Inhibitory Effect of PPARδ Agonist GW501516 on Proliferation of Hypoxia-induced Pulmonary Arterial Smooth Muscle Cells by Regulating the mTOR Pathway.

OBJECTIVE: This study aimed to investigate the effects of the peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by hypoxia, in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling. METHODS: PASMCs were incubated with different concentrations of GW501516 (10, 30, 100 nmol/L) under the hypoxic condition. The proliferation was determined by a CCK-8 assay. The cell cycle progression was analyzed by flow cytometry. The expression of PPARδ, S phase kinase-associated protein 2 (Skp2), and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting. Then PASMCs were treated with 100 nmol/ L GW501516, 100 nmol/L mammalian target of rapamycin (mTOR) inhibitor rapamycin and/or 2 µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs. RESULTS: The presented data demonstrated that hypoxia reduced the expression of PPARδ in an oxygen concentration- and time-dependent manner, and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle. In accordance with these findings, GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs. Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation, arresting the cell cycle, regulating the expression of Skp2 and p27, and inactivating mTOR in hypoxia-exposed PASMCs. Moreover, MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs. CONCLUSION: GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.

[Effects of GW501516 on the proliferation of pulmonary artery smooth muscle cells induced by hypoxia and its mechanisms].

Objective: To investigate the effects of the peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the proliferation of primary rat proliferation of pulmonary artery smooth muscle cells ( PASMCs ) induced by hypoxia, in order to discover new drugs for the treatment and prevention of pulmonary vascular remodeling. Methods: The PASMCs in the control group were cultured with 21% oxygen, while the PASMCs in the hypoxia group were cultured with 3% oxygen to induce cell proliferation. PASMCs were incubated with GW501516 at the concentrations of 10, 30 and 100 nmol/L under hypoxic conditions for different time points (12, 24, and 48 h) to find out the appropriate concentrations of GW501516 for inhibition the proliferation. PASMCs were incubated with 100 nmol/L GW501516 and ( or ) protein kinase B (AKT) agonist SC79 for 24 h to explore related mechanisms of GW501516 in regulating the proliferation. The proliferation and DNA synthesis were determined by CCK-8 and BrdU kit. The cell cycle progression was analyzed by flow cytometry. The mRNA expressions of Cyclin D1 and the cyclin kinase inhibitor p27(p27) were measured by quantitative real-time PCR (RT-PCR). The expressions of PPARδ, total and phosphorylated forms AKT and glycogen synthase kinase 3ß (GSK3ß) were detected by Western blot. Results: Compared with the hypoxia group, PASMCs incubated with different concentrations of GW501516 (10, 30, 100 nmol/L) for 12, 24, 48 h under hypoxic conditions could inhibit the proliferation and DNA synthesis, and the greatest level of suppression of proliferation was induced by GW501516 at the concentration of 100 nmol/L(P＜0.05 or P＜0.01). Compared with the control group, the expression of PPARδ was upregulated markedly in PASMCs incubated with 100 nmol/L GW501516 for 24 h,while hypoxia could downregulate the expression of PPARδ significantly(P＜0.01). Compared with the hypoxia group, 100 nmol/L GW501516 blocked the proliferation and DNA synthesis of PASMCs significantly(P＜0.01), increased the proportion of PASMCs in G0 /G1 phase while decreased the proportion of PASMCs in S phase and G2 /M phase(P＜0.05 or P＜0.01), markedly downregulated the mRNA expression of cyclin D1 and upregulated the mRNA expression of p27(P＜0.01), significantly inhibited the protein expressions of phosphorylated AKT and GSK3ß(P＜0.01). Compared with the 100 nmol/L GW501516 hypoxia group, AKT agonist SC79 reversed all the above effects of 100 nmol/L GW501516 on hypoxia stimulated PASMCs(P＜0.05 or P＜0.01). Conclusion: GW501516 inhibits hypoxia induced proliferation in PASMCs via inactivating AKT/GSK3ß signaling pathway.

The prognostic significance of electrocardiography findings in patients with coronavirus disease 2019: A retrospective study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has reached a pandemic level. Cardiac injury is not uncommon among COVID-19 patients. We sought to describe the electrocardiographic characteristics and to identify the prognostic significance of electrocardiography (ECG) findings of patients with COVID-19. HYPOTHESIS: ECG abnormality was associated with higher risk of death. METHODS: Consecutive patients with laboratory-confirmed COVID-19 and definite in-hospital outcome were retrospectively included. Demographic characteristics and clinical data were extracted from medical record. Initial ECGs at admission or during hospitalization were reviewed. A point-based scoring system of abnormal ECG findings was formed, in which 1 point each was assigned for the presence of axis deviation, arrhythmias, atrioventricular block, conduction tissue disease, QTc interval prolongation, pathological Q wave, ST-segment change, and T-wave change. The association between abnormal ECG scores and in-hospital mortality was assessed in multivariable Cox regression models. RESULTS: A total of 306 patients (mean 62.84 ± 14.69 years old, 48.0% male) were included. T-wave change (31.7%), QTc interval prolongation (30.1%), and arrhythmias (16.3%) were three most common found ECG abnormalities. 30 (9.80%) patients died during hospitalization. Abnormal ECG scores were significantly higher among non-survivors (median 2 points vs 1 point, p < 0.001). The risk of in-hospital death increased by a factor of 1.478 (HR 1.478, 95% CI 1.131-1.933, p = 0.004) after adjusted by age, comorbidities, cardiac injury and treatments. CONCLUSIONS: ECG abnormality was common in patients admitted for COVID-19 and was associated with adverse in-hospital outcome. In-hospital mortality risk increased with increasing abnormal ECG scores.

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/R­induced excessive myocardial autophagy through activating the phosphatidylinositol 3­kinase (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 kinase­MB (CK­MB) and cardiac troponin I (cTnI) were measured by enzyme­linked immunosorbent assay. Additionally, the protein levels of light chain (LC) 3Ⅱ, Beclin1, phosphorylated (p)­mTOR, p­Akt and p­PI3K were determined by western blot analysis. Hesperidin pretreatment significantly decreased the myocardial infarct size, myocardial damage and serum levels of CK­MB and cTnI. Furthermore, the expression levels of LC3Ⅱ and Beclin1 were significantly downregulated and the expression levels of p­mTOR, p­Akt and p­PI3K 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.

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.

MicroRNA­451 protects against cardiomyocyte anoxia/reoxygenation injury by inhibiting high mobility group box 1 expression.

High mobility group box 1 (HMGB1) protein serves an important role in myocardial ischemia/reperfusion (I/R) injury. MicroRNAs (miRNAs) are a group of small non­coding RNAs that regulate numerous signaling pathways involved in myocardial I/R injury. The present study aimed to investigate whether miR­451 protects against cardiomyocyte anoxia/reoxygenation (A/R) injury by attenuating HMGB1 expression. Neonatal rat ventricular cardiomyocytes were prepared and then subjected to A/R injury. The effect of upregulation or downregulation of miR­451 on cell viability, apoptosis, superoxide dismutase (SOD) activity, and the expression of cleaved­caspase­3 and HMGB1 were measured accordingly. A luciferase assay was performed to further confirm whether miR­451 can directly recognize the 3'­untranslated region of HMGB1 in HEK293 cells. The expression of miR­451 was significantly decreased in the cardiomyocytes during A/R, and upregulation of miR­451 led to increased miR­451 expression (P<0.05). Upregulation of miR­451 significantly attenuated the loss of cardiomyocyte viability (P<0.05) and increased the intracellular levels of SOD during A/R (P<0.05). Furthermore, upregulation of miR­451 significantly decreased the apoptosis of cardiomyocytes during A/R (P<0.05). The HMGB1 mRNA and protein expression levels were significantly downregulated in the Ad­miR­451 group compared with those in the A/R group (P<0.05). In addition, upregulation of miR­451 reduced its translocation from the nucleus to the cytoplasm. The luciferase assay confirmed that HMGB1 mRNA is a direct target of miR­451 in cardiomyocytes. In conclusion, the present study suggested that upregulation of miR­451 could protect against A/R­induced cardiomyocyte injury by inhibiting HMGB1 expression.

Vagus nerve stimulation attenuates myocardial ischemia/reperfusion injury by inhibiting the expression of interleukin-17A.

Interleukin (IL)-17A has an important role in myocardial ischemia/reperfusion (I/R) injury, and vagal stimulation (VS) has been demonstrated to exert cardioprotective effects. The present study aimed to investigate the effects of VS on a rat model of myocardial I/R injury, and detected an association between VS and IL-17A. Anesthetized rats underwent VS (2 msec; 10 Hz) or were treated with anti-IL-17A neutralized monoclonal antibodies (mAbs) (200 µg; iv), and subjected to ischemia for 30 min prior to 4 h reperfusion. The following parameters were measured: Infarct size; lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD) and caspase-3 activity levels; tumor necrosis factor (TNF)-α and IL-6 expression levels; and the percentage of terminal deoxynucleotidyl-transferase mediated dUTP nick-end labeling (TUNEL) positive cells. High mobility group box 1 protein (HMGB1) and IL-17A expression levels were assessed by immunoblotting. Following 4 h reperfusion, VS was able to significantly decrease the infarct size and the activity levels of LDH and CK (P<0.05). Furthermore, VS administration significantly suppressed the increased MDA and decreased SOD activity levels, and significantly reduced caspase-3 activity and the percentage of TUNEL-positive cells (P<0.05). Treatment with anti-IL-17A mAbs demonstrated the same effects as VS. Furthermore, VS was able to significantly inhibit the increased expression levels of TNF-α, IL-6, HMGB1 and IL-17A induced by I/R (P<0.05). The results of the present study suggested that VS may attenuate myocardial I/R injury by reducing the expression of inflammatory cytokines, oxidative stress and the apoptosis of cardiomyocytes. Furthermore, VS may induce cardioprotective effects, which may be associated with the inhibition of IL-17A expression.