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Objective@#To investigate the effects of oxidized low-density lipoprotein/β2 glycoproteinⅠ/β2 glycoproteinⅠantibody (oxLDL/β2GPⅠ/β2GPⅠ-Ab) complex on the phenotypic transformation and lipid transpoters on the surface of rat thoracic aorta smooth muscle cell line (A7r5), and their correlation with toll-like receptor 4 (TLR4) signaling pathway. @*Methods@#A7r5 cells were stimulated by oxLDL, oxLDL/β2GPⅠ complex, oxLDL/β2GPⅠ-Ab complex, β2GPⅠ/β2GPⅠ-Ab complex and oxLDL/β2GPⅠ/β2GPⅠ-Ab complex respectively, and then total RNA and protein were collected. The expressions of α-smooth muscle actin (α-SMA), macrophage surface marker CD68, galectin-3 (LGALS3), scavenger receptor class B member 3 (CD36) and ATP-binding cassette transporter A1/G1 (ABCA1/ABCG1) were detected by real-time quantitative PCR (RT-qPCR), western blot and immunofluorescence (IF) respectively. The roles of TLR4 and its downstream signaling molecules in the phenotypic transformation and expression changes of lipid transporters of A7r5 cells induced by oxLDL/β2GPⅠ/β2GPⅠ-Ab complex were investigated by the pretreatment of TLR4 blocker TAK-242 (5 μmol/L) or c-Jun N-terminal kinases 1/2 (JNK 1/2) blocker SP600125 (90 nmol/L). @*Results@#The oxLDL/β2GPⅠ/β2GPⅠ-Ab complex significantly increased the levels of CD68 and LGALS3, and decreased the level of α-SMA, while TAK-242 could reverse this phenomenon. The oxLDL/β2GPⅠ/β2GPⅠ-Ab complex could promote the expression of CD36 and inhibit the expression of ABCA1/ABCG1, while TAK-242 and SP600125 could reverse this process. @*Conclusion@#The oxLDL/β2GPⅠ/β2GPⅠ-Ab complex promotes the phenotypic transformation of A7r5 cells to macrophage-like cells, regulates the expression of lipid transport-related molecules and enhances the ability of lipids transport into cells. TLR4 and JNK1/2 are closely related to this process.
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Objective To evaluate the effect of autophagy inhibitor 3-methyladenine (3-MA) on phenotype transformation and proliferation of rat pulmonary arterial smooth muscle cells (PASMCs).Methods Cultured PASMCs were treated with different concentrations of 3-MA (low-dose group,2.0 mmol/L;middle-dose group,4.0 mmol/L;high-dose group,8.0 mmol/L;control group,0 mmol/L).The protein expression of LC3 Ⅱ,OPN and Vimentin was detected by Western blotting.Cell proliferation was detected by MTT assay.Results The autophagy of PASMCs was decreased with the increase of the concentration of 3-MA.Compared with the control group,significantly down-regulated protein expression of LC3 Ⅱ,OPN and Vimentin was observed in 3-MA-treated cells,with significantly lower proliferation activity.Conclusion The autophagy inhibitor 3-MA significantly down-regulated the expression of LC3 Ⅱ,OPN and Vimentin in PASMCs,with inhibiting the proliferation of PASMCs.
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Objective To compare the effects of cobalt chloride (CoCl2) induced chemical hypoxia and hypoxia environment on the proliferation,migration and phenotype transformation of rat primary pulmonary artery fibroblasts (PAFs).Methods Primary PAFs were isolated and cultured.Cells were stimulated by CoCl2,or hypoxia cell culture (1% O2) was used to stimulate and induce PAFs.Then the effects of CoCI2 and hypoxia environment on PAFs were compared by CCK-8 assay,scratch assay,transwell assay,phenotype marker protein expression and PI3K/Akt signaling pathway protein expression.Results Compared with the control group,100 μmol/mL CoCl2 stimulation had no significant effect on the cell proliferation activity,cell migration ability and phenotype transformation ability of PAFs (P>0.05);while 1% O2 could significantly improve the cell proliferation and migration activities of PAFs as well as the upregulation of α-SMA expression (P<0.05).Conclusion There exist differences of effects between CoCl2 induced chemical hypoxia and hypoxia environment on promoting cell proliferation, cell migration and phenotype transformation in PAFs.
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Objective@#To investigate the effects of hypoxia on the phenotype transformation of human dermal fibroblasts to myofibroblasts and the mechanism.@*Methods@#The third passage of healthy adult human dermal fibroblasts in logarithmic phase were cultured in DMEM medium containing 10% fetal bovine serum for the following five experiments. (1) In experiments 1, 2, and 3, cells were divided into normoxia group and hypoxia group according to the random number table, with 10 dishes in each group. Cells of normoxia group were cultured in incubator containing 21% oxygen, while those of hypoxia group with 1% oxygen. At post culture hour (PCH) 0 and 48, 5 dishes of cells were collected from each group, respectively. mRNA expressions of markers of myofibroblasts including alpha smooth muscle actin (α-SMA), type Ⅰ collagen, and type Ⅲ collagen of cells were determined with real time fluorescent quantitative reverse transcription polymerase chain reaction in experiment 1. Protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen of cells were determined with Western blotting in experiment 2. The protein expression of nuclear factor-kappa B (NF-κB) of cells was determined with Western blotting in experiment 3. (2) In experiment 4, cells were divided into normoxia group, hypoxia group, and hypoxia+ pyrrolidine dithiocarbamate (PDTC) group according to the random number table, with 5 dishes in each group. Cells in the former two groups were treated the same as those in experiment 1. Cells in hypoxia+ PDTC group were treated the same as those in hypoxia group plus adding 4 mL PDTC with a final molarity of 10 μmol/L in the culture medium. At PCH 48, the protein expression of NF-κB of cells was determined with Western blotting. (3) In experiment 5, cells were divided into normoxia group, hypoxia group, hypoxia+ PDTC group, and normoxia+ PDTC group according to the random number table, with 5 dishes in each group. Cells in the former three groups were treated the same as those in experiment 4. Cells in normoxia+ PDTC group were treated the same as those in normoxia group plus adding 4 mL PDTC with a final molarity of 10 μmol/L in the culture medium. At PCH 48, protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen of cells were determined with Western blotting. Data were processed with analysis of variance of factorial design, one-way analysis of variance, and LSD-t test.@*Results@#(1) Compared with those of normoxia group at corresponding time point, mRNA expressions and protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen and the protein expression of NF-κB in fibroblasts of hypoxia group were not changed obviously at PCH 0 (with t values from -1.21 to 2.04, P values above 0.05), while mRNA expressions and protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen and the protein expression of NF-κB significantly increased at PCH 48 (with t values from -12.57 to -3.44, P values below 0.01). (2) At PCH 48, the protein expression of NF-κB in fibroblasts of hypoxia group was 0.83±0.12, significantly higher than that of normoxia group (0.17±0.06, t=-16.96, P<0.001). The protein expression of NF-κB in fibroblasts of hypoxia+ PDTC group was 0.31±0.08, significantly lower than that of hypoxia group (t=12.73, P<0.001). (3) At PCH 48, protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen in fibroblasts of hypoxia group were 0.73±0.09, 1.25±0.10, and 1.16±0.07, respectively, significantly higher than those of normoxia group (0.14±0.06, 0.87±0.08, and 0.77±0.13, respectively, with t values from 9.24 to 11.24, P values below 0.001). The protein expression of α-SMA in fibroblasts of normoxia+ PDTC group was 0.24±0.07, significantly higher than that of normoxia group (t=4.22, P<0.01). Protein expressions of type Ⅰ collagen and type Ⅲ collagen in fibroblasts of normoxia+ PDTC group were 0.25±0.06 and 0.32±0.11, respectively, significantly lower than those of normoxia group (with t values respectively -4.31 and -3.88, P values below 0.01). Protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen in fibroblasts of hypoxia+ PDTC group were 0.09±0.08, 0.38±0.12, and 0.47±0.08, respectively, significantly lower than those of hypoxia group (with t values from 11.78 to 22.98, P values below 0.001).@*Conclusions@#Hypoxia can significantly up-regulate the expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen in human dermal fibroblasts, which may promote the phenotype transformation of fibroblasts to myofibroblasts, and this is likely to be associated with the activation of NF-κB signal pathway.
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ABSTRACT:Objective To verify whether rosuvastatin can inhibit homocysteine (Hcy)‐induced rat aortic vascular smooth muscle cell (VSMC ) phenotype transformation and its potential mechanism .Methods The primary culture and identification of rat VSMCs were conducted , using VSMCs in passage4‐7 for the following experiments .The VSMCs were divided into 4 groups:control group ,Hcy (100μmol/L) group ,Hcy+ rosuvastatin group ,and Hcy+ rapamycin group .MTT was used to investigate the proliferation of VSMCs .Transwell chambers and wound healing were employed to test the migration ability of VSMCs . ICC was used to detect the VSMCs'morphology .Western blotting was used to investigate the expressions of SM‐actin ,SM‐MHC ,calponin ,OPN ,and p‐P70S6K1 in VSMCs of each group .Results Compared with those in control group , the proliferation and migration ability of VSMCs were significantly increased in Hcy modulation group (P< 0 .01) .The expressions of SM‐MCH and calponin increased but those of OPN and p‐P70S6K1 decreased in Hcy group compared with control group (P<0 .01) .The expression of SM‐actin did not significantly differ between the groups .Compared with those in Hcy modulation group , the proliferation and migration ability of VSMCs were significantly decreased in rosuvastatin and rapamycin groups ( P<0 .01) .The expressions of SM‐MCH and calponin increased while OPN and p‐P70S6K1 expressions decreased in rosuvastatin and rapamycin groups compared with Hcy group ( P< 0 .01 ) . Conclusion Hcy can induce the dedifferentiation of VSMCs ,and rosuvastatin can inhibit this effect of Hcy .Its potential mechanism may be realized via the mTOR/P70S6K1 signal pathway .
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OBJECTIVE To investigate the effect ofγ-secretase inhibitor N-[N-(3,5-difluorophen?acetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)on phenotypic transformation and matrix accu?mulation induced by aristolochic acid(AA) in renal tubular epithelial cells(NRK-52E)and explore the mechanism. METHODS NRK-52E cells were divided stochastically into normal cell control group,AA 10 mg·L-1 group and AA 10 mg·L-1+DAPT 1 and 10μmol·L-1 group. After 24 h,the mRNA expressions of Notch1,Jagged1,Numb,E-cadherin,transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),bone morphogenic protein 7 (Bmp7),typeⅠ a1 (Col1a1) and Ⅲ collagens a1 (Col3a1)were quantified by quantitative real-time RT-PCR. The protein expressions of Notch1,Jagged1,α-SMA,and Col3a1 in NRK-52E cel s were detected by immunofluorescence staining. RESULTS In NRK-52E cells,AA enhanced the expression of TGF-β1,α-SMA and Col3a1 mRNA(P<0.05),reduced the expression of E-cadherin mRNA(P<0.05),up-regulated the mRNA expression of Notch1 mRNA(P<0.01)and Jagged1(P<0.05),and down-regulated the mRNA expression of Numb mRNA(P<0.05) compared with normal cell control group,indicating that phenotypic transformation and matrix accumu?lation occurred in AA-treated NRK-52E cells,accompanied by activated Notch signaling. Treatment with DAPT inhibited Notch signaling by decreasing the expression of Notch1 and Jagged1 (P<0.05),and increasing the expression of Numb mRNA(P<0.05). Furthermore, DAPT also down-regulated the expression levels of TGF-β1,α-SMA,Col1a1 and Col3a1 mRNA(P<0.05), and up-regulated the expression level of Bmp7 and E-cadherin mRNA(P<0.05) compared with AA group,suggesting that DAPT inhibited phenotypic transformation and matrix accumulation in AA-treated NRK-52E cells. CONCLUSION AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells,which is inhibited by DAPT treatment. The possible mechanism is that DAPT suppresses the activation of Notch signaling,resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.
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AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .
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Objective To determine the efficacy of berberine in the treatment of non-alcoholic steatohepatitis ( NASH) , and to investigate the regulating effect on macrophage phenotype transformation in hepatic tissue on methionine -choline deficiency (MCD) diet induced NASH mice.Methods Fourty male C57BL/6 mice were randomly divided into 4 groups (10 mice per group): the normal group (fed with normal diet), the NASH model group (fed with MCD diet), rosiglitazone treatment group (30mg/kg) and berberine treatment group (150mg/kg).Drugs were adopted in the preventive intervention method for 2 weeks.The hepatic histopathological method was adopted to evaluate the drug therapeutic effect.The serum levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-6, and IL-10 were examined with ELISA method.M1 and M2 phenotype were detected by flow cytometry .Results The results showed berberine improved the degree of hepatic histopathology .Berberine not only reduced the level of TNF-α, but also increased the level of IL-10 in serum on NASH mice significantly ( P <0.05 ) . Flow cytometry data indicated that berberine decreased M 1 type macrophages and increased M 2 type macrophages in liver tissue .The ratio of M1/M2 was significantly decreased in berberine and rosiglitazone treated group ( P <0.01 ) .Conclusion Berberine may improve the hepatic pathological process in MCD diet induced NASH model possibly through modulating macrophage phenotype transformation , i.e.The ratio of M2 type is more than M1 type in hepatic tissue , and increasing anti-inflammatory cytokines .
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AIM:To explore the effect of extracellular signal-regulated kinase 1/2 ( ERK1/2) on the vascular adventitial remodeling in a hypertension rat model .METHODS:The rats were randomly divided into control group , mini-pump infusion of saline group and mini-pump infusion of angiotensin II ( Ang II) group as the hypertension model .The sys-tolic pressure and vascular morphology of the rats were examined .Adventitial fibroblasts were treated with Ang II , PD98059 ( ERK1/2 inhibitor) and Ang II+PD98059.The catalase ( CAT) expression in the cells was detected by Western blotting . RESULTS:Compared with control group and mini-pump infusion of saline group , the systolic pressure and carotid media thickness (stained by HE) in mini-pump infusion of Ang II group were significantly increased (P<0.01).Meanwhile, ar-tery morphology in mini-pump infusion of Ang II group had obviously changed with a significant occurrence of pathological vascular remodeling .The result of Western blotting showed that the expression of CAT in the adventitial fibroblasts treated with Ang II+PD98059 was much higher than that in the cells treated with Ang II alone (P<0.05), indicating that down-regulation of CAT induced by Ang II was restored by ERK 1/2 signaling pathway .CONCLUSION:Ang II down-regulates CAT through ERK1/2 pathway and promotes cell phenotype transformation , which lead to pathological vascular remodeling .
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ObjectiveTo study the influence of different culture conditions in vitro on phenotype, proliferation and cytoskeletal proteins expression of vascular smooth muscle cells(VSMCs). Methods The cultured VSMCs from rat aorta were divided into six groups: P2 control,P2 starvation,P4 control,P4 starvation,P6 control and P6 starvation. The proliferating cells were labeled by 5-bromodeoxyuridine (5-BrdU); The mRNA expression of smooth muscle 22 alpha (SM22α) was detected by reverse transcription-polymerase chain reaction method (RT-PCR); The cytoskeletal proteins including SMα-actin,β-Tubulin and Desmin were observed through immunohistochemical staining. Results With the increase of cell passage, cytoskeletal proteins expression of VSMCs decreased,cellular organs increased and secretory vesicles were abundant; in serum-free cultured cells mitochondria increased and electron density enhanced in cytoplasm of VSMCs.On the contrary the expression of SMα-actin decreased, and the expression of SMα-actin increased. The expression of β-Tublin and Desmin decreased more obviously, and at 6 passages failed to express. Conclusion The conditioned medium and serum-free had the different effects on the phenotype,proliferation and cytoskeleton of VSMCs in different passage, and there was internal relationship among them. The internal relationship played an important role in the maintaining of cell morphology, contractile function and vascular remodeling. The disappearance of expression of β-Tubulin and desmin might have important biological significance.
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Objective To study the effect of hypoxial endothelia cell conditional medium(HECCM)on the proliferation of pulmonary arterial muscle cell(PASMC).Methods MTT assay was used to test the proliferation,immuno-cytochemistry was used to identify the expression of ?-SM-actin.Results(1)HECCM could promote the proliferation of PASMC,down-regulate their expression of ?-SM-actin.(2)DDPH could up-regulate the expression of ?-SM-actin in PASMC which was time-dependant.Conclusions DDPH could reverse the phenotype transformation of PASMC exposed to HECCM,the action was time-dependant.After some time DDPH could reversly transform PASMC to the normal contractile phenotype.
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AIM:To provide evidence for the molecular mechanism of homocysteine (Hcy) as an independent risk factor in atherosclerosis (AS) by investigating the effect of Hcy on phenotype transformation and proliferation of vascular smooth muscle cells(VSMCs) in rats.METHODS:After treated with different concentrations of Hcy for 24 h,the cultured VSMCs were assayed for cell proliferation rate by MTT method,cell cycle by flow cytometry,the expression of SM22-? mRNA by semi-quantitative RT-PCR and the observation of morphological characteristics and the phenotype transformation by transmission electron microscopy.RESULTS:Hcy increased the cell proliferation rate and gradually reduced the proportion of the cells in G0 /G1 phase.Hcy down-regulated the expression of SM22? mRNA and the most significant effect was observed at concentration of 1 000 ?mol/L.The observations of transmission electron microscopy revealed an abundant endoplasmic reticulum,Golgi's complex,loose nucleus and puffy chromatin in VSMCs treated with high concentration of Hcy.CONCLUSION:Hcy promotes the proliferation and phenotype transformation of VSMCs simultaneously.