Effects And Mechanisms Of SM_22α On The Transformation Of Rat Vascular Adventitial Fibroblasts Induced By SWCNTs

Background:Since the 21th century, the nanoscience progessed leaps and bounds, and the engineered nanomaterials have been mass-produced and widely applied. Simultaneously, people are increasingly exposed to kinds of manufactured nanoparticles. Though the biological effects of some nanomaterials have been already assessed, information on vasculotoxic is limited and the direct and indirect effect on the cardiovascular system and the molecule mechanisms of nanomaterials toxicology is identified. The cardiovascular diseases are the kind of graveness disease. People's health and life were harmly influenced by these kinds of disease, so carry out the nanomaterials safety assessment is very essential and significance.Single-walled carbon nanatubes (SWCNTs) is one of the VIP members in nanomaterials. Because of its'unique physical and chemical properties, SWCNTs were used in drug delivery, medical imaging, diagnostics, and engineering technology. That means the blood and blood vessel might be the cardiovascular toxicity potential organ. Nano- toxicology confirmed that the ultra-fine particles can go through the lung-blood barricade, and participate in the whole blood cycle. The prior studies proved that the vessel proliferation dues to the functional disturbance of the vascular endothelial cell (VEC) and the vascular smooth muscle cell (VSMC), ignored the effects of the vascular adventitial fibroblasts cell. While the latest findings show the vascular adventitial not only the supporting structure, but also unified the nerves-endocrine-immune function. It is a complicating construction and function part of vessel, plays an important role in vessel proliferation. In this study, we choose the vascular adventitial fibroblasts cell as the target cell.SM22αis one of the markers of cell differentiation. There is VAFs only in the normal vascular adventitial, when the vascular adventitial damaged, VAF allaxis to myofibroblast (MF), the identification is that SM22αappears in kytoplasm. When the VAF cells transformation into MF, it begins to migrate to endomembrane, and proliferate, to become cell component of the new endomembrane. The molecular biology mechanism of the transformation of the VAF to MF is not clear. The form of the MF has a close relationship with lots of growth factor and cytokine, in which the transferring growth factorβ1 (TGF-β1) is the closest factor. Therefore, we presume that the expression of SM22αis controlled by the TGF-β1 signal transduction system and the Smad family.The purpose of this study is approaching the biology pathway of the damaging that SWCNTs to VAF cells, explore effects and mechanisms of SM22αon the transformation of rat vascular adventitial fibroblasts induced by SWCNTs.Methods:1. Primary culture the VAF cells by collagenaseⅠenzyme digestion, identified by morphology and immunology;2. cellular level: 1) Establish the in vitro exposure model after detected cell survival by MTT and WST; 2) Determination the cellular oxidative damage(GSH, SOD, MDA); 3) The change of the ultrastructure of VAF cells using the transmission electron microscopy.3. Molecular level: Effects of SWCNTs on VAF cells, the expression of SM22αand TGF-β1 content in different exposure conditions. 1) The SM22α, TGF-β1 and TGF-β1RⅡexpression changes in mRNA levels by Real-time quantitative PCR. 2) Detected the SM22αand TGF-β1 expression at the protein level using Western-blot.Results: 1. Successfully cultured rat vascular adventitial fibroblasts: cells were spindle-shaped or irregular quadrilateral, vimentin positive immunocytochemical staining, desmin staining andⅧfactor was negative, combined with the negative control, can be determined the cells is vascular adventitial fibroblasts;2. Cellular level:1) Cell viability: MTT experiments showed that when the dose of 100μg/mL, the cell survival rate was 41.0%, significantly lower than the control group (72.3%), (P<0.05); WST -1 experiments showed that when the dose was 50μg/mL, the cell survival rate was 51.2%, significantly lower than the control group (77.2%) (P <0.05).2) Oxidative damage: GSH: when the dose of 3.2μg/mL, GSH content was 0.73U/mg protein, was significantly lower than the control group (0.90U/mg protein), (P <0.05); SOD : When the dose of 12.5μg/mL, SOD activity was 9.61 U / mg protein, was significantly lower than the control3) Group (14.24U/mg protein), was significant difference (P <0.05); MDA: When the dose of 3.2μg/mL , MDA content was 0.30 nmol/mg protein, was significantly higher (0.23nmol/mg protein), was significant difference (P <0.05).4) Changes of cell ultrastructure: Transmission electron microscopy structure shows, SWCNTs can penetrate the cell membrane into the cytoplasm, thus making damage to the cells. The group of 50μg/mL dose exposured 24h shows cellular edema, and some had necrosis, incomplete membrane, nucleus shrink, endoplasmic reticulum, flocks, mitochondria becomes round, shallow substrate, shorter crest or even disappear. Some mitochondria changes into small vesicular structures, apparato reticulare was damaged, there is flat pouch bag and flat expansion, big bubbles and small bubbles collapse.3. Molecular level1) Real-time fluorescence quantitative PCR: After exposed SWCNTs, compared with control group, the SM22αexpression level decreased, and TGF-β1 and TGF-β1RⅡexpression increase.2) Western-blot: After exposed SWCNTs, compared with control group, the SM22αand TGF-β1 expression increase, and TGF-β1 expression in a dose-response relationship, with the dose increased, TGF-β1 expression also increased.Conclusions:1. The cellular level results showed that, The SWCNTs can made damage to vascular adventitial fibroblasts. After exposure SWCNTs, Shows decreased cell viability, ultrastructural changes and oxidative stress.2. The molecular level showed that the SM22αexpression is controlled by the TGF-β1 signaling pathway which mediated by the Smad, SWCNTs can activate TGF-β1 signaling pathway, phosphorylation the Rsmad protein, and in the synergistic effect of Smad4, resulting in the intracellular SM22αincreased expression, VAF cells translate into MF cells, then proliferation, through into the basal membrane and migrate to the membrane, remodeling the blood vessel tissue.