Evaluation of Overexpressed Cannabinoid 1 Receptor in Liver Fibrosis Tissues via Detecting CB1R Antagonist, AM251, by MALDI-TOF MS in Vitro

程 俊嘉(Chun-Chia Cheng), 何 愛生(Ai-Sheng Ho), 羅 彩月(Tsai-Yueh Luo), 張 榮善, 張 甄(Tiong Cheng), 張 君照(Chun-Chao Chang)

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Liver fibrosis leads to liver cirrhosis and even cancer progressively, but can be prevented through early detection. A reliable tissue biomarker is desirable in order to design a non-invasive diagnosing tool to complement invasive liver biopsy. Presently many studies have reported that the cannabinoid 1 receptor (CB1R) is associated with liver fibrosis, and therefore it is possible to measure the expression of CB1R for detecting the stages of liver fibrosis. The aim of this study was to measure the protein expression of CB1R via detecting its antagonist, AM251, utilizing biomedical techniques and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer in vitro. Methods: The rats were treated with/without 0.04% thioacetamide to induce each stage of liver fibrosis (F0-F4) which was determined by hematoxylin and eosin (H&E) staining according to METAVIR Classification. Immunohistochemistry was used to detect the expression and position of CB1R. Fluorescent microscopy was used to observe the binding capacity of tocriflour 1117 (T1117), a fluorescent analogue of AM251, to CB1R. MALDI-TOF MS was used to measure the amount of AM251 after incubating with liver fibrosis tissue sections. Results: CB1R overexpressed in the liver fibrosis tissues and increased from F0 to F4 stage. T1117 binding assay showed that AM251 could interact with CB1R specifically. Moreover, the quantification analysis of MALDITOF MS indicated that AM251 could present the amount of CB1R in each stage of liver fibrosis with increase from F0 to F4 (p<0.05). Conclusion: We showed that CB1R overexpressed in liver fibrosis tissues as a reliable biomarker. The data imply that in vitro measurement of CB1R antagonist, AM251, may help distinguish the stages of liver fibrosis. Furthermore, we suggest that AM251 may be used as a detecting ligand and applied in nuclear imaging technique for diagnosing liver fibrosis.Background: Liver fibrosis leads to liver cirrhosis and even cancer progressively, but can be prevented through early detection. A reliable tissue biomarker is desirable in order to design a non-invasive diagnosing tool to complement invasive liver biopsy. Presently many studies have reported that the cannabinoid 1 receptor (CB1R) is associated with liver fibrosis, and therefore it is possible to measure the expression of CB1R for detecting the stages of liver fibrosis. The aim of this study was to measure the protein expression of CB1R via detecting its antagonist, AM251, utilizing biomedical techniques and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer in vitro. Methods: The rats were treated with/without 0.04% thioacetamide to induce each stage of liver fibrosis (F0-F4) which was determined by hematoxylin and eosin (H&E) staining according to METAVIR Classification. Immunohistochemistry was used to detect the expression and position of CB1R. Fluorescent microscopy was used to observe the binding capacity of tocriflour 1117 (T1117), a fluorescent analogue of AM251, to CB1R. MALDI-TOF MS was used to measure the amount of AM251 after incubating with liver fibrosis tissue sections. Results: CB1R overexpressed in the liver fibrosis tissues and increased from F0 to F4 stage. T1117 binding assay showed that AM251 could interact with CB1R specifically. Moreover, the quantification analysis of MALDITOF MS indicated that AM251 could present the amount of CB1R in each stage of liver fibrosis with increase from F0 to F4 (p<0.05). Conclusion: We showed that CB1R overexpressed in liver fibrosis tissues as a reliable biomarker. The data imply that in vitro measurement of CB1R antagonist, AM251, may help distinguish the stages of liver fibrosis. Furthermore, we suggest that AM251 may be used as a detecting ligand and applied in nuclear imaging technique for diagnosing liver fibrosis.
Original languageEnglish
Pages (from-to)199-206
Number of pages8
JournalGastroenterological Journal of Taiwan
Volume30
Issue number2
DOIs
Publication statusPublished - Jun 1 2013

Keywords

  • 肝纖維化
  • 類大麻受體
  • 質譜技術
  • liver fibrosis
  • AM251
  • cannabinoid 1 Receptor (CB1R)
  • matrix assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF MS)

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