Role of IGF-Ⅱ in proliferation and migration of hepatocellular carcinoma Huh7 cells

AIM: To observe the effects of insulin-like growth factorⅡ (IGF-Ⅱ) at different expression levels on hepatocellular carcinoma Huh7 cell proliferation and migration, and to explore the role of IGF-Ⅱ in the development of HCC. METHODS: The Huh7 cells were transfected with the over-expression plasmid pcDNA3.1(+)- IGF-Ⅱ or RNA interference plasmid pLVX-shRNA-IGF-Ⅱ by Lipofectamine 2000. The quantitative real-time PCR and Western blotting were used to verify the expression of IGF-II. The biological behaviors of the Huh7 cells were analyzed by CCK-8 assay, plate clone formation assay, cell scratch test and Transwell chamber experiment.RESULTS: Over-expression of IGF-II promoted the growth and migration of hepatocellular carcinoma cells (P < 0.05), and the cell proliferation was significantly inhibited in the Huh7 cells with low IGF-II expression (P < 0.05).CONCLUSION: IGF-II is involved in the regulation of biological behavior of hepatocellular carcinoma Huh7 cells in vitro, which may play a promoting role in the development of hepatocellular carcinoma.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbonate on the proliferation and apoptosis in K562 cells

AIM: To investigate the effect of pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB on the proliferation and apoptosis of K562 cells and to explore the anti-tumor mechanism of PDTC.METHODS: Trans AMTM NF-κB p65 kit was used to detect the activity of p65 in K562 cells treated by PDTC. The effect of PDTC on the proliferation of K562 cells was measured by WST-1 method. DNA damage was detected by single cell gel electrophoresis (comet assay). The procaspase-3 and activated protein level of caspase-3 were detected by Western blotting.RESULTS: The activity of p65 in K562 cells was inhibited after treated by PDTC (P<0.01). Simultaneously the cell proliferation was significantly inhibited in a dose-and time-dependent manner (P<0.01). The degree of DNA damage in K562 cells treated with PDTC at concentrations of 25 μmol/L, 50 μmol/L or 100 μmol/L was more severe than that in control. The rates of comet cells in the PDTC-treated groups (43.50％, 84.00％, 95.63％) were significantly higher than those in control (9.75％, P<0.01), and it was also dose-dependent. The expression of procaspase-3 and activated caspase-3 protein were detected in the cytoplasm of the K562 cells treated by PDTC by Western blotting.CONCLUSION: NF-κB plays an important role in regulating cell proliferation and apoptosis in K562 cells. PDTC inhibits NF-κB activity and elevates the expression of caspase-3, which is related to increase in cell apoptosis.     

Effect of caveolin-1 and ERK1/2 on 17β-estradiol-induced inhibition of VSMC proliferation

AIM: To investigate the effect of caveolin-1 and phosphorylation of ERK1/2 on 17β-estradiol (E2) induced inhibition of vascular smooth muscle cells (VSMCs). METHODS: The proliferation in cultured VSMCs was determined by using ［3H］-thymidine incorporation. The expressions of caveolin-1, MKP-1 and ERK1/2 phosphorylation were measured by Western blotting. The expression of caveolin-1 mRNA was measured by RT-PCR. RESULTS: Exposed to fetal calf serum (FCS) for 24 h, the increase in proliferation of VSMCs was detected by ［3H］-thymidine incorporation. Pretreatment with various concentrations of E2 for 24 h inhibited VSMC proliferation induced by FCS. The results of Western blotting and RT-PCR showed that pretreated with 17β-estradiol for 24 h reserved the decrease in caveolin-1 induced by FCS. Western blotting results further proved that the expression of MKP-1 was significantly increased and the expression of ERK1/2 phosphorylation was decreased after incubated with 17β-estradiol. CONCLUSION: 17β-estradiol increases caveolin-1 and MKP-1 expressions, and decreases ERK1/2 phosphorylation, leading to the inhibition of VSMC proliferation.     

Effects of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1α) and stromal cell-derived factor 1 (SDF-1) in pulmonary artery endothelial cells (PAECs) of SD rats and to investigate the role of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells to PAECs. METHODS: Immunomagnetic beads were used to separate and purify the CD34⁺/CXCR4⁺ progenitor cells derived from the peripheral circulation of SD rats. The expression of HIF-1α and SDF-1 in PAECs exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) was detected by immunofluorescence, Western blotting and ELISA. The migration index and adhesion rate were measured in the progenitor cells, which were subjected to the following different treatments: (1) normoxia (21% O₂); (2) hypoxia 12 h; (3) hypoxia 12 h +HIF-1α inhibitor (2ME2); (4) hypoxia 12 h+SDF-1 neutralizing antibody; (5) hypoxia 12 h+2ME2+SDF-1 neutralizing antibody.RESULTS: The expression of HIF-1α and SDF-1 in PAECs was effectively induced by the hypoxic exposure, and both of them reached the peak levels after 12 h of hypoxic treatment (P<0.01), while administration of 2ME2 decreased the hypoxia-induced SDF-1 expression (P<0.05). Treatment of the PAECs with 2ME2 or SDF-1 neutralizing antibody attenuated the migration index and adhesion rate of progenitor cells to the PAECs (P<0.05). CONCLUSION: There is a HIF-1α/SDF-1 signaling axis in hypoxia-exposed PAECs, which may play a crucial role in the migration and adhesion of progenitor cells to PAECs.     

Effect of neuregulins on mtp53 and HIF-1α in MDA-MB-231 cells

AIM: To explore the effect and significance of neuregulins /ErbB2 receptor signal transduction pathway on mtp53 and hypoxia-iducible factor-1α (HIF-1α) in none-overexpression ErbB2 breast cancer cell MDA-MB-231. METHODS: The expression of neuregulin was detected by immunocytochemistry and Western blotting. MDA-MB-231 cells were treated with ErbB2 kinase inhibitor AG825. Proliferation was measured by MTT assay. The cell cycle and apoptosis were determined by flow cytometry. The expressions of mtp53 and HIF-1α were detected by Western blotting. The mRNA expression of HIF-1α was detected by RT-PCR. RESULTS: MDA-MB-231 cells expressed a relative higher level of neuregulin. In the results of Western blotting, the positive reaction band was found in 44 kD which coincides with the molecular weight of neuregulin. When MDA-MB-231 cells were treated with AG825, the proliferation was inhibited in time and dose dependent manners (P<0.01). The cell cycle was arrested in G0/G1 phase (P<0.05). The apoptosis rate was increased (P<0.05). The protein expression levels of mtp53 and HIF-1α were decreased (P<0.05), and the mRNA level of HIF-1α was also decreased (P<0.05).CONCLUSION: Our study indicates that neuregulins are synthesized in MDA-MB-231 cells as transmembrane proteins. Neuregulins activate ErbB2 receptor signal transduction pathway by ligand autocrine or paracrine actions, and play an important role in proliferation of none-overexpression ErbB2 breast cancer cell MDA-MB-231. Proliferation and survivorship, and inhibition apoptosis can be induced with up-regulation of mtp53 and HIF-1α level.     

Effect of Notch1/Hes1 signaling pathway on proliferation and differentiation of AECⅡ by regulating expression of C/EBPα

AIM: To investigate whether Notch1/Hes1 signaling pathway regulates the expression of CCAAT/enhancer binding protein alpha (C/EBPα), thus affecting the proliferation and differentiation of type Ⅱ alveolar epithelial cells (AECⅡ). METHODS: Human AECⅡ were cultured in vitro, and randomly divided into control group, activator group (adding Notch pathway activator Jagged1 protein, 500 μg/L) and inhibitor group (adding Notch inhibitor DAPT, 10 μmol/L). AECⅡ in each group were collected after 24 h of intervention. The expression of Notch1, Hes1 and C/EBPα at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell proliferation ability of the AECⅡ was measured by living cell counting and CCK-8 assay. The cell cycle distribution and differentiation of the AECⅡ were analyzed by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly increased in activator group (P<0.05), AECⅡ entered G₂/M phase from S phase, the proliferation of AECⅡ was increased, and the differentiation of AECⅡ was reduced (P<0.05). The mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly reduced in inhibitor group (P<0.05), the cell cycle of AECⅡ cells was arrested in G₀/G₁ phase, the proliferation of AECⅡ cells was reduced, and the differentiation of AECⅡ cells was increased (P<0.05). CONCLUSION: Notch1/Hes1 signaling pathway regulates the expression of C/EBPα and affects the proliferation and differentiation of AECⅡ.     

Effect of IL-1β and IL-6 on expression of nerve growth factor in human nucleus pulposus cells

AIM: To determine the effect of interleukin-1β(IL-1β)and IL-6 on the expression of nerve growth factor (NGF) by human nucleus pulposus (NP) cells. METHODS: The NP cells from the normal disc of operative patients with scoliosis were isolated, cultured and identified. After 7 days preculture, the NP cells were treated with IL-1β (10 μg/L, 50 μg/L) or IL-6 (10 μg/L, 50 μg/L) for 48 h in the experimental groups and 0.3% PBS was used in the control groups. The expression of NGF was detected by the methods of immunohistochemistry, semi-quantitative RT-PCR and Western blotting.RESULTS: The NP cells were chondrocyte-like cellular morphology with positive staining of toluidine blue, safranine O and anti-collagen II antibody. The NP cells cultured in monolayer showed immunoreactivity to NGF either in control condition or in experimental group. IL-1β and IL-6 up-regulated the mRNA expression of NGF and the protein production of NGF. The effect of this up-regulation was higher by treating with IL-6 than by treating with IL-1β in the same concentration.CONCLUSION: Our results demonstrate that the proinflammatory cytokines IL-1β and IL-6 stimulate the production of NGF in NP cells. The effect of IL-6 is more significant than that of IL-1β.     

Effect of Ad-14-3-3σ on microRNA expression in different radioresistant nasopharyngeal carcinoma cells

AIM: To discuss the effect of Ad-14-3-3σ to microRNA (miRNA) in different radioresistant nasopharyngeal carcinoma (NPC) cells, CNE-1 and CNE-2, and study the relationship between the discrepancy of miRNA and radiosensitivity of NPC.METHODS: Ad-14-3-3σ was transfected to CNE-1 and CNE-2 cells, and then miRNAs were detected by Paraflo microfluidic microRNA chip. Hybridization images were collected using a laser scanner and the signals were normalized using a LOWESS filter. The effect of Ad-14-3-3σ to miRNAs and the relationship between the discrepancy of miRNA and radiosensitivity of NPC were studied according to Targetscan3.1 database (http://www.targetsan.org) after analyzing data.RESULTS: After treated by Ad-14-3-3σ, comparing to CNE-2 cells, there are 37 miRNAs changed remarkably, including 17 over-expression microRNAs and 20 under-expression microRNAs in CNE-1 cells. 6 miRNAs that one detective value was more than 1 000 and 3 folds than the other were hsa-miR-152,hsa-miR-205,hsa-miR-203,hsa-miR-7,hsa-miR-636 and hsa-miR-100.CONCLUSION: Ad-14-3-3σ can change the expression of miRNAs in different radioresistant nasopharyngeal carcinoma, and some miRNAs have relevance to carcinoma and radiosensitivity.     

Effect of rosiglitazone on SREBP-1 and TGF-β1 expressions and accumulation of ECM in renal tubular cells of Wistar rats treated with high fat diet

AIM: To study the effect of high fat diet on the expression of sterol regulatory element biding protein-1 (SREBP-1) and transforming growth factor β1 (TGF-β1) in renal tubular cells and rosiglitazone intervention. METHODS: Wistar rats were treated with high fat diet and rosiglitazone for 3 months. The serum glucose, serum insulin and serum triglyceride were detected. Oil Red O staining was used to observe the renal lipid deposit and Masson staining was for the detection of ECM accumulation. SREBP-1, TGF-β1 and FN protein were determined by the methods of immunohistochemistry and Western blotting. SREBP-1 mRNA was detected by in situ hybridization. RESULTS: Rosiglitazone prevented effectively the increase in serum glucose, serum insulin and serum triglyceride resulted from high fat diet. High fat diet led to lipid droplet formation in renal tubular cells and interstitial ECM accumulation, which was decreased by rosiglitazone treatment. Compared to normal rats, SREBP-1 protein and SREBP-1 mRNA showed high expressions in high fat diet rats that were lowered by rosiglitazone. The precursor segment and mature segment of SREBP-1 protein were decreased by 27.39% and 27.32%. Similarly, the high expressions of TGF-β1 and FN protein in kidney of high fat diet rats were also prevented by rosiglitazone intervention. Compared to high fat diet rats, the expression of TGF-β1 in rosiglitazone treatment rats was lowered by 19.14%. CONCLUSION: Rosiglitazone prevents effectively the over-expression of SREBP-1 and TGF-β1 in renal tubular cells, and decreases lipid accumulation and ECM production in rats fed with high fat diet.     

Effects of endothelial progenitor cell-conditioned medium on proliferation and differentiation of type Ⅱ alveolar epithelial cells exposed to hyperoxia

AIM: To investigate the effect of hyperoxia exposure on the paracrine function of endothelial progenitor cells (EPCs), and to explore the effects of paracrine factors of EPCs on the proliferation and differentiation of type Ⅱ alveolar epithelial cells (AECⅡ) exposed to hyperoxia. METHODS: Bone marrow-derived mononuclear cells were isolated and cultured in EGM-2MV medium for 7~10 d to obtain and identify EPCs. EPCs were cultured in room air (RA) or 60% O₂. The normoxia EPC-conditioned medium (E-CM-RA) and hyperoxia EPC-conditioned medium (E-CM-O₂) were collected. The levels of VEGF, FGF10, PDGF-BB and EGF in E-CM-RA and E-CM-O₂ were detected by ELISA. AECⅡ from adult rats were isolated, purified and cultured for 2 d, then divided into RA group, O₂ group, O₂+E-CM-RA group and O₂+E-CM-O₂ group. The proliferation of AECⅡ was detected by MTT assay and cell counting. The mRNA expression of SP-C and AQP5 was quantified by real-time PCR. RESULTS: The expression of VEGF and FGF10 in E-CM-O₂ group decreased significantly compared with E-CM-RA group (P<0.01). There were significant differences in AECⅡ viability and number among the 4 groups at 12 h, 24 h, 2 d and 3 d (P<0.01). Compared with RA group, AECⅡ viability and number in O₂ group decreased significantly at 12 h, 24 h, 2 d and 3 d (P<0.05). The AECⅡ viability and number in O₂+E-CM-RA group were significantly higher than those in O₂ group at 12 h, 24 h, 2 d and 3 d (P<0.05). However, no significant difference in AECⅡ viability and number between O₂+E-CM-O₂ group and O₂ group at 12 h, 2 d and 3 d was observed. There were significant differences in the mRNA expression of SP-C and AQP5 in the 4 groups at 24 h, 2 d and 3 d (P<0.01). Compared with RA group, the mRNA expression of SP-C in O₂ group was significantly inhibited (P<0.01), but the mRNA expression of AQP5 was promoted (P<0.01) at 24 h, 2 d and 3 d. Compared with O₂ group, the mRNA level of SP-C in O₂+E-CM-RA group and O₂+E-CM-O₂ group (P<0.05) at 24 h, 2 d and 3 d was increased, and the mRNA expression of AQP5 (P<0.01) at 2 d and 3 d was inhibited.CONCLUSION: EPCs secrete VEGF and FGF10, and hyperoxia impairs this paracrine function. Hyperoxia exposure inhibits AECⅡ proliferation and the mRNA expression of SP-C, but promotes the mRNA expression of AQP5. EPC-conditioned medium improves the proliferation of hyperoxia-exposed AECⅡ, and inhibits the transformation of AECⅡ. Hyperoxia exposure impairs the paracrine function of EPCs, and weakened the effects of E-CM-O₂ on AECⅡ.     

Effect of breviscapin on Ito and IK1 channel current in isolated ventricular myocytes of rats

AIM: The effects of breviscapin on transient outward potassium current (I_to) and inward rectifier potassium current (I_K1) channel in isolated ventricular myocytes of rats were determined. The mechanism of breviscapin at the ionic channel level was explored. METHODS: Single ventricular myocyte of rats was isolated by enzymatic dissociation. The whole-cell patch-clamp recording technique was used to record the change of I_to and I_K1 channel current influenced by breviscapin. RESULTS: Breviscapin decreased the I_to channel current in a dose-dependent and voltage-dependent manner in ventricular myocytes of rats. (1) The current-voltage curve was significantly decreased. Breviscapin at concentrations of 0.02, 0.05, 0.08, 0.10 g·L^-1 respectively decreased I_to current density (10.07±0.50)%, (27.47±2.36)%, (42.72%±2.50)% and (56.09±5.60)%. I_to was inhibited in a voltage-dependent manner, showing a significant attenuating effect at test potentials from 0 to + 50 mV. (2) The I_to activation curve, the activation curve and the recovery curve were not altered. (3) Breviscapin did not affect I_K1. CONCLUSION: Breviscapin concentration-dependently decreases I_to channel current in ventricular myocytes of rat.     

Effect of TNF-α on production and activation of caspase-3 in primary rat renal proximal tubule cells

AIM: To investigate the production and activation of caspase-3 in primary rat renal proximal tubule cells in response to tumor necrosis factor-α(TNF-α) and the implication of nuclear factor-κB (NF-κB) in the process. METHODS: Isolated rat renal proximal tubule cells (PTCs) from male adult Sprague Dawley rats were treated with TNF-α according to the indicated time courses. A specific NF-κB inhibitor, Bay11-7082, was used alone or as a pretreatment for 1 h followed by exposure to TNF-α for 24 h. The protein levels of cleaved caspase-3, caspase-3, I-κBα, phosphorylated I-κBα, and GAPDH were detected by Western blotting using specific antibodies. RESULTS: The protein level of cleaved caspase-3 relative to caspase-3 was significantly increased in the presence of TNF-α for 6 h, 12 h, and 24 h. Protein levels of caspase-3 were significantly decreased by 12 h and returned to baseline by 24 h in the presence of TNF-α. Treatment with Bay11-7082 for 25 h alone or pretreatment with Bay11-7082 for 1 h followed by addition of TNF-α for 24 h caused a remarkable reduction in both cleaved caspase-3 and caspase-3 as compared to control and TNF-α treated groups. An increase in phosphorylated I-κBα was observed from 15 min to 60 min after treatment with TNF-α at a dose of 10 μg/L in PTCs. CONCLUSION: NF-κB is not only associated with the activation of caspase-3 but also the production of caspase-3 in primary rat renal proximal tubule cells in response to TNF-α.     

Effect of interfering TGF-β receptor Ⅱ expression on viability,differentiation and apoptosis of NB4 cells

AIM: To evaluate the effect of interfering TGF-β receptor Ⅱ (TβRⅡ) expression on the viability and differentiation of human acute promyelocytic leukemia NB4 cells induced by all-trans retinoic acid (ATRA) and their apoptosis induced by arsenic trioxide (ATO). METHODS: The technique of lentivirus-mediated RNA interference was used to obtain stable NB4 cells with TβRⅡ knockdown, named TβRⅡ-shRNA NB4 cells. CCK-8 assay was used to detect the viability of TβRⅡ-shRNA NB4 cells. The expression level of CD11b was analyzed by flow cytometry, and Wright-Giemsa staining was used to detect the effects of ATRA on the differentiation of TβRⅡ-shRNA NB4 cells. Double staining (Annexin V-FITC/PI) and AO/EB staining were used to detect the effects of ATO on the apoptosis of TβRⅡ-shRNA NB4 cells. RESULTS: The viability of TβRⅡ-shRNA NB4 cells was significantly higher than that of NB4 parental cells. The differentiation was induced in TβRⅡ-shRNA NB4 cells and NB4 parent cells by treatment with ATRA at different concentration (0.01, 0.02, 0.04, 0.08, 0.1 μmol/L) for 96 h. The differentiation rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells in a dose-dependent manner. ATO induced apoptosis of TβRⅡ-shRNA NB4 cells and NB4 parent cells at different concentrations (2, 4 and 8 μmol/L) for 24 h. The apoptotic rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells dose-dependently. At the concentration of 8 μmol/L for 24 h, the apoptotic rates in TβRⅡ-shRNA NB4 cells and NB4 cells were (49.15±2.05)% and (66.85±2.41)%, respectively (P<0.01). CONCLUSION: Down-regulation of TβRⅡ increases the viability of NB4 cells, inhibits NB4 cell differentiation induced by ATRA, and also inhibits apoptosis induced by ATO.     

Effects of simvastatin on proliferation of esophageal cancer cells through NF-κB p65 pathway

AIM To investigate the effects of simvastatin （SIM） on the proliferation of esophageal cancer Eca109 cells through NF-κB p65 pathway. METHODS Esophageal cancer Eca109 cells were cultured in vitro and treated with SIM at different concentrations （2 , 4, 8, 16 and 32 μmol/L）. The proliferation of Eca109 cells was measured by MTT assay and plate colony formation assay. The proliferation-related protein levels of cyclin D1, c-Myc, NF-κB p65 （p65） and IκB-α in the esophageal cancer Eca109 cells were determined by Western blot. ELISA was used to detect the levels of tumor necrosis factor α （TNF-α） and interleukin-6 （IL-6） in the culture supernatant of Eca109 cells. RESULTS Compared with control group, the inhibitory effects of SIM at 2 , 4 , 8 and 16 μmol/L on the viability of Eca109 cells were increased in a concentration-dependent and time-dependent manners （P<0.05）. Simultaneously, the inhibitory rates of SIM at 16 and 32 μmol/L on the viability of Eca109 cells showed no significant difference （P>0.05）. The inhibitory rates of SIM at 16 μmol/L on the viability of esophageal cancer cells was 50.61% at 48 h, which was closed to half of the inhibitory dose IC₅₀, and it was used as the optimum concentration and time for follow-up experiments. Compared with control group, the plate colony formation rate of Eca109 cells, the protein levels of cyclinD1, c-Myc, nuclear p65, TNF-α and IL-6 in 8 and 16 μmol/L groups were decreased, while the levels of cytosolic p65 and IκB-α proteins were increased （P<0.05）. No significant difference of plate colony formation rate in Eca109 cells, and the protein levels of cyclin D1, c-Myc, nuclear and cytoplasmic p65, IκB-α, TNF-α and IL-6 between 16 μmol/L SIM group and 32 μmol/L SIM group was observed （P>0.05）. CONCLUSION Simvastatin inhibits the proliferation of esophageal cancer Eca109 cells, and its mechanism may be related to up-regulation of IκB-α, down-regulation of cyclinD1 and c-myc, inhibition of p65 nuclear translocation, and the expression of TNF-α and IL-6 downstream of NF-κB p65 pathway.     

Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.     

Effects of IOX1 on proliferation,apoptosis and extracellular matrix-related protein expression in LX2 cells induced by TGF-β

AIM To investigate the effects of histone demethylase inhibitor IOX1 (5-carboxy-8-hydroxyquinoline) on the proliferation, apoptosis and extracellular matrix (ECM)-related protein expression in transforming growth factor-β (TGF-β)-induced human hepatic stellate LX2 cells. METHODS The proliferation and apoptosis of the LX2 cells were determined by real-time cell analysis and flow cytometry, respectively. The level of histone H3 lysine 9 dimethylation (H3K9me2) and the protein expression of ECM-related molecules [α-smooth muscle actin (α-SMA), collagen type I (Col I), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1)] in the LX2 cells were detected by Western blot. RESULTS Treatment with IOX1 at 50~300 μmol/L significantly inhibited LX2 cell proliferation, and 300 μmol/L IOX1 significantly promoted the apoptosis of the LX2 cells. In addition, different concentrations of IOX1 increased the levels of H3K9me2 and MMP-1, and down-regulated the expression of α-SMA, Col I and TIMP-1 in TGF-β-induced LX2 cells (P<0.05). CONCLUSION Treatment with IOX1 inhibits the proliferation of LX2 cells induced by TGF-β, promotes the cell apoptosis, and regulates the synthesis and metabolism of ECM by elevating H3K9me2 level, thus attenuating hepatic fibrosis.     

Effect of inhibiting nuclear factor-kappa B on TNF-α-induced expression of angiotensinogen and production of angiotensinⅡ in glomerular mesangial cells

AIM: To study the effect of inhibiting nuclear factor-kappa B (NF-κB) activity on the expression of angiotensinogen (AGT) and the production of angiotensinⅡ (AngⅡ) induced by tumor necrosis factor-α (TNF-α) in glomerular mesangial cells (MCs) of SD rats. METHODS: The MCs of SD rats were isolated and divided into three groups as follows: control; MCs treated with TNF-α, and the MCs treated with TNF-α + pyrrolidinedithiocarbamate (PDTC). The activity of nuclear factor-kappa B was measured by electrophoretic mobility shift assay. The expression of AGT was determined by RT-PCR for mRNA and Western blotting for protein. The concentration of angiotensinⅡ in supernatant was measured by RIA. RESULTS: The NF-κB activity in the MCs treated with TNF-α (20.67±9.14)×102 μg/cell was significantly higher than that in control cells ［(8.25±4.35)×102 μg/cell, P<0.01］ and the MCs treated with TNF-α+PDTC ［(7.20±4.57)×102 μg/cell, P<0.01］, and no significant difference was found between control and the MCs treated with TNF-α＋PDTC (P>0.05). The AGT mRNA level in the MCs treated with TNF-α (0.27±0.05) was higher than that in the control cells (0.20±0.05, P<0.05), and no significant difference was observed when compared with that in the MCs treated with TNF-α＋PDTC (0.22±0.06, P>0.05). The expression of AGT protein in the MCs treated with TNF-α (0.60±0.19) μg/cell was higher than that in the control ［(0.37±0.15)μg/cell, P＜0.05］ and the MCs treated with TNF-α＋PDTC ［(0.37±0.17)μg/cell, P<0.05］, and no significance was found between the MCs treated TNF-α＋PDTC and the control (P>0.05). The AngⅡ level in supernatant of cultured MCs treated with TNF-α ［(9.73±2.38)×10-5 ng·L-1/cell］ was significantly higher than that in the control ［(7.50±1.51)×10-5 ng·L-1/cell, P<0.05］ and in the MCs treated with TNF-α＋PDTC ［(6.94±1.46)×10-5 ng·L-1/cell, P<0.05］, however, the difference between the MCs treated with TNF-α＋PDTC and the control was of no significance (P>0.05). CONCLUSION: TNF-α activates the NF-κB in glomerular MCs, induces the AGT expression and the production of AngⅡ. Inhibition of NF-κB decreases the AGT expression and the production of AngⅡ. Therefore, the effects of TNF-α on AGT and AngⅡ may be mediated by NF-κB.     

Effects of rhTGF-β1 and TGF-β1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro

AIM: To investigate the effects of rhTGF-β₁ and TGF-β₁ gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro. METHODS: Cell growth induced by various concentrations of rhTGF-β₁ was determined by MTT proliferation assay. Under the induction of liposomes, recombinant pSecTag2-TGF-β₁MP vectors were transferred into the corneal endothelial cells. Morphological changes of transfected cells were observed by HE staining. The expression levels of TGF-β₁ were assessed by ELISA. Cell cycle analysis was assessed by flow cytometry. DNA fragment analysis was used to confirm the presence of apoptosis. RESULTS: rhTGF-β₁ in concentrations of 5-20 μg/L showed a significant suppressive effect on the proliferation of corneal endothelial cells, 0.5-1 μg/L had no effect, 0.05-0.1 μg/L facilitated cell growth, as compared with negative controls. The morphous of transfected corneal cells had no significant abnormality compared with normal cells. According to the result of ELISA, the concentration of TGF-β₁ in the supernatant was calculated to be (98±3) ng/L. Flow cytometry assay showed that S and G₂/M phase of transfected cells decreased significantly compared with that of control group, but the cell cycle recovered normally after adding 10 μg/L EGF into the culture medium. Agarose electrophoresis didn′t show marked ladders in transfected group. CONCLUSION: Effects of rhTGF-β₁ on the proliferation of corneal endothelial cells are different with various concentrations. TGF-β₁ gene transfection shows suppressive effect on the proliferation of cultured corneal endothelial cells, but does not induce cell apoptosis. EGF is the antagonist of this suppressive effect.