By study on the effect of anisodamine on lipopolysaccharide- induced expression of tissue factor(TF) in vascular endothelial cells(EC) ,the mechanism of anisodam ine antithrom bosis,as well as in the treatm ent of b...By study on the effect of anisodamine on lipopolysaccharide- induced expression of tissue factor(TF) in vascular endothelial cells(EC) ,the mechanism of anisodam ine antithrom bosis,as well as in the treatm ent of bacteraemic shock was investigated.Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion m ethod.TF activity was measured in the lysates of HUVEC by using a single step clotting assay.Specific m RNA expression was detected by Northern blotting.In order to evaluate a possible contribution of the nuclear factor (NF) -κB pathway on the effects observed,electrophoretic mobility shift assays(EMSA) were performed using nuclear extracts from HU VECs and NF- κB- binding oligonucleotides.The results showed that treatment of HUVEC with L PS resulted in a significant increase in TF activity.Anisodamine dose- dependently inhibited L PS- induced upregulation of TF.These effects was also confirm ed on the level of specific TF m RNA expression by Northern blotting.Furtherm ore,EMSA showed that anisodamine com pletely abolished L PS- induced NF-κB DNA binding activity in nuclear ex- tracts from HUVECs treated with L PS together with anisodamine.The results suggest thataniso- damine counteracts endothelial cell activation by inhibiting L PS- induced TF expression in these cells.Its interference with the NF- κB pathway might- at least in part- contribute to this effect. The ability of anisodamine to counteract L PS effect on endothelial cells m ight be one underlying m echanism explaining its antithrombosis and efficacy in the treatment of bacteraemic shock.展开更多
Purpose: The aim of the study is to reveal associations between NF-κB, HIF-1alpha, VEGF expres-sions, proteasome and calpain activities with tumor progression in patients with kidney cancers and to find molecular par...Purpose: The aim of the study is to reveal associations between NF-κB, HIF-1alpha, VEGF expres-sions, proteasome and calpain activities with tumor progression in patients with kidney cancers and to find molecular parameters, associated with the effective pazopanib therapy. 93 patients with clear cell kidney cancers are included in investigation. 26 patients with disseminated kidney cancer have the pazopanib therapy. Methods: Transcription factors, VEGF, VEGFR2 and p-m-TOR expression are measured by ELISA kits. Proteasome and calpain activity are determined using specific fluorogenic substrate. Results: It is found the increase of NF-κB, HIF-1 expression in cancer tissues followed the hematogenic metastasis development. Coefficient NF-κB р65/р50 and VEGF expression are increased in cancer tissues with single metastasis and are decreased in cancer tissues with multiple ones. It is observed in the low proteasome activity in metastatic cancer tissues. The partial cancer regression is revealed in 29.6% of patients treated with pazopanib, cancer stabilization—in 61.5% of patients and cancer progression—in 11.5% of patients. The increased level of transcription factors NF-κB, HIF-1, growth factor VEGF and high proteasome activity in cancer tissues before targeted therapy are associated with the effective treatment. It is obtained the significant decrease of investigated markers after pazopanib application in metastatic kidney cancer patients. Conclusion: Coefficient NF-κB р65/р50, VEGF expression and proteases activities are the potential prognostic molecular markers of hematogenic metastasis development in kidney cancers. NF-κB, HIF-1 and VEGF levels can be considered as additional molecular markers predicting the effective pazopanib therapy.展开更多
The role of progesterone in the Toll-like receptor 4 （TLR4）-MyD88-dependent signaling pathway in pre-eclampsia was studied. Peripheral blood mononuclear cells （PBMCs） from pre-eclampsia （PE） patients were subjec...The role of progesterone in the Toll-like receptor 4 （TLR4）-MyD88-dependent signaling pathway in pre-eclampsia was studied. Peripheral blood mononuclear cells （PBMCs） from pre-eclampsia （PE） patients were subjected to primary culture, and stimulated with different concentra- tions of progesterone （0, 10^-8, 10^-6, and 10^-4 mol/L）. The mRNA expression of TLR4, MyD88 and nu- clear factor-kappaB （NF-κB） was detected by using real-time PCR. The Ikappa-B protein expression was detected by using Western blotting. The expression of tumor necrosis factor-or （TNF-α and inter- leukin-6 （IL-6） in the supernatant was determined by using ELISA. With the concentrations of proges- terone increasing, the mRNA expression levels of TLR4, MyD88 and NF-κB in 2^△△CT value were sig- nificantly decreased, and the IkappaB protein expression levels were significantly increased. The TNF-α and IL-6 expression showed a downward trend when the progesterone concentration increased, and there were significant differences among all of the groups （P〈0.05）. It was suggested that progesterone can inhibit the TLR4-MyD88-dependent signaling pathway in PE significantly and benefit for the preg- nancy.展开更多
基金This projectwas supported byagrantfrom National Natu-ral Sciences Foundation of China (No. 39730 2 2 0 ) andWuhan Chenguang Program (No. 985 0 0 30 86)
文摘By study on the effect of anisodamine on lipopolysaccharide- induced expression of tissue factor(TF) in vascular endothelial cells(EC) ,the mechanism of anisodam ine antithrom bosis,as well as in the treatm ent of bacteraemic shock was investigated.Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion m ethod.TF activity was measured in the lysates of HUVEC by using a single step clotting assay.Specific m RNA expression was detected by Northern blotting.In order to evaluate a possible contribution of the nuclear factor (NF) -κB pathway on the effects observed,electrophoretic mobility shift assays(EMSA) were performed using nuclear extracts from HU VECs and NF- κB- binding oligonucleotides.The results showed that treatment of HUVEC with L PS resulted in a significant increase in TF activity.Anisodamine dose- dependently inhibited L PS- induced upregulation of TF.These effects was also confirm ed on the level of specific TF m RNA expression by Northern blotting.Furtherm ore,EMSA showed that anisodamine com pletely abolished L PS- induced NF-κB DNA binding activity in nuclear ex- tracts from HUVECs treated with L PS together with anisodamine.The results suggest thataniso- damine counteracts endothelial cell activation by inhibiting L PS- induced TF expression in these cells.Its interference with the NF- κB pathway might- at least in part- contribute to this effect. The ability of anisodamine to counteract L PS effect on endothelial cells m ight be one underlying m echanism explaining its antithrombosis and efficacy in the treatment of bacteraemic shock.
文摘Purpose: The aim of the study is to reveal associations between NF-κB, HIF-1alpha, VEGF expres-sions, proteasome and calpain activities with tumor progression in patients with kidney cancers and to find molecular parameters, associated with the effective pazopanib therapy. 93 patients with clear cell kidney cancers are included in investigation. 26 patients with disseminated kidney cancer have the pazopanib therapy. Methods: Transcription factors, VEGF, VEGFR2 and p-m-TOR expression are measured by ELISA kits. Proteasome and calpain activity are determined using specific fluorogenic substrate. Results: It is found the increase of NF-κB, HIF-1 expression in cancer tissues followed the hematogenic metastasis development. Coefficient NF-κB р65/р50 and VEGF expression are increased in cancer tissues with single metastasis and are decreased in cancer tissues with multiple ones. It is observed in the low proteasome activity in metastatic cancer tissues. The partial cancer regression is revealed in 29.6% of patients treated with pazopanib, cancer stabilization—in 61.5% of patients and cancer progression—in 11.5% of patients. The increased level of transcription factors NF-κB, HIF-1, growth factor VEGF and high proteasome activity in cancer tissues before targeted therapy are associated with the effective treatment. It is obtained the significant decrease of investigated markers after pazopanib application in metastatic kidney cancer patients. Conclusion: Coefficient NF-κB р65/р50, VEGF expression and proteases activities are the potential prognostic molecular markers of hematogenic metastasis development in kidney cancers. NF-κB, HIF-1 and VEGF levels can be considered as additional molecular markers predicting the effective pazopanib therapy.
文摘The role of progesterone in the Toll-like receptor 4 （TLR4）-MyD88-dependent signaling pathway in pre-eclampsia was studied. Peripheral blood mononuclear cells （PBMCs） from pre-eclampsia （PE） patients were subjected to primary culture, and stimulated with different concentra- tions of progesterone （0, 10^-8, 10^-6, and 10^-4 mol/L）. The mRNA expression of TLR4, MyD88 and nu- clear factor-kappaB （NF-κB） was detected by using real-time PCR. The Ikappa-B protein expression was detected by using Western blotting. The expression of tumor necrosis factor-or （TNF-α and inter- leukin-6 （IL-6） in the supernatant was determined by using ELISA. With the concentrations of proges- terone increasing, the mRNA expression levels of TLR4, MyD88 and NF-κB in 2^△△CT value were sig- nificantly decreased, and the IkappaB protein expression levels were significantly increased. The TNF-α and IL-6 expression showed a downward trend when the progesterone concentration increased, and there were significant differences among all of the groups （P〈0.05）. It was suggested that progesterone can inhibit the TLR4-MyD88-dependent signaling pathway in PE significantly and benefit for the preg- nancy.
