ABSTRACT
Objective To investigate the neuroprotective effect and antioxidation mechanism of tianmagouteng decoction on retinal ganglion cells (RGCs) in optic nerve crush model.Methods Optic nerve crush models were established using optic nerve clamping method in the right eyes of 50 SPF male Wistar rats.The rats were randomized into model group (17 rats),1.2 g/ml tianmagouteng decoction group (16 rats) and 2.4 g/ml tianmagouteng decoction group (17 rats).Ttianmagouteng decoction at the dose of 1.2 g/(ml· d) or 2.4 g/(ml · d) was intragastrically administered 2 hours after onset of optic nerve damage (10 ml/kg),respectively,based on grouping for consecutive 28 days,and equal volume of distilled water was used in the same way in the model group.Fluoro-gold (FG,3 μl,3％) was bilaterally injected into superior colliculus to retrogradely label RGCs 23 days following modeling.The rats were sacrificed at day 28 and retinal flatmounts were prepared to count RGCs.Retinal glutathione peroxidase (GSH-Px) activity in rat retinas was detected by chemical colorimetric analysis.Results The survival rate of RGCs was (59.67 ± 9.85) ％,(71.33 ± 9.14) ％ and (73.63 ± 8.33) ％ in the model group,1.2 g/ml tianmagouteng decoction group and 2.4 g/ml tianmagouteng decoction group,respectively,showing a significant difference among the three groups (F =5.322,P =0.014),and the survival rate of RGCs was evidently higher in the 1.2 g/ml tianmagouteng decoction group and 2.4 g/ml tianmagouteng decoction group than that in the model group (P =0.023,0.006).The GSH-Px activity was (222.20±76.67),(311.30 ±46.93) and (473.65 ± 117.73)μmol/(s · mg) in the model group,1.2 g/ml tianmagouteng decoction group and 2.4 g/ml tianmagouteng decoction group,respectively,with a significant difference among the three groups (F =20.005,P ＜ 0.001),and the GSH-Px activity in the 2.4 g/ml tianmagouteng decoction group is considerably increased as compared to the model group and 1.2 g/ml tianmagouteng decoction group (P ＜ 0.001;P =0.001).Conclusions Tianmagouteng decoction plays a neuroprotective effect on RGCs after optic nerve damage in rat,which may be achieved by improving the activity of the GSH-Px in retina,suggesting that antioxidation probably be one of the neuroprotective mechanisms of tianmagouteng decoction.
ABSTRACT
Background Collapsin response mediator protein-2 (CRMP-2) can promote the growth of axons,but CRMP-2 occurs hyperphosphorylation under the induction of cyclin-dependent kinase-5 (CDK5) after central nervous system injury,which leads to the collapse of the growth cone and hinders the repair of nervous system.Being a central nervous system tissue,whether the expressions of CRMP-2 and its phosphorylated protein (p-CRMP-2) change after optic nerve injury are rarely studied.Objective This study was to investigate the dynamic changes of CRMP-2 and p-CRMP-2 expressions in injured optic nerve tissue.Methods Forty-eight 8-or 9-week-old BALB/c mice were randomly divided into the sham operation group and postoperative 3-,7-and 14-day group.Optic nerves were exposed and clamped at retrobulbar 2 mm for 10 seconds in the right eyes during the surgery in the postoperative 3-,7-and 14-day groups,and the same operation was performed except the clamp of optic nerve in the sham operation group.The optic nerve tissue was obtained from the eyes 3,7 and 14 days after surgery.The relative expression levels of CRMP-2 mRNA and CRMP-2,p-CRMP-2 and CDK5 proteins in the tissue were detected by real-time fluorescence quantitative PCR and Western blot,respectively.The use and care of the experimental animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals of Third Military Medical University.Results No significant differences were found in the expression levels of CRMP-2 mRNA and CRMP-2 protein among the sham operation group and postoperative 3-,7-and 14-day groups (CRMP-2 mRNA:F =2.971,P =0.097;C RMP-2 protein:F=1.202,P =0.370).The relative expression levels of p-CRMP-2 protein in the optical nerve were 0.001±0.000,0.064±0.003,0.136±0.005 and 0.346±0.012,and those of CDK5 protein were 0.440±0.009,0.723±0.011,0.874±0.015 and 0.952±0.019 in the sham operation group and postoperative 3-,7-and 14-day groups respectively,showing statistically significant differences among them (p-CRMP-2:F=445.600,P ＜ 0.001;CDK5:F=186.600,P＜0.001),and the relative expression levels of p-CRMP-2 and CDK5 protein were evidently higher in the optical nerve tissue in the postoperative 3-,7-and 14-day groups than those in the sham operation group (all at P＜0.01).Conclusions There are not significant changes in the expression level of CRMP-2 in the BALB/c mice after optic nerve injury.However,the expression levels of p-CRMP-2 and CDK5 proteins are gradually upregulated as the extending of injured time.