CX3CR1 deficiency aggravates brain white matter injury and affects expression of the CD36/15LO/NR4A1 signal.

OBJECTIVE: To study the effects of CX3CR1 on white matter injury, neurofunction, recognition, and expression of the CD36/15LO/NR4A1 signal in mice with traumatic brain injury (TBI). METHODS: CX3CR1GFP/GFP, CX3CR1GFP/+ and C57BL/6 male mice were randomly divided into 3 groups. We used a controlled cortical impact (CCI) to establish a TBI model and T2wt MRI to detect the TBI lesion. FA and DTI allowed for quantitative evaluation of the structural integrity of white matter tracts. Several behavior tests were used to investigate nerve function; a computer-based tracing system was used to trace and analyze dendrites and cell bodies of microglia and astrocytes in the peri-lesional brain areas. We also used RT-PCR and western blot to detect the effect of CX3CL1/CX3CR1 axis on CD36/15LO/NR4A1 signal. RESULTS: The fractional anisotropy (FA) at the corpus callosum area of brain was decreased at 3 days post TBI, the average lesion volume CX3CR1GFP/GFP group was increased, and the neurologic deficit scores of mice of Cx3Cr1GFP/+ and wild-type groups were significantly increased compared to Cx3Cr1GFP/GFP group mice. In the Corner turn test, TBI induced impairments in forelimb function that were more severe than Cx3Cr11GFP/+ and wild-type TBI mice. We operated the Y-maze at 3 days post-TBI and the NOR test at 28 days after TBI. There was a significant TBI effect induced in decreased percentage entries into the novel arm in Cx3Cr1GFP/+ and wild-type TBI mice, compared with Cx3Cr1GFP/GFP; Cx3Cr1GFP/+. Wild-type mice showed decreased exploration time in new objects compared with Cx3Cr1GFP/GFP. Those two behavior tests demonstrated that Cx3Cr1 knock-out increased the damage caused by TBI to memory. In the tail suspension and force swimming tests, there was no significant difference between those three groups. CD36 increased in Cx3Cr1GFP/GFP compared with the other three groups at 3 days after TBI. TBI inhibited the expression of NR4A1 at 3 d after damage. Cx3Cr1 deficiency can induce high expression of 15LO, this was unaffected by TBI. CONCLUSION: CX3CR1 deletion can enhance white matter injury. It increased the expression of CD36 and 15LO and increased expression of NR4A1. The lack of CX3CR1 can affect the recovery of nerve function.

Expression of Notch and Wnt/ß-catenin signaling pathway in acute phase severe brain injury rats and the effect of exogenous thyroxine on those pathways.

BACKGROUND: With the rapid development of economy, transportation and industry, the incidence of severe traumatic brain injury (sTBI) is rising rapidly, which is one of the main traumatic diseases threatening human life. It is very difficult for sTBI patients to regenerate and repair the central nervous and recover the brain function. Moreover, no effective neuroprotective drug has been found in the treatment of sTBI patients. Seeking drugs to promote nerve repair has become a hot and difficult problem. It is widely accepted that thyroxine is one of the essential hormones in the human body, which not only promotes the growth and development of the nervous system, but also plays an important role in maintaining adult brain function. There are many reports of modern research on thyroxine, mainly focusing on the changes of thyroid hormone levels and their effects on the prognosis after injury. Besides, most of them are observed in clinical cases. Currently, there are few dynamic experimental studies about observing whether thyroxine can promote the repair of central nervous system at different stages after sTBI. In our previous experiment, we found that Wnt/ß-catenin signaling pathway, whose functions are opposite to Notch signaling pathway, can be further activated by exogenous thyroxine in rats with sTBI. As a result, we are interested in the expression of Notch and Wnt/ß-catenin signaling pathway in acute phase sTBI rats and the effect of thyroxine on those pathways. OBJECTIVE: To investigate expression of Notch and Wnt/ß-catenin signaling pathway in acute phase severe brain injury rats and the effect of thyroxine on those pathways by observing dynamically Notch and Wnt/ß-catenin signaling pathway, NSS, GFAP, S100B, Bcl-2, Bax, etc. METHODS: 108 rats were randomly divided into Group A (normal control group), Group B (normal-thyroxine group), Group C (TBI group), Group D (TBI+ low-dose thyroxine group), Group E (TBI + moderate-dose thyroxine) and Group F (TBI + high-dose thyroxine) with 18 rats in each group. The animal model was established according to Feeney's free-fall method, and administered with thyroxine or physiological saline at 6 h after sTBI. Six rats in each group were randomly killed on the 1st, 3rd and 7th days after intragastric administration. The changes of brain pathology and NSS were observed. The level of Wnt3a, ß-catenin, Notch1 and Hes1 mRNA was detected by RT-PCR method, and the level of GFAP and S100B protein in serum was detected by ELISA. The expression of Bcl-2 and Bax was detected by immunohistochemistry. RESULTS: (1) There was no significant change in brain pathology and NSS in groups A and B, but the changes of brain pathology and NSS in group D, E and F were significantly less than those in group C, especially in groups E and F. (2) RT-PCR showed that there was no change in the expression of Wnt3a mRNA, ß-catenin mRNA, Notch1 and Hes1 mRNA in groups A and B. Compared with group C, the expression of Wnt3a mRNA and ß-catenin mRNA in group D increased significantly on the 7th day after sTBI, especially in groups E and F; expression of Notch1 and Hes1 mRNA in groups D, E and F increased gradually with time, especially in group F. (3) ELISA showed that Compared with group C, GFAP and S100B in group D did not change significantly at 3 time points, GFAP in groups E and F decreased gradually with time and reached the lowest value on the 7th day, and S100B in groups E and F decreased gradually with time, especially in group F. (4) Compared with group C, the expression of BCL-2 in brain tissue of groups D, E and F increased gradually with time, and peaked on the 7th day, and the increase of E and F was more obvious. The expression of Bax in brain tissue of group D, E and F decreased gradually with time. CONCLUSION: Exogenous thyroxine has no effect on Notch and Wnt/ß-catenin signaling pathway in normal rats. After TBI, exogenous thyroxine can activate Notch and Wnt/ß-catenin, and have a synergistic effect on the repair of central nervous system, which may be related to the up-regulation of Notch and Wnt/ß-catenin signaling pathway mRNA expression and the increase of BDNF and NGF, and resist apoptosis in the brain of sTBI rats.

[Effects of Xiao-Cheng-Qi decoction on brain edema and inflammatory factors 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-1ß (IL-1ß) in brain tissue were detected by Western blotting. Serum TNF-α and IL-1ß 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-1ß in brain tissue, and the contents of TNF-α, IL-1ß 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-1ß 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/ß-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-1ß (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-1ß in brain tissue of XCQD medium and high dose groups also significantly decreased compared with sTBI model group [TNF-α (TNF-α/ß-actin): 0.90±0.24, 0.79±0.35 vs. 1.17±0.15; IL-1ß (IL-1ß/ß-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-1ß 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-1ß.

[Effect of Huoxue Huayu decoction on Wnt/ß-catenin signal pathway expression in rats with severe traumatic brain injury].

OBJECTIVE: To observe the dynamic of neurological severity scores (NSS) and the expressions of Wnt/ß-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 ß-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 ß-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 ß-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 ß-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, ß-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, ß-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.

Effect of icariin on early ß-defensin-2 and T cell subsets in rats after tracheotomy. / ·ARTICLES··è®º 著·.

OBJECTIVES: To investigate the effect of icariin (ICA) on early ß-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 ß-defensin-2 mRNA in lung tissue was detected by RT-PCR. The content of ß-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 ß-defensin-2 mRNA and ß-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 (P<0.05). The content of ß-defensin-2 in peripheral blood of group B decreased gradually, and the content of ß-defensin-2 in 168 h was significantly lower than that in 24 h (P<0.05), but there was no significant difference between group B and group A (P>0.05). The level of CD3+ T cells in peripheral blood was significantly lower than that in the group A (P<0.05), but their was no significant difference in CD4+ and CD8+ T cells compared with group A (P>0.05). After ICA intervention in group C: lung tissue, alveolar lavage fluid, peripheral blood serum ß-defensin-2 content, and peripheral blood CD3+ and CD4+ T cell levels were gradually increased, significantly higher than those in the group B (P<0.05). CD8+ T cell level was significantly lower than that in the group A at 24 h (P<0.05), the CD4+/CD8+ ratio was significantly higher at 168 h than those in the group A or B (both P<0.01). CONCLUSIONS: ICA can improve the early lung immune function in rats with tracheotomy, which might be related to up-regulation of ß-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.

[Diammonium glycyrrhizinate promotes the regeneration and repair of central nervous system in rats with severe traumatic brain injury by Wnt/ß-catenin signaling pathway].

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/ß-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, ß-catenin, glycogen synthetase kinase-3ß (GSK-3ß) 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 ß-catenin were almost not expressed, the mRNA expressions of GSK-3ß 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 ß-catenin in hippocampus, the contents of BDNF and NGF in serum were significantly increased, and the mRNA expressions of GSK-3ß and Axin were significantly decreased. The mRNA expressions of Wnt3a and ß-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-3ß 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, ß-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-3ß 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. CONCLUSIONS: 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/ß-catenin signaling pathway and the reconstruction of nerve tissue in SGZ of hippocampus.

Characterization and determination of antioxidant components in the leaves of Camellia chrysantha (Hu) Tuyama based on composition-activity relationship approach.

Camellia chrysantha (Hu) Tuyama (CCT), an ornamental plant possessing antioxidant activity, has been infused as tea and drank for its health benefits. The antioxidant components in CCT, however, had not been clearly characterized. To quickly identify the antioxidant constituents of CCT, a composition-activity relationship strategy based on ultra high-pressure liquid chromatography coupled with linear ion trap hybrid orbitrap mass spectrometry and orthogonal partial least-squares method has been applied. As a result, 16 variables were found to make significant contributions to the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Six of them were identified as catechin (1), epicatechin (5), vitexin (8), isovitexin (10), quercetin-7-O-ß-D-glucopyranoside (12) and kaempferol (16). The strength of 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity was found to be 12 > 1 > 5 > 16 > 8 > 10 by validation test. Meanwhile, a liquid chromatography-electrospray ionization-mass spectrometry method was established for quantitative determination of six marker compounds in CCT samples from different preparations. The validation of the method, including linearity, sensitivity (limitation of detection and limitation of quantification), repeatability, precision, stability, and recoveries, was carried out and demonstrated to meet the requirements of quantitative analysis. This is the first report on the comprehensive characterization and determination of chemical constituents in CCT by ultra high-pressure liquid chromatography coupled with linear ion trap hybrid orbitrap mass spectrometry. The results indicate that the composition-activity relationship approach may be a useful method for the discovery of active constituents in natural plants and the quality control of medicinal herbs.

The effects of Rhizoma drynariae on interleukin-2 and T-lymphocyte levels in rats after severe head injury.

OBJECTIVE: To investigate the effects of Rhizoma drynariae on the levels of interleukin-2(IL-2) and T-lymphocyte subset in rats with severe head injury (SHI). METHODS: 72 Sprague Dawley (SD) rats were randomly divided into 3 groups: control group, model group, and R. drynariae group. The experimental group received intragastrical infusions of with aqueous R. drynariae extract four hours after SHI while the other groups were administered with equivalent volumes of physiological saline. Infusions were administered to the 3 groups once a day for 7 d. IL-2 and T-lymphocyte (CD3, CD4, CD8) levels were measured at 24, 72, and 168 h after initial infusion. RESULTS: The levels of IL-2 and CD4 T cells reduced obviously after 24 h in the model group (P<0.05), but recovered to the levels of the control group after 72 h, and remained elevated after 168 h. In the R. drynariae group, IL-2 and CD4 levels were did not decrease while the level of CD8 T cells was reduced significantly (P<0.05). CONCLUSIONS: R. drynariae can protect against immune dysfunction or improve immunity in rats with severe head injury (SHI).

[Effects of jianwei yuyang granule on inflammatory reaction and NF-kappaB expression in rat gastric mucosa of ulcer healing and recurrence].

OBJECTIVE: To observe the effects of Jianwei Yuyang granule (JWYY) on inflammatory reaction and NF-kappaB expression in rat gastric mucosa of ulcer healing and recurrence. METHOD: Gastric ulcer was induced in rat by acetic acid according Okeba's method with some modification and the recurrence model was induced by IL-1beta. Pathohistology of ulcer healing and recurrence was observed. Density of inflammatory cell infiltrating regenerative mucosa, NF-kappaB protein and mRNA expression were measured. RESULT: JWYY had effects on improving the quality of ulcer healing, reducing the rate of ulcer recurrence, decreasing the density of inflammatory cell infiltrating regenerative mucosa and suppressing the activation and expression quantity of NF-kappaB protein and mRNA. CONCLUSION: JWYY may promote the ulcer healing and prevent the recurrence of the gastric ulcer by suppressing the activation of NF-kappaB and the following inflammatory reaction.