BACKGROUND: Studies indicated that lipid peroxidation due to increase of free radical is the key factor of ischemia/reperfusion injury.Shinyleaf pricklyash root extracts, rutaceae plant, is bitter in taste, no stimulation, which has the effects of promoting qi, relieving pain, promoting blood circulation by removing blood stasis, dispelling wind and dredging collaterals and antioxidation.OBJECTIVE: To observe the influence of nitidine chloride on myocardial ischemia/reperfusion injury in rats and analyze its mechanism.DESIGN: Randomized controlled study.SETTING: Departmentof Pharmacology and Department of Chemistry,Guangxi Medical University.MATERIALS: A total of 60 healthy Wistar rats were selected, half male and half female, with the body mass of 250-300 g. Nitidine chloride was provided by Department of Chemistry, Guangxi Medical University, batch number 20050609. MS4000U biological signal quantitative record analysis system, 722N evident spectrophotometer, hydrochloric acid verapamil (batch number 020701, 2 mL in each), malondialdehyde (MDA) and superoxide dismutase (SOD) kit were purchased from Guangzhou Longfeida Technology Co., Ltd., Shanghai Precision Scientific Instruments Corporation, Shanghai Harvest Pharmaceutical Co, Ltd. and Nanjing Jiancheng Bioengineering Institute, respectively. Hitachi 7170A full automatic biochemistry analyzer was also applied.METHODS: The experiment was performed at the Department of Pharmacology and Department of Chemistry, Guangxi Medical University between June 2004 and May 2006. ①Totally 60 healthy Wistar rats with normal ECG (half male and half female) were randomly divided into 6 groups:sham operation group, model group, 2, 1, 0.5 mg/kg nitidine chloride groups, positive control group with 10 rats in each group. The rats in the sham operation group received threading without deligation, and 90 minutes later the experiment was accomplished. Other 50 rats received left anterior descending branch of coronary artery deligation, ischemia for 30 minutes reperfusion for 60 minutes. 2 mg/kg verapamil, 2,1,0.5 mg/kg, 5 mL/kgnitidine chloride, saline of the same volume were injected into femoral vein in rats of the positive control group, different doses nitidine chloride groups and model group, respectively 10 minutes before deligating left anterior descending branch of coronary artery. ②Monitoring was conducted successively with standard limb Ⅱ lead ECG when performing reperfusion. Type,incidence rate and duration of cardiac arrhythmia were recorded within 60minutes. Change of ST segment was also recorded after reperfusion for 15minutes and 60 minutes. ③At the end of experiment, serum myocardial enzymology indexes were measured wi th full automatic biochemistry analyzer.MDA content and SOD activity in myocardial tissues were examined with thiobarbituric acid(TBA) method and xanthine oxidase (XOD) method, respectively. ④Measurement data and enumeration data between two groups were compared with t test and x2 test. MAIN OUTCOME MEASURES: Occurrence of cardiac arrhythmia, ECG ST segment elevation, change of serum myocardial enzymology indexes, MDA content and SOD activity of myocardial tissues in rats of each group.RESULTS: A total of 60 rats were involved in the result analysis. ①Degree of cardiac arrhythmia and ECG ST segment elevation of rats: The emergency time of cardiac arrhythmia in 1 and 2 mg/kg nitidine chloride groups was significantly later than that in the model group (P ＜ 0.05,0.01). The duration of cardiac arrhythmia in the 1 and 2 mg/kg nitidine chloride groups and positive control group was obviously shorter than that in the model group (P ＜ 0.05-0.01). The incidence rates of various kinds of cardiac arrhythmia were markedly less than those in the model group (P ＜ 0.01). The degree of ST segment elevation at reperfusion for 15 and 60 minutes was remarkably lower than that in the model group (P ＜ 0.05-0.01). ②Serum myocardial enzyme level: It was significantly higher in the model than the sham operation group after 60-minute myocardial ischemia and reperfusion (P?.01). Activity of myocardial enzyme in the 1 and 2 mg/kg nitidine chloride groups was remarkably lower than that in the model group (P ＜ 0.01,P ＜ 0.05). The level of myocardial enzyme decreased with the increase of nitidine chloride. It was lower significantly in the positive control group than the model group (P ＜ 0.05-0.01 ). ③SOD activity of myocardial tissues: It was markedly lower in the model group than the sham operation group after 60-minute myocardialischemia and reperfusion (P ＜ 0.01); It was dramatically higher than in the 1 and 2 mg/kg nitidine chloride groups than the model group (P ＜ 0.01). The activity also increased with the increase of nitidine chloride. ④MDA content of myocardial tissues: It was distinctly higher in the model group than the sham operation group after myocardial ischemia reperftsion for 60 minutes (P ＜ 0.01). It was remarkably lower in the 1 and 2 mg/kg nitidine chloride groups than the model group (P ＜ 0.01). The content decreased with the increase of nitidine chloride. It was obviously lower in the positive control group than the model group (P ＜ 0.05 ).CONCLUSION: ①1 and 2 mg/kg nitidine chloride can reduce the incidence rate of cardiac arrhythmia in rats with myocardial ischemia and reperfusion, postpone the emergence time of cardiac arrhythmia and shorten its duration, decrease the degree of ST segment elevation after reperfusion for 15 minutes and 60 minutes, which have similar effect with verapamil.② 1 and 2 mg/kg nitidine chloride can reduce the release of myocardial enzyme, relieve the severity of oxygen-derived free radicals injury, and has the effect of protecting myocardial injury during ischemia-reperfusion, in which represents a dose-dependent effect.

Objective To explore the protective effect of thyroxin on severe traumatic brain injury of brain tissue by observing the effect of thyroxin on neuronal apoptosis,serum neuronal specific enolase(NSE),interleukin-6 (IL-6) and serum FT3 and FT4.Methods A total of 90 SD rats was randomly divided into control group,model group,low dosage of levothyroxine sodium tablets group,moderate dosage of levothyroxine sodium tablets group and high dosage of levothyroxine sodium tablets group,18 rats in each group.The animal model was reproduced by referring to Feeney's free fall impact modeling.Intragastric administration was performed at 6 h after injury.The levels of neuronal apoptosis and serum NSE,IL-6,FT3 and FT4 were detected by TUNEL method,ELISA method and radioimmunoassay at 24,72,168 h after intragastric administration.Results (1) After severe traumatic brain injury,the levels of serum FT3 and FT4 were under the normal and the level of FT4 was decreased to the lowest at 168 h.Thyroxine could increase the levels of FT3 and FT4.(2) Significant neuronal apoptosis was observed in rats with severe craniocerebral injury,and the apoptosis continued until 168 h.Moderate and high dose of thyroxine could improve neuronal apoptosis within 24 h,while low dose of thyroxine changed within 168 h.(3) The levels of serum NSE and IL-6 were increased significantly in rats after severe traumatic brain injury until 168 h,and they could be decreased by moderate and high dose of thyroxine within 72 h.Conclusion Exogenous thyroxine can protect brain tissue in rats with severe traumatic brain injury.

Objective:To observe the effects of the Chinese medicine prescription Xiao-Cheng-Qi decoction (XCQD) on acute brain edema and inflammatory factors in rats with severe traumatic brain injury (sTBI).Methods:A total of 108 male Sprague-Dawley (SD) rats were divided into control group, sham operation group, sTBI model group, and XCQD low, medium, high dose groups by random number table method, with 18 rats in each group. sTBI rat model was prepared according to the modified Freeney method. At 6 hours after injury, the XCQD low, medium, and high dose groups were given XCQD 1.80, 2.78, and 4.59 g/kg by gavage, respectively, and the other three groups were given the same amount of normal saline, once a day for 3 days. After 3 days of injury, rats in each group were sacrificed after the modified neurologic severity score (mNSS) assessed. Pathological changes of brain tissue were observed under light microscope after hematoxylin eosin (HE) staining, water content of brain tissue was measured by dry-wet specific gravity method, and the expressions of aquaporin 4 (AQP4), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in brain tissue were detected by Western blotting. Serum TNF-α and IL-1β levels were detected by enzyme linked immunosorbent assay (ELISA).Results:Compared with the normal group, the mNSS score of rats increased significantly, the structure of brain tissue was disordered, and pathological changes appeared such as inflammation, edema, pyknosis of nerve nuclei, water content, the protein expressions of AQP4, TNF-α and IL-1β in brain tissue, and the contents of TNF-α, IL-1β in serum were significantly increased. After XCQD intervention, the above indexes were significantly improved. Compared with sTBI model group, the mNSS score of XCQD medium and high dose groups significantly decreased (6.94±1.16, 6.88±1.02 vs. 8.61±1.09, both P < 0.05), and the pathological changes such as brain edema and inflammation were alleviated. Brain tissue water content, AQP4 protein expression and contents of serum TNF-α, IL-1β in XCQD low, medium, and high dose groups significantly decreased compared with sTBI model group [brain tissue water content: (78.25±0.71)%, (77.62±0.44)%, (76.70±0.74)% vs. (80.08±0.66)%; the expression of brain AQP4 protein (AQP4/β-actin): 0.86±0.13, 0.84±0.22, 0.65±0.13 vs. 1.08±0.14; serum TNF-α (ng/L): 106.34±15.07, 95.75±17.26, 89.00±17.36 vs. 141.96±29.47; serum IL-1β (ng/L): 90.41±12.88, 72.82±13.51, 71.32±16.79 vs. 128.57±22.56, respectively, all P < 0.05]. The protein expressions of TNF-α,IL-1β in brain tissue of XCQD medium and high dose groups also significantly decreased compared with sTBI model group [TNF-α (TNF-α/β-actin): 0.90±0.24, 0.79±0.35 vs. 1.17±0.15; IL-1β (IL-1β/β-actin): 0.91±0.21, 0.68±0.28 vs. 1.23±0.08, respectively, all P < 0.05]. Brain tissue water content, the expression of brain AQP4 protein, the levels of brain tissue and serum IL-1β in XCQD high dose group improved more significant than those of XCQD low dose group. Conclusions:XCQD can alleviate the acute brain edema in sTBI rats, and it is dose-dependent. The mechanism may be relevant to reduce the secondary inflammatory response of sTBI by inhibiting the expression of inflammatory factors TNF-α and IL-1β.

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.

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.

OBJECTIVES: To investigate the effect of icariin (ICA) on early β-defensin-2 and T cell subsets in rats after tracheotomy. METHODS: A total of 54 SPF male Sprague-Dawley rats were randomly divided into a normal control group (group A), a model group (group B), and a model+ICA treatment group (group C), with 18 rats in each group. A tracheotomy intubation model of the B and C group was prepared. After 6 h of surgery, ICA intervention was given to group C. Groups A and B were given the same amount of normal saline. Lung tissue, alveolar lavage fluid and peripheral blood were taken at 24 h, 72 h and 168 h, respectively. The expression of rat β-defensin-2 mRNA in lung tissue was detected by RT-PCR. The content of β-defensin-2 in alveolar lavage fluid and peripheral blood serum was detected by ELISA. The content of peripheral blood T cell subsets (CD3, CD4, CD8) was detected by flow cytometry, and the ratio of CD4/CD8 was calculated. RESULTS: After tracheotomy, the levels of β-defensin-2 mRNA and β-defensin-2 in lung tissue from the group B were increased significantly at 24 h, then they were decreased gradually, and decreased most significantly at 168 h (<0.05). The content of β-defensin-2 in peripheral blood of group B decreased gradually, and the content of β-defensin-2 in 168 h was significantly lower than that in 24 h (<0.05), but there was no significant difference between group B and group A (>0.05). The level of CD3 T cells in peripheral blood was significantly lower than that in the group A (<0.05), but their was no significant difference in CD4 and CD8 T cells compared with group A (>0.05). After ICA intervention in group C: lung tissue, alveolar lavage fluid, peripheral blood serum β-defensin-2 content, and peripheral blood CD3 and CD4 T cell levels were gradually increased, significantly higher than those in the group B (<0.05). CD8 T cell level was significantly lower than that in the group A at 24 h (<0.05), the CD4/CD8 ratio was significantly higher at 168 h than those in the group A or B (both <0.01). CONCLUSIONS ICA can improve the early lung immune function in rats with tracheotomy, which might be related to up-regulation of β-defensin-2 in lung tissue and alveolar lavage fluid, concomitant with increases in CD3 and CD4 T cells and CD4/CD8 ratio in peripheral blood while reduction in CD8 cells.
Animals ; Flavonoids ; Male ; Rats ; Rats, Sprague-Dawley ; T-Lymphocyte Subsets ; Tracheotomy ; beta-Defensins

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.