| Background Arterial calcification(AC) is a kind of vascular disease, commonly regarded as the bad effect on diabetes(TD2), chronic kidney disease(CKD) and chronic inflammatory disease, and is likely to result in a worse condition and a higher death rate. It is closely related to the complications like damage to blood vessel elasticity caused by diabetes and uremia as well as restenosis after PCI, and also highly associated with the incidence and morality of cardiovascular disease. With some typical symptoms, if arterial calcification cannot be cured in time, it will cause a series of vascular diseases. In addition, it may lead to some diseases, such as chronic inflammatory disease, calcific aortic valve disease and uremia, increasing the risk of heart diseases. Therefore, the prevention and treatment of vascular calcification is of great significance for the primary disease progressing.So far, there are few effective methods to cure AC in clinical medicine. In the past ten years, it has been discovered that osteore-gulin stimulates or inhibits AC, for collagen Matrix Gla Protein(MGP) on which Vitamin K(VK) depends is one of the strongest inhibitors to AC in the body. The active MGP inhibits the transformation of vascular smooth muscle cell(VSMC) to osteoblast like cell and stops the calcification of vessels by slowing down the calcium saturation and calcium salt deposition in the vascular wall. Thus, VSMC transformation into osteobalst like cell becomes the cellular basis and the key process of AC. However, the molecular mechanism in the calcification of VSMC needs to be illustrated further.Growth Arrest-Specific Protein 6(Gas 6), which can be inhibited by VK during the growth of protein once being activated, it will inhibit the apoptosis of VSMC and push them transferring to the damaged areas. But we cannot know how much Gas6 is needed and what role it plays in the reversal of AC. For this reason, the following questions need be answered: ① When warfarin(WFN) induces the content of calcium in arterial vascular to the high level in experimental rats, can the VK2 supplement reverse the formed calcification? ② During the process of calcification, what happens to the biological reactions such as oxidative stress injury, osteogenesis transformation and VSMC apoptosis? Does it have something to do with calcification? ③ What level of Gas6, Axl, Akt and Bcl-2 perform in the calcified aortic tissue of lab rats? Can VK2 retards vascular smooth muscle cells calcification by restoring Gas6-Axl-Akt-Bcl-2 anti-apoptosis pathway? The questions above are remained to be studied further.Objectives(1)To establish AC model and make clear that VK2’ action on calcification and osteogenesis express induced by WFN.(2)To investigate the expression of reactive oxygen species(ROS), serum alkaline phosphatase(ALP) activity and apoptosis level in calcification organization, and analyze the calcification about apoptosis.(3)To clarify the expression of Gas6, Axl, Akt and Bcl-2 protein in WFN-induced calcification, and explore its mechanism relating to them.Methods(1)10 W SD male rats received WFN(3 mg/g) and VK1(1.5 mg/g) feed for 6 weeks was regarded as calcification successful model. On the basis of calcification model, then VK2(100 μg/g) feed for 6 weeks was treating calcification.(2)The calcification rats were randomly divided into five groups: control group( 0 W、6 W、12 W) 6 W calcification group, 12 W calcification group, 6 W calcification + 6 W normal group, 6 W calcification + 6 W VK2 group. Then detecting the calcification、ROS、ALP activity、apoptosis and Gas6, Axl, p-Akt and Bcl-2 protein level at 6 week after WFN-induced.(3)O-cresol peptide complex ketone colorimetric method and Von Kossa and alizarin red staining was used to detect the calcification formation.(4)Frozen biopsy detects rat aorta ROS expression.(5)Heart(right ventricle) blood tests serum ALP’ activity.(6)Using doable immunofluorescent labeling with TUNEL and DAPI, VSMC apoptosis in rats was observed with a laser confocal microscope.(7)Mitochondria morphology changing was observed with transmission electron microscopy.(8)Gas6, Axl, p-Akt and Bcl-2 protein levels were detected by western blotting.Results(1)The arterial calcium induced by WFN for 12 weeks is increasing linearly. High VK2 intake not only blocking the progress of further calcium accumulation but also leading to more than 39 % reduction to previously accumulated arterial calcium precipitates within 6 weeks( 60 % as compared with the 12-week calcification time point, P < 0.01). And the preformed calcium salts of 44 % had been removed.(2)ROS activity of the aorta tissue calcification group than control group increased obviously, ROS activity of VK2 intervention treatment group compared with calcification model group decreased significantly.(3)Compared with those in calcification group, count of calcium nodus, aorta calcium depositions, ALP activity were reduced in VK2 intervention(P < 0.01). Compared with those in calcification group,ALP activity and calcium deposits is consistent with that about.(4)Apoptosis in calcification group significantly increased than the control group, apoptosis of VK2 intervention groups decreased significantly in the calcification group. Calcification group’ apoptosis increased significantly than normal group(P < 0.01). Apoptosis of VK2 intervention groups decreased significantly than calcification group(P < 0.01). Pearson correlation analysis about the apoptosis and calcification has significant correlation(R2 = 0.8853, P < 0.0001).(5)Transmission electron microscope shows mitochondria swelling and partly dissolving in calcification group, mitochondrial cristae partly disappearing. But the swelling and dissolving of mitochondrial were disappearing in VK2 intervention groups.(6)Comparing with control group, cell apoptosis rate and the protein level of Gas6、Axl、p-Akt and Bcl-2 were increased in calcification group( P < 0.01). Comparing with calcification group, cell apoptosis rate and the protein level of Gas6, Axl, p-Akt and Bcl-2 were decreased in VK2 intervention groups(P < 0.01).Conclusions High-dose VK2 has inhibiting and/or reversible effect on WFN-induced AC in rats. Some studies have shown that VK2 can activate phosphorylation of Akt, promote the expression of Bcl-2 through up-regulating expression of Gas6 and Axl. Then the apoptosis and calcification on the cultured aortic SMCs induced by WFN in rats can also be suppressed significantly. The study helps us to define the apoptosis signal pathway of Gas6-Axl-Akt-Bcl-2 mediated by Gas6, which can significantly increase the active transport capacity of VSMC and decrease the apoptosis. Therefore calcification of the arteries can be efficiently inhibited and reversed. The researching result clearly illustrates the treating strategy and theoretical foundation solving calcified lesions with Gas6 in the arterial calcified disease. |
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