Plasma miR-218a-5p as a biomarker for acute cholestatic liver injury in rats and investigation of its pathophysiological roles.

MicroRNAs (miRNA) have received considerable attention as potential biomarkers for drug-induced liver injury. We recently reported that the plasma levels of miR-143-3p and miR-218a-5p increased in severe cholestasis in rats. This study aimed to investigate whether these miRNAs increase in a severity-dependent manner and to elucidate their pathophysiological roles in cholestasis. Male Sprague-Dawley rats were orally administered different doses of α-naphthylisothiocyanate or 4,4-methylenedianiline to induce acute cholestasis. They were also orally administered acetaminophen or thioacetamide to induce hepatocellular injury. We found that plasma miR-143-3p and miR-218a-5p levels increased in a dose-dependent manner in cholestatic rats but not in hepatocellular injury. Bioinformatic analysis provided putative target genes of hsa-miR-218-5p, rno-miR-218a-5p, and mmu-miR-218-5p, among which GNAI2, PPP1CB, and PPP2R5A were experimentally validated as their direct target genes in human cholangiocyte line MMNK-1. Proliferation of MMNK-1 cells was significantly suppressed after overexpression of miR-218-5p and transduction of siRNAs for GNAI2, PPP1CB, and PPP2R5A. In the cholestatic livers of rats, Ppp1cb and Ppp2r5a expression levels decreased, whereas Gnai2 expression levels increased compared with those in vehicle-treated rats, suggesting that Ppp1cb and Ppp2r5a may be under the control of miR-218a-5p in vivo. In conclusion, our data suggest that miR-218(a)-5p is involved in the suppression of cholangiocyte proliferation by inhibiting the expression of PPP1CB and PPP2R5A, thereby contributing to the pathogenesis of cholestasis; and miR-218a-5p leaks into the plasma probably from damaged cholangiocytes in a severity-dependent manner in rats. Therefore, miR-218a-5p overexpression could be one of the underlying mechanisms of acute cholestatic liver injury in rats.

Profiling of Microbiota at the Mouth of Bottles and in Remaining Tea after Drinking Directly from Plastic Bottles of Tea.

It has been speculated that oral bacteria can be transferred to tea in plastic bottles when it is drunk directly from the bottles, and that the bacteria can then multiply in the bottles. The transfer of oral bacteria to the mouth of bottles and bacterial survival in the remaining tea after drinking directly from bottles were examined immediately after drinking and after storage at 37 °C for 24 h. Twelve healthy subjects (19 to 23 years of age) were asked to drink approximately 50 mL of unsweetened tea from a plastic bottle. The mouths of the bottles were swabbed with sterile cotton, and the swabs and the remaining tea in the bottles were analyzed by anaerobic culture and 16S rRNA gene sequencing. Metagenomic analysis of the 16S rRNA gene was also performed. The mean amounts of bacteria were (1.8 ± 1.7) × 104 colony-forming units (CFU)/mL and (1.4 ± 1.5) × 104 CFU/mL at the mouth of the bottles immediately after and 24 h after drinking, respectively. In contrast, (0.8 ± 1.6) × 104 CFU/mL and (2.5 ± 2.6) × 106 CFU/mL were recovered from the remaining tea immediately after and 24 h after drinking, respectively. Streptococcus (59.9%) were predominant at the mouth of the bottles immediately after drinking, followed by Schaalia (5.5%), Gemella (5.5%), Actinomyces (4.9%), Cutibacterium (4.9%), and Veillonella (3.6%); the culture and metagenomic analyses showed similar findings for the major species of detected bacteria, including Streptococcus (59.9%, and 10.711%), Neisseria (1.6%, and 24.245%), Haemophilus (0.6%, and 15.658%), Gemella (5.5%, and 0.381%), Cutibacterium (4.9%, and 0.041%), Rothia (2.6%, and 4.170%), Veillonella (3.6%, and 1.130%), Actinomyces (4.9%, and 0.406%), Prevotella (1.6%, and 0.442%), Fusobacterium (1.0%, and 0.461%), Capnocytophaga (0.3%, and 0.028%), and Porphyromonas (1.0%, and 0.060%), respectively. Furthermore, Streptococcus were the most commonly detected bacteria 24 h after drinking. These findings demonstrated that oral bacteria were present at the mouth of the bottles and in the remaining tea after drinking.