| Objective Establishing pulmonary arterial hypertension (PAH) induced by high pulmonary blood flow rats model by abdominal shunt surgery, to explore effects of proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) by a special TMEM16A inhibitor (T16Ainh-A01).Methods thirty Sprague Dawley rats were randomly divided into normal group, sham group and shunt group. Shunt group established by abdominal aorta-inferior vena cava shunting surgery was left to right shunt PAH model induced by high pulmonary blood flow; animals in sham group underwent abdominal surgery, except for the shunt; normal group received no treatment. All rats were measured pulmonary artery pressure after 11 weeks. Lung tissue was stained by HE and structures of pulmonary arteries were seen under light microscopy; primary cultured PASMCs were separated by method of tissue block anchorage and primary cultured and identified by methods of cell morphology, immunocytochemistry and immunofluorescence; immunofluorescence assay verified expression of TMEM16A in PASMCs among groups; PASMCs among three groups were respectively intervened by 10μM T16Ainh-A01 or 20μM T16Ainh-A01 or 50μM T16Ainh-A01 for 24h, and detected the state of cell proliferation by CCK-8 assay; PASMCs above were intervened by 20μM T16Ainh-A01 or 50μM T16Ainh-A01 for 24h and to explore changes of cell migration numbers with transwell chamber assay.Results (1) pulmonary arterial pressure:respectively compared with pulmonary artery systolic, diastolic and mean pressure of normal and sham group, those of shunt group were all significantly increased, the differences were all statistically significant (P<0.01). Compared with sham group, pulmonary artery systolic, diastolic and mean pressure of normal group had no difference and there was no significant (P>0.05). Information above pointed out successfully established animal models.(2) Pathology of lung tissue by HE staining revealed small pulmonary arterial wall of shunt group was obviously thickening and luminal stenosis, while normal and sham group had no those pathological changes.(3) PASMCs tended to be long spindle and nuclear oval and partially overlapping regions of cells grew in a typical "peak-valley" mode under light microscope. Immunology results showed characteristic filaments of PASMCs paralleled to cell long axis were stained brown and red fluorescent marked filament were showed and nuclear non-staining.(4) TMEM16A marked with green fluorescence were visible and nuclear non-staining among three groups, while green fluorescence in PASMCs of shunt group was more brighter and larger wide range than that of normal and sham group.(5) Cell Proliferation:comparison of PASMCs viability (%) among groups intervened by 10μuM T16Ainh-A01 for 24h showed no significantly difference (P>0.05); Intervened by 20μM T16Ainh-A01 for 24h, comparison of that among groups were significantly different (P<0.05):compared with normal group (92.21±1.25), shunt group (88.82±1.92) was significantly decreased, there was statistically significantly different (P<0.05), while compared with sham group (90.42±3.12), shunt group was not significantly decreased, there was no statistically difference (P>0.05) and difference between normal and sham group was not significant(p>0.05); Intervened by 50μM T16Ainh-A01 for 24h, cell viability (%) among groups had significantly difference(P<0.01):compared with that of normal group (87.41±3.27) or sham group (83.73±4.05), shunt group (74.48±5.95) was significantly decreased and there both had statistical differences (P<0.01) and compared with normal, sham group had mild decreased and there was statistical difference(P<0.05).(6) Cell migration:Intervened by 20μM T16Ainh-A01 for 24h among groups, compared with migrating cell numbers (n) of non-intervention normal group (83.75±1.26) and non-intervention sham group (88.75±2.21), intervention normal group (76.25±2.87) and intervention sham group (81.75±2.06) decreased respectively and differences were both statistically significant (P<0.05), and compared with migration cell number (n) of non-intervention shunt group (123.75±3.77), intervention shunt group (92.25±4.27) significantly decreased and difference was statistically significant (P<0.01). Meanwhile compared with inhibition rate (%) of normal group (8.98±2.21) or sham group (7.85±2.88), shunt group (25.32±5.72) was more obvious and there existed statistical difference (P<0.01), while normal and sham group showed no significant difference (P>0.05); condition exchanged into 50μM T16Ainh-A01, compared with migrating cell numbers (n) of non-intervention normal group (86.25±4.40) and non-intervention sham group (86.25±4.57), intervention normal group (63.00±8.49) and intervention sham group (64.00±8.83) decreased respectively and differences were both statistically significant (P<0.05), similarly migration cell numbers (n) of intervention shunt group (72.25±9.05) was significantly decreased, compared with non-intervention group (135.75±6.23) and difference was statistically significant (P<0.01). Meanwhile inhibition rate (%) of shunt group (46.78±6.31), compared with normal group (27.10±7.64) or sham group (25.92±8.15), remain more obvious and difference was statistically significant (P<0.01), while normal and sham group showed no significant difference (P>0.05).Conclusion (1) TMEM16A overexpressed in PASMCs of PAH rats may play an important role in pulmonary vascular remodeling, and thus participate in PAH induced by high pulmonary blood flow on regulating PASMCs proliferation and migration. (2) PASMCs proliferation and migration of PAH rats induced by high pulmonary blood flow were inhibited by T16Ainh-A01 in vitro by inhibiting TMEM16A and that provided a new horizon on treatment of PAH. |
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