Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: Sildenafil attenuated the hypoxia-induced downregulation of PPARγ expression and inhibited the hypoxia-induced upregulation of TRPC and Ki67 expression in HPASMCs. (A) HPASMCs were identified by immunofluorescence staining for α-SMA in cells grown under normoxic conditions. (B) Western blot and (C) RT-qPCR analysis of the increase in PPARγ protein and mRNA expression in HPASMCs induced by different concentrations of sildenafil under hypoxic conditions. Each of the four groups were treated with 0, 1, 10 or 50 nM sildenafil. (D) Western blot and (E) RT-qPCR analysis of the sildenafil-mediated attenuation of hypoxia-induced downregulation of PPARγ expression and sildenafil-mediated inhibition of hypoxia-induced upregulation of TRPC and Ki67 expression in HPASMCs. There were three experimental groups: i) The normoxic control group (60 h), ii) the hypoxia group (60 h, 4% O 2 ), and iii) the hypoxia + sildenafil group (60 h, 4% O 2 , 50 nM sildenafil). Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; SMA, smooth muscle actin; TRPC, transient receptor potential canonical; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Inhibition, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: A PPARγ inhibitor (GW9662) inhibited the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs under hypoxic conditions. (A) Western blot and (B) RT-qPCR analysis of the inhibitory effects of different concentrations of a PPARγ inhibitor (GW9662) on PPARγ protein expression in HPASMCs under hypoxic conditions. Cells were treated with either 0, 1 or 10 nM GW9662. (C) Western blot and (D) RT-qPCR analysis of the inhibitory effect of a PPARγ inhibitor (GW9662) on the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs under hypoxic conditions. There were four groups: i) The hypoxic control group (60 h, 4% O 2 ), ii) the hypoxia + sildenafil group (60 h, 4% O 2 ), iii) the hypoxia + GW9662 group (60 h, 4% O 2 , 10 nM), and (iv) the hypoxia + sildenafil + GW9662 group (60 h, 4% O 2 , 10 nM). Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control, # P>0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; TRPC, transient receptor potential canonical; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: si-PPARγ reversed the sildenafil-induced downregulation of TRPC and Ki67 expression under hypoxic conditions. (A) Western blot and (B) RT-qPCR analysis of the downregulation of PPARγ protein expression in HPASMCs transfected with si-PPARγ under normoxic conditions. There were four groups: si-NC, siR-PP1, siR-PP2 and siR-PP3. (C) Western blot and (D) RT-qPCR analysis of the reversal of the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs induced by PPARγ siRNA under hypoxic conditions. There were four groups: i) The hypoxic control group (60 h, 4% O 2 ), ii) the hypoxia + sildenafil group (60 h, 4% O 2 ), iii) the hypoxia + siR-PP1 group (60 h, 4% O 2 ), and iv) the hypoxia + sildenafil + siR-PP1 group. Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; siRNA, small interfering RNA; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control, Standard Deviation, Small Interfering RNA, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Respiratory Research

Article Title: STAT6 deficiency mitigates the severity of pulmonary arterial hypertension caused by chronic intermittent hypoxia by suppressing Th2-inducing cytokines

doi: 10.1186/s12931-024-03062-z

Figure Lengend Snippet: IL-4 promotes the proliferation of hPASMCs. The concentration of IL-4 ( A ) and IL-13 ( B ) in serum from STAT6 -/- and WT mice after 6 weeks of normoxia or CIH exposure ( n = 6 per group). The concentration of IL-4 ( C ) and IL-13 ( D ) in BALF from STAT6 -/- and WT mice after 6 weeks of normoxia or CIH exposure ( n = 6 per group). ( E ) CCK8 analysis of hPASMCs with IL-4 treatment under concentration of 0, 0.5, 1,10, and 100 ng/ml. ( F ) CCK8 analysis of hPASMCs with IL-13 treatment under concentration of 0, 0.5, 1,10, and 100 ng/ml. ( G ) The percentage of Ki67-positive cells in 10ng/ml IL-13 or IL-4 treated hPASMCs. Data are presented as the mean ± SD. No significant difference is indicated by ns. Significant differences are presented as *( p < 0.05), **( p < 0.01), ***( p < 0.001) and determined by Student’s t-test unless specified. hPASMCs: human pulmonary artery smooth muscle cells; Nor: normoxic; CIH: chronic intermittent hypoxia; WT: wide type; BALF: bronchoalveolar lavage fluid

Article Snippet: hPASMCs were obtained from iCell Bioscience, Inc., Shanghai.hPASMCs were cultured at 37 °C in a humidified incubator with 5% CO 2 using PriMed-iCell-004-LS medium (iCell Bioscience, China).Cells at passages 2–5 were used for further investigation.

Techniques: Concentration Assay

Journal: Respiratory Research

Article Title: STAT6 deficiency mitigates the severity of pulmonary arterial hypertension caused by chronic intermittent hypoxia by suppressing Th2-inducing cytokines

doi: 10.1186/s12931-024-03062-z

Figure Lengend Snippet: IL-4 increased p-STAT6 expression in hPASMCs. ( A ) Western blotting was performed to assess the expression of the STAT6 and p-STAT6 in hPASMCs treated with 10ng/ml IL-4 or IL-13 for 24 h. ( B ) Quantitative analysis of relative expression analysis of STAT6 and p-STAT6 using ImageJ. ( C ) Western blotting was used to detect the expression of STAT6 and p-STAT6 in hPASMCs. ( D - E ) Quantitative analysis of STAT6 and p-STAT6 in hPASMCs using ImageJ. ( F ) Under Nor or IH conditions, hPASMCs were treated with 10 ng/ml IL-4 for 48 h. Cell proliferation was assessed using the CCK8 assay. ( G - H ) Knockdown or overexpression of STAT6 expression in hPASMCs, treat with 10 ng/ml IL-4 and culture for 48 h, then measure hPASMCs proliferation under different conditions using the CCK8 assay. Data are presented as the mean ± SD. No significant difference is indicated by ns. Significant differences are presented as *( p < 0.05), **( p < 0.01), ***( p < 0.001) and determined by Student’s t-test unless specified. Nor normoxia, IH intermittent hypoxia

Article Snippet: hPASMCs were obtained from iCell Bioscience, Inc., Shanghai.hPASMCs were cultured at 37 °C in a humidified incubator with 5% CO 2 using PriMed-iCell-004-LS medium (iCell Bioscience, China).Cells at passages 2–5 were used for further investigation.

Techniques: Expressing, Western Blot, CCK-8 Assay, Knockdown, Over Expression