Heme oxygenase decreases the generation of ROS in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells

AIM:To investigate the role of heme oxygenase (HO) in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells.METHODS:(1) Western blotting analysis was carried out to detect protein level of HO-1 in the tissues.(2) ［3H］-TdR, ［3H］-leucine incorporation was measured in cultured vascular smooth muscle cells.(3) 2,7-dichlorofluorescin diacetate (DCFH-DA) as an index was used to determine the cellular reactive oxygen species (ROS) level.RESULTS:(1) No significant difference in HO-1 protein expression level between AngⅡ-stimulated and control groups was observed, but HO-1 protein level in Hemin-induced group was higher than that in other two groups (P<0.01).No significant increase in HO-1 protein expression was found in zinc-protoporphyrin IX (ZnPPIX) group.(2) After AngⅡ stimulation, ［3H］-TdR and ［3H］-leucine incorporations of vascular smooth muscle cells (VSMCs) were increased.Hemin inhibited this increase.The higher concentration of Hemin, the more significant was the inhibitory effect.On the contrary, ZnPPIX promoted the increase in the effect of AngⅡ by inhibiting HO.(3) Fluorescence intensity in AngⅡ group was obviously higher than that in control groups (P<0.01).Compared with AngⅡ group, Hemin group decreased 62.7%, but ZnPPIX group increased 39.5%.CONCLUSION:Hemin induces HO-1 expression and inhibits the effect of AngⅡ to stimulate proliferation and hypertrophy of VSMCs.The mechanism may be related to its inhibition of ROS production.     

Endothelin receptor antagonist BQ123 inhibits calcification in vascular smooth muscle cells induced by β-glycerophosphate

AIM:To observe the effect of endothelin receptor antagonist (BQ123) on calcification in rat vascular smooth muscle cells (VSMCs) in vitro. METHODS:Calcification of cultured rat VSMCs was produced by incubation with β-glycerophosphate. Calcium content, Ca2+ deposition and alkaline phosphatase activity were analyzed to estimate the extent of calcification. The DNA synthesis was detected by ［3H］ -TdR and ［3H］-Leu incorporation. Osteopontin (OPN) mRNA was measured by competitive quantitative RT-PCR. Content of ET was measured by radioimmunoassay (RIA). RESULTS:The results showed that compared with the control, the content of calcium, ［45Ca2+］ uptake and alkaline phosphatases activities in calcified VSMCs increased by 118%, 174% and 7-fold (all P<0.01), respectively. The expression of OPN mRNA in calcified VSMCs was up-regulated by 86% (P<0.01). The calcified VSMCs grew rapidly, in which ［3H］-TdR and ［3H］-Leu were elevated by 71% and 35%. The content of ET in calcified VSMCs medium was increased by 35% as compared with control. Furthermore, calcified VSMCs plus BQ123 groups obviously relieved degree of calcification, of which calcium content, Ca2+ deposition and alkaline phosphatase activities were 33%, 37%, 40% lower than those in calcified VSMCs (P<0.01), respectively. The expression of OPN mRNA was down-regulated by 25% (P<0.01) and significantly inhibited VSMCs proliferation. CONCLUSION:BQ123 reduces VSMCs calcification, suggesting that ET promotes calcification in VSMCs mainly by ET/ ETA receptor pathway.     

NKT cells inhibit Ang II-induced phenotypic transformation of mouse vascular smooth muscle cells by down-regulating PDGFRβ

AIM To investigate the role of natural killer T (NKT) cells in the phenotypic transformation of vascular smooth muscle cells induced by hypertension in mice. METHODS The hypertension model in wild-type and CD1d gene knockout mice was established by continuous infusion of angiotensin II (Ang II) at a dose of 490 ng·kg^-1·min^-1 for 14 d, and the NKT cell specific agonist α-galactosylceramide (α-GC) was given at a dose of 100 μg/kg in wild-type mice. The blood pressure of the mice was monitored by non-invasive tail cuff method. The thickness of aortic wall was measured by HE staining, and the degree of aortic fibrosis was observed by Masson staining. The expression levels of α-smooth muscle actin (α-SMA), osteopontin (OPN) and platelet-derived growth factor receptor β (PDGFRβ) in aortic tissue were detected by Western blot. RESULTS Compared with control group, the blood pressure, the aortic wall thickness, the degree of aortic fibrosis, the secretory marker protein OPN expression and the PDGFRβ expression were all increased significantly after infusion of Ang II for 14 d (P<0.05), while the expression of contractile marker protein α-SMA was decreased (P<0.05). The above changes were aggravated after CD1d gene knockout (P<0.05), but were attenuated after administration of α-GC (P<0.05). CONCLUSION NKT cells reduces Ang II-induced phenotypic transformation of mouse vascular smooth muscle cells by reducing the expression of PDGFRβ, increasing the expression of contractile protein and decreasing the expression of secretory protein.     

Effects of angiotensinⅡ and angiotensin-(1-7) on expression of(pro) renin receptor in rat vascular smooth muscle cells

AIM: To observe the effects of angiotensinⅡ (AngⅡ) and angiotensin-(1-7) on the expression of (pro)renin receptor in rat vascular smooth muscle cells.METHODS: The cultured VMSCs were randomly divided into control group, AngⅡ group, Ang-(1-7) group, AngⅡ+losartan (an AT₁ receptor antagonist) group, AngⅡ+PD123319(an AT₂ receptor antagonist) group and CGP42112A (an AT₂ receptor agonist)group. The expression of (P)RR at protein and mRNA levels was detected by Western blotting and real-time PCR,respectively.RESULTS: Compared with control group, AngⅡ distinctly increased and Ang-(1-7) decreased the expression of (P)RR mRNA and protein in VMSCs in a dose-dependent manner (P<0.01). Compared with AngⅡ group, losartan did not inhibit the expression of (P)RR mRNA and protein in VMSCs induced by AngⅡ (P>0.05), but PD123319 did (P<0.01). CGP42112A also induced the expression of (P)RR protein and mRNA in VMSCs (P<0.01).CONCLUSION: AngⅡ induces the expression of (P)RR in VMSCs by AT₂. However, Ang-(1-7) inhibits the expression of (P)RR in VMSCs.     

Aβ1-40 induces inflammation and phenotypic switching via activation of MAPKs signaling pathway in vascular smooth muscle cells

AIM To investigate the effects of amyolid β-protein 1-40 （Aβ1-40） on inflammation, viability, migration and phenotypic switching in vascular smooth muscle cells （VSMCs）, and to analyze the underlying mechanisms. METHODS The VSMCs were treated with Aβ1-40 at different concentration gradients for appropriate time. CCK-8 and Transwell assays were performed to evaluate the viability and migration ability of VSMCs. The levels of inflammatory factors including interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）, phenotypic switching-related proteins including α?smooth muscle actin （α?SMA）, osteopontin （OPN） and Krüppel?like factor 4 （KLF4）, and mitogen-activated protein kinases （MAPKs） signaling pathway-related proteins including p-p38 MAPK, p-ERK1/2 and p-JNK were determined by Western bolt. RESULTS After Aβ1-40 treatment, the levels of inflammatory factors IL-1β and TNF-α in the VSMCs were significantly increased （P<0.05）, and the expression of phenotypic switching-related proteins was altered, as indicated by down-regulation of α?SMA and up-regulation of OPN and KLF4 （P<0.05）. Treatment with Aβ1-40 within a certain concentration range promoted the viability and migration of the VSMCs. In addition, the protein levels of p-p38 MAPK, p-ERK1/2 and p-JNK were significantly increased by Aβ1-40 treatment （P<0.05）. Furthermore, pretreatment with specific inhibitors of MAPKs pathway significantly reduced the levels of IL-1β and TNF-α （P<0.05）, and inhibited the phenotypic switching, as indicated by up-regulation of α?SMA and down-regulation of OPN and KLF4 （P<0.05）. CONCLUSION Treatment with Aβ1-40 induces the inflammation and phenotypic switching in VSMCs via activation of MAPKs signaling pathway.     

Effect of miR-206 over-expression on proliferation and migration of pap?illary thyroid carcinoma K1 cells

AIMTo determine the effect of microRNA-206 (miR-206) on proliferation and migration of human papillary thyroid carcinoma K1 cells and to explore its possible mechanism. METHODSThe expression of miR-206 in the K1 cells was detected by RT-qPCR. The cell viability was detected by CCK-8 assay. The number of viable K1 cells was counted by the method of Trypan blue exclusion. The migration ability of K1 cells was detected by Transwell chamber migration assay. Bioinformatics software was used to predict the target gene of miR-206. The targeting relationship between miR-206 and c-Met was verified by dual-luciferase reporter assay. The protein levels of c-Met, p-Met, AKT, p-AKT, mTOR and p-mTOR were determined by Western blot. RESULTSAfter the K1 cells were transfected with miR-206 mimic transiently, the relative expression of miR-206 in treatment group was significantly higher than that in blank group and negative control group (P<0.01). The results of CCK-8 assay and Trypan blue exclusion assay showed that the proliferation ability of K1 cells in treatment group transfected with miR-206 mimic was significantly inhibited compared with other groups (P<0.01). The results of Transwell assay showed that the number of migratory K1 cells in treatment group was lower than that in blank group and negative control group (P<0.01). Moreover, our results demonstrated that miR-206 directly targeted c-Met and repressed the activation of downstream AKT/mTOR signaling pathway. CONCLUSION miR-206 over-expression inhibits the proliferation and migration abilities of papillary thyroid carcinoma K1 cells, and its mechanism may be related to the inhibition of c-Met/AKT/mTOR signaling pathway.     

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

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

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

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

Protective effects of multi-enzyme Ⅱ on vascular endothelial cell injury induced by hyperlipidemia serum

AIM and METHODS: The protective effects of multi-enzyme Ⅱ was studied on cultured endothelial cells which was injuried by hyperlipidemia serum. RESULTS: Hyperlipidemia serum increased ICAM-1 expression on the surface of endothelial cells, and decreased NO₂^- release significantly (P＜0.01). ICAM-1 expression could be reduced and NO₂^- release could be enhanced markedly by multi-enzyme Ⅱ (P＜0.01). CONCLUSION: Multi-enzyme Ⅱ had an obvious protective effect on vascular endothelial cells which was injuried by hyperlipidemia seurm. Multi-enzyme Ⅱ could clean out oxide free radicals effectively because it had the acitive structure of both SOD and CAT.     

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

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

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

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

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

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

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

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