Objective To study the effects of lipopolysaccharide(LPS) on the expression of toll like receptor 4 (TLR4), reactive oxygen species(ROS) and on the proliferation of cells as well as secretion of six proinflammmatory cytokines including TNF-α, IL-1, IL-6, IL-8, IL-10, IL-12 levels in SKOV3 cells. And to explore the mechanism of SKOV3 cells in regulation. Methods Cultured primary SKOV3 cells were stimulated with different concentrations of LPS (0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml and 20 μg/ml) for 4 h, the TLR4 expression in SKOV3 cells were examined by flow cytometry;1 μg/ml LPS stimulated SKOV3 for 4 h, 8 h, 12 h, 24 h respectively, the TLR4 expression and cell cycle in SKOV3, cell proliferation, ROS level as well as cells and TNF-α and IL-1, IL-6, IL-8, IL-10, IL-12 levels in the culture medium were assayed by flow cytometry, MTT, CBA assay respectively. Results LPS with different concentrations of LPS stimulation in-duced an increased TLR4 expression, however, the expression was reduced when LPS concentration up to 10 μg/ml. LPS stimulation for 4 h, 8 h induced an increased TLR4 expression and cell proliferation. Stimulated for 24 h, however, the TLR4 expression and cell growth were inhibited in S period. Meanwhile, LPS stimulation for 4 h, 8 h, 12 h, 24 h induced a higher ROS secretion in comparison with control group. LPS stimulation induced a stronger cytokine response in comparison with control group, as demonstrated by the production of TNF-α, IL-1, IL-6, IL-8 secretion in cultured SKOV3 cells, while IL-10 and IL-12 with low expression have no obvious difference in the all medium samples. Conclusion TLR4 expression, cell proliferation, ROS and proin-flammmatory cytokine secretion could be induced in SKOV3 through LPS stimulation. The study provide new ex-periment evidences for human ovarian cells SKOV3 immunity regulation and inflammation reaction to promote cells inhibition after LPS stimulation.

Objective To investigate the relationship between Helicobacter pylori (H. pylori) infection and concentration of reactive oxygen species (ROS) in AGS cells. Methods AGS cells were cultured with either Hp11638 (CagA~+ , VacA~+ ) extract or Hp11638 mutant (CagA~+ , VacA~-) extract for 48 hours, then the cells and supernatants were collected. The concentration of ROS in AGS cells was measured by flow cytometry. The eytochrome C reduction was detected by spectrophotometer at 550 nm. Results The ROS levels in the AGS cells were correlated with two H. pylori strains in a concentration- and time-dependent manner. The ROS levels in AGS cells treated with Hp11638 extract in different concentrations or times were correspondingly higher than those treated with Hp11638 mutant extract. Similar results were found in examination of cytochrome C reduction. Conclusion The elevation of ROS in AGS cells is related to effects of H. pylori proteins, and the VaeA protein involves in the process.

Objective To search for a biomarker from the serum of Schistosoma japonicum infected rabbits for early diagnosis of schistosomiasis. Methods The sera were obtained from different periods of the infected rabbits. The serum proteins were generated by WCX kit (Bruker Daltonics GmBH) and analyzed by the technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results In the mass range from 1 000 to 12 000 Da, sixty-three proteins were captured by WCX kit. ClinProTools software was used to find the differential expressed proteins. The result revealed 7 distinct proteins compared with normal serum. Among them,1 787 Da protein expression was increased (P

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 impact of acute Toxoplasma gondii infection on cerebral proteins and nerve growth in mice by 2D electrophoresis. Methods The cerebral proteins from C57BL/6J mice infected with Toxoplasma gondii and normal paired mice were extracted. The discrepant proteins were checked by 2D electrophoresis. Isoelectric focusing was determined as the first direction (immobilized pH gradient gel 3-10) ,SDS-PAGE as the second direction to execute 2D electrophoresis, and PDQuest 1.0 software was used to analyze 2D electrophoretogram. Results The protein spots in Toxoplasma gondii infected mice and normal paired mice were (132 ±10) and (170 ± 13) , respectively. After the analysis by PDQuest 1. 0 software, only 19 protein spots were found to express in infected mice and only 37 protein spots were found to express in normal paired mice. Additionally, the obvious quantitative changes in a part of proteins of the cerebrum in the both group occurred. Conclusion There are obvious changes in cerebral proteins from mice with acute Toxoplasma gondii infection, which provids useful clues for studying the cerebral proteins injury in acute Toxoplasma gondii infected mice and the new cure drug.

To investigate the effect of Toxoplasma gondii infection upon the expression of brain-derived neurotrophic factor (BDNF) mRNA and N-methyl-D-aspirate receptor (NMDA) subunits NR2A and NR2B,Wistar rats of 4 weeks old were randomly divided into 3 groups with 10 rats in each group in which 2 mL suspensions of T.gondii tachyzoits in the concentrations of 2×10~7/mL and 2×10~5/mL were injected intra-peritoneally to rats in group A and group B respectively, serving as the experimental groups, while 2 mL of sterile physiologic saline was injected intra-peritoneally in group C serving as the control group. Four weeks after injection, the expressions of BDNF mRNA and BDNF protein in the brain tissues were detected by in situ hybridization and immunohistochemical assay and the expressions of NR2A and NR2B immune activity in the hippocampal CA1,CA3 and DG were investigated by using computer-assisted image analysis system. Compared with the control group, the expression of BDNF protein in the hippocampus of the experimental groups was significantly enhanced [(64.27±23.18), (50.39±19.34) vs (44.68±22.74)/mm~2,P＜0.05]. In addition, the increased expressions of BDNF mRNA in the hippocampus of the experimental groups were also demonstrated [(0.13±0.02), (0.12±0.02) vs (0.09±0.01); P＜0.05]. In the expression of the NR2A protein, their expressions in group A and B of rats were significantly lower than that of group C in CA3 (P＜0.05),but there was no significant change in CA1 and DG. In the expression of NR2B protein, the expressions in group A and B were also lower than that of group C in CA1 and CA3, and had no significant change in DG. It is evident that the expressions of BDNF mRNA and BDNF protein in hippocampal tissues were significantly increased following chronic infection with T.gondii, supporting the hypothesis that BDNF may be involved in the intrinsic neuro-protective mechanism.

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 establish high performance liquid chromatography(HPLC)characteristic chromatograms of Xinqingduyin Granules(composed of Taraxaci Herba,Lonicerae Japonicae Flos,Chrysanthemi Indici Flos,etc.)and content determination of chicory acid and glycyrrhizic acid,and to optimize the preparation process of Xinqingduyin Granules.Methods Using the characteristic chromatograms of Xinqingduyin and the retention rate of chicory acid and glycyrrhizic acid as indexes,we carried out orthogonal experiment to optimize the extraction process of Xinqingduyin,and studied the concentration process.The molding process of Xinqingduyin Granules was conducted by screening the types and dosage of auxiliary materials,then three batches of pilot experiments were carried out.Results HPLC characteristic chromatograms of Xinqingshuyin Granules and the determination methods of chicory acid and glycyrrhizic acid were established.The optimal preparation technology was as follows:8 times amount of water was added,the drug was decocted for 3 times,with 1 hour per time.After the extract was concentrated under reduced pressure at 80℃,the appropriate amount of steviol glycoside and lactose was added into the extract and mixed.One-step granulation and packaging were adopted.The retention rates of chicoric acid and glycyrrhizic acid in the 3 batches of Xinqingduyin Granules,which were prepared on the pilot scale,were(54.56±1.63)%and(54.96±1.08)%,and the rate of finished product was(87.47±0.49)%,respectively.The quality is uniform,and the characteristic map of Xinqingduyin Granules showed high similarity with that of decoction prepared from the same batch of slices.Conclusion The optimized preparation technology is reasonable,feasible and reproducible.This preparation can be used to obtain the granule with similar materials of Xinqingduyin decoction.

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.

Excessive theta (θ) frequency oscillation and synchronization in the basal ganglia (BG) has been reported in elderly parkinsonian patients and animal models of levodopa (L-dopa)-induced dyskinesia (LID), particularly the θ oscillation recorded during periods when L-dopa is withdrawn (the off L-dopa state). To gain insight into processes underlying this activity, we explored the relationship between primary motor cortex (M1) oscillatory activity and BG output in LID. We recorded local field potentials in the substantia nigra pars reticulata (SNr) and M1 of awake, inattentive resting rats before and after L-dopa priming in Sham control, Parkinson disease model, and LID model groups. We found that chronic L-dopa increased θ synchronization and information flow between the SNr and M1 in off L-dopa state LID rats, with a SNr-to-M1 flow directionality. Compared with the on state, θ oscillational activity (θ synchronization and information flow) during the off state were more closely associated with abnormal involuntary movements. Our findings indicate that θ oscillation in M1 may be consequent to abnormal synchronous discharges in the BG and support the notion that M1 θ oscillation may participate in the induction of dyskinesia.