Study On Anti-oxidative Stress Effects Of Bilberry Extract In Eyes

Object:The study aims to investigate the effects of bilberry extract (BE) on the functions of antioxidants, antioxidant enzymes and mitochondria in eyes under oxidative stress of mice. Furthermore, the possible mechanism was also studied.Method:(1) The contents of bilberry extract were analyzed through HPLC/ESI-MS, and its antioxidative effect in vitro was evaluated with oxygen radical absorbance capacity (ORAC) and DPPH methods.(2) Oxidative stress damage in eyes of mice was induced after 18 h restraint stress. The aminotransferase (ALT) level in plasma was determined by Reitman-Frankel method. The malondialdehyde (MDA) content was determined by TBA colorimetric method with a MDA kit. The ORAC level was determined by automated multifunction microplate reader. The contents of glutathione (GSH) and vitamin C (Vit C) were determined by HPLC. The nitric oxide (NO) level was determined by Griess method. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were determined by colorimetric method with SOD and GPx kits, respectively. The expression of inducible nitric oxide synthase (iNOS), copper/zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and GPx mRNAs was semiquantitatively assessed by the method of reverse transcription-polymerase chain reaction (RT-PCR).(3) Uveitis in mice was induced 24 h after footpad lipopolysaccharide (LPS) injection. The levels of NO, MDA, ORAC, GSH and Vit C were determined. Furthermore, the expression of iNOS, CuZnSOD, MnSOD and GPx mRNAs was examined using RT-PCR.(4) The activities of respiratory chain complexes in eyes of mice were examined through automated multifunction microplate reader, and the expression of related genes, such as Ndufsl, Ndufs4, Ndufs5, Ndufs6, SDHA, SDHB and COXI-â…¡mRNAs in eyes of mice was examined using RT-PCR.(5) We examined the expression of NF-kB p65 and IKBa mRNAs using RT-PCR and detected the expression of NF-kB p65 protein by Western Blotting.Result:(1) Our experiments showed that bilberry extract contained 15 kinds of anthocyanins which account for 42.04% of the extract by HPLC/ESI-MS analysis. In addition, both ORAC and DPPH method indicated that bilberry extract had good antioxidative capacity in vitro.(2) Our experiments showed that 18h restraint stress could induce oxidative stress damage in eyes. Compared with restraint stress group, the levels of MDA and NO decreased, the levels of ORAC, GSH and Vit C increased and the activities of SOD and GPx elevated in eyes of restraint+BE.100 mg/kg and restraint+BE.200 mg/kg groups. Moreover, further study showed that the expression of iNOS mRNA decreased and the expression of CuZnSOD, MnSOD and GPx mRNAs increased in eyes of restraint+BE.100 mg/kg and restraint+BE.200 mg/kg groups. (3) Our experiments showed that footpad injection of LPS could induce uveitis. Compared with LPS group, the levels of NO and MDA decreased, the levels of ORAC, GSH and Vit C increased and the activities of SOD and GPx elevated in eyes of LPS+BE.100 mg/kg and LPS+BE.200 mg/kg groups. Moreover, further study showed that the expression of iNOS mRNA decreased and the expression of CuZnSOD, MnSOD and GPx mRNAs increased in eyes of LPS+BE.100 mg/kg and LPS+BE.200 mg/kg groups.(4) Administration of bilberry extract (100 mg/kg and 200 mg/kg) increased the activities of some respiratory chain complexes and elevated the expression of related genes in eyes under oxidative stress of mice.(5) Administration of bilberry extract (100 mg/kg and 200 mg/kg) reduced the expression of NF-kB p65 mRNA and protein in eyes under oxidative stress of mice and reduced the activity of NF-kB p65, which may account for the decreased expression of iNOS mRNA, the increased expression of genes related with respiratory chain complexes, and the increased expression of CuZnSOD, MnSOD and GPx mRNAs.Conclusion:Bilberry extract showed protective effects against oxidatice stress damage in eyes of mice, and the effects of bilberry extract (100 mg/kg and 200 mg/kg) were dose-dependent. Furthermore, the mechanism of its protective effects may be attributed to scavenging free radicals, maintaining the levels of antioxidants and antioxidant enzymes and protecting mitochondrial function in eyes.Novel Points:(1) The protective effects of bilberry extract against oxidative stress damage in eyes of mice and its mechanism were studied at level of animals by methods and techniques of biochemistry and molecular biology.(2) Oxidative stress in eyes selectively decreased the activities and gene expression of the first and second respiratory chain complexes.(3) Bilberry extract improved the status of oxidative stress in eyes of mice to some extent through modulating the activity of transcription factor NF-kB.