| Cardiovascular disease is the most serious disease frequently threatening people’s lives and health. Atherosclerosis is the major pathological basis of cardiovascular disease. In the process of formation and development of atherosclerosis, the interaction of several growth regulating factors, cytokines and chemokines expanding the inflammatory response becomes the key pathology aspects of atherosclerosis. Protein tyrosine phosphorylation and dephosphorylation play a variety of significant role in numerous cell intracellular transduction signaling pathways, which is a reversible dynamic accommodation process. SHP2, known as PTPN11 is an important component of PTP. SHP2 acts as downstream signal of many growth regulating factors and tyrosine kinases, playing an integrant role in multiple cell events for multiple functions including migration, differentiation, survival and metabolic regulation. PHPS1, known as a potent and cell-permeable inhibitor, is specific for SHP2 over the closely related tyrosine phosphatases Shp1 and PTP1 B. PHPS1 inhibits SHP2-dependent cellular events. The relationship between PHPS1 and AS has not been claimed.Objective: The purpose of this study was to determine whether theproatherosclerotic or atheroprotective effects of PHPS1 in vivo situation. We therefore evaluated the effect of the level of phosphorylation on development of atherosclerosis in LDL receptor–deficient mice.Method: Preparation of Animals: Ldlr-/- Mice were received high-fat for 4wk diet containing 1.25% cholesterol chow. PHPS1(phenylhydrazonopyrazolone sulfonate 1), SHP2 inhibitor, was purchased by Sigma-Aldrich. PHPS1 was dissolved in saline with 0.5% DMSO. 30 mice were randomly divided into 3 groups, respectively named Atherosclerosis group(AS), AS+Vehicle group. Mice of AS+PHPS1 group were intraperitoneally(i.p) injected with 3mg/kg PHPS1. Mice of AS+Vehicle group were received an equal volume of saline with 0.5% DMSO.Detection indexes and measures :(1) Weights and blood glucose: Detect the weight and blood glucose once a week.(2) Blood lipid: Serum levels of TC, TG, LDL-C and HDL-C were measured at the end of 4 weeks treatment. Mice were fasted for 12 h blood was collected from the retro-orbital sinus. Serum was centrifuged and stored at-80 until analysis. Serum TG and TC were measured by enzymatic determination. LDL-C was measured by selective precipitation method.(3)The area of atherosclerosis lesions were analyzed by oil red O staining. Both en face of the aorta and aortic root in cryostat sections were stained. Respectively use Movat staining, immunohistochemistry, Sirius red staining to analysis the degree of plaque progression plaques, the percentage of smooth muscle cells and collagen ratio.(4)RT-PCR Analyses: Total RNA from whole murine heart was isolated from isolate with the Trizol reagent. Detection of the level of IL-1, IL-6, IL-10, TNF-α, TGF-β, and other cytokines m RNA in aortic vascular.(5)Western blot analysis: Western blot analysis the protein phosphorylation of SHP2 signal pathway.Result: There was no notable difference between the AS, AS+Vehicle and AS+PHPS1 group in weight, blood glucose, total cholesterol, triacylglycerol and LDL-cholesterol after 4 weeks. PHPS1 significantly decrease the atherosclerosis burden not only from en face of the aorta, but also aortic root, while not change the composition of plaque. Compared with the other groups, the level of IL-1β in AS+PHPS1 group was significantly lower. Western blot analysis reveals PHPS1 can decrease the level of P-ERK expression.Conclusion: The study demonstrated that the effect of PHPS1 on atherosclerosis in Ldlr-/- mice was independent on the mice weight, blood glucose total cholesterol, triacylglycerol and LDL-cholesterol. PHPS1 could inhibit the progression of atherosclerosis lesions in Ldlr-/- mice. Such effects were shown to associate with down-regulation of IL-1β. In addition to such mechanism, the decrease the level of P-ERK might also involve in the protective effects. |
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