Chongshan Dai

Colistin-Induced Nephrotoxicity in Mice Involves the Mitochondrial, Death Receptor, and Endoplasmic Reticulum Pathways

ABSTRACT Nephrotoxicity is the dose-limiting factor for colistin, but the exact mechanism is unknown. This study aimed to investigate the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in colistin-induced nephrotoxicity. Mice were intravenously administered 7.5 or 15 mg of colistin/kg of body weight/day (via a 3-min infusion and divided into two doses) for 7 days. Renal function, oxidative stress, and apoptosis were measured. Representative biomarkers involved in the mitochondrial, death receptor, and endoplasmic reticulum pathways were investigated, and the key markers involved in apoptosis and autophagy were examined. After 7-day colistin treatment, significant increase was observed with blood urea nitrogen, serum creatinine, and malondialdehyde, while activities of superoxide dismutase (SOD) and catalase decreased in the kidneys. Acute tubular necrosis and mitochondrial dysfunction were detected, and colistin-induced apoptosis was characterized by DNA fragmentation, cleavage of poly(ADP-ribose) polymerase (PARP-1), increase of 8-hydroxydeoxyguanosine (8-OHdG), and activation of caspases (caspase-8, -9, and -3). It was evident that colistin-induced apoptosis involved the mitochondrial pathway (downregulation of Bcl-2 and upregulation of cytochrome C [cytC] and Bax), death receptor pathway (upregulation of Fas, FasL, and Fas-associated death domain [FADD]), and endoplasmic reticulum pathway (upregulation of Grp78/Bip, ATF6, GADD153/CHOP, and caspase-12). In the 15-mg/kg/day colistin group, expression of the cyclin-dependent kinase 2 (CDK2) and phosphorylated JNK (p-JNK) significantly increased (P < 0.05), while in the 7.5-mg/kg/day colistin group, a large number of autophagolysosomes and classic autophagy were observed. Western blot results of Beclin-1 and LC3B indicated that autophagy may play a protective role in colistin-induced nephrotoxicity. In conclusion, this is the first study to demonstrate that all three major apoptosis pathways and autophagy are involved in colistin-induced nephrotoxicity.

Lycopene Attenuates Colistin-Induced Nephrotoxicity in Mice via Activation of the Nrf2/HO-1 Pathway

ABSTRACT Nephrotoxicity is the major dose-limiting factor for the clinical use of colistin against multidrug-resistant (MDR) Gram-negative bacteria. This study aimed to investigate the protective effect of lycopene on colistin-induced nephrotoxicity in a mouse model. Fifty mice were randomly divided into 5 groups: the control group (saline solution), the lycopene group (20 mg/kg of body weight/day administered orally), the colistin group (15 mg/kg/day administered intravenously), the colistin (15 mg/kg/day) plus lycopene (5 mg/kg/day) group, and the colistin (15 mg/kg/day) plus lycopene (20 mg/kg/day) group; all mice were treated for 7 days. At 12 h after the last dose, blood was collected for measurements of blood urea nitrogen (BUN) and serum creatinine levels. The kidney tissue samples were obtained for examination of biomarkers of oxidative stress and apoptosis, histopathological assessment, and quantitative reverse transcription-PCR (qRT-PCR) analysis. Colistin treatment significantly increased concentrations of BUN and serum creatinine, tubular apoptosis/necrosis, lipid peroxidation, and heme oxygenase 1 (HO-1) activity, while the treatment decreased the levels of endogenous antioxidant biomarkers glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). Notably, the changes in the levels of all biomarkers were attenuated in the kidneys of mice treated with colistin by lycopene (5 or 20 mg/kg). Lycopene treatment, especially in the colistin plus lycopene (20 mg/kg) group, significantly downregulated the expression of NF-κB mRNA (P < 0.01) but upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1 mRNA (both P < 0.01) in the kidney compared with the results seen with the colistin group. Our data demonstrated that coadministration of 20 mg/kg/day lycopene can protect against colistin-induced nephrotoxicity in mice. This effect may be attributed to the antioxidative property of lycopene and its ability to activate the Nrf2/HO-1 pathway.

Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells

Abstract Quinocetone (QCT), a new quinoxaline 1,4-dioxides, has been used as antimicrobial feed additive in China. Potential genotoxicity of QCT was concerned as a public health problem. This study aimed to investigate the protective effect of curcumin on QCT-induced oxidative stress and genotoxicity in human hepatocyte L02 cells. Cell viability and intracellular reactive oxygen species (ROS), biomarkers of oxidative stress including superoxide dismutase (SOD) activity and glutathione (GSH) level were measured. Meanwhile, comet assay and micronucleus assay were carried out to evaluate genotoxicity. The results showed that, compared to the control group, QCT at the concentration ranges of 2–16 μg/mL significantly decreased L02 cell viability, which was significantly attenuated with curcumin pretreatment (2.5 and 5 μM). In addition, QCT significantly increased cell oxidative stress, characterized by increases of intracellular ROS level, while decreased endogenous antioxidant biomarkers GSH level and SOD activity (all p < 0.05 or 0.01). Curcumin pretreatment significantly attenuated ROS formation, inhibited the decreases of SOD activity and GSH level. Furthermore, curcumin significantly reduced QCT-induced DNA fragments and micronuclei formation. These data suggest that curcumin could attenuate QCT-induced cytotoxicity and genotoxicity in L02 cells, which may be attributed to ROS scavenging and anti-oxidative ability of curcumin. Importantly, consumption of curcumin may be a plausible way to prevent quinoxaline 1,4-dioxides-mediated oxidative stress and genotoxicity in human or animals.

Furazolidone induces apoptosis through activating reactive oxygen species-dependent mitochondrial signaling pathway and suppressing PI3K/Akt signaling pathway in HepG2 cells.

Furazolidone (FZD), a synthetic nitrofuran with a broad spectrum of antimicrobial activities, has been shown to be genotoxic and potentially carcinogenic in several types of cells. However, the proper molecular mechanisms of FZD toxicity remain unclear. This study was aimed to explore the effect of FZD on apoptosis in HepG2 cells and uncover signaling pathway underlying the cytotoxicity of FZD. The results showed that FZD induced apoptosis in HepG2 cells in a dose-dependent manner characterized by nuclei morphology changes, cell membrane phosphatidylserine translocation, poly (ADP-ribose) polymerase (PARP) cleavage and a cascade activation of caspase-9 and -3. FZD could enhance reactive oxygen species (ROS) generation, up-regulate Bax/Bcl-2 ratio, disrupt mitochondrial membrane potential (MMP) and subsequently cause cytochrome c release. Both ROS scavenger (N-acetyl cysteine, NAC) and caspase inhibitors suppressed FZD-induced apoptosis. Furthermore, NAC attenuated FZD-induced ROS generation and mitochondrial dysfunction. Meanwhile, FZD treatment inhibited both the activation and expression of Akt, and PI3K/Akt inhibitor LY294002 promoted FZD-induced apoptosis. On the contrary, PI3K/Akt activator insulin-like growth factor-1 (IGF-1) attenuated lethality of FZD in HepG2 cells. In conclusion, it is first demonstrated that FZD-induced apoptosis in HepG2 cells might be mediated through ROS-dependent mitochondrial signaling pathway and involves PI3K/Akt signaling.

Curcumin Ameliorates Furazolidone-Induced DNA Damage and Apoptosis in Human Hepatocyte L02 Cells by Inhibiting ROS Production and Mitochondrial Pathway

Furazolidone (FZD), a synthetic nitrofuran derivative, has been widely used as an antibacterial and antiprotozoal agent. Recently, the potential toxicity of FZD has raised concerns, but its mechanism is still unclear. This study aimed to investigate the protective effect of curcumin on FZD-induced cytotoxicity and the underlying mechanism in human hepatocyte L02 cells. The results showed that curcumin pre-treatment significantly ameliorated FZD-induced oxidative stress, characterized by decreased reactive oxygen species (ROS) and malondialdehyde formation, and increased superoxide dismutase, catalase activities and glutathione contents. In addition, curcumin pre-treatment significantly ameliorated the loss of mitochondrial membrane potential, the activations of caspase-9 and -3, and apoptosis caused by FZD. Alkaline comet assay showed that curcumin markedly reduced FZD-induced DNA damage in a dose-dependent manner. Curcumin pre-treatment consistently and markedly down-regulated the mRNA expression levels of p53, Bax, caspase-9 and -3 and up-regulated the mRNA expression level of Bcl-2. Taken together, these results reveal that curcumin protects against FZD-induced DNA damage and apoptosis by inhibiting oxidative stress and mitochondrial pathway. Our study indicated that curcumin may be a promising combiner with FZD to reduce FZD-related toxicity in clinical applications.

Quinocetone triggered ER stress-induced autophagy via ATF6/DAPK1-modulated mAtg9a trafficking

The present study is undertaken to explore quinocetone-induced autophagy and its possible mechanism. Western blotting and green fluorescence protein (GFP)-LC3 vector transfection were performed to determine the ratio of LC3 conversion and its subcellular localization. Results revealed that the quinocetone induced autophagy in time- and dose-dependent manners. Besides, we tested the expressions of immunoglobulin heavy chain binding protein (BiP) and C/EBP homologous protein (CHOP) and the transcription of BiP, HerpUD, and sec24D by western blotting and RT-PCR, respectively. Results showed that quinocetone also induced endoplasmic reticulum (ER) stress during quinocetone-induced autophagy. Furthermore, we observed the cleavage of ATF6, the phosphorylation of MRLC, and the expression of death-associated protein kinase (DAPK1) by western blotting; the transcription of DAPK1 by RT-PCR; and the subcellular localization of ATF6 and mAtg9 by immunofluorescence. These results suggest that quinocetone stimulates the MRLC-mediated mAtg9 trafficking, which is critical for autophagosome formation, via the ATF6 upregulated expression of DAPK1. Last, we generated ATF6 and DAPK1 stable knockdown HepG2 cell lines and found that the conversion ratios of LC3 were decreased upon the treatment of quinocetone. Together, we propose that quinocetone induces autophagy through ER stress signaling pathway-induced cytoskeleton activation.

GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

Olaquindox, a quinoxaline 1,4-dioxide derivative, is widely used as a feed additive in many countries. The potential genotoxicity of olaquindox, hence, is of concern. However, the proper mechanism of toxicity was unclear. The aim of the present study was to investigate the effect of growth arrest and DNA damage 45 alpha (GADD45a) on olaquindox-induced DNA damage and cell cycle arrest in HepG2 cells. The results showed that olaquindox could induce reactive oxygen species (ROS)-mediated DNA damage and S-phase arrest, where increases of GADD45a, cyclin A, Cdk 2, p21 and p53 protein expression, decrease of cyclin D1 and the activation of phosphorylation-c-Jun N-terminal kinases (p-JNK), phosphorylation-p38 (p-p38) and phosphorylation-extracellular signal-regulated kinases (p-ERK) were involved. However, GADD45a knockdown cells treated with olaquindox could significantly decrease cell viability, exacerbate DNA damage and increase S-phase arrest, associated with the marked activation of p-JNK, p-p38, but not p-ERK. Furthermore, SP600125 and SB203580 aggravated olaquindox-induced DNA damage and S-phase arrest, suppressed the expression of GADD45a. Taken together, these findings revealed that GADD45a played a protective role in olaquindox treatment and JNK/p38 pathways may partly contribute to GADD45a regulated olaquindox-induced DNA damage and S-phase arrest. Our findings increase the understanding on the molecular mechanisms of olaquindox.

Effect of GADD45a on olaquindox-induced apoptosis in human hepatoma G2 cells: Involvement of mitochondrial dysfunction.

Olaquindox, a quinoxaline 1, 4-dioxide derivative, has been widely used as a feed additive for promoting animal growth in China. The aim of present study was to investigate the effect of grow arrest and DNA damage 45 alpha (GADD45a) on olaquindox-induced apoptosis in HepG2 cells. The result showed that olaquindox induced the decrease of cell viability in a dose dependent manner. Compared to the control group, olaquindox treatment at 400 and 800μg/mL increased the expression level of GADD45a protein and reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (MMP), and subsequently increased the expression of Bax while decreased the expression of Bcl-2, leading to the release of cytochrome c (Cyt c). However, knockdown of GADD45a enhanced olaquindox-induced ROS production, disrupted MMP and subsequently caused Cyt c release, then further increased olaquindox- induced cell apoptosis by increasing the activities of caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP). In conclusion, the results revealed that GADD45a played a critical role in olaquindox-induced apoptosis in HepG2 cells, which may embrace the regulatory ability on the mitochondrial apoptosis pathway.

Involvement of the activation of Nrf2/HO-1, p38 MAPK signaling pathways and endoplasmic reticulum stress in furazolidone induced cytotoxicity and S phase arrest in human hepatocyte L02 cells: modulation of curcumin

Abstract Furazolidone (FZD) is extensively used as the antiprotozoal and antibacterial drug in clinic. The previous study has shown that curcumin pretreatment could improve FZD induced cytotoxicity by inhibiting oxidative stress and mitochondrial apoptotic pathway. The current study aimed to investigate the potential roles of endoplasmic reticulum (ER) stress, p38 mitogen-activated protein kinases (p38 MAPK) signaling pathway in curcumin against FZD cytotoxicity by using human hepatocyte L02 cells. The results showed that curcumin could markedly attenuate FZD induced cytotoxicity. Compared with FZD alone group, curcumin pretreatment significantly reduced the expression of phospho (p)-p38, cyclin D1, p-checkpoint kinase 1 (ChK1) and breast cancer associated gene 1 (BRCA1) protein, followed to attenuate S phase arrest. Meanwhile, curcumin pretreatment prevented FZD induced ER stress, evidenced by the inhibition of glucose-regulated protein 78 and DNA damage inducible gene 153/C/EBP-homologous protein (GADD153/CHOP) protein expression. Moreover, compared with the control, FZD exposure activated the protein and mRNA expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1), which were further activated by curcumin treatment. These results reveal that curcumin could prevent FZD induced cytotoxicity and S phase arrest, which may involve the activation of Nrf2/HO-1 pathway and the inhibition of p38 MAPK pathway and ER stress.

Minocycline attenuates colistin-induced neurotoxicity via suppression of apoptosis, mitochondrial dysfunction and oxidative stress

Background Neurotoxicity is an adverse effect patients experience during colistin therapy. The development of effective neuroprotective agents that can be co-administered during polymyxin therapy remains a priority area in antimicrobial chemotherapy. The present study investigates the neuroprotective effect of the synergistic tetracycline antibiotic minocycline against colistin-induced neurotoxicity. Methods The impact of minocycline pretreatment on colistin-induced apoptosis, caspase activation, oxidative stress and mitochondrial dysfunction were investigated using cultured mouse neuroblastoma-2a (N2a) and primary cortical neuronal cells. Results Colistin-induced neurotoxicity in mouse N2a and primary cortical cells gives rise to the generation of reactive oxygen species (ROS) and subsequent cell death via apoptosis. Pretreatment of the neuronal cells with minocycline at 5, 10 and 20 μM for 2 h prior to colistin (200 μM) exposure (24 h), had an neuroprotective effect by significantly decreasing intracellular ROS production and by upregulating the activities of the anti-ROS enzymes superoxide dismutase and catalase. Minocycline pretreatment also protected the cells from colistin-induced mitochondrial dysfunction, caspase activation and subsequent apoptosis. Immunohistochemical imaging studies revealed colistin accumulates within the dendrite projections and cell body of primary cortical neuronal cells. Conclusions To our knowledge, this is first study demonstrating the protective effect of minocycline on colistin-induced neurotoxicity by scavenging of ROS and suppression of apoptosis. Our study highlights that co-administration of minocycline kills two birds with one stone: in addition to its synergistic antimicrobial activity, minocycline could potentially ameliorate unwanted neurotoxicity in patients undergoing polymyxin therapy.