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1.
AIM: To investigate the effects of high mobility group A2(HMGA2) gene knockdown on the cell viability, apoptosis, collagen synthesis and oxidative stress of human embryonic lung fibroblast (HELF) induced by transforming growth factor-β1 (TGF-β1). METHODS: The HELF were divided into blank group, TGF-β1 group,negative control (NC) group and HMGA2 siRNA(si-HMGA2) group. The protein levels of HMGA2, AKT and p-AKT were determined by Western blot. The cell viability and apoptotic rate was analyzed by MTT assay and flow cytometry,respectively. The mRNA expression of collagen I (COL-Ⅰ) and COL-Ⅲ was detected by RT-qPCR. DCFH-DA was used to detect the content of reactive oxygen species (ROS). RESULTS: Compared with blank group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in TGF-β1 group were significantly increased, but the apoptotic rate and ROS level were significantly decreased (P<0.05). Compared with TGF-β1 group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in si-HMGA2 group were significantly decreased, but the apoptotic rate and ROS level were significantly increased (P<0.05). CONCLUSION: Knockdown of HMGA2 gene expression decreases the viability and collagen synthesis, and promotes apoptosis and ROS production of human embryonic lung fibroblasts induced by TGF-β1. The mechanism may be related to down-regulation of PI3K/AKT signaling pathway.  相似文献   

2.
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.  相似文献   

3.
AIM： To explore the protective effect of carnosine (Car) on cardiomyocytes with high glucose (HG)-induced injury. METHODS：Rat H9c2 cardiomyocytes were cultured in vitro and divided into three groups: normal control (NC) group, HG group and Car pretreatment (Car+HG) group. The survival rate of H9c2 cells was measured by MTT assay. Intracellular level of reactive oxygen species (ROS) was detected by fluorescent probe DCFH-DA. The protein expression of caspase-8, caspase-9 and caspase-3 was determined by Western blotting. RESULTS：The survival rate of H9c2 cells decreased with the increases in glucose concentration and time, while pretreatment with 20 mmol/L Car could increase the survival rate significantly (P<0.05). The intracellular level of ROS in HG group was significantly increased compared with NC group (P<0.05), while that in Car+HG group was significantly decreased compared with HG group (P<0.05). The expression of caspase-8, caspase-9 and caspase-3 proteins in HG group was significantly increased compared with NC group (P<0.05). Compared with HG group, the expression of caspase-9 and caspase-3 was significantly decreased in Car+HG group (P<0.05), but the expression of caspase-8 did not obviously change (P>0.05). CONCLUSION：Carnosine can protect H9c2 cells against the injury of oxidative stress and apoptosis induced by high glucose.  相似文献   

4.
AIM:To investigate the effect of apelin-13 on nicotine-induced apoptosis of cardiomyocytes and its potential molecular mechanism. METHODS:Rat H9c2 cells were treated with nicotine (10 μmol/L) to induced apoptosis. Flow cytometry was used to detect apoptotic rate. Western blot was used to determined the expression of related proteins. RESULTS:Compared with control group, nicotine treatment significantly increased the apoptotic rate of the H9c2 cells (P<0.01), and the protein levels of apoptosis-related proteins Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with nicotine group, apelin-13+nicotine significantly decreased the apoptotic rate of the H9c2 cells (P<0.01) and the the protein levels of Bax and cleaved caspase-3, but markedly increased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with apelin-13+nicotine group, apelin-13+nicotine+PI3K/Akt inhibitor LY294002 significantly increased the apoptotic rate of the H9c2 cells (P<0.01) and the protein levels of Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt and p-PI3K (P<0.05). CONCLUSION:Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells through PI3K/Akt signaling pathway.  相似文献   

5.
AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by LipofectamineTM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca2+ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca2+ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca2+ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.  相似文献   

6.
AIM: To investigate the protective effect of astragaloside IV (ASIV) on angiotensin II (Ang II)- induced apoptosis of H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were treated with different concentrations of Ang II and ASIV. The effects of Ang II and ASIV on the viability of H9c2 cells was measured by CCK-8 assay. The optimum concentration of Ang II was 1 μmol/L and the concentrations of ASIV were 25, 50 and 100 μmol/L. The H9c2 cells was divided into 6 groups:control group, ASIV group, Ang II group, Ang II+ASIV (25 μmol/L) group, Ang II+ASIV 50 (μmol/L) group and Ang II+ASIV (100 μmol/L) group. The morphological changes of the H9c2 cells were observed under inverted phase-contrast microscope. Apoptosis was detected by TUNEL assay. The generation of reactive oxygen species (ROS) was detected by DCFH-DA staining. The protein expression of Bax, Bcl-2, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was determined by Western blot. H9c2 cardiomyocytes were transfected with negative control shRNA (NC) or Nrf2-shRNA (shRNA), and the cells were divided into 8 groups:NC+control group, NC+AngⅡgroup, NC+ASIV group, NC+AngⅡ+ASIV group, shRNA+control group, shRNA+AngⅡgroup, shRNA+ASIV group and shRNA+AngⅡ+ASIV group. ROS level was detected by ROS detection kit. The protein expression of Nrf2 and HO-1 was determined by Western blot. RESULTS: Ang II decreased the viability of H9c2 cells in a concentration-dependent manner (P<0.05). ASIV reversed the effect of Ang II on the viability of H9c2 cells in a concentration-dependent manner (P<0.05). Compared with control group, the apoptotic rate, the level of ROS and the protein expression of Bax in Ang II group were increased significantly, while the protein expression of Bcl-2, Nrf2 and HO-1 was decreased significantly (P<0.05). Compared with Ang II group, ASIV reversed the increase in apoptotic rate of H9c2 cells induced by Ang II in a concentration-dependent manner, reduced ROS level, down-regulated the protein expression of Bax and up-regulated the protein expression of Bcl-2, Nrf2 and HO-1 (P<0.05). After shRNA transfection, the effects of ASIV decreasing ROS production induced by Ang II and up-regulating the expression of Nrf2 and HO-1 were eliminated. CONCLUSION: ASIV protects H9c2 cardiomyocytes from apoptosis induced by Ang II, which may be related to reducing ROS generation and mediating the Nrf2/HO-1 signaling pathway.  相似文献   

7.
AIM:To observe the effect of p53 agonist nutlin-3 on the proliferation, apoptosis and expression of extracellular matrix in the glomerular mesangial cells (MCs) cultured in high glucose, and to explore its possible mechanism. METHODS:After successful modeling of diabetic nephropathy (DN), PAS staining was performed on the kidney tissues to observe pathological changes. In addition, the p53 expression in the kidney tissue was detected by immunohistochemical staining. The mesangial cell SV40 was cultured in vitro and divided into mannitol (Motl) group, normal glucose (NG) group, high glucose (HG) group, high glucose plus nutlin-3 (HG+Nut) group and nutlin-3 control (Nut) group. The cell viability was detected by CCK-8 assay, and the living cell counting was performed by the method of Trypan blue exclusion. The apoptosis was analyzed by flow cytometry. The protein levels of collagen type IV (Col-IV), p53, p-p53, Bcl-2 and Bax were determined by Western blot. RESULTS:The significant increases in the proliferation of mesangial cells and the levels of p53 in renal tissue of diabetic nephropathy mice were observed. The results of CCK-8 assay showed that high glucose promoted the viability of mesangial cells, and nutlin-3 at 40 μmol/L inhibited the viability of mesangial cells in high glucose environment. Western blot analysis showed that the protein levels of p53, Bax and p-p53 were significantly increased (P<0.05), and the protein levels of Bcl-2 and Col-IV was decreased in HG+Nut group compared with HG group (P<0.05). Furthermore, the ratio of Bax/Bcl-2 was greater than 1. The results of flow cytometry showed that compared with HG group, nutlin-3 promoted the apoptosis of mesangial cells in high glucose environment, the apoptotic rate was significantly increased (P<0.05).CONCLUSION:High glucose promotes the proliferation of mesangial cells. p53 agonist inhibits the viability and promotes apoptosis of mesangial cells under high glucose.  相似文献   

8.
FU Liang  PAN Rui  CHEN Zhao 《园艺学报》2019,35(4):606-613
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.  相似文献   

9.
ATM: To explore whether the C-reactive protein (CRP) level in microinflammation state induces the apoptosis of renal tubular epithelial cells. METHODS: HK-2 cells were stimulated with recombinant human CRP. Annexin-FITC-PI staining and flow cytometry were used to detect the percentage of apoptotic cells. Morphology observation of apoptosis was assessed by Hoechst 33258 staining. Caspase-3 activity was measured by a colorimetric assay. The expression of apoptotic gene bax and anti-apoptotic gene bcl-2 at mRNA levels was determined by real-time PCR. RESULTS: CRP induced apoptosis of HK-2 cells in a time- and dose-dependent manner. The maximal apoptotic effect of CRP concentration was 10 mg/L CRP at concentration of 20 mg/L. CRP treatment was associated with the characteristic morphological features of apoptosis such as condensation, fragmentation or margination of nuclear chromatin. CRP exposure increased caspase-3 activity, up-regulated the mRNA expression of Bax and down-regulated the mRNA expression of Bcl-2. CONCLUSION: Slightly increased CRP level has the potential to induce apoptosis of renal tubular cells.  相似文献   

10.
AIM: To investigate the effect of reactive oxygen species (ROS) on the apoptosis of HepG2 cells induced by artesunate. METHODS: The effect of artesunate on the viability of HepG2 cells was measured by MTT assay. The morphological changes of the apoptotic cells were observed by the method of Hoechst 33258 fluorescence staining.The apoptosis of HepG2 cells was analyzed by flow cytometry. DCFH-DA was used to detect the changes of ROS generation during the process of apoptosis. The protein levels of Bax, Bcl-2, cleaved caspase-3 and cytochrome C (Cyt C) were determined by Western blot. HepG2 cells were pretreated with apocynin and then Western blot was used to detect the expression of p47phox and p22phox, and ROS changes were analyzed by flow cytometry. RESULTS: Compare with control group, the cell viability was obviously inhibited after treatment with artesunate for 24 h (P<0.05). The nuclei were densely stained, and the proportion of apoptotic cells was increased (P<0.05). ROS was increased significantly (P<0.05). The results of Western blot demonstrated that the expression level of Bax was increased, Bcl-2 was decreased, the ratio of Bax/Bcl-2 was increased, and the protein levels of cleaved caspase-3 and Cyt C were increased. Pretreatment with apocynin reduced the expression of p47phox and p22phox and the generation of ROS in the artesunate treatment group. CONCLUSION: Artesunate induces the apoptosis of HepG2 cells. The possible mechanism may be related to the increase in the generation of ROS.  相似文献   

11.
AIM To observe the effect of Jinlida combined with Tongxinluo on the apoptosis of renal microvascular endothelial cells in the high-glucose environment, and to explore their mechanism of protecting renal microvascular endothelial cells. METHODS The renal microvascular endothelial cells cultured in vitro were divided into control group, high glucose group, Tongxinluo group, Jinlida group, and Tongxinluo+Jinlida group. After intervention for 24 h, CCK-8 assay was used to measurethe cell viability. The apoptotic rate and reactive oxygen species (ROS) level were measured by flow cytometry. Western blot was used to determine the protein levels of protein kinase R-like endoplasmic reticulum kinase (PERK), phosphorylated PERK (p-PERK), glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), caspase-12 and Bcl-2. RESULTS Compared with control group, the viability of renal microvascular endothelial cells in the high-glucose environment was decreased, the apoptotic rate, the ROS level and the protein levels of PERK, p-PERK, GRP78, CHOP and caspase-12 were increased, while Bcl-2 protein was decreased (P<0.05). In comparison with high glucose group, the viability of renal microvascular endothelial cells in Tongxinluo, Jinlida and Tongxinluo+Jinlida groups was increased to varying degrees, the apoptotic rate, the ROS level and the protein levels of PERK, p-PERK, GRP78, CHOP and caspase-12 were decreased, while Bcl-2 protein was increased (P<0.05). Compared with Jinlida group and Tongxinluo group, the improvement of each index in Jinlida+Tongxinluo group was statistically significant. CONCLUSION Jinlida and Tongxinluo attenuate the damage of renal microvascular endothelial cells in the high-glucose environment, and the mechanism may be related to the reduction of endoplasmic reticulum stress-mediated apoptosis pathway. The combined application of Jinlida and Tongxinluo synergistically enhances the protective effect of the drugs on the renal microvascular endothelial cells.  相似文献   

12.
AIM: To investigate the effect of calcium-regulated heat stable protein 1 (CARHSP1) gene expression on the viability, apoptosis and expression of interleukin-6 (IL-6) and C-reactive protein (CRP) in vascular endothe-lial cells induced by hypoxia.METHODS: The protein expression of CARHSP1 was detected by Western blot in atherosclerotic plaques. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia, and the cells were divided into normal culture group, hypoxia group, hypoxia+CARHSP1-siRNA group and hypoxia+pcDNA3.1-CARHSP1 group. The viability and apoptotic rate of the HUVECs were measured by CCK-8 assay and flow cytometry, respectively. The mRNA expression of IL-6 and CRP was detected by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot.RESULTS: The protein expression of CARHSP1 in atherosclerotic plaques was significantly higher than that in control group (P<0.05). Hypoxia significantly increased the expression of CARHSP1. The cell viability and the protein expression of Bcl-2 were significantly lower in hypoxia group than those in normal culture group (P<0.05). The apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were significantly higher than those in normal culture group (P<0.05). Compared with hypoxia group, the cell viability and protein expression of Bcl-2 were significantly increased in hypoxia+CARHSP1-siRNA group, while the apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were decreased significantly (P<0.05). The cell viability and protein expression of Bcl-2 were decreased significantly in hypoxia+pcDNA3.1-CARHSP1 group, while the apoptotic rate and the protein le-vels of IL-6, CRP, cleaved caspase-3 and Bax were increased significantly (P<0.05).CONCLUSION: The expression of CARHSP1 is increased in atherosclerotic plaques, and inhibition of CARHSP1 expression improves the viability, reduces the apoptosis, and down-regulates the expression of IL-6 and CRP in the HUVECs. Over-expression of CARHSP1 exerts the opposite effect.  相似文献   

13.
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14.
AIM: To investigate the effect of vitamin D3 up-regulated protein 1 (VDUP-1) on apoptosis of renal tubular epithelial cells induced by high glucose and its mechanism. METHODS: Human renal proximal tubular epithelial cell line HK-2 was treated with high glucose. The mRNA and protein levels of VDUP-1 in HK-2 cells were detected by real-time PCR and Western blot. HK-2 cells were transfected with VDUP-1 small interfering RNA (siRNA). Real-time PCR and Western blot were used to detect the inhibitory effect. The HK-2 cells were treated with high glucose, and the change of VDUP-1 expression was detected. The apoptosis was analyzed by flow cytometry. The activities of caspase-3 and caspase-9 in the cells were measured. The tumor necrosis factor-α (TNF-α) content in the culture supernatant was examined by ELISA. The key proteins of Sonic hedgehog (Shh) signaling pathway, Patched 1 (Ptch1), Smoothened (Smo), zinc finger protein Gli2 and Shh, were determined by Western blot. The HK-2 cells were treated with exogenous Shh, and the levels of Ptch1, Smo and Gli2 were detected by Western blot. After the HK-2 cells with VDUP-1 silencing were treated with exogenous Shh and high glucose, the apoptosis was analyzed by flow cytometry, the activities of caspase-3 and caspase-9 in the cells were examined, and the TNF-α content in culture supernatant was measured by ELISA. RESULTS: High levels of VDUP-1 mRNA and protein were observed in the HK-2 cells treated with high glucose. The mRNA and protein levels of VDUP-1 were decreased in the HK-2 cells transfected with VDUP-1 siRNA(P<0.05). Compared with the normally cultured cells, the apoptotic rate of HK-2 cells was increased after high glucose treatment, and the activities of caspase-3 and caspase-9 and the content of TNF-α were also significantly increased (P<0.05). After down-regulation of VDUP-1 expression by siRNA transfection, the apoptotic rate of HK-2 cells decreased after high glucose treatment, and the activities of caspase-3 and caspase-9, and the content of TNF-α were also significantly decreased (P<0.05). The protein levels of Ptch1, Smo, Gli2 and Shh were decreased after high glucose culture, while down-regulation of VDUP-1 partly antagonized the effect of high glucose on the expression of Ptch1, Smo, Gli2 and Shh in the HK-2 cells. Exogenous Shh promoted the expression of Ptch1, Smo and Gli2, and inhibited the apoptosis of the HK-2 cells induced by high glucose. Exogenous Shh and down-regulation of VDUP-1 synergistically inhibited high glucose-induced apoptosis of the HK-2 cells. CONCLUSION: Down-regulation of VDUP-1 expression inhibits high glucose-induced apoptosis and release of TNF-α in renal tubular epithelial cells by activating Shh signaling pathway.  相似文献   

15.
AIM: To investigate the effect of hydrogen sulfide (H2S) on high glucose （HG）-induced injury of the mouse podocyte cell line MPC5. METHODS: The cultured MPC5 cells were randomly divided into 4 groups: HG group, normal glucose (NG) group, NG+DL-propargylglycine (PPG) group, and HG+NaHS group. After treated for a certain time, the cells were collected for further detection. The expression of zonula occludens-2 (ZO-2), nephrin, β-catenin and cystathionine γ-lyase (CSE) was determined by Western blotting. RESULTS: High glucose significantly reduced the expression of nephrin, ZO-2 and CSE (P<0.05), while the level of β-catenin was elevated obviously (P<0.05), all in a time-dependent manner. NG+PPG inhibited the levels of ZO-2 and nephrin significantly (P<0.05), and increased the level of β-catenin (P<0.05), all in a PPG concentration-dependent manner. HG+NaHS induced a more significant increase in the levels of ZO-2 and nephrin as compared with HG group (P<0.01), whereas a severe reduction of β-catenin in HG+NaHS group was observed as compared with HG group. Compared with NG group, the expression of ZO-2 and nephrin was decreased obviously, and the level of β-catenin was increased in HG+NaHS group. CONCLUSION: Down-regulation of CSE contributes to hyperglycemia-induced podocyte injury. Exogenous H2S protects against hyperglycemia-induced podocyte injury, possibly through up-regulation of ZO-2 and subsequent suppression of Wnt/β-catenin pathway.  相似文献   

16.
AIM To investigate the effects of carboxy terminus of heat shock protein 70-interacting protein (CHIP) on high glucose (HG)-induced vascular endothelial cell injury. METHODS Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with 5.5 mmol/L glucose (normal glucose， NG) or 25.5 mmol/L glucose (HG) for 24 h. Down-regulation of CHIP expression by RNA interference was conducted. Before the experiment, mannitol was used to eliminate the interference of osmotic pressure. Subsequently, the cells was divided into 4 groups: NG+siRNA NC group, NG+siRNA CHIP group, HG+siRNA NC group, and HG+siRNA CHIP group. Additionally, MTT assay and TUNEL staining were used to detect the viability and apoptosis. The level of endothelin-1 (ET-1) was measured by ELISA, and the level of reactive oxygen species (ROS) was detected by fluorescence probe dihydroethidium. The level of nitric oxide (NO), and the activity of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in the cells were detected by their respective kits. The mRNA expression of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) was detected by RT-qPCR. The protein levels of CHIP, NADPH oxidase (NOX) 2, NOX4, p38, p65, p-p38, p-p65, Bax and Bcl-2 were determined by Western blot. RESULTS Compared with NG+siRNA NC group, the cell viability was decreased, the apoptosis rate, the mRNA expression of IL-8 and MCP-1, and the level of ROS were increased (P<0.05), the activity of SOD was decreased (P<0.05), while the levels of ET-1, NO and iNOS and the protein levels of p-p38, p-p65 and Bax were increased in HG+siRNA NC group (P<0.05). Compared with HG+siRNA NC group, the inflammatory response, the oxidative stress, the apoptosis rate, and the protein levels of p-p38, p-p65 and Bax were significantly increased in HG+siRNA CHIP group (P<0.05). CONCLUSION Down-regulation of CHIP expression aggravates HG-induced vascular endothelial cell injury.  相似文献   

17.
AIM: To investigate the protective effects of DAPT on rat model with atherosclerotic (AS) ischemic brain stroke by blocking Notch pathway. METHODS: SD rats (n=24) were randomly divided into control group and model group, and the rats in model group were fed high-fat diet for 6 weeks to establish the AS model. The AS rats were randomly divided into 3 groups (n=6 in each group):AS-sham group, AS rats with ischemia (AS-ishemia) group, and DAPT administration (AS-ishemia-DAPT) group. The histopathological changes of carotid aorta were observed by HE staining. The serum levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by automatic biochemical analyzer. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by ELISA. The protein levels of Notch1 and Hes1 in rat artery, and nuclear factor-κB (NF-κB) and Toll-like receptor 4 (TLR4) in rat brain were determined by Western blot. RESULTS: Notch signaling pathway inhibitor DAPT significantly reduced intimal thickening, vascular stenosis and the formation of AS plaque. Compared with AS-ischemia group, the serum levels of lipids and inflammatory factors were decreased significantly in AS-ischemia-DAPT group, and the protein levels of Notch1 and Hes1 in the rat carotid artery and NF-κB and TLR4 protein expression in rat brain were also decreased significantly (P<0.05). CONCLUSION: Blocking Notch pathway by DAPT not only improves the blood lipid levels, but also inhibits the serum inflammatory cytokine release and NF-κB/TLR4 pathway activation.  相似文献   

18.
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19.
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 LipofectamineTM 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.  相似文献   

20.
AIM: To study the expression of Jagged2/Notch3 signaling molecules in pulmonary vascular wall of pulmonary hypertensive rats induced by monocrotaline. METHODS: SD rats were randomly divided into normal control group (C group,n=15), solvent control group (S group,n=15) and monocrotaline model groups (M group,n=15). The model of pulmonary hypertension was established by a single subcutaneous injection of monocrotaline (50 mg/kg). The rats in S group were given a single subcutaneous injection of the same dose of solvent. After 4 weeks, the pulmonary vascular remodeling was assessed by HE staining, and the mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) were determined by right heart catheterization. The expression of Jagged2/Notch3/Hes5 molecules in the pulmonary vascular wall was detected by immunohistochemical method and real-time PCR. RESULTS: Compared with S group and C group, the percentage of medial wall thickness of smaller arteries in model group increased significantly (P<0.01). The levels of mPAP and RVSP in M group were significantly higher than those in S group and C groups (P<0.01). The results of real-time PCR showed that the expression of Jagged2, Notch3 and Hes5 was significantly increased in M group compared with S group and C group. The data from immunohistochemical detection indicated that Jagged2 mainly expressed in the intima of small lung artery, Notch3 and Hes5 mainly expressed in the medial smooth muscle cells. Compared with S group and C group, the expression of Jagged2 and Notch3 was significantly increased in the lung small arteries of M group. CONCLUSION: The activation of Jagged2/Notch3 signaling pathway might play an important role in the formation of pulmonary hypertension.  相似文献   

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