How do different arsenic species affect the joint toxicity of perfluorooctanoic acid and arsenic to earthworm Eisenia fetida: A multi-biomarker approach.

Perfluorooctanoic acid (PFOA) and arsenic are widely distributed pollutants and can coexist in the environment. However, no study has been reported about the effects of different arsenic species on the joint toxicity of arsenic and PFOA to soil invertebrates. In this study, four arsenic species were selected, including arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA), and dimethylarsinate (DMA). Earthworms Eisenia fetida were exposed to soils spiked with sublethal concentrations of PFOA, different arsenic species, and their binary mixtures for 56 days. The bioaccumulation and biotransformation of pollutants, as well as eight biomarkers in organisms, were assayed. The results indicated that the coexistence of PFOA and different arsenic species in soils could enhance the bioavailability of arsenic species while reducing the bioavailability of PFOA, and inhibit the arsenic biotransformation process in earthworms. Responses of most biomarkers in joint treatments of PFOA and As(III)/As(V) showed more significant variations compared with those in single treatments, indicating higher toxicity to the earthworms. The Integrated Biomarker Response (IBR) index was used to integrate the multi-biomarker responses, and the results also exhibited enhanced toxic effects in combined treatments of inorganic arsenic and PFOA. In comparison, both the biomarker variations and IBR values were lower in joint treatments of PFOA and MMA/DMA. Then the toxic interactions in the binary mixture systems were characterized by using a combined method of IBR and Effect Addition Index. The results revealed that the toxic interactions of the PFOA/arsenic mixture in earthworms depended on the different species of arsenic. The combined exposure of PFOA with inorganic arsenic led to a synergistic interaction, while that with organic arsenic resulted in an antagonistic response. Overall, this study provides new insights into the assessment of the joint toxicity of perfluoroalkyl substances and arsenic in soil ecosystems.

[Shenxiong Glucose Injection inhibits H_2O_2-induced apoptosis of H9c2 cells by activating PI3K/AKT pathway].

The aim of this paper was to explore the mechanism of Shenxiong Glucose Injection antagonizing apoptosis of H9 c2 cells induced by H_2O_2. H9 c2 cells were pretreated with 1. 7%,3. 4% and 6. 8% Shenxiong Glucose Injection,and then H_2O_2 was introduced to induce apoptosis in vitro. Cell viability was detected by MTS assay,morphological changes of apoptosis were observed by AO/EB fluorescence staining,apoptosis rate was detected by Annexin/PI method,cell expression profile was detected by gene chip technology,the mRNA of PIK3 CA,Bcl-2,Bax,caspase-3 and GAPDH were detected by qRT-PCR,the protein expression levels of PIK3 CA,AKT,P-AKT,Bcl-2,Bax and caspase-3 were detected by Western blot,and the contents of LDH and MDA were detected by kit. The results showed that Shenxiong Glucose Injection of different concentrations significantly increased the viability of H9 c2 cells treated with H_2O_2( P<0. 01),and reversed H_2O_2-induced apoptosis( P< 0. 01). The microarray experiments showed that 138 genes were altered in H9 c2 cells after treatment with Shenxiong Glucose Injection. The differential expression fold of PIK3 CA associated with PI3 K/AKT pathway was 3. 59. The results of qRT-PCR and Western blot showed that Shenxiong Glucose Injection could down-regulate the mRNA and protein expression levels of caspase-3( P<0. 01),up-regulate the mRNA and protein expression level of PIK3 CA and Bcl-2( P<0. 01),and up-regulate the phosphorylation levels of AKT( P<0. 01) in H_2O_2-treated H9 c2 cells. The protective effect of Shenxiong Glucose Injection on H_2O_2 cells injury was significantly inhibited by LY294002,a PI3 K/AKT pathway inhibitor. The results suggested that Shenxiong Glucose Injection may inhibit H_2O_2-induced H9 c2 cells apoptosis by regulating PI3 K/AKT signaling pathway.

AEBP1 exacerbates myocardial ischemia-reperfusion injury via inhibition of IκBα.

Myocardial ischemia/reperfusion injury (MIRI) remains a main reason for death after cardiovascular diseases. Up-regulation of adipocyte enhancer binding protein 1 (AEBP1) has been found in ischemic cardiomyopathy patients. However, its influence and detailed mechanisms in MIRI are obscure. In this study, expression of target molecules was determined by RT-qPCR and Western blotting. Cell viability and apoptosis were evaluated by CCK-8 and TUNEL. Inflammatory cytokine levels were assessed by ELISA. Myocardial function and pathological changes were examined by echocardiography and HE staining. Cardiac infarct size was determined by TTC staining. Our data indicated that oxygen-glucose deprivation/reoxygenation (OGD/R) resulted in high expression of AEBP1, while low expression of IκBα in cardiomyocytes. In vitro data indicated that AEBP1 knockdown increased viability, inhibited apoptosis, and inflammation in H9c2 cells under OGD/R. AEBP1 interacted with IκBα to cause IκBα degradation, and facilitated the nuclear translocation of NF-κB. Moreover, IκBα silencing attenuated siAEBP1-medaited inhibition in inflammation and apoptosis of OGD/R-treated H9c2 cells, suggesting that IκBα was involved in the pro-inflammatory action of AEBP1. Finally, deficiency of AEBP1 mitigated MIRI in rats through IκBα/NF-κB pathway. Taken together, AEBP1 exacerbated MIRI through repressing IκBα expression to trigger NF-κB-mediated inflammation.

Shenxiong glucose injection inhibits oxidative stress and apoptosis to ameliorate isoproterenol-induced myocardial ischemia in rats and improve the function of HUVECs exposed to CoCl2.

Background: Shenxiong Glucose Injection (SGI) is a traditional Chinese medicine formula composed of ligustrazine hydrochloride and Danshen (Radix et rhizoma Salviae miltiorrhizae; Salvia miltiorrhiza Bunge, Lamiaceae). Our previous studies and others have shown that SGI has excellent therapeutic effects on myocardial ischemia (MI). However, the potential mechanisms of action have yet to be elucidated. This study aimed to explore the molecular mechanism of SGI in MI treatment. Methods: Sprague-Dawley rats were treated with isoproterenol (ISO) to establish the MI model. Electrocardiograms, hemodynamic parameters, echocardiograms, reactive oxygen species (ROS) levels, and serum concentrations of cardiac troponin I (cTnI) and cardiac troponin T (cTnT) were analyzed to explore the protective effect of SGI on MI. In addition, a model of oxidative damage and apoptosis in human umbilical vein endothelial cells (HUVECs) was established using CoCl2. Cell viability, Ca2+ concentration, mitochondrial membrane potential (MMP), apoptosis, intracellular ROS, and cell cycle parameters were detected in the HUVEC model. The expression of apoptosis-related proteins (Bcl-2, Caspase-3, PARP, cytoplasmic and mitochondrial Cyt-c and Bax, and p-ERK1/2) was determined by western blotting, and the expression of cleaved caspase-3 was analyzed by immunofluorescence. Results: SGI significantly reduced ROS production and serum concentrations of cTnI and cTnT, reversed ST-segment elevation, and attenuated the deterioration of left ventricular function in ISO-induced MI rats. In vitro, SGI treatment significantly inhibited intracellular ROS overexpression, Ca2+ influx, MMP disruption, and G2/M arrest in the cell cycle. Additionally, SGI treatment markedly upregulated the expression of anti-apoptotic protein Bcl-2 and downregulated the expression of pro-apoptotic proteins p-ERK1/2, mitochondrial Bax, cytoplasmic Cyt-c, cleaved caspase-3, and PARP. Conclusion: SGI could improve MI by inhibiting the oxidative stress and apoptosis signaling pathways. These findings provide evidence to explain the pharmacological action and underlying molecular mechanisms of SGI in the treatment of MI.

Antioxidant defense system responses, lysosomal membrane stability and DNA damage in earthworms (Eisenia fetida) exposed to perfluorooctanoic acid: an integrated biomarker approach to evaluating toxicity.

Perfluorooctanoic acid (PFOA) is one of the most representative perfluoroalkyl substances and has garnered intense human and ecological health concerns due to its ubiquity in the environment, bio-accumulative nature and potential toxicological effects. In this study, an artificial soil containing PFOA was used to evaluate the biological toxicity of PFOA to earthworms Eisenia fetida. Six kinds of oxidative stress biomarkers, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO), as well as lysosomal membrane stability (LMS) and DNA damage in earthworms were detected after exposure to 0, 10, 20, 40, 80 and 120 mg kg-1 PFOA in the soil for 3, 7, 14, 28, and 42 days. The results of multi-biomarker responses indicated that PFOA can induce various adverse effects on earthworms, including growth inhibition, oxidative stress and genotoxicity, resulting in lipid membrane peroxidation, decreased lysosomal membrane stability and DNA damage. LPO, LMS and DNA damage all presented dose- and time-dependent relationships. An integrated biomarker response (IBR) index was applied to summarize the multi-biomarker responses to star plots, and the IBR value was calculated as the area of the plots to indicate the integrated stress of PFOA on earthworms. The IBR index showed that the integrated stress induced by PFOA increased markedly throughout the exposure period, exhibiting a concentration-related and exposure time-related effect. The graphical changing trend of the IBR star plots, along with the multi-biomarker responses, suggested that the biomarkers of the antioxidant defense system in earthworms are sufficiently sensitive for short-term PFOA biomonitoring programs, while the bioindicators that indicate actual damage in organisms are more suitable to be employed in long-term monitoring programs for the risk assessment of PFOA. This is the first study evaluating the biological toxicity of PFOA by using an integrated biomarker approach. Our results showed that PFOA can potentially damage soil ecosystems, which provides valuable information for chemical risk assessment of PFOA in the soil environment and early warning bioindicators of soils contaminated by PFOA.

Shenxiong glucose injection inhibits H2O2-induced H9c2 cell apoptosis by activating the ERK signaling pathway.

BACKGROUND: Shenxiong glucose injection (SGI) is a traditional Chinese medicine injection composed of water extract of Salvia miltiorrhiza and Ligustrazine hydrochloride. SGI has shown strong antioxidant and anti-apoptotic properties. However, the mechanisms underlying its anti-apoptotic effect need to be addressed. METHODS: H9c2 cell apoptosis model was established by treatment of hydrogen peroxide (H2O2). Cell survival rates were examined by MTS assay, cell apoptosis rates were determined by flow cytometry, levels of intracellular ROS were assessed by ROS kit, proteome phosphorylation was determined by phosphoproteomic analysis, and extracellular signal-regulated kinase (ERK), phosphorylated ERK, phosphorylated c-Jun, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, and cleaved caspase-3 were examined by Western blot. RESULT: SGI showed protective effects against H2O2-induced reduced cell viability, elevated ROS, and increased apoptosis in H9c2 cells. Phosphorylation proteomics detected a total of 3369 proteins with 78 protein of upregulated phosphorylation and 104 protein of downregulated phosphorylation. Kyoto Encyclopedia Genes and Genomes pathway analyses of differentially phosphorylated proteins showed that the ERK pathway, the downstream pathway of the focal adhesion pathway related to apoptosis, was highly enriched, and the phosphorylation levels of ERK and c-Jun were confirmed by Western blot. In addition, the ERK pathway inhibitor PD98059 significantly inhibited the anti-apoptotic effect of SGI. CONCLUSION: SGI antagonizes H2O2-induced cell apoptosis by activating the ERK pathway.

[Establishment of Flp-InTM CHO cell lines with double expression of CYP2A13 and MRP2].

Objective To construct a double transfected Flp-InTM CHO cell line stably expressing both cytochrome P450 family 2 subfamily A member 13(CYP2A13) and multidrug resistance-associated protein 2(MRP2). Methods We constructed the recombinant plasmids of pCMV6-NEO-CYP2A13 and pcDNA5-MRP2. The pCMV6-NEO-CYP2A13 recombinant plasmid was first transfected into Flp-InTM CHO cells, and CYP2A13-Flp-InTM CHO cells with higher CYP2A13 activity were screened using limiting dilution method and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) cytotoxicity assay. Thereafter, pcDNA5-MRP2 was transfected into CYP2A13-Flp-InTM CHO cells. The expression levels and activities of CYP2A13 and MRP2 in the double transfected cells and normal cells were detected by real-time quantitative PCR, Western blot analysis and NNK cytotoxicity assay in order to screen Flp-InTM CHO cells with stable expression of CYP2A13 and MRP2. Results Compared with non-transfected cells, the expression of CYP2A13 and the sensitivity of NNK toxicity in CYP2A13-Flp-InTM CHO cells increased. The expression of CYP2A13 and MRP2 in CYP2A13/MRP2-Flp-InTM CHO cells also increased significantly. Compared with CYP2A13-Flp-InTM CHO cells, CYP2A13/MRP2-Flp-InTM CHO cells showed no significant difference in CYP2A13 expression; the expression of MRP2 increased while the sensitivity of NNK toxicity decreased significantly. Conclusion The double transfected cell model of CYP2A13 and MRP2 has been successfully established, which lays the foundation for the study of in situ activation of respiratory carcinogens.