Role of PI3K/Akt signaling pathway in P12 cells apoptosis induced by β-amyloid peptide

AIM:To investigate the role of PI3K/Akt signaling pathway on cell apoptosis induced by β-amyloid peptide in PC12 cells. METHODS: The viability of PC12 cells and the levels of p-AktSer473 and p-GSK-3βSer9 were detected by MTT and Western blotting, respectively. Cell apoptosis was determined by Annexin V-PI staining. RESULTS: Aβ25-35 treatment decreased the viability of PC12 cells in a time-depended manner (P<0.05). Annexin V-PI staining demonstrated that Aβ25-35 induced PC12 cells apoptosis in a time-depended manner (P<0.05). Western blotting showed that Aβ25-35 induced an immediately decreased expression of p-AktSer473 and p-GSK-3βSer9 at 30 min (P<0.05), but increased at 3 h and decreased again at 6 h. However, after 12 h of peptide treatment, the p-GSK-3βSer9 increased while the p-AktSer473 remained decrease. CONCLUSION:PI3K/Akt signaling pathway is involved in cell apoptosis induced by β-amyloid peptide, which provides a new clue for the study of pathogenesis and management of AD.     

Senescence of endothelial cells and gene expression associated with apoptosis induced by angiotensinⅡ

AIM: To study the senescence of human umbilical vein endothelial cells (HUVECs) and Bcl-2, Bax gene expression associated with apoptosis induced by angiotensinⅡ (AngⅡ).METHODS: HUVECs were cultured in vitro and the cell viability was observed by methyl thiazolyl tetrazolium (MTT). HUVECs were intervened by AngⅡ and valsartan (AngⅡ type 1 receptor blocking) and divided into 3 groups: the control group, AngⅡ group (stimulated with AngⅡ10^-6mol/L for 48 h), valsartan group (valsartan was added to cells 1 h before 10^-6mol/L AngⅡ treatment). β-gal staining and cell cycle analysis were used to identify the cell aging status. Morphologic changes and percentage of apoptosis were assayed with Hoechst33258 under fluorescent microscope. The expressions of Bcl-2 and Bax, and the apoptosis-associated genes were detected by immunocytochemical staining, RT-PCR and Western blotting. RESULTS: The cell viability by AngⅡ-induced cells was (81.9%±4.1)%, the positive cell number of β-gal staining was significantly higher in AngⅡ-induced cells (80.10%±6.81)% than that in the control cells. The cell cycle was at G₀-G₁(91.36%±6.45)%, the apoptotic cells significantly increased (31.84±2.86)% under fluorescent microscope. In valsartan group, Bcl-2 mRNA and protein expression increased markedly (P<0.05), but Bax mRNA and protein expression decreased evidently (P<0.05) compared to those in the AngⅡ group.CONCLUSION: Cell apoptosis is possibly an important factor for endothelial cell senescence and vascular aging induced by AngⅡ. One of its molecular mechanisms might be associated with decreasing the expression level of Bcl-2 and increasing that of Bax, which regulate the imbalance between mRNA and protein expression of Bcl-2 and Bax. Valsartan improves endothelial cell aging.     

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.     

Antidepressant-like effect of N-palmitoylethanolamide by prefrontal cortex PPARα pathway in a rat model

AIM: To investigate the role of cortical peroxisome proliferator-activated receptor α (PPARα) in the regulation of depression-like behavior in the rats by N-palmitoylethanolamide (PEA). METHODS: A rat model of chronic unpredictable mild stress (CUMS) was established. The rats (n=70) were randomly divided into normal control group, CUMS model group, CUMS+ fluoxetine (10 mg/kg) group, CUMS+ PEA (2.5, 5 and 10 mg/kg) groups and CUMS+ PEA (10 mg/kg)+ MK886 (3 mg/kg) group. On the 8th day during CUMS, the drugs were continuously admi-nistered for 28 d. The body weight and the related behavioral changes in the open-field test and sucrose consumption test were monitored every week. On the 36th day, some of the brain tissues from the rats were fixed in 4% formalin solution for histomorphological and immunohistochemical observations to determine the number and morphological changes of prefrontal cortex (PFC) neurons and the protein expression of synaptophysin (SYP). Other brain tissues were quickly removed, PFC was separated and weighed, and Western blot and RT-PCR were used to detect the expression of PPARα at protein and mRNA levels in the PFC of rats. RESULTS: Compared with CUMS model group, PEA increased the body weight gain, the sucrose preference rate, and the locomotion time and distance in the open-field test, and shortened the immobility time in the open-field test. PEA increased the weight of PFC, the percentage of PFC/brain weight and the number of neurons in PFC, and improved the morphological changs of the neurons. PEA also up-regulated the protein expression of SYP in PFC, and down-regulated the expression of PPARα at mRNA and protein levels in the PFC of CUMS model rats (P<0.05). In addition, compared with PEA (10 mg/kg) group, MK886 significantly reduced the body weight gain of the rats, the percentage of sucrose preference and the locomotion distance in the open-field test, and increased the immobility time in the open-field test on the 35th day during CUMS. The number of neurons SYP expression in PFC tissues were decreased, and the expression of PPARα at protein and mRNA levels was increased in MK886 group. CONCLUSION: PEA may antagonize the depression-like behavior of rats by regulating the PPARα pathway in PFC, improving synaptic plasticity of PFC and protecting the neurons.     

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.     

PPARγ affects epithelial-mesenchymal transition in renal tubular epithelial cells under high glucose by regulating AKT/FAK signaling pathway through PTEN

AIM To investigate the role of peroxisome proliferator-activited receptor γ （PPARγ） in the regulation of PTEN/AKT/FAK signaling pathway and epithelial-mesenchymal transition （EMT） in renal tubular epithelial cells grown in high-glucose environment. METHODS Renal tubular epithelial cells （NRK52E cells） cultured in high glucose were used as an in vitro model system. PPARγ was over-expressed or knocked down in these cells, and its effect on PTEN expression was determined by RT-qPCR, immunofluorescence and Western blot. The changes of EMT-related proteins were also measured. The PPARγ inhibitor GW9662 and the PPARγ agonist rosiglitazone were used along with PTEN over-expression or knockdown to determine whether the effects of PPARγ were mediated through PTEN. RESULTS PPARγ over-expression resulted in the increased expression of PTEN at mRNA and protein levels, the up-regulation of E-cadherin, and the down-regulation of vimentin and α-SMA. Knockdown of PPARγ expression reduced the mRNA and protein levels of PTEN, down-regulated E-cadherin, and up-regulated vimentin and α-SMA （P<0.05）. Treatment of the NRK-52E cells with GW9662 decreased PTEN expression and increased the protein levels of p-AKT （Thr308）, FAK and p-FAK （Tyr397）. These effects were rescued by PTEN over-expression. Treatment of the NRK-52E cells with rosiglitazone increased PTEN expression and decreased the protein levels of p-AKT （Thr308）, FAK and p-FAK （Tyr397）. These effects were rescued by PTEN knockdown. These changes were all statistically significant （P<0.05）. CONCLUSION PPARγ regulates the mRNA and protein expression of PTEN in renal tubular epithelial NRK52E cells, and affects EMT in renal tubular epithelial cells. The regulation of AKT/FAK signaling pathway by PPARγ is primarily mediated by PTEN.     

Effect of ERK1/2/c-Fos signal pathway on angiotensin-(1-7) inhibiting proliferation of rat glomerular mesangial cell strain induced by angiotensin Ⅱ

AIM: To investigate the effect of ERK1/2/c-Fos signal pathway during angiotensin-(1-7) inhibiting proliferation of rat glomerular mesangial cell strain (GMCS) induced by angiotensin Ⅱ. METHODS: Rat glomerular mesangial cells (GMC) were co-cultured with angiotensin Ⅱ and different doses of angiotensin-(1-7). The numbers of GMC were evaluated by crystal violet staining. The amounts of p-ERK1/2 and c-Fos expressions were detected by Western blotting. RESULTS: Angiotensin- (1-7) showed its inhibitory effects on GMC number increasing induced by angiotensin Ⅱ as well as the amounts of p-ERK1/2 and c-Fos expressions in a concentration dependent manner. CONCLUSION: ERK/c-Fos signal pathway is involved in the inhibitory effects of angiotensin-(1-7) on angiotensin Ⅱ -induced GMC proliferation.     

Effect of PPAR γ-ACAT1 pathway on macrophage foam cell formation induced by Chlamydia pneumoniae

AIM: To investigate the expressions of peroxisome proliferator-activated receptor γ (PPAR γ) and acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT1), and to discuss the mechanisms and pathways of Chlamydia pneumoniae (C.pn)-induced macrophage foam cell formation. METHODS: THP-1-derived macrophages were incubated for 48 h with or without C.pn (1×105 to 1×106 IFU) and/or rosiglitazone (1 to 20 μmol/L), a specific PPAR γ agonist. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellular cholesterol ester were detected by enzyme-fluorescence. PPAR γ, ACAT1 mRNA and protein expressions were determined by RT-PCR and Western blotting, respectively. RESULTS: THP-1-derived macrophages infected with C.pn at concentration of 5×105 and 1×106 IFU resulted in the large accumulation of lipid droplets and the ratio of cholesteryl ester (CE) to total cholesterol (TC) was much higher than 50%when co-incubated with low density lipoprotein (LDL). C.pn up-regulated the expressions of ACAT1 mRNA and protein, and down-regulated the expressions of PPAR γ mRNA and protein in a concentration-dependent manner (P<0.05). Rosiglitazone (10, 20 μmol/L) markedly suppressed the accumulation of lipid droplets and CE by C.pn. Moreover, rosiglitazone inhibited the up-regulation of ACAT1 mRNA and protein expression by C.pn infection in a concentration-dependent manner (P<0.05). CONCLUSION: C.pn induces macrophage foam cell formation by up-regulating ACAT1 expression via PPARγ pathway, which may provide new evidences for the development and progression of atherosclerosis initiated by C.pn infection.     

Role of retinoic acid X receptor in regulation of autophagy in rat alveolar type Ⅱ epithelial cells induced by hypoxia/reoxygenation

AIM To investigate the regulatory effect of retinoic acid X receptor （RXR） on autophagy induced by hypoxia/reoxygenation （H/R） in rat alveolar type Ⅱ epithelial cells （AEC Ⅱ） and its molecular mechanism. METHODS AEC Ⅱ were cultured in normoxia. The cells growing to logarithmic growth phase were randomly divided into 5 groups： （1） control （Con） group： cells were cultured for 30 h under normal operation; （2） H/R group： cells were cultured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h; （3） DMSO group： cells were pretreated 1.5 h with medium containing less than 0.1% DMSO before modeling, and the rest were treated the same as the H/R group; （4） 9-cis-retinoic acid （9-RA） group： cells were pretreated for 1 h with 9-RA （100 nmol/L） before hypoxia; （5） HX531 group： cells were treated with 9-RA （100 nmol/L） for 0.5 h, then treatment with HX531 （2.5 μmol/L） for 1 h. CCK-8 assay was used to detect the cell viability. Immunofluorescence staining was used to observe the expression of RXRα. Transmission electron microscope was used to observe the changes of intracellular ultrastructure, and the mRNA expression of adenosine AMP-activated protein kinase （AMPK）, beclin 1, LC3, mammalian target of rapamycin （mTOR） and P62 was detected by RT-PCR. Western blot was used to detected the protein levels of p-AMPK, beclin 1, LC3-Ⅱ, p-mTOR and P62. RESULTS Compared with Con group, the cell viability in H/R, DMSO, 9-RA and HX531 groups were significantly decreased. The mRNA expression of AMPK, beclin 1 and LC3 was significantly increased, and the protein levels of p-AMPK, beclin 1 and LC3-Ⅱ were also increased. The mRNA expression of mTOR and P62 was decreased, and the protein levels of p-mTOR and P62 were also decreased （P<0.05）. The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R, DMSO and HX531 groups （P<0.05）, and no significant difference among H/R, DMSO and HX531 groups was observed （P>0.05）. CONCLUSION H/R induces autophagy of AEC Ⅱ. Activating RXR reduce the damage of AEC Ⅱ cells induced by H/R, and its mechanism may be related to the inhibition of autophagy.     

PCDH10 inhibits proliferation of breast cancer MDA-MB-231 cells by NF-κB/cyclin D1 signaling pathway

AIM To investigate the inhibitory effect of protocadherin 10 （PCDH10） on proliferation of human breast cancer cells and its possible mechanism. METHODS RT-PCR was used to measure the mRNA expression levels of PCDH10 in 4 human breast cancer cell lines and normal mammary epithelial MCF-10A cells. The breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed by recombinant lentivirus （pLV-PCDH10） infection, and blank control （blank） group and negative control （pLV-NC） group were also set up. The cell proliferation ability was detected using methyl thiazolyl tetrazolium （MTT） and colony formation experiments. The cell cycle was detected by flow cytometry. Western blot was used to determine the expression of cyclin D1, cyclin-dependent kinase 4 （CDK4）, nucear factor-κB p65 subunit （NF-κB p65） and NF-κB inhibitor α （IκBα）. RESULTS The mRNA expression levels of PCDH10 in 4 breast cancer cell lines were lower than that in normal mammary epithelial MCF-10A cells （P<0.05）. A breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed successfully. Compared with negative control group, PCDH10 overexpression significantly inhibited cell proliferation, induced cell cycle arrest at G₁ phase, down-regulated the expression of cyclin D1 and CDK4, and decreased phosphorylation of NF-κB p65 and IκBα（P<0.05）. CONCLUSION PCDH10 inhibits the proliferation and blocks cell cycle progression of breast cancer cells by targeting NF-κB/cyclin D1 signaling pathway.     

CYP450 epoxygenase/EET pathway attenuates adipose inflammation of obese mice through HIF-1α

AIM To investigate the effects of cytochrome P450 （CYP450） epoxygenase/epoxyeicosatrienoic acid （EET） pathway on insulin resistance in obese mice, and to explore the possible mechanisms. METHODS High-fat diet-induced obesity model was established in C57BL/6Cnc mice, and the obese mice were randomly divided into 3 groups, including obesity group （treated with saline; n=10）, EET group （treated with 11,12-EET; n=10） and EET inhibitor 14,15-epoxyeicosa-5（Z）-enoic acid （EEZE） group （n=10）. Normal C57BL/6Cnc mice （n=10） treated with saline served as control. Protein expression of CYP2J2 （one of CYP450 epoxygenases） and hypoxia-inducible factor-1α （HIF-1α） was measured by Western blot. Vessel-like structure was detected by immunofluorescence staining. The serum levels of insulin, tumor necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-6 and monocyte chemoattractant protein-1 （MCP-1） were measured by ELISA. RESULTS In obese mice, homeostasis model assessment of insulin resistance （HOMA-IR） values were increased, the protein level of CYP2J2 was reduced, and the protein level of HIF-1α was increased in adipose tissues as compared with the controls （P<0.05）. The serum levels of MCP-1, IL-1β, IL-6 and TNF-α were also significantly increased in obese mice （P<0.05）. After treatment with 11, 12-EET, the HOMA-IR values were decreased compared with vehicle-treated obese mice, HIF-1α expression levels were decreased in the adipose tissue, and the serum levels of MCP-1, IL-1β, IL-6 and TNF-α were reduced （P<0.05）. Immunohistochemical results of adipose tissue from vehicle-treated obese mice showed a marked decrease in vessel-like structures （CD31-positive） compared with normal control mice （P<0.05）. EET treatment significantly increased the newly formed vessel-like structures in the visceral adipose tissues of obese mice as compared with vehicle-treated obese mice （P<0.05）. CONCLUSION High-fat diet-induced obesity and insulin resistance are closely related to the CYP450 pathway. Exogenous EETs effectively decrease obesity-induced insulin resistance possibly through pro-angiogenesis and attenuation of hypoxia and inflammation.     

Proapoptotic mechanism and changes of endogenous TGF-β1 in NB4 cells induced by exogenous TGF-β1

AIM: To study the effects of transforming growth factor-β1 (TGF-β1) on cell apoptosis, cell cycle, production of endogenous TGF-β1, expressions of P27Kip1, cyclin E and bcl-2 mRNA levels in NB4 cells. METHODS: Apoptotic morphological changes were observed by Wright-Giemsa staining. Cell cycle and apoptosis were detected with flow cytometry. Semiquantitative RT-PCR was used to examine the mRNA levels of endogenous TGF-β1, P27Kip1, cyclin E and bcl-2. RESULTS: TGF-β1 significantly restrained the growth and promoted the apoptosis of NB4 cells. The blockage of NB4 cells treated by TGF-β1 at concentration of 5 μg/L was in G1 phase. Endogenous TGF-β1 mRNA expression in NB4 cells was up-regulated when the concentration of exogenous TGF-β1 was <5 μg/L. Meanwhile, the expression of endogenous TGF-β1 mRNA was down-regulated when the concentration of exogenous TGF-β1 was 10 μg/L. After treated with TGF-β1 at concentration of 5 μg/L, P27Kip1 mRNA expression in NB4 cells was up-regulated, cyclin E and bcl-2 were reduced. CONCLUSION: TGF-β1 is able to induce apoptosis and cell cycle distribution abnormally in NB4 cells by (1) Up-regulation of endogenous TGF-β1, so that NB4 cells was induced into apoptosis through consequently high expression of P27Kip1. (2) TGF-β1 may lead to cell cycle arrest by inhibiting the expression of cyclin E directly, or by inhibiting the activity of cyclin E through the increased expression of P27Kip1. (3) Down-regulation of bcl-2 induces apoptosis of NB4 cells.