Effect of calcitonin gene-related peptide on myocardin expression and phenotypic switch in vascular smooth muscle cells

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on myocardin expression and phenotypic switch in vascular smooth muscle cells (VSMCs). METHODS: VSMCs were obtained by aortic tissue adherent culture and treated with angiotensin Ⅱ (AngⅡ), AngⅡ + CGRP or AngⅡ + CGRP + CGRP_8-37. The protein expression of myocardin and the phenotypic proteins of the VSMCs was detected by Western blot. RESULTS: The expression of myocardin in cultured VSMCs showed downregulation along with time expansion. The protein level of myocardin was higher at 48 h and 72 h than that at baseline in the cultured VSMCs (P<0.05). However, the myocardin was lower at 48 h and 72 h than that at baseline after treatment with CGRP in cultured VSMCs (P<0.05). Furthermore, at 48 h in cultured VSMCs, the myocardin decreased along with α-smooth muscle actin (α-SMA) (P<0.05), and osteopontin (OPN) increased (P<0.05) in AngⅡ group compared with control group. After treatment with CGRP, the levels of myocardin and α-SMA become higher (P<0.05) but OPN was lower (P<0.05) in CGRP group than those in AngⅡ group. CGRP_8-37 abrogated CGRP-induced increase in myocardin and α-SMA and decrease in OPN in CGRP_8-37 group compared with CGRP group. CONCLUSION: CGRP may regulate the phenotypic switch of the VSMCs and maintain the cells in contractile phenotype through the upregulation of myocardin protein, which may be accomplished by the combination of CGRP and its receptor.     

Cepharanthine induces miRNA expression in human lung adenocarcinoma LTEP-a-2 cells

AIM: To observe the effect of cepharanthine on human lung adenocarcinoma LTEP-a-2 cell growth, and to explore the changes of related microRNA (miRNA) expression in the cells. METHODS: LTEP-a-2 cells were treated with cepharanthine at concentrations of 0 μmol/L, 10 μmol/L, 20 μmol/L and 40 μmol/L. The growth inhibition rate was detected by MTT assay, and the cell morphological changes were observed under light microscope. The cell apoptosis was analyzed by flow cytometry. The expression of let-7c, miR-34a and miR-34b was measured by real-time PCR. RESULTS: Cepharanthine inhibited the cell activity of LTEP-a-2 cells in a dose-dependent manner. With the increase in cepharanthine concentration, the pyknosis of the cells was visible under the inverted microscope. Flow cytometry analysis found that different concentrations of cepharanthine induced the increase in the apoptotic rates of LTEP-a-2 cells. The results of real-time PCR showed that the cepharanthine also increased the expression of let-7c, miR-34a and miR-34b. CONCLUSION: Cepharanthine inhibits the growth of LTEP-a-2 cells, and induces apoptosis. Cepharanthine increases the expression of let-7c, miR-34a and miR-34b, indicating that these miRNAs in LTEP-a-2 cells has the function as tumor suppressor genes.     

High glucose induces H9c2 cardiomyocyte hypertrophy through Ca2+-CaN-NFAT3 signaling pathway

AIM: To study the morphological changes of cardiac H9c2 cells during the developmental process of fetal rat. METHODS: Embryonic rat heart-derived H9c2 cells were maintained in DMEM supplemented with 10% fetal bovine serum. The H9c2 cells were plated at a density of 6000 cells/cm and divided into 5 groups: H9c2 cells were treated with 5 mmol/L glucose, 25 mmol/L glucose, 50 mmol/L glucose, Norvasc (25 nmol/L)+25 mmol/L glucose, or Norvasc (25 nmol/L)+50 mmol/L glucose for 48 h. The morphology of H9c2 cells was observed. The cell surface area was measured by Image-Pro Plus 6.1 software. Fluorescence spectrophotometry was used to detect the concentration of intracellular calcium ion ([Ca²⁺]_i)in the cardiomyocytes. The concentration of CaN in the cell was measured by ELISA. The mRNA expression of CaNAβ, NFAT3 and β-MHC in the cells was detected by real-time PCR. The protein levels of CaNAβ, NFAT3 and β-MHC in cultural H9c2 cells were detected by Western blot. RESULTS: The mean area of the cells, the mean fluorescence value of [Ca²⁺]_i and the concentration of CaN in 25 mmol/L glucose group were higher than those in 5 mmol/L glucose group, and those were lower than those in 50 mmol/L glucose group. After treated with Norvasc, those results decreased significantly. The expression of CaNAβ, NFAT3 and β-MHC at mRNA and protein levels in 25 mmol/L glucose group was higher than those in 5 mmol/L glucose group, but was lower than those in 50 mmol/L glucose group. The expression of CaNAβ, NFAT3 and β-MHC at mRNA and protein levels decreased significantly in Norvasc treatment group. CONCLUSION: Ca²⁺-CaN-NFAT3 signaling pathway is perhaps involved in high glucose-induced H9c2 cardiomyocyte hypertrophy.     

Identification of ATTM as a novel H2S donor and investigation of its protective effect on HaCaT skin cells

AIM: To investigate the ability of a metal complex ammonium tetrathiomolybdate (ATTM) to release H₂S and its cytoprotective effect on an oxidative injury model. METHODS: Released H₂S was absorbed in a reaction flask from ATTM dissolved in the cell medium. Staining with dichlorodihydrofluorescein diacetate or rhodamine 123 followed by photofluorography was conducted for the observation of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ_m) levels, respectively. Cell viability and release of lactate dehydrogenase (LDH) from the cells were measured with commercial kits. RESULTS: Similar to another H₂S donor GYY4137, ATTM had an ability to release H₂S in the cell medium in a dose-dependent manner. Treatment of human skin HaCaT cells with ATTM at concentrations of 25~400 μmol/L didn't significantly alter cell viability. Exposure of the cells to ultraviolet rays or a ROS donor H₂O₂ increased the intracellular ROS levels. Treatment with 400 μmol/L H₂O₂ significantly reduced the viability of HaCaT cells (P<0.01). However, before the treatment with H₂O₂, pretreatment with ATTM at 100 and 200 μmol/L markedly prevented the H₂O₂-induced cell injury (P<0.01). In addition, the treatment with H₂O₂ triggered ΔΨ_m loss (P<0.01) and LDH release from the cells (P<0.01). Prior to suffering from H₂O₂ injury, the preconditioning with 200 μmol/L ATTM significantly improved ΔΨ_m levels (P<0.05) and attenuated LDH release from the cells (P<0.01).CONCLUSION: ATTM is capable of releasing H₂S and protecting human skin cells against oxidative injury.     

Effect of oxidative stress-induced autophagy on proliferation and apoptosis of MSCs

AIM: To investigate whether oxidative stress is able to induce autophagy in mesenchymal stem cells (MSCs), and to explore the effects of autophagy on MSC proliferation and apoptosis under oxidative stress circumstance as well as the underlying mechanism for promoting the therapeutic effects of transplanted MSCs on treating diabetes mellitus erectile dysfunction (DMED). METHODS: Hydrogen peroxide (H₂O₂) was applied to simulate the oxidative stress circumstance. The effects of H₂O₂ at concentration of 0, 50, 100, 200, 400 μmol/L on the viability of MSCs were tested by the method of Trypan blue exclusion and MTT assay respectively . The methods of MTT assay, Western blot and transmission electron microscope (TEM) were used to explore the effects of H₂O₂ on MSC apoptosis and autophagy. RESULTS: The proliferation of MSCs was obviously inhibited by H₂O₂ in a dose-dependent manner (P<0.01) and the 50% inhibiting concentration (IC₅₀) was (384.58±16.89) μmol/L. H₂O₂ induced apoptosis and autophay of MSCs. The proliferation rate of MSCs was suppressed by H₂O₂ significantly (P<0.05), with a further decline by blockade of autophagy (P<0.05) whereas increased by blockade of apoptosis (P<0.05). H₂O₂ induced MSCs apoptosis obviously (P<0.05), with an augment of apoptosis (P<0.05) by blockade of autophagy. Furthermore, the H₂O₂ increased expression of cleaved caspase-3 and cleavage of poly ADP-ribose polymerase 1 (PARP1), Which were decreased by apoptosis blockade whereas were enhanced by blockade of autopahgy. CONCLUSION: Oxidative stress plays a dual role in MSC survival, which induces MSC apoptosis and autophagy. Moreover, blockade of autophagy intensifies MSC apoptosis. Therefore, it is a promising method to ameliorate the effects of stem-cell based therapy on DMED by enhancing protective autophagy to increase the survival rate of transplanted MSCs against oxidative stress circumstance caused by diabetes mellitus.     

Radiosensitizing effect of baicalin on human cervical carcinoma HeLa cells

AIM: To investigate the effect of baicalin on the radiosensitization of HeLa cells. METHODS: The cell activity was determined by MTT assay. The radiosensitivity of HeLa cells was detected by colony formation assay. The cell cycle was analyzed by flow cytometry. The protein levels of Akt, p-Akt, Bad and p-Bad were examined by Western blot. RESULTS: The cell growth of the HeLa cells was inhibited by baicalin dose-dependently and IC₅₀ was 43.65 mg/L. The results of colony formation assay showed that combination of 8 mg/L baicalin and radiotherapy further improved survival curve and decreased the value of D0 and Dq, as compared with radiotherapy alone (P<0.05). Furthermore, baicalin enhanced the effect of radiotherapy on cell cycle, as evidenced by the increase in cell percentage in G₂/M phase (P<0.05). Additionally, after incubation with baicalin, radiotherapy-induced phosphorylation of Akt and Bad were further augmented (P<0.05). CONCLUSION: Baicalin augments radiosensitivity of HeLa cells through the inhibition of cell cycle transition and activation of PI3K/Akt signaling pathway.     

Effects of liquid crystal/PU composite substrate on osteogenic differentiation of rBMSCs

AIM: To explore the effect of the elastic modulus and sizes of liquid crystal (LC) phases on osteogenic differentiation based on OPC/PU composite substrate by mimicking the microenvironment in rat bone mesenchymal stem cells (rBMSCs). METHODS: A series of composite substrates with different elastic modulus were constructed via modulation of LC content in the composites. The surface phase structure was observed by polarized microscopy, and the mechanical property was measured by a universal material testing machine. Furthermore, the laser confocal microscope was employed to observe the spreading, polarization and the cytoskeleton arrangement of the rBMSCs. The proliferation of rBMSCs was evaluated by CCK-8 assay. The specific mRNA expression of osteogenic differentiation such as collagen Ⅰ, and osteopontin on the composite membranes was detected by real-time PCR. RESULTS: The size and number of LC phase increased and the elastic modulus of the composite substrates decreased with the increase of the LC content. The rBMSCs exhibited better characteristics of initial adhesion, spreading and proliferation on the OPC10-PU and OPC30-PU in the early and medium culturing. The rBMSCs displayed higher expression of collagen Ⅰ and osteopontin on the OPC10-PU in the early and medium osteogenic induction, while the high expression of these osteogenic genes occured on the OPC30-PU and OPC50-PU in later osteogenic induction. The emphasis of genetic expression was switched from collagen Ⅰ in the early and medium osteogenic induction to osteopontin in the later stage. CONCLUSION: When the content of LC remained low in the composite substrates, rBMSCs mainly responded to the mechanical stimuli induced by substrate stiffness and exhibited distinguished cellular behaviors; with the increase in the LC content, rBMSCs had strong interactions with LC by sensing the viscoelasticity of LC, probably resulted from the contribution of both substrate stiffness and the viscoelasticity of LC phase.     

Salvia extract promotes angiogenesis of myocardium in rats with myocardial infarction

AIM: To determine the effect of salvia extract on angiogenesis of the myocardium in the rats with myocardial infarction (MI) and to analyze its possible mechanism. METHODS: Left coronary artery of Sprague-Dawley rats was ligated to establish a MI model. The rats were randomly divided into MI model group, 3 different dose groups of salvia (10, 20 and 40 mg·kg^-1·d^-1), and sham operation group. Each group consisted of 8 rats. The rats in all treatment groups were orally administered with the salvia extract, and the rats in MI group and sham operation group were fed with the same volume of saline. The rats were sacrificed 4 weeks later. The hemodynamic changes of the rats were determined, and the segmental heart samples were used for morphological observation by hematoxylin and eosin staining, Masson staining, or electron microscopic analysis. The expression of vascular endothelial growth factor (VEGF) and cluster of differentiation 34 (CD34) was analyzed according to immunohistochemistry. RESULTS: Compared with sham operation group, the morphological changes of the myocardium in MI group were disordered, part of myocardial cell outline disappeared, and obvious fibrosis in the necrosis myocardial tissue and fuzzy or disappearing microvascular ultrastructure were also observed. Compared with MI group, the number of new microvessels in all the treatment groups increased obviously, and the morphological changes of the endothelial cells were relatively complete according to electron microscopy. Compared with sham operation group, the protein expression of VEGF and CD34 in the cytoplasm of the myocardial tissues in MI group increased only a little. Compared with MI group, the protein expression of VEGF and CD34 in the cytoplasm of the myocardial tissues in all treatment groups increased significantly (P<0.01). CONCLUSION: Salvia extract obviously promotes angiogenesis of the myocardial tissues in the rats after myocardial infarction.     

Promoting effects of activated Nodal signal pathway on definitive endoderm induction from mouse embryonic stem cells

AIM: To study the process of promoting mouse embryonic stem cells (ESC) to specify to definitive endoderm by up-regulating of Nodal signal pathway in order to find the best cultivated systems of differentiation of mouse ESC to definitive endoderm cells. METHODS: The cells were divided into different groups based on the culture medium: ESC group (serum-free medium + LIF), natural differentiation group (serum-free medium) and activin A group (serum-free medium +50μg/L activin A). The cells and the sterilized coverslips with cells were collected at 1, 3, 5 and 7 d of the cultivation. The proportion of CXCR4⁺ cells was detected by flow cytometry. The expression of CXCR4 was determined by immunocytochemical method, and the protein expression of OCT4 and CXCR4 was detected by Western blot. RESULTS: The proportion of CXCR4⁺ cells showed no dramatic change in ESC group along with the extending of cultivation day, while there were gradually increased in natural differentiation group and activin A group and the highest level was observed at 5 d. Among the 3 groups, the proportion of CXCR4⁺ cells at 5 d was the highest in activin A group. The brown or tan staining in the cells observed under microscope was considered as positive CXCR4 by immunocytochemistry. The protein levels of OCT4 and CXCR4 in ESC group along with the extending of cultivation days was observed. The expression levels of OCT4 were gradually decreased in the cells in natural differentiation group and activin A group, while those of CXCR4 were gradually increased with the highest level at 5 d. It was highest in the cells in activin A group. CONCLUSION: The proportion of definitive endoderm was the highest at 5 d of the induction during in vitro mouse ESC differentiation. Up-regulation of Nodal signal pathway by adding activin A at the early stage of mouse ESC differentiation promotes mouse ESC to specify to definitive endoderm with CXCR4 molecular marker.     

miR-193b enhances cytotoxicity of doxorubicin by targeting Mcl-1 in breast cancer

AIM: To investigate the effect of microRNA(miR)-193b on doxorubicin therapy in breast cancer in vitro.METHODS: miR-193b level in plasma was detected by real-time PCR in the patients with breast cancer or the healthy controls. MTT assay was performed to measure the inhibitory effect of miR-193b plus doxorubicin on the growth of MDA-MB-231 cells. Bioinformatics, real-time PCR and Western blot were performed to determine whether the expression of Mcl-1 was regulated by miR-193b. Mcl-1 expression vector was constructed, and the role of Mcl-1 vector toward miR-193b plus doxorubicin-induced cytotoxicity in MDA-MB-231 cells was observed by MTT assay.RESULTS: Down-regulation of miR-193b was found in breast cancer patients. The miR-193b plus doxorubicin group showed a higher growth inhibition than cisplation group in MDA-MB-231 cells. The expression of Mcl-1 at both mRNA and protein levels was down-regulated after miR-193b transfection. The growth inhibition of MDA-MB-231 cells treated with miR-193b plus doxorubicin was significantly decreased after the transfection of Mcl-1 expression vector.CONCLUSION: miR-193b sensitizes doxorubicin-induced cytotoxicity by targeting Mcl-1 in breast cancer.