Lactate Dehydrogenase Production Research Articles

Clinical value of BRE-AS1 in myocardial infarction and its role in myocardial infarction-induced cardiac muscle cell apoptosis

Objectives. The aim of this study was to investigate the expression of long non-coding RNA (lncRNA) brain and reproductive organ-expressed protein (BRE) antisense RNA 1 (BRE-AS1) in patients with acute myocardial infarction (AMI) and its effect on ischemia/reperfusion (I/R)-induced oxidative stress and apoptosis of cardiomyocytes. Methods. Serum BRE-AS1 levels in patients with AMI was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic and prognostic values of BRE-AS1 were evaluated. H9c2 cells were treated with hypoxia/reoxygenation to establish an in vitro myocardial infarction cell model. The levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). Levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were determined by commercial kits. Cell counting kit-8 (CCK-8) and flow cytometry were used to evaluate the cell viability and cell apoptosis. Results. The expression of BRE-AS1 in serum of patients with AMI is upregulated, which shows the clinical diagnostic value for AMI. In the I/R injury cell model, the knockout of BRE-AS1 can significantly alleviate the increase in TNF-α, IL-1β, and IL-6 levels, inhibit the production of LDH and MDA, increase the activities of SOD and GSH-Px, promote the cell viability and suppress cell apoptosis. Conclusions. Abnormally elevated BRE-AS1 has a high diagnostic value for AMI as well as a prognostic value for major adverse cardiovascular events (MACEs). The elevation of BRE-AS1 promoted oxidative stress injury and cell apoptosis in vitro.

Characterization of Escherichia coli Strains for Novel Production of Plasmodium ovale Lactate Dehydrogenase.

Malaria is one of the most prevalent diseases worldwide with high incidence and mortality. Among the five species that can infect humans, Plasmodium ovale morphologically resembles Plasmodium vivax, resulting in misidentification and confusion in diagnosis, and is responsible for malarial disease relapse due to the formation of hypnozoites. P. ovale receives relatively less attention compared to other major parasites, such as P. falciparum and P. vivax, primarily due to its lower pathogenicity, mortality rates, and prevalence rates. To efficiently produce lactate dehydrogenase (LDH), a major target for diagnosing malaria, this study used three Escherichia coli strains, BL21(DE3), BL21(DE3)pLysS, and Rosetta(DE3), commonly used for recombinant protein production. These strains were characterized to select the optimal strain for P. ovale LDH (PoLDH) production. Gene cloning for recombinant PoLDH production and transformation of the three strains for protein expression were performed. The optimal PoLDH overexpression and washing buffer conditions in nickel-based affinity chromatography were established to ensure high-purity PoLDH. The yields of PoLDH expressed by the three strains were as follows: BL21(DE3), 7.6 mg/L; BL21(DE3)pLysS, 7.4 mg/L; and Rosetta(DE3), 9.5 mg/L. These findings are expected to be highly useful for PoLDH-specific diagnosis and development of antimalarial therapeutics.

Effects of Hericium erinaceus polysaccharide in porcine IPEC-J2 intestinal epithelial cells against apoptosis induced by oxidative stress

This study was intended to investigate whether Hericium erinaceus polysaccharides (HEP) prevent oxidative stress and apoptosis of intestinal porcine epithelial cells from jejunum (IPEC-J2 cells) induced by hydrogen peroxide (H2O2). Crude HEP were extracted and purified by chromatography. The ultraviolet and infrared spectra and monosaccharide composition of HEP were analyzed. Reactive oxygen species (ROS) generation was quantified by flow cytometry method, and lactate dehydrogenase (LDH) and malondialdehyde (MDA) production were determined by TBARS. Also, apoptosis was analyzed by flow cytometry method and the apoptosis-related regulatory molecules were determined by microplate or western blotting method. Our results showed that pretreatment of IPEC-J2 cells with HEP significantly scavenged ROS and reduced LDH and MDA production. HEP also reduced apoptosis and kept polarity of the mitochondrial membrane potential. Moreover, HEP increased the content of caspase-3 and PARP, and protein expression of Bcl-2, while inhibited Bax and Bad and reduced the content of caspase-9 and release of CytC. Meanwhile, HEP inhibited the protein expression of TNFR1, FAS, and FASL, and decreased the content of caspase-8. The results indicated that HEP had a protective effect against oxidative stress in IPEC-J2 cells and the underlying mechanism was reducing apoptosis via mitochondrial and death receptor pathways.

Ruscogenin regulates endogenous antioxidation in dopamine neurons by activating Keap1/Nrf2/HO-1 pathway

Purpose: To investigate the effect of ruscogenin (RUS) on cell viability, lipid peroxidation and mitochondrial dysfunction in a Parkinson’s disease (PD) model.Methods: The neuroblastoma cell line SH-SY5Y was modified with 1-methyl-4-phenylpyridine (MPP+) to establish a PD model. RUS (1 or 10 μM) was used to treat MPP+ induced SH-SY5Y cells. Cell viability was assessed using CCK-8 assay. The concentrations of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GPx) were determined by enzyme-linked immunosorbent assay (ELISA). ATP production, Ca2+ concentration and JC-1 were quantified using commercial kits. The expression levels of tyrosine hydroxylase (TH), Keap1, Nrf2 and HO-1 were evaluated by western blot analysis.Results: RUS protected cell viability, reduced LDH production, and elevated TH expression in MPP+- modified SH-SY5Y cells. RUS promoted the release of SOD, CAT and GPx, but suppressed MDA production. Furthermore, RUS enhanced ATP metabolism, decreased Ca2+ leakage and maintained mitochondrial function. RUS also repressed Keap1 expression but increased Nrf2 and HO-1 levels.Conclusion: RUS enhances cell viability while alleviating cytotoxicity, lipid peroxidation and mitochondrial dysfunction in dopamine neurons through the activation of Keap1/Nrf2/HO-1 signaling pathway. Thus, RUS is a promising therapeutic candidate for PD treatment.

BRAP silencing protects against neuronal inflammation, oxidative stress and apoptosis in cerebral ischemia-reperfusion injury by promoting PON1 expression.

BRCA1 associated protein (BRAP) participates in the regulation of myocardial infarction and atherosclerosis. But the function of BRAP in cerebral ischemia-reperfusion (CIR) injury has not been elucidated yet. BRAP expression in PC12 cells in response to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment was examined with Western blot assay. PC12 cells underwent OGD/R-treatment and were subsequently transfected with pcDNA-BRAP or sh-BRAP, followed by determination of viability, lactate dehydrogenase (LDH) production, apoptosis, inflammatory cytokine secretion, and oxidative stress marker protein levels. Paraoxonase 1 (PON1) promoter methylation was evaluated with methylation-specific PCR assay. the effect of BRAP/PON1 axis on CIR injury was investigated by rescue experiments. Additionally, sh-BRAP was injected into a middle cerebral artery occlusion (MCAO) rat model, and the changes of neurological damage were evaluated. BRAP overexpression exacerbated OGD/R-induced viability reduction, LDH production, apoptosis, inflammatory cytokine secretion and oxidative stress in PC12 neuronal cells. In contrast, BRAP silencing showed the opposite results. Mechanistically, BRAP reduced PON1 expression by promoting DNA methyl transferase1 (DNMT1)-mediated PON1 promoter methylation. PON1 silencing reversed BRAP-mediated neuroprotection. Additionally, BRAP silencing alleviated CIR-induced neurological damage in MCAO rats. BRAP silencing suppressed OGD/R-induced neuronal apoptosis, inflammation, and oxidative stress, and alleviated CIR-induced neurological damage in MCAO rats through facilitating PON1 expression.

Acai Berry (Euterpe sp.) Extracts Are Neuroprotective against L-Glutamate-Induced Toxicity by Limiting Mitochondrial Dysfunction and Cellular Redox Stress.

Aberrant accumulation of the neurotransmitter L-glutamate (L-Glu) has been implicated as a mechanism of neurodegeneration, and the release of L-Glu after stroke onset leads to a toxicity cascade that results in neuronal death. The acai berry (Euterpe oleracea) is a potential dietary nutraceutical. The aim of this research was to investigate the neuroprotective effects of acai berry aqueous and ethanolic extracts to reduce the neurotoxicity to neuronal cells triggered by L-Glu application. L-Glu and acai berry effects on cell viability were quantified using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and effects on cellular bioenergetics were assessed via quantitation of the levels of cellular ATP, mitochondrial membrane potential (MMP), and production of reactive oxygen species (ROS) in neuroblastoma cells. Cell viability was also evaluated in human cortical neuronal progenitor cell culture after L-Glu or/and acai berry application. In isolated cells, activated currents using patch-clamping were employed to determine whether L-Glu neurotoxicity was mediated by ionotropic L-Glu-receptors (iGluRs). L-Glu caused a significant reduction in cell viability, ATP, and MMP levels and increased ROS production. The co-application of both acai berry extracts with L-Glu provided neuroprotection against L-Glu with sustained cell viability, decreased LDH production, restored ATP and MMP levels, and reduced ROS levels. Whole-cell patch-clamp recordings showed that L-Glu toxicity is not mediated by the activation of iGluRs in neuroblastoma cells. Fractionation and analysis of acai berry extracts with liquid chromatography-mass spectrometry identified several phytochemical antioxidants that may have provided neuroprotective effects. In summary, the acai berry contains nutraceuticals with antioxidant activity that may be a beneficial dietary component to limit pathological deficits triggered by excessive L-Glu accumulations.

Prevention of Cell Proliferation Signaling Via RNA Interference of c-myc Gene Expression in Colon Cancer In-vitro

Colon cancer is a cancer begins in the large intestine (colon). Its aggression is due to late diagnosis, so poor prognosis and higher mortality rates are reported. Colon cancer has become a thorny research area that requires more examination of cellular pathways involved in its emergency. Based on this, we sought to check the biological role of cMyc in colon cancer cell proliferation through targeting its expression by a respective siRNA using CaCo-2 cell lines. Notably, the cytotoxic potential of cMyc knockdown was monitored by cell imaging using an inverted microscope, the production of lactate dehydrogenase (LDH) and viability of the transfected cells using MTT assay. The relative gene expression of c-myc and other related factors such as Raf-1 was investigated using quantitative real-time PCR (qRT-PCR). ELISA assay has been used to achieve the production of pro and anti-inflammatory cytokines released from transfected cells. Interestingly, the transfection with a new designed siRNA antagonist cMyc markedly decreased CaCo-2 cell viability rate in a dose-dependent manner compared with the transfection with specific siRNA antagonist Luciferase gene, which served as control. Likewise, number of survived cells significantly reduced following transfection with siRNA against c-myc, while the relative production of LDH significantly increased. Importantly, the cytotoxic effect of the transfection protocol showed neglected influence in normal colon epithelial cells (NCM-460 cell lines) indicating the selective regulation of colon cancer cell proliferation by transfection with siRNA targeting c-myc. Noteworthy, the knockdown efficiency of c-myc showed more than 80% inhibition of its expression in CaCo-2 cells transfected with the siRNA antagonist c-myc indicated by qRT-PCR. The relative gene expression of Raf-1 significantly decreased in c-myc knockdown cells, while the relative expression of both P53 and Caspase 3, as cell death indicators, significantly upregulated compared with control-transfected cells. Furthermore, the transfection with siRNA targeting c-myc markedly increased the production of interleukin 1 alpha (IL-1α ) and IL-1β, as pro-inflammatory cytokines, while decreased the production of IL- 4 and IL-10, as anti-inflammatory cytokines. These data provide evidence for the involvement of c-myc gene expression in colon cancer cell proliferation and immune response during cancer development.

Icariin protects cerebral neural cells from ischemia‑reperfusion injury in an invitro model by lowering ROS production and intracellular calcium concentration.

Ischemia is one of the major causes of stroke. The present study investigated the protection of cultured neural cells by icariin (ICA) against ischemia-reperfusion (I/R) injury and possible mechanisms underlying the protection. Neural cells were isolated from neonatal rats and cultured in vitro. The cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD-R) as an I/R mimic to generate I/R injury, and were post-OGD-R treated with ICA. Following the treatments, cell viability, apoptosis, reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and Ca2+ concentration were assessed using Cell Counting Kit-8 assay, flow cytometry, CyQUANT™ LDH Cytotoxicity Assay, H2DCFDA and SOD colorimetric activity kit. After OGD-R, considerable I/R injury was observed in the neural cells, as indicated by reduced cell viability, increased apoptosis and increased production of ROS and LDH (P<0.05). Cellular Ca2+ concentration was also increased, while SOD activity remained unchanged. Post-OGD-R ICA treatments increased cell viability up to 87.1% (P<0.05) and reduced apoptosis as low as 6.6% (P<0.05) in a concentration-dependent manner. The treatments also resulted in fewer ROS (P<0.05), lower extracellular LDH content (440.5 vs. 230.3 U/l; P<0.05) and reduced Ca2+ increase (P<0.05). These data suggest that ICA protects the neural cells from I/R injury in an in vitro model through antioxidation activity and maintaining cellular Ca2+ homeostasis. This function may be explored as a potential therapeutic strategy for ischemia-related diseases after further in vivo studies.

Lectroacupuncture stimulation at CV4 sites prevents breast cancer-induced osteoporosis by activating the Wnt/β-catenin signaling pathway

Osteoporosis is a common condition among breast cancer patients, characterized by decreased bone density due to chemotherapy and hormone therapy. However, the application of electroacupuncture (EA) stimulation at Guanyuan (CV4) sites has been found to be beneficial in enhancing bone mineral density. In this study, we aimed to investigate the efficacy of EA stimulation at CV4 sites in breast cancer-induced osteoporosis and elucidate the potential underlying mechanisms. A breast cancer model in mice was established, EA stimulation at CV4 sites was performed, and hematoxylin-eosin (HE) stain was conducted to assess the bone mineral density and trabecular bone volume of femur tissues. Additionally, Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify and visualize osteoclasts. Furthermore, Western blots were used to evaluate the expression levels of various proteins, including c-src, Cathepsin K (CtSK), matrix metalloproteinase 9 (MMP9), Collagen 1A1 (COL1A1), Osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), Wnt3a and β-catenin, and Enzyme Linked Immunosorbent Assay (ELISA) to measure the concentration of Alkaline phosphatase (ALP), NO synthase 2 (NOS2), glutathione reductase (GSR), superoxide dismutase (SOD), reactive oxygen species (ROS) and lactate dehydrogenase (LDH) in the serum. The results revealed that EA stimulation in the breast cancer model resulted in higher bone mineral density and trabecular bone volume, along with a reduction in the number of osteoclasts. EA stimulation at CV4 site significantly decreased the protein levels of c-src, CtSK and MMP9, while also reducing ROS and LDH production. Conversely, it promoted the expression of COL1A1, OPN, RUNX2, Wnt3a and β-catenin. Additionally, it elevated the concentration of ALP, NOS2, GSR and SOD in the serum. Overall, EA stimulation at the CV4 site may inhibit bone loss and promote bone differentiation by activating the Wnt/β-catenin pathway, indicating its potential use for breast cancer-induced osteoporosis treatment.

Anticancer Properties of N,N-dibenzylasparagine as an Asparagine (Asp) analog, Using Colon Cancer Caco-2 Cell Line.

This study was conducted to investigate the potential anticancer properties of N, N-dibenzyl asparagine (NNDAsp), as an Asparagine (Asp) analog, using colon cancer Caco-2 cell and the normal NCM-460 cell line. Cell viability rate and levels of produced lactate dehydrogenase (LDH) were achieved upon treatment with NNDAsp compared to Asp treatment using MTT assay and LDH production kit. The protein expression profile of asparagine synthetase (ASNS) was achieved by using ELISA and flow cytometry assay. The levels of released inflammatory cytokines, including interleukin-1 alpha (IL-1α) and IL-1 beta (IL-β), were monitored using an ELISA assay. Our findings showed significant inhibition of colon cancer cell proliferation accompanied by a high level of produced LDH in a dose-dependent of an NNDAsp treatment without detectable toxic effect in normal cells. Interestingly, NNDAsp showed competitive inhibition of ASNS protein expression, in almost 3% of stained cancer cells, compared to 18% and 35% of untreated cells and cells pre-treated with Asp, respectively. Likewise, the concentration of ASNS protein was dramatically depleted in a dose and time-dependent of NNDAsp treatment in comparison with Asp treatment indicated by ELISA assay. Furthermore, as an apoptotic indicator, the expression of P53 and Caspase 3 (Caps3) was significantly increased in Caco-2 cells treated with NNDAsp at both RNA and protein levels. In contrast, their expression was markedly depleted in Asp-treated cells. In addition, the expression of both IL-1α and IL-1 β was markedly increased in Caco-2 cells in a dose and time-dependent of NNDAsp exogenous treatment. Moreover, targeting of ASNS by the Asp analog, NNDAsp, was further confirmed by the docking analysis of inhibitors ligands and crystal structure of ASNS protein. These data provide evidence for the effectiveness of NNDAsp in cancer treatment via selective degradation of ASNS protein expression in colon cancer cells.

Lycium barbarum polysaccharide protects rats and cardiomyocytes against ischemia/reperfusion injury via Nrf2 activation through autophagy inhibition.

The irreversible loss of cardiomyocytes is mainly the result of ischemic/reperfusion (I/R) myocardial injury, leading to persistent heart dysfunction and heart failure. It has been reported that Lycium barbarum polysaccharide (LBP) has protective effects on cardiomyocytes, but the specific mechanism is still not completely understood. The present study examined the protective role of LBP in myocardial I/R injury. Rats were subjected to myocardial I/R injury and LBP treatment. Moreover, rat myocardial H9C2 cells exposed to hypoxia/reoxygenation (H/R) were used to simulate cardiac injury during myocardial I/R process and were exposed to LBP, rapamycin (an autophagy activator) or nuclear factor-erythroid factor 2-related factor 2 (Nrf2) transfection. Morphological examination, histopathological examination and echocardiography were used to determine the cardiac injury after I/R injury. Cell viability and apoptosis were determined via MTT and flow cytometry assays, respectively. The levels of lactate dehydrogenase (LDH), creatine kinase (CK), cardiac troponin T (cTnT), IL-1β, IL-6, TNF-α, malondialdehyde (MDA) and superoxidase dismutase (SOD) in rat serum, hearts and/or cells were assessed using ELISAs. The expression levels of Beclin 1, LC3II/LC3I, P62 and Nrf2 were analyzed via reverse transcription-quantitative PCR and western blotting. The results demonstrated that LBP improved heart function and repaired cardiomyocyte damage in I/R model rats, as well as reduced the production of cTnT, CK, LDH, IL-1β, IL-6 and TNF-α. The in vitro study results indicated that LBP increased cell viability, the apoptosis rate, and the levels of SOD and P62, as well as reduced the levels of LDH, CK, IL-1β, IL-6, TNF-α, MDA, Beclin 1 and LC3-II/LC3-I in H/R-injured H9C2 cells. Moreover, LBP promoted Nrf2 nuclear translocation, but decreased Nrf2 expression in the cytoplasm. Rapamycin exacerbated the aforementioned effects in H/R injured H9C2 cells, and partially reversed LBP-induced effects. Overexpressing Nrf2 counteracted I/R-induced effects and partially resisted rapamycin-induced effects. These findings demonstrated that LBP exhibited a cardiac protective effect on the ischemic myocardium of rats after reperfusion and attenuated myocardial I/R injury via autophagy inhibition-induced Nrf2 activation.

Bone marrow-mesenchymal stem cell-derived exosomal microRNA-141 targets PTEN and activates β-catenin to alleviate myocardial injury in septic mice

Background Mesenchymal stem cells (MSCs) and their derived exosomes have shown potentials in the control of myocardial dysfunction. This study aimed to reveal the function of bone marrow (BM)-MSC-derived exosomes in sepsis-induced myocardial injury and the molecular mechanism. Methods BM-MSC-derived exosomes were obtained and identified. A mouse model with sepsis was induced by cecalligation puncture (CLP) and treated with exosomes. The myocardial function of mice, the production of creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH) in serum, the phosphorylation of a key myocardial contractility-related protein phospholamban (PLB), and the pathological changes in the myocardial tissues were examined. A microRNA (miRNA) microarray analysis was performed to examine the candidate miRNAs carried by the exosomes. Rescue experiments were conducted to validate the involvement of miR-141. Results CLP treatment led to sepsis and notably reduced the myocardial function in mice. Further treatment of BM-MSC-derived exosomes alleviated the CLP-induced myocardial impairment, production of CK-MB and LDH, and inflammatory infiltration and cell apoptosis in mouse myocardial tissues, and restored the PLB phosphorylation. miR-141 was the most upregulated miRNA in the myocardial tissues after exosome treatment. Downregulation of miR-141 blocked the myocardium-protective functions of the exosomes. miR-141 was found to bind to and suppress PTEN expression, which further enhanced the activity of β-catenin. Conclusion This study suggested that BM-MSC derived exosomes ameliorates myocardial injury in septic mice through conveying miRNA-141 and regulating the PTEN/β-catenin axis, and exosomes may serve as promising tools for the management of myocardial injury induced by sepsis or other factors.

Effect of polysaccharides from seeds of Vaccaria segetalis in alleviating urinary tract infection induced bladder injury by inhibiting NLRP3 inflammasome

To study the mechanism of polysaccharides from seeds of Vaccaria segetalis( PSV) in the treatment of bacterial cystitis through the NLRP3 inflammasome pathway. The rat model of urinary tract infection was used and treated with PSV,and the urine and bladders were collected. The level of interleukin-10( IL-10) in rat urine was detected by enzyme linked immunosorbent assay( ELISA). Western blot and immunofluorescence staining were used to detect the expressions of sonic hedgehog( SHH) and NLRP3 inflammasome [NOD-like receptor thermoprotein domain 3( NLRP3),apoptosis associated speck like protein( ASC) and pro-caspase-1]. The expression of Toll-like receptor pathway was detected by RT-PCR. The death of 5637 cells induced by uropathogenic Escherichia coli( UPEC) and lactate dehydrogenase( LDH) release were evaluated using live/dead staining. The results showed that in the rat bladder,the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors were significantly up-regulated,and NLRP3 inflammasomes were significantly activated by UPEC infection. The administration with PSV could significantly increase the concentration of IL-10 in urine,inhibit the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors in bladder,and inhibit the activation of NLRP3 inflammasomes. A large number of 5637 cells were dead after UPEC infection and caused LDH production. PSV could significantly inhibit the death of 5637 cells and the release of LDH. In conclusion,PSV could inhibit the expression and activation of NLRP3 inflammasomes by inhibiting the Toll-like receptor pathway,thereby mitigating the bladder injury.

Effect of Cryptotanshinone on Measures of Rat Cardiomyocyte Oxidative Stress and Gene Activation Associated with Apoptosis

Background: Oxidative stress is a key factor that results in cardiomyocyte apoptosis and cardiovascular diseases. Cryptotanshinone (CTS), one of the major bioactive constitutes extracted from the root of the plant Salvia miltiorrhizaBunge, has been widely studied for various disease treatments. However, the roles of CTS on cardiomyocytes remain unclear. Methods: In the present study, neonatal rat cardiomyocytes were pretreated with CTS for 4 h before being exposed to H₂O₂. Cell viability for the cells with or without pretreatment with CTS before exposure to H₂O₂ was evaluated by the MTT assay. Production of lactate dehydrogenase (LDH), nitric oxide (NO), prostaglandin E₂ (PGE₂), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxides (GSH-Px) was quantified by corresponding detection kits. The mRNA levels of Bcl-2 antiapoptotic and Bax-like proapoptotic genes were quantified with RT-PCR. Production of reactive oxygen species (ROS) was qualified and quantified with a dichlorofluorescein diacetate cellular ROS detection assay kit. The extracellular signal-related kinase (ERK) phosphorylation and nuclear factor κB (NF-κB) activation were measured by Western blot. Results: Our results revealed that the CTS pretreatment could enhance cell viability and promote Bcl-2 antiapoptotic gene expression. Additionally, CTS could abolish the H₂O₂-induced production of NO, LDH, and PGE₂. Consistent with these findings, CTS could inhibit ROS and MDA production and promote SOD, CAT, and GSH-Px activities. Mechanistically, CTS may achieve these processes by inhibiting ERK and NF-κB signal pathways. Conclusion: CTS protects cardiomyocytes against the H₂O₂-induced cellular injuries through ERK and NF-κB inactivation and ROS scavenging. Therefore, CTS is a promising reagent against ROS-induced cardiomyopathy.

Gastrodin prevents homocysteine-induced human umbilical vein endothelial cells injury via PI3K/Akt/eNOS and Nrf2/ARE pathway.

In this study, we investigated the protective effects of gastrodin (Gas) against homocysteine‐induced human umbilical vein endothelial cell (HUVEC) injury and the role of the phosphoinositide 3‐kinase (PI3K)/threonine kinase 1 (Akt)/endothelial nitric oxide synthase (eNOS) and NF‐E2‐related factor 2 (Nrf2)/antioxidant response element (ARE) pathways. We stimulated cells with homocysteine (1 mmol/L, 24 hours) and tested the effects of gastrodin (200‐800 μg/mL) on cell viability and the production of malondialdehyde (MDA), lactate dehydrogenase (LDH) and reactive oxygen species (ROS). Then, Nrf2 distribution in the cytoplasm and nucleus as well as the expression of enzymes downstream of Nrf2 was determined. Furthermore, we analysed the expression of bax, bcl‐2 and cleaved caspase3, and assessed the involvement of the PI3K/Akt/eNOS pathway by Western blots. Finally, we tested the vasoactive effect of gastrodin in thoracic aortic rings. The results showed that gastrodin decreased MDA, LDH and ROS production and increased cell viability, NO production and relaxation of thoracic aortic rings. Moreover, the protective effects of Gas on NO production and relaxation of thoracic aortic rings were blocked by L‐NAME but enhanced by Cav‐1 knockdown, and MK‐2206 treatment abolished the effect of Gas on the ROS. In addition, treatment with gastrodin increased Nrf2 nuclear translocation, thus enhancing the expression of downstream enzymes. Finally, gastrodin increased the expression of PI3K, p‐Akt, and eNOS and decreased Cav‐1 protein expression. In conclusion, our study suggested that gastrodin may protect HUVECs from homocysteine‐induced injury, and the PI3K/Akt/eNOS and Nrf2/ARE pathways may be responsible for the efficacy of gastrodin.

Differential effects of cisplatin on cybrid cells with varying mitochondrial DNA haplogroups.

BackgroundDrug therapy yields different results depending on its recipient population. Cisplatin, a commonly used chemotherapeutic agent, causes different levels of resistance and side effects for different patients, but the mechanism(s) are presently unknown. It has been assumed that this variation is a consequence of differences in nuclear (n) DNA, epigenetics, or some external factor(s). There is accumulating evidence that an individual’s mitochondrial (mt) DNA may play a role in their response to medications. Variations within mtDNA can be observed, and an individual’s mtDNA can be categorized into haplogroups that are defined by accumulations of single nucleotide polymorphisms (SNPs) representing different ethnic populations.MethodsThe present study was conducted on transmitochondrial cytoplasmic hybrids (cybrids) that possess different maternal-origin haplogroup mtDNA from African (L), Hispanic [A+B], or Asian (D) backgrounds. Cybrids were created by fusing Rho0 ARPE-19 cells (lacking mtDNA) with platelets, which contain numerous mitochondria but no nuclei. These cybrid cells were cultured to passage five, treated with cisplatin, incubated for 48 h, then analyzed for cell metabolic activity (tetrazolium dye (MTT) assay), mitochondrial membrane potential (JC-1 assay), cytotoxicity (lactate dehydrogenase (LDH) assay), and gene expression levels for ALK, BRCA1, EGFR, and ERBB2/HER2.ResultsResults indicated that untreated cybrids with varying mtDNA haplogroups had similar relative metabolic activity before cisplatin treatment. When treated with cisplatin, (1) the decline in metabolic activity was greatest in L (27.4%, p < 0.012) < D (24.86%, p = 0.0001) and [A+B] cybrids (24.67%, p = 0.0285) compared to untreated cybrids; (2) mitochondrial membrane potential remained unchanged in all cybrids (3) LDH production varied between cybrids (L >[A+B], p = 0.0270). (4) The expression levels decreased for ALK in L (p < 0.0001) and [A+B] (p = 0.0001) cybrids but not in D cybrids (p = 0.285); and decreased for EGFR in [A+B] cybrids (p = 0.0246) compared to untreated cybrids.ConclusionOur findings suggest that an individual’s mtDNA background may be associated with variations in their response to cisplatin treatment, thereby affecting the efficiency and the severity of side effects from the treatment.

Neuroprotection against cerebral ischemia/reperfusion by dietary phytochemical extracts from Tibetan turnip (Brassica rapa L.)

Ethnopharmacological relevanceThe Tibetan turnip (Brassica rapa L.) has a wide array of medicine properties including heat-clearing, detoxifying and anti-hypoxia as listed in the famous centuries-old Tibetan medicine classic “The Four Medical Tantras”. Evidence-based medicine also indicated the anti-hypoxic effect of turnips, suggesting a potential link to neuroprotective effect on ischemic stroke. This thereby enables turnips to serve as a novel nontoxic agent in related treatment. Aim of the studyThis study aimed to investigate the neuroprotective effect and elucidate the mechanism of aqueous extract of turnip (AET) on cerebral ischemia/reperfusion. Materials and methodsThe experimental models of cerebral ischemia included transient middle cerebral artery occlusion/reperfusion (MCAO) in C57BL/6J mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in HT-22 cells. Long-term effect of AET on infarct volume was evaluated by microtubule-associated protein 2 (MAP2) immunofluorescence 28 days after MCAO, and on neurofunctional outcomes determined by rotarod, grid walking, and cylinder tests in the meantime. Efficacy of AET was determined by the cell viability, the release of lactate dehydrogenase (LDH) and reactive oxygen species (ROS) in neurons. The underlying mechanism of AET rescued OGD/R cells were characterized by PI3K, Akt and mTOR expressions, which were further used to validate AET's role in the pathway. ResultsAET can reduce cerebral infarct volume and ameliorate behavioral deficits of MCAO/R mice dose-dependently. In vitro experiment further demonstrated that suitable concentrations of AET inhibited ROS, LDH production and restored mitochondrial expression induced by OGD/R. AET pretreatment can reverse the OGD/R-induced decreased level of phosphorylation of PI3K, Akt, mTOR, whereas this effect was blocked in the LY294002 (PI3K inhibitor) treatment group. ConclusionsAET improved the survival of OGD/R-injured HT-22 cells by activating the PI3K/Akt/mTOR pathway. Based on the results above, aqueous extract of turnip has a protective effect on focal cerebral ischemic injury.

Hepatoprotective Effects of Streptococcus thermophilus LM1012 in Mice Exposed to Air Pollutants.

In this study, we explored whether the use of Streptococcus thermophilus LM1012 (TL-LM1012) as a safe probiotic exerts hepatoprotective effects by suppressing oxidative stress and inflammation in vitro and alleviating aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) production in vivo. In a series of safety tests, TL-LM1012 was found to have a negative response to hemolysis and biogenic amines, as well as susceptibility to antibiotics. TL-LM1012 protected cell viability and suppressed cytotoxicity by inhibiting oxidative stress and induced heme oxygenase-1 and superoxide dismutase activity in a dose-dependent manner in diesel exhaust particulate matter (DEPM)-treated HepG2 cells. Moreover, proinflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-6, and IL-1β, were suppressed in DEPM-treated splenocytes. In DEPM-treated mice, oral administration of TL-LM1012 regulated AST, ALT, and LDH production in the serum after 14 days of treatment. These findings indicate that TL-LM1012, a safe probiotic, provides a potent preventive or therapeutic effect against liver disease caused by air pollution.

Helix B Surface Peptide Protects Cardiomyocytes From Hypoxia/Reoxygenation-induced Autophagy Through the PI3K/Akt Pathway.

Helix B surface peptide (HBSP) is a newly discovered tissue-protective erythropoietin derivative that provides benefits after myocardial ischemia/reperfusion. This study explores the cardioprotective effects of HBSP in myocardial cells in response to hypoxia/reoxygenation injury and its potential mechanism. In this study, rat ventricular (H9c2) cell cultures were established and pretreated with HBSP. H9c2 cardiomyocytes were randomly assigned to the control, H/R, H/R + LY294002 (a PI3K inhibitor), HBSP + H/R, and HBSP + H/R + LY294002 groups. The pretreated cardiomyocytes underwent H/R, and the cardiomyocytes were monitored for viability through a CCK-8 assay, whereas flow cytometry was used to test cell apoptosis. Orgotein Superoxide Dismutase (SOD) and lactate dehydrogenase (LDH) expression were monitored by SOD and LDH kits, respectively. The expression of LC3 autophagosomes was determined by immunocytochemistry. The expression of LC3II/LC3I, p-Mammalian Target of Rapamycin (mTOR) mTOR, mTOR, Beclin 1, p-PI3K, PI3K p-Akt, and Akt was determined by Western blotting. HBSP increased cell viability and reduced SOD and LDH production, and it also reduced H/R-induced cell apoptosis. Moreover, the expression of the autophagy-related proteins (LC3II/LC3I) was inhibited by HBSP, whereas the expression of p-PI3K, p-Akt, and p-mTOR was enhanced. However, the PI3K inhibitor (LY294002) notably abolished these effects in H9c2 cells. HBSP inhibits excessive autophagy and apoptosis induced by H/R by activating the PI3K/Akt pathway. HBSP may potentially be a therapeutic intervention for myocardial ischemia/reperfusion injury.

Longxuetongluo capsule alleviates lipopolysaccharide-induced neuroinflammation by regulating multiple signaling pathways in BV2 microglia cells.

Longxuetongluo capsule (LTC), derived from the total phenolic compounds of Chinese dragon's blood, is now used in the treatment of ischemic stroke in convalescence. The aim of this study is to explore the neuroprotective effect of LTC from the perspective of neuroinflammation. Cell viability and lactate dehydrogenase (LDH) release were measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and LDH assay kit. Proinflammatory mediators and cytokines production including Nitric Oxide (NO), prostaglandin E2, (PGE2), interleukin (IL-β), IL-6, and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA) assay. In addition, western blot was used to detect the expression of inflammatory proteins associated with the mitogen-activated protein kinases (MAPKs), janus kinase/signal transducer and activator of tranions (JAK/STAT), nuclear transcription factor κB (NF-κB), and nuclear factor erythroid-2-related actor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathways. Moreover, immunofluorescence assay and electrophoretic mobility shift assays (EMSA) were performed to determine the Nrf2 translocation and the binding-DNA activity of NF-κB, respectively. LTC at 0.5 to 2 μg/mL significantly increased cell viability and decreased LDH, NO, PGE2, IL-1β, IL-6, and TNF-α production in oxygen-glucose deprivation/reoxygenation (OGD/R) and lipopolysaccharide (LPS)-induced BV2 microglia cells. Meanwhile, LTC not only decreased the protein expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) but also down-regulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38, and up-regulated HO-1 expression via nuclear translocation of Nrf2. LTC can significantly inhibit the phosphorylation of JAK1/STAT3 and reduce the translocation of NF-κB from cytosol to nucleus as well as the binding-DNA activity. PC12 cell pretreated with LTC-condition medium (CM) significantly alleviated LPS-induced neurotoxicity and increased PC12 cell viability in a dose-dependent manner. The present study showed that LTC exhibited a strong antineuroinflammatory activity and neuroprotective effects on LPS-stimulated BV2 microglial cells and PC12 cells.