| Objective: To investigate the neuroprotective effect of anti-apoptosis and anti-inflammatory of EGCG in rat models of fluid percussion brain injury by observing its effect on Nrf2, caspase-3, caspase-12 and inflammatory cytokines.Methods:1 24 SD adult male rats were randomly divided into three groups(n=8): Sham operation group(Sham group),Traumatic brain injury with Vehicle group(TBI+Vehicle group) and Traumatic brain injury with administration of EGCG group(TBI+EGCG group). After general anesthesia, animals from TBI+Vehicle group and TBI+EGCG group were made skull bone window at the right of sagittal suture and the back of coronal suture 5mm, and treated with fluid impact. At the same time, animals of TBI+Vehicle group received intraperitoneal injection of 1ml physiological saline while those of the TBI+EGCG group received intraperitoneal injection of EGCG with the dose of 50mg/kg+1ml physiological saline. For the rats of sham group, only bone window was drilled after general anesthesia and not treated with fluid impact.2 After 24 h, all the animals were evaluated according to the modified Neurological Severity Scores(mNSS) and the brains tissue were quickly harvested after overdose hyperanesthesia. 100 mg of brain tissue was collected from the front of damage area for the measurement of the water content. Across the damage area, coronal slices of approximately 3mm thick, were cut and fixed with paraformaldehyde. The slices were stained with HE for histological observation. 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 Modified Neurological Severity ScoresAll the rats which were subjected to fluid percussion brain injury have hypermyotonia of all Limbs, apnea, heart rate increased, roachback, piloerection, forelimb flexion and hind legs tetany imediately. Compared with the sham group, the mNSS score of rats from the TBI+Vehicle group at 24 h after injury was much higher(9.500±0.926). And the TBI+EGCG group was also rised(7.875±0.835). However, the score of rats from TBI+EGCG group was lower than that of the TBI+Vehicle group, and the difference between two groups was statistically significant(P<0.05).2 Brain water contentElliot formula was used to measure the content of water in brain tissue. Compared with the sham group, the brain water content of rats from the TBI+Vehicle group at the 24 h after injury was significantly higher(83.305±0.539), and the TBI+EGCG group was the same to it(80.846±0.550).While water content of rats from TBI+EGCG group was much lower than that of the TBI+Vehicle group, and the difference between the two groups was statistically significant(P<0.05).3 Histological observation by HE stainingUnder the microscope, the structure of brain tissue of sham group were integrity, and the cells were arranged orderly, membranes were intact without edema or bleeding.The rats from TBI+Vehicle group at 24 h after injury were perifocal punctate hemorrhage and edema, around which the brain tissue was highly loose with the edematous neural cells. The volume of neural cells increased with bigger space around neurons, the inflammatory cells infiltrated and the glial cell proliferated. Animal brains of TBI+EGCG group had the same histological characteristics as those of TBI+Vehicle group, but their cellular edema was significantly reduced.4 Immunohistochemistry(for Nrf2,Caspase-3 and Caspase-12 protein)The expression of Nrf2 protein: Compared with the sham group, the positive AOD of Nrf2 from the other two groups were significantly increased. Surprisedly, the positive AOD of TBI+EGCG group(0.391±0.060)was much higher than that of the TBI+Vehicle group(0.282±0.055), and the difference between them was statistically significant(P<0.05).The expression of caspase-3 protein: Compared with the sham group, the positive AOD of caspase-3 from the other two groups were significantly increased. Howevere, the positive AOD of TBI+EGCG group(0.264±0.029) was much lower than that of the TBI+Vehicle group(0.371±0.038), and the difference between them was statistically significant(P<0.05).The expression of caspase-12: Compared with the sham group, the positive AOD of caspase-12 from the other two groups were significantly increased. Howevere, the positive AOD of TBI+EGCG group(0.324±0.046) was much lower than that of the TBI+Vehicle group(0.426±0.036), and the difference between them was statistically significant(P<0.05).5 Apoptotic cell detection by TUNELIn the sham group there were only a small number of the TUNEL-positive cells. Compared with the sham group, TUNEL-positive cells in rats from the TBI+Vehicle group and the TBI+EGCG group significantly increased. The apoptotic index of the TBI+EGCG group(21.374±2.887)was much more lower than the TBI+Vehicle group(29.548±2.919), and the difference between them was statistically significant(P <0.05).6 Western Blot(for nuclear Nrf2,cytoplasmic Nrf2,Caspase-3 and Caspase-12 protein)The expression of nuclear Nrf2 protein: Compared with the sham group, the expression of nuclear Nrf2 in the TBI+Vehicle group and the TBI+EGCG group increased significantly. The expression of TBI+Vehicle group(0.337±0.058) were relatively lower than TBI+EGCG group(0.554 ± 0.052).The difference between them was statistically significant(P <0.05).The expression of cytoplasmic Nrf2 protein: Compared with the sham group, the expression of cytoplasmic Nrf2 in the TBI+Vehicle group and the TBI+EGCG group decreased significantly. The expression of TBI+Vehicle group(0.537±0.032)were relatively higher than TBI+EGCG group(0.405±0.040).The difference between them was statistically significant(P <0.05).The expression of caspase-3 protein: Compared with the sham group, the expression of caspase-3 in the TBI+Vehicle group and the TBI+EGCG group increased significantly. The expression of TBI+Vehicle group(0.814±0.028)were relatively higher than TBI+EGCG group(0.664±0.023).The difference was statistically significant(P <0.05).The expression of caspase-12 protein: Compared with the sham group, the expression of caspase-12 in the TBI+Vehicle group and the TBI+EGCG group increased significantly. The expression of TBI+Vehicle group(0.764±0.030)were relatively higher than TBI+EGCG group(0.545 ± 0.018).The difference was statistically significant(P <0.05).7 ELISA(for IL-1?,IL-6,TNF-? expression)The expression of IL-1?: Compared with the sham group, the expression of IL-1? in the other two groups increased significantly. However, IL-1? expression of TBI+EGCG group(576.078±55.986) was relatively lower than that of TBI+Vehicle group(809.422±68.577) and the difference between them was statistically significant(P <0.05).The expression of IL-6: Compared with the sham group, the expression of IL-6 in the other two groups increased significantly. However, IL-6 expression of TBI+EGCG group(251.603±9.500) was relatively lower than the TBI+Vehicle group(306.207±13.524) and the difference between them was statistically significant(P <0.05).The expression of TNF-?: Compared with the sham group, the expression of TNF-? in the other two groups increased significantly. However, TNF-? expression of TBI+EGCG group(23.167±4.068) was relatively lower than the TBI+Vehicle group(31.800±2.103) and the difference between them was statistically significant(P <0.05).Conclusions:1 The expressions of Nrf2, caspase-3, caspase-12 and inflammtory cytokines were increased in rats following fluid percussion brain injury, indicating that they may play an important role in the secondary traumatic brain injury.2 EGCG has a neuroprotective effect with anti-apoptosis and anti-inflammatory on rats following fluid percussion brain injury, by way of increasing the activity of Nrf2, and reducing the expression of caspase-3, caspase-12 and inflammatory cytokines. |
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