Oxymatrine inhibits cell migration and invasion in non-small-cell lung cancer by down-regulating Toll-like receptor 4 / 中国病理生理杂志

AIM:To investigate the therapeutic effect of oxymatrine on non-small-cell lung cancer(NSCLC) A549 cells and a xenograft mouse model,and to explore the underlying molecular mechanisms. METHODS:The effect of oxymatrine on the A549 cell viability was assessed by CCK-8 assay. After the A549 cells were treated with Toll-like re?ceptor 4(TLR4)stimulator lipopolysaccharide(LPS)and oxymatrine(5,10 and 15 mmol/L),the mRNA and protein ex?pression levels of TLR4 and myeloid differentiation factor 88(MyD88)were analyzed by RT-qPCR and Western blot,re?spectively. The migration and invasion abilities of the cells were measured by Transwell assay,and the mRNA and protein expression levels of matrix metalloproteinases-2(MMP-2),MMP-9 and vascular endothelial growth factor(VEGF)were also determined. A xenograft model in nude mice was utilized to evaluate the effect of oxymatrine on tumor growth. RE?SULTS:Oxymatrine inhibited the viability of A549 cells,decreased LPS-induced expression of TLR4,MyD88,MMP-2, MMP-9 and VEGF in A549 cells,and suppressed LPS-increased migration and invasion abilities of A549 cells. In the xe?nograft model,oxymatrine both reduced tumor growth and inhibited TLR4 expression in the tumor. CONCLUSION:Oxy?matrine exerts anti-tumor properties in NSCLC in vitro and in vivo by down-regulating the TLR4/MyD88 signaling pathway, suggesting that oxymatrine can be a potential therapeutic agent for NSCLC.

Síndrome de disfunção de múltiplos órgãos induzida por sepse: estudo experimental em ratos / Sepsis inducing syndrome of multiple organ dysfunction: an experimental study in rats

RACIONAL: A principal causa de morte em pacientes com sepse em cirurgia é a síndrome de disfunção de múltiplos órgãos. Assim, modelos experimentais que simulem alterações orgânicas da sepse em humanos são necessários. OBJETIVO: Apresentar dois modelos que induzem a síndrome de disfunção de múltiplos órgãos e comparar as alterações induzidas por inoculação endovenosa de 36UE de lipopolissacarídeo ou célula viável de Escherichia coli, em relação à: mortalidade e sobrevivência; nível de lipopolissacarídeo; liberação de fator de necrose tumoral alfa; alterações hematológicas e das funções hepática e renal. MÉTODO: Este estudo teve duração de sete dias e utilizou-se nele 50 ratos Wistar machos, divididos em três grupos: controle, lipopolissacarídeo e Escherichia coli. Os grupos experimento eram inoculados e separados em dois subgrupos, com inoculação a cada 24 ou 48 horas. No sétimo dia eram procedidas coletas de sangue e análise histopatológica de fígado, rins e pulmões. RESULTADOS: Houve sobrevivência de dez animais no grupo controle; zero no bacteriano de 24 horas e seis no de 48 horas; dez no lipopolissacarídeo de 24 horas e seis no de 48 horas. Nos grupos experimentais, os níveis de lipopolissacarídeo, fator de necrose tumoral alfa, leucócitos, plaquetas, bastonetes e as alterações renais e hepáticas foram superiores ao grupo controle. Houve alterações histopatológicas no grupo bacteriano. CONCLUSÃO: Os dois modelos de sepse induziram síndrome de disfunção de múltiplos órgãos, contudo a administração de 36UE de endotoxina a cada 48 horas pode ser utilizada com vantagens sobre os demais por não induzir morte em número significativo durante o período de sete dias.

BACKGROUND: The leading cause of death in patients with sepsis in surgery is syndrome of multiple organ dysfunction. Thus, experimental models that simulate organic changes of sepsis in humans are required. AIM: To present two models that induce the syndrome of multiple organ dysfunction and to compare, the changes induced, by intravenous injection of lipopolysaccharide or cell 36UE of viable Escherichia coli in relation to mortality and survival, level of lipopolysaccharide, release of tumor necrosis factor alpha ; hematological, liver and kidney function. METHOD: The study lasted seven days and it was used on it 50 male Wistar rats divided into three groups: control, lipopolysaccharide and Escherichia coli. The experimental groups were inoculated and divided into two subgroups, with inocuation with 24 or 48 hours. On the seventh day were proceeded blood collection and histopathologic analysis of liver, kidneys and lungs. RESULTS: There was a survival of ten animals in the control group; zero in bacteria group of 24 hours and six in 48 hours; ten of lipopolysaccharide in 24 hours and six in 48 hours. In the experimental groups, levels of endotoxin, tumor necrosis factor alpha, leukocytes, platelets, renal and liver levels were higher than the control group. There were histopathological changes in the bacterial group. CONCLUSION: The two models of sepsis induced multiple organ dysfunction syndrome; yet the administration 36UE endotoxin every 48 hours could be utilized in advantage over the other for not induce death in significant numbers during the period of seven days.

Changes of cervical vagal efferent discharge induced by lipopolysaccaride in rats / 第三军医大学学报

Objective To investigate the changes of cervical vagal efferent discharge induced by lipopolysaccaride in rats. Methods Ten rats were randomly divided into two groups, receiving lipopolysaccaride (5 mg/kg) or normal saline through intravenous injection. Frequency of cervical vagal efferent discharge was recorded at 30 min, 1, 2 and 4 h after the injection. Results The frequency of cervical vagal efferent discharge was significantly increased after LPS injection (P

Interleukin-1β,Tumor Necrosis Factor-α and Lipopolysaccharide Induce Expression of Monocyte Chemoattractant Protein-1 in Calf Aortic Smooth Muscle Cells / 华中科技大学学报（医学）（英德文版）

To investigate whether interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) induce expression of monocyte chemoattractant protein-1 (MCP-1)mRNA and protein in calf aortic smooth muscle cells(SMCs), calf aortic SMCs were cultured by a substrate-attached explant method. The cultured SMCs were used between the third to the fifth passage. After the cells became confluent, the SMCs were exposed to 2 ng/ml IL- 1β, 20 ng/mlTNF-lα and 100 ng/ml LPS respectively, and the total RNA of SMCs which were incubated for 4h at 37℃ were extracted from the cells by using guanidinium isothiocyanate method. The expression of MCP-1 mRNA in SMCs was detected by using dot blotting analysis using a probe of γ-32p-end-labelled 35-mer oligonucleotide. After a 24-h incubation, the media conditioned by the cultured SMCs were collected. The MCP-1 protein content in the conditioned media was determined by using sandwich ELISA. The results were as follows: Dot blotting analysis showed that the cultured SMCs could express MCP-1 mRNA. After a 4-h exposure to IL-Iβ, TNF-α and LPS, the MCP-1 mRNA expression in SMCs was increased (3.6-fold, 2.3-fold and 1.6-fold, respectively).ELISA showed that the levels of MCP-1 protein in the conditioned media were also increased (2.9-fold, 1.7-fold and 1.1-fold, respectively ). The results suggest that calf aortic SMCs could express MCP-1 mRNA and protein. IL-1β and TNF-α can induce strong expression of MCP- 1mRNA and protein, and the former is more effective than the latter.

Interleukin-1β,Tumor Necrosis Factor-α and Lipopolysaccharide Induce Expression of Monocyte Chemoattractant Protein-1 in Calf Aortic Smooth Muscle Cells / 华中科技大学学报（医学）（英德文版）

To investigate whether interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) induce expression of monocyte chemoattractant protein-1 (MCP-1)mRNA and protein in calf aortic smooth muscle cells(SMCs), calf aortic SMCs were cultured by a substrate-attached explant method. The cultured SMCs were used between the third to the fifth passage. After the cells became confluent, the SMCs were exposed to 2 ng/ml IL- 1β, 20 ng/mlTNF-lα and 100 ng/ml LPS respectively, and the total RNA of SMCs which were incubated for 4h at 37℃ were extracted from the cells by using guanidinium isothiocyanate method. The expression of MCP-1 mRNA in SMCs was detected by using dot blotting analysis using a probe of γ-32p-end-labelled 35-mer oligonucleotide. After a 24-h incubation, the media conditioned by the cultured SMCs were collected. The MCP-1 protein content in the conditioned media was determined by using sandwich ELISA. The results were as follows: Dot blotting analysis showed that the cultured SMCs could express MCP-1 mRNA. After a 4-h exposure to IL-Iβ, TNF-α and LPS, the MCP-1 mRNA expression in SMCs was increased (3.6-fold, 2.3-fold and 1.6-fold, respectively).ELISA showed that the levels of MCP-1 protein in the conditioned media were also increased (2.9-fold, 1.7-fold and 1.1-fold, respectively ). The results suggest that calf aortic SMCs could express MCP-1 mRNA and protein. IL-1β and TNF-α can induce strong expression of MCP- 1mRNA and protein, and the former is more effective than the latter.

Bacterial Endotoxin, Lipopolysaccaride, and Induced Activation of Stress-Activated Protein Kinase and Generation of Nitric Oxide

BACKGROUND: Previously, it has been suggested that lipopolysaccaride (LPS) stimulates the activation of the transcriptional factor activator protein (AP-1) which is in part regulated by activation of the c-Jun N-terminal kinase (JNK) / stress-activated protein kinase (SAPK) in the murine macrophage cell line RAW 264.7. METHODS: Consistent with this notion, we find that treatment of LPS on RAW 264.7 cells induces the generation of nitric oxide (NO) and results in the activation of JNK and treated with NO donors and NO inhibitors. RESULTS: NO donors including sodium nitroprusside (1 mM), GSNO (0.2 mM), or SNAP (0.5 mM) treatment of the macrophage cell line markedly induces the activation of JNK. However NGMMA (2 mM), a competitive inhibitor of NO, does not inhibit the activation of JNK induced by LPS. SIN-1, NO, and superoxide donor induce an activation of JNK that is slightly decreased by treatment with sodium dismutase whereas the activation of JNK is significantly augmented by adding sodium dismutase with catalase. C2 ceramide suppresses the generation of NO induced by LPS, but significantly increases the activity of JNK in vivo. LPS can induce the activation of JNK at 30 min after stimulation in RAW 264.7 cells. Exposure to SNP does not affect the enzymatic activity of JNK, immunoprecipitates, JNK, and c-Jun N-terminal proteins. CONCLUSIONS: These data suggest that even though NO is one of the major activators of JNK induced by LPS, there is, at least, an NO-independent JNK activation, signaling a pathway for LPS. Also, there may be an undefined NO-sensitive JNK-regulator (s) in vivo.

The Effects of Intraventricular Injection of Lipopolysaccharide on the Periventricular Neural Tissue

Ventriculitis is one of the most serious complication of the ventriculoperitoneal shunt, which may cause intelligence deterioration in children. The purpose of this study is to investigate the mechanism of the neural damage in lipopolysacciride(LPS)-induced ventriculitis in rat. Ventriculitis was induced by intraventricular injection of 1mg/Kg LPS in rat. H & E and Tunel stains were done on the day 1, 2 and 14 to access the microscopic changes of the periventricular tissue and apoptosis, respectively. TNF-alpha and IL-1beta mRNA expressions were studied using RT-PCR. HRP was injected into the femoral vein and electron microscopic examinations were performed to access the BBB changes. Light microscopic examination one day after LPS injection revealed neutrophilic infiltration, which diminished on day 4, and disappeared on 14. Tunel stain revealed apoptosis on day 1 and 4. TNF-alpha and IL-1beta were expressed on day 1, and diminished progressively thereafter. HRP histochemical electron microscopic examination revealed accumulation of HRP reaction in the interstitial space around the brain parenchyma. These findings suggest the opening of the BBB and increased capillary permeability in the periventricular tissue in the LPS induced ventriculitis. This can possibly damage the periventricular neural tissue. TNF-alpha and IL-1beta seemed to play an important role in the opening of the BBB.