The Effect Of SL Extract On The Regulation Of Low Shear Stress In Vascular Endothelial Barrier Function

With the development of society, cardiovascular disease has become the number one disease in threating human life. Atherosclerosis is the common pathological basis of cardiovascular disease. Although the pathogenesis of atherosclerosis has not been clear, the initiating factor of As has got the general consensus that vascular endothelial injury. Vascular endothelial injury led directly to endothelial barrier dysfunction, which induce the occurrence and development of As. Blood flow shear stress has a crucial role in the lead to vascular endothelial barrier dysfunction.Hemodynamic shear stresses have been shown to play an important role in atherosclerotic plaque localization. Specifically, regions of arteries exposed to relatively lower FSS conditions (less than4dyn/cm2) are prone to these plaque formations, while regions exposed to higher FSS conditions (greater than15dyn/cm2) are atheroprotected. Vascular endothelial cells directly perceive the change of hemodynamic shear stresses. The lower shear stress can induce cytoskeletal remodeling, and damage of endothelial barrier function, which further results in blood lipid and other substances more easily through the endothelial cells and promote the formation of As.F-actin is the major contractile proteins which regulate cytoskeletal remodeling. F-actin rearrangement is dependent on myosin light chain (MLC) phosphorylation. Myosin light chain kinase (MLCK) and ROCK/ROK/Rho kinase (Rho kinase, ROCK) are two important kinase used to regulate MLC phosphorylation. Research confirms that hemodynamic shear stresses acting on osteoblast contraction have two ways which are Ca2+dependent (MLCK pathway) and non Ca2+dependent. Then, the hemodynamic shear stress acting on umbilical artery endothelial cytoskeletal rearrangement whether also have Ca+dependent and non Ca2+dependent need to confirm by further research.SL extraet was an effect therapy to treat As with the combination of Chinese traditional medicine theory, the damage reaction theory and pharmacology achievements. Our lab’s former experiments found that SL extract can improve endothelial function and has a good control effect on As. But the improvement of endothelial function whether is by means of inhibiting on change or reorganization of endothelial cytoskeleton has not been reported up.Therefore, the study aims at researching SL extract inhibiting cell skeleton reconstruction. Taking the key protein which induces cytoskeletal remodeling-MLC phosphorylation as the breakthrough point the study focuses on the two key pathways of SL extract inducing phosphorylation of MLC on dielectric:MLCK intervention pathway and ROCK kinase pathway. From the hemodynamic shear stress point of view, the Ca+dependent and non Ca+dependent effect of SL extract inhibits cell skeleton reconstruction is analyzed, and SL extract inhibition mechanism of As occurrence is studied.Research methods and content1. Study on the role of SL extract effect on the formation of rabbit carotid atherosclerosis low shear stress mediatedFirstly, the local low shear stress in atherosclerosis rabbit model was established by adopting carotid artery cannula method. In the model established in2weeks,4weeks and8weeks, electromagnetic flowmeter were used to detect the cannula proximal and the flow of blood to the side, and full automatic biochemical analyzer is used to detect serum total cholesterol, blood viscosity, low density lipoprotein cholesterol(LDL-C), high density lipoprotein cholesterol(HDL-C) in different time. Then, HE staining is adopted to observe vascular pathology, and the pathological changes of morphology is observed by scanning electron microscope.2. Study on the mechanism of SL extract on low shear force mediated effect on formation of rabbit carotid atherosclerosisWestern blot was used to detect vascular proximal, contralateral MLC and the level of MLC phosphorylation in2weeks,4weeks,8weeks, and detect expression of MLCK and ROCK/ROK/Rho kinase. Then, MLCK protein and ROCK protein expression level at were detected by immunohistochemistry.3. Study on the stress mediated human umbilical artery endothelial cell calcium influx of cut SL extract interventionBioflux1000controlled shear stress of microfluidic channels in cultured human umbilical artery endothelial cells. NC shear flow system loaded with different shear stress, and changes of free calcium in cells by laser scanning confocal microscope were observed in real-time after immunofluorescence staining. Then, at the premise of the calcium channel blocker Nicardipine intervention, effect of SL extract on low shear stress dependence of calcium influx is analyzed.4. Study on mechanism of SL extract effect on cytoskeletal rearrangements shear stress mediatedBioFlux1000high-throughput microfluidic system cultured and loaded shear stress, then, laser confocal microscope was used to observe the changes of cytoskeleton rearrangement after immunofluorescent staining.Then, the expression changes of F-actin, MLCK, ROCK, MLC, p-MLC were analyzed. The influent of MLCK specific inhibitor ML-7, ROCK specific inhibitor of Y-27632, a calcium channel blocker Nicardipine on F-actin, MLCK, ROCK, MLC, p-MLC shear stress mediated and the effect of SL extract were observed.Study Results1. Carotid atherosclerosis rabbit model induced by low shear stress was replicated successfully:casing proximal formed local low shear stress, while contralateral shear stress was normal. SL extract has a certain improvement on local blood flow, and dose-dependently increased vascular end blood flow near the heart, improved the shear stress of blood flow. SL extract can reduce the blood lipid levels in rabbit model of As, and inhibit the formation of intimal thickening and plaques. At the same time, using scanning electron microscope, it was observed that SL extract on low shear stress induced vascular endothelial lesion has inhibitory effect.2. SL extracts could inhibit of expression of MLCK and ROCK time-dependently in the proximal. At the same time, the level of MLC phosphorylation also has obvious inhibitory effect. The progress of MLCK in the distribution of vessels with time increased gradually. At prophase, it is mainly in endometrial expression, and many in the expression of media and adventitia from4to8weeks, while the expression of ROCK distribution mainly concentrated in the media.3. Low shear stress can be increased effectively mediated by cytosolic free calcium in HUAEC cells, and its effect is time-dependent. The increasing effect of HUAEC intracellular free calcium is mainly related to in flow of extracellular calcium. SL extract can inhibit calcium extracellular flow induced by the low shear stress.4. Cytoskeletal remodeling cell induced by low shear stress is related to the level of MLC phosphorylation. Under low shear stress, F-actin, MLCK, ROCK, p-MLC/MLC expression level increased, and SL extract can flip the effect. SL extracts inhibiting the cytoskeleton rearrangement function mainly related with Ca+dependent MLCK pathway. The inhibition of ROCK pathway of non Ca2+dependent is lower than MLC phosphorylation of Ca2+dependent.Conclusion1. Inhibition from cytoskeleton rearrangement could be one of important mechanisms which SL extract intervenes vascular endothelial barrier dysfunction of low shear stress mediated cell.2. Ca2+dependent pathway may be an important signal way of SL extract to inhibit cytoskeletal remodeling.