Humans express two ACAT (acyl-CoA:cholesterol acyltransferase) genes, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed, whereas ACAT2 is primarily expressed in intestinal mucosa and plays an important role in intestinal cholesterol absorption. To investigate the molecular mechanism(s) responsible for the tissue-specific expression of ACAT2, we identified five cis-elements within the human ACAT2 promoter, four for the intestinal-specific transcription factor CDX2 (caudal type homeobox transcription factor 2), and one for the transcription factor HNF1alpha (hepatocyte nuclear factor 1alpha). Results of luciferase reporter and electrophoretic mobility shift assays show that CDX2 and HNF1alpha exert a synergistic effect, enhancing the ACAT2 promoter activity through binding to these cis-elements. In undifferentiated Caco-2 cells, the ACAT2 expression is increased when exogenous CDX2 and/or HNF1alpha are expressed by co-transfection. In differentiated Caco-2 cells, the ACAT2 expression significantly decreases when the endogenous CDX2 or HNF1alpha expression is suppressed by using RNAi (RNA interference) technology. The expression levels of CDX2, HNF1alpha, and ACAT2 are all greatly increased when the Caco-2 cells differentiate to become intestinal-like cells. These results provide a molecular mechanism for the tissue-specific expression of ACAT2 in intestine. In normal adult human liver, CDX2 expression is not detectable and the ACAT2 expression is very low. In the hepatoma cell line HepG2 the CDX2 expression is elevated, accounting for its elevated ACAT2 expression. A high percentage (seven of fourteen) of liver samples from patients affected with hepatocellular carcinoma exhibited elevated ACAT2 expression. Thus, the elevated ACAT2 expression may serve as a new biomarker for certain form(s) of hepatocellular carcinoma.
A multiplex real-time PCR (qPCR) assay was developed for simultaneous detection and differentiation of the three most important Campylobacter species in chickens. Three novel sets of PCR primers and TaqMan probes were designed to amplify the unique DNA sequences within the hipO, cdtA, and pepT genes which are specific to Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, respectively. To avoid competition in the multiple target amplifications, the concentrations of primers and probes were optimized. By using the optimized qPCR conditions together with a minor-groove binding probe of pepT, amplification efficiency greater than 92% and detection sensitivity of 38 genome copies/reaction have been achieved for all three targets. The assay was highly specific for C. jejuni, C. coli, and C. lari with testing of 33 Campylobacter strains and 20 non-Campylobacter strains. In chicken samples spiked with known quantities of Campylobacter cells, the assay was able to detect 1 CFU/ g after a 24-h enrichment. Application of the assay in food was further evaluated using 21 fresh chicken samples obtained from local supermarkets. The results revealed that, after a 24-h or 48-h enrichment, 14 samples (66.7%)were positive for C. jejuni, five samples (23.8%) were positive for C. coli, and none of the samples was contaminated by C. lari. Taken together, the multiplex qPCR assay combined with an enrichment step is a sensitive, species-specific, and non-labor-intensive method suitable for rapid detection of C. jejuni, C. coli, and C. lari in chicken samples.
Bicyclol is a synthetic antihepatitis drug with antioxidative property. The present study was performed to investigate the effect of bicyclol on liver regeneration after partial hepatectomy in rats. Bicyclol (300 mg/kg) was given to rats subjected to 70% hepatectomy three times before operation. At 6, 24, and 48 h after resection, samples were collected for the measurement of serum alanine aminotransferase (ALT), total bilirubin (TBil), hepatic glycogen, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH). Moreover, liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling, proliferation index, and histopathological examination were evaluated at 48 h after hepatectomy. As a result, bicyclol significantly increased regeneration rate, mitotic index (MI), PCNA labeling index, and proliferation index in PH rats. Additionally, bicyclol remarkably inhibited the elevation of serum ALT and TBil levels, alleviated the formation of liver MDA, restored impaired antioxidant SOD and GSH, increased hepatic glycogen content, and also attenuated hepatic vacuolar degeneration. These results suggested that bicyclol had a beneficial effect on liver regenerative capacity of the remnant liver tissue after hepatectomy, probably due to its antioxidative property.
Bicyclol had a remarkable protective effect on hepatic I/R injury, which was partially due to inhibiting the expression of TLR4 and NF-kappaB via its ability to attenuate oxidative stress and endotoxin.
Endoplasmic reticulum (ER) stress is known to be involved in the development of several metabolic disorders, including non-alcoholic fatty liver disease (NAFLD). Tetracycline can cause hepatic steatosis, and ER stress may be involved in tetracycline-induced fatty liver. Our previous study showed that bicyclol has been proven to protect against tetracycline-induced fatty liver in mice, and ER stress may also be involved in bicyclol's hepatoprotective effect. Therefore, this study was performed to investigate the underlying mechanisms associated with ER stress and apoptosis, by which bicyclol attenuated tetracycline-induced fatty liver in mice. Bicyclol (300 mg/kg) was given to mice by gavage 3 times. Tetracycline (200 mg/kg, intraperitoneally) was injected at 1 h after the last dose of bicyclol. At 6 h and 24 h after single dose of tetracycline injection, serum ALT, AST, TG, CHO and hepatic histopathological examinations were performed to evaluate liver injuries. Hepatic steatosis was assessed by the accumulation of hepatic TG and CHO. Moreover, hepatic apoptosis and ER stress related markers were determined by TUNEL, real-time PCR, and western blot. As a result, bicyclol significantly protected against tetracycline-induced fatty liver as evidenced by the decrease of elevated serum transaminases and hepatic triglyceride, and the attenuation of histopathological changes in mice. In addition, bicyclol remarkably alleviated hepatic apoptosis and the gene expression of caspase-3, and increased the gene expression of XIAP. The gene expressions of ER stress-related markers, including CHOP, GRP78, IRE-1α, and ATF6, which were downregulated by bicyclol pretreatment in tetracycline-injected mice. These results suggested that bicyclol protected tetracycline-induced fatty liver partly due to its ability of anti-apoptosis associated with ER stress.
During the aging process, dimers of dietary vitamin A accumulated in retinal pigment epithelium (RPE) cells. Vitamin A dimer-mediated photooxidation resulted in RPE apoptosis, which is associated with age-related degenerative disease of retina, leading to blindness. It has been reported that proanthocyanidin-rich grape seed extract reduces oxidative stress in the eye. In this study, we investigated the underlying mechanism of photooxidation-induced apoptosis inhibition by procyanidins B2 (PB2), one of the main components of grape seed proanthocyanidin. To mimic vitamin A dimer-mediated photooxidation, ARPE-19 cells that accumulated vitamin A dimer, A2E, were used as a model system. Exposure of A2E loaded ARPE-19 cells to blue light induced ER stress and resulted in significant apoptosis. Pretreatment of blue light-exposed A2E containing ARPE-19 cells with PB2 inhibited apoptosis, increased the ratio of Bcl-2/Bax in the mitochondria, attenuated ROS and cytochrome c release, and decreased caspase cleavage. Additionally, PB2 inhibited the phosphorylation of ER stress markers elF2α and IRE1α and reduced CHOP expression. Moreover, PB2 inhibition of apoptosis is dependent on the UPR chaperone GRP78, indicating PB2 inhibits vitamin A dimer-mediated apoptosis in RPE cells by activating the UPR.
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