1 inhibition.

IL-23 Promotes Myocardial I/R Injury by Increasing the Inflammatory Responses and Oxidative Stress Reactions

Background/Aims: Inflammation and oxidative stress play an important role in myocardial ischemia and reperfusion (I/R) injury. We hypothesized that IL-23, a pro-inflammatory cytokine, could promote myocardial I/R injury by increasing the inflammatory response and oxidative stress. Methods: Male Sprague-Dawley rats were randomly assigned into sham operated control (SO) group, ischemia and reperfusion (I/R) group, (IL-23 + I/R) group and (anti-IL-23 + I/R) group. At 4 h after reperfusion, the serum concentration of lactate dehydrogenase (LDH), creatine kinase (CK) and the tissue MDA concentration and SOD activity were measured. The infarcte size was measured by TTC staining. Apoptosis in heart sections were measured by TUNEL staining. The expression of HMGB1 and IL-17A were detected by Western Blotting and the expression of TNF-α and IL-6 were detected by Elisa. Results: After 4 h reperfusion, compared with the I/R group, IL-23 significantly increased the infarct size, the apoptosis of cardiomyocytes and the levels of LDH and CK (all P < 0.05). Meanwhile, IL-23 significantly increased the expression of eIL-17A, TNF-α and IL-6 and enhanced both the increase of the MDA level and the decrease of the SOD level induced by I/R (all P<0.05). IL-23 had no effect on the expression of HMGB1 (p > 0.05). All these effects were abolished by anti-IL-23 administration. Conclusion: The present study suggested that IL-23 may promote myocardial I/R injury by increasing the inflammatory responses and oxidative stress reaction.

Downregulation of microRNA-17-5p improves cardiac function after myocardial infarction via attenuation of apoptosis in endothelial cells

MicroRNA-17-5p (miR-17-5p) was indicated to suppress the formation of blood vessels, which is associated with cardiac function after myocardial infarction. In this study, the relationship between miR-17-5p and cardiac function was researched. Human umbilical vein endothelial cells were infected with adenoviruses. Apoptosis was determined by Annexin V-7AAD/PI. Real-time RT-PCR was used to evaluate miR-17-5p and ERK levels. Western blotting was used to determine the levels of ERK, the anti-apoptosis protein bcl-2 and apoptosis proteins, including bax, caspase 3, and caspase 9. An in vivo acute myocardial infarction (AMI) model was established in SD male rats. Heart function was evaluated by echocardiography prior to inducing AMI and after 7 and 28 days later. The heart was removed to perform histological examination, real-time RT-PCR, and western blotting, as described above. The result indicated that the ERK pathway was activated by miR-17-5p downregulation and an increase in the level of the anti-apoptosis protein bcl-2; however, the levels of apoptosis proteins (bax/caspase 3/caspase 9) were decreased. The results were completely reversed when miR-17-5p was up-regulated. At 7 and 28 days after the induction of AMI, in the miR-17-5p inhibition group, the infarction areas and collagen fibers were decreased, apoptosis in cardiac tissues was inhibited, and the endothelial growth process was promoted. Therefore, MiR-17-5p silencing protects heart function after AMI through decreasing the rate of apoptosis and repairing vascular injury.

Inhibition of autophagy via activation of PI3K/Akt/mTOR pathway contributes to the protection of hesperidin against myocardial ischemia/reperfusion injury

Hesperidin has been reported to attenuate myocardial ischemia/reperfusion (I/R) injury; however, its effect on autophagy during myocardial I/R and the underlying mechanism remains unknown. The present study aimed to investigate whether hesperidin inhibited I/R-induced excessive myocardial autophagy through activating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. Male adult rats were pretreated with hesperidin for a total of 3 days prior to ischemia in the absence or presence of LY294002, a PI3K inhibitor, and then subjected to ischemia for 30 min followed by reperfusion for 4 h. Myocardial infarct size was measured by Evans blue/triphenyltetrazolium chloride staining. Hematoxylin and eosin staining was used for observing the histological changes in the heart, and the serum levels of creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) were measured by enzyme-linked immunosorbent assay. Additionally, the protein levels of light chain (LC) 3II, Beclin1, phosphorylated (p)-mTOR, p-Akt and p-PI3K were determined by western blot analysis. Hesperidin pretreatment significantly decreased the myocardial infarct size, myocardial damage and serum levels of CK-MB and cTnI. Furthermore, the expression levels of LC3II and Beclin1 were significantly downregulated and the expression levels of p-mTOR, p-Akt and p-PI3K were markedly upregulated by hesperidin. However, the aforementioned effects as a result of hesperidin were significantly reversed by the presence of LY294002. These results demonstrated that hesperidin reduced myocardial I/R injury by suppressing excessive autophagy. Activation of the PI3K/Akt/mTOR pathway contributed to the inhibitory effect of hesperidin on excessive autophagy.

Promoting effects of IL‑23 on myocardial ischemia and reperfusion are associated with increased expression of IL‑17A and upregulation of the JAK2‑STAT3 signaling pathway

Interleukin (IL)-23, as a novel pro-inflammatory cytokine, is important in several inflammatory diseases, including myocardial ischemia and reperfusion (I/R) injury, however, the underlying mechanism remains to be elucidated. The present study was designed to investigate the specific role of IL-23 in myocardial I/R injury, and whether the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2-STAT3) signaling pathway, one of the important downstream signaling pathways of IL-23, and the IL-17A downstream pro-inflammatory cytokine, were involved. Anesthetized rats underwent different treatments with adenovirus (Ad) vectors (Ad-GFP, Ad-IL-23, Anti-IL-23 or Ad-IL-23+AG490) and were then subjected to ischemia for 30 min prior to 4 h reperfusion. The effects of the upregulation and downregulation of IL-23 on myocardial injury, inflammatory responses in myocardial tissue, and myocardial apoptosis were measured accordingly. In addition, the levels of phosphorylated (P-)JAK2 and P-STAT3 were measured to assess the activity of the JAK2-STAT3 signaling pathway. The results demonstrated that there was an increased expression of IL-23 in the myocardial tissue exposed to myocardial I/R injury (P<0.05). The upregulation of IL-23 significantly increased the infarct size and the expression levels of lactate dehydrogenase and creatine kinase (P<0.05). The upregulation of IL-23 significantly increased inflammatory responses, as reflected by the high expression levels of IL-17A, IL-6, tumor necrosis factor-α in the myocardial tissues (P<0.05). Furthermore, the upregulation of IL-23 significantly facilitated the decrease in the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein ratio, and the increases in the myocardial apoptotic index and expression of caspase-3 induced by myocardial I/R (P<0.05). IL-23 also activated the JAK2-STAT3 signaling pathway, upregulating the expression levels of P-JAK2 and P-STAT3 in the myocardial tissues (P<0.05). Treatment with AG490, an inhibitor of JAK2-STAT3, partially attenuated the pro-inflammatory and pro-apoptotic effects of IL-23 (P<0.05). The results of the present study suggested that IL-23 aggravated myocardial I/R injury by promoting inflammatory responses and myocardial apoptosis, which may be associated with high expression levels of IL-17A and upregulation of the JAK2-STAT3 signaling pathway.

The HMGB1‑IL‑17A axis contributes to hypoxia/reoxygenation injury via regulation of cardiomyocyte apoptosis and autophagy

Both the high‑mobility group box 1 protein (HMGB1) and interleukin (IL)‑17A serve important roles in myocardial ischemia and reperfusion injury. The purpose of the present study was to evaluate whether HMGB1 could induce IL‑17A secretion and lead to cardiomyocyte hypoxia/reoxygenation (H/R) injury. Neonatal rat cardiomyocytes were treated with HMGB1‑neutralizing antibody, IL‑17A‑neutralizing antibody, recombinant HMGB1 (rHMGB1) and recombinant IL‑17A (rIL‑17A), respectively. Cell viabilities, lactate dehydrogenase and creatine kinase levels were measured. Apoptotic cells were assessed by flow cytometry. The expression of HMGB1, IL‑17A, microtubule‑associated proteins 1A/1B light chain 3B (LC3), Beclin‑1, B‑cell lymphoma (Bcl)‑2 and Bcl‑2‑associated X protein were assessed by western blot analysis. The results demonstrated that HMGB1 significantly increased the expression of IL‑17A. HMGB1 or IL‑17A antibody significantly ameliorated H/R‑induced cell injury and improved the cell viability. In contrast, rHMGB1 or rIL‑17A aggravated cell injury and inhibited the cell viability. Furthermore, cardiomyocytes were treated with HMGB1 or IL‑17A antibody significantly increased Bcl‑2 protein expression and had fewer apoptotic cells, whereas rHMGB1 or rIL‑17A‑treated cardiomyocytes markedly decreased Bcl‑2 protein expression and had more apoptotic cells. Moreover, HMGB1 or IL‑17A antibodies significantly inhibited H/R induced autophagy dysfunction (as determined by the inhibition of Beclin‑1 expression, a lower ratio of LC3‑II to LC3‑I), whereas rHMGB1 or rIL‑17A may promote cardiomyocyte autophagy. Together, these results suggested that the HMGB1‑IL‑17A axis contributes to H/R injury via regulation of cardiomyocyte apoptosis and autophagy.