Behavioral and Molecular Analysis of Antioxidative Potential of Rosmarinic Acid Against Methamphetamine-induced Augmentation of Casp3a mRNA in the Zebrafish Brain

Methamphetamine (MA) acts as a powerful oxidant agent, while Rosmarinic Acid (RA) is an effective herbal antioxidant. Oxidative stress-mediated by MA results in apoptosis, and caspase-3 is one of the critical enzymes in the apoptosis process. MA can epigenetically alter gene regulation. In this paper, to investigate the effects of RA on MA-mediated oxidative stress, changes in the level of casp3a mRNA were demonstrated in zebrafish.

The animals were grouped in 3 treatment conditions for the behavioral test: control, MA, MA pretreated by RA, and 6 treatment conditions for the molecular test: control, RA, MA, MA co-treated with RA, MA co-treated with RA/ZnO/chitosan nanoparticle, and ZnO/chitosan nanoparticle. Then molecular and behavioral investigations were carried out, and critical comparisons were made between the groups. MA solution was prepared with a concentration of 25 mg/L, and RA solution was prepared by DPPH test with the antioxidant power of about 97%. Each solution was administered by immersing 20 zebrafish for 20 minutes, once per day for 7 days. The level of casp3a mRNA was quantified by using qRT-PCR. One-sided trapezoidal tank diving test was applied to study behavioral alterations.

The qPCR analysis demonstrated the high potential of RA/ZnO/chitosan in counteracting the MA-mediated elevation in casp3a mRNA level. Based on the diving test results of MA-treated fish, MA was found to be anxiolytic compared to the control. While the resulted diving pattern of the MA-treated animals pretreated by RA was novel and different from both the control and MA-treated groups.

The potential of RA combined with a suitable nanoparticle against MA-induced oxidative stress was supported. The high efficiency of ZnO/chitosan in increasing RA penetration to the brain cells was evident. MA at a dose of 25 mg/L is anxiolytic for zebrafish. However, the molecular mechanisms involved in these processes should be studied.