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L-carnitine ameliorates bile duct ligation induced liver fibrosis via reducing the nitrosative stress in experimental animals: preclinical evidences.

ABSTRACT: Bile duct ligation (BDL) has been extensively used in studying the mechanisms of fibrogenesis and anti-fibrotic drugs. Considering the liver regenerative capacity and the diverse results from BDL, the present study aimed to evaluate the protective effect of L-carnitine on bile duct ligation-induced liver fibrosis in experimental rats. Rats were randomly divided into seven groups (n = 6). The bile duct was ligated and serum aspartate transaminase (AST), alanine transaminase (ALT), total bilirubin and albumin, hepatic hydroxyproline (HP), reduced glutathione (GSH), and malondialdehyde (MDA) and cytokines were measured. iNOS expression was measured by using Western blot and finally, liver tissue was processed for histopathological analysis (H&E staining)". The level of iNOS was increased in the control group, whereas a decrease in the level of iNOS was found in the L-carnitine treated group. In the present study, we found that bile duct ligation in rats showed an increase in body and liver weight, while treatment with carnitine showed normal body and liver weight. Serum AST, ALT, total bilirubin, HP, GSH, MDA, and cytokines were increased in bile duct ligated rats. In addition, L-carnitine treated rats showed a reduction in oxidative stress as well as inhibiting the release of cytokines in a dose-dependent manner and showed protection against bile duct ligation. The study concludes that L-carnitine has a protective effect against the liver fibrosis induced by bile duct ligation.

SUBMITTER: Nimbalkar V

PROVIDER: S-EPMC8642613 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Male Sprague-Dawley rats weighing 250-300 g were purchased from Japan SLC Co. (Shizuoka, Japan). The animals were housed under a daily controlled 12-h light and 12-h dark cycle at 23 °C with free access to rat chow (Japan SLC Co.) and water for 1 week prior to the experiments. Rats were laparotomized under ether anesthesia in each experiment, and the common bile duct of each rat was ligated between the liver hilus and small intestine. Control animals underwent a sham operation with exposure but without ligation of the common bile duct. After the CBDL or sham operation, animals were allowed access to food and water ad libitum. Groups of three rats were sacrificed under ether anesthesia at 1, 3 and 12 hr after the CBDL or sham operation. At the time of sacrifice, the right lateral liver lobe was removed and flash-frozen in liquid nitrogen and stored at -80 °C. Total RNA isolated from frozen livers using TRIzol reagent (Invitrogen Co., Carlsbad, CA) was mixed to minimize variation among animals. Poly(A) RNA was purified using Oligotex-dT30 (Takara Shuzo Co., Ltd., Kyoto, Japan) in accordance with manufacturer’s instructions. Fluorescence-labeled probes were prepared by reverse transcription using a superscript II reverse transcriptase (Invitrogen Co.) and cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has undergone CBDL and from those that has undergone a sham operation were labeled with cyanine-3 and cyanine-5, respectively, and vise varsa. Fluorescence-labeled probes were purified using a MinElute PCR Products Purification Kit (Quiagen GmbH, Hilden, Germany) in accordance with manufacturer’s instructions. Each purified probe was suspended in hybridization buffer containing 1.6 mg/mL poly(A) (Roche Diagnostics, Basel, Switzerland) and yeast tRNA (Roche Diagnostics), 0.67 mg/mL herring sperm, 16% 20 x SSC and 0.3% SDS and applied to a cDNA microarray containing 1,800 rat genes on a slide glass (Asahi Technoglass Co., Tokyo, Japan). Three house-keeping genes (GAPDH, HPRT and b-actin) and rat unrelated traits (Lambda DNA) were also spotted on the slide as internal positive and negative controls, respectively. Hybridization was carried out twice to eliminate any dye bias. In one experiment, duplicate slides were hybridized with probes derived from CBDL and control rats that had been labeld with cyanine-3 and cyanine-5, respectively. In a replicate experiment, other duplicate slides were hybridized with probes derived from CBDL rats and control rats that had been labeled with cyanine-5 and cyanine-3, respectively (color swap). Then, the slides were cover-slipped and incubated in a sealed chamber (Asahi Technoglass Co.) for 16 hrs under a humidified (65 °C) condition. After being washed in low-stringency buffer (2 x SSC and 0.1% SDS), high-stringency buffer (0.2 x SSC and 0.1% SDS) and 0.2 x SSC and then rinsing with 99.5% ethanol, the slide was dried by centrifugation at low speed and used for scanning. Fluorescence was scanned by using a ScanArray 4000 (Packard BioChip Technologies, Billerica, MA) and quantified by using QuantArray Software (Packard BioChip Technologies, Billerica, MA). Keywords: time-course

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L-carnitine ameliorates bile duct ligation induced liver fibrosis via reducing the nitrosative stress in experimental animals: preclinical evidences.

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