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Objective:To observe the dynamic of neurological severity scores (NSS) and the expressions of Wnt/β-catenin signaling pathway, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in rats with severe traumatic brain injury (sTBI), and to explore the effect of Huoxue Huayu decoction.Methods:A total of 126 Sprague-Dawley (SD) rats were randomly divided into seven groups by random number table with 18 rats in each group, namely control group (normal saline 2 kg/L), model group (normal saline 2 kg/L), brain protolysate group (BP group, 5.6 g/kg), Taohong Siwu decoction group (TH group, 10.2 g/kg), Xuefu Zhuyu decoction group (XF group, 15.6 g/kg), Tongqiao Huoxue decoction group (TQ group, 9.6 g/kg) and Buyang Huanwu decoction group (BY group, 28.7 g/kg). The sTBI rat model was reproduced by modified Feeney free fall method, and the rats in the control group were not treated with trauma. The rats in each group were intragastrical administered with corresponding drugs at 6 hours after injury, and the NSS scores were evaluated on the 1st, 3rd and 7th days after injury. After the hippocampus was harvested, the mRNA expressions of Wnt3a and β-catenin were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the positive expressions of BDNF and NGF were detected by immunohistochemistry.Results:Compared with the control group, the rats in the model group showed obvious symptoms of craniocerebral injury at 1 day after injury, which was manifested as significantly increased NSS score, up-regulated mRNA expressions of Wnt3a and β-catenin, and increased positive expressions of BDNF and NGF, which indicated that the sTBI rat model was successfully prepared and presented a certain self-repair ability with the extension of time. Compared with the model group, NSS scores in the XF group, TQ group and BY group significantly decreased at 1 day after injury (6.6±1.5, 6.1±2.0, 5.7±2.4 vs. 9.4±1.5, all P < 0.05); however, the NSS scores in the BP group and TH group decreased significantly at 7 days after injury, and the NSS scores in the TQ group and BY group decreased more significantly than those in other drug groups. Compared with the model group, mRNA expressions of Wnt3a and β-catenin in the hippocampus of the BP group increased significantly at 1 day and 3 days after injury, respectively, and continued to increase with the extension of time. The mRNA expression levels of Wnt3a and β-catenin in the four groups of Huoxue Huayu decoction fluctuated to varying degrees from 1 day to 3 days after injury, but they were significantly higher than those in the model group at 7 days after injury, and the increase was more significant in the BY group [Wnt3a mRNA (2 -ΔΔCt): 154.7±4.1 vs. 17.4±1.0, β-catenin mRNA (2 -ΔΔCt): 17.05±0.45 vs. 2.74±0.13, both P < 0.05], and the second was the TQ group [Wnt3a mRNA (2 -ΔΔCt): 126.6±2.8 vs. 17.4±1.0, β-catenin mRNA (2 -ΔΔCt): 8.70±1.19 vs. 2.74±0.13, both P < 0.05]. Compared with the model group, the positive expressions of BDNF and NGF in the BP group increased significantly at 1 day after injury, but decreased after 3 days after peak. The positive expressions of BDNF and NGF in the four Huoxue Huayu decoction groups fluctuated to varying degrees from 1 day to 3 days after injury, but they were significantly higher than those in the model group at 7 days after injury, among which, the positive expressions of BDNF and NGF in the TQ group and BY group were significantly higher than those in the model group at 1 day after injury [BDNF positive cells (cells/MP): 56.4±6.2, 61.6±7.0 vs. 37.4±2.0, NGF positive cells (cells/MP): 58.4±5.0, 62.4±4.4 vs. 53.4±3.6, all P < 0.05], the increase amplitude at 7 days after injury was more significant than those in the other groups. Conclusions:Taohong Siwu decoction, Xuefu Zhuyu decoction, Tongqiao Huoxue decoction and Buyang Huanwu decoction have curative effect on the nerve regeneration and repair of rats with sTBI at acute stage, but the intensity of the effect is different. Buyang Huanwu decoction and Tongqiao Huoxue decoction have a fast and better effect.
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Objective To observe the effects of diammonium glycyrrhizinate (DG) on nerve regeneration repair in rats with severe traumatic brain injury (STBI) from the perspective of Wnt/β-catenin signaling pathway. Methods Seventy-two Sprague-Dawle (SD) male rats were randomly divided into normal group, STBI model group, ganglioside (GA) treatment group and DG treatment group. The STBI animal model was reproduced referring to modified Feeney free fall impact model. No injury was made in normal group. Six hours after modeling, monosialotetrahexosylganglioside sodium injection and DG injection were injected via tail vein of rats in GA treatment group and DG treatment group respectively, once a day for 7 days. Normal group and STBI model group were given the same amount of normal saline. Six rats in each group were sacrificed on the 1st, 3rd and 7th day after the challenge for neurological severity score (NSS), and then the blood of abdominal aorta was drawn and brain tissue was harvested. The contents of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in serum were detected by enzyme linked immunosorbent assay (ELISA). The pathological changes of sub-granular zone (SGZ) were observed under light microscope after hematoxylin eosin (HE) staining. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of Wnt3a, β-catenin, glycogen synthetase kinase-3β (GSK-3β) and Axin. Results ① There was no neurological deficit in the normal group and NSS was 0. NSS score of rats increased significantly on the first day after modeling, and then decreased gradually over time. NSS of the rats treated with GA and DG were significantly lower than that of the STBI model rats (score: 7.33±2.07, 6.17±2.23 vs. 9.33±1.63, both P < 0.01). Though NSS gradually decreased over time, the differences were still statistically significant on the 7th day (score: 2.67±0.82, 1.00±0.00 vs. 6.17±2.23, both P < 0.01), and NSS of DG treatment group was significantly lower than that of GA treatment group. ② In SGZ of rats, cells were arranged in a compact and orderly way in the normal group, but neurons and tissues were damaged and destroyed at different time points in the STBI model group. After either GA or DG treatment, the damage of nerve tissue was improved gradually over time, and the effect of DG was more obvious.③ In the normal group, the mRNA expressions of Wnt3a and β-catenin were almost not expressed, the mRNA expressions of GSK-3β and Axin were higher, and the contents of BDNF and NGF in serum were less. On the 1st day after STBI, the mRNA expressions of Wnt3a and β-catenin in hippocampus, the contents of BDNF and NGF in serum were significantly increased, and the mRNA expressions of GSK-3βand Axin were significantly decreased. The mRNA expressions of Wnt3a and β-catenin in the hippocampus and the contents of BDNF and NGF in serum were significantly higher than those in the model group 1 day after GA or DG was added, the mRNA expressions of GSK-3β and Axin were significantly decreased, and the effect of DG was more significant than that of GA [Wnt3a mRNA (2-ΔΔCt):3.51±0.14 vs. 2.93±0.05, β-catenin mRNA (2-ΔΔCt): 1.90±0.08 vs. 1.75±0.04, BDNF (ng/L): 4.06±0.55 vs. 3.16±0.64, NGF (ng/L): 9.53±1.08 vs. 7.26±0.43, GSK-3βmRNA (2-ΔΔCt): 0.75±0.01 vs. 0.79±0.01, Axin mRNA (2-ΔΔCt): 0.74±0.02 vs. 0.76±0.02, all P < 0.05]. It was gradually increasing or decreasing over time and the difference was still statistically significant up to the 7th day. Conclusion DG can promote the recovery of nerve function in rats with STBI, and its mechanism may be related to the regeneration of nerve cells proliferation and differentiation by Wnt/β-catenin signaling pathway and the reconstruction of nerve tissue in SGZ of hippocampus.
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Objective@#To observe the effects of diammonium glycyrrhizinate (DG) on nerve regeneration repair in rats with severe traumatic brain injury (STBI) from the perspective of Wnt/β-catenin signaling pathway.@*Methods@#Seventy-two Sprague-Dawle (SD) male rats were randomly divided into normal group, STBI model group, ganglioside (GA) treatment group and DG treatment group. The STBI animal model was reproduced referring to modified Feeney free fall impact model. No injury was made in normal group. Six hours after modeling, monosialotetrahexosylganglioside sodium injection and DG injection were injected via tail vein of rats in GA treatment group and DG treatment group respectively, once a day for 7 days. Normal group and STBI model group were given the same amount of normal saline. Six rats in each group were sacrificed on the 1st, 3rd and 7th day after the challenge for neurological severity score (NSS), and then the blood of abdominal aorta was drawn and brain tissue was harvested. The contents of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in serum were detected by enzyme linked immunosorbent assay (ELISA). The pathological changes of sub-granular zone (SGZ) were observed under light microscope after hematoxylin eosin (HE) staining. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of Wnt3a, β-catenin, glycogen synthetase kinase-3β (GSK-3β) and Axin.@*Results@#① There was no neurological deficit in the normal group and NSS was 0. NSS score of rats increased significantly on the first day after modeling, and then decreased gradually over time. NSS of the rats treated with GA and DG were significantly lower than that of the STBI model rats (score: 7.33±2.07, 6.17±2.23 vs. 9.33±1.63, both P < 0.01). Though NSS gradually decreased over time, the differences were still statistically significant on the 7th day (score: 2.67±0.82, 1.00±0.00 vs. 6.17±2.23, both P < 0.01), and NSS of DG treatment group was significantly lower than that of GA treatment group. ② In SGZ of rats, cells were arranged in a compact and orderly way in the normal group, but neurons and tissues were damaged and destroyed at different time points in the STBI model group. After either GA or DG treatment, the damage of nerve tissue was improved gradually over time, and the effect of DG was more obvious.③ In the normal group, the mRNA expressions of Wnt3a and β- catenin were almost not expressed, the mRNA expressions of GSK-3β and Axin were higher, and the contents of BDNF and NGF in serum were less. On the 1st day after STBI, the mRNA expressions of Wnt3a and β- catenin in hippocampus, the contents of BDNF and NGF in serum were significantly increased, and the mRNA expressions of GSK-3βand Axin were significantly decreased. The mRNA expressions of Wnt3a and β- catenin in the hippocampus and the contents of BDNF and NGF in serum were significantly higher than those in the model group 1 day after GA or DG was added, the mRNA expressions of GSK-3β and Axin were significantly decreased, and the effect of DG was more significant than that of GA [Wnt3a mRNA (2-ΔΔCt): 3.51±0.14 vs. 2.93±0.05, β- catenin mRNA (2-ΔΔCt): 1.90±0.08 vs. 1.75±0.04, BDNF (ng/L): 4.06±0.55 vs. 3.16±0.64, NGF (ng/L): 9.53±1.08 vs. 7.26±0.43, GSK-3β mRNA (2-ΔΔCt): 0.75±0.01 vs. 0.79±0.01, Axin mRNA (2-ΔΔCt): 0.74±0.02 vs. 0.76±0.02, all P < 0.05]. It was gradually increasing or decreasing over time and the difference was still statistically significant up to the 7th day.@*Conclusion@#DG can promote the recovery of nerve function in rats with STBI, and its mechanism may be related to the regeneration of nerve cells proliferation and differentiation by Wnt/β-catenin signaling pathway and the reconstruction of nerve tissue in SGZ of hippocampus.
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Objective To investigate the effect of exogenous thyroxine on the regeneration and repair of central nervous system in rats with severe traumatic brain injury.Methods A total of 108 male SD rats were randomly divided into Group A (normal group),Group B (thyroxine group),Group C (TBI group),Group D [TBI + low dose of thyroxine (6.5 μg/kg) group],Group E [TBI + medium dose of thyroxine (10 μg/kg) group)] and Group F [TBI +high dose of thyroxine (15.4 μg/kg)] by random number table method,with 18 rats in each group.The animal model was established according to the Feeney method.Groups A and C were given saline and thyroxine 6 hours after TBI.At 1 day,3 days and 7 days after injury,six rats were selected from each group according to random number table method for brain tissue harvest.The pathological changes of brain tissues were observed,and the expressions of Wnt3a and β-catenin,brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were detected by RT-PCR and immunohistochemistry respectively.Results (1) There were no pathological changes in brain tissue in Groups A and B.The pathological changes in brain tissue of TBI rats in Groups D,E and F were significantly mitigated than those in Group C,and the effect of Groups E and F were more obvious.(2) RT-PCR showed that the expressions of Wnt3a and β-catenin mRNA in hippocampus of Group D at 7 days after injury were significantly higher [(6.46±2.02) vs.(4.08 ±1.06);(13.53 ±1.64) vs.(2.11 ±0.63);P＜0.05);Wnt3a mRNA expression of Groups E and F were increased significantly at 3 days and 7 days after injury [(5.45 ± 1.54) vs.(2.78±1.04),(8.59±1.88) vs.(4.08 ±1.06),(5.40±1.38) vs.(2.78±1.04),(8.63±1.74) vs.(4.08± 1.06);P ＜ 0.05)].The expressions of β-catenin mRNA were increased significantly at 1 day,3 days,and 7 days after injury [(3.10 ± 0.59) vs.(1.45±0.28),(5.28±0.62) vs.(1.45±0.28),(4.12±0.43) vs.(1.64± 0.31),(8.78±0.92) vs.(1.64±0.31),(23.80±2.38)vs.(2.11±0.63),(25.94±3.79) vs.(2.11 ± 0.63);P ＜ 0.05)].The expressions of Wnt3a and β-catenin in hippocampus of Group B did not change significantly.(3) Immunohistochemical staining showed that the expression of BDNF and NGF in hippocampus of B did not change significantly after thyroxine treatment,but that of BDNF and NGF in Group D did not change significantly after treatment.The expressions of BDNF and NGF of Groups E and F were significandy higher than that in Group C and reached the peak at 3 days after injury [BDNF:(77.3 ± 5.4) vs.(55.0 ± 5.6),(78.0 ± 7.8) vs.(55.0±5.6);NGF:(83.7±5.8) vs.(62.2±10.3),(86.8±4.8) vs.(62.2 ±10.3);P＜0.05].Conclusions Exogenous thyroxine has no effect on the Wnt/β-catenin pathway in normal rats.But exogenous thyroxine can significantly promote the centralis regeneration and repair in TBI rats,which might be associated with its role in upregnlating the expression of mRNA and increasing the secretion of BDNF and NGF.