[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and lig...[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.展开更多
Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of nefer...Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition（IC50） being 26.15 μmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 μmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.展开更多
Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of...Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I,the small product fragment,from pcDNA3-L539fs/47,was subcloned into the big fragment of pEGFP-C2-HERG under T4 ligase.pEGFP-C2-L539fs/47 was identified by agarose gel electrophoresis and sequencing.pcDNA3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into HEK293 cells by Lipofect,respectively.The expression of fusion protein in HEK293 cells was detected through immunofluorescence,laser confocal imaging scanning in vivo,Western blot and PCR.Results:Mutation region cDNA fragment(about 1 kb) and target vector fragment(about 7.2 kb) were ligated after purification and gel recovery.Agarose gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47,constructed approximately 8.2 kb,sequencing consistent with template gene.The transfection efficiency of recombinant plasmid by fluorescence microscopy was more than60%.Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD,the expression of pEGFP-C2 fusion protein size of approximately 90 KD.The L539fs/47 gene expression in HEK293 cells was significant by PCR analysis.Confocal laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of HEK293 cells.Conclusion:pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in HEK293 cells,which laid a foundation for the further study on L539fs/47.展开更多
This study investigated the conjoined cellular oxidative damage of human embryo kidney 293T（HEK293T） cells induced by cadmium chloride（CdCl2） and nanometer titanium dioxide（nano-TiO2）.RT-PCR technique was used t...This study investigated the conjoined cellular oxidative damage of human embryo kidney 293T（HEK293T） cells induced by cadmium chloride（CdCl2） and nanometer titanium dioxide（nano-TiO2）.RT-PCR technique was used to detect the expressions of Heme oxygenase-1（HO-1） and 8-oxoguanine DNA glycosylase（OGG1）.The activities of superoxide dismutase（SOD） and catalase enzyme（CAT） and concentrations of reactive oxygen species（ROS） and maldondialdehyde（MDA） were measured by different approaches.The results showed that CdCl2 and nano-TiO2 at a low concen-tration of 0.75 total toxic unit（TU） exerted an additive effects on HO-1 gene expression,CAT activities and MDA concentrations.When the total TU was increased to 1 or 1.25 TU,the interaction was syner-getic.Moreover,the mixture with high proportion of CdCl2 produced an additive effect on the OGG1 gene expression,and the interaction was changed to be synergetic when the concentration of CdCl2 was lower than or equal to that of nano-TiO2.Synergetic effects of CdCl2 and nano-TiO2 on cellular oxida-tive damage of HEK293T cells were found as indicated by the changes in the SOD activities and ROS concentrations.It was concluded that CdCl2 and nano-TiO2 exerts synergistic effects on the cellular oxidative damage of HEK293T cells,and the sensitivity of these indicators of oxidative damage varies with the proportion of CdCl2 and nano-TiO2 in the mixture.展开更多
Hydrogen peroxide（H_2O_2） and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene（C_（60）） is critical fo...Hydrogen peroxide（H_2O_2） and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene（C_（60）） is critical for scavenging oxygen free radicals originated from cell metabolism, and reduced glutathione（GSH） is another important endogenous antioxidant. In this study, a novel water-soluble reduced glutathione fullerene derivative（C_（60）-GSH） was successfully synthesized, and its beneficial roles in protecting against H_2O_2-induced oxidative stress and apoptosis in cultured HEK 293 T cells were investigated. Fourier Transform infrared spectroscopy and 1H nuclear magnetic resonance were used to confirm the chemical structure of C_（60）-GSH. Our results demonstrated that C_（60）-GSH prevented the reactive oxygen species（ROS）-mediated cell damage. Additionally, C_（60）-GSH pretreatment significantly attenuated H_2O_2-induced superoxide dismutase（SOD） consumption and malondialdehyde（MDA） elevation. Furthermore, C_（60）-GSH inhibited intracellular calcium mobilization, and subsequent cell apoptosis via bcl-2/bax-caspase-3 signaling pathway induced by H_2O_2 stimulation in HEK 293 T cells. Importantly, these protective effects of C_（60）-GSH were superior to those of GSH. In conclusion, these results suggested that C_（60）-GSH has potential to protect against H_2O_2-induced cell apoptosis by scavenging free radicals and maintaining intracellular calcium homeostasis without evident toxicity.展开更多
Objective:To investigate the role of water-soluble extract of Salvia fruticosa(Creek sage)(S.fruticosa) leaves in reducing both intrinsic cellular and H_2O_2-induced DNA oxidation in cultured human embryonic kidney 29...Objective:To investigate the role of water-soluble extract of Salvia fruticosa(Creek sage)(S.fruticosa) leaves in reducing both intrinsic cellular and H_2O_2-induced DNA oxidation in cultured human embryonic kidney 293 cells.S.fruiicosa.native to the Eastern-Mediterranean basin,is widely used as a medicinal herb for treatment of various diseases.Methods:Dried leaves of 5.fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations.Each mL of the preparation contained(7.1±1.0)mg of extract.HEK-293 cells were incubated in one set with S.fruticosa extract in the presence of 0.1 mmol/L H_2O_2,and in the other set with the addition of the extract alone.The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivarization of 8-oxoguanine moieties.The fluorescence was measured using flow cytometry technique.Results:Cells incubated 3 h with 150μL extract and exposed to 0.1 mmol/L H_2O_2 showed lower intensity of fluorescence,and thus lower DNA oxidation.Moreover,cells incubated 3 h with 100μl.of the extract showed lower intensity of fluorescence,and thus lower intrinsic cellular DNA oxidation compared to control(without S.fruticosa).Conchisions:The results from this study suggest that the water-soluble extract of S.fruticosa leaves protects against both H_2O_2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.展开更多
The nonstructural protein 1 （NS1） of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon （IFN）-mediated antiviral response. We en...The nonstructural protein 1 （NS1） of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon （IFN）-mediated antiviral response. We engineered an NS1-expressing 293 （293-NS1） cell line with no response to IFN stimulation. Compared with the parental 293 cells,展开更多
The acute and chronic effects of sodium nitroprusside (SNP) are well characterized for vascular smooth muscle cells (VSMC). Stimulation of soluble guanylyl cyclase (sGC) gives a rapid elevation of intracellular cGMP l...The acute and chronic effects of sodium nitroprusside (SNP) are well characterized for vascular smooth muscle cells (VSMC). Stimulation of soluble guanylyl cyclase (sGC) gives a rapid elevation of intracellular cGMP levels and relaxation of VSMC. The antiproliferative effect of SNP needs days to develop. In the present study human embryonic kidney (HEK 293) cells were used to study the growth after repeated exposure to SNP. A dose-dependent antiproliferative effect was evident and after 5 days with an IC50 value of 108 μM. Cyclic GMP was able to mimic the antiproliferative effect of SNP on HEK293 cells. When cGMP (1000 μM) was added to the cell culture medium for 5 days the cell densities were reduced with 37% below baseline and cGMPin increased from 5.3 to 195 pmol/107cells. The interaction with the non-selective PDE (cyclic nucleotide phosphodiesterase) inhibitor 3-isobutyl-1-methylxanthine (IBMX) was tested after three days. IBMX alone (1000 μM) reduced cell densities with 48% and elevated cGMPin (from 5.2 to 9.3 pmol/107cells). The effect of 10 μM SNP was reinforced on proliferation (from 13% to 90%) and elevation of cGMP levels (from 7.6 to 13.5 pmol/107cells). A corresponding effect was observed after addition of 1000 μM cGMP and 1000 μM IBMX for 3 days. The antiproliferative effect of cGMP increased from 30% to 89% and the cGMPin increased from 240 to 480 pmol/107cells. However, additional mechanisms exist for the antiproliferative effect of SNP. One of these is the intracellular oxidative effect which includes production of S-nitrosoglutathione. The fall in ratios between GSH and GSSG from 260 to 85 after 100 μM SNP exposure is compatible with such a mechanism since cGMP (1000 μM) added to the culture medium did not change the ratio. This study shows that the antiproliferative effects of SNP on HEK293 cells are mediated through cGMP-dependent and cGMP-independent mechanisms. The concentration-dependent effects develop over time. HEK293 cells had an efficient efflux system for cGMP and the use of inside-out vesicles (IOVs) showed high affinity ATP-dependent cGMP transport with a Km value of 2.3 μM. The antiproliferative effect of SNP was correlated to cGMPex/in.展开更多
Objective This study investigated the impact of occupational mercury(Hg) exposure on human gene transcription and expression, and its potential biological mechanisms.Methods Differentially expressed genes related to H...Objective This study investigated the impact of occupational mercury(Hg) exposure on human gene transcription and expression, and its potential biological mechanisms.Methods Differentially expressed genes related to Hg exposure were identified and validated using gene expression microarray analysis and extended validation. Hg-exposed cell models and PTEN lowexpression models were established in vitro using 293T cells. PTEN gene expression was assessed using qRT-PCR, and Western blotting was used to measure PTEN, AKT, and PI3K protein levels. IL-6 expression was determined by ELISA.Results Combined findings from gene expression microarray analysis, bioinformatics, and population expansion validation indicated significant downregulation of the PTEN gene in the high-concentration Hg exposure group. In the Hg-exposed cell model(25 and 10 μmol/L), a significant decrease in PTEN expression was observed, accompanied by a significant increase in PI3K, AKT, and IL-6 expression.Similarly, a low-expression cell model demonstrated that PTEN gene knockdown led to a significant decrease in PTEN protein expression and a substantial increase in PI3K, AKT, and IL-6 levels.Conclusion This is the first study to report that Hg exposure downregulates the PTEN gene, activates the PI3K/AKT regulatory pathway, and increases the expression of inflammatory factors, ultimately resulting in kidney inflammation.展开更多
文摘[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.
文摘Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition（IC50） being 26.15 μmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 μmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.
基金Supported by the National Natural Science Foundation of China (No. 30800473)
文摘Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I,the small product fragment,from pcDNA3-L539fs/47,was subcloned into the big fragment of pEGFP-C2-HERG under T4 ligase.pEGFP-C2-L539fs/47 was identified by agarose gel electrophoresis and sequencing.pcDNA3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into HEK293 cells by Lipofect,respectively.The expression of fusion protein in HEK293 cells was detected through immunofluorescence,laser confocal imaging scanning in vivo,Western blot and PCR.Results:Mutation region cDNA fragment(about 1 kb) and target vector fragment(about 7.2 kb) were ligated after purification and gel recovery.Agarose gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47,constructed approximately 8.2 kb,sequencing consistent with template gene.The transfection efficiency of recombinant plasmid by fluorescence microscopy was more than60%.Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD,the expression of pEGFP-C2 fusion protein size of approximately 90 KD.The L539fs/47 gene expression in HEK293 cells was significant by PCR analysis.Confocal laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of HEK293 cells.Conclusion:pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in HEK293 cells,which laid a foundation for the further study on L539fs/47.
基金the National Investigation Projects of Soil Pollution supported by a grant from Ministry of Environmental Protection of the People's Republic of China
文摘This study investigated the conjoined cellular oxidative damage of human embryo kidney 293T（HEK293T） cells induced by cadmium chloride（CdCl2） and nanometer titanium dioxide（nano-TiO2）.RT-PCR technique was used to detect the expressions of Heme oxygenase-1（HO-1） and 8-oxoguanine DNA glycosylase（OGG1）.The activities of superoxide dismutase（SOD） and catalase enzyme（CAT） and concentrations of reactive oxygen species（ROS） and maldondialdehyde（MDA） were measured by different approaches.The results showed that CdCl2 and nano-TiO2 at a low concen-tration of 0.75 total toxic unit（TU） exerted an additive effects on HO-1 gene expression,CAT activities and MDA concentrations.When the total TU was increased to 1 or 1.25 TU,the interaction was syner-getic.Moreover,the mixture with high proportion of CdCl2 produced an additive effect on the OGG1 gene expression,and the interaction was changed to be synergetic when the concentration of CdCl2 was lower than or equal to that of nano-TiO2.Synergetic effects of CdCl2 and nano-TiO2 on cellular oxida-tive damage of HEK293T cells were found as indicated by the changes in the SOD activities and ROS concentrations.It was concluded that CdCl2 and nano-TiO2 exerts synergistic effects on the cellular oxidative damage of HEK293T cells,and the sensitivity of these indicators of oxidative damage varies with the proportion of CdCl2 and nano-TiO2 in the mixture.
基金supported by the Nature Science Foundation Committee Projects of China(No.30470425)
文摘Hydrogen peroxide（H_2O_2） and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene（C_（60）） is critical for scavenging oxygen free radicals originated from cell metabolism, and reduced glutathione（GSH） is another important endogenous antioxidant. In this study, a novel water-soluble reduced glutathione fullerene derivative（C_（60）-GSH） was successfully synthesized, and its beneficial roles in protecting against H_2O_2-induced oxidative stress and apoptosis in cultured HEK 293 T cells were investigated. Fourier Transform infrared spectroscopy and 1H nuclear magnetic resonance were used to confirm the chemical structure of C_（60）-GSH. Our results demonstrated that C_（60）-GSH prevented the reactive oxygen species（ROS）-mediated cell damage. Additionally, C_（60）-GSH pretreatment significantly attenuated H_2O_2-induced superoxide dismutase（SOD） consumption and malondialdehyde（MDA） elevation. Furthermore, C_（60）-GSH inhibited intracellular calcium mobilization, and subsequent cell apoptosis via bcl-2/bax-caspase-3 signaling pathway induced by H_2O_2 stimulation in HEK 293 T cells. Importantly, these protective effects of C_（60）-GSH were superior to those of GSH. In conclusion, these results suggested that C_（60）-GSH has potential to protect against H_2O_2-induced cell apoptosis by scavenging free radicals and maintaining intracellular calcium homeostasis without evident toxicity.
基金Supported by Cleveland State University and Jordan University of Science and Technologygrant number 20130097
文摘Objective:To investigate the role of water-soluble extract of Salvia fruticosa(Creek sage)(S.fruticosa) leaves in reducing both intrinsic cellular and H_2O_2-induced DNA oxidation in cultured human embryonic kidney 293 cells.S.fruiicosa.native to the Eastern-Mediterranean basin,is widely used as a medicinal herb for treatment of various diseases.Methods:Dried leaves of 5.fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations.Each mL of the preparation contained(7.1±1.0)mg of extract.HEK-293 cells were incubated in one set with S.fruticosa extract in the presence of 0.1 mmol/L H_2O_2,and in the other set with the addition of the extract alone.The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivarization of 8-oxoguanine moieties.The fluorescence was measured using flow cytometry technique.Results:Cells incubated 3 h with 150μL extract and exposed to 0.1 mmol/L H_2O_2 showed lower intensity of fluorescence,and thus lower DNA oxidation.Moreover,cells incubated 3 h with 100μl.of the extract showed lower intensity of fluorescence,and thus lower intrinsic cellular DNA oxidation compared to control(without S.fruticosa).Conchisions:The results from this study suggest that the water-soluble extract of S.fruticosa leaves protects against both H_2O_2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.
基金supported by China Mega-Project for Infectious Disease(2014ZX10004002-004-001)National Natural Science Foundation of China(31500152)National Key Technology R&D Program(2014BAI13B04)
文摘The nonstructural protein 1 （NS1） of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon （IFN）-mediated antiviral response. We engineered an NS1-expressing 293 （293-NS1） cell line with no response to IFN stimulation. Compared with the parental 293 cells,
文摘The acute and chronic effects of sodium nitroprusside (SNP) are well characterized for vascular smooth muscle cells (VSMC). Stimulation of soluble guanylyl cyclase (sGC) gives a rapid elevation of intracellular cGMP levels and relaxation of VSMC. The antiproliferative effect of SNP needs days to develop. In the present study human embryonic kidney (HEK 293) cells were used to study the growth after repeated exposure to SNP. A dose-dependent antiproliferative effect was evident and after 5 days with an IC50 value of 108 μM. Cyclic GMP was able to mimic the antiproliferative effect of SNP on HEK293 cells. When cGMP (1000 μM) was added to the cell culture medium for 5 days the cell densities were reduced with 37% below baseline and cGMPin increased from 5.3 to 195 pmol/107cells. The interaction with the non-selective PDE (cyclic nucleotide phosphodiesterase) inhibitor 3-isobutyl-1-methylxanthine (IBMX) was tested after three days. IBMX alone (1000 μM) reduced cell densities with 48% and elevated cGMPin (from 5.2 to 9.3 pmol/107cells). The effect of 10 μM SNP was reinforced on proliferation (from 13% to 90%) and elevation of cGMP levels (from 7.6 to 13.5 pmol/107cells). A corresponding effect was observed after addition of 1000 μM cGMP and 1000 μM IBMX for 3 days. The antiproliferative effect of cGMP increased from 30% to 89% and the cGMPin increased from 240 to 480 pmol/107cells. However, additional mechanisms exist for the antiproliferative effect of SNP. One of these is the intracellular oxidative effect which includes production of S-nitrosoglutathione. The fall in ratios between GSH and GSSG from 260 to 85 after 100 μM SNP exposure is compatible with such a mechanism since cGMP (1000 μM) added to the culture medium did not change the ratio. This study shows that the antiproliferative effects of SNP on HEK293 cells are mediated through cGMP-dependent and cGMP-independent mechanisms. The concentration-dependent effects develop over time. HEK293 cells had an efficient efflux system for cGMP and the use of inside-out vesicles (IOVs) showed high affinity ATP-dependent cGMP transport with a Km value of 2.3 μM. The antiproliferative effect of SNP was correlated to cGMPex/in.
基金supported by the Jiangsu Province’s Outstanding Medical Academic Leader Program [CXTDA2017029]the Jiangsu Provincial Key Medical Discipline [ZDXK202249].
文摘Objective This study investigated the impact of occupational mercury(Hg) exposure on human gene transcription and expression, and its potential biological mechanisms.Methods Differentially expressed genes related to Hg exposure were identified and validated using gene expression microarray analysis and extended validation. Hg-exposed cell models and PTEN lowexpression models were established in vitro using 293T cells. PTEN gene expression was assessed using qRT-PCR, and Western blotting was used to measure PTEN, AKT, and PI3K protein levels. IL-6 expression was determined by ELISA.Results Combined findings from gene expression microarray analysis, bioinformatics, and population expansion validation indicated significant downregulation of the PTEN gene in the high-concentration Hg exposure group. In the Hg-exposed cell model(25 and 10 μmol/L), a significant decrease in PTEN expression was observed, accompanied by a significant increase in PI3K, AKT, and IL-6 expression.Similarly, a low-expression cell model demonstrated that PTEN gene knockdown led to a significant decrease in PTEN protein expression and a substantial increase in PI3K, AKT, and IL-6 levels.Conclusion This is the first study to report that Hg exposure downregulates the PTEN gene, activates the PI3K/AKT regulatory pathway, and increases the expression of inflammatory factors, ultimately resulting in kidney inflammation.
