Effect of HMGA2 gene on high glucose-induced apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway

AIM:To investigate the effect of HMGA2 down-regulation on apoptosis and Notch signaling pathway in renal tubular epithelial cells exposed to high glucose (HG). METHODS:D-glucose at 5, 10, 20 and 30 mmol/L was used to stimulate human renal tubular epithelial HK-2 cells for 2 h, and D-glucose at 30 mmol/L was used to stimulate the HK-2 cells for 10 min, 60 min and 120 min. The protein expression of HMGA2 was determined by Western blot. The HK-2 cells were divided into normal glucose (NG) group, HG group, HG+si-HMGA2 group and HG+NC group, in which siRNA was transfected by Lipofectamine^TM 2000 for 48 h. Flow cytometry was used to analyze the apoptotic rate, reactive oxygen species (ROS) assay kit was used to detect ROS content, and Western blot was used to detect the protein levels of Notch1, Hes1 and Bcl-2. The HK-2 cells were treated with the Notch signaling pathway inhibitor DAPT, and then the cells were divided into HG group, HG+DAPT group and HG+si-HMGA2+DAPT group. The apoptotic rate was analyzed by flow cytometry. RESULTS:Exposure of the HK-2 cells to D-glucose at different concentrations for different time significantly increased the expression of HMGA2 (P<0.05). Compared with NG group, the protein expression of HMGA2, Notch1 and Hes1 in HG group was increased, the expression of Bcl-2/Bax was decreased, the apoptotic rate was increased, and the content of ROS was increased obviously (P<0.05). Compared with HG group, the protein expression of HMGA2, Notch1 and Hes1 of HG+si-HMGA2 group was decreased, the expression of Bcl-2/Bax was increased, the apoptotic rate was decreased, and the content of ROS was decreased significantly (P<0.05). The apoptotic rate in HG+DAPT group was significantly lower than that in HG group, while the apoptotic rate in HG+si-HMGA2+DAPT group was significantly lower than that in HG+DAPT group (P<0.05). CONCLUSION:Down-regulation of HMGA2 expression inhibits the apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway and decreasing ROS production.     

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.     

Intervention effects of apyrase on silica-induced pulmonary fibrosis in mice

AIM: To investigate the effect of apyrase on the experimental silicosis. METHODS: C57BL/6 male mice were randomly divided into control group, silica treatment group, silica+apyrase group and silica+NS group. A mouse model of lung fibrosis was induced by crystalline silica particles (50 mg/kg, via oropharyngeal instillation), and were sacrificed at 3 h, 7 d, 14 d and 28 d. Apyrase was delivered by oropharyngeal aspiration at the same time and 4 h after silica challenge. The lung indexes were calculated and the concentration of ATP was detected by bioluminescent assay. The mRNA expression levels of collagen type Ⅰ(Col Ⅰ), collagen type Ⅲ (Col Ⅲ) and transforming growth factor β1 (TGF-β1) were examined by real-time PCR. The protein levels of TGF-β1 in bronchoalveolar lavage fluid were measured by ELISA. RESULTS: The elevated lung index and collagen levels showed that silicosis model was established successfully. Compared with silica group, apyrase treatment significantly alleviated silica-induced inflammation, reduced inflammation score on day 7, and decreased the lung index, collagen volume fraction and the mRNA expression of Col Ⅰand Col Ⅲ on day 28. Treatment with apyrase effectively down-regulated the mRNA levels of TGF-β1 in the lung tissues and TGF-β1 protein levels in bronchoalveolar lavage fluid on day 7.CONCLUSION: Apyrase attenuates the pulmonary inflammation and fibrosis of silicosis, which may be related with down-regulation of ATP and TGF-β1 in the lung tissues.     

Effect of KLF6 gene on apoptosis of THP-1 cell-derived macrophages

AIM: To investigate the effects of Kruppel-like factor 6 (KLF6) over-expression on the viability, apoptosis, reactive oxygen species (ROS) level and AKT signaling pathway of THP-1 cell-derived macrophages. METHODS: Human monocyte cell line THP-1 was induced to differentiate into macrophages by phorbol myristate acetate (PMA), and the macrophages were randomly divided into pcDNA3.1 group, oxidized low-density lipoprotein (ox-LDL) group, ox-LDL+pcDNA3.1 group and ox-LDL+pcDNA3.1-KLF6 group. pcDNA3.1 was transfected according to Lipofectamine^TM 2000 Kit. The cell viability, apoptotic rate and ROS level were detected by MTT assay, flow cytometry with Annexin V-FITC/PI double staining and H₂DCF-DA probing, respectively. The protein levels of Bcl-2, Bax and p-AKT were determined by Western blot. RESULTS: After pcDNA3.1-KLF6 was transfected into the macrophages, the expression of KLF6 was increased significantly (P<0.05). ox-LDL significantly inhibited the viability of the macrophages, induced apoptosis and ROS production, up-regulated the protein expression of Bax, and down-regulated the protein levels of Bcl-2 and p-AKT (P<0.05). Over-expression of KLF6 significantly reduced the effects of ox-LDL on cell viability, apoptosis, ROS level and the protein levels of Bcl-2, Bax and p-AKT (P<0.05). CONCLUSION: KLF6 significantly reduces the apoptosis of THP-1 cell-derived macrophages induced by ox-LDL, which may be related to the reduction of ROS level and activation of AKT signaling pathway.     

Role of HMGA2 in epithelial-mesenchymal transition of gastric cancer cells

AIM:To investigate the role of HMGA2 in the epithelial-mesenchymal transition (EMT) in gastric cancer cells. METHODS:The expression of HMGA2 in human gastric cancer cell lines with different degrees of differen-tiation (MKN45, MKN28 and SGC7901) and immortalized human gastric epithelial cell line GES-1 was determined by Western blot and RT-qPCR. pcDNA3.0-HMGA2 plasmid was transfected into the MKN28 cells by liposome method. Transfection of si-HMGA2 interference fragments into MKN45 cells was also performed. The transfection efficiency was evaluated by Western blot and RT-qPCR. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the cell viability were measured by CCK-8 assay. The effects of HMGA2 over-expression in the MKN28 cells on the cell migration and invasion abilities were detected by wound healing and Transwell invasion assays. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the expression of EMT-related markers E-cadherin, N-cadherin, vimentin at mRNA and protein levels were determined by RT-qPCR and Western blot. The changes of Wnt/β-catenin signaling pathway-related molecules in the MKN28 cells with HMGA2 over-expression were also determined by RT-qPCR. RESULTS:The expression levels of HMGA2 were quite different in different differentiation levels of gastric cancer cells (P<0.05). The increased expression level of HMGA2 in MKN28 cells inhibited the cell viability (P<0.05), while the decreased expression level of HMGA2 in MKN45 cells promoted the cell viability (P<0.05). The increased expression level of HMGA2 in MKN28 cells promoted cell migration and invasion (P<0.05), changed the expression of EMT-related markers (P<0.05), while the decreased expression level of HMGA2 in the MKN45 cells changed the expression of EMT-related markers (P<0.05). The increased expression level of HMGA2 in the MKN28 cells significantly increased the mRNA levels of β-catenin in the Wnt/β-catenin pathway and the downstream molecules c-Myc and cyclin D1 (P<0.05). CONCLUSION:HMGA2 is closely related to the migration and invasion abilities of gastric cancer cells. Moreover, it promotes the EMT process of gastric cancer cells by activating Wnt/β-catenin pathway.     

Expression of programmed cell death factor 4 in pulmonary fibrosis model

AIM:To detect the expression of programmed cell death protein 4 (PDCD4) in pulmonary fibrosis model and its effect on cell viability and pulmonary fibrosis indicators, and to explore its mechanism. METHODS:The expression level of PDCD4, α-smooth muscle actin (α-SMA) and collagen type I (COL-I) in human embryonic lung fibroblasts cell line HFL-1 group and HFL-1+TGF-β1 group were detected by Western blot and RT-qPCR. The plasmid pEZ-M03-PDCD4 and empty vector pEZ-M03 were transfected into myofibroblasts (MB), and the protein expression level of PDCD4 was detected by Western blot. The protein levels of p-AKT and AKT in blank control group, pEZ-M03-PDCD4 group, pEZ-M03 group and LY294002 group and the expression of cell cycle-related proteins c-Myc and cyclin D1 were determined by Western blot. The effect of PDCD4 on the viability of MB was measured by CCK-8 assay. The hydroxyproline content in the culture supernatant of HFL-1 cells and transfected MB was detected by hydroxyproline digestion method. The expression of PDCD4 in the lung tissues of the mice in model group and control group was detected by Western blot. RESULTS:Compared with HFL-1 group, the expression of α-SMA and COL-I at mRNA and protein levels in HFL-1+TGF-β1 group was significantly increased (P<0.01), the mRNA expression of PDCD4 was not significantly changed, while PDCD4 protein was significantly down-regulated (P<0.01). Compared with blank control group and pEZ-M03 group, the protein expression of PDCD4 in pEZ-M03-PDCD4 group was significantly increased (P<0.05), the protein expression of c-Myc and cyclin D1 was significantly decreased (P<0.05), the cell viability was also significantly inhibited (P<0.01), and the content of hydroxyproline in the culture supernatant was significantly reduced (P<0.05). Compared with blank control group, the protein levels of p-AKT were significantly decreased in pEZ-M03-PDCD4 group and LY294002 group, and no significant difference between blank control group and pEZ-M03 control group was observed. Compared with control group, PDCD4 expression was decreased in model group (P<0.01).CONCLUSION:PDCD4 is low expressed in the process of pulmonary fibrosis. Over-expression of PDCD4 inhibits the viability of MB, decreases the content of hydroxyproline, and inhibits the PI3K/AKT signaling pathway.     

Pirfenidone inhibits TGF-β1-induced differentiation of myofibroblast in human lung fibroblast populations

ATM: To investigate the effect of pirfenidone on transforming growth factor-β1 (TGF-β1)-induced fibroblast-to-myofibroblast transition in vitro. METHODS: The cell viability was measured by MTT assay. The proliferation of human lung fibroblasts (HLFs) was detected by EdU incorporation. Migratory and invasive abilities were measured by Boyden chamber assay. The α-smooth muscle actin (α-SMA) protein expression was determined by Western blot and immunofluorescence. The mRNA expression of α-SMA and type Ⅰ and Ⅲ collagens was evaluated by RT-qPCR. RESULTS: Pirfenidone at different concentrations (0.1, 0.2, 0.3, 0.5 and 0.8 mg/L) had no cytotoxic effect on the HLFs, and pirfenidone at 0.2 mg/L was used for the intervention. Pretreatment of the HLFs with 0.2 mg/L pirfenidone prior to TGF-β1 not only markedly suppressed the changes of proliferation, migration, invasion and reorganization of actin cytoskeleton in the HLFs (P<0.01﹚,but also down-regulated the expression of α-SMA and type C and Ⅲ collagens triggered by TGF-β1 ﹙P<0.05﹚.CONCLUSION: Pirfenidone has an inhibitory effect on TGF-β1-induced activated cell functions and fibroblast-to-myofibroblast transition in HLFs.     

Over-expression of SIRT1 gene inhibits high glucose-stimulated H9c2 cardiomyocyte apoptosis and ROS level through PI3K/AKT signaling pathway

AIM: To investigate the effects of silent information regulator 1 (SIRT1) over-expression on the apoptosis and the level of reactive oxygen species (ROS) in high glucose-induced H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were transfected with empty plasmid (pcDNA3.1-NC) and SIRT1 over-expression plasmid (pcDNA3.1-SIRT1), and then stimulated by high glucose. The H9c2 cells were divided into control group, high glucose group, high glucose + pcDNA3.1-NC group and high glucose + pcDNA3.1-SIRT1 group. The expression of SIRT1 at mRNA and protein levels in each group was determined by qPCR and Western blot. The viability of the cells was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The protein levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, AKT and phosphorylated AKT were examined by Western blot. RESULTS: SIRT1 was significantly decreased in high glucose-induced H9c2 cardiomyocytes, the cell viability was significantly decreased compared with control group, while the ROS levels and apoptotic rate were increased, and the phosphorylated PI3K and AKT protein levels were down-regulated (P<0.05). Over-expression of SIRT1 significantly promoted the viability of H9c2 cardiomyocytes induced by high glucose, decreased the ROS levels and apoptotic rate, and up-regulated phosphorylated PI3K and AKT protein levels (P<0.05). CONCLUSION: SIRT1 over-expression reverses the decrease in the viability of high glucose-stimulated H9c2 cardiomyocytes, and the increases in apoptotic rate and oxidative stress by regulating PI3K/AKT signaling pathway.     

Regulatory effect of NOX-4 on PI3K signaling pathway in TGF-β1-induced collagenⅠsynthesis from lung cancer cells

AIM: To investigate the regulatory effect of NADPH oxidase-4 (NOX-4) on PI3K signaling pathway in transforming growth factor-β1 (TGF-β1)-induced collagen type I (collagen I)synthesis from lung cancer cells and the mechanisms. METHODS: Human lung cancer A549 cells were cultured in vitro and stimulated with TGF-β1. The expression of NOX family and collagen family at mRNA and protein levels as well as the PI3K class I catalytic subunits and the activation of PI3K signaling pathway was measured. A549 cells were pre-treated with NOX-4 inhibitor diphenyleneiodonium (DPI), and the expression of collagen I at mRNA level as well as the PI3K class I catalytic subunits and the activation of PI3K signaling pathway was measured upon TGF-β1 stimulation. RESULTS: TGF-β1 stimulated the expression of NOX-4 and collagen I at mRNA and protein levels as well as the expression of PIK3CD and the activation of PI3K signaling pathway at a dose-and time-dependent manner. NOX-4 inhibitor DPI partly reversed TGF-β1-induced collagen I expression. Inhibition of NOX-4 down-regulated the degree of TGF-β1-stimulated activation of PI3K signaling pathway without effect on the expression of PIK3CD. CONCLUSION: NOX-4 participates in TGF-β1-induced collagenⅠsynthesis from lung cancer cells via regulating the activation of PI3K signaling pathway. TGF-β1/NOX-4/PI3K signaling pathway axis acts as a regulatory role in collagenⅠsynthesis from lung cancer cells.     

Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via ERK-mediated autophagy inhibition

AIM: To explore the effect of Pycnogenol on transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cell activation. METHODS: Cultured LX-2 cells were treated with 5 μg/L TGF-β1 and different concentrations (0, 10, 25 and 50 mg/L) of Pycnogenol. The viability of the LX-2 cells under the conditions with or without autophagy inhibitor 3-MA and ERK inhibitor PD98059 was determined by MTT assay. The protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2 and ERK1/2 were detected by Western blot. RESULTS: Compared with control group, 5 μg/L TGF-β1 treatment elevated the cell viability, and increased the protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2, and ERK1/2 in the LX-2 cells (P<0.05). However, these effects were reversed by Pycnogenol pretreatment in a dose-dependent manner and the inhibitory effect of 50 mg/L Pycnogenol was the most significant in the LX-2 cells (P<0.05). Furthermore, compared with TGF-β1 group, pretreatment with 50 mg/L Pycnogenol, 5 mmol/L 3-MA or 20 μmol/L PD98059 downregulated TGF-β1-induced cell viability and the protein levels of α-SMA and LC3-Ⅱ/Ⅰ in the LX-2 cells (P<0.05). CONCLUSION: Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via p-ERK and autophagy inhibition.     

Effects of soluble transforming growth factor-β type Ⅱ receptor on cardiac functions after myocardial infarction in rats

AIM: To observe the effects of soluble transforming growth factor-β type Ⅱ receptor (sTGFβRⅡ) on cardiac functions after myocardial infarction (MI) in rats. METHODS: MI was induced in Sprague-Dawley (SD) rats by ligating the left anterior descending coronary artery. The rats surviving to the third day after MI were included in the study and randomly divided into MI group, pAd-sTGFβRⅡ group (transfected with recombinant adenovirus vector expressing the extracellular domain gene of TGF-βRⅡ), vector group and sham group. Four weeks later, the heart rate (HR), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD) and ejection fraction (EF) were evaluated by echocardiograms. The expression of sTGFβRⅡ in myocardial tissues was observed under fluorescence microscope by frozen sectioning, and the expression of typeⅠ and Ⅲ collagens was observed by Sirius red-saturated picric acid staining. The expression of matrix metalloproteinase 9 (MMP-9) at mRNA and protein levels was determined by RT-PCR and immunohistochemical method. The activity of MMP-9 was assayed by gelatin zymography. RESULTS: Compared with sham group, HR, LVEDD, LVESD, typeⅠ and Ⅲ collagen, mRNA and protein of MMP-9, and the activity of MMP-9 increased significantly (P<0.01), and EF decreased (P<0.01) in MI group and vector group. Compared with MI group, EF was increased (P<0.01), but HR, LVEDD, LVESD, typeⅠ and Ⅲ collagen, mRNA and protein expression of MMP-9 and the activity of MMP-9 decreased significantly (P<0.01) in pAd-sTGFβRⅡ group, and all the parameters above were still higher than those in sham group. CONCLUSION: sTGFβRⅡ intervention improves the cardiac functions after MI by inhibiting TGF-β-mediated MMP-9 expression.     

Lentiviral vector CV072-mediated Mfn2 over-expression inhibits hepatic stellate cell activation

AIM:To construct a lentiviral vector carrying mitofusin 2 (Mfn2), and to investigate the inhibitory effect of Mfn2 on the activation of rat hepatic stellate cells and its mechanism of reducing the formation of hepatic fibrosis-related factors. METHODS:The lentiviral over-expression vector CV072-pCMV-Mfn2-EGFP containing Mfn2 was constructed and transfected into the hepatic stellate cells. The expression of green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was evaluated. The protein levels of Bax, Bcl-2, cleaved caspase-3, α-SMA, TGF-β1, Smad2 and Smad3 were detected by Western blot. The levels of type I collagen, type Ⅲ collagen and type IV collagen in the cell culture supernatants were determined by ELISA. RESULTS:Compared with control group, the apoptosis of the hepatic stellate cells transfected with lentivirus over-expression vector CV072-pCMV-Mfn2-EGFP was increased, and the protein levels of proapoptotic molecules Bax and cleaved caspase-3 were increased (P<0.01). TGF-β1/Smad pathway-related proteins TGF-β1, p-Smad2 and p-Smad3 were decreased, and the levels of fibrosis-related proteins α-SMA, type I collagen, type Ⅲ collagen and type IV collagen were decreased (P<0.01). CONCLUSION:Transfection of lentiviral over-expression vector CV072-pCMV-Mfn2-EGFP effectively inhibits hepatic stellate cell activation in vitro and may reduce the production of hepatic fibrosis-related factors by inhibiting TGF-β1/Smad pathway.     

Effects of endogenous carbon monoxide on the expression of transforming growth factor-beta 3 and type Ⅰ collagen of pulmonary artery in rats under hypoxia

AIM: To explore the impact of endogenous carbon monoxide (CO) on the expression of transforming growth factor-beta 3 (TGF-β₃) and type Ⅰcollagen in pulmonary artery of rats under hypoxia. METHODS: In the model of rats under hypoxic pulmonary hypertension, the measurement of pulmonary artery mean pressure (PAMP) and carboxyhemoglobin (HbCO) formation within pulmonary tissue homogenates was performed. TGF-β₃ and collagen Ⅰexpressions were detected by immunohistochemical assay. The expressions of TGF-β₃, type Ⅰ procollagen mRNA, and tissue inhibitor of metalloproteinase -1 (TIMP-1) mRNA were detected by in situ hybridization. RESULTS: ZnPP significantly increased PAMP and markedly decreased HbCO formation within lung tissue homogenates in rats under hypoxia( P< 0.01). Meanwhile, ZnPP promoted the expression of TGF-β₃ and collagen Ⅰprotein in pulmonary arteries in rats under hypoxia ( P< 0.01). ZnPP obviously elevated the expressions of TGF-β₃ mRNA, type Ⅰ procollagen mRNA, and TIMP-1 mRNA in pulmonary arteries in rats under hypoxia ( P< 0.01). CONCLUSION: Endogenous CO plays an important role in decreasing collagen synthesis and promoting degradation in pulmonary artery of rats under hypoxia by inhibiting the expression of TGF-β₃.     

Effect of integrin β1 on multidrug resistance in gastric cancer SGC-7901 cells

AIM: To study the effect of integrin β1 on multidrug resistance in gastric cancer and its possible mechanisms. METHODS: The expression of integrin β1 at mRNA and protein levels in the SGC-7901 cells and SGC-7901/DDP cells was determined by qPCR and Western blot. The expression of integrin β1 in the SGC-7901/DDP cells was silenced by antisense oligodeoxynucleotide. The cell viability was detected by the CCK-8 assay, the cell apoptosis were analyzed by flow cytometry, and the protein levels of integrin β1, Bcl-2/Bax, cleaved caspase-3/caspase-3, cytochrome C (Cyt-C) and p-AKT/AKT were determined by Western blot.RESULTS: The expression of integrin β1 at both mRNA and protein levels was significantly upregulated in SGC-7901/DDP cells. The expression of integrin β1 was increased in SGC-7901 cells treated with chemotherapeutic agents such as cisplatin, paclitaxel and 5-fluorouracil. Knockdown of integrin β1 induced apoptosis of SGC-7901/DDP cells with an increased sensitivity to the chemotherapeutic agents. Meanwhile, knockdown of integrin β1 downregulated the protein levels of Bcl-2/Bax, p-AKT^Ser473 and p-AKT^Thr308, while promoted the release of Cyt-C and upregulated the protein level of cleaved caspase-3. CONCLUSION: Knockdown of integrin β1 increases the sensitivity of SGC-7901/DDP cells to the chemotherapeutic agents, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of AKT pathway by inhibiting phosphorylations of AKT at Ser473 and Thr308.     

Effect of FGFR1 expression knock-down on biological characteristics of infantile hemangioma endothelial cells

AIM: To study the effect of fibroblast growth factor receptor 1 (FGFR1) expression knock-down on the viability, apoptosis, invasion and migration of infantile hemangioma endothelial cells (HemECs). METHODS: FGFR1 was down-regulated by FGFR1 small interfering RNA (si-FGFR1) transfection. The viability of the cells was measured by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry and the invasion and migration abilities were determined by Transwell assay. The protein levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and phosphorylated AKT (p-AKT) were examined by Western blot. RESULTS: Transfection of si-FGFR1 into HemECs had significant effects on inhibiting cell viability (P<0.05), promoting apoptosis (P<0.05), and decreasing cell invasion and migration abilities (P<0.05). The results of Western blot showed that knockdown of FGFR1 gene expression in the cells reduced the protein levels of PI3K and p-AKT (P<0.05), and had no significant effect on AKT protein level. CONCLUSION: Knock-down of FGFR1 expression changes the biological characteristics of endothelial cells in infantile hemangiomas by regulating PI3K/AKT signaling pathway.     

Inhibitory effect of oxymatrine on high glucose-induced rat renal tubular epithelial-mesenchymal transition

AIM:To investigate the inhibitory effect of oxymatrine (OM) on high glucose-induced rat renal tubular epithelial-mesenchymal transition (EMT). METHODS:The rat renal tubular epithelial NRK52E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+different concentrations of OM groups and high glucose+0.50 g/L OM dynamic observation group. The expression of TGF-β1, Smad7, α-SMA and E-cadherin at mRNA and protein levels was detected by real-time PCR and Western blotting. The viability of NRK52E cells was determined by MTT assay. RESULTS:(1) Compared with control group, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose group gradually increased, and Smad7 protein and E-cadherin mRNA and protein gradually reduced, but the mRNA expression of Smad7 gradually increased. (2) Compared with high glucose group, as increases in OM doses, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose+different concentrations of OM groups gradually reduced, and Smad7 protein and E-cadherin mRNA and protein gradually increased, but the mRNA expression of Smad7 had no significant change. (3) Compared with high glucose group, the expression of TGF-β1 and α-SMA at mRNA and protein levels was significantly reduced, the expression of E-cadherin at mRNA and protein levels significantly increased, and the protein expression of Smad7 significantly increased, but the mRNA expression of Smad7 had no significant change in high glucose+0.50 g/L OM dynamic observation group. CONCLUSION: In NRK52E cells, oxymatrine inhibits high glucose induced EMT by down-regulating TGF-β1 and up-regulating Smad7, thus preventing the fibrosis effect of TGF-β1/Smads signaling.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.     

Effect of KLF4 on viability,apoptosis of colorectal cancer cells and chemosensitivity to cisplatin

AIM:To investigate the effect of Krüppel-like factor 4 (KLF4) on the viability, apoptosis and cisplatin chemosensitivity of colorectal cancer cells. METHODS:KLF4 expression in colorectal cancer cell lines Caco2, SW480 and HCT116 was detected by Western blot. The SW480 cells were divided into pcDNA3.1 group (transfected with pcDNA3.1 empty plasmid), pcDNA3.1-KLF4 group (transfected with pcDNA3.1-KLF4 expression plasmid) and pcDNA3.1-KLF4+cisplatin group (treated with 1 mg/L cisplatin for 48 h after pcDNA3.1-KLF4 was transfected into SW480 cells). The protein levels of KLF4, p-IκBα, cyclin D1 and survivin were determined by Western blot. The cell viability was measured by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry. The content of reactive oxygen species(ROS) was measured by DCFH-DA probe. RESULTS:The expression of KLF4 in the colorectal cancer cells were significantly lower than that in the human colon mucosal epithelial NCM460 cells (P<0.05). Compared with pcDNA3.1 group, the protein expression of KLF4 in pcDNA3.1-KLF4 group was significantly increased (P<0.05). Compared with pcDNA3.1 group, the cell viability and the protein expression of cyclin D1 and survivin were significantly decreased, and the apoptotic rate, the content of ROS and the protein level of p-IκBα were significantly increased in pcDNA3.1 group (P<0.05). Compared with pcDNA3.1-KLF4 group, the cell viability and the expression of cyclin D1 and survivin proteins were significantly decreased, and the apoptotic rate, the content of ROS and the protein level of p-IκBα were significantly increased in pcDNA3.1-KLF4+cisplatin group (P<0.05). CONCLUSION:Upregulation of KLF4 gene expression in colorectal cancer cells reduces the cell viability, induces apoptosis and increases the chemosensitivity of the cells to cisplatin. The mechanism may be related to the enhancement of intracellular ROS content and down-regulaton of the phosphorylation level of IκBα, the key molecule of NF-κB signaling pathway.