UPLC-Q-TOF/MS Based Metabolomics Approach to Study the Hepatotoxicity of Cantharidin on Mice.

Cantharidin is the major bioactive compound extracted from the blister beetle, a traditional Chinese medicine, and has been proved to be a natural component with widely antitumor activity. However, clinical application of cantharidin is relatively restricted due to its potential toxic effects, especially hepatotoxicity. Although cantharidin-induced liver injury has been reported, the underlying molecular mechanisms remain unclear. In the present study, an UPLC-Q-TOF/MS based metabolomics approach combined with blood biochemical analysis, histopathological examination, and cell apoptosis assay were used to investigate the mechanisms of cantharidin-induced hepatotoxicity. A total of 54 significantly changed metabolites and 14 disturbed metabolic pathways were identified in the cantharidin exposed groups. Among them, four metabolites (oxidized glutathione, glutathione, 3-sulfinoalanine, and deoxycholic acid 3-glucuronide) were selected based on their high impact value and potential biological function in the process of liver injury post cantharidin treatment. Our study provides a deeper understanding of the mechanisms of cantharidin-induced hepatotoxicity and may contribute to reduce the liver injury and gain more effective and safe clinical use of cantharidin. In addition, our results also demonstrated that cantharidin could impair multiple biological processes in liver, and future studies will be necessary to reveal the detailed molecular mechanisms of cantharidin-induced hepatotoxicity.

An essential function for MKP5 in the formation of oxidized low density lipid-induced foam cells.

The uptake of oxidized low density lipoprotein (ox-LDL) by macrophages usually leads to the formation of lipid-laden macrophages known as "foam cells," and this process plays an important role in the development of atherosclerosis. Ox-LDL activates mitogen-activated protein kinase (MAP) kinases and nuclear factor (NF)-κB, and activations of p38 and NF-κB are important for the formation of foam cells. MAP kinase phosphatase (MKP) 5 is a member of the dual specificity phosphatases (DUSPs) family that can selectively dephosphorylate activated MAPKs to regulate innate and adaptive immune responses. However, the role of MKP5 in the formation of foam cells remains unknown. Here, we found that stimulation of ox-LDL induces the expression of MKP5 in macrophages. MKP5 deficiency blocked the uptake of ox-LDL and the formation of foam cells. Further analysis revealed that deletion of MKP5 reduced the ox-LDL-induced activation of NF-κB. Also, MKP5 deficiency markedly inhibited the production of TNF-α, but enhanced the levels of TGF-ß1 in ox-LDL-stimulated macrophages. Moreover, inhibition of NF-κB by p65 RNAi significantly reduced foam cell formation in macrophages from WT mice relative to MKP5-deficient mice. Thus, MKP5 has an essential role in the formation of foam cells through activation of NF-κB, and MKP5 represents a novel target for the therapeutic intervention of atherosclerosis.

Effect of overexpression of human SR-AI on oxLDL uptake and apoptosis in 293T cells.

Type I class A macrophage scavenger receptor (SR)-AI plays an important role in foam cell formation and in apoptosis in atherosclerosis, however the mechanism remains unclear. Therefore, we generated a pEGFP-C1-SR-AI plasmid construct for transient transfection of 293T human embryonic kidney cells and observed if SR-AI expression led: (i) to foam cell formation or apoptosis; and (ii) to expression of apoptosis-related genes Bcl-2 and Bak-1 in cells treated with oxidized low-density lipoprotein (oxLDL). The pEGFP-C1 (empty vector) transfected cell line was used as a control. Transfection efficiency of each group was >90% and transfected cells expressed functional SR-AI protein. Binding and uptake of 3,3'-dioctadecylindocarbocyanine-labeled oxLDL (DiI-oxLDL) were verified by flow cytometry; increases in the rate of oxLDL binding and uptake were observed in pEGFP-C1-SR-AI transfected 293T cells and incubation with oxLDL also led to increased apoptosis (≈50%) compared with controls. A decrease in Bcl-2 and an increase in Bak-1 mRNA and protein expression were observed in pEGFP-C1-SR-AI transfected cells compared with controls. We conclude that transient over-expression of SR-AI leads to an increase in oxLDL uptake and binding in a non-macrophage cell line. In addition, over-expression of SR-AI induced non-macrophage cell apoptosis via downregulation of Bcl-2 and upregulation of Bak-1 expression. We conclude that the 293T cell expression described here is a model for foam cell formation. These results may form the basis of further research into SR-AI structure and function (including lipoprotein uptake, apoptosis modulation and adhesion), which may give an insight into the progression of atherosclerosis in vivo.

Inhibitory effects of genistein on metastasis of human hepatocellular carcinoma.

AIM: To investigate the inhibitory effects of genistein on metastasis of MHCC97-H hepatocellular carcinoma cells and to explore the underlying mechanism. METHODS: MHCC97-H hepatocellular carcinoma cells were exposed to genistein. A cell attachment assay was carried out in a microculture well pre-coated with fibronectin. The invasive activity of tumor cells was assayed in a transwell cell culture chamber, and cell cycle and apoptosis were evaluated by a functional assay. In addition, the expression and phosphorylation of FAK were detected by Western blotting. In situ xenograft transplantation of hepatocellular carcinoma was performed in 12 nude mice and lung metastasis of hepatocellular carcinoma was observed. RESULTS: Genistein significantly inhibited the growth of MHCC97-H cells in vitro. Adhesion and invasiveness of MHCC97-H cells were inhibited in a concentration-dependent fashion, and the inhibitory effect of genistein was more potent in the 10 microg/mL and 20 microg/mL genistein-treated groups. Genistein caused G(0)/G(1) cell cycle arrest, an S phase decrease, and increased apoptosis. The expression and phosphorylation of FAK in MHCC-97H cells were significantly decreased. In situ xenograft transplantation of hepatocellular carcinoma was also significantly suppressed by genistein. The number of pulmonary micrometastatic foci in the genistein group was significantly lower compared with the control group (12.3 +/- 1.8 vs 16.6 +/- 2.6, P < 0.05). CONCLUSION: Genistein appears to be a promising agent in the inhibition of metastasis of hepatocellular carcinoma.

[Effects of Astragalus membranaccus on cardiac function and SERCA2a gene expression in myocardial tissues of rats with chronic heart failure].

OBJECTIVE: To investigate the effects of Astragalus membranaccus (As) on cardiac function and SERCA2a gene expression in left ventricular tissues of rats with chronic heart failure. METHODS: Heart failure was induced by clipping the abdominal aorta 60 male SD rats were divided into four groups: sham-operated (Sham), aortic stenosis (Model), Model + As (20 g/kg) and Model + Captopril (0.05 g/kg). The drugs were administered orally from the 13th week after surgery. Rats were examined after 12 weeks' treatment with drugs. The parameters of hemodynamics including LVSP, LVEDP, and +/- LVdp/dt(max) were measured. SERCA2a mRNA and protein expressions in left ventricular tissues were determined by half-quantitative RT-PCR and Western blot normalized to abundance of GAPDH mRNA and portein, respectively. RESULTS: LVSP and LVEDP were obviously enhanced (P < 0.01 or P < 0.001) in model rats in vivo. Both Captopril and As prevented the increase of LVSP (P < 0.05 or P < 0.01) and LVEDP (P < 0.05 or P < 0.01). RT-PCR and Western blot results demonstrated that SERCA2a gene expression was downregulated (P < 0.05) significantly in model group compared with sham group. As upregulated SERCA2a gene expression (P < 0.05), whereas Captopril had no effect on that. CONCLUSION: As can ameliorate abnormity of cardiac function, especially diastoilc function in rats with pressure overload-induced heart failure, and that may be partly related to its up-regulation of SERCA2a gene expressions in left ventricular tissues.

[Effect of astragalus on calcium accumulation and SERCA2a gene expression in myocardial tissues in rats with pressure overload-induced left ventricular hypertrophy].

OBJECTIVE: To investigate the effect of astragalus (As) on calcium accumulation and SERCA2a gene expression in left ventricular tissues in rats with pressure overload-induced cardiac hypertrophy. METHOD: cardiac hypertrophy was induced by clipping the abdominal aorta in rats. Male SD rats were allocated to six groups: sham-operrated (Sham), aortic stenosis (Model), model +As-L (5 g x kg(-1) x d(-1)), model+As-M (10 g x kg(-1) x d(-1)), model+As-H (20 g x kg(-1) x d(-1)) and model + captopril (0.05 mg x kg(-1) x d(-1), a positive control). The drugs were administered orally from the 13 th week after surgery. Rats were examined after 12 week treatment with drugs. The cardiac hypertrophy was evaluated by left ventricular mass index (LVMI, left ventricular weight/ body weight). The calcium content in left ventricular tissue was measured by atomic absorption spectrometry. SERCA2a mRNA and protein expressions in left ventricular tissues were determined by half-quantitative RT-PCR and Western blot normalized to abundance of GAPDH mRNA and protein, respectively. RESULT: The increase of LVMI was dose-dependently lessened by As (P < 0.01, P < 0.001). The effect of As-H was similar to that of Captopril. As markedly attenuated calcium accumulation in myocardial tissure (P < 0.01). RT-PCR and Western blot results demonstrated that SERCA2a gene expressions were downregulated (P < 0.05) significantly in model group compared with sham group. As-H upregulated SERCA2a gene expressions (P < 0.05), whereas Captopril had no effect on that. CONCLUSION: The inhibition of As on left ventricular hypertrophy induced by pressure overload in rats may partly contribute to its attenuation of calcium accumulation and up-regulation of SERCA2a gene expressions in left ventricular tissues.

[Effects of 2-chlorophenol-acclimation on microbial community structure in anaerobic granular sludge].

The microbial community structure in 2-chlorophenol-acclimated anaerobic granular sludge and inoculating sludge were analyzed by 16S rDNA-based approach. Total DNA was extracted directly from the inoculating sludge and 2-CP-acclimated anaerobic sludge, and then amplified by polymerase chain reaction (PCR) technique with the specific primer pair ARC21F/ARC958R for Archaea and 31F/907R for Acidobacteria respectively. The positive PCR products were cloned and sequenced. The sequences analysis shows that there exist common Archaea in both sludge, including Methanothrix soehngenii, Methanosaeta concilii and uncultured euryarchaeote etc. Some special Archaea appear in the 2-CP-acclimated sludge, such as Methanobacterium aarhusense, Methanobacterium curvum and Methanobacterium beijingense etc. Others originally existed in the inoculating sludge disappear after acclimation. Common Acidobacteria are found in both sludge, including uncultured bacterium, uncultured Acidobacterium and unknown Actinomycete (MC 9). Some special microbes originally existed in the inoculating sludge, such as Desulfotomaculum sp. 176, uncultured Deltaproteobacterium n8d and uncultured hydrocarbon seep bacterium etc. disappear after acclimation, and uncultured Holophaga/Acidobacterium, uncultured Acidobacteria bacterium and unidentified Acidobacterium are found after 2-CP-acclimation.