Study On The Protection Mechanism Of The Lemongrass Essential Oil Induced Damage By Benzo[a]pyrene In Mice Brain

Objective:The aim of this paper is to detect some indicators of oxidative stress in mice brain tissues that subchronicly exposed by the benzo[a]pyrene(B[a]P) and affected by the lemongrass essential oil(LEO),and to determine the levels of 3-hydroxyl-benzo[a]pyrene(3-OHB[a]P) and(+)-anti-benzo[a]pyrene diolepoxide-DNA adducts((+)-anti-BPDE-DNA)in mice brain tissues and blood, and to measure the levels of p53 gene and bcl-2 gene expression, to study the variation and to illustrate the protection mechanism of the LEO to mice brain tissues damage induced by B[a]P, and to furher explore the possible mechanism of the LEO that is used for cancer chemoprevention.Method:(1) Established a contamination model and divided experimental groups:SPF kunming mouse were randomly divided into 9 groups, with 32 animals per group,with a weigh of 20-22 g, free access to water. B[a]P with a purity of 96% was dissolved in olive oil at a concentration of 6.25 mg/mL and administered to the mice at a dosage of 50 mg/kg body weight by gavage, which amounted to 0.2 mL of B[a]P for each animal. LEO was dissolved in water at three different concentrations: 0.05%, 0.1%,and 0.25% in aqueous solution, and the animals were allowed to inhale the LEO preparation for 30 minutes every day. After B[a]P subchronic exposure for three months, mice were treated eyeball blood, and blood was collected an EP tube which previously added anticoagulant and mixed the blood.Taked blood plasma and extracted whole blood DNA from specimens, then mice were sacrificed by dislocation of thecarotid artery and removed the brain tissues, placed in saline wash, dried surface water on brains, the brains were divided into 2 parts, one wet weighed, added saline to make tissue homogenates, another part was stored.The samples were stored at-80 ℃ until analysis.(2) Developed an analytical method for determining the levels of 3-OHB[a]P and(+)-anti-BPDE-DNA in mice blood and brain tissues by high performance liquid chromatography with fluorescence detection of the internal standard.(3) Applicated brain tissues homogenates and coomassie detection kits, superoxide dismutase(SOD)kits, catalase(CAT) kits, malondialdehyde(MDA) kits, total antioxidant capacity(T-AOC) test kits and mouse 8-hydroxy-2’-deoxyguanosine(8-OHdG) enzyme-linked immunosorbent assay kits to measure the levels of SOD, CAT, MDA, T-AOC and8-OHdG in brain tissues.(4) Applied real-time quantitative PCR, Western blotting and immunohistochemistry to detect the levels of p53 gene and bcl-2 gene expression, to further explain the protection mechanism of LEO to mice brain damage induced by B[a]P.Results:(1) A reliable model of mice B[a]P subchronic exposure was established.LEO as an effective chemopreventive method was applied to the mice brain tissues damage induced by B[a]P.(2) Compared with normal group, B[a]P alone treatment increase the contents of 3-OHB[a]P and(+)-anti-BPDE-DNA(P <0.05); Compared with B[a]P group, B[a]P and LEO gradually increase the the contents of 3-OHB[a]P(P <0.05)and gradually decrease(+)-anti-BPDE-DNA(P<0.05) in three LEO protection groups.(3) Oxidative stress enzymes experiments: Compared with normal group, only B[a]P treatment reduce the levels of SOD, CAT and T-AOC(P <0.05), and increase the contents of MDA, 8-OHdG(P <0.05); Compared with the B[a]P group, B[a]P and LEO increase the levels of SOD, CAT and T-AOC(P <0.05), and reduce the contents of MDA, 8-OHdG(P <0.05) in three LEO protection groups.(4) The molecular biology experiments: Compared with normal group, B[a]P alone treatment upregulate the level of p53 gene expression in brain tissues(P <0.05), as well as upregulate bcl-2 gene expression(P <0.05); Compared with the B[a]P group, B[a]P and LEO upregulate the level of p53 gene expression(P <0.05) and downregulate the level of bcl-2 geneexpression in three LEO protection groups(P <0.05).Conclusion: LEO can reduce DNA damage in mice brain tissues induced by B[a]P,which suggest that LEO has an antioxidant function and has a protective effect for mice brain damage induced by B[a]P, its mechanism may be upregulate the levels of p53 gene expression and downregulate bcl-2 gene expression.