Decreased LDH Release Research Articles

Lnc-PXMP4-2-4 alleviates myocardial cell damage by activating the JAK2/STAT3 signaling pathway

PurposeThe aim of this study was to investigate the protective effect of long non-coding lnc-PXMP4-2-4 on myocardial cell damage caused by acute myocardial infarction (AMI). MethodsPeripheral blood mononuclear cells (PBMC) were collected from 24 patients with AMI on the day of admission, the first day after percutaneous coronary intervention (PCI) and the third day after surgery, and 24 patients with clinical control group. Real-time quantitative PCR(QRT-PCR) was used to detect the expression of related genes. Then in human cardiomyocytes (AC16), Cell Counting Kit-8 (CCK-8) was used to determine cell viability, lactate dehydrogenase release assay (LDH) was used to determine the release of lactate dehydrogenase, PCR was used to detect the expression of genes, cell death was detected by flow cytometry, and the expression of related proteins was measured by Western blot. The effect of lnc-PXMP4-2-4 was further studied by silencing and overexpressing lnc-PXMP4-2-4. ResultsCompared with clinical control group, the expression of lnc-PXMP4-2-4 in PBMC of AMI patients was significantly higher than it. Compared with pre-operation, the expression of lnc-PXMP4-2-4 was significantly up-regulated on day 1 after PCI, and recovered to pre-operation level on day 3 after surgery. In AC16 cells, lnc-PXMP4-2-4 inhibited the proliferation of AC16, promoted the release of LDH and increased cell death, aggravated the cardiomyocyte injury caused by H2O2, and inhibited the expression of JAK2 and STAT3 mRNA and protein. The up-regulation of lnc-PXMP-4-2-4 had the opposite effect. In addition, the inhibition of the signal pathway by JAK2/STAT3 pathway inhibitor AG490 partially weakened the enhanced viability of AC16 cells, decreased LDH release and apoptosis induced by lnc-PXMP4-2-4 overexpression, increased Bcl-2 expression and down-regulated Bax expression. ConclusionTherefore, we conclude that lnc-PXMP4-2-4 protects cardiomyocytes from injury by activating the JAK2/STAT3 signaling pathway.

#4904 EPAC1- MEDIATED CAMP SIGNALLING PROMOTES CELLULAR ENERGY ADAPTATIONS IN PODOCYTES TO PROTECT FROM GLOMERULONEPHRITIS

Abstract Background and Aims Many efforts are made to identify new therapeutic targets to slow down, prevent or even reverse Chronic Kidney Disease (CKD) progression. One of the therapeutic approaches is activating the renoprotective cAMP pathway, especially by stimulation of its downstream effector, the protein kinase A (PKA). PKA was considered as the unique cAMP effector, however, the exchange factor directly activated by cAMP 1 (EPAC1) has been recently identified as a novel, PKA-independent, mediator of cAMP signalling. EPAC1 is a guanidine exchange factor that promotes the exchange of GDP for GTP regulating important cellular functions. Epac1 activation exerts a renoprotective effect during acute kidney injury, via maintenance of epithelial adhesion and protection from oxidative stress. However, the role of EPAC1 in CKD remains poorly understood. Here we aim to determine the role of EPAC1 in CKD progression. Method Nephrotoxic serum glomerulonephritis (NTS-GN) is induced in genetically modified mice with total and conditional EPAC1 deletion. Then isolated glomeruli from mice with conditional EPAC1 deletion in podocytes are analysed by RNA-sequencing. The main metabolic energy pathways are studied in podocytes in vitro under oxidative stress exposure in the presence or absence of an EPAC1 agonist. Results Following the induction of NTS-GN, mice with genetic deletion of EPAC1 show aggravated renal disease, characterized by increased proteinuria, glomerular damage, tissue inflammation and fibrosis compared to wild-type mice. Conversely, pharmacological activation of EPAC1, with the agonist 8-pCPT-2-OMe-cAMP, delays NTS-GN progression. Since in human and wild type mouse kidney tissues we observe EPAC1 expression in podocytes, mice with conditional deletion of EPAC1 in podocytes (Nphs2Cre:epac) are generated. Similar to the whole-body knockout, mice with EPAC1 deletion in podocytes show increased renal damage and worsened disease progression compared to control mice. RNA-sequencing analysis of glomeruli isolated from these mice show that gene expression of proteins linked to the pathway of glycolysis are abolished in early stage of NTS-GN (day 4). These data suggest that EPAC1-mediated activation of glycolysis in podocytes is essential to limit GN progression. This is substantiated by the in vitro experiments in podocytes, in which EPAC1 activation under oxidative stress promotes glycolysis with cellular energy production independently from mitochondrial respiration. The EPAC1-mediated glycolysis protected podocytes by increasing cell viability, decreasing LDH release and activating the AKT pathway. Conclusion Our results suggest a protective role of podocytes-derived EPAC1 against the development of GN through cellular energetic adaptations based on metabolic switch to glycolysis. Activating the cAMP-EPAC1 signalling axis could represent a therapeutic option to delay the development of CKD. Further investigations are needed to define its relevance in human CKD.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomal KLF4 Alleviated Ischemic Stroke Through Inhibiting N6-Methyladenosine Modification Level of Drp1 by Targeting lncRNA-ZFAS1

Ischemic stroke hasbecome a serious public health problem that causes high rates of death and disability. Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have shown promising therapeutic results in IS, while the underlying mechanisms need further investigation. Cell and mice models were established through oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and middle cerebral artery occlusion (MCAO)/reperfusion. Exosomes were isolated from BMSCs. Related gene and protein expression was measured by qRT-PCR, Western blotting, and immunofluorescence analysis. The biological functions of treated cells and tissues were analyzed by MTT, ELISA, JC-1, flow cytometry, TTC staining, or TUNEL staining. The interaction of KLF4/lncRNA-ZFAS1 promoter and lncRNA-ZFAS1/FTO was measured by ChIP, dual-luciferase reporter, or RIP assays. The m6A levels of Drp1 were measured by MeRIP-PCR. Mitochondrial staining and transmission electron microscopy (TEM) were used to evaluate the mitochondrial morphology in N2a cells and brain tissues. BMSC-derived exosomes increased the viability of neuronal cells treated with OGD/R while decreasing LDH release, oxidative stress, mitochondrial injury, and apoptosis. Furthermore, these effects were abolished by knockdown of exosomal KLF4. KLF4 increased lncRNA-ZFAS1 through binding to its promoter. LncRNA-ZFAS1 overexpression suppressed the m6A levels of Drp1 and reversed the promoting effect of exosomal KLF4 silencing on mitochondrial injury and the imbalance of mitochondrial dynamics by targeting FTO. Exosomal KLF4 alleviated the infarct area, neuronal injury, and apoptosis in MCAO mice through lncRNA-ZFAS1/FTO/Drp1 axis. BMSC-derived exosomal KLF4 promoted lncRNA-ZFAS1 expression to repress Drp1 m6A modification by targeting FTO, thus reducing mitochondrial dysfunction and alleviating neuronal injury in ischemic stroke.

Surface grafting of sericin onto thermoplastic polyurethanes to improve cell adhesion and function

Thermoplastic polyurethane (TPU) membrane has super physical-mechanical properties and biocompatibility, but the surface is inert and lack of active groups which limit its application in cell culture. Silk sericin (SS) can improve cell adhesion, proliferation, growth and metabolism. In this paper, SS was grafted onto the surface of TPU membrane by -NH2 bridge to build a high efficiency cell culture membrane. The FT-IR spectrum results indicated SS was grafted by chemical bond. According to the SEM and AFM results, we found that the grafting of SS reduced the water contact angle by 43.31% and increased the surface roughness by about four times. When TPU-SS was used for HepG2 cell culture, the cell adhesion rate of TPU-SS was significantly higher than that of the general TCPS cell culture plate, and the cell proliferation rate was close to that of TCPS. FDA/EB staining showed that HepG2 cells remained a better cellular growth behavior. HepG2 cells had higher cell vitality including the albumin secretion and the intracellular total protein synthesis. Grafting SS maintained the stability of cell and significantly decreased the cytotoxicity by decreased LDH release. In conclusion, SS grafting is beneficial to cell culture in vitro, and provides a key material for bioartificial liver culture system.

MiR-342-5p protects neurons from cerebral ischemia induced-apoptosis through regulation of Akt/NF-κB pathways by targeting CCAR2

ObjectivesIschemic stroke causes high morbidity, mortality and health burden in the world. MiR-342-5p was associated with Alzheimer's disease and cardio-protection. Herein, we aimed to reveal effects of miR-342-5p on cerebral ischemia injury as well as novel targets for stroke. Materials and methodsAgomiR-342-5p was intracerebroventricularly injected into the middle cerebral artery occlusion (MCAO) mouse models to evaluate functions of miR-342-5p on cerebral ischemia. RT-qPCR and western blot assays were used to evaluate genes expression. Oxygen-glucose deprivation (OGD) was used as an in vitro model for ischemia. Viability and apoptosis ratio of neurons was evaluated by CCK-8, LDH release detection, and flow cytometry. The potential targets of miR-342-5p were predicted by Targetscan, and their interaction was confirmed by luciferase assay. ResultsThe intervention of miR-342-5p effectively attenuated ischemic injury in MCAO mice. MiR-342-5p overexpression could protect neurons against OGD-induced injury, as revealed by increased cell viability and BCL2 expression, and decreased LDH release, apoptosis ratio, and BAX expression in OGD-induced neurons. Mechanically, miR-342-5p could directly bound with CCAR2 to inhibit its expression. Overexpressing CARR2 aggravated the OGD-induced injury of neurons, which was partly restrained by overexpressing miR-342-5p reversed. Furthermore, miR-342-5p/CARR2 axis regulates Akt/NF-κB signaling pathway in vitro as well as in vivo cerebral ischemia models. ConclusionsMiR-342-5p inhibited neuron apoptosis by regulating Akt/NF-kB signaling pathway via CCAR2 suppression. Our findings revealed the neuroprotection of miR-342-5p in cerebral ischemia.

Abstract 113: Protection Of Cardiomyocytes Against Hypoxia/reoxygenation Injury: Interaction Of Poloxamer 188 And Endothelial Cells

Administration of Poloxamer (P) 188, a copolymer-based cell membrane stabilizer, significantly improves myocardial ischemia/reperfusion (IR) injury through its ability to protect cell membranes. Cell-to-cell interactions between endothelial cells (ECs) and cardiomyocytes (CMs) further protect CM from IR injury. Thus, we established a co-culture model of ECs and CMs and could previously demonstrate that the mere presence of ECs potentiates cardioprotection as evidenced by dose-dependent attenuation of LDH release from CMs caused by hypoxia/reoxygenation (HR). Here, we sought to determine if P188 can further potentiate the attenuation of LDH release in the presence of different ECs densities. Mouse CMs cultured in wells underwent 24 h hypoxia followed by 2 h reoxygenation. Mouse coronary artery ECs were plated in well-inserts at densities of 25,000, 50,000, or 100,000 per well which were placed into the CM wells. P188 at 0, 30, 100, 300, or 1,000 μM was administered upon reoxygenation. Statistics: ANOVA and SNK, alpha 0.05. ECs did not have a protective effect by themselves when a low number of ECs (25,000 per well) were present, but P188 could reduce LDH release in CMs after HR injury in a dose-dependent manner (lower panel). When a high number of ECs (100,000 per well) were co-cultured with CMs, ECs themselves exerted a protective effect while P188 did not further decrease LDH release significantly (upper panel). An intermediate number of ECs (50,000 per well) did not produce significant results in either direction (center panel). Our data suggests that the EC-mediated protection of CMs depends on EC density; furthermore, while at low density the role of ECs was limited and allowed P188 to promote additional protection against HR injury, at high density ECs appeared to saturate cardioprotection and blunt additional P188’s CM protection. These findings suggest an important, yet not additive interaction between the protection conferred by ECs and that by P188.

Resveratrol, novel application by preconditioning to attenuate myocardial ischemia/reperfusion injury in mice through regulate AMPK pathway and autophagy level.

Myocardial ischemia/reperfusion injury (MI/RI) is the main cause of deaths in the worldwide, leading to severe cardiac dysfunction. Resveratrol (RSV) is a polyphenol plant‐derived compound. Our study aimed to elucidate the underlying molecular mechanism of preconditioning RSV in protecting against MI/RI. Mice were ligated and re‐perfused by the left anterior descending branch with or without RSV (30 mg/kg·ip) for 7 days. Firstly, we found that RSV pretreatment significantly alleviated myocardial infarct size, improved cardiac function and decreased oxidative stress. Furthermore, RSV activated p‐AMPK and SIRT1, ameliorated inflammation including the level of TNF‐α and IL‐1β, and promoting autophagy level. Moreover, neonatal rat ventricular myocytes (NRVMs) and H9c2 cells with knockdown the expression of AMPK, SIRT1 or FOXO1 were used to uncover the underlying molecular mechanism for the cardio‐protection of RSV. In NRVMs, RSV increased cellular viability, decreased LDH release and reduced oxidative stress. Importantly, Compound C(CpC) and EX527 reversed the effect of RSV against MI/RI in vivo and in vitro and counteracted the autophagy level induced by RSV. Together, our study indicated that RSV could alleviate oxidative stress in cardiomyocytes through activating AMPK/SIRT1‐FOXO1 signallingpathway and enhanced autophagy level, thus presenting high potential protection on MI/RI.

Anfibatide alleviates inflammation and apoptosis via inhibiting NF-kappaB/NLRP3 axis in ischemic stroke

Recent evidence suggests that Nod-like receptor protein-3 (NLRP3) inflammasome is a key mediator of inflammatory response and can induce the activation of apoptosis signaling pathways in ischemic stroke. In this research, we assessed the effects of anfibatide (ANF) on inflammatory and apoptosis in cerebral ischemic injury and the potential mechanisms. Middle cerebral artery occlusion (MCAO) model was established on male Sprague-Dawley rats to induce cerebral ischemia/reperfusion (I/R) injury in vivo. Primary cortical neurons (PCN) cells were exposed to oxygen-glucose deprivation and reintroduction (OGD/R) to mimic cerebral I/R injury in vitro. The results showed that ANF markedly alleviated infarct volume, neurological deficit and neurobehavioral impairment in MCAO/R rats, enhanced cell viability and decreased LDH release in PCN after OGD/R. The number of TUNEL-positive cells, Bax, cleaved-caspase-3, p-IκBα, p-p65, NLRP3, ASC, cleaved caspase-1, IL-β and IL-18 proteins expression were significantly upregulated in the cortex of MCAO/R rats and PCN exposed to OGD/R, NLRP3 and caspase-1 mRNA levels were also evidently elevated. Bcl-2 protein expression significantly decreased in the cortex of MCAO/R rats. Treatment with ANF obviously inhibited the expression of p-IκBα, p-p65, NLRP3, ASC, cleaved caspase-1, Bax and cleaved-caspase-3, promoted the expression of Bcl-2, then decreased the TUNEL-positive cell number and the level of inflammatory cytokines (IL-β and IL-18) in cerebral ischemia reperfusion in vito and in vitro. Our findings suggest that ANF exerts effects of alleviating inflammation and apoptosis through inhibiting NF-kappaB/NLRP3 axis. ANF is a potential candidate for treating cerebral I/R injury.

New Antibacterial Chloro-Containing Polyketides from the Alga-Derived Fungus Asteromyces cruciatus KMM 4696.

Six new polyketides acrucipentyns A–F (1–6) were isolated from the alga-derived fungus Asteromyces cruciatus KMM 4696. Their structures were established based on spectroscopic methods. The absolute configurations of acrucipentyn A was assigned by the modified Mosher’s method and ROESY data analysis. Acrucipentyns A–E were identified to be the very first examples of chlorine-containing asperpentyn-like compounds. The cytotoxic and antimicrobial activities of the isolated compounds were examined. Acrucipentyns A–F were found as antimicrobial agents, which inhibited sortase A enzyme activity, bacterial growth and biofilm formation of Staphylococcus aureus and decreased LDH release from human keratinocytes HaCaT in S. aureus skin infection in an in vitro model.

The Protective Effects of Hydrogen Sulfide New Donor Methyl S-(4-Fluorobenzyl)-N-(3,4,5-Trimethoxybenzoyl)-l-Cysteinate on the Ischemic Stroke.

In this paper, we report the design, synthesis and biological evaluation of a novel S-allyl-l-cysteine (SAC) and gallic acid conjugate S-(4-fluorobenzyl)-N-(3,4,5-trimethoxybenzoyl)-l-cysteinate (MTC). We evaluate the effects on ischemia-reperfusion-induced PC12 cells, primary neurons in neonatal rats, and cerebral ischemic neuronal damage in rats, and the results showed that MTC increased SOD, CAT, GPx activity and decreased LDH release. PI3K and p-AKT protein levels were significantly increased by activating PI3K/AKT pathway. Mitochondrial pro-apoptotic proteins Bax and Bim levels were reduced while anti-apoptotic protein Bcl-2 levels were increased. The levels of cleaved caspase-9 and cleaved caspase-3 were also reduced in the plasma. The endoplasmic reticulum stress (ERS) was decreased, which in turns the survival rate of nerve cells was increased, so that the ischemic injury of neurons was protected accordingly. MTC activated the MEK-ERK signaling pathway and promoted axonal regeneration in primary neurons of the neonatal rat. The pretreatment of MEK-ERK pathway inhibitor PD98059 and PI3K/AKT pathway inhibitor LY294002 partially attenuated the protective effect of MTC. Using a MCAO rat model indicated that MTC could reduce cerebral ischemia-reperfusion injury and decrease the expression of proinflammatory factors. The neuroprotective effect of MTC may be due to inhibition of the over-activation of the TREK-1 channel and reduction of the current density of the TREK1 channel. These results suggested that MTC has a protective effect on neuronal injury induced by ischemia reperfusion, so it may have the potential to become a new type of neuro-ischemic drug candidate.

Z-Guggulsterone alleviates renal fibrosis by mitigating G2/M cycle arrest through Klotho/p53 signaling

Chronic kidney disease (CKD) has become a major public health problem worldwide. Renal fibrosis is considered to be the final outcome and potential therapeutic target of CKD. Z-Guggulsterone (Z-GS), an active compound derived from Commiphora mukul, has been proved to be effective in various diseases. The present study was aimed to evaluate the effect and mechanism of Z-GS on renal fibrosis. Unilateral ureteral obstruction (UUO) mice and hypoxia-induced HK-2 cells were used to simulate renal fibrosis, respectively. The mice and cells were treated with different doses of Z-GS to observe the pharmacological action. Results demonstrated that Z-GS lightened renal function and histopathological injury induced by UUO. Z-GS also alleviated renal fibrosis in mice by inhibiting the expressions of α-SMA, TGF-β, and Collagen Ⅳ. Besides, Z-GS delayed G2/M cycle arrest by promoting the expressions of CDK1 and CyclinB1. Experiments in vitro indicated that Z-GS increased cell viability while decreased LDH release in hypoxia-induced HK-2 cells. In addition, fibrosis and G2/M cycle arrest induced by hypoxia in HK-2 cells were retarded by Z-GS. The study of its possible mechanism exhibited that Z-GS increased the level of Klotho and inhibited p53 level. Nevertheless, the effect of Z-GS on Klotho/p53 signaling was reversed by siRNA-Klotho. Moreover, siRNA-Klotho eliminated the effects of Z-GS on G2/M cycle arrest and fibrosis. Taken together, this study clarified that Z-GS alleviated renal fibrosis and G2/M cycle arrest through Klotho/p53 signaling. People who have suffered CKD may potentially benefit from treatment with Z-GS.

Artemisinin attenuated ischemic stroke induced cell apoptosis through activation of ERK1/2/CREB/BCL-2 signaling pathway in vitro and in vivo.

Ischemic stroke is characterized by the presence of both brain ischemic and reperfusion-induced injuries in the brain, leading to neuronal dysfunction and death. Artemisinin, an FDA-approved antimalarial drug, has been reported to have neuroprotective properties. However, the effect of artemisinin on ischemic stroke is not known. In the present study, we investigated the effect of artemisinin on ischemic stroke using an oxygen-glucose deprivation/reperfusion (OGD/RP) cellular model and a mouse middle cerebral artery occlusion (MCAO) animal model and examined the underlying mechanisms. The obtained results revealed that a subclinical antimalarial concentration of artemisinin increased cell viability and decreased LDH release and cell apoptosis. Artemisinin also attenuated the production of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (Δψm). Importantly, artemisinin attenuated the infarction volume and the brain water content in the MCAO animal model. Artemisinin also improved neurological and behavioural outcomes and restored grasp strength and the recovery of motor function in MCAO animals. Furthermore, artemisinin treatment significantly inhibited the molecular indices of apoptosis, oxidative stress and neuroinflammation and activated the ERK1/2/CREB/BCL-2 signaling pathway. Further validation of the involved signaling pathway by the ERK1/2 inhibitor PD98059 revealed that inhibiting the ERK1/2 signaling pathway or silencing ERK1/2 reversed the neuroprotective effects of artemisinin. These results indicate that artemisinin provides neuroprotection against ischemic stroke via the ERK1/2/CREB/BCL-2 signaling pathway. Our study suggests that artemisinin may play an important role in the prevention and treatment of stroke.

In vitro neuroprotective effects of allicin on Alzheimer’s disease model of neuroblastoma cell line

Background/Aim: Alzheimer's disease is a progressive disorder that causes atrophy and neuronal death in the brain. Currently, there is not any effective therapy for Alzheimer's disease. The current research was designed to investigate the beneficial effects of allicin on Alzheimer’s disease in SHSY-5Y cells in vitro and elucidating the neuroprotective mechanism of allicin. Methods: Human neuroblastoma cell line (SH-SY5Y) was differentiated with retinoic acid to conduct the in vitro Alzheimer’s Disease model. Amyloid β1-42 protein was applied to the cells for 24 hours (2.5 µM) to induce cytotoxicity. Allicin was applied to the cell cultures in a wide spectrum dose (10 μM, 50 μM, 100 μM) to investigate neuroprotective effect against amyloid β for 24 hours. MTT and LDH analyses were performed to assess the cell viability. MDA and ROS concentrations and SOD activity were analyzed to determine the oxidative stress. Moreover, the effects of allicin on the caspase-3 expression in amyloid β induced neurotoxicity were determined by the RT-PCR analysis. Results: Amyloid β markedly decreased cell viability of SH-SY5Y in MTT analysis and elevated LDH levels. In contrast, in MTT analysis, the allicin markedly increased cell viability, indicating that allicin induces cell proliferation. Moreover, in LDH analysis, allicin treatment markedly decreased LDH release. Exposure to amyloid β markedly increased MDA and ROS levels, in comparison with the control. Moreover, amyloid β decreased activity of SOD in SH-SY5Y cells. Allicin markedly balanced out the amyloid β-induced MDA and ROS generation. In the same pathway, allicin increased activity of SOD in amyloid β -exposed SH-SY5Y cells. The caspase 3 expression was increased in amyloid β group in comparison to the control group and allicin markedly lowered the expression of caspase-3 levels. Conclusion: The beneficial effects of allicin on amyloid β -induced neurotoxicity on SH-SY5Y cells were reported for the first time in terms of cell viability, oxidative stress and apoptosis.

Microtubule affinity regulating kinase 4 promoted activation of the NLRP3 inflammasome-mediated pyroptosis in periodontitis

ABSTRACT Background Microtubule dynamics plays a crucial role in the spatial arrangement of cell organelles and activation of the NLRP3 inflammasome. Purpose This study aimed to explore whether microtubule affinity regulating kinase 4 (MARK4) can be a therapeutic target of periodontitis by affecting microtubule dynamics and NLRP3 inflammasome-mediated pyroptosis in macrophages. Materials and Methods The NLRP3 inflammasome-related genes and MARK4 were measured in the healthy and inflamedhuman gingival tissues. Bone marrow-derived macrophages (BMDMs) were infected with Porphyromonas gingivalis,while the MARK4 inhibitors (OTSSP167 and Compound 50) and small interference RNAwere utilized to restrain MARK4. Apoptosis-associated speck-like protein (ASC)speck was detected by confocal, and levels of interleukin-1β (IL-1β), as wellas IL-18, were assessed by ELISA. Results Increased staining and transcription of MARK4, NLRP3, ASC, and Caspase-1 were observed in the inflamed gingiva. P. gingivalis infection promoted MARK4 expression and the NLRP3 inflammasome in BMDMs. Inhibition of MARK4 decreased LDH release, IL-1β and IL-18 production, ASC speck formation, and the pyroptosis-related genes transcription. Furthermore, MARK4 inhibition reduced microtubule polymerization and acetylation in P. gingivalis-infected BMDMs. Conclusions MARK4 promoted NLRP3 inflammasome activation and pyroptosis in P. gingivalis-infected BMDMs by affecting microtubule dynamics. MARK4 inhibition might be a potential target in regulating the NLRP3 inflammasome during periodontitis progress.

Melatonin Alleviates LPS-Induced Pyroptotic Cell Death in Human Stem Cell-Derived Cardiomyocytes by Activating Autophagy.

Endotoxemia in sepsis remains a problem due to a lack of effective strategies. Our previous studies have demonstrated that melatonin (Mel) protects against ischemic heart injury and arteriosclerosis. However, its role in endotoxemia-exposed cardiomyocytes remains poorly understood. This study explored, for the first time, the protective effect of Mel on the pyroptosis of human stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to lipopolysaccharide (LPS). Our results showed that treatment with 1 μM or 10 μM Mel for 12 h significantly improved 1 μg/ml LPS-induced hiPSC-CM injuries, as reflected by drastically decreased LDH release and increased cell viability, which was accompanied by the overt induction of autophagy. Specifically, Mel profoundly alleviated LPS-induced cell pyroptosis, as evidenced by decreased propidium iodide (PI) and active caspase-1 double-positive cell rates; suppressed the expression of NLRP3, cleaved caspase-1 (activated form of caspase-1), and GSDMD-NT (functional N-terminal fragment of GSDMD) expression; and inhibited the production of the cleaved IL-1β and cleaved IL-18 cytokines. Additionally, double-membrane autophagosomes were observed in LPS-injured hiPSC-CMs treated with 1 μM or 10 μM Mel. The hiPSC-CMs treated with LPS exhibited considerably fewer acidic vesicles (as revealed by LAMP1 staining) and autophagosomes (as revealed by LC3-II staining); however, Mel reversed this outcome in a dose-dependent manner. Furthermore, coincubation with rapamycin (an autophagy activator) or 3-MA (an autophagy inhibitor) accentuated and attenuated the antipyroptotic actions of Mel, respectively. Collectively, our findings demonstrate that Mel shields hiPSC-CMs against pyroptosis during endotoxemia by activating autophagy.

Salidroside attenuates CoCl2-simulated hypoxia injury in PC12 cells partly by mitochondrial protection

Salidroside has been shown to exert neuroprotective effects against hypoxia. However, its mitochondrial protective mechanisms still remain elusive. The present study aimed to explore the mitochondrial protection of salidroside on PC12 cells and the involved mechanisms. The hypoxic injury of PC12 cells was triggered by CoCl2 stimulus. The contents of LDH release, SOD, GSH-PX, Na+-K+-ATPase, ATP, NAD+ and NADH were determined by using commercial biochemical kits. Clark-type oxygen electrode and Seahorse XFe24 analyzer were employed to evaluate cell respiration and measure oxygen consumption rate (OCR), respectively. Mitochondrial swelling and mitochondrial membrane potential (MMP) were measured by using isolated mitochondria from the brain tissue of mice. The proteins expression of cleaved Caspase-3, HIF-1α, ISCU1/2, COX10 and PFKP were tested by immunofluorescence and Western blot. While the genes expression of Caspase-3, HIF-1α, ISCU1/2, COX10 and miR-210 were tested by quantitative real-time PCR (qRT-PCR) analysis. Salidroside alleviated CoCl2-induced oxidative stress in PC12 cells as evidenced by increased cell viability, decreased LDH release and elevated GSH-PX and SOD activities. Salidroside could inhibit apoptosis by suppressing the level of cleaved Caspase-3 and Caspase-3. The enhanced mitochondrial energy synthesis by salidroside treatment was evidenced by the increases of Na+-K+-ATPase activity, ATP content, NAD+/NADH ratio, cellular respiration and OCR. In addition, salidroside could reduce mitochondrial swelling and MMP dissipation in isolated mitochondria. The results of immunofluorescence, Western blot and qRT-PCR analyses further revealed that salidroside raised the level of HIF-1α, ISCU1/2, COX10, and miR-210. Collectively, salidroside can reverse CoCl2-simulated hypoxia injury in PC12 cells partly by mitochondrial protection via inhibiting oxidative stress event, anti-apoptosis and enhancing mitochondrial energy synthesis.

Inhibition of miR-134-5p protects against kainic acid-induced excitotoxicity through Sirt3-mediated preservation of mitochondrial function

Epilepsy is a neurological disorder which is characterized by brain hyper-excitability and manifests as seizure. Due to its complicated pathogenesis, treatment for epilepsy still remains a huge challenge for neurology in the whole world. MciroRNA-134 (miR-134) is one kind of miRNAs which was firstly found abundant in synapses. In this study, we tried to unveil the role of inhibiting MciroRNA-134-5p (miR-134-5p) in excitotoxicity induced by kainic acid (KA) in the hippocampal neurons (HT22) cells. The results showed that treatment of KA increased the expression of miR-134-5p significantly and caused marked neuron excitotoxicity, evidenced by risen cell death rate, higher LDH release and aggravated cell viability. After suppressing miR-134-5p expression via transfecting HT22 cells with miR-134-5p antisense (Anti-134), cell viability was promoted obviously, along with decreased LDH release and cell death rate. In addition, KA-induced lipid peroxidation, cytochrome c release and mitochondrial ROS generation were also attenuated by Anti-134. The level of Sirtuin 3 (Sirt3) and its downstream antioxidant enzymes, such as mitochondrial superoxide dismutase 2 (SOD2), isocitrate dehydrogenase 2 (IDH2) and glutathione peroxidase (GSH-Px), were significantly higher in Anti-134 group compared with the control and scramble group. After inhibiting Sirt3 expression with SiRNA targeting Sirt3 (Si-Sirt3) and 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP), the positive role of Anti-134 was apparently reversed. In conclusion, this research highly suggests that inhibition of miR-134-5p could protect neurons from KA-induced excitotoxicity through Sirt3-mediated preservation of mitochondrial function.

Cardioprotective effect of Butanol Fraction of Rivea ornata against H2O2 Induced Oxidative Stress in H9c2 Cells: Role of its Phenolic Content and Antioxidant Effect

Rivea ornata used traditionally for various disease of heart. However, its protective effect against oxidative stress induced injury is unclear and unproved. So, we attempted to investigate the protective role of butanol fraction of Rivea ornata on H9c2 cardiomyocytes against oxidative stress induced by H2O2. Ethanol extract and its fractions such as n-hexane, ethyl acetate and butanol fractions were assessed for its phytochemical composition, total phenolic and flavonoid content and assessed in vitro antioxidant activity by using established assays such as DPPH, H2O2 neutralization activity, ferric reduction assays and total antioxidant capacity. Among all the extracts/fractions butanol fraction found with high phenolic (82.4 ± 4.61 mg of GAE/g) and flavonoid (105 ± 4.09 mg of QE/g) content as well as possess strong antioxidant abilities evident by comparative lowest IC50 values for DPPH (41.80 µg/mL), H2O2 neutralization assay (35.39 µg/m), highest ferric reduction at 120µg/mL and high antioxidant capacity equivalent to quercetin (72.5±2.98 µg/mL). By considering these results, only butanol fraction assessed for its protective effect H2O2 induced oxidative stress on H9c2 cardiac cell lines was assessed by MTT, LDH assay and ROS expression studies. Butanol fraction at 25µg/mL maintain H9c2 cell lines viability (in MTT assay), cell integrity (decreased LDH release) and decreased ROS expression (decrease in percentage mean fluorescence). These findings are proved antioxidant rich polyphenolic butanol fraction protective effect against oxidative stress injury observed with H2O2 by maintain cell viability, cell integrity and ROS expression.

Inhibition of GPR4 attenuates SH-SY5Y cell injury in cerebral ischemia/reperfusion via anti-apoptotic pathways.

Cerebral ischemia/reperfusion injury (CIRI) can lead to increased vascular endothelial permeability and blood-brain barrier damage in patients with stroke. G protein-coupled receptor 4 (GPR4) is a functional pH sensor that plays a key role in renal ischemia-reperfusion-induced apoptosis. However, whether GPR4 has a role in cerebral ischemia remains to be further studied. Our study found that after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment, the levels of GPR4 and CHOP in SH-SY5Y cells were significantly increased, which was accompanied by a decrease in cell viability, and an increase in LDH release and apoptosis. After knockdown of GPR4 using shRNA, CHOP levels in SH-SY5Y cells were also decreased, which unexpectedly increased cell activity and decreased LDH release and apoptosis rate. Interestingly, CHOP overexpression reversed the effect of GPR4 knockdown, suggesting that OGD/R-induced CIRI may involve endoplasmic reticulum stress-related apoptosis. In conclusion, our study provided a basis for further research on the mechanism of CIRI.

Caspase 3/GSDME-dependent pyroptosis contributes to chemotherapy drug-induced nephrotoxicity

Chemotherapy drug-induced nephrotoxicity limits clinical applications for treating cancers. Pyroptosis, a newly discovered programmed cell death, was recently reported to be associated with kidney diseases. However, the role of pyroptosis in chemotherapeutic drug-induced nephrotoxicity has not been fully clarified. Herein, we demonstrate that the chemotherapeutic drug cisplatin or doxorubicin, induces the cleavage of gasdermin E (GSDME) in cultured human renal tubular epithelial cells, in a time- and concentration-dependent manner. Morphologically, cisplatin- or doxorubicin-treated renal tubular epithelial cells exhibit large bubbles emerging from the cell membrane. Furthermore, activation of caspase 3, not caspase 9, is associated with GSDME cleavage in cisplatin- or doxorubicin-treated renal tubular epithelial cells. Meanwhile, silencing GSDME alleviates cisplatin- or doxorubicin-induced HK-2 cell pyroptosis by increasing cell viability and decreasing LDH release. In addition, treatment with Ac-DMLD-CMK, a polypeptide targeting mouse caspase 3-Gsdme signaling, inhibits caspase 3 and Gsdme activation, alleviates the deterioration of kidney function, attenuates renal tubular epithelial cell injury, and reduces inflammatory cytokine secretion in vivo. Specifically, GSDME cleavage depends on ERK and JNK signaling. NAC, a reactive oxygen species (ROS) inhibitor, reduces GSDME cleavage through JNK signaling in human renal tubular epithelial cells. Thus, we speculate that renal tubular epithelial cell pyroptosis induced by chemotherapy drugs is mediated by ROS-JNK-caspase 3-GSDME signaling, implying that therapies targeting GSDME may prove efficacious in overcoming chemotherapeutic drug-induced nephrotoxicity.