Dapagliflozin Protects H9c2 Cells Against Injury Induced by Lipopolysaccharide via Suppression of CX3CL1/CX3CR1 Axis and NF-κB Activity.

BACKGROUND: Dapagliflozin, a selective Sodium-glucose cotransporter-2 (SGLT2) inhibitor, has been shown to play a key role in the control and management of metabolic and cardiac diseases. OBJECTIVE: The current study aims to address the effects of dapagliflozin on the expression of fractalkine (FKN), known as CX3CL1, and its receptors CX3CR1, Nuclear factor-kappa B(NF-κB) p65 activity, Reactive oxygen species (ROS), and inflammation in LPS-treated H9c2 cell line. METHODS: H9c2 cells were cultured with lipopolysaccharide (LPS) to establish a model of LPS-induced damage, and then, subsequently were treated with dapagliflozin for 72 h. Our work included measurement of cell viability (MTT), Malondialdehyde (MDA), intracellular ROS, tumor necrosis factor-α (TNF-α), NF-κB activity, and expression of CX3CL1/CX3CR1. RESULTS: The results showed that LPS-induced reduction of cell viability was successfully rescued by dapagliflozin treatment. The cellular levels of MDA, ROS, and TNF-α, as an indication of cellular oxidative stress and inflammation, were significantly elevated in H9c2 cells compared to the control group. Furthermore, dapagliflozin ameliorated inflammation and oxidative stress through the modulation of the levels of MDA, TNF-α, and ROS. Correspondingly, dapagliflozin reduced the expression of CX3CL1/CX3CR1, NF-κB p65 DNA binding activity, and it also attenuated nuclear acetylated NF-κB p65 in LPS-induced injury in H9c2 cells compared to untreated cells. CONCLUSION: These findings shed light on the novel pharmacological potential of dapagliflozin in the alleviation of LPS-induced CX3CL1/CX3CR1-mediated injury in inflammatory conditions such as sepsis-induced cardiomyopathy.

Sulforaphane modulates CX3CL1/CX3CR1 axis and inflammation in palmitic acid-induced cell injury in C2C12 skeletal muscle cells.

Studies have shown that sulforaphane (SFN) has potent anti-inflammatory and free radical scavenging effects on obesity and associated disorder such as diabetes, polycystic ovary syndrome, and metabolic syndrome. fractalkine (CX3CL1) and its receptor, CX3CR1, play an important role in muscle metabolism by improving insulin-sensitizing effects. Here, in this study we examined the SFN effect on CX3CL1 and its receptor, CX3CR1, in C2C12 myotubes in palmitic acid (PA)-induced oxidative stress and inflammation. The results showed that PA (750 µM) evoked lipotoxicity as a reduction in cell viability, increased IL-6 and TNF-α expression, and enhanced reactive oxygen species (ROS). However, SFN pretreatment attenuated the levels of, IL-6 and TNF-α in C2C12 myotubes exposure to PA. Moreover, SFN pretreatment up-regulated nuclear factor erythroid related factor 2 (Nrf2) /heme oxygenase-1(HO-1) pathway protein in C2C12 cells as indicated by a decrease in ROS levels. Interestingly, PA also caused an increase in CX3CL1 and CX3CR1 expression that SFN abrogated it. We also found the protective effect of SFN agonist PA-induced lipotoxicity with promotes in UCP3 gene expression in C2C12 cells. Collectively, these findings suggest that SFN hampers the PA-induced inflammation in C2C12 cells by modulation of the Nrf2/HO-1 pathway and CX3CL1/CX3CR1 axis and may propose a new therapeutic approach to protect against obesity-associated disorders in skeletal muscle cells.

Human Amnion Membrane Proteins Prevent Doxorubicin-Induced Oxidative Stress Injury and Apoptosis in Rat H9c2 Cardiomyocytes.

Doxorubicin (DOX) is widely used as an effective chemotherapy agent in cancer treatment. Cardiac toxicity in cancer treatment with DOX demand urgent attention and no effective treatment has been established for DOX-induced cardiomyopathy. It has been well documented that human amniotic membrane proteins (AMPs), extracted from amnion membrane (AM), have antioxidant, anti-apoptotic, and cytoprotective properties. Therefore, in this study, we aimed to investigate the protective effects of AMPs against cardiotoxicity induced by DOX in cultured rat cardiomyocyte cells (H9c2). DOX-induced cell injury was evaluated using multi-parametric assay including thiazolyl blue tetrazolium bromide (MTT), the release of lactic dehydrogenase (LDH), intracellular Ca2+ , reactive oxygen species (ROS) levels, cellular antioxidant status, mitochondrial membrane potential (ΔΨm), malondialdehyde (MDA), and NF-κB p65 DNA-binding activity. Moreover, expression profiling of apoptosis-related genes (P53, Bcl-2, and Bax) and Annexin V by flow cytometry were used for cell apoptosis detection. It was shown that AMPs pretreatment inhibited the cell toxicity induced by DOX. AMPs effectively attenuated the increased levels of LDH, Ca2+ , ROS, and MDA and also simultaneously elevated the ΔΨm and antioxidant status such as superoxide dismutase (SOD) and Catalase (CAT) in pretreated H9c2 cardiomyocytes. Besides, the activity of NF-kB p65 was reduced and the p53 and Bax protein levels were inhibited in these myocardial cells subjected to DOX. These findings provide the first evidence that AMPs potently suppressed DOX-induced toxicity in cardiomyocytes through inhibition of oxidative stress and apoptosis. Thus, AMPs can be a potential therapeutic agent against DOX cardiotoxicity.

1, 25-Dihydroxyvitamin D3 activates Apelin/APJ system and inhibits the production of adhesion molecules and inflammatory mediators in LPS-activated RAW264.7 cells.

BACKGROUND: 1, 25-Dihydroxyvitamin D3 (1, 25(OH)2D3), an active form of vitamin D3, plays a crucial role in the mitigation of inflammation damage. Recent studies have revealed that apelin and its receptor (apelin/APJ system) could significantly ameliorate LPS-induced inflammation-response. This investigation aimed to appraise the effects of 1, 25(OH)2D3 on the apelin/APJ system and production of adhesion molecules and inflammatory mediators in LPS-activated RAW264.7 macrophage cells. METHODS: Murine RAW264.7 cells were pretreated with 1, 25(OH)2D3, followed stimulation with LPS (1 µg/mL) for 24 h. The effect of 1, 25(OH)2D3 on LPS-induced cell injury was determined by MTT assay, whereas, enzyme-linked immunosorbent assay (ELISA), qPCR and western blotting were used to evaluate cytokine production and apelin/APJ system expression. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein expression were measured by flow cytometry. RESULTS: The levels of IL-1ß, IL-6, and TNF-α cytokines were significantly increased by incubation with LPS. LPS also increased the protein expression of adhesion molecules, including VCAM-1 and ICAM-1. However, pretreatment with 1, 25(OH)2D3 markedly inhibited LPS-induced production of inflammatory cytokines and adhesion molecules. Moreover, we found that 1, 25(OH)2D3 could induced the apelin/APJ system expression. Further experiments demonstrated the significant increase of apelin/APJ system expression at both the protein and mRNA levels in LPS-activated cells when pretreated with 1, 25(OH)2D3. CONCLUSION: Taken together, our results indicated that 1, 25(OH)2D3 confers an anti-inflammatory effect through a likely mechanism involving a reduction in pro-inflammatory mediators and adhesion molecules via up-regulation of the apelin/APJ system in RAW264.7 cells.

Nrf2 activation and down-regulation of HMGB1 and MyD88 expression by amnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cells.

BACKGROUND: human Amniotic Membrane (hAM) extracts contain bioactive molecules such as growth factors and cytokines. Studies have confirmed the ability of hAM in reduction of post-operative dysfunction in patients with cardiac surgery. However, the function of Amniotic Membrane Proteins (AMPs), extracted from hAM, against hypoxia-induced H9c2 cells injury have never been investigated. In this study, we aimed to appraise the protective impact of AMPs on H9c2 cells under hypoxia condition. METHODS: Cardiomyocyte cells were pre-incubated with AMPs and subjected to 24 h hypoxia to elucidate its effects on expression of Nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1(HO-1). Furthermore, the high mobility group box-1 (HMGB1) and Myeloid differentiation primary response 88 (MyD88) expressions were detected by qPCR and western-blotting. The mitochondrial membrane potential (ΔΨm) was estimated by JC-1 using fluorescent microscopy and fluorimetry. Moreover, the cell apoptosis and intracellular calcium levels were measured by flow cytometry. RESULTS: Pre-treatment of AMPs resulted in significant induction in cell viability and decreased the LDH release under hypoxic condition in H9c2 cells. Accordingly, these protective effects of AMPs were associated with a reduction in apoptosis rates and intracellular Ca2+, meanwhile, ΔΨm was increased. Pre-treatment with AMPs resulted in degradation of HMGB1 and MyD88 levels and depicted pro-survival efficacy of AMPs against hypoxia-induced cell damage through induction of HO-1 and Nrf2. CONCLUSION: The data indicated that AMPs mediated HO-1 regulation by Nrf2 activation and plays critical protective effects in hypoxia-induced H9c2 injury in vitro by the inhibition of myocardial HMGB1 and MyD88 inflammatory cascade.

Copper and zinc in stage I multiple myeloma: relation with ceruloplasmin, lipid peroxidation, and superoxide dismutase activity.

Background The main aim of this study was to assess the serum levels of copper (Cu), zinc (Zn) with lipid peroxidation, Cu/Zn superoxide dismutase (Cu/Zn SOD) activity, and ceruloplasmin (Cp) in multiple myeloma (MM) patients. Materials and methods The study was conducted in 34 MM patients at stage I. Serum Cu and Zn levels were measured by atomic absorption spectrometry. Also, spectrophotometric assays of malondialdehyde (MDA) levels in addition to Cp and Cu/Zn SOD were quantitated. Results The results showed a significant decrease in the serum Zn levels in patients with MM (p < 0.0001). Also, serum Cu levels were significantly higher (p < 0.0001). However, the serum Cu/Zn ratio was significantly higher in the cancer patients (p < 0.0001). A significant difference was observed in the patients group compared with the control group according to the Cu/Zn SOD activity (p < 0.0001). Moreover, serum levels of Cp and MDA were significantly increased in patients (p < 0.0001, both). Conclusions The elevated levels of serum Cu and MDA with a decrease in Zn and Cu/Zn SOD might explain the increased oxidative stress in MM disease. As the high Cu level was observed in MM patients, therefore, Cu levels should be concentrated in the pathogenesis and progression of MM disease.

Amniotic membrane extracted proteins protect H9c2 cardiomyoblasts against hypoxia-induced apoptosis by modulating oxidative stress.

Protection of the cardiac cell against hypoxia-induced cell damage is one of the main approaches to preventing cardiovascular disease. Earlier studies have shown the cardioprotective effect of the human Amniotic Membrane (hAM) in animal model of cardiac injury. However, the effect of Amniotic Membrane Proteins (AMPs), extracted from hAM, on myocardial hypoxia injury remains unclear. So, our study aimed to investigate the protective effect of AMPs against hypoxia-induced cardiomyocytes apoptosis. H9c2 cardiomyocytes were pre-treated with AMPs followed by 24 h in hypoxia condition. Cell viability and apoptotic induction were detected by MTT and PI staining assay. Furthermore, the reactive oxygen species (ROS) generation, caspase-3 activity and malondialdehyde (MDA) were measured using the relevant kits. Moreover, apoptosis associated molecules and NF-kB p65 subunit, the master regulator of inflammation; expression was measured by western blotting. Our results indicated that AMPs increased the cellular viability of H9c2 cells during hypoxia and attenuated apoptotic induction. AMPs reduced hypoxia-induced ROS generation and as indicated by decreased MDA content. Moreover, AMPs decreased Bax/Bcl-2 ratios followed by reduction the caspase-3 activity; and further repressed the phosphorylated NF-kB p65. Altogether, suggesting that AMPs offers cardioprotective effects to H9c2 cell in hypoxia condition by modulating the gene involved in apoptosis and reducing oxidative stress and inflammatory response.

A methodological approach for production and purification of polyclonal antibody against dog IgG.

Antibodies are a class of biomolecules that has an important role in the immune system and lots of applications in biotechnological methods and in pharmaceutics. Production and purification of antibodies in laboratory animals is one of the first ways to manufacture of these prominent tools. The obtained antibodies from these process could be used in various types of bioassay techniques such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay, etc. Also, antibodies employed in diagnostics applications in humans and other animals in order to detect specific antigens. In this study, we aimed to produce and purify anti-dog IgG via immunizing rabbits with dog IgG in combination with Freund's adjuvant. Polyclonal IgG were purified by ion exchange chromatography and then the purified antibody was labeled with horse radish peroxidase (HPR). Direct ELISA was used to determine the optimum titer and cross-reactivity of HRP conjugated IgG. The purity of various IgG preparations and the optimum dilution of prepared HRP conjugated IgG, respectively, was about 95.00% and 1:8000. This study showed that efficiency ion-exchange chromatography could be an appropriate method for purification of IgG antibodies. This antibody could be a useful tool for future dog immune diagnosis tests. This product characterization shown here sets the foundations for future work on dog IgGs.

Decreasing serum homocysteine and hypocholesterolemic effects of Bovine lactoferrin in male rat fed with high-cholesterol diet.

Introduction: Lipid metabolism disorder or hyperlipidemia is known as a risk factor for cardiovascular disease, the increase in serum homocysteine and leptin are associated with atherosclerotic disease. The purpose of the present study was to examine the effects of bovine lactoferrin (bLF) on serum homocysteine (Hcy), apolipoproteinA-I (ApoA-I) and B (ApoB), leptin and lipid profile changes in high-cholesterol-diet (HCD) fed rats. Methods: The Healthy Adult Sprague-Dawley (SD) male rats were randomly assigned into three experimental groups. Each group consisted of eleven male rats including control group, HCD rats and hypercholesterolemic rats, which were treated with bLF (HCD+bLF). bLF was given by gavage (200 mg/kg/d). After 4 weeks of feeding and overnight fasting, total blood samples were collected. Results: The results showed the elevated level of Hcy, leptin, total cholesterol, low density lipoprotein cholesterol (LDL-C), ApoB and decrease in ApoA-I in non-treated HCD group compared to the control rats. Administration of bLF significantly ameliorated the Hcy and leptin levels with decrease in LDL-C and total cholesterol in rats fed with a high-cholesterol diet. bLF also tended to increase low serum concentration of ApoA-I and high density lipoprotein cholesterol (HDL-C) in HCD rats. Meanwhile, upon bLF-treated rats, there was a significant decrease in ApoB in HCD group. Conclusion: The findings indicated that bLF can improve the alteration of serum Hcy, leptin, apolipproteins and lipid changes in male rats fed with high-cholesterol diet. So, bLF can counteract with HCD elicited hyper-homocysteinemia and hyper-leptinemia, suggesting it to have the useful therapeutic potential in patients with atherosclerosis and lipid disorder.

Bovine lactoferrin ameliorates antioxidant esterase activity and 8-isoprostane levels in high-cholesterol-diet fed rats.

The main aim of the present study was to show the effect of bovineLactoferrin (bLF), an 80 kD iron-binding glycoprotein, its application on antioxidant esterase activities and 8-isoprostane changes in high-cholesterol-diet fed (HCD-Fed) rats. The 44 adult Sprague-Dawley male rats were randomly assigned into four experimental groups. They were randomly assigned into four equivalent groups (n = 11). The groups included the control group which was fed with normal diet, bLF group, the third group which were made hypercholesterolemia by being fed with high cholesterol diet, and the last group which consisted of hypercholesterolemia rats treated with bLF (HCD + bLF) for 4 weeks (200 mg.kg-1 per day wt. dissolved in 0.9% normal saline).After 4 weeks, the serum Paraoxonase1 (PON1), Arylesterase (ARE) activity and 8-isoprostane with lipid profile were measured. Upon treatment with the bLF, the decrease in LDL-Cholesterol (LDL-C), Glucoses, Triglyceride (TG) and Total-Cholesterol (TC) levels and an increase in HDL-Cholesterol (HDL-C) level were observed. The co-administration of bLf for 4 weeks had decreased the 8-isoprostane levels significantly (P < 0.001) (86.36 ± 7.1 vs 117.18 ± 8.62) when compared to hypercholesterolemia-induced rats. Also, the Atherogenic Index (AI) in HCD + bLF group showed a significant decrease as compared to the HCD group (P < 0.001) (0.37 ± 0.07 vs 0.57 ± 0.09). The results indicated that bLF was effective against oxidative stress by its ability to increase PON1 activity and reduce the lipid peroxidation in high-cholesterol-fed rats.

The Study of Extracellular Protein Fractions of Probiotic Candidate Bacteria on Cancerous Cell Line.

INTRODUCTION: Probiotics are live microorganisms, habituated in the human intestine, which have a beneficial effect on our health. In spite of many reports about the anticancer effect of these bacteria in in-vivo and in-vitro, their mechanisms of action are not completely understood. The goal of this study was to compare the extracellular fractions of Lactobacillus casei and L. paracasei on the anti proliferation and apoptosis induction in K562 cell line. MATERIALS AND METHODS: L. casei and L. paracasei were cultured in MRS broth medium. Then extracellular secretions were collected and after enrichment, analyzed by electrophoresis. Fractionation were determined by gel filtration chromatography using sephadex G100 column, and the anticancer properties were evaluated. RESULTS: The results of SDS-PAGE showed various molecular weight of fractionated proteins of L. casei and L. paracasei. Bioactivity assessment illustrated that anti proliferative effects on K562 cells is dose and time dependent and the cytotoxic effects was parallel with protein concentration and the increase of time from 36 to 72 hours. CONCLUSION: Regarding the cell cytotoxicity results, the fractionated extracellular proteins of L. casei and L. paracasei have significant effects in inhibition of cancer cell proliferation. However, more study is needed to  better elucidate the  mechanisms of extracted proteins, and its effect on other human cancer cell lines.

A methodological approach for purification and characterization of human serum albumin.

As the most predominant protein in plasma, albumin is synthesized in the liver. Given to various applications of albumin as biopharmaceutical agent, the annual demand for it is 500 tons in the world, which is the highest in the biomedical solutions demand ranking. There exist different procedures for production of albumin. The aim of this study was the purification of human serum albumin (HSA) using immunoaffinity chromatography. After immunization of rabbits, passive immunodiffusion and indirect ELISA tests were applied for assessment of polyclonal antibody production against HSA. Purification was performed by ion exchange chromatography (IEC) and protein G affinity chromatography. The produced anti-HSA IgG was attached to the CNBR-activated Sepharose and applied for albumin purification from human serum. Western blotting (WB) analysis and heat-induced insolubility were performed for functional and stability measurement assessment of immunoaffinity purified HSA, respectively. The optimum titer of anti-HSA determined by indirect ELISA was 256000. The SDS-PAGE showed that the purity rate of albumin was approximately 98% and WB confirmed the HSA functionality. Also, the heat-induced insolubility of immunoaffinity purified HSA was the same as the commercial HSA. Affinity chromatography using produced polyclonal antibody would be a robust method for purification of HSA.