The dedifferentiation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of vascular remodeling-related disease. 11,000 g for 10 min and protein concentrations were determined by BCA method following the manufacturers protocol. Alizarin Equal amounts of proteins were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore, MA, USA). Blots were incubated for 1 h at room temperature with 5% skimmed milk in TBST, and then incubated with primary antibodies for p-p38 (#4631), p38 (#9212), p-ERK Alizarin (#4370), ERK (#4695), p-JNK (#9255), JNK (#9252, all obtained from Cell signaling technology, Danvers, MA, USA), NLRP3 (ab4207), procaspase-1 (ab179515), IL-1 (ab20478), -SMA (ab7817), Osteopontin (ab8448), SM22 (ab14106, all obtained from Abcam, Cambridge, UK) at 4C overnight. -actin (bsm-33036M, Bioss antibodies, Woburn, MA, USA) were served as an internal reference protein. Subsequently, membranes were incubated in horseradish peroxidase-conjugated secondary antibodies (Beyotime). The blot was developed with an enhanced chemiluminescence reagent kit (Beyotime) and quantification of band intensity was carried out using Quantity One 5.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Immunofluorescence evaluation Cells were harvested in 8-well chamber slides (Millipore). After treatment, VSMCs had been collected and set with 4% paraformaldehyde accompanied by permeabilization with 0.1% Triton X-100 for Alizarin 15 min. Subsequently, 1% bovine serum albumin (BSA) in PBST was utilized to stop unspecific locations for 30 min at area temperature. For increase immunofluorescence staining, paraffin-embedded tissue were lower into 5 m areas utilizing a cryostat (Leica, Solms, Germany), deparaffinized, rehydrated, and put through antigen retrieval then. Tissues areas or cell slides had been incubated with major antibodies at 4C right away, accompanied by incubation with supplementary antibodies for one hour at area temperatures. The nuclei had been counterstained for visualization with DAPI. The principal antibodies used had been anti–SMA (ab7817), Osteopontin (ab8448), NLRP3 (ab4207), IL-1 (ab20478). The supplementary antibodies used were Alexa Fluor 488-, Alexa Fluor 594-conjugated anti-immunoglobulin G (Abcam). Images were captured under a Nikon Eclipse Ti-U fluorescence microscope (400). Statistical analysis Data from individual experiments were represented as mean standard deviation. Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Prism Software, San Diego, Alizarin USA). Comparisons between groups were made by one-way ANOVA analysis, followed with Tukeys post hoc analysis. A value 0.05 was considered statistically significant. Results SXBX pill inhibits HHcy-induced dedifferentiation of VSMCs in vivo Firstly, we analyzed plasma Hcy and lipid levels of experimental mice. Plasma total cholesterol, triglycerides and LDL-C levels were significantly higher in mice fed with HFD compared to the control group. However, treatment with SXBX pill had no influence on plasma lipids. Similarly, plasma Hcy levels were obviously increased in the HFD group compared with the control group. However, dietary SXBX pill had no significant effect on Hcy levels in mice fed with HFD (Table 1). These data indicate that SXBX pill had no effect on HFD-induced hyperlipidaemia or hyperhomocysteinemia on LDLR-/- mice. Due to the crucial role of VSMCs Cdh5 dedifferentiation in vascular remodeling, we subsequently investigated the effects of SXBX pill on hyperhomocysteinemia-related vascular remodeling in the aorta of mice. Immunofluorescence double staining experiments showed that -SMA was highly expressed in the medial layers of the aorta in the control group and decreased in the HFD group. However, SXBX pill treatment abolished HFD-induced reduction in -SMA expression. However, OPN expression was rarely detected in medial VSMCs in the control groups and its expression was upregulated in the aorta of mice received HFD. Whereas SXBX tablet treatment markedly reduced OPN appearance (Body 1A). To quantitatively check out the consequences of SXBX tablet in the obvious adjustments of markers of VSMCs dedifferentiation, both RT-qPCR and traditional western blotting verified that HFD induced downregulation of SM22 and -SMA appearance, and induced upregulation of OPN appearance. On the other hand, treatment with low or high SXBX tablet considerably abolished HFD-induced alteration of above markers (Body 1B and ?and1C).1C). Collectively, above outcomes confirmed that SXBX tablet could inhibit HFD-related VSMCs dedifferentiation and em in vitro /em , simply because confirmed by reversing Hcy-induced decreased SM22a and a-SMA and increased OPN appearance. In addition, Tablet suppressed Hcy-activated VSMCs via inhibiting proliferation and migration SXBX. Although SXBX tablet had no impact on HFD induced hyperlipidemia in LDLR-/- mice, our data indicated that SXBX tablet may be served as a highly effective agent for inhibiting Hcy-induced dedifferentiation of VSMCs. We therefore additional Alizarin investigate the mechanism by which SXBX tablet influences the experience of VSMCs. Taking into consideration Hcy triggers vascular inflammation and atherosclerosis, we focused on NLRP3, which has been identified as the cellular.