Objective To evaluate the relationship between the mechanism of spinal monocyte chemoattractant protein-1 (MCP-1)-mediated maintenance of chronic pathological pain and synaptic transmission in spinal dorsal horns of rats.Methods Female Sprague-Dawley rats,aged 2-3 weeks after birth,weighing 150-210 g,were studied.The experiment was performed in 2 parts.Experiment Ⅰ Eighteen Sprague-Dawley rats were randomly divided into 2 groups (n =9 each) on 7 days after intrathecal catheters were inserted:phosphate buffer solution (PBS) group and MCP-1 group.PBS 10 μl was intrathecally injected in group PBS,and PBS 10 μ1 containing 100 ng MCP-1 was intrathecally injected in group MCP-1.The mechanical pain threshold was measured at 30 and 60 min before intrathecal injection,and 30,60,90,120,150 and 180 min and 1,2 and 3 days after intrathecal injection.Experiment Ⅱ The transverse spinal cord slices were prepared,and substantia gelatinosa neurons were selected for whole-cell patch-clamp recording.Electrophysiological recording was performed at 1 h of incubation with artificial cerebrospinal fluid (ACSF) and immediately after adding MCP-1:for excitatory synaptic transmission recording,MCP-1 (final concentration 100 nmol/L),N-methyl-D-aspartate (NMDA,final concentration 100 μmol/L) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA,final concentration 20 μmol/L) were added to ACSF,and spontaneous excitatory postsynaptic currents (sEPSCs),AMPA receptors-mediated currents and NMDA receptors-mediated currents were recorded;for inhibitory synaptic transmission recording,MCP-1 (final concentration 100 nmol/L) and γ-aminobutyric acid (GABA,final concentration 1 mmol/L) were added to ACSF,and spontaneous inhibitory postsynaptic currents (sIPSCs) and GABA receptors-mediated currents were recorded.Results Compared with group PBS,the mechanical pain threshold was significantly decreased at 30 min-2 days after intrathecal injection in group MCP-1 (P＜0.01).Compared with those at 1 h of incubation with ACSF,the frequency and amplitude of sEPSCs were significantly increased,the amplitude of NMDA receptors-and AMPA receptors-mediated currents were increased,the frequency and amplitude of sIPSCs were decreased,and the amplitude of GABA receptors-mediated currents was decreased immediately after adding MCP-1 (P＜0.05).Conclusion MCP-1 enhances excitatory synaptic transmission through enhancing the function of NMDA and AMPA receptors in the posterior substantia gelatinosa neurons of the spinal cord;MCP-1 weakens inhibitory synaptic transmission through inhibiting GABA receptor function,which may be involved in MCP-l-mediated maintenance of chronic pathological pain in rats.

Objective To study the effects of musk on the expressions of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) in the rat model of skull bone defect.Methods We constructed the bone defect model by dental drilling into the full skull of 300 SD rats (150 males and 150 females).The model animals were divided with completely random method into model group and drug group,with 150 in each.The two groups were further divided according to drug administration time into 7,14 and 28 d groups,respectively,with 50 in each.The drug group received perfusion of natural musk every day (4.2 mg/100 g) while the model group received perfusion of normal saline of the same volume every day.FGF-2 mRNA and EGF mRNA expressions in skull bone defect were determined using Real-time fluorescent quantitative PCR method.Results EGF mRNA expression at 7 d and 14 d was higher in the drug group than in the model group,but with no significant difference.EGF mRNA expression at 28 d decreased to the lowest level,with a significant difference (P＜0.05).FGF-2 mRNA expression in the drug group reached the highest at 7 d,with a significant difference (P＜0.05),and decreased at 14 and 28 d without significant difference.Conclusion Musk administered at different time points can effectively promote the healing rate of the bone defect area of the rat skull,and the mechanism of this repair is mainly related to the increased FGF-2 mRNA expression and the decreased EGF mRNA expression.

Objective To investigate the biological significance of differentially expressed proteins from human primary lung adenocarcinoma with lymph node metastasis adenocarcinoma (LNM AdC) and without metastasis (non-LNM AdC) according to clinical diagnosis of lymph node metastasis and distant metastasis,with bioinformatics approach.Methods Cytoscape software was used to analyze a functional enrichment analysis and a protein-protein interaction network from differentially expressed proteins from LNM AdC and non-LNM AdC.Results The top biological processes were related to glucose catabolic process,hexose catabolic process,monosaccharide catabolic process,alcohol catabolic process,and cellular carbohydrate catabolic process.The top molecular functions were related to phospholipase inhibitor activity,lipase inhibitor activity,calcium-dependent phospholipid binding,phosphlipase A2 inhibitor activity,and lipid binding.A protein-protein interaction network of differentially expressed proteins was generated with literature data.Conclusions This bioinformatics analysis demonstrated that glucose catabolic process,alcohol catabolic process,calcium-dependent phospholipid binding,phosphlipase A2 inhibitor activity,ACTB,ANXA1,ANXA2,ANXA3,VCP,NPM1,KRT1,and SUMO4 are significantly associated with a lung adenocarcinoma.These network data provide new insights into the metastasis mechanisms of human lung adenocarcinoma.

BACKGROUND:In recent years, in-depth studies that single Chinese herbs or extracts, compound traditional Chinese medicine and medicated serum are used to regulate the directional differentiation of bone marrow mesenchymal stem cels into myofibroblasts, chondrocytes, osteoblasts, myocardial cels and nerve cels, which have become a highlight in the tissue engineering research. OBJECTIVE:To review the latest progress in the directional differentiation of bone marrow mesenchymal stem cels induced by Chinese herbs or their extracts. METHODS:The first author searched the CNKI, Wanfang and PubMed databases using the keywords of “Chinese herb, directional differentiation, mesenchymal stem cels” in Chinese and English, respectively, to retrieve relevant articles published from January 2010 to January 2016. Repetitive articles or those with no originality were eliminated. Totaly 99 articles were searched initialy, and then 43 articles were included in result analysis. RESULTS AND CONCLUSION:As the strongest seed cels in the bone differentiation system, bone marrow mesenchymal stem cels have a wide range of directional differentiation potential, and highlight the important value in combination with Chinese herbs for clinical treatment of various refractory diseases, especialy for treatment of metabolic bone diseases, bone defects, nonunion and delayed union, which is not only conducive to in-depth, multi-angle studies on effects and mechanisms of Chinese herbs, but also to clinical treatment of various refractory diseases using bone marrow mesenchymal stem cels. Cite this article:Li N, Li YF, Xie XW, Song M, Xu SH, Li DP.Directional differentiation of bone marrow mesenchymal stem cel induced by traditional Chinese Medicine. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):135-139.

Objective To investigate the protective effect of rosiglitazone on the rats with high altitude pulmonary edema.Methods Thirty-six SD rats were randomly (random number) divided into 6 groups (n =6 each):control group (Control),hypobaric hypoxia model group (HH),rosiglitazone groups (RSG) which were administered with 3 different doses [RSG-L:5 mg/ (kg · d),RSG-M:10 mg/ (kg·d),RSG-H:20 mg/ (kg· d)],dexamethasone group [Dex,4 mg/ (kg· d)].Rats were injected intraperitoneally with different doses of rosiglitazone (RSG),dexamethasone (Dex) or vehicle (Control and HH) for 3 days before placed in simulated altitude of 6 000 m hypobaric hypoxia animal chamber where the temperature and pressure were constant.After 72 h in the chamber,each rat was anesthetized.The water content of lung was determined with wet/dry weight ratio.Bronchoalveolar lavage fluid was measured by bradford method.The contents of GSH was measured by micro-ezymed labeled method.The contents of MDA was measured by TBA method.The enzymatic activities of SOD was measured by WST-1 method.The changes of the TNF-α,IL-6 and IL-10 in serum were determined by ELISA.Light microscope was used to observe the pathological changes of lung tissue.Results Compared with Control group,the wet/dry weight ratio of lung (5.08 ± 0.24) and total protein content of BALF (351.06 ± 44.55) μg/mL increased significantly (P ＜ 0.01) in HH group.There were red blood cells in the alveolar and interstitium,pink fluid exudation in the alveolar,the alveolar septum enhancement,and a large number of inflammatory cell infiltration;the SOD activity (10.65 ± 0.94) U/mgprot and the content of GSH (1.63 ±0.20) μmol/gprot in lung tissue were significantly decreased (P ＜ 0.01),the contents of MDA (2.1 5 ± 0.18) nmol/mgprot increased significantly (P ＜ 0.01),TNF-o (56.92 ± 2.87) pg/mL and IL-6 (217.80 ±48.01) pg/mL levels in serum were significantly increased (P ＜0.01),and IL-10 (76.85 ± 16.72) pg/mL level decreased (P ＜ 0.05).Compared with the HH group,the wet/dry ratio of lung and total protein content of BALF in different doses of rosiglitazone group significantly decreased (P ＜ 0.01),the pathological changes of the lung tissue was significantly improved,SOD activity and the content of GSH in lung tissue was significantly increased (P ＜ 0.01),the content of MDA decreased (P ＜ 0.01),The levels of TNF-α and IL-6 in serum were significantly decreased (P ＜ 0.01),while the IL-10 level was significantly increased (P ＜ 0.01).Conclusion Rosiglitazone could protect the high altitude pulmonary edema by alleviating the oxidative stress and inflammatory response.

Objective To prepare the anti-TLR4 C-terminal domain monoclonal antibody and investigate its effect in treatment of sepsis.Methods TLR4 C-terminal polypeptide (amino acid sequence:368-579,named as TLR4-C) was obtained through prokaryotic expression and Sephacryl S-100 gel purification,and then was used to immunize female Balb/c mice (6-8 weeks old).After cell fusion,antibody screening and purification,monoclonal antibody specific for the C terminal of TLR4 was obtained.Specificity of monoclonal antibody was detected by Western blot and cell immunofluorescence.In vitro antibody activity test,NR8383 was cultured for 1 h with adding antibody (100 μg/ml) and then 12 h after adding lipopolysaccharide (LPS) (10 ng/ml),and level of tumor growth factor (TNF)-α in the culture medium was tested by ELISA.In vivo septic animal experiment,40 SD rats were assigned to control antibody group (n =20) and anti-TLR4 monoclonal antibody group (n =20) according to the random number table.Each group was rejected 50 mg/kg corresponding antibodies via caudal vein for 1 h,and then LPS (10 mg/kg) via intraperitoneal injection for 4 h.Blood samples from caudal vein of ten rats in each group were collect to test the serum level of TNF-α.The rest rats in each group were used to measure the animal survival rate within 72 h.Results Three highly specific anti-TLR4 monoclonal antibodies were obtained and could combined with TLR4-C and TLR4 holoprotein.In vitro cell activity study indicated only one monoclonal antibody could obviously inhibit the release of TNF-α.In vivo animal experiment showed serum TNF-α level in anti-TLR4-C antibody group was (1.54 ± 0.18) ng/ml,significantly lower than (0.51 ± 0.10) ng/ml in antibody control group (P ＜ 0.01).Animal survival rate in anti-TLR4-C antibody group was 70％,higher than 30％ in antibody control group (P ＜ 0.05).Conclusion Anti-TLR4-C monoclonal antibodies have great capacity to neutralize TLR4 and good protective effect on LPS-induced sepsis.

Objective To investigate the effect of high altitude hypoxia on chronic inflammation in rats.Methods Forty SD rats were randomly divided into 4 groups: control group (Con),chronic inflammation group (CI),high altitude hypoxia group (HH),high altitude hypoxia+chronic inflammation group (HH+CI).Rats in CI group were injected with lipopolysaccharide (LPS) (0.5 mg/kg) through the caudal vein twice a week for 4 weeks.Rats in HH+CI group were treated just as CI group was,but together with HH group rats were settled in a hypoxic environment of 6000 m altitude for three days.Pathological changes in lung tissues were observed by hematoxylin eosin stain.The peripheral white blood cell count and classification were measured.The levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in serum and lung tissues were detected by enzyme-linked immunosorbent assay (ELISA).Changes in IL-6 expression in rat lung tissues were observed by Western blotting.Results After LPS and high altitude hypoxia exposure,inflammatory cells infiltration and alveolar capillary expansion were observed in rats' lung tissue.Compared with Con group,not only the peripheral white blood cell count,but also the level of IL-6 and TNF-α in serum and lung tissue increased in CI and HH group(P<0.01).IL-6 expression levels observed by Western blotting were also increased in HH and CI group(P<0.01).High altitude hypoxia and chronic inflammation interacted(P<0.01).The peripheral white blood cell count was higher in HH+CI group than in other groups,and IL-6 and TNF-α expressions in lung tissue were increased(P<0.05).Conclusion An LPS-induced chronic inflammation model in rats is successfully obtained,and high altitude hypoxia could aggravate chronic inflammation.

Objective Toinvestigatetheexpressionof SIRT1anditsassociationwith clinicopathologic features in breast carcinoma with type 2 diabetes mellitus.MethodsThe expression of SIRT1 in 30 breast cancer with type 2 diabetes mellitus,65 samples of breast cancer without diabetes mellitus and 18 samples of corresponding normal breast tissues was investigated using immunohistochemistry.ResultsThe positive rate of SIRT1 in breast cancer tissues was significantly higher than that breast cancer with type 2 diabetes mellitus and normal breast tissue (P ＜0.05). In breast cancer with type 2 diabetes mellitus group.The positive rate of SIRT1 was significantly higher than that normal breast tissue(P ＜0.05).The expression of SIRT1 was positively correlated with the number of lymph node(P =0.011),pTNM tumor stage (P =0.028), p53 (P =0.003) and Her-2 (P =0.031) in breast cancer with type 2 diabetes mellitus group. The expression level of SIRT1 in lymph node-positive group was higher than that in lymph node-negative group (P ＜0.05).The expression level of SIRT1 was in lymph node-positive group was higher than that in lymph node-negative group(P ＜0.05).ConclusionSIRTI was up-regulated in breast cancer with type 2 diabetes mellitus,but its expression was lower than that breast cancer without diabetes mellitus,and was associated with the progression of diabetes mellitus. SIRT1 was positively correlated with lymph node, pTNM tumor stage,p53 and Her-2, SIRT1 may be a novel biological parameter to evaluate the malignant degree of breast carcinoma and to predict prognosis of breast cancer.

Objective To obtain the recombinant corticotropin releasing hormone (CRH) protein with soluble, high purity protein through optimizing prokaryotic expression condition and purifying glutathione thiol transferase (GST)-CRH protein. Methods To detect the expression of soluble CRH protein through grope of the host strain GST-CRH temperature of induction expression, the host strain concentration (OD600), IPTG concentration and induction time, the purification of GST-CRH was performed by GST-CRH agarose gel. Western Blot assay was used for the expression identification of the target protein. Results The optimal conditions for the induction of CRH protein were determined: temperature of 30 ℃, IPTG induced concentration 0.1 mmol/L, bacteria density (OD600) 0.8, the induction time of 8 hours, purified GST-CRH>95% fusion protein was obtained. Conclusion The optimal expression conditions of GST-CRH are obtained, and the soluble protein of high purity GST-CRH is also obtained.

Objective To investigate the mechanism of oxidative damage caused by lipopolysaccharide (LPS)induced sepsis in rat brain.Methods The rats were randomly divided into control group and model group (low LPS group and high LPS group).Twenty-four hours after the modeling,the rats were sacrificed before their brain tissue was taken out and prepared for the test.The changes in malondialdehyde (MDA),superoxide dismutase (SOD),glutathione peroxidase (GSH-Px),total antioxidant capacity (T-AOC),hydrogen peroxide (H2 O2 )and succinate dehydrogenase (SDH)were detected.The expression level of JNK and Nrf2 protein in brain tissue was detected by qRT-PCR and Western blotting. Results Compared with the control group,the MDA,SOD,GSH-px,T-AOC,H2O2 and SDH level increased significantly in the model group,and the difference in expressions of JNK and Nrf2 was statistically significant (P <0.05). Conclusion The LPS induced septic oxidative brain damage model in rats is successfully established,and the process may be regulated through the Nrf2 and JNK signal pathways.