Protection of glandular acinar cells from autoimmune-induced damage would be of significant clinical benefit to Sjogren's syndrome (SS) patients. EGCG (the most abundant green tea polyphenol) possesses anti-apoptotic, anti-inflammatory, and autoantigen-inhibitory properties. To investigate if EGCG can protect against certain autoimmune-induced pathological changes in the salivary glands of the non-obese diabetic (NOD) mouse model for SS-like symptoms, animals were provided with either water or water containing 0.2% EGCG. At the age of 8, 16 and 22 weeks, samples were collected for pathological and serological analysis. Massive lymphocyte infiltration was observed in the salivary glands of the water-fed group at the age of 16 weeks, while the EGCG group showed significantly reduced lymphocyte infiltration. By 22 weeks of age, animals fed with water demonstrated elevated levels of apoptotic activity within the lymphocytic infiltrates, and high levels of serum total anti-nuclear antibody, in comparison with the animals fed with EGCG. Remarkably, proliferating cell nuclear antigen (PCNA) and Ki-67 levels in the salivary glands of NOD animals fed with water were significantly elevated in comparison to BALB/c control mice; in contrast, PCNA and Ki-67 levels in EGCG-fed NOD animals were similar to BALB/c controls. These results indicate that EGCG is able to protect the NOD mouse submandibular glands from autoimmune-induced inflammation, and reduces serum autoantibody levels. Abnormal proliferation, rather than apoptosis, appears to be a characteristic of the NOD mouse gland that is normalized by EGCG. The evidence suggests that EGCG could ultimately be used to delay or manage SS-like autoimmune disorders.
Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the gold standard for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope labeled metabolites such as acyl-coenzyme A thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell media with commercially available [13C3 15N1]-pantothenic acid, mammalian cells exclusively incorporated [13C3 15N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope labeled CoA and acyl-CoAs from [13C3 15N1]-pantothenate using Stable Isotope Labeling by Essential nutrients in Cell culture (SILEC) in Pan6 deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof-of-concept for generating other labeled metabolites in yeast mutants.
Biliatresone is an electrophilic isoflavone isolated from Dysphania species plants that has been causatively linked to naturally occurring outbreaks of a biliary atresia (BA)-like disease in livestock. Biliatresone has selective toxicity for extrahepatic cholangiocytes (EHC) in zebrafish larvae. To better understand its mechanism of toxicity, we performed transcriptional profiling of liver cells isolated from zebrafish larvae at the earliest stage of biliatresone-mediated biliary injury, with subsequent comparison of biliary and hepatocyte gene expression profiles. Transcripts encoded by genes involved in redox stress response, particularly those involved in glutathione (GSH) metabolism, were among the most prominently upregulated in both cholangiocytes and hepatocytes of biliatresone-treated larvae. Consistent with these findings, hepatic GSH was depleted at the onset of biliary injury, and in situ mapping of the hepatic GSH redox potential using a redox-sensitive GFP (roGFP) biosensor showed that it was significantly more oxidized in EHC both before and after treatment with biliatresone. Pharmacological and genetic manipulation of GSH redox homeostasis confirmed the importance of GSH in modulating biliatresone-induced injury as GSH depletion sensitized both EHC and the otherwise resistant intrahepatic cholangiocytes (IHC) to the toxin, whereas replenishing GSH level via N-acetylcysteine administration or activation of nuclear factor, erythroid 2-like 2 (Nrf2), a transcriptional regulator of GSH synthesis, inhibited EHC injury. Conclusion: These findings strongly support redox stress as a critical contributing factor in biliatresone-induced cholangiocyte injury, and suggest variations in intrinsic stress responses underlie the susceptibility profile. Insufficient antioxidant capacity of EHC may be critical to the early pathogenesis of human BA.
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