Effects of 2.3,5.6-Tetramethylpyrazine on alcohol-induced injury in liver cells and on the early life stages of zebrafish (Danio rerio Hamilton 1822)

Abstract

Alcohol liver disease (ALD) is a major health problem referring to the collection of liver damage caused by excessive alcohol intake. It encompasses three stages of increasing aggressiveness – steatosis, steatohepatitis, and cirrhosis. This study aimed to assess the hepatoprotective effects of 2,3,5,6-Tetramethylpyrazine (TMP) against ALD using histopathological analysis of zebrafish livers subjected to different exposure groups. TMP is compound that has been mainly used for the treatment of cardio- and cerebrovascular diseases due to its antioxidant, anti-inflammatory, and anti-apoptotic properties. Three TMP concentrations (40, 60, and 80 mg/L TMP) were used for this part of the study. Results showed that TMP was able to dose-dependently decrease mean scores for the four parameters diagnostic of ALD – steatosis, inflammation, cell death, and ballooning degeneration. These scores were comparable to those of the untreated group (no ethanol + no treatment) and positive control (ethanol + Hepasil DTXTM), with all groups’ scores being statistically different from those of the negative control (ethanol + no treatment) (p<0.05). Other anomalies, namely, cholestasis, vessel congestion, and hemorrhage were noted only for the ethanol group, but not for other groups. These imply the high efficacy of TMP in terms of hepatoprotection. To further gauge the plausibility of TMP as liver treatment, its toxicity to the early development of embryos was evaluated using the Zebrafish Embryotoxicity Test (ZFET). Analysis of ZFET suggests that TMP is also non-toxic or non-teratogenic at concentrations used for liver treatment. Percent mortalities in the TMP groups (20 mg/L to 100mg/L), as assessed by lethal endpoints (i.e. coagulation, non-detachment of tail, non-formation of somites, and non-detection of heartbeat), were minimal and not statistically different with that of the negative control (reconstituted water), but were statistically different to the positive control (3.5% ethanol). The occurrences of sublethal endpoints (i.e. yolk sac edema, pericardial edema, spinal curvature, abnormal heart rate, and body length) were generally comparable to those in the negative control and statistically different from the positive control. This initial investigation on TMP, therefore, provided justification for its plausibility as a hepatoprotective compound against ALD.