Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) leads to enhanced activity of cardiac fibroblasts (CFs) and abnormal deposition of extracellular matrix proteins, resulting in cardiac fibrosis. Tartrate-resistant acid phosphatase 5 (ACP5) has been shown to promote cell proliferation and phenotypic transition. However, it remains unclear whether ACP5 is involved in the development of cardiac fibrosis after MI. The present study aimed to investigate the role of ACP5 in post-MI fibrosis and its potential underlying mechanisms. METHODS: Clinical blood samples were collected to detect ACP5 concentration. Myocardial fibrosis was induced by ligation of the left anterior descending coronary artery. The ACP5 inhibitor, AubipyOMe, was administered by intraperitoneal injection. Cardiac function and morphological changes were observed on Day 28 after injury. Cardiac CFs from neonatal mice were extracted to elucidate the underlying mechanism in vitro. The expression of ACP5 was silenced by small interfering RNA (siRNA) and overexpressed by adeno-associated viruses to evaluate its effect on CF activation. RESULTS: The expression of ACP5 was increased in patients with MI, mice with MI, and mice with Ang II-induced fibrosis in vitro. AubipyOMe inhibited cardiac fibrosis and improved cardiac function in mice after MI. ACP5 inhibition reduced cell proliferation, migration, and phenotypic changes in CFs in vitro, while adenovirus-mediated ACP5 overexpression had the opposite effect. Mechanistically, the classical profibrotic pathway of glycogen synthase kinase-3ß (GSK3ß)/ß-catenin was changed with ACP5 modulation, which indicated that ACP5 had a positive regulatory effect. Furthermore, the inhibitory effect of ACP5 deficiency on the GSK3ß/ß-catenin pathway was counteracted by an ERK activator, which indicated that ACP5 regulated GSK3ß activity through ERK-mediated phosphorylation, thereby affecting ß-catenin degradation. CONCLUSION: ACP5 may influence the proliferation, migration, and phenotypic transition of CFs, leading to the development of myocardial fibrosis after MI through modulating the ERK/GSK3ß/ß-catenin signaling pathway.

Meteorin-like (METRNL) attenuates hypertensive induced cardiac hypertrophy by inhibiting autophagy via activating BRCA2.

Hypertension, a prevalent cardiovascular ailment globally, can precipitate numerous complications, notably hypertensive cardiomyopathy. Meteorin-like (METRNL) is demonstrated to possess potential protective properties on cardiovascular diseases. However, its specific role and underlying mechanism in hypertensive myocardial hypertrophy remain elusive. Spontaneously hypertensive rats (SHRs) served as hypertensive models to explore the effects of METRNL on hypertension and its induced myocardial hypertrophy. The research results indicate that, in contrast to Wistar-Kyoto (WKY) rats, SHRs exhibit significant symptoms of hypertension and myocardial hypertrophy, but cardiac-specific overexpression (OE) of METRNL can partially ameliorate these symptoms. In H9c2 cardiomyocytes, METRNL suppresses Ang II-induced autophagy by controlling the BRCA2/Akt/mTOR signaling pathway. But when BRCA2 expression is knocked down, this effect will be suppressed. Collectively, METRNL emerges as a potential therapeutic target for hypertensive cardiomyopathy.

Elabela inhibits TRAF1/NF-κB induced oxidative DNA damage to promote diabetic foot ulcer wound healing.

Diabetic foot ulcer (DFU) is a serious complication of diabetes. Elabela (ELA), a ligand of apelin receptor (APJ), was shown to promote angiogenesis and suppress inflammation. This study aimed to illustrate the role of ELA in DFU wound healing. A whole-skin defect model was constructed using db/m and db/db mice to observe the effects of ELA on wound healing. The function of ELA in endothelial cells cultured in high glucose medium was investigated. Administration of ELA in peri-wound area of db/db mice accelerated wound closure and reduced inflammatory infiltration. Indicators of DNA damage, elevated reactive oxygen species (ROS) levels and tail DNA amounts, were downregulated by ELA but compromised after TRAF1 overexpression. ELA-mediated inhibition of NF-κB phosphorylation improved cell migration and angiogenesis, which were blocked by APJ silencing. The findings imply that ELA suppresses TRAF1-mediated NF-κB signal activation, reducing ROS-related oxidative DNA damage and improving protection of endothelial function.

Elabela, a Novel Peptide, Exerts Neuroprotective Effects Against Ischemic Stroke Through the APJ/miR-124-3p/CTDSP1/AKT Pathway.

Elabela (ELA), which is the second endogenous peptide ligand of the apelin receptor (APJ) to be discovered, has been widely studied for potential use as a therapeutic peptide. However, its role in ischemic stroke (IS), which is a leading cause of disability and death worldwide and has limited therapeutic options, is uncertain. The aim of the present study was to investigate the beneficial effects of ELA on neuron survival after ischemia and the underlying molecular mechanisms. Primary cortical neurons were isolated from the cerebral cortex of pregnant C57BL/6J mice. Flow cytometry and immunofluorescence showed that ELA inhibited oxygen-glucose deprivation (OGD) -induced apoptosis and axonal damage in vitro. Additionally, analysis of the Gene Expression Omnibus database revealed that the expression of microRNA-124-3p (miR-124-3p) was decreased in blood samples from patients with IS, while the expression of C-terminal domain small phosphatase 1 (CTDSP1) was increased. These results indicated that miR-124-3p and CTDSP1 were related to ischemic stroke, and there might be a negative regulatory relationship between them. Then, we found that ELA significantly elevated miR-124-3p expression, suppressed CTDSP1 expression, and increased p-AKT expression by binding to the APJ receptor under OGD in vitro. A dual-luciferase reporter assay confirmed that CTDSP1 was a direct target of miR-124-3p. Furthermore, adenovirus-mediated overexpression of CTDSP1 exacerbated neuronal apoptosis and axonal damage and suppressed AKT phosphorylation, while treatment with ELA or miR-124-3p mimics reversed these effects. In conclusion, these results indicated that ELA could alleviate neuronal apoptosis and axonal damage by upregulating miR-124-3p and activating the CTDSP1/AKT signaling pathway. This study, for the first time, verified the protective effect of ELA against neuronal injury after ischemia and revealed the underlying mechanisms. We demonstrated the potential for the use of ELA as a therapeutic agent in the treatment of ischemic stroke.

Protective properties of extracellular vesicles in sepsis models: a systematic review and meta-analysis of preclinical studies.

BACKGROUND: Multiple preclinical studies have reported a beneficial effect of extracellular vesicles (EVs), especially mesenchymal stem cells derived EVs (MSC-EVs), in the treatment of sepsis. However, the therapeutic effect of EVs is still not universally recognized. Therefore, we conducted this meta-analysis by summarizing data from all published studies that met certain criteria to systematically review the association between EVs treatment and mortality in animal models of sepsis. METHODS: Systematic retrieval of all studies in PubMed, Cochrane and Web of Science that reported the effects of EVs on sepsis models up to September 2022. The primary outcome was animal mortality. After screening the eligible articles according to inclusion and exclusion criteria, the inverse variance method of fixed effect model was used to calculate the joint odds ratio (OR) and 95% confidence interval (CI). Meta-analysis was performed by RevMan version 5.4. RESULTS: In total, 17 studies met the inclusion criteria. Meta-analysis of those studies showed that EVs treatment was associated with reduced mortality in animal models of sepsis (OR 0.17 95% CI: 0.11,0.26, P < 0.001). Further subgroup analysis showed that the mode of sepsis induction, the source, dose, time and method of injection, and the species and gender of mice had no significant effect on the therapeutic effect of EVs. CONCLUSION: This meta-analysis showed that MSC-EVs treatment may be associated with lower mortality in animal models of sepsis. Subsequent preclinical studies will need to address the standardization of dose, source, and timing of EVs to provide comparable data. In addition, the effectiveness of EVs in treating sepsis must be studied in large animal studies to provide important clues for human clinical trials.

Peptide hormone ELABELA promotes rat bone marrow-derived mesenchymal stem cell proliferation and migration by manipulating the cell cycle through the PI3K/AKT pathway under the hypoxia and ischemia microenvironment

BACKGROUND: Mesenchymal stem cells (MSCs) are emerging as a potential candidate for stem cell transplantation to repair myocardial tissue in myocardial infarctions (MI). However, there are some pivotal limitations such as poor survival and low migration capacity of MSCs in hypoxic and ischemic microenvironments of MI. Our previous work verified that ELABELA (also abbreviated as ELA), a peptide hormone, could play a role as a growth factor and prolong the life span of rat bone marrow-derived mesenchymal stem cells (RAT BM-MSCs) under hypoxic and ischemic conditions. Nevertheless, the influence of ELA on the cell cycle, proliferation, and migration remains elusive. This study will further explore the improvement of the biological functions of ELA-treated RAT BM-MSCs, so as to provide a reference for improving the efficacy of RAT BM-MSCs in MI. METHODS: Rat BM-MSCs were isolated from 80 to 120 g Sprague Dawley rats by flushing femurs and tibias under the aseptic condition. RAT BM-MSCs of the third passage were divided into control group, hypoxic/ischemic (H/I) group, ELA group, ELA-LY group and LY group. RAT BM-MSCs were cultured under normoxia in control group. In H/I group, RAT BM-MSCs were exposed to hypoxia (1% O2) and serum deprivation for 24 h. RAT BM-MSCs in ELA group were treated with 5 µM ELA prior to the H/I exposure for 24 h. The PI3K/AKT inhibitor, LY294002 (50 µM), was used in ELA-LY group and LY group to observe the effect of ELA on PI3K/AKT activation. Cell proliferation ability was examined by CCK-8. Cell cycle was assessed with flow cytometry. Cell migration was evaluated by Transwell assay. Expression levels of total-AKT, phosphorylated-AKT, and cell cycle-associated proteins were examined by Western blotting. RESULTS: ELA-treated RAT BM-MSCs exhibited significantly higher proliferation ability, cell viability, and migration under H/I conditions. The cell cycle analysis showed that an increased proportion of cells in the S and G2/M phases of the cell cycle were observed in ELA-treated RAT BM-MSCs. The addition of ELA activated the PI3K/AKT signaling pathway. Additionally, upon treating with the inhibitor of the PI3K/AKT signaling pathway, ELA-triggered proliferation, cell viability, and migration were abrogated. CONCLUSIONS: ELA can be used to enhance the proliferation ability, cell viability, and migration of RAT BM-MSCs through the PI3K/AKT signaling pathway and alleviate cell cycle arrest at the G0/G1 phase under hypoxic and ischemic injury. Thus, this study provides a promising strategy that ELA may help to optimize the mesenchymal stem cell-based therapy in MI.

LncRNAs Participate in Post-Resuscitation Myocardial Dysfunction Through the PI3K/Akt Signaling Pathway in a Rat Model of Cardiac Arrest and Cardiopulmonary Resuscitation.

In this study, we aimed to explore the role of lncRNAs in post-resuscitation myocardial dysfunction in a rat model of CA-CPR. A rat model of CA-CPR was constructed using a VF method. Myocardial functions, including cardiac output (CO), ejection fraction (EF), and myocardial performance index (MPI), were evaluated at the baseline, and 1, 2, 3, 4, and 6 h after resuscitation. A high throughput sequencing method was used to screen the differentially expressed lncRNAs, miRNAs, and mRNAs, which were further analyzed with bioinformatics. In addition, relationships between the molecules involved in the PI3K/Akt signaling pathway were explored with ceRNA network. Compared with the sham group, EF was significantly reduced and MPI was increased at the five consecutive time points in the CA-CPR group. 68 lncRNAs were upregulated and 40 lncRNAs were downregulated in the CA-CPR group, while 30 miRNAs were downregulated and 19 miRNAs were upregulated. Moreover, mRNAs were also differentially expressed, with 676 upregulated and 588 downregulated. GO analysis suggested that genes associated with cell proliferation, cell death and programmed cell death were significantly enriched. KEGG analysis showed that the PI3K/Akt, MAPK and Ras signaling pathways were the three most-enriched pathways. Construction of a ceRNA regulatory network indicated that LOC102549506, LOC103689920, and LOC103690137 might play important roles in the regulation of the PI3K/Akt signaling pathway in the CA-CPR treated rat. Taken together, LncRNAs, including LOC102549506, LOC103689920 and LOC103690137, might participate in post-resuscitation myocardial dysfunction by functioning as ceRNAs and regulating the PI3K/Akt signaling pathway.

ELABELA ameliorates hypoxic/ischemic-induced bone mesenchymal stem cell apoptosis via alleviation of mitochondrial dysfunction and activation of PI3K/AKT and ERK1/2 pathways.

BACKGROUND: Mesenchymal stem cells (MSCs) have exerted their brilliant potential to promote heart repair following myocardial infarction. However, low survival rate of MSCs after transplantation due to harsh conditions with hypoxic and ischemic stress limits their therapeutic efficiency in treating cardiac dysfunction. ELABELA (ELA) serves as a peptide hormone which has been proved to facilitate cell growth, survival, and pluripotency in human embryonic stem cells. Although ELA works as an endogenous ligand of a G protein-coupled receptor APJ (Apelin receptor, APLNR), whether APJ is an essential signal for the function of ELA remains elusive. The effect of ELA on apoptosis of MSCs is still vague. OBJECTIVE: We studied the role of ELABELA (ELA) treatment on the anti-apoptosis of MSCs in hypoxic/ischemic (H/I) conditions which mimic the impaired myocardial microenvironment and explored the possible mechanisms in vitro. METHODS: MSCs were obtained from donated rats weighing between 80~120 g. MSCs were exposed to serum-free and hypoxic (1% O2) environments for 24 h, which mimics hypoxic/ischemic damage in vivo, using serum-containing normoxic conditions (20% O2) as a negative control. MSCs that were exposed to H/I injury with ELA processing were treated by 5 µM of ELA. Cell viability and apoptosis of MSCs were evaluated by CCK8 and flow cytometry, respectively. Mitochondrial function of MSCs was also assessed according to mitochondrial membrane potential (MMP) and ATP content. The protein expression of key kinases of the PI3K/AKT and ERK1/2 signaling pathways involving t-AKT, p-AKT, t-ERK1/2, and p-ERK1/2, as well as apoptosis-related protein expression of Bcl-2, Bax, and cleaved Caspase 3, were monitored by Western blot. RESULTS: We found that ELA treatment of H/I-induced MSCs improved overall cell viability, enhanced Bcl/Bax expression, and decreased Caspase 3 activity. ELA inhibited H/I-induced mitochondrial dysfunction by increasing ATP concentration and suppressing the loss of mitochondrial transmembrane potential. However, this anti-apoptotic property of ELA was restrained in APJ-silenced MSCs. Additionally, ELA treatment induced the phosphorylation of AKT and ERK, while the blockade of PI3K/AKT and ERK1/2 pathways with respective inhibitors, LY294002 and U0126, suppressed the action of ELA. CONCLUSION: ELA positively affected on the survival of MSCs and exhibited anti-apoptotic characteristics when exposed to hypoxic/ischemic condition in vitro. Also, the function of ELA was correlated with the APJ receptor, reduced mitochondrial damage, and activation of the PI3K/AKT and ERK1/2 signal axes.

Mild hypothermia protects hippocampal neurons from oxygen-glucose deprivation injury through inhibiting caspase-3 activation.

Mild hypothermia (MH) is thought to be one of the most effective therapeutic methods to treat hypoxic-ischemic encephalopathy (HIE) after cardiac arrest (CA). However, its precise mechanisms remain unclear. In this research, hippocampal neurons were cultured and treated with mild hypothermia and Ac-DEVD-CHO after oxygen-glucose deprivation (OGD). The activity of caspase-3 was detected, in order to find the precise concentration of Ac-DEVD-CHO with the same protective role in OGD injury as MH treatment. Western blot and immunofluorescence staining were conducted to analyze the effects of MH and Ac-DEVD-CHO on the expressions of caspase-3, caspase-8, and PARP. The neuronal morphology was observed with an optical microscope. The lactic acid dehydrogenase (LDH) release rate, neuronal viability, and apoptotic rate were also detected. We found that MH (32 °C) and Ac-DEVD-CHO (5.96 µMol/L) had equal effects on blocking the activation of caspase-3 and the OGD-induced cleavage of PARP, but neither had any effect on the activation of caspase-8, which goes on to activate caspase-3 in the apoptotic pathway. Meanwhile, both MH and Ac-DEVD-CHO had similar effects in protecting cell morphology, reducing LDH release, and inhibiting OGD-induced apoptosis in neurons. They also similarly improved neuronal viability after OGD. In conclusion, caspase-3 serves as a key intervention point of the key modulation site or regulatory region in MH treatment that protects neuronal apoptosis against OGD injury. Inhibiting the expression of caspase-3 had a protective effect against OGD injury in MH treatment, and caspase-3 activation could be applied to evaluate the neuroprotective effectiveness of MH on HIE.

[Effect of autophagy regulator on the injury of rat hippocampal neurons induced by oxygen-glucose deprivation].

OBJECTIVE: To explore the effect of autophagy regulator on the injury of rat hippocampal neurons induced by oxygen-glucose deprivation (OGD). METHODS: Rat hippocampal neurons were cultivated in primary and subjected to OGD to simulate neuronal hypoxic ischemia injury for 2 hours or 6 hours followed by reperfusion for 12 hours with or without 3-methyladenine (3-MA, 20 µmol/L) or rapamycin (0.2 µmol/L). The morphology of neurons was observed with optical microscope. The expression of autophagy-related protein (LC3, P62) and apoptosis-related protein (cleaved caspase-3) were assessed by Western Blot analysis. The apoptosis of neurons was detected by flow cytometry, the release rate of lactate dehydrogenase (LDH) was calculated by automatic biochemical analyzer, and the cell activity was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay. RESULTS: Compared with the control group, the expression of LC3 II/I (gray value: 3.091±0.160, 3.422±0.186 vs. 0.256±0.021), cleaved caspase-3 (gray value: 0.230±0.025, 0.440±0.051 vs. 0.050±0.007), neuronal apoptotic rate, LDH release rate [(38.50±4.15)%, (59.60±5.65)% vs. (12.40±1.32)%] were increased, while the expression of P62 (gray value: 0.290±0.025, 0.120±0.026 vs. 0.450±0.040), neuronal activity [(71.40±7.23)%, (42.80±4.12)% vs. (100.30±2.30)%] were decreased at 2 hours or 6 hours after OGD (all P < 0.05). When the time of OGD was 2 hours and it was combined with 3-MA, the expression of LC3 II/I (gray value: 2.281±0.121), the neuronal activity [(51.10±5.73)%] were decreased, while the expression of P62 and cleaved caspase-3 (gray scale: 0.410±0.037, 0.330±0.027, respectively), neuronal apoptotic rate, the injury of neurons [LDH release rate: (47.30±4.43)%] were increased (all P < 0.05). When the time of OGD was 2 hours and it was combined with rapamycin, the expression of LC3 II/I (gray value: 3.689±0.214), the neuronal activity [(85.30±8.56)%] were increased, while the expression of P62 and cleaved caspase-3 (gray value: 0.170±0.040, 0.090±0.096, respectively), neuronal apoptotic rate, the injury of neurons [LDH release rate: (24.30±2.14)%] were decreased (all P < 0.05). On the contrary, when the time of OGD was 6 hours and it was combined with 3-MA, the expression of LC3 II/I and cleaved caspase-3 (gray value: 3.021±0.178, 0.240±0.017), neuronal apoptotic rate, the injury of neurons [LDH release rate: (36.60±3.45)%] were decreased, while the expression of P62 (gray value: 0.350±0.060), the neuronal activity [(59.70±6.13)%] were increased (all P < 0.05). When the time of OGD was 6 hours and it was combined with rapamycin, the expression of LC3 II/I and cleaved caspase-3 (gray value: 3.923±0.201, 0.590±0.062), neuronal apoptotic rate, the injury of neurons [LDH release rate: (71.20±7.81)%] were increased, while the expression of P62 (gray value: 0.070±0.008), the neuronal activity [(27.30±2.12)%] were decreased (all P < 0.05). CONCLUSIONS: The enhancement of autophagy has protective effect on neurons under the condition of mild OGD, while it can aggravate the injury of neurons induced by a long-time OGD.

Mild hypothermia protects hippocampal neurons against oxygen-glucose deprivation/reperfusion-induced injury by improving lysosomal function and autophagic flux.

Mild hypothermia has been proven to be useful to treat brain ischemia/reperfusion injury. However, the underlying mechanisms have not yet been fully elucidated. The present study was undertaken to determine whether mild hypothermia protects hippocampal neurons against oxygen-glucose deprivation/reperfusion(OGD/R)-induced injury via improving lysosomal function and autophagic flux. The results showed that OGD/R induced the occurrence of autophagy, while the acidic environment inside the lysosomes was altered. The autophagic flux assay with RFP-GFP tf-LC3 was impeded in hippocampal neurons after OGD/R. Mild hypothermia recovered the lysosomal acidic fluorescence and the lysosomal marker protein expression of LAMP2, which decreased after OGD/R.Furthermore, we found that mild hypothermia up-regulated autophagic flux and promoted the fusion of autophagosomes and lysosomes in hippocampal neurons following OGD/R injury, but could be reversed by treatment with chloroquine, which acts as a lysosome inhibitor. We also found that mild hypothermia improved mitochondrial autophagy in hippocampal neurons following OGD/R injury. Finally,we found that chloroquine blocked the protective effects of mild hypothermia against OGD/R-induced cell death and injury. Taken together, the present study indicates that mild hypothermia protects hippocampal neurons against OGD/R-induced injury by improving lysosomal function and autophagic flux.

Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/ß-catenin signaling pathway in hippocampal neurons.

Mild hypothermia is thought to be one of the most effective therapies for cerebral ischemia/reperfusion injuries. Our previous research revealed that mild hypothermia inhibits the activation of caspase-3 and protects against oxygen glucose deprivation/reoxygenation (OGD/R)-induced injury in hippocampal neurons. However, the mechanisms behind the activation of caspase-3 remain unclear. The aims of this study were to determine whether the protective effects of mild hypothermia were exerted through the Wnt/ß-catenin signaling pathway. We found that, under OGD/R conditions, the pathway was down regulated, but mild hypothermia induced the reactivation of the Wnt/ß-catenin signaling pathway, which had been suppressed by OGD/R injury. Mild hypothermia also caused the down regulation of the expression of apoptosis promoting proteins (Bax cleaved caspase-3), up-regulated the expression of apoptosis inhibiting proteins (Bcl-2), and ameliorated OGD/R injury-induced apoptosis. The protective effects of mild hypothermia were blocked by DKK1 (an antagonist of the canonical Wnt signaling pathway). Taken together, these results indicate that the Wnt/ß-catenin signaling pathway mediates the protective effects of mild hypothermia against OGD/R-induced apoptosis. Our study provides evidence that mild hypothermia reactivates the Wnt/ß-catenin signaling pathway, which is suppressed by OGD/R injury, in hippocampal neurons and protects neurons from OGD/R-induced apoptosis via the reactivation of the Wnt/ß-catenin signaling pathway, ultimately suggesting that mild hypothermia could have therapeutic effects on OGD/R-induced apoptosis.