| Objectives Explore whether citrus peel extract(GL) has a therapeutic effect on rats suffered traumatic brain injury(TBI) and its mechanism.Methods 1 Model preparation and drug administration. 120 Sprague-Dawley(SD) male rats screened out by Morris water maze platform test were randomly divided into three groups, Sham group, TBI group and GL group, each group had 1d, 3d, 7d, 14 d, 21 d, 5time points after TBI, each group at each time point eight rats. Sham rats were performed craniotomy only without hit, the other two groups of rats were prepared with CCI. After TBI, the rats were gavaged with intervention agent once a day, Sham group and TBI group: vehicle(saline containing 15% ethanol) 1.0ml, GL group: GL1.0 ml. 2Experimental methods and indicators. After TBI, the rats in each group were carried m NSS test at 1d, 3d, 7d, 14 d, 21 d, five time points. From the 15 th day to the 18 th day after TBI, 21 d time point rats of all groups were carried Morris water maze place navigation test, which lasted 4 days, then the escape latency and swimming distance at the 18 th day after TBI were statistically analysised for detecting the spatial learning ability in rats, the 19 th day after TBI, one time of Morris water maze spatial probe test was carried for detecting rats spatial memory. The rats were sacrificed at corresponding time points, lesions and HE staining was observed, IHC method was used to detect the expression of GFAP, OX-42, TNF-α and VEGF, Western blot method to detect the expression of Neu N, COX-2, NF-κB(P65) and TLR4. 3 Statistical analysis. Image Pro Plus image analysis software was used to detect the expression of GFAP, OX-42, VEGF and TNF-α staining semi-quantitatively. The results were expressed by sum value of the IOD. Western blot results of Neu N, COX-2, NF-κB(P65) and TLR4 were analyzed by Image Lab, we used the Int ratio of target protein to the corresponding internal reference(GAPDH or Lamin A/C) to reflect the expression level of target facter. Experimental data obtained was expressed as mean ± standard deviation(`x±s), then was analysised with SPSS17.0 for one-way ANOVA analysis, with P<0.05 indicating a statistically significant difference.Results 1 m NSS test. 1, 4, 7, 14 days after TBI, m NSS score of both TBI and GL group rats significantly increased, while compared with TBI group rats, the rats of GL group have a lower m NSS score, the difference was statistically significant(P<0.05). 2 Morris water maze. Compared with Sham group rats, two other groups of rats behaved poorly in both place navigation test and spatial probe test. While compared to the rats of the TBI group, the rats of GL group have a better behavior, the difference was statistically significant(P<0.05). 3 Injury lesion. 7 days after TBI, rats of Sham group have no significant injury lesion; in the right parietal lobe center of TBI rats brain, a lesion can be easily found, wirh irregular margin, and a certain amount of visible congestion; compared to the TBI group, each rat in GL group can be seen a limited injury lesion in the same position, with significantly fewer congestion. 4 HE staining. The injured side of the cerebral cortex was seen a apparent injury lesion, brain parenchyma damaged, partly lost,injury lesion almost throughout the cerebral cortex, with cingulate cortex and hippocampus involving in, cingulate gyrus cells loosely arranging, a clearly visible damage notch on the outside edge of hippocampus. Cortical hierarchy is unclear, with unclear boundaries of gray and white matter, cell edema and sheet-like necrosis peri injury lesion, neutrophils and a small amount of monocytes- macrophage infiltration. 5IHC staining. 1 day after TBI, glial cells of injuried rats activated, the level of GFAP,OX-42, TNF-α and VEGF expression significantly increased, OX-42 and TNF-α peaked at 1 day after TBI, GFAP and VEGF peaked at 3 days after TBI. Intervention with GL,GFAP, OX-42 and TNF-α expression was significantly reduced, the level of VEGF expression further increased. 6 Western blot. 1 day after TBI, rats of TBI group have a significant increase in the expression of COX-2, NF-κB(P65) and TLR4. Intervention with GL significantly reduced the expression level of COX-2, NF-κB(P65) and TLR4.While the Neu N expression of TBI group significantly decreased, compared with TBI group, GL group expressed Neu N increasingly.Conclusions 1 Citrus peel extract(GL) can obviously improve the nerve damage and cognitive dysfunction in TBI rats. 2 Citrus peel extract(GL) can reduce the expression of GFAP and OX-42 in TBI rats, inhibit glial cell activation. 3 Citrus peel extract(GL) can reduce the expression of some inflammatory facters, including COX-2, NF-κB(P65) and TLR4, inhibit inflammatory reaction. 4 Citrus peel extract(GL) can play a neuroprotective role by both increasing the production of VEGF and reducing neuronal cell damage. |
PDF Full Text Request