| Objective: To observe the anti-apoptosis and anti-inflammatory effect of atorvastatin on rat models of fluid percussion brain injury by observing its effect on Nrf2, caspase-3, caspase-12 and inflammatory cytokines.Methods: 48 health adult male SD rats, weighting about 250 g, were seldomly divided into four groups: Sham operation group(Sham+vehicle), Traumatic brain injury group(TBI+vehicle group), Low doses of atorvastatin group(TBI+LD statin group) and High doses of atorvastatin group(TBI+HD statin group). Animals of TBI+LD statin group and TBI+HD statin group after establishing models were lavaged with atorvastatin of 5?/? and 10?/?respectively. The TBI+vehicle group, TBI+LD statin group and TBI+HD statin group were subjected to fluid percussion. All animals were envaluated with modified neurological severity scores(m NSS) after 24 hours and decapitated, and the brains were harvested. Cortical lesion volume were determined by TTC staining. 100 mg brain tissues were taken from the front of the injury area, and their water content were measured by dry-wet weight method; About 4mm thick coronal slices across the brain injury area were fixed with 4% paraformaldehyde for 24 hours, and then were stained with HE for the observation of morphology. The expressions of nuclear and cytoplasmic Nrf2 protein, caspase-3 and caspase-12 were investigated by immunohistochemical staining and the neuronal apoptosis by TUNEL. Brain tissue collected from posterior edge of impact site were quckly put into the liquid nitrogen, and Western Blot was employed to measure the expression of caspase-3, caspase-12, Nrf2 and ELISA to the expression of IL-1??IL-6?TNF-?.Results: 1 m NSS of animals from all groups All the rats which were subjected to fluid percussion brain injury had hypermyotonia of all limbs, apnea, heart rate increased, roachback, piloerection, forelimb flexion and hind legs tetany imediately. Compared with Sham+vehicle group, the m NSS score of rats from the TBI+Vehicle group at 24 h after injury was obviously higher; Surprisingly, m NSS of animals from TBI+LD statin group and TBI+HD statin group decreased significately, compared to those in the TBI+vehicle group(9.167±0.753, 8.167±0.753, 11.000±0.894, P<0.05), and the difference between two groups was statistically significant(P<0.05). 2 Brain water content Compared with Sham+vehicle group, brain water content of rats from the TBI+Vehicle group at 24 h after injury was obviously higher(78.044±0.781,83.014±0.816, P<0.05); Surprisingly, brain water content of animals from TBI+LD statin group and TBI+HD statin group decreased significately, compared to those in the TBI+vehicle group(82.801±0.667, 80.837±0.644, 83.014±0.816, P<0.05), and the difference between two groups was statistically significant(P<0.05). 3 Cortical lesion volume by TTC staining Compared with Sham+vehicle group, cortical lesion volume of rats from the TBI+Vehicle group at 24 h after injury was obviously higher; Surprisingly, cortical lesion volume of animals from TBI+LD statin groupand TBI+HD statin group decreased significately, compared to those in the TBI+vehicle group(49.55±3.916, 44.31±1.781, 61.04±4.426,P<0.05),and the difference between two groups was statistically significant(P<0.05). 4 The expression expression of Caspase-3, Caspase-12 and Nrf2 by Immunohistochemistry Compared with Sham+vehicle group, the positive AOD of caspase-3and caspase-12 around fluid percussion area of rats from the TBI+Vehicle group at 24 h after injury was obviously higher; Surprisingly, the positive AOD of caspase-3 and caspase-12 from TBI+LD statin group and TBI+HD statin group decreased significately, compared to those in the TBI+vehicle group, and the difference between two groups was statistically significant(P<0.05). Compared with Sham+vehicle group, the positive AOD of Nrf2 around fluid percussion area of rats from the TBI+Vehicle group at 24 h after injury was obviously higher; Surprisingly, the positive AOD of Nrf2 from TBI+LD statin group and TBI+HD statin group increased significately, compared to those in the TBI+vehicle group, and the difference between two groups was statistically significant(P<0.05). 5 Histological observations HE staining of brain tissue slices under the microscope: in the brain tissue slices of Sham+vehicle group, structure was integrated, cells arranged orderly, nuclear membrane integrity, the nucleus was clear, no edema, hemorrhage; TBI+vehicle group, fluid percussion area appeared dotted hemorrhage and edema, the surrounding structure of fluid percussion area was highly porous, cell height edema, neuron clearance increased obviously, degeneration of neurons, the cell volume increased obviously, cytoplasm, cell cavity became apparent, around with inflammatory cells infiltrated and glial cell proliferated. The degree of brain edema are reduced, the range of brain edema and inflammatory cells less in the TBI+LD statin group and TBI+HD statin group compared to those in the TBI+vehicle group. Effect is more apparent in the TBI + HD statin group. 6 The protein expression of nuclear and cytoplasmic Nrf2, caspase-3 and caspase-12 by Western Blot Compared with Sham+vehicle group, the nucleoprotein expression of Nrf2 around fluid percussion area of rats from the TBI+Vehicle group at 24 h after injury was obviously higher; Surprisingly, the nucleoprotein expression of Nrf2 from TBI+LD statin group and TBI+HD statin group increased significately, compared to those in the TBI+vehicle group, and the difference between two groups was statistically significant(P<0.05). Compared with Sham+vehicle group, the cytoplasmprotein expression of Nrf2 around fluid percussion area of rats from the TBI+Vehicle group at 24 h after injury was lower; Surprisingly, the cytoplasmprotein expression of Nrf2 from TBI+LD statin group and TBI+HD statin group decreased significately, compared to those in the TBI+vehicle group, and the difference between two groups was statistically significant(P<0.05). Compared with Sham+vehicle group, the protein level of caspase-3 and caspase-12 around fluid percussion area of rats from the TBI+Vehicle group at 24 h after injury was obviously higher; Surprisingly, the protein level of caspase-3 and caspase-12 from TBI+LD statin group and TBI+HD statin group decreased significately, compared to those in the TBI+vehicle group, and the difference between two groups was statistically significant(P<0.05). 7 Neural apoptosis by TUNEL Animals of Sham+vehicle group had almost no positive cell.After fluid percussion, compared with Sham+vehicle group,apoptosis Index of rats from the TBI+Vehicle group at 24 h after injury was obviously higher(0.265±0.022,0.726±0.032,P<0.05); Surprisingly, apoptosis Index from TBI+LD statin group and TBI+HD statin group decreased significately, compared to those in the TBI+vehicle group(0.649±0.041,0.526±0.035,0.726±0.032,P<0.05), and the difference between two groups was statistically significant(P<0.05). 8 Detecte the level of IL-1?,IL-6,TNF-? by ELISA Compared with Sham+vehicle group, the level of IL-1?,TNF-?,IL-6 around fluid percussion area of rats from the TBI+Vehicle group at 24 h after injury was obviously higher; Surprisingly, the level of IL-1?,TNF-?,IL-6 from TBI+LD statin group and TBI+HD statin group decreased significately, compared to those in the TBI+vehicle group, and the difference between two groups was statistically significant(P<0.05).Conclusions: 1 The expressions of caspase-12, caspase-3, Nrf2 and inflammtory cytokines were elevated in rats following fluid percussion brain injury. 2 Atorvastatin had a neuroprotective effect with anti-apoptosis and anti-inflammatory on rats following fluid percussion brain injury, by way of increasing the activity of Nrf2. 3 Neuroprotective effect of atorvastatin was dose-dependent. |
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