Increased Lactate Dehydrogenase Release Research Articles

Ulinastatin inhibits NLRP3-induced apoptosis in a PD cell model.

BackgroundEmerging evidence suggests that inflammation induced by the inflammasome plays a crucial role in the course of Parkinson’s disease (PD). Ulinastatin (UTI) has shown significant anti-inflammatory effects. However, few studies have examined whether UTI protects neurons through its anti-inflammatory effects in PD. The purpose of this study is to determine whether UTI exerts neuroprotection in a PD cell model and to explore the mechanisms.MethodsSH-SY5Y cells and nerve growth factor (NGF)-treated PC12 cells were used to establish MPP+ induced PD cell models. Cells were pre-treated with UTI, then cell viabilities were detected using the MTT assay. Lactate dehydrogenase (LDH) release was detected using the LDH release assay kit. Inflammatory factors such as IL-1β, IL-6, and TNF-α were detected using ELISA. The expression levels of TH, NLRP3, caspase-1, ASC, IL-1β, and IL-18 were measured using western blotting, and DA release was detected using HPLC. A NLRP3 activator Nigericin was used to verify the effect of NLRP3 in the neuroprotective mechanism of UTI.ResultsWe observed decreased cell viability, increased apoptosis, and increased inflammatory factors such as IL-1β, IL-6, and TNF-α in the MPP+ induced PD model. We also found decreased DA secretion and TH expression, as well as increased NLRP3, caspase-1, ASC, IL-1α, and IL-18 expression in the MPP+ induced PD model. These changes were significantly attenuated by UTI pre-treatment in a dose dependent manner. NLRP3 activator Nigericin markedly increased LDH release, accelerated apoptosis, increased inflammation levels and decreased DA secretion and TH expression, suggesting that Nigericin eliminated the neuroprotective effect of UTI on MPP+ treated cells.ConclusionsOur data demonstrated that UTI pre-treatment performed a neuroprotective role in the MPP+ induced PD cell models by inhibiting the NLRP3 pathway.

Nephrotoxic Potential of the Three Dichlorobenzene Isomers in Isolated Kidney Cells from Male Fischer 344 Rats

Dichlorobenzenes (DCBs) are commonly used as chemical intermediates in the manufacture of a wide range of agricultural, industrial and pharmaceutical products. Previous studies have shown that 1,4-DCB can induce nephrotoxicity in male rats characterized as α2u-globulin nephropathy. Initially, 1,4-DCB binds to α2u-globulin at extra-renal sites. The 1,4-DCB-protein complex then accumulates in kidney and causes protein droplet formation, ultimately leading to cell death and/or eventually tumor formation. The purpose of this study was to explore the direct cytotoxic effects of the three DCB isomers in isolated kidney cells (IKCs) from male Fischer 344 rats. IKCs (~4 million/ml, 3 ml) were incubated with a DCB (0.25-1.0 mM) or dimethyl sulfoxide (DMSO, vehicle) for 30 or 60 min with shaking at 37oC under a 95% oxygen/5% carbon dioxide atmosphere. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release. In separate experiments, IKCs were pretreated with an antioxidant (ascorbate, 1.0 mM; N-acetyl-L-cysteine, 2.0 mM; glutathione, 1.0 mM) prior to the addition of 1,4-DCB (0.5 mM) and followed by a 60 min co-incubation. 1,4-DCB increased LDH release at all times and concentrations tested. 1,3-DCB increased LDH release at 0.5 mM or greater at both time points, while 1,2-DCB only increased LDH release at 0.5 mM or greater at 60 min. Thus, the order of decreasing nephrotoxic potential was 1,4-DCB > 1,3- DCB > 1,2-DCB. Pretreatment with any of the antioxidants tested attenuated 1,4-DCB nephrotoxicity. These results indicate that DCBs can induce nephrotoxicity in vitro without the presence of extra-renal α2u-globulin, 1,4-DCB is the most potent nephrotoxicant among the three DCB isomers in IKCs, and free radicals play a role in DCB nephrotoxicity.

LncRNA OIP5-AS1 accelerates ox-LDL-treated HUVECs injury by NF-κB pathway via miR-30c-5p.

Oxidized low-density lipoprotein (ox-LDL) could induce endothelial injury and played a vital role in the progression and development of atherosclerosis. This study aimed to investigate the role of Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in ox-LDL-induced human umbilical vascular endothelial cells (HUVECs) injury and the potential mechanisms. Cell proliferation and apoptosis were evaluated by Cell Counting Kit-8 (CCK-8) assay and flow cytometry assay, respectively. The levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO) were detected by corresponding detection kits, respectively. Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression of OIP5-AS1 or microRNA-30c-5p (miR-30c-5p) in HUVECs. Binding between OIP5-AS1 and miR-30c-5p was predicted through bioinformatics analysis and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Western blot was used to analyze p-IκB, IκB, p-p65 and p65 levels. In HUVECs, exposure to ox-LDL led to a decrease in cell viability and an increase in LDH release and apoptosis with concomitant enhancement of oxidative stress, as evidenced by increased ROS and MDA generation, as well as decreased SOD activity and NO levels, while OIP5-AS1 knockdown or miR-30c-5p upregulation could rescue these effects above. Mechanically, OIP5-AS1 functioned as a sponge of miR-30c-5p. OIP5-AS1-induced injury and apoptosis, oxidative stress and activation of NF-κB pathway were reversed by miR-30c-5p in ox-LDL-treated HUVECs. OIP5-AS1 contributed to ox-LDL-treated HUVECs injury by activation of NF-κB pathway via miR-30c-5p.

Hesperidinalleviates bupivacaineanesthesia-induced neurotoxicity in SH-SY5Y cells by regulating apoptosis and oxidative damage.

Local anesthetic with bupivacaine (BV) administration may cause severe neurotoxicity and neurological complications in developing neurons. Any substances that can mitigate the toxic effects of BV are of great importance in surgical procedures and pain management. The present study attempted to investigate if hesperidin (HN) could inhibit or prevent BV-induced neurotoxicity in SH-SY5Y cells. Exposure of BV at 5 mM resulted in a significant decrease of cell viability and a remarkable increase of lactate dehydrogenase release via the induction of apoptosis and production of reactive oxygen species. Moreover, a loss of mitochondrial membrane potential, decreased Bcl-2 protein expression, as well as increased expression of cytoplasmic cytochrome c, Bax, and cleaved caspase-3 protein was also observed in BV-stimulated SH-SY5Y cells. In addition, BV stimulation impaired the balance of oxidation-reduction and inflammation system, as evidenced by the increased malondialdehyde content, decreased superoxide dismutaseand catalaseactivity, and reduced level of reduced glutathione, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α. However, these iatrogenic changes were all reversed by the HN (5, 10, and 20 μM) supplement for 48 h in a concentration-dependent manner. In conclusion, HN can protect SH-SY5Y cells against BV-stimulated neurotoxicity via the inhibition of apoptosis, oxidative stress, and inflammation response. The present findings suggested that HN may be an effective alternative agent to inhibit or prevent BV-induced neurotoxicity in human patients.

Apoptosis-related gene expression induced by Colombian propolis samples in canine osteosarcoma cell line.

Background and Aim:Osteosarcoma (OSA) is the most common bone tumor in canines and humans. This study aimed to assess the cytotoxic and apoptotic effects of Colombian propolis samples on a canine OSA cell line (OSCA-8) by evaluating the expression of BCL-2, BAX, CASPASE 9, CASPASE 8, and TNFR1 genes involved in the apoptosis pathway.Materials and Methods:After treating the cells with five Colombian propolis samples (Usm, Met, Fus, Sil, and Caj), we evaluated cell viability and lactate dehydrogenase (LDH) release. Early and late apoptosis was determined by flow cytometry using annexin V/propidium iodide. Furthermore, the effects of three selected samples on gene expression were analyzed by real-time polymerase chain reaction.Results:The Colombian propolis samples reduced OSCA-8 cell viability and increased LDH release. All samples induced apoptosis significantly and upregulated BCL-2 and CASPASE 8 expression. Usm and Sil increased BAX expression, Met and Sil induced CASPASE 9 expression, and Usm increased TNFR1.Conclusion:Colombian propolis samples exhibited cytotoxic and apoptotic effects on canine OSA cells, and CASPASE 8 upregulation indicated apoptosis induction by the extrinsic pathway.

Cytotoxicity of adducts formed between quercetin and methylglyoxal in PC-12 cells

Quercetin (Que) or quercetin-containing food stuffs are widely incorporated in bakery foods for improving food texture and health effects, and scavenging reactive aldehydes, such as methylglyoxal (MGO) that exhibits various deleterious effects including contribution to neurodegeneration. This study aimed to investigate the cytotoxicity of the adducts formed between quercetin and MGO resulted from the incorporation of quercetin in foods. Two highly-purified adducts (Que-mono-MGO and Que-di-MGO) were found to display higher cytotoxicity than their precursor MGO and quercetin. They elevated apoptosis via upregulation of expression of apoptotic markers, including p-P38, cleaved caspase-9 and -3, and pro-apoptotic Bax. They induced mitochondrial dysfunction via decreasing mitochondrial membrane potential and increasing lactate dehydrogenase release. Moreover, they attenuated levels of p-Akt, Nrf2, NQO-1, and HO-1, proving that they induced neurodegeneration apoptosis through mitochondria-mediated signaling pathways (PI3K-Akt and Nrf2-HO-1/NQO-1). These findings indicated that the safety consequence of MGO after scavenged by polyphenols needs to be concerned.

Irigenin alleviates angiotensin II-induced oxidative stress and apoptosis in HUVEC cells by activating Nrf2 pathway.

Endothelial dysfunction is closely related to various cardiovascular diseases. Oxidative stress and apoptosis are involved in the progress of endothelial dysfunction. Irigenin (IR) has antioxidative properties. We investigated IR as a novel therapy for angiotensin II (Ang II)-induced endothelial dysfunction and explored the potential mechanisms of IR. After human umbilical vein endothelial cell lines (HUVECs) were treated with Ang II (100, 200, 300 and 400 nmol/L) alone, IR (2.5, 5, 10, 20 and 40 μmol/L) alone or Ang II plus IR for 24 h, HUVECs viability, lactate dehydrogenase (LDH), apoptosis, oxidative stress, apoptosis-related protein and nuclear factor E2-related factor 2 (Nrf2) levels were detected by Cell Counting Kit (CCK)-8 assay, enzyme-linked immunosorbent assay, flow cytometry and western blot. Transfection rate of Nrf2 was detected by western blot. In the next rescue experiment, we used silent Nrf2 (siNrf2) to verify the previous experimental results. Different concentrations' Ang II repressed HUVECs viability and increased LDH release, and different concentrations' IR did not affect HUVECs viability or LDH release. Furthermore, IR elevated cell viability and Nrf2 level, inhibited LDH release, apoptosis, oxidative stress and apoptosis-related protein levels in Ang II-induced HUVECs. More important, siNrf2 suppressed the expression of Nrf2, and siNrf2 abrogated the protective effect of IR on Ang II-induced Nrf2 expression, cell viability, LDH activity, oxidative stress generation and apoptosis-related protein in HUVECs. IR protected HUVECs from Ang II-induced oxidative stress and apoptosis injury by activating Nrf2 pathway.

Febuxostat Prevents the Cytotoxicity of Propofol in Brain Endothelial Cells.

Background and purpose: A high risk of brain injury has been reported with the usage of general anesthetics such as propofol in infants. Experimental data indicated that oxidative stress and inflammation are involved in the neurotoxicity induced by propofol. Febuxostat is a novel anti-gout agent recently reported to exert an anti-inflammatory effect. The present study aims to investigate the protective property of febuxostat against the cytotoxicity of propofol in brain endothelial cells as well as the underlying preliminary mechanism. Methods: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to screen the optimized incubation concentration of febuxostat. bEnd.3 brain endothelial cells were stimulated with 2% propofol in the presence or absence of febuxostat (10, 20 μM) for 24 h. The lactate dehydrogenase (LDH) release assay was conducted to detect cytotoxicity. The reactive oxygen species (ROS) levels were evaluated using dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining, and the concentration of reduced glutathione (GSH) was determined using a commercial kit. The expressions of TNF-α, IL-6, IL-12, CXCL-1, PDPN, CXCL8, VCAM-1, and E-selectin were determined using a quantitative real-time polymerase chain reaction (qRT-PCR) and an enzyme-linked immunosorbent assay (ELISA). Western blot and qRT-PCR were utilized to determine the expressions of COX-2 and KLF6. The production of PGE2 was evaluated by ELISA. Results: First, increased LDH release induced by propofol was significantly suppressed by febuxostat. The oxidative stress (elevated ROS levels and decreased GSH level) induced by propofol was alleviated by febuxostat. Second, the upregulated inflammatory factors (TNF-α, IL-6, and IL-12), pro-inflammatory chemokines (CXCL-1, PDPN, and CXCL8), adhesion molecules (VCAM-1 and E-selectin), and inflammatory mediators (COX-2 and PGE2) induced by propofol were greatly downregulated by febuxostat. Lastly, the expression of KLF6 was significantly suppressed by propofol but greatly elevated by febuxostat. Conclusion: Febuxostat prevented the cytotoxicity of propofol in brain endothelial cells by alleviating oxidative stress and inflammatory response through KLF6.

Schizandrin B attenuates hypoxia/reoxygenation injury in H9c2 cells by activating the AMPK/Nrf2 signaling pathway.

Schizandrin B exhibits prominent antioxidant and anti-inflammatory effects, and plays an important role in ameliorating myocardial ischemia/reperfusion injury. However, the underlying protective mechanisms remain to be elucidated. The aim of the present study was to explore the cardioprotective effects of schizandrin B against hypoxia/reoxygenation (H/R)-induced H9c2 cell injury, focusing on the role of the adenosine monophosphate-activated protein kinase (AMPK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in this process. The results showed that schizandrin B attenuated the H/R-induced decrease in cell viability and the increase in lactate dehydrogenase release, as well as the apoptosis rate in H9c2 cells. Schizandrin B also mitigated H/R-induced oxidative stress, as illustrated by the decrease in intracellular reactive oxygen species generation, malondialdehyde content and NADPH oxidase 2 expression, and the increase in antioxidant enzyme superoxide dismutase and glutathione peroxidase activities. In addition, schizandrin B reversed the H/R-induced upregulation of pro-inflammatory cytokines [interleukin (IL)-1β (IL-1β) tumor necrosis factor-α, IL-6 and IL-8] and the downregulation of anti-inflammatory cytokines (transforming growth factor-β and IL-10) in the culture supernatant. Notably, schizandrin B increased the expression of Nrf2, NAD(P)H: Quinone oxidoreductase (NQO-1) and heme oxygenase-1 (HO-1) in H/R-treated H9c2 cells, activating the Nrf2 signaling pathway. The cardioprotection of schizandrin B against H/R injury was inhibited by Nrf2 knockdown induced byNrf-2-specific small interfering RNA (siRNA; si-Nrf2) transfection. Furthermore, schizandrin B enhanced phosphorylated (p)-AMPK expression, while AMPK knockdown induced by AMPK-specific siRNA(si-AMPK) transfection remarkably eliminated schizandrin B-induced cardioprotection and reduced Nrf2 expression in H/R-treated H9c2 cells. Taken together, these results suggested that schizandrin B exerts cardioprotection on H/R injury in H9c2 cells due to its antioxidant and anti-inflammatory activities via activation of the AMPK/Nrf2 pathway.

The influence of hyperglycemia on the safety of ultrasound in retinal pigment epithelial cells.

Ultrasound (US) assisted drug delivery is receiving interest in treating posterior eye diseases, such as diabetic retinopathy due to its ability to maximize drug penetration into difficult to reach tissues. Despite its promise, the technique has only been investigated using healthy cell and tissue models, with no evidence to date about its safety in active disease. As a result, the aim of this study was to evaluate the safety of US administration in vitro in retinal pigment epithelial cells under normal and high glucose conditions. US protocols within the presently accepted safety threshold were applied and their influence on cell membrane and tight junction integrity as well as intracellular inflammation was evaluated using lactate dehydrogenase (LDH), zona occludens-1 (ZO-1), fluorescein isothiocyanate (FITC)-dextran dye leak and nuclear factor-kappaB (NF-κB) assays, respectively. Under high glucose conditions, US application increased LDH release and resulted in loss of ZO-1 labeling at 2 h; however, normal levels were restored within 24 h. US within its safety parameters did not induce any FITC-dextran dye leak or NF-κB nuclear translocation in normal or high glucose conditions. In conclusion, our results suggest that while high glucose conditions increase cell susceptibility to US-mediated stress, basal conditions can be restored within 24 h without long-lasting cell damage.

YBX1 Protects against Apoptosis Induced by Oxygen-Glucose Deprivation/Reoxygenation in PC12 Cells via Activation of the AKT/GSK3β Pathway

Reperfusion therapies for ischaemic stroke can induce secondary injury accompanied by neuronal death. The Y-box binding protein 1 (YBX1), an oncoprotein, is critical for regulating tumour cell proliferation and apoptosis. Thus, we wanted to know whether YBX1 could regulate neuronal cell apoptosis caused by cerebral ischaemia/reperfusion (I/R). We established a model of cerebral I/R-induced injury in vitro by oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and determined YBX1 expression using Western blot. Next, the effect of YBX1 on the apoptosis and viability of OGD/R-treated PC12 cells was evaluated by flow cytometry, MTT assay, and Western blot. Besides, the release of lactate dehydrogenase (LDH) and the activity of catalase (CAT) and superoxide dismutase (SOD) were detected to evaluate oxidative stress of PC12 cells induced by OGD/R. The regulatory roles of YBX1 in the AKT/GSK3β pathway were examined by Western blot. As a result, OGD/R treatment down-regulated YBX1 expression in PC12 cells. YBX1 over-expression attenuated the growth inhibition and apoptosis of PC12 cells induced by OGD/R. Besides, the increase of LDH release and the decrease of SOD and CAT activities caused by OGD/R were reversed by YBX1 over-expression. Moreover, YBX1 over-expression could activate the AKT/GSK3β pathway in OGD/ R-treated PC12 cells. Therefore, YBX1 could protect against OGD/R-induced injury in PC12 cells through activating the AKT/GSK3β signalling pathway, and thus YBX1 has the potential to become a therapeutic target for cerebral I/R-induced injury.

Inhibition of microRNA-495 inhibits hypoxia-induced apoptosis in H9c2 cells via targeting NFIB.

Acute myocardial infarction (AMI) is a serious cardiovascular disease that threatens human life. MicroRNA is considered to be an important participant in the pathophysiology of AMI. This article focused on the role of microRNA-495 (miR-495) in regulating apoptosis after myocardial infarction (MI) and its underlying mechanisms. H9c2 cells were cultured in an incubator containing 1% O2 to establish a cell model of MI. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was utilized to detect miR-495 expression in H9c2 cells. The effects of miR-495 and NFIB on hypoxia-treated H9c2 cells were observed by Western blot, lactate dehydrogenase (LDH) detection, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, flow cytometry, and terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) staining. Luciferase reporter gene experiment was used to prove the regulatory relationship between miR-495 and NFIB. Hypoxia induced injury to H9c2 cells, which was manifested by decreased cell viability, increased LDH release, increased pro-apoptotic proteins (Bax, Cleaved Caspase-3) expression, decreased anti-apoptotic protein (Bcl-2) expression, and increased in the rate of apoptosis and TUNEL positive cells. MiR-495 expression was remarkably increased in H9c2 cells treated with hypoxia. Inhibiting miR-495 expression markedly alleviated the hypoxia-induced injury in H9c2 cells, while silencing NFIB aggravated the hypoxia-induced damage. In addition, NFIB was confirmed to be the target of miR-495. MiR-495 expression was increased in hypoxia-treated H9c2 cells. Silencing miR-495 could significantly inhibit hypoxia-induced apoptosis of H9c2 cells by targeting NFIB.

Inhibition of autophagy aggravates molybdenum-induced mitochondrial dysfunction by aggravating oxidative stress in duck renal tubular epithelial cells

Excessive molybdenum (Mo) has adverse effects on animals. To elucidate the effects of autophagy on Mo-induced nephrotoxicity, the duck renal tubular epithelial cells were cultured in medium in absence and presence of (NH4)6Mo7O24.4H2O (0, 480, 720, 960 μM Mo), 3-Methyladenine (3-MA) (2.5 μM), and the combination of Mo and 3-MA for 12 h. After 12 h exposure, the MDC staining, morphologic observation, LC3 puncta, cell viability, autophagy-related genes mRNA and proteins levels, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) level, antioxidant indices, mitochondrial membrane potential (MMP), mitochondrial mass, mitochondrial respiratory control ratio (RCR) and oxidative phosphorylation rate (OPR) were determined. The results showed that excessive Mo exposure significantly elevated the number of autophagosome and LC3 puncta, upregulated Beclin-1, Atg5, LC3A and LC3B mRNA levels, and LC3II/LC3I and Beclin-1 protein levels, decreased mTOR, p62 and Dynein mRNA levels and p62 protein level. Besides, co-treatment with Mo and 3-MA dramatically increased LDH release, ROS level, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents as well as cell dam age, reduced cell viability, the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), MMP, mitochondrial mass, mitochondrial RCR and OPR compared to treatment with Mo alone. Taken together, these results suggest that excessive Mo exposure can induce autophagy in duck renal tubular epithelial cells, inhibition of autophagy aggravates Mo-induced mitochondrial dysfunction by regulating oxidative stress.

The Effects of Targeted Temperature Management on Oxygen-Glucose Deprivation/Reperfusion-Induced Injury and DAMP Release in Murine Primary Cardiomyocytes

Introduction. Ischemia/Reperfusion (I/R) is a primary cause of myocardial injury after acute myocardial infarction resulting in the release of damage-associated molecular patterns (DAMPs), which can induce a sterile inflammatory response in the myocardial penumbra. Targeted temperature management (TTM) after I/R has been established for neuroprotection, but the cardioprotective effect remains to be elucidated. Therefore, we investigated the effect of TTM on cell viability, immune response, and DAMP release during oxygen-glucose deprivation/reperfusion (OGD/R) in murine primary cardiomyocytes. Methods. Primary cardiomyocytes from P1-3 mice were exposed to 2, 4, or 6 hours OGD (0.2% oxygen in medium without glucose and serum) followed by 6, 12, or 24 hours simulated reperfusion (21% oxygen in complete medium). TTM at 33.5°C was initiated intra-OGD, and a control group was maintained at 37°C normoxia. Necrosis was assessed by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activation. OGD-induced DAMP secretions were assessed by Western blotting. Inducible nitric oxide synthase (iNOS), cytokines, and antiapoptotic RBM3 and CIRBP gene expressions were measured by quantitative polymerase chain reaction. Results. Increasing duration of OGD resulted in a transition from apoptotic programmed cell death to necrosis, as observed by decreasing caspase-3 cleavage and increasing LDH release. DAMP release and iNOS expression correlated with increasing necrosis and were effectively attenuated by TTM initiated during OGD. Moreover, TTM induced expression of antiapoptotic RBM3 and CIRBP. Conclusion. TTM protects the myocardium by attenuating cardiomyocyte necrosis induced by OGD and caspase-3 activation, possibly via induction of antiapoptotic RBM3 and CIRBP expressions, during reperfusion. OGD induces increased Hsp70 and CIRBP releases, but HMGB-1 is the dominant mediator of inflammation secreted by cardiomyocytes after prolonged exposure. TTM has the potential to attenuate DAMP release.

Z-ligustilide reduces cisplatin-induced nephrotoxicity via activation of NRF2/HO-1 signaling pathways

Purpose: To investigate the effect of Z-ligustilide (Z-lig) on cisplatin-induced nephrotoxicity and examine whether NRF2 signaling mediates the underlying mechanism of action.Methods: Human proximal tubular epithelial cells (HK-2) were pretreated with 20 or 100 μM Z-lig for 2 h, followed by 10 μM cisplatin treatment for 24 h. Cell viability was measured using (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A commercial kit was used todetermine lactate dehydrogenase (LDH) release. Apoptosis was determined by flow cytometry while Western blotting was used to evaluate protein levels. Levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) were assessed by enzymelinked immunosorbent assay (ELISA).Results: Cisplatin decreased HK-2 cell viability and increased LDH release, while Z-lig increased cell viability and decreased LDH release in a dose-dependent manner (p < 0.05). Moreover, Z-lig reduced cisplatin-induced apoptosis (p < 0.01), and alleviated cellular oxidative stress caused by cisplatin (p < 0.05). Furthermore, Z-lig activated NRF2/HO-1 signaling in cells treated with cisplatin (p < 0.05).Conclusion: Z-lig reduces cisplatin-induced nephrotoxicity via activation of NRF2/HO-1 signaling. Thus, Z-lig is a potential drug for the treatment of nephrotoxicity caused by cisplatin.
 Keywords: Z-ligustilide, Cisplatin, Nephrotoxicity, Oxidative stress, Apoptosis, Nuclear factor erythroid 2-related factor 2, Heme oxygenase-1

Aloperine attenuates high glucose-induced oxidative injury in Schwann cells via activation of NRF2/HO-1 pathway

Purpose: To determine the involvement of nuclear factor erythroid 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) in the action of aloperine on Schwann cell injury caused by high glucose (HG).Methods: Cell viability was determined using MTT assay while the release of lactate dehydrogenase (LDH) was determined by biochemical assay. Apoptosis was assessed using flow cytometry, while the levels of malondialdehyde (MDA) were determined by Annexin V-FIT staining. Glutathione Stransferase (GST), glutathione peroxidase (GPX), and reactive oxygen species (ROS) were determined using enzyme-linked immunosorbent assay.Results: Treatment with HG suppressed RSC96 cell viability and increased LDH release, while aloperine reversed these results (p < 0.05). Apoptosis of RSC96 cells was induced by HG stimulation, but was abolished by aloperine. The levels of ROS, MDA, and GST were enhanced in cells followingtreatment with HG, but was reversed by aloperine (p < 0.05). The decreased level of GPX caused by HG in RSC96 cells was elevated by aloperine. Moreover, aloperine upregulated NRF2 and HO-1 in RSC96 cells treated with HG (p < 0.05).Conclusion: Aloperine attenuates HG-induced oxidative injury in Schwann cells via activation of NRF2/HO-1 pathway, suggesting its potential as a potent drug for the management of diabetic peripheral neuropathy.
 Keywords: Aloperine, Schwann cells, High glucose, Oxidative stress, NRF2, HO-1

Exposure to mono (2-ethylhexyl) phthalate facilitates apoptosis and pyroptosis of human endometrial microvascular endothelial cells through NLRP3 inflammasome.

Mono (2-ethylhexyl) phthalate (MEHP) is a major metabolite of di (2-ethylhexyl) phthalate (DEHP). This study aimed to observe the toxic effect of MEHP on human endometrial microvascular endothelial cells (HEMECs) and its potential molecular mechanism. HEMECs were exposed to different concentrations of MEHP (0, 50, 100, and 200 nM). Cell viability and apoptosis were assessed by cell counting kit-8 (CCK-8) and flow cytometry assays. Western blot was performed to examine the expression of apoptosis-related proteins (Bcl-2, Bax, and Caspase-3). Moreover, the expression of pyroptosis-related Caspase-1 was detected by western blot and immunofluorescence assays. Lactate dehydrogenase (LDH) release levels were evaluated in HEMECs treated with MEHP and/or Caspase-1 inhibitor Ac-YVAD-CHO. After exposure to MEHP, NLRP3 expression was examined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot. LDH release and apoptosis levels were tested in HEMECs induced by MEHP and/or siNLRP3. MEHP significantly induced cell viability and inhibited apoptosis for HEMECs, with a concentration-dependent manner. Furthermore, Bcl-2/Bax ratio was distinctly reduced and Caspase-3 expression was increased in HEMECs after exposure to MEHP. Western blot and immunofluorescence results confirmed that MEHP markedly augmented Caspase-1 expression in HEMECs. Furthermore, LDH release levels were fortified in HEMECs treated with MEHP, which were improved following cotreatment with Ac-YVAD-CHO. At the mRNA and protein levels, NLRP3 expression was prominently increased in HEMECs exposed to MEHP. NLRP3 knockdown markedly ameliorated the increase in LDH release and apoptosis induced by MEHP exposure in HEMECs. Our findings suggested that exposure to MEHP facilitates apoptosis and pyroptosis of HEMECs through NLRP3 inflammasome.

Oxidative stress-dependent frataxin inhibition mediated alcoholic hepatocytotoxicity through ferroptosis

Alcoholic liver disease (ALD) is one of the severe liver diseases, resulting in high morbidity and mortality. However, frataxin, a mitochondrial protein mainly participating in iron homeostasis and oxidative stress, remains uncertain in the pathogenesis of ALD. In the present study, the role of frataxin in ALD was investigated. Ethanol (100 mM) decreased frataxin expression at 48 and 72 h in HepG2. Dramatically, in HepG2 overexpressing cytochrome P450 2E1 (HepG2CYP2E1+/+), frataxin level was down-regulated with ethanol stimulation at 12 h. Moreover, chronically feeding ethanol to mice via Lieber-DeCarli liquid diet (30 % of total calories) for 15 weeks significantly inhibited frataxin expression. Ferroptosis signature proteins were dysregulated, accompanied by mitochondrial damage of morphology, enhanced malondialdehyde and decreased glutathione in the liver, as well as accumulation of reactive oxygen species and mitochondrial labile iron pool in primary hepatocytes. Notably, proteomics screening of frataxin deficient-HepG2 further suggested frataxin was associated with ferroptosis. Furthermore, the ferroptosis inhibitor ferrostatin-1 blocked the increase of lactate dehydrogenase release by ethanol in HepG2CYP2E1+/+. Most importantly, frataxin deficiency enhanced ferroptosis driven by ethanol via evaluating the levels of lactate dehydrogenase, cell morphological changes, mitochondrial labile iron pool, and lipid peroxidation. Conversely, restoring frataxin alleviated the sensitivity to ferroptosis. In addition, frataxin overexpression mitigated the sensitivity of ethanol-induced ferroptosis in HepG2CYP2E1+/+. Collectively, our study revealed that frataxin-mediated ferroptosis contributed to ALD, highlighting a potential therapeutic strategy for ALD.

KD025 Shifts Pulmonary Endothelial Cell Bioenergetics and Decreases Baseline Lung Permeability.

KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.

In vitro immune responses of bovine mammary epithelial cells induced by Escherichia coli, with multidrug resistant extended-spectrum β-lactamase, isolated from mastitic milk

Bovine mastitis is an inflammatory condition of mammary glands causing huge economic losses for dairy industries. Infection with extended-spectrum β-lactamase (ESBL)-producing sequence types (ST) 410-Escherichia coli (ESBL-ST410 E. coli) is considered a leading cause of bovine mastitis in China. However, pathogenic effects of these strains in an in vitro model, e.g. bovine mammary epithelial cells (bMECs), are unknown. Therefore, our objectives were to explore pathogenesis (adhesion and invasion, inflammation, oxidative stress and apoptosis) of ESBL-E. coli (highly prevalent in bovine mastitis) in bMECs. Non-pathogenic E. coli DH5α and a prototypical E. coli P4 were included as negative and positive controls, respectively. The bMECs were infected with our isolated ST410 strains, plus DH5α and P4, with assessment of the following end points: adhesive and invasive capabilities; lactate dehydrogenase (LDH) activities; inflammatory responses, including concentrations of interleukin-1β (IL-1β), IL-6, IL-10 and tumor necrosis factor-α; oxidative stress including intracellular reactive oxygen species production, malondialdehyde concentrations, activities of glutathione peroxidase and superoxide dismutase; and apoptosis. All ST410 strains had greater adhesive and invasive capabilities and increased LDH release, with varying degrees of inflammatory responses, oxidative stress and apoptosis compared to blank and DH5α groups, similar to P4-infected bMECs. In particular, ST410(4) was more likely than the other 3 isolates to adhere to and invade bMECs and increase LDH activities, cytokine release, oxidative stress and apoptosis. Thus, ST410 isolates had pathogenic manifestations of adhesive and invasive capabilities; furthermore, they induced inflammation, oxidative stress and apoptosis in bMECs. Finally, ST410(4) was the most pathogenic strain.