Exogenous Hydrogen Sulfide Intervention In Human Umbilical Vein Smooth Muscle Cell Calcification Mechanism Research

Objectives On the basis of exploring successfully the culture method which the tissueblock repeat adherent, replication the HUSMCs calcification models, and explore theintervention mechanism of calcification HUSMCs.Method1. The method of tissue block repeat adherent in primary cells cultureUsing modified human umbilical vein tissue explant method，after primary culture cellsand then explanted the tissue in another new flask continuing culturing.The cells wereobserved using inverted microscopes for morphological, and identified human umbilicalvein smooth muscle cells by α-actin.2. The effects and underlying mechanism of H₂S on HUSNCs calcification2.1Using NaHS intervention on the basis of successfully replicated HUSMCscalcification model, and experimental group were randomly divided into6groups （n=6）:the control group （CON）, calcification group （CAL）, pure NaHS group （NaHS）, NaHSwith low-dose in calcified nutrient solution （NaHS-L group）, NaHS with moderate dosein the calcified nutrient solution （NaHS-M Group）, NaHS with high-dose in calcifiednutrient solution group （NaHS-H Group）.2.2Calcium deposition in HUSMCs were detected by Von Kossa staining, and calculatedVon Kossa staining by micro-image analysis system; the total calcium contents werecalculated by ultraviolet spectrophotometry; the alkaline phosphatase （ALP） activitywere calculated by automated enzyme-linked immunosorbent; Activity of transforminggrowth factorβ1（TGFβ1） in the nutrient solution were analysised by enzyme-linked immunosorbent assay, determineing the change of the level of TGFβ1in HUSMCs. TheTGFβ1and core binding factor α1（Cbfα1） mRNA expression were detected by real-timefluorescence quantitative PCR. The expression of osteopontin HUSMCs were analyzedby Western-blot.ResultsThe effects and underlying mechanism of H₂S on HUSNCs calcification1. H₂S can significantly reduce the calcification of the HUSMCs. Compared with CALVon Kossa staining of NaHS-L group, NaHS-M group, NaHS-H group showed brownnodules were significantly reduced （P＜0.05）; Compared with CAL group, thepercentage of positive cells in calcification, the calcium content and ALP levels ofNaHS-L group, NaHS-M group, NaHS-H group was significantly reduced respectively（P＜0.05）;2. Compared with the CAL group, TGFβ1levels of NaHS-L group, NaHS-M group,NaHS-H group were reduced respectively （P＜0.05）; expression of Cbfα1, TGFβ1mRNA and osteopontin in HUSMCs were decreased respectively （P＜0.05）.ConclusionThe role of H₂S inhibited HUSMCs calcification through influenced TGFβ1-Smads-Cbfα1signaling, weakening the key signaling molecule TGFβ1, Cbfα1expression andreducing osteopontin contents.