| BackgroundGiven the small diameter and low-flow velocity of microvessels, they are prone to be occluded by blood clots, atheromatous fragments or other circulating debris. The mechanisms that have been discovered to keep the microvascular patency are hemodynamic forces, fibrinolytic system and angiophagy (AP), which lead to embolus washout, dissolution or extravasation respectively. Angiophagy involves the engulfment of emboli by the endothelium and emboli translocation through the vessel wall into the perivascular space, the blocked vessel achieves recanalization eventually. It is a ubiquitous mechanism of recanalization, the engulfment process began as early as 1h after embolization in mice, which would block hemodynamic clearance and disrupt clot fibrinolysis, however. Indirect evidence of transvascular embolus extrusion was also found in humans. The precise mechanisms underlying it are still unknown. Some possible mechanisms involved in engulfment process are integrins, actin cytoskeletal remodeling and so on. For the embolus extravasation process, it is likely to require the remodeling of the perivascular extracellular matrix and/or tight junction, in which ZO-1, CD31, collagen Ⅳ and matrix metalloproteinases (MMPs) may be involved.Atherosclerosis (AS) is a chronic inflammatory disease of blood vessels, which is characterized by the narrowing of blood vessels due to the growth of an atherosclerotic plaque, vascular endothelial cells (VEC) injury is a critical pathology initiating and driving AS progression. Endothelial cells (ECs) are major determinant of vascular tone, leukocyte adhesion, and vascular smooth muscle cells (VSMCs) proliferation. VEC dysfunction may occur well before the structural manifestation of overt vascular disease such as AS. Given that angiophagy is a protective effect of VEC to clear microemboli and keep vascular patency, it is still unclear that whether the capacity of angiophagy and embolus extravasation of microvessels will be affected in AS disease, and how it is.Qing-Nao-Tong-Luo Recipe is an experiential prescription of Zhang Xue-wen, one of Chinese Medicine Masters in Shan’xi University of Chinese Medicine, China, for transient ischemic attack (TIA) and stroke prevention. The recipe was composed of 12 compoments including Semen Cassiae (Cao jue ming), Ligusticum Wallichii (Chuan xiong), Red Paeony Root (Chi shao), Hauthorn (Shan zha), Redix Salviae Miltiorrhizae (Dan shen), Magnetite (Ci shi), Flos Chrysanthemi (Ju hua), Lobed Kudzuvine Root (Ge gen), Lumbricus (Di long), Herba Siegesbeckiae (Xi xian cao), Radic Cyathulae (Chuan niu xi), Hirudo (Shui zhi). The recipe had effect on regulating blood and vessels, as well as clearing away the liver-fire. Clinical research indicated that patients with transient ischemic attack (TIA) had lower expression of CD41 and lower recurrence rate while appropriately being treated with the QNTLR. Study also showed that the recipe had a certain effect on improving mild cognitive impairment in both clinical and experimental practice. Ligustrazine has been widely used in the treatment of cerebral and cardiac ischemia, the underlying mechanisms of it on neural protection are related to its anti-oxidant, anti-apoptotic and anti-inflammatory effects. As AS is a disease of blood and vessels, does QNTL receipe have effect on it and the angiophagy, and what the mechanisms are. In this study, we used ApoE gene knockout mice to build AS model to explore the regulation of QNTL recipe on cerebral angiophagy in AS mice and the mechanisms.Objectives1. To explore the regulation of QNTL recipe and TMP on cerebral angiophagy in AS mice.2. To explore the effect of QNTL recipe and TMP on blood lipid levels and oxidative stress injury in blood and brain tissue.3. To study the mechanisms of angiophagy and the mechanism of QNTL recipe on angiophagy, the expression of ZO-1, CD31, MMP2/9 in AS and the effect of QNTL recipe and TMP on them was detected.Methods1. Animal groups and drug administration:Adult male apoE knockout mice, weighting 18-22g, aged 8-9 weeks, were fed adaptively for 1 week, and then fed with high cholesterol diet for 9 weeks. Adult male C57BL/6 wild type mice, with the same genetic background as the aopE knockout mice and matched by weight and age, were fed with common diet for 9 weeks to be the control group. At the end of the ninth week, apoE knockout mice were randomly divided into four groups:the atherosclerotic model group, atorvastatin calcium treated group (ATC group), QNTLR treated group (QNTLR group), ligustrazine hydrochloride treated group (TMP group). Atorvastatin (3mg/kg/d) and QNTLR (30g/kg/d) was administered separately to the ATC group and QNTLR group by gastrogavage for 4 weeks. Ligustrazine hydrochloride (12mg/kg/d) was administered to the TMP group via intraperitoneal injection. The model and control groups received equivalent volumes of distilled water as the other groups for 4 weeks. The administration dosage was according to clinical practical and human rat equivalent dosage conversion. Every group received corresponding diet continuously during the administration period.2. Preparation of fluorescent microemboli and Embolization surgery: Heterologous fibrin-rich clots were generated by collecting blood from the left ventricle of C57BL/6 mice, which had the same genetic background as the apoE knockout ones. After standing for 24 h at 4℃, blood turned into clots. The clots were rinsed and fragmented by sonication, then weighted and suspended in sodium bicarbonate buffer. Texas Red -X, succinimidyl ester was dissolved in DMSO. The reactive dye solution was added to the clots and they were incubated for 4 h at RT away from light with continuous shaking. Labeled fibrin fragments were washed with 70% ethyl alcohol for three times, then filtered through sieves and resuspended in 0.01 M PBS. After 4 weeks intervention, every mouse would undergo an embolization surgery. Briefly, mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (350mg/kg), and a ventral midline incision was made in the neck. The left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were isolated carefully. Blood flow of the left ECA was interrupted temporarily by sutures. A small incision was made in the CCA and a syringe needle attached to a syringe filled with 200 u 1 fluorescent emboli at the desired concentrations was inserted into the CCA. The injection period was over 2 minutes. The needle was removed and the CCA was ligatured. Ligating sutures of ECA were removed. The surgical incision was sutured and mice were carefully monitored.3. Angiophagy rate assessment:Forty-eight hours after embolization, mice received an intravenous injection of fluorescein isothiocyanate (FITC)-conjugated Lycopersicon esculentum lectin which circulated for 15 minutes before sacrificed. Brain tissues were cryosectioned (50 μm thick), and the slices were imaged with confocal microscope to quantify extravasation. Emboli (in red) were classified by whether they remained within the lumen (in green) or were outside of it. Angiophagy rate was calculated as the number of extravasation event divided by the total number of clots counted.4. Blood lipid levels measurement:48h after microemboli injection, blood was sampled and supernatant was collected. Blood lipid levels (including TC, TG, LDL-C, HDL-C) were mesured by Cobas8000 biochemical analyzer.5. Measurement of SOD, MDA, LPO in serum and brain: 48h after microemboli injection. The supernatant of blood and brain was collected, respectively. Activity of SOD was measured by WET-1 method. The content of MDA was determing by TBA method, and the content of LPO was measured directly. The detection procedure was in strict accordance with the kit operating instructions.6. Immunofluorescence staining of ZO-1 and CD317. Western-blot analysis of ZO-1, CD31, MMP-2 and MMP-98. Gelation zymography of brain tissueResults1. QNTL could promote angiophagy:Forty-eight hours after embolization, brain was sampled and frozen sections was made by freezing microtome. Angiophagy rate assessment indicated that:compared with the control group, angiophagy rate of the model group decreased significantly, that of QNTL group and TMP group was higher than the model group.2. QNTL could regulate the blood lipid levels of AS mice:After high-fat feeding for 13 weeks and receving corresponding intervention for 4 weeks, blood lipid levels was measured. The results showed that:Compared with the control group, the model group had higher level of TC, TG and LDL-C, as well as lower level of HDL-C, which indicated that lipid metabolic disorder was induced in apolipoprotein E (ApoE) gene knockout mice with high-fat feeding. Compared with the model group, the ATC group and QNTLR group had lower levels of TC, TG, LDL-C, and the levels of TG and LDL-C in TMP group was significantly decreased.3. QNTL had the effect of anti-oxidative stress:Compared with the control group, the content of MDA and LPO in serum and brain of the model group increased and the vitality of SOD decreased significantly. QNTL could reduce the production of MDA and LPO, enhance the vitality of SOD. TMP could also reduce the production of MDA and enhance the vitality of SOD.4. The protein expression of ZO-1 and CD31:The protein expression of ZO-1 and CD31 was detected by immunofluorescence staining and western-blot analysis. Fluorescent immunohistochemical results indicated that compared with the control group, the expression of ZO-1 and CD31 in AS model group decreased significantly, QNTL and TMP could improve the expression, and QNTL had better results. Semi-quantitative analysis of ZO-1 and CD31 protein expression by western-blot showed same results.5. The results of MMP-2 and MMP-9 protein expression and gelatin activity: In order to explore the correlation between angiophagy and gelatin, western-blot analysis was used to detecte the protein expression of MMP-2 and MMP-9, it indicated that:the protein expression of MMP-2 and MMP-9 significantly increased in model group, QNTL and TMP treatment could reduced the expression of them. The result of tissue zymography also indicated that the activity of MMP-9 increased, QNTL and TMP could reduce it. The activity of MMP-2 had no obvious diffecence between the groups.Conclusion1. Angiophagy reduced significantly in AS model group, while the treatment with QNTL and TMP intervention could promote the embolus extravasation, improve angiophagy ability.2. Because the ApoE gene knockout mice lack the ability to clear the lipoprotein cholesterol, hypercholesterolemia, which is the character of lipid metabolic disorder, was induced by high-fat feeding for 13 weeks. QNTL and ATC administration could regulate lipid metabolism and improve blood lipid metabolism disorders. TMP showed moderate effect on blood lipid metabolism disorders improvement.3. A lot of free radicals were produced in AS, SOD was consumed largely and the vitality droped, the production of peroxide materials like MDA and LPO increased, which indicated oxidative stress damage of vascular endothelial cells. QNTL and ATC intervention could enhance the vitality of SOD and reduce the production of MDA and LPO to relieve endothelial injury; TMP could improve SOD vitality and reduce MDA content.4. The protein expression of CD31 and ZO-1 decreased, which indicated the barrier function of tight junction between endothelial cells and the permeability of BBB was damaged, the administration of QNTL and TMP could up-regulate the expression of ZO-1 and CD31 and protect the tight junction.5. The protein expression of MMP-9 and MMP-2 and the enzyme activity of MMP-9 significantly increased, extracellular matrix degraded. While QNTL and ligustrazine could lower the gelatin enzyme activity, alleviate the degradation of extracellular matrix.6. QNTL recipe could promote angiophagy by regulating blood lipid levels, anti-oxidative stress and improve the permeability of BBB in AS mice. |
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