Attenuation Of Atherosclerotic Lesions In Diabetic Apolipoprotein E-deficient Mice Using Gene Silencing Of Macrophage Migration Inhibitory Factor

第一部分(英文)Part oneThe Study of Pro-atherosclerotic Effects on ApoE-/-Mice combined Streptozotocin-induced Diabetes with Chow DietBackground and objective:Lacking the ability to clear very low density lipoprotein cholesterol (VLDL-C) and lipoprotein particles produced by chyle particles, ApoE-/-mice are susceptible to atheromatous plaque. Experiments showed that the atherosclerotic lesion from ApoE-/-mice fed on chow diet appears to be light and developed slowly, while aggravated with time. However, ApoE-/-mice fed on high-fat diet tends to have early and intense atherosclerotic lesions.Therefore, ApoE-/-mice model fed on high-fat diet is widely used in the basic research of AS. However, animal model for diabetic AS experimental study is relatively scarce. Former diabetic models such as Glut4-/-mice, MKR transgenic mice, Fatless-A-ZIP/F1mice are failed to meet the needs of the current scientific research. In recent years, the STZ induced diabetic ApoE-/-mice hane been widely used for the basic researches. Animal Models of Diabetic Complications Consortium (AMDCC), together with some formal studies affirmed the feasibility of this kind of model too. While former researchers usually used a high-fat diet feeding instead of the chow diet. But high-fat feeding could greatly increase the TC in mice, which is not very obvious in clinical diabetic AS. Stimulation simultaneously with high blood glucose and high TC is not suitable for the study of mechanism and intervention in diabetic associated AS disease. Therefore, high-fat diet combined with STZ (Streptozotocin) induced diabetic ApoE-/-mice model couldn’t reflect the true state of clinical atherosclerotic patients with diabetes. Based on the above reasons, we intended to observe the effects of STZ induced diabetic ApoE-/-mice combined with chow diet on atherosclerotic lesions.Method:1. Construct diabetic ApoE-/-mice model75ApoE-/-mice (male,8weeks old,22±2g) were purchased from Peking University (Beijing, China) and confirmed by PCR genotyping. Animals were randomly divided into five per cage and housed at the Animal Care Center of Qilu Hospital of Shandong University. Mice were kept on a12-hr light/12-hr dark cycle, at a constant temperature (24℃) with access to standard mouse chow diet (5%fat,0.02%cholesterol with no cholic acid) and water ad libitum for15weeks until sacrifice. All the mice were allowed to acclimatize for2weeks before the study started. Intraperitoneal injecting of multiple low dose STZ diluted by citrate buffer (pH4.5, final concentration:1%,) at dose of45mg kg-1May-1to the ApoE-/-mice (10week old) for five days. ApoE-/-mice (n=36) injected with vehicle alone (citrate buffer, pH4.5) for five days were served as non-DM control. In the following four weeks, blood glucose was dynamicly monitored using a One Touch Ultra glucometer (Johnson&Johnson), and only the mice with continuous blood glucose levels>15mmol/L were recruited to the DM group (n=37). And all mice were euthanized8weeks later for further study.2. Serum Lipids and Glucose Measurement In order to test the level of glucose concentration, Total Cholesterol (TC), Triglyceride (TG), Low-Density Lipoprotein Cholesterol (LDL-C) and High-Density Lipoprotein Cholesterol (HDL-C), blood samples were got before and after the STZ injection as well as before anesthesia. Body weight was tested at the same time.3. Glucose Tolerance Test (GTT) Three days before euthanasia, intraperitoneal GTT was performed on6hr fasted mice.6mice of each group were intraperitoneally injected with glucose (2g/kg body weight,20%glucose solution). Tail vein blood samples were respectively obtained at0,10,30,60,90and120minutes after the glucose load, and blood glucose concentration was also measured using a One Touch Ultra glucometer (Johnson&Johnson).The Area Under the Curve (AUC) was then determined using the linear method of the trapezoid rule.4. Tissue Preparation After the mice were anesthetized with pentobarbital sodium, open the chest and expose the heart. Puncture the left ventricular apex with1.0mL of syringe to get the blood. Then perfuse the heart with cold Phosphate Buffered Saline (PBS) for5minutes, followed by perfusion fixation with phosphate buffered4%paraformaldehyde for another7minutes. The heart containing the aortic root, together with the artery from aortic arch to left and right common iliac artery were rapidly fixed and harvested. The adventitial side of the aorta was carefully dissected free from fat and muscle. The frozen cross-sections of the aortic sinus embedded in freezing microtome, while the paraffin cross-sections of the aortic sinus embedded in paraffin wax sectioned by paraffin slicing machine, which were mounted on slides for histological and immunohistochemical staining.5. Histopathological Staining Total aortae were cleaned of excess fat under a dissecting microscope and subsequently stained with Oil Red O. Mice sample slice was stained with Hematoxylin and Eosin (H&E), Oil Red O, Masson trichrome and Picrosirus Red stain using a standard protocol for the plaque area, lipid, total collagen and collagen I.6. Immunohistochemical Staining For the immunohistochemical analyses, sections were tested with a-SMA, Endomucin, MCP-1, TIPM-1or MMP-2according to the protocols. 7. Enzyme-Linked Immuno Sorbent Assay (ELISA) Plasma from each mouse was prepared and stored at-80℃until use. IL-6was measured using a commercially available ELISA kit according to the Kit instructions.8. Quantitative real-time PCR Total RNA was extracted from the aortae with the TRIzol reagent, according to the manufacturer’s instructions. Total RNA concentration was quantified by spectrophotometry and reverse-transcribed into cDNA. Real-time PCR was performed to determine the gene expression of MMP-9, MCP-1, TNF-a, by using SYBR Green Technology, and the mouse housekeeping gene P-actin was applied as an internal control. The data were analyzed by the2-△△CT method. All experiments were repeated for at least three times (see table1).9. Western blot analysis Fresh tissues were snap-frozen in liquid nitrogen, pulverized, and resuspended in ice-cold lysis buffer. Protein concentrations were determined with the the BCA protein assay method. Solubilized protein was separated by electrophoresis and transferred to0.22μm PVDF membranes. After nonspecific binding was blocked membranes were respectively probed with the specific antibodies (a-SMA; MAC; Collagen I; p-actin).10. Statistical Analysis All data are presented as means±SEM and were analyzed by paired t test or one-way ANOVA. A value of P<0.05was considered statistic significant. Data were analyzed by using SPSS18.0software.Results:1. Suceesfully construct diabetic ApoE-/-mice and the mice fed on chow diet showed the deteriorate glucose intoleranceCompared to non-diabetic controls,14week old diabetic mice (3weeks after STZ injection) had higher blood glucose level. And this hyperglycemia lasted until the end of the study (12weeks of diabetes). Based on it, we further performed a study on whether the STZ induced hyperglycemia has an effect on glucose intolerance by GTT. The GTT results suggested a deterioration of glucose intolerance because of STZ-induced hyperglycemia. Whereas, we could conclude that the STZ induced diabetes was successful.2. Diabetic ApoE-/-mice fed on chow diet showed the exacerbated lipid metabolismBefore the STZ injection, there were no significant differences in body weight, blood glucose, TG and TC levels between the two groups. All hyperglycemia ApoE-/-mice gradually lost body weight after the STZ injection. At the end of the study, the body weight of diabetic mice remained significantly lower than that of non-diabetic mice, which is consistent with the previous report. Compared to non-diabetic controls,14week old diabetic mice (3weeks after STZ injection) had higher plasma cholesterol level. Blood TC of diabetic mice also kept in higher level in comparison with the non-DM mice. However, there was no statistical significance in blood LDL-C and HDL-C level during the whole experiment. Body weight and biochemical parameters are described in Table2.3. Diabetic ApoE-/-mice fed on chow diet showed the increased atherosclerosis lesionsIn the condition of hyperglycemia, the ratio of plaque area/vessel area was significantly increased in the aortic sinus, carotid artery, and abdominal aorta, even in the proximal aorta showed in the En-Face oil red O staining. Compared with the non-diabetic ApoE-/-mice, the DM ApoE-/-mice also showed a larger burden plaque, accompanied by the remarkably increased ratio of plaque area/total cross-sectional vessel wall area of aortic root. 4. Diabetic ApoE-/-mice fed on chow diet showed the modified composition of atheromatous plaqueAtheromatous plaque chiefly includes the contents of lipid, macrophage, SMC and collagen, especially collagen I. Diabetic mice showed the increased contents of lipid, collagen I, SMC and macrophage using immunohistochemical staining. So STZ induced-hyperglycosemia accelerated the process of atheromatous plaque in ApoE-/-mice. Meantime, the protein level of macrophages, SMCs and collagen I detected by Western blot showed similar changes. In addition, our immunohistochemical staining showed the decreased Endothelial Cells (ECs) in the plaques of the diabetic mice, Immunohistochemical staining showed that exposure to high glucose could significantly decrease the expression of TIMP-1and increased the content of MMP-2in aortic tissue of ApoE-/-mice. RT-PCR results showed the increased expression of MMP-9mRNA in the aorta of diabetic mice. Interestedly, the ratio of MMP-2/TIPM-1, which is chiefly attributed to matrix composition in plaque, showed significant increase in the diabetic mice compared with that in the non-diabetic mice.5. Diabetic ApoE-/-mice fed on chow diet showed the increased expression of inflammation cytokines:IL-6, TNF-a and MCP-1IL-6, TNF-a and MCP-1are important inflammation cytokines that are engaged in the development of atherosclerosis, which can exacerbate it. In this study, we detected the higher protein and mRNA level of MCP-1in diabetic mice than that in non-diabetic mice. In addition, the serum IL-6level was significantly higher in the diabetic mice, compared with that in the non-DM control group. Moreover, the mRNA expression of TNF-a also showed consistent results. Conclusion:STZ induced diabetes combined with chow diet can aggravate atherosclerotic lesions in ApoE-/-mice by increasing lipid, macrophage, smooth muscle cell, collagen content as well as inflammation status. 第二部分(英文)Part twoAttenuation of Atherosclerotic Lesions in Diabetic Apolipoprotein E-Deficient Mice using Gene Silencing of Macrophage Migration Inhibitory FactorBackground and objective:Diabetes mellitus (DM) patients with coronary artery disease (CAD) have become a major public health concern because of its high morbidity and increasing mortality rate. Macrophage Migration Inhibitory Factor (MIF) involves in the pathogenesis of atherosclerosis (AS) and the increased plasma MIF levels in DM patients are associated with a risk for AS. Here we will detect the effect of MIF gene in streptozotocin (STZ)-induced diabetic apolipoprotein E deficient mice (ApoE-/-mice). STZ-induced diabetic ApoE-/-mice were randomly divided into three groups: Ad-MIFi, Ad-EGFP and normal saline (NS) groups (n≥33each). Non-diabetic ApoE-/-mice were served as controls. Ad-MIFi or Ad-EGFP was injected repeatedly4weeks later. The results showed diabetic ApoE-/-mice had. aggravated atherosclerotic lesion, compared with non-diabetic mice. MIF gene interference attenuated atherosclerotic lesions, accompanied by decreased macrophages, lipid, collagen I content and SMCs deposition and inflammatory cytokine production, improved glucose intolerance and plasma cholesterol level, decreased ratio of MMP-2/TIMP-1. Meantime, an increased expression of MIF and its ligand CD74was detected in the human coronary artery with DM and CAD patient. Above all, MIF gene interference inhibits atherosclerotic lesions and increases plaque stability in STZ induced diabetic ApoE-/-mice, thus providing a novel and promising approach to the treatment of DM associated atherosclerosis. Methods:1. Construct Diabetic ApoE-/-mice Model150ApoE-/-mice (male,8weeks old,22±2g) were purchased from Peking University (Beijing, China) and confirmed by PCR genotyping. Animals were randomly divided into five per cage and housed at the Animal Care Center of Qilu Hospital of Shandong University. Mice were kept on a12-hr light/12-hr dark cycle, at a constant temperature (24℃) with access to standard mouse chow diet (5%fat,0.02%cholesterol with no cholic acid) and water ad libitum for another15weeks until sacrifice. All mice were allowed to acclimatize for2weeks before the study started. Intraperitoneal injecting of multiple low dose STZ diluted by citrate buffer (pH4.5, final concentration:1%,) at dose of45mg kg-1May-1to the ApoE-/-mice (10week old) for five days. ApoE-/-mice (n=36) injected with vehicle alone (citrate buffer, pH4.5) for five days were served as non-DM control. In the following four weeks, blood glucose was dynamicly monitored using a One Touch Ultra glucometer (Johnson&Johnson), and only the mice with continuous blood glucose levels>15mmol/L were recruited to the DM group (n=110). These diabetic mice with steady plasma glucose level (exactly15week old) were then randomly divided into3groups:Ad-MIFi (n=36), Ad-EGFP (n=37) and normal saline (NS)(n=37) groups. Ad-MIFi (4×109Pfu), Ad-EGFP (4×109Pfu) diluted to a total volume of100μL or100μL NS was respectively injected into the tail vein of each mouse in three intervention DM groups. The tail vein injection was repeated4weeks later. And all mice were euthanized8weeks later for further study.2. Serum Lipids and Glucose Measurement In order to test the level of glucose concentrations, Total Cholesterol (TC), Triglycerides (TG), Low-Density Lipoprotein Cholesterol (LDL-C) and High-Density Lipoprotein Cholesterol (HDL-C), blood samples were got before and after the STZ injection as well as before anesthesia. Body weights were tested at the same time. 3. Glucose Tolerance Test, GTT Three days before euthanasia, intraperitoneal Glucose Tolerance Test (ipGTT) was performed on6hr fasted mice.6mice of each group were intraperitoneally injected with glucose (2g/kg body weight,20%glucose solution). Tail vein blood samples were respectively obtained at0,10,30,60,90and120minutes after the glucose load, and blood glucose concentration was also measured using a One Touch Ultra glucometer (Johnson&Johnson).The Area Under the Curve (AUC) was then determined using the linear method of the trapezoid rule.4. Tissue Preparation After the mice were anesthetized with pentobarbital sodium, open the chest and expose the heart. Puncture the left ventricular apex with1.0mL of syringe to get the blood. Then perfuse the heart with cold Phosphate Buffered Saline (PBS) for5minutes, followed by perfusion fixation with phosphate buffered4%paraformaldehyde for another7minutes. The heart containing’the aortic root, together with the artery from aortic arch to left and right common iliac artery were rapidly fixed and harvested. The adventitial side of the aorta was carefully dissected free from fat and muscle. The frozen cross-sections of the aortic sinus were embedded in freezing microtome, while the paraffin cross-sections of the aortic sinus were embedded in paraffin wax sectioned by paraffin slicing machine, which were mounted on slides for histological and immunohistochemical staining.5. Histopathological Staining Total aortae were cleaned of excess fat and muscle under a dissecting microscope and subsequently stained with Oil Red O. Mice sample slice was stained with Hematoxylin and Eosin (H&E), Oil Red O, Masson trichrome and Picrosirus Red stain using a standard protocol for the plaque area, lipid, total collagen and collagen I.6. Immunohistochemical staining For the immunohistochemical analyse, sections were tested with MIF, CD74, α-SMA, Endomucin, MCP-1, TIPM-1and MMP-2according to the protocols. 7. Enzyme-linked Immuno Sorbent Assay (ELISA) Plasma from each mouse was prepared and stored at-80℃until use. MIF and IL-6were measured using commercially available ELISA kits according to the Kit instructions.8. Quantitative real-time PCR Total RNA was extracted from the aortae with the TRIzol reagent, according to the manufacturer’s instructions. Total RNA concentration was quantified by spectrophotometry and reverse-transcribed into cDNA. Real-time PCR was performed to determine the gene expression of MIF, CD74, MMP-9, MCP-1, TNF-a by using SYBR Green Technology, and the mouse house keeping gene β-actin was applied as an internal control. The data were analyzed by the2-△△CT method. All experiments were repeated for at least three times.See table1.9. Western blot Analysis Fresh tissues were snap-frozen in liquid nitrogen, pulverized, and resuspended in ice-cold lysis buffer. Protein concentrations were determined with the the BCA protein assay method. Solubilized protein was separated by electrophoresis and transferred to0.22um PVDF membranes. After nonspecific binding was blocked membranes were respectively probed with the specific antibodies (MIF; CD74; a-SMA; MAC; Collagen; β-actin).10. Histopathological Staining for Human Left Anterior Descending (LAD) LAD coronary arteries were got from corpse of AS patients or diabetic AS patients. Human LAD sample slice was stained with H&E, Oil Red O, Masson trichrome and Picrosirus Red stain using a standard protocol for the plaque area. For the immunohistochemical analyse, sections were tested with a-SMA, macrophage, CD74or MIF according to the protocols.11. Statistical Analysis All data are presented as means±SEM and were analyzed by paired t test or one-way ANOVA. A value of P<0.05was considered statistic significant. Data were analyzed using SPSS18.0software. Results:1. STZ-induced diabetic ApoE-/-mice and adenovirus transfection in vivo Compared with non-diabetic controls,14week old diabetic mice (3weeks after STZ injection) had hyperglycemia. And this hyperglycemia lasted until the end of the study (12weeks of diabetes). The upregulated expression of MIF in atherosclerotic lesions of diabetic ApoE-/-mice was detected using immunohistochemistry, compared with non-DM control. RT-PCR assay also confirmed the increased MIF mRNA level in the DM group. Meantime, Western blot assay further supported the upregulated serum MIF protein expression. Moreover, the plasma MIF level was significantly increased in the DM mice. As a ligand of MIF, CD74protein and mRNA level significantly upregulated after STZ induced-hyperglycosemia, detected by the immunohistochemistry, Western blot and RT-PCR analysis, respectively. As expected, the mRNA and protein level of MIF was down-regulated after the adenovirus-mediated MIF gene interference using immunohistochemistry, Western blot and RT-PCR analysis, respectively. What’s more, MIF concentration in the serum was significantly descended, while there were no differences between DM-NS group and DM-Ad-EGFP group, suggesting the adenovirus itself did not influence the endogenous MIF expression. In the DM-Ad-MIFi group, CD74protein and mRNA expression significantly reduced in comparison with that in the DM control group. However, MIF gene interference has no influence on Jab-1signaling pathway. These results suggested a successful diabetic ApoE-/-mouse model induced by STZ injection and an efficient in vivo transfection of adenovirus by siRNA.2. MIF gene silence improved atherosclerosis lesionsIn this study, DM-Ad-MIFi led to the decreased plaque in the proximal aorta. Total plaque area of aortae and the local AS lesions of aortic root in DM-Ad-MIFi group were significantly decreased in comparison with that in DM-Ad-EGFP group. Thus, MIF gene interference significantly inhibited the AS lesions.3. MIF gene interference improved lipid metabolism and glucose intoleranceOur data showed no significant effects of MIF gene interference on body weight of the diabetic mice. In this study, MIF gene interference decreased blood glucose, TC and TG level. Based on it, we further performed a study on whether MIF has an effect on glucose intolerance by GTT. The GTT results suggested an improvement of glucose intolerance after MIF gene interference. However, there was no statistical significance in blood LDL-C and HDL-C level during the whole experiment. Body weight and biochemical parameters are described in Table2.4. MIF gene interference influenced the composition of atheromatous plaqueAtheromatous plaque chiefly includes the contents of lipid, macrophage, SMC and collagen, especially collagen I. The lipid content, total collagen, collagen Ⅰ, SMC, together with macrophage significantly reduced in response to the low MIF level in the DM-Ad-MIFi group. So MIF gene interference could change the composation of atheromatous plaque by decreasing the contents mentioned above. Meantime, the protein level of macrophages, SMCs and collagen I detected by Western blot also showed similar changes. In addition, our immunohistochemical staining showed the accumulated Endothelial Cells (ECs) and the improved integrality of endothelium in the plaques of the DM-Ad-MIFi mice.5. MIF gene interference inhibited the expression of inflammation cytokines: IL-6, TNF-a and MCP-1IL-6, TNF-a and MCP-1are important inflammation cytokines that are engaged in the development of atherosclerosis, which can exacerbate it. In this study, MIF gene interference almost restored MCP-1protein and mRNA level back to normal. In addition, the serum IL-6level was descended after MIF gene interference. Moreover, TNF-a mRNA expression also showed consistent results. So MIF gene interference could inhibit the production of inflammation cytokines like MCP-1, IL-6and TNF-a.6. MIF gene interference attenuated the expression of plaque matrix-related factor:MMP-2, MMP-9and TIMP-1Immunohistochemical staining showed the reduced MMP-2but increased TIMP-1content in the DM-Ad-MIFi group. What’s more, MMP-9mRNA level was also reduced in the DM-Ad-MIFi group tested by RT-PCR. Interestedly, the ratio of MMP-2/TIPM-1, which is chiefly attributed to matrix composition in the plaque, showed significant decrease after MIF gene interference.7. Increased MIF and CD74expression in CAD patients with diabetesImmunohistochemical staining demonstrated MIF over expressed in the left anterior descending branch (LAD) from CAD patients with diabetes, compared with the patients with CAD alone. Moreover, CD74expression significantly ascended. H&E staining showed a larger plaque area in CAD patients with DM. The increased collagen contents and SMCs were also observed in the diabetic atherosclerosis plaques. Whenever in the CAD patients or the CAD patients with DM, there were few macrophages in atherosclerotic plaques. While the contents of macrophages increased slightly in CAD patients with DM. These results indicated larger and vulnerable plaques existed in diabetic atherosclerosis patients, compared with that in the patients with CAD alone.Conclusion:MIF gene slience attenuates the progression of AS in STZ induced diabetic ApoE-/-mice fed on chow diet.