Objective To observe the expression of zinc finger protein A20(A20), NF-κB and related inflammatory factors before and after lipopolysaccharide (LPS) stimulates degeneration of rabbit intervertebral disc nucleus pulposus cells.Methods The normal and degenerative nucleus pulposus cells were isolated and cultured, then divided into normal group,degenerative group,LPS stimulation group and NF-κB inhibition group.HE staining observe the morphological changes of nucleus pulposus and annulus fibrosus,immunohistochemistry was used to detect the expression of A20,NF-κB/p65 and COL-Ⅱ.Real-time PCR was employed to analyze the expression of A20,IL-1β,TNF-α,NF-κB and COL-Ⅱ,Western blot was used to observe the A20 protein,p65 and COL-Ⅱexpression in the four groups, and TNF-α, IL-1β in cell supernatant was determined by ELISA.Results The number of nucleuspulposus cells significantly decreased, aggregation occured in the degenerative group.COL-Ⅱ was obvious lower and A20, p65 significantly higher than that in normal group by immunohistochemical staining.Compared with the normal group,A20,TNF-α,IL-1β,p65 expression was significantly increased and COL-Ⅱ decreased in the mRNA and protein levels in degenerative group.Above indexes changed more significant in LPS stimulation group than in degenerative group.The expression of A20, TNF-α, IL-1β, p65 in the NF-κB inhibitor group was lower than that in the LPS group, and the expression of type Ⅱ collagen increased(P<0.05).Conclusions Intervertebral disc inflammatory response is closely related to the development of intervertebral disc degeneration, A20 may play an important role.

In order to clarify material basis of effective parts of liangxue tongyu prescription, blood-heat and blood-stasis rat model induced by dry yeast was established. The changes of rectal temperature, blood viscosity and plasma viscosity were used to evaluate the cooling-blood and activating-blood effects of liangxue tongyu prescription and its parts. Compared with the model group, the extract from liangxue tongyu prescription, its volatile oil and n-butanol part could significantly reduce rectal temperature (P<0.01), and also reduce blood viscosity and plasma viscosity to various degrees (P<0.01 or P<0.05). So volatile oil and n-butanol part were primarily identified as effective parts of liangxue tongyu prescription. By using GC-MS with normalization method of area to analyze volatile oil of liangxue tongyu prescription, 70 compounds were identified, accounting for about 92.54%, mainly as β-asarone, paeonol, α-asarone and shyobunone. 42 compounds such as peony glycosides, tannins, and iridoid glycosides were identified by HPLC-MS techniques and standard comparison. The study determined the effective parts of liangxue tongyu prescription and clarified the chemical composition providing the foundation for further studies on material basis of liangxue tongyu prescription.
Acetophenones ; chemistry ; Animals ; Anisoles ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Gas Chromatography-Mass Spectrometry ; Oils, Volatile ; chemistry ; Rats ; Tannins ; chemistry

Objective To investigate the effect of CTLA4-Ig chimera protein on mice mortality, histopathological changes, viral fiters, expression of CTLA4 protein on infiltrated T lymphocyte and the balance of Thl/Th2 in mice myocarditis caused by coxsackie virus B3 (CVB3). Methods A total of 106 four to six week-old male BALB/c mice were used in the experiments, which were divided into CTLA4-Ig group (n = 16), CVB3 group (n=40), IgG group (n =40) and normal control group(n = 10) randomly. The mice in CVB3 group, IgG group and CTLA4-Ig group were inoculated intraperitoneally with 0. 15 ml CVB3 and the mice in norreal control group with 0. 15 ml Eagle. The mice in IgG group and CTLA4-1g group were inoculated with IgG (0. I mg/kg) and CTLA4-Ig(0. 1 mg/kg) at 6 h and 72 h post inoculation(p, i. ), respectively, The surplus mice in each group were sacrificed at day 7 p.i. Light microscope was used to quantify the inflammation. The expression of CVB3 mRNA in mycardium were semi-quantified by real-time quantitative polymerase chain reaction (RQ-PCR). The expression of CTLA4 protein were analyzed by immunohistochemistry. The levels of IL-2, IL-4 and 1FN-γ in serum were measured by ELISA. Results The mice mortality, histopathological score and CVB3 mRNA in CTLA4-Ig group were lower than that in CVB3 group ( P < 0.05, P < 0.01, P < 0. 05, respectively). The expression of CTLA4 was significantly increased in CTLA4-Ig therapy group (P < 0.05 ). The serum level of IFN-γ of mice in CVB3 group were significantly higher than that in normal control group( P < 0.01 ). The serum level of IL-4 of mice in CVB3 group were much lower than that in normal control group( P < 0.01 ). The serum level of IL-2 in CVB3 group had no statistical significance with that in normal control group ( P > 0.05 ). The serum level of IFN-γ in mice of CTLA4-Ig group were much lower than that in CVB3 group ( P <0.01 ) and lgG group (P < 0. 01 ). The serum level of IL-4 of mice in CTLA4-Ig group were significantly higher than that in CVB3 group (P<0.01) and IgG group (P<0.01). The serum level of IL-2 in CTLA4-Ig group had no statistical significance with that in CVB 3 control group and lgG group ( P > 0. 0 5 ) . Conclusion CTLA4-Ig may relieve inflammation and reduce mice mortality by blocking the costimulation signals for T lymphocyte activation and reinforcing Th2 response.

OBJECTIVETo explore the effects of stromal interaction molecule 1 (STIM1) on the expression of apoptosis-related proteins in prostate cancer PC-3 cells.

METHODSWe transfected the lentivirus vector STIM1-pGCSIL-GFP carrying STIM shRNA into human hormone-independent prostate cancer PC-3 cells, and 3 days later observed the transfection efficiency by fluorescence microscopy. At 7 days after transfection, we determined the expression of STIM1 in the PC-3 cells by RT-PCR and Western blot and those of apoptosis-related proteins Bcl-2, Bax, survivin and activated Caspase-3 by Western blot.

RESULTSAt 3 days, inverted microscopy revealed a transfection efficiency of > 80%. At 7 days, the STIM1 expression was significantly inhibited at both mRNA and protein levels. The Bcl-2/Bax rate was remarkably decreased as compared with that of the control group (0. 31 vs 1.24 ) , and the survivin expression was markedly reduced, 0. 14 times that of the relative expression in the control. However, the Caspase-3 cleavage was significantly activated, 1.52 times that of the control (P <0.05).

CONCLUSIONSTIM1 can be regarded as an oncogene in prostate cancer PC-3 cells. Inhibition of its expression can induce PC-3 cell apoptosis by reducing the Bcl-2/Bax rate, decreasing the survivin expression, and activating the Caspase-3 pathway.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Male ; Membrane Proteins ; genetics ; Neoplasm Proteins ; genetics ; Prostatic Neoplasms ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Small Interfering ; genetics ; Stromal Interaction Molecule 1 ; Transfection ; bcl-2-Associated X Protein ; metabolism

Aim To investigate the up-regulation of miR-22 through Wnt pathway inhibits the proliferation,migration and invasion in human gastric MGC803 cells induced by diallyl disulfide(DADS).Methods The effects of proliferation,migration,and invasion of gastric cancer cells were evaluated by MTT,wound-healing and invasion assays.Online prediction software was applied to search the target gene of miR-22.Luciferase report gene assay was used to assess the target genes Wnt-1 of miR-22.The expressions of Wnt-1,β-catenin and TCF-4 were tested by qRT-PCR and Western blot,respectively.Results MTT showed that DADS and miR-22 notably decreased the proliferation compared with control group(P<0.05).Wound-healing assay showed that DADS and miR-22 could significantly inhibit the migration of MGC803 cells compared with the control group, especially in miR-22+DADS(P<0.05). Invasion assay showed that DADS and miR-22 could markedly inhibit the invasion of MGC803 cells compared with the control group, especially in miR-22+DADS(P<0.05). Online prediction software to search the target gene exhibited that Wnt-1 may be a target gene of miR-22. Luciferase report gene assay disclosed that Wnt-1 was identified as a direct target of miR-22. Qrt-PCR showed that the expression of Wnt-1 Mrna was respectively down-regulated by DADS and miR-22 compared withcontrol group, especially in miR-22+DADS(P<0.05). Western blot exhibited that DADS and miR-22 obviously suppressed the expressions of Wnt-1, β-catenin and TCF-4 proteins, especially in miR-22+DADS(P<0.05).Conclusion Up-regulation of miR-22 through Wnt pathway can remarkably suppress the proliferation, migration and invasion in MGC803 cells by DADS.

Objective To investigate the effect of ketamine on depression-like behaviors at different developmental stages of offspring rat exposed to prenatal restraint stress (PRS).MethodsPregnant SD rats were randomly divided into control group (n=6) and PRS group (n=8).The dams of PRS group received three times(45 minutes/time)restraint stress every day.The anxiety-like and depression-like behaviors of the offsprings of the two groups were tested in the stage of juvenile,adolescence and early adulthood.Then the antidepressant effect of ketamine on prenatal stress rats at different developmental stages was observed.ResultsIn the open-field test,the time in the central area of the offspring rats in PRS group at different developmental stages (juvenile(2.50±0.43)s,adolescence(9.17±1.05)s,early adulthood(8.33±0.92)s) were significantly lower than those of the control group((8.33±1.05)s,(19.17±1.06)s,(18.83±1.30)s,all P<0.05).In the forced swimming test,the immobility time in the offspring rats of PRS group at the different developmental stages (juvenile(192.50±10.82)s,adolescence(182.75±10.12)s,early adulthood(199.88±9.20) s)were significantly higher than those of control group((76.00±19.00)s,(96.30±12.91)s,(108.30±10.98)s,all P<0.05).Ketamine could quickly and strongly reduce the immobility time of the offsprings exposed to PRS in the stage of adolescence and early adulthood (P<0.01),but the effect was weaker in the juvenile offsprings (P<0.05).ConclusionPRS leads to persistent anxiety-like and depression-like behavior in offsprings and ketamine exerts a good antidepressant effect on the offspring rats in the stage of adolescence and early adulthood.

Objective To find the relationship between nuclear factor kappB (NF-κB) activation and cell apoptosis.Methods Degenerative human nucleus pulposus cells were cultured in vitro.Useing CCK-8 to observe the proliferative effect of LPS on the nucleus pulposus cells in vitro, in the concentration of 100, 200, 500 and 1 000 μg/mL and choose the most apropriate concentration.The experiment was divided into blank control group, LPS(500 μg/mL)groups, and NF-κB inhibitor(BAY11-7082)plus LPS(500 μg/mL)group,Annexin V-FITC/PI flow cytometry and Hoechest33258 staining was used to analyze apoptosis.The expression of cleaved caspase-3,PARP,P65,P-P65 proteins were detected by Western blot respectively.Results When LPS was 500 μg/mL, the cell vitality was obviously declined.Compared with the blank control group, cell apoptosis rate of the LPS group is increasing (P<0.05), and the expression of P-P65,cleaved caspase-3, cleaved PARP were alsohigher (P< 0.05).Compared with the LPS group, cell apoptosis rate of the NF-κB inhibitor plus LPS group is significantly lower (P<0.05) and the expression of P-P65,cleaved caspase-3,cleaved PARP were also lower (P< 0.05).Conclusions NF-κB signaling pathway may be associated with the nucleus pulposus cell apoptosis in disc degeneration.

OBJECTIVETo investigate a possible mechanism responsible for anti-apoptotic effects of melatonin and provide theoretical evidences for clinical therapy.

METHODSIschemia-reperfusion mediated neuronal cell injury model was constructed in cerebellar granule neurons (CGNs) by deprivation of glucose, serum and oxygen in media. After ischemia, melatonin was added to the test groups to reach differential concentration during reperfusion. DNA fragmentation, mitochondrial transmembrane potential, mitochondrial cytochrome c release and caspase-3 activity were observed after subjecting cerebellar granule neurons to oxygen-glucose deprivation (OGD).

RESULTSThe results showed that OGD induced typical cell apoptosis change, DNA ladder and apoptosis-related alterations in mitochondrial functions including depression of mitochondrial transmembrane potential (its maximal protection ratio was 73.26%) and release of cytochrome c (its maximal inhibition ratio was 42.52%) and the subsequent activation of caspase-3 (its maximal protection ratio was 59.32%) in cytoplasm. Melatonin reduced DNA damage and inhibited release of mitochondrial cytochrome c and activation of caspase-3. Melatonin can strongly prevent the OGD-induced loss of the mitochondria membrane potential.

CONCLUSIONOur findings suggested that the direct inhibition of mitochondrial pathway might essentially contribute to its anti-apoptotic effects in neuronal ischemia-reperfusion.

Animals ; Apoptosis ; Blotting, Western ; Caspase 3 ; Caspases ; metabolism ; Cerebellum ; pathology ; Cytochromes c ; metabolism ; Cytoplasm ; metabolism ; DNA Fragmentation ; Glucose ; metabolism ; Immunoblotting ; Melatonin ; metabolism ; pharmacology ; Membrane Potentials ; Mitochondria ; metabolism ; Neurons ; metabolism ; Nitric Oxide Synthase Type I ; metabolism ; Oxygen ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion ; Reperfusion Injury ; Time Factors

OBJECTIVETo evaluate the efficacy and safety of Solitaire(TM) AB neurovascular stenting-assisted coil embolization for patients with wide-necked or dissecting aneurysms.

METHODSThe clinical results and prognosis from a consecutive series of 38 patients with 40 wide-necked or dissecting aneurysms aneurysms who treated by Solitaire(TM) AB neurovascular stenting-assisted coil embolization from August 2010 to January 2012 was retrospectively analyzed. There were 12 male and 26 female patients, the age was 21 - 78 years (mean 55 years). Thirty-one cases were confirmed wide-neck aneurysms and 9 cases were dissection aneurysms by DSA. Acute subarachnoed hemorrhage due to the rupture of aneurysms was seen in 28 cases (according Hunt-Hess scale, 1 case of Class I, 20 cases of Class II, 4 cases of Class III, 3 cases of Class IV), 1 case was traumatic intracranial aneurysm, 1 case was misdiagnosed during the operation of pituitary adenoma by the approach of transsphenoid, and unruptured aneurysms were seen in 8 cases. The aneurysms were located at the posterior communicating segment of internal carotid artery (21 cases), the supraclinoid segment of internal carotid artery (6 cases), the cavernous segment of internal carotid artery (3 cases), the anterior communicating artery (1 case), and the vertebral artery (9 cases). The patients were performed DSA and Glasgow outcome score (GOS) to evaluate the prognosis 6 months after surgery.

RESULTSForty stents were used and all remodeling device were achieved successful position. Owing to acute thrombosis in 3 patients, the stents were retrieved successfully. The proportion of patients in whom Raymond class 1 occlusion was obtained in 31 cases (77.5%), Raymond class 2 occlusion in 5 cases (12.5%) and Raymond class 3 occlusion in 4 cases (10.0%). The follow-up was 3 to 12 months (median 6 months). The results of DSA indicated none of the patients' anuerysm was recurred; and GOS was applied to evaluate the prognosis of patients after 3 months. Of 38 patients, 34 recovered well, 3 moderately disabled, 1 patient died.

CONCLUSIONSIt is safe to embolize aneurysms with Solitaire(TM) AB neurovascular stenting-assisted coil; meanwhile, the stents can be retrieved when acute thrombosis to reduce the complications.

Adult ; Aged ; Aneurysm, Dissecting ; therapy ; Cerebral Angiography ; Embolization, Therapeutic ; instrumentation ; Female ; Humans ; Intracranial Aneurysm ; therapy ; Male ; Middle Aged ; Retrospective Studies ; Stents ; Young Adult

UNLABELLEDTo screen and analyze the apoptosis- and proliferation-related genes in human nasopharyngeal carcinoma (NPC).

METHODSAccording to gene ontology classification, the abnormal expressions of the genes related to cell apoptosis and proliferation were identified in the NPC gene chip data. The cell apoptosis- and proliferation-related genes expressed in each of the 3 stages, as defined by the tree model for the pathogenesis and progression of NPC, were screened, and with literature review, their distribution in the tree model were analyzed.

RESULTSNineteen genes related to cell apoptosis were found in NPC, among which 9 were down-regulated (such as DNASE1L3) and located in the chromosome deletion regions, and 10 were up-regulated (such as DEDD) in the chromosome amplification regions. Twenty-one cell proliferation-related genes were identified, including 8 down-regulated genes (such as TUSC2) in the chromosome deletion regions and 13 up-regulated ones (such as EMP1) in the chromosome amplification regions. In the chromosome deletion regions, the down-regulated cell apoptosis-related genes participated mostly in inducing and regulating cell apoptosis, and the up-regulated cell proliferation-related genes in the chromosome amplification regions were mostly associated with the positive regulation of cell proliferation.

CONCLUSIONNPC occurs possibly through two pathways by inhibiting cell apoptosis or by promoting excessive cell proliferation.

Apoptosis ; genetics ; Cell Proliferation ; Chromosome Deletion ; Down-Regulation ; Gene Expression Profiling ; Humans ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Oligonucleotide Array Sequence Analysis ; Up-Regulation