Rat Myocardial Tissue Research Articles

Restoration of NRF2 attenuates myocardial ischemia reperfusion injury through mediating microRNA‐29a‐3p/CCNT2 axis

Accumulated studies have been implemented for comprehending the mechanism of myocardial ischemia reperfusion injury (MI/RI). Nuclear factor erythroid-2 related factor 2 (NRF2)-mediated transcription activity in MI/RI has not been completely interpreted from the perspective of microRNA-29a-3p (miR-29a-3p) and cyclin T2 (CCNT2). Therein, this study intends to decode the mechanism of NRF2/miR-29a-3p/CCNT2 axis in MI/RI. Rat MI/RI models were established by right anterior descending coronary artery ligation. Rats were injected with NRF2 or CCNT2 overexpression plasmids or miR-29a-3p agomir to explore their effects on MI/RI. Hypoxia/reoxygenation (H/R) cardiomyocytes were established and transfected with restored NRF2 or miR-29a-3p or CCNT2 for further exploration of their roles. NRF2, miR-29a-3p, and CCNT2 expression in myocardial tissues in rats with MI/RI and in cardiomyocytes in H/R injury were detected. ChIP assay verified the relationship between miR-29a-3p and NRF2, and the bioinformatics software and dual-luciferase reporter experiment verified the interaction between miR-29a-3p and CCNT2. NRF2 and miR-29a-3p were down-regulated while CCNT2 was up-regulated in myocardial tissues in rats with MI/RI and in H/R-treated cardiomyocytes. Restoration of NRF2 or miR-29a-3p improved hemodynamics and myocardial injury and suppressed serum inflammation and cardiomyocyte apoptosis via CCNT2 in rats with MI/RI. Upregulation of NRF2 or miR-29a-3p inhibited LDH and CK-MB activities, oxidative stress, and apoptosis and promoted viability of cardiomyocytes with H/R injury. NRF2 bound to the promoter of miR-29a-3p and CCNT2 was targeted by miR-29a-3p. This study elucidates that up-regulating NRF2 or miR-29a-3p attenuates MI/RI via inhibiting CCNT2, which may renew the existed knowledge of MI/RI-related mechanism and provide a novel guidance toward MI/RI treatment.

MicroRNA-23a reduces lipopolysaccharide-induced cellular apoptosis and inflammatory cytokine production through Rho-associated kinase 1/sirtuin-1/nuclear factor-kappa B crosstalk.

Background:MicroRNAs are closely associated with the progression and outcomes of multiple human diseases, including sepsis. In this study, we examined the role of miR-23a in septic injury.Methods:Lipopolysaccharide (LPS) was used to induce sepsis in a rat model and H9C2 and HK-2 cells. miR-23a expression was evaluated in rat myocardial and kidney tissues, as well as H9C2 and HK-2 cells. A miR-23a mimic was introduced into cells to identify the role of miR-23a in cell viability, apoptosis, and the secretion of inflammatory cytokines. Furthermore, the effect of Rho-associated kinase 1 (ROCK1), a miR-23a target, on cell damage was evaluated, and molecules involved in the underlying mechanism were identified.Results:In the rat model, miR-23a was poorly expressed in myocardial (sham vs. sepsis 1.00 ± 0.06 vs. 0.27 ± 0.03, P < 0.01) and kidney tissues (sham vs. sepsis 0.27 ± 0.03 vs. 1.00 ± 0.06, P < 0.01). Artificial overexpression of miR-23a resulted in increased proliferative activity (DNA replication rate: Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 34.13 ± 3.12 vs. 12.94 ± 1.21 vs. 13.31 ± 1.43 vs. 22.94 ± 2.26, P < 0.05; HK-2 cells: 15.17 ± 1.43 vs. 34.52 ± 3.46 vs. 35.19 ± 3.12 vs. 19.87 ± 1.52, P < 0.05), decreased cell apoptosis (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 11.39 ± 1.04 vs. 32.57 ± 2.29 vs. 33.08 ± 3.12 vs. 21.63 ± 2.35, P < 0.05; HK-2 cells: 15.17 ± 1.43 vs. 34.52 ± 3.46 vs. 35.19 ± 3.12 vs. 19.87 ± 1.52, P < 0.05), and decreased production of inflammatory cytokines, including interleukin-6 (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 59.61 ± 5.14 vs. 113.54 ± 12.30 vs. 116.51 ± 10.69 vs. 87.69 ± 2.97 ng/mL; P < 0.05, F = 12.67, HK-2 cells: 68.12 ± 6.44 vs. 139.65 ± 16.62 vs. 143.51 ± 13.64 vs. 100.82 ± 9.74 ng/mL, P < 0.05, F = 9.83) and tumor necrosis factor-α (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 103.20 ± 10.31 vs. 169.67 ± 18.84 vs. 173.61 ± 15.91 vs. 133.36 ± 12.32 ng/mL, P < 0.05, F = 12.67, HK-2 cells: 132.51 ± 13.37 vs. 187.47 ± 16.74 vs. 143.51 ± 13.64 vs. 155.79 ± 15.31 ng/mL, P < 0.05, F = 9.83) in cells. However, ROCK1 was identified as a miR-23a target, and further up-regulation of ROCK1 mitigated the protective function of miR-23a in LPS-treated H9C2 and HK-2 cells. Moreover, ROCK1 suppressed sirtuin-1 (SIRT1) expression to promote the phosphorylation of nuclear factor-kappa B (NF-κB) p65, indicating the possible involvement of this signaling pathway in miR-23a-mediated events.Conclusion:Our results indicate that miR-23a could suppress LPS-induced cell damage and inflammatory cytokine secretion by binding to ROCK1, mediated through the potential participation of the SIRT1/NF-κB signaling pathway.

MKRN1 Ubiquitylates p21 to Protect against Intermittent Hypoxia-Induced Myocardial Apoptosis.

Although chronic intermittent hypoxia- (IH-) induced myocardial apoptosis is an established pathophysiological process resulting in a poor prognosis for patients with obstructive sleep apnea syndrome, its underlying mechanism remains unclear. This study is aimed at exploring the role of makorin ring finger protein 1 (MKRN1) in IH-induced myocardial apoptosis and elucidating its molecular activity. First, the GSE2271 dataset was downloaded from the Gene Expression Omnibus database to identify the differentially expressed genes. Then, an SD rat model of IH, together with rat cardiomyocyte H9C2 and human cardiomyocyte AC16 IH models, was constructed. TUNEL, Western blot, and immunohistochemistry assays were used to detect cell apoptosis. Dihydroethidium staining was conducted to analyze the concentration of reactive oxygen species. In addition, RT-qPCR, Western blot, and immunohistochemistry were performed to measure the expression levels of MKRN1 and p21. The direct interaction between MKRN1 and p21 was determined using coimmunoprecipitation and ubiquitination analysis. MKRN1 expression was found to be downregulated in IH rat myocardial tissues as well as in H9C2 and AC16 cells. Upregulated expression of MKRN1 in H9C2 and AC16 cells alleviated the IH-induced reactive oxygen species production and cell apoptosis. Mechanistically, MKRN1 promoted p21 protein ubiquitination and the proteasome pathway degradation to negatively regulate p21 expression. Thus, MKRN1 regulates p21 ubiquitination to prevent IH-induced myocardial apoptosis.

Upregulation of miR-335 reduces myocardial injury following myocardial infarction via targeting MAP3K2.

Acute myocardial infarction (AMI) is a serious cardiovascular disease with a high incidence worldwide and the main cause of sudden cardiac death. The aim of this article was to study the protective role of miR-335 in myocardial infarction (MI) and the underlying molecular mechanism. Thirty Sprague Dawley (SD) rats were randomly divided into sham group, MI + NC group and MI + agomiR-335 group. The expression of miR-335 in rat myocardium was detected by quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Western blot was performed to detect the expression of TNF-α, IL-6, IL-1β, Caspase-3, Cleaved Caspase-3 (C-Caspase-3) and MAP3K2 in rat myocardium. On the 7th day of the establishment of the rat MI model, a high-resolution small animal ultrasound system was utilized to detect the cardiac function of the rats, and the heart tissues and blood samples of the rats were collected. The corresponding kits were purchased to detect the contents of LDH, CK-MB, MDA and SOD in rat serum, and HE staining was employed to observe the morphology of rat myocardial tissue. The expression of miR-335 in myocardial infarcted zones and border zones of MI rats decreased significantly. The upregulation of miR-335 remarkably inhibited myocardial inflammation and apoptosis after MI, thus improving the cardiac function of MI rats. Compared with the sham group, the MAP3K2 expression in the MI + NC group was observably increased, while the overexpression of miR-335 could inhibit the expression of this protein. Through the Luciferase reporter gene experiment, we found that miR-335 could directly target MAP3K2. The expression of miR-335 decreased in myocardial tissue after MI, and the overexpression of miR-335 reduced myocardial damage by inhibiting oxidative stress, inflammation, and apoptosis via targeting MAP3K2, thereby improving the cardiac function of MI rats.

Ultrasound‑targeted microbubble destruction‑mediated Galectin‑7‑siRNA promotes the homing of bone marrow mesenchymal stem cells to alleviate acute myocardial infarction in rats.

Bone marrow mesenchymal stem cells (BMSCs) are accepted as a form of cellular therapy to improve cardiac function following acute myocardial infarction (AMI). The present study was performed to investigate the synergistic effect of ultrasound-targeted microbubble destruction (UTMD)-mediated Galectin-7-small interfering (si)RNA with the homing of BMSCs for AMI. The rat model of AMI was established, followed by identification of BMSCs. Rats with AMI received BMSC transplantation, BMSC transplantation + UTMD + siRNA negative control, or BMSC transplantation + UTMD + Galectin-7-siRNA. The cardiac function, hemodynamics indexes, degree of myocardial fiber injury and expression of apoptosis-related proteins in myocardial tissues of rats were detected. The homing of BMSCs was observed, and the indexes of myocardial microenvironment and the TGF-β/Smads pathway-related proteins in myocardial tissues were determined. AMI rats treated with UTMD-mediated Galectin-7-siRNA exhibited improved cardiac function and hemodynamics-related indices, decreased myocardial fiber injury and apoptotic cells, as well as enhanced homing ability of BMSCs, improved myocardial microenvironment, and suppressed TGF-β1/Smads pathway activation. In conclusion, the present study demonstrated that UTMD-mediated Galectin-7-siRNA treatment could enhance the homing ability of BMSCs, thus alleviating AMI in rats.

Peroxiredoxin2 (Prdx2) Reduces Oxidative Stress and Apoptosis of Myocardial Cells Induced by Acute Myocardial Infarction by Inhibiting the TLR4/Nuclear Factor kappa B (NF-κB) Signaling Pathway.

BackgroundPeroxiredoxin2 (Prdx2) is an endogenous peroxidase and has been found to reduce the oxidative burden in cells and thereby reduce cell damage and apoptosis. Therefore, the purpose of this study was to investigate the effect of Prdx2 on the oxidative level and apoptosis of myocardial cells after acute myocardial infarction (AMI).Material/MethodsWe constructed an AMI model for Sprague-Dawley rats by ligating the left anterior descending coronary artery. We determined the effect of Prdx2 on AMI by detecting changes in Prdx2 in myocardial tissue via western blot and qRT-PCR. In addition, we used recombinant Prdx2 protein to treat rats and detect changes in oxidative stress and apoptosis in rat myocardial tissue to verify the protective effect of Prdx2 on the rat heart.ResultsThe protein and mRNA expression of Prdx2 in myocardial tissue of rats in the AMI group was significantly lower than that in the control group. The oxidative and apoptotic levels of myocardial tissue in Prdx2-administered rats were significantly improved compared to the non-administered group, which was manifested by a decrease in reactive oxygen species (ROS) levels and a decrease in the expression of the caspase family. In addition, Prdx2 also inhibited p65 phosphorylation in myocardial tissues and inhibited TLR4/NF-κB signaling pathway activity.ConclusionsThe expression of Prdx2 was decreased in myocardial tissue after AMI. Prdx2 can reduce apoptosis and ROS caused by AMI by inhibiting the TLR4/NF-κB signaling pathway, thereby reducing myocardial injury caused by AMI.

MiR-145-5p promotes myocardial cell apoptosis in rats with myocardial infarction through PI3K/Akt signaling pathway.

The aim of this study was to explore the influence of micro ribonucleic acid (miR)-145-5p on myocardial cell apoptosis in rats with myocardial infarction (MI) through the phosphatidylinositol 3-hydroxy kinase/protein kinase B (PI3K/Akt) pathway. In this study, Sprague-Dawley rats were used as research objects to establish the acute MI model in vivo. Infarction tissues and non-infarction tissues were both collected from rats. The expression level of miR-145-5p was determined using quantitative Polymerase Chain Reaction (qPCR), and the pathological changes in myocardial tissues of rats were observed through hematoxylin-eosin (HE) staining. In addition, H9c2 rat myocardial cells were cultured under hypoxia or normal oxygen concentration to simulate hypoxia in MI tissues. The changes in the expression of miR-145-5p in H9c2 cells in normal oxygen and hypoxia were determined. Meanwhile, the ratio of apoptotic cells to viable cells, and the changes in the expressions of proteins B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), Caspase-3 and Caspase-9 were evaluated through flow cytometry assay and Western blotting, respectively. The expression levels of crucial proteins in the PI3K/Akt pathway were measured as well. Additionally, H9c2 cells were transfected with miR-145-5p control and miR-145-5p mimic to evaluate cell apoptosis. QPCR results revealed that the expression level of miR-145-5p was substantially elevated in MI tissues (p<0.05). HE results indicated that the soma exhibited deformation after MI, suggesting that there were more necrotic and apoptotic cells. Compared with those cultured under normal oxygen concentration, H9c2 cells cultured in hypoxia environment exhibited significantly upregulated expression level of miR-145-5p, downregulated expression level of anti-apoptosis protein Bcl-2, upregulated level of pro-apoptosis protein Bax, activated Caspase-3 and Caspase-9, and downregulated expression level of functional proteins in the PI3K/Akt pathway (p<0.05). Furthermore, the expression levels of apoptosis-associated proteins significantly rose in H9c2 cells transfected with miR-145-5p mimic compared with those transfected with miR-145-5p control, showing statistically significant differences (p<0.05). MiR-145-5p is notably raised in MI tissues of rats. After infarction, there are evidently more apoptotic myocardial cells. The expression of miR-145-5p is markedly elevated in H9c2 rat myocardial cells in hypoxia. Compared with those cultured in normal oxygen, H9c2 cells cultured in hypoxia showed increased apoptosis. The apoptosis of myocardial cells transfected with miR-145-5p mimic is notable higher than that of myocardial cells transfected with miR-145-5p control. Moreover, the expressions of active Akt and PI3K proteins decrease remarkably. The results of this study demonstrate that miR-145-5p inactivates the PI3K/Akt pathway to promote the apoptosis of MI cells.

Analysis of the differential expression profile of miRNAs in myocardial tissues of rats with burn injury.

Fifteen percent third-degree burn rat model was used to identify miRNAs that are markers of burn injury-induced myocardial damage. Cardiac tissues were evaluated to determine miRNA profile sequencing. Pearson's correlation analysis was used between miRNAs and injury markers. ROC curve analysis was used to estimate miRNA's sensitivity and specificity for the diagnosis of myocardial damage caused by burn injury. The sequencing analysis revealed 23 differentially expressed miRNAs. Pearson's correlation analysis revealed that rno-miR-190b-3p and C5b9, rno-miR-341, rno-miR-344b-3p and TnI, rno-miR-344b-3p and CK-MB were significantly positively correlated, respectively. ROC curve analysis demonstrated that rno-miR-341, rno-miR-344b-3p, and rno-miR-190b-3p exhibited high sensitivity and specificity for the diagnosis of myocardial damage caused by burn injury. In conclusion, our results suggest that rno-miR-341, rno-miR-344b-3p, and rno-miR-190b-3p have the potential to be used as sensitive and specific biomarkers to diagnose myocardial damage caused by burn injury.

Trigonoside II mitigates sepsis-induced myocardial injury via reduction in oxidative stress and regulation of TLR- 4/NF-kB inflammatory pathway

Purpose: To investigate the protective effect of trigonoside II against sepsis-induced myocardial injury in rats, and the mechanism involved.&#x0D; Methods: Adult male Sprague Dawley rats (n = 30) weighing 200 - 230 g (mean weight = 215 ± 15 g) were used for this study. The rats were randomly assigned to 3 groups (10 rats/group): sham, cecal ligation puncture (CLP), and trigonoside II. Rats in the treatment group received trigonoside II at a dose of 2 mg/kg intraperitoneally (i.p.) at 3, 12 and 24 h post-surgery. Sepsis was induced using CLP method. Lactate dehydrogenase (LDH) and creatine kinase (CK-MB) activities, and hemodynamic functions were determined in the rats. The levels of interleukin (IL)-1β and IL-6, and tumor necrosis factor α (TNF-α) were assayed in rat serum. Oxidative stress and myocardial cell apoptosis were determined by measuring malondialdehyde (MDA) levels, while activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and myeloperoxidase (MPO), as well as levels of expression of bax, bcl-2 and caspase-3 were also assessed.&#x0D; Results: Treatment of myocardial injury rats with trigonoside II led to significant reductions in the activities of LDH, CK-MB and MPO, and decreases in levels of IL-1β, IL-6 and TNF-α (p &lt; 0.05). It also significantly reversed the effects of sepsis on rat hemodynamic functions (p &lt; 0.05). Trigonoside IItreatment significantly reduced MDA levels in rat myocardial tissues, but significantly increased SOD and GPx activities (p &lt; 0.05). It significantly down-regulated protein expressions of NF-kB and TLR-4 in myocardial tissues (p &lt; 0.05). The number of apoptotic cells and activity of caspase-3 were significant increased in myocardial tissues of rats in CLP group, when compared with sham group, but were reduced significantly in myocardial tissues of trigonoside II-treated rats (p &lt; 0.05). Similarly, trigonoside II treatment down-regulated the protein expressions of caspase-3 and bax, but upregulated bcl-2 protein expression in the rat myocardial tissues (p &lt; 0.05).&#x0D; Conclusion: The results of this study indicate that trigonoside II confers protection on sepsis-induced myocardial injury via reduction in oxidative stress and regulation of TLR-4/NF-kB inflammatory pathway.&#x0D; Keywords: Cecal ligation puncture, Myocardial injury, Oxidative stress, Sepsis, Trigonoside II

Calcium sensing receptor involving in therapy of embryonic stem cell transplantation alleviates acute myocardial infarction by inhibiting apoptosis and oxidative stress in rats.

Objective(s):The aims of the present study were to investigate the expression of calcium sensing receptor (CaSR) at different times in acute myocardial infarction (AMI) rat myocardial tissue after mouse embryonic stem cells (mESCs) transplantation treatment and to assess its effects on apoptosis and oxidative stress of cardiomyocytes. Materials and Methods:The AMI rats were treated with mESCs, Calindol (a CaSR agonist) and Calhex231 (a CaSR inhibitor). Serum measurements, Echocardiographic analysis and TUNEL assay were performed. Myocardial ultrastructure changes were viewed by electron microscopy. Additionally, western blotting was used to detect the protein expressions.Results:Compared to the sham group, it was found that the expression levels of CaSR, caspase-3, cytoplasmic cytochrome C (cyt-C) and Bcl2-associated x (Bax), and the levels of Malondialdehyde (MDA) were significantly increased in both AMI and AMI + mESCs + Calindol groups with the development of myocardial infarction. Furthermore, the ultra-microstructure of cardiomyocyte was highly damaged, the expression levels of mitochondrial cyt-C and B-cell lymphoma 2 (Bcl-2) were significantly decreased, and there was decreased activity of superoxide dismutase (SOD). However, the combination of Calhex231 and mESCs transplantation could inhibit these changes. Conclusion:Our results suggested that CaSR expression in myocardial tissue of AMI rats was increased over time, and that Calhex231 could enhance the efficacy of ESCs transplantation for the treatment of AMI, which would be a new therapeutic strategy for the treatment of AMI.

Using ultrasound three-dimensional speckle tracking technology to explore the role of SIRT1 in ventricular remodeling after myocardial infarction.

To investigate the role of SIRT1 in ventricular remodeling after myocardial infarction using ultrasound three-dimensional speckle tracking (3D-STI). Fifty-eight patients with acute myocardial infarction diagnosed in the Second Affiliated Hospital of Qiqihar Medical College from June 2015 to July 2017 were enrolled in the study. They were divided into ventricular remodeling group and ventricular non-remodeling group. Fifty-eight healthy people underwent physical examination were controls. 3D-STI was used to detect end-diastolic ventricular septal thickness (LVST), end-diastolic left ventricular posterior wall thickness (LVPWT), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular ejection fraction (LVEF), systolic peak radial strain (PRS). SIRT1 expression levels in peripheral blood samples of the 3 groups were measured. Rats with acute myocardial infarction were treated with SIRT1 agonist. After 4 weeks, LVEDV, LVESV, LVEF, stroke volume (SV) were recorded by three-dimensional ultrasound; rat myocardial tissue protein was extracted, and SIRT1 and TGF-β, α-SMA, Vimentin and other fibrosis indicators were detected to explore the effects of SIRT1 on ventricular remodeling and myocardial fibrosis. At the time of initial diagnosis, SIRT1 level in healthy group > non-ventricular remodeling group > remodeling group (p<0.05); at the return visit, SIRT1 levels in the remodeling group and non-ventricular remodeling group were significantly elevated (p<0.05), but that in the remodeling group was significantly lower than that in the non-ventricular group (p<0.05). The expression level of SIRT1 in H9c2 hypoxia-reperfusion cell model control group > SIRT agonist treatment model group > model group. In summary, SIRT1 in the peripheral blood is negatively correlated with the degree of ventricular remodeling. The expression of SIRT1 in myocardial tissue is related to the cardiac morphology expansion and relief of reduced function in vivo after acute myocardial infarction. Up-regulation of SIRT1 expression in cell models can reduce cardiomyocyte apoptosis and inhibit cardiomyocyte fibrosis. SIRT1 has a good application prospect in predicting and treating myocardial infarction and delaying ventricular remodeling.

Exploration of the effect of salvianolate on myocardial infarction in rats based on tandem mass tags

Salvianolate is a compound from traditional Chinese medicine widely used in the treatment of various cardiovascular diseases. This study explored the effects of salvianolate on myocardial infarction and used tandem mass tags (TMT) to discover differentially expressed proteins. Male Sprague Dawley rats were randomly divided into the sham operation group, model group, and salvianolate group. The myocardial infarction model was established by ligating the left anterior descending coronary artery while the sham group had a sham operation. The rats were intraperitoneally injected with 2 ml of 5% glucose once a day, with 48.438 mg/kg/d salvianolate for the rats in the salvianolate group. After 4 weeks, the rats’ hemodynamics were measured to evaluate cardiac function, and Masson staining assessed the area of myocardial infarction. TMT analysis was performed and validated by western blot. Salvianolate improved cardiac function after myocardial infarction, reduced the myocardial infarction area, and protected the myocardial tissue. 100 differentially expressed proteins were identified between the sham operation and model groups, salvianolate reversed the expression of 25 of those proteins, that were mainly involved in the metabolism of extracellular collagen matrix and the response to growth factor stimulation. Type I collagen, type V collagen, chymase, β-myosin heavy chain, and A-Raf differential expression were consistent in western blotting. In conclusion, salvianolate had a protective effect on myocardial tissues of rats with myocardial infarction. Several proteins including type I collagen, type V collagen, chymase, β-myosin, and A-Raf may be salvianolate targets for treatment of myocardial infarction.

Berberine protects myocardial injury and cardiac dysfunction in a septic rat model

Objectives: To investigated whether berberine could ameliorate septic cardiomyopathy in a rat model of sepsis and it's mechanisms. Methods: SD rats were divided into 3 groups: sepsis group (LPS group), rats were intraperitoneal injected of LPS (10 mg/kg); Berberine intervention group (Ber group), Ber (50 mg/kg, one time per day) was gavage fed 3 days before intraperitoneally injection of lipopolysaccharides (LPS); control group (Con group), rats were gavage fed with double distilled water (2 ml/100 g, one time per day) 3 days before intraperitoneal injection of normal saline (1 ml/100 g). LPS group and the Ber group was further divided into 3 subgroups (n=6), and the follow-up experiments were conducted at 6 h, 24 h and 48 h after LPS injection (of which 48 h subgroup rats were gavage fed with Ber/saline at 24 h). Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of change of left ventricular pressure (±dp/dtmax) were monitored, the level of cardiac troponin T (cTnT), tumor necrosis factor (TNF)-α and interleukin (IL)-1β was detected by ELISA method, HE staining of myocardial tissues was done to observe myocardial injury; Western blotting method was used to detect the expression of toll-like receptor 4(TLR4) protein in rat myocardial tissue, the level of myocardial cell nucleus protein p65 was detected to reflects the degree of NF-κB activation. The correlation of factors was analyzed with Pearson correlation analysis. Results: Pre-treatment with berberine stabilized cardiac hemodynamics and improved the systolic function and diastolic function in the heart of LPS-induced rats, as evidenced by the partial recovery of the reduced±dp/dtmax and LVSP, as well as the decreased LVEDP. Compared with the LPS group, the Ber group showed improved myocardial injury, as demonstrated by decreased cTnT at each time point. HE staining results showed that berberine decreased inflammatory cell infiltration and LPS-induced cell swelling. These effects were observed early at 6 hours, severe at 24 hours, and become more serious at 48 hours after LPS injection. Further, TLR4 and NF-κB p65 subunits, which were the two key factors of the TLR4/NF-κB signaling, were upregulated in the LPS group and attenuated in the Ber group. Consistently, the expression levels of the downstream cytokines TNF-α and IL-1β were lower in the Ber group than those in the LPS group (all P<0.05). Myocardial injury markers were positively correlated with the markers of TLR4/NF-κB signals and the downstream host inflammatory factors (all P<0.05). Conclusions: Berberine can improve myocardial injury and cardiac function in sepsis rats, the mechanism is considered to be related to that it can inhibit the activation of TLR4/NF-κB signaling pathway induced by LPS and further reducing the production of TNF-α and IL-1β.

FULLEROL alleviates myocardial ischemia-reperfusion injury by reducing inflammation and oxidative stress in cardiomyocytes via activating the Nrf2/HO-1 signaling pathway.

Myocardial ischemia-reperfusion injury (MIRI) is myocardial tissue injury caused by blood supply returning to myocardial tissue after ischemia or hypoxia. The purpose of this study was to investigate the protective effect of FULLEROL on myocardial tissue in MIRI rats and its mechanism. We use rats and ligate their left anterior descending coronary artery to make a MIRI model, and we also subcutaneously injected some MIRI rats with FULLEROL daily for two weeks before modeling. We determined the effects of model building and the therapeutic effect of FULLEROL on MIRI by detecting the changes of myocardial tissue morphology, myocardial injury markers and cardiac function in rats. In addition, we detected the expression changes of inflammatory factors and antioxidative molecules in rat myocardial tissue and serum to determine the effect of FULLEROL on inflammation and oxidative stress in myocardial tissue. Finally, we detected the activity of the Nrf2/HO-1 signaling pathway in rat cardiomyocytes to determine the mechanism of action of FULLEROL. The structure of myocardial tissue in MIRI rats was remarkably damaged, and the range of myocardial infarction was increased. In addition, the concentrations of creatine kinase and lactate dehydrogenase were increased, and the heart function was reduced, while FULLEROL could reverse these conditions. In addition, FULLEROL was found to reduce the concentration of the inflammatory factors in rat myocardial tissue and serum, and to increase the expression of antioxidative molecules in myocardial tissue. The Nrf2/HO-1 signaling pathway was found to be related to MIRI and FULLEROL could increase the activity of the Nrf2/HO-1 signaling pathway in cardiomyocytes. FULLEROL can alleviate MIRI by promoting the activity of the Nrf2/HO-1 signaling pathway to reduce the expression of inflammatory factors in rats and increase the antioxidative capacity of cardiomyocytes.

Qishen Huanwu capsule reduces pirarubicin-induced cardiotoxicity in rats by activating the PI3K/Akt/mTOR pathway

One of the common adverse reactions to anthracyclines, a group of chemotherapeutics, is cardiotoxicity. Cancer patients receiving anthracycline-based chemotherapeutic regimens often discontinue treatment due to cardiotoxicity. How to prevent and reduce the cardiotoxicity of anthracyclines is one of the hot topics in the field of onco-cardiology. Traditional Chinese medicine can reduce the toxic side effects of chemotherapeutics. The present study aimed to investigate the protective effect of Qishen Huanwu capsule (QSHWC) on pirarubicin (THP)-induced myocardial injury in rats and the underlying mechanisms. Forty-eight male Sprague-Dawley (SD) rats were randomly divided into six groups: control group, THP, low-dose QSHWC, moderate-dose QSHWC, high-dose QSHWC, and LY294002 [phosphatidylinositol 3-kinase (PI3K) inhibitor] (n=8 each). Echocardiographic examination was performed to examine heart structure and function. Hematoxylin and eosin (HE) staining was conducted to examine histopathological changes in myocardial tissue. Immunofluorescence staining was carried out to examine the expression of the autophagosome-specific marker protein microtubule-associated protein 1 light chain 3 (LC3). Western blot was performed to analyze the expression of LC3-I, LC3-II, PI3K, phosphorylated (p)-PI3K, protein kinase B (Akt), p-Akt, mammalian target of rapamycin (mTOR), and p-mTOR. Compared with the control group, the THP group had a higher left ventricular end-systolic diameter (LVESD), lower left ventricular ejection fraction (LVEF), lower left ventricular fractional shortening (LVFS), and inferior heart function. In addition, compared with the control group, the THP group had significantly higher LC3 protein expression, a significantly higher LC3-II/LC3-I ratio (P<0.05), and significantly lower p-PI3K, p-Akt, and p-mTOR (P<0.05). QSHWC attenuated the THP-induced decline in heart function, downregulated LC3 protein in rat myocardial tissue, decreased the LC3-II/LC3-I ratio, and increased p-PI3K, p-Akt, and p-mTOR. In the LY294002 group, the above effects of QSHWC were reversed. QSHWC alleviated THP-induced myocardial injury. The underlying mechanism was related to the activation of the PI3K/Akt/mTOR pathway and the mitigation of the excessive autophagy of cardiomyocytes caused by THP.

T follicular helper cells are elevated in a rat model of autoimmune myocarditis.

Myocarditis is an inflammatory disease of the myocardium that is associated with immune dysfunction. Earlier studies have suggested that T helper 1/2 cell imbalance plays an important role in the development of myocarditis, but the role of T follicular helper (Tfh) cells in the development of autoimmune myocarditis has not previously been reported. Here, we investigated this involvement by using a rat model of experimental autoimmune myocarditis (EAM). Inflammatory cell infiltration, myocardial structure destruction and tissue necrosis were observed in EAM myocardial tissues, and the percentages of CD4+CXCR5+ Tfh cells and CD19+ B cells were both significantly higher in spleen and myocardial tissues of the EAM model as compared with the control group. Furthermore, the expression levels of interleukin‐21, CXCL13 and myosin antibody were significantly higher in the serum of rats with EAM compared with the control group on days 14 and 35 after immunization. Fourteen or 35 days after immunization, the expression levels of interleukin‐21 and CXCL13 were both significantly higher in myocardial tissues of rats with EAM as compared with the control group. Our findings suggest that Tfh cell balance is disrupted during the pathological process of autoimmune myocarditis.

Overexpression of Peroxisome Proliferator-Activated Receptor γ Coactivator 1-α Protects Cardiomyocytes from Lipopolysaccharide-Induced Mitochondrial Damage and Apoptosis.

Mitochondrial damage is considered one of the main pathogenetic mechanisms in septic cardiomyopathy. Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) is critical for maintaining energy homeostasis in different organs and in various physiological and pathological states. It is also a key regulator gene in mitochondrial metabolism. In this study, we investigated whether regulation of the PGC-1α gene had protective effects on septic cardiomyopathy. We developed a rat model of septic cardiomyopathy. H9c2 myocardiocytes were treated with lipopolysaccharide (LPS) and PGC-1α expression measured. PGC-1α-overexpressing lentivirus was used to transfect H9c2 cells. ZLN005 was used to activate PGC-1α. The effect of the inhibition of PGC-1α expression on myocardial cell injury and its underlying mechanisms were also explored. Cell viability was measured by CCK-8 assay. Mitochondrial damage was determined by measuring cellular ATP, reactive oxygen species, and the mitochondrial membrane potential. An apoptosis analysis kit was used to measure cellular apoptosis. Mitochondrial DNA was extracted and real-time PCR performed. LC3B, mitochondrial transcription factor A (TFA), P62, Bcl2, and Bax were determined by immunofluorescence. LC3B, TFA, P62, Parkin, PTEN-induced putative kinase 1, and PGC-1α proteins were determined by Western blotting. We found mitochondrial damage and apoptotic cells in the myocardial tissue of rats with septic cardiomyopathy and in LPS-treated cardiomyocytes. PGC-1α expression was decreased in the late phase of septic cardiomyopathy and in LPS-treated cardiomyocytes. PGC-1α activation by ZLN005 and PGC-1α overexpression reduced apoptosis in myocardiocytes after LPS incubation. PGC-1α gene overexpression alleviated LPS-induced cardiomyocyte mitochondrial damage by activating mitochondrial biogenesis and autophagy functions. Our study indicated that mitochondrial damage and apoptosis occurred in septic cardiomyopathy and LPS-treated cardiomyocytes. The low expression level of PGC-1α protein may have contributed to this damage. By activating the expression of PGC-1α, apoptosis was reduced in cardiomyocytes. The underlying mechanism may be that PGC-1α can activate mitochondrial biogenesis and autophagy functions, reducing mitochondrial damage and thereby reducing apoptosis.

Simvastatin relieves myocardial ischemia/reperfusion injury in rats through hedgehog signaling pathway.

The aim of this study was to explore the effect of simvastatin (Sim) on myocardial ischemia/reperfusion (I/R) injury in rats and to elucidate the possible underlying mechanism. Our findings might help to provide a certain reference for the clinical prevention and treatment of myocardial I/R injury. A total of 60 male Sprague-Dawley (SD) rats were randomized into three groups using a random number table, including: Sham group (n=20), I/R group (n=20) and I/R + Sim group (n=20). The I/R injury model was successfully established in rats via ligation of the left anterior descending coronary artery (LAD), followed by reperfusion. Before operation, the rats in I/R + Sim group were administered with Sim at 10 mg/kg/d through oral gavage for 7 d. Cardiac ejection fraction (EF) (%) and fractional shortening (FS) (%) of rats in each group were detected using echocardiography. 2,3,5-triphenyltetrazolium chloride (TTC) staining was performed to measure the myocardial infarction (MI) area in each group. Collagen deposition in myocardial tissues of rats in each group was detected by Masson's trichrome staining. The apoptosis level of myocardial cells and fibroblasts in myocardial tissues of rats in each group were evaluated via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The level of reactive oxygen species (ROS) in myocardial tissues of rats in each group was determined using fluorescent probes. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was conducted to measure the expression levels of pro-inflammatory cytokines interleukin (IL)-1β and monocyte chemoattractant protein-1 (MCP-1) in myocardial tissues of rats in each group. Furthermore, the effects of Sim on the hedgehog signaling pathway-associated proteins were detected using Western blotting. Sim significantly alleviated I/R-induced cardiac dysfunction in rats and increased EF (%) and FS (%) (p<0.05). Meanwhile, it also evidently mitigated the MI caused by I/R and reduced the infarction area (p<0.05). According to the Masson's trichrome staining results, I/R + Sim group exhibited remarkably declined myocardial interstitial collagen deposition compared with I/R group (p<0.05). ROS-sensitive fluorescent staining showed that Sim notably reversed the increase of ROS expression and the decrease of myocardial oxidative stress induced by I/R (p<0.05). Finally, Western blotting results revealed that Sim dramatically restrained the protein expressions of sonic hedgehog (SHH), patched 1 (PTC1) and glioma-associated oncogene homolog 1 (GLI1) (p<0.05). Sim can significantly relieve myocardial I/R injury in rats. The possible underlying mechanism may be related to its inhibition on the hedgehog signaling pathway.

LncRNA HULC alleviates HUVEC inflammation and improves angiogenesis after myocardial infarction through down-regulating miR-29b.

The aim of this study was to investigate the long non-coding RNA (lncRNA) HULC in promoting angiogenesis after myocardial infarction (MI) and to further investigate its possible mechanism. Twenty-four male Sprague Dawley (SD) rats were randomly divided into two groups, namely, operation group and control group. The rats in the operation group were induced by ligation of the left anterior descending coronary artery, while those in control group received the same surgery without ligating the blood vessels. Seven days after the operation, the myocardial tissues of rats were collected to detect HULC expression by quantitative real-time polymerase chain reaction (RT-PCR). At the same time, the expression of HULC in primary myocardial cells and cardiac microvascular endothelial cells were induced by hypoxia. A hypoxia model was constructed in HUVEC cells, and the effects of HULC were explored by RT-PCR, Western blot Technology (WB), Cell Counting Kit-8 (CCK8) assay, EdU staining, Tube-like structure formation experiments. Thereafter, HULC downstream miRNAs were verified by Luciferase, pull-down, and RNA IP experiments. Similarly, the effects of miR-29b on HUVEC were verified by RT-PCR, WB, CCK8 assay, EdU staining, and tube-like structure formation experiments, respectively. RT-PCR detection results showed that the expression of HULC in myocardial tissues was down-regulated after MI, and the expression of HULC in cardiac microvascular endothelial cells was decreased under hypoxia-induced inflammation. In addition, the overexpression of HULC in HUVEC cells could inhibit the expressions of inflammatory factors (IL-1, IL-6 and IL-8) and promote angiogenesis (increased cell viability, increased tube-like structure formation, and increased cell proliferation). Through Dual-Luciferase reporter gene experiments, it was found that HULC could directly target miR-29b. At the same time, miR-29 overexpression significantly reversed the effects of HULC on cell viability, pro-inflammatory cytokines, and angiogenesis. LncRNA HULC protects HUVEC cells from hypoxia-induced inflammation damage by interacting with miR-29b and inhibiting its expression, and it can also promote angiogenesis.

Kallistatin alleviates heart failure in rats by inhibiting myocardial inflammation and apoptosis via regulating sirt1.

Heart failure (HF) is the loss of myocardial structure and function caused by various congenital or acquired heart diseases. This study explored the new target of treatment of HF by investigating the effect of Kallistatin (KS) on inflammation and apoptosis of myocardial tissue in HF rats. We used doxorubicin to induce rat HF, and determined the success rate of modeling by detecting changes in rat heart weight and body weight, cardiac function and histology. We used two different doses (1 mg/kg, 2 mg/kg) of KS intraperitoneally injected rats and detected changes in inflammation and apoptosis of rat myocardial tissue by enzyme-linked immunosorbent assay (ELISA), Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunohistochemical staining. Changes in the expression of sirt1 were also detected. In addition, we cultured rat myocardial cell line, H9c2 cells, and used siRNA-sirt1 to inhibit sirt1 in H9c2 cells to clarify the mechanism of KS regulating myocardial cells. The body weight of HF rats treated with KS decreased while the heart weight increased. KS has also been found to reduce the concentration of brain natriuretic polypeptide (BNP) in rat serum. The results of echocardiography showed that KS effectively relieved the cardiac function of HF rats. Inflammatory factors (interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α) and pro-apoptotic molecules (caspase3/9 and Bax) in the serum and myocardial tissue of rats treated with KS were also significantly reduced. The inhibition of sirt1 in H9c2 cells significantly reduced the anti-apoptotic effect of KS on H9c2 cells. KS reduces the inflammation and apoptosis of myocardial tissue in HF rats by promoting the expression of sirt1, thereby alleviating HF-induced myocardial injury.