| BackgroundHigh blood pressure and type2diabetes in China are the most common chronic disease in older adults, and have a close relationship. There are about10%of hypertensive patients with diabetes or abnormal glucose tolerance, in which the incidence of diabetes is2.5times of normal hypertensive patients. There are at least one-third of patients with diabetes mellitus and hypertension,while the prevalence rate of hypertension is running at70%～80%in patients with diabetes mellitus and renal damage. This kind of patients with the special type of high blood pressure have obvious metabolic disorder and more serious target organ damage, and antihypertensive agents used to show lower effect.Vascular complications are the most common complications of diabetes, in which abnormal lipid metabolism, high blood sugar, insulin resistance, and glycosylated hemoglobin play an important role. And advanced glycation end products (Advanced Glycation End Products AGEs) can cause the pathological changes of body tissues and organs in direct or indirect way. AGEs refer to a stable covalent adduct which macromolecules such as proteins, lipids or nucleic acid react spontaneously with glucose or other reducing monosaccharides without enzymes to produce. The reaction process is called non-enzymatic glycation reaction (Maillard Reaction). Formation of AGEs is a slow process. The free amino group on saccharides and carbonyl on the reducing sugar were condensated rapidly to form an unstable Schiff without enzymes, then Schiff base become a product of the ketone amine after Amadori rearrangement. The above two process are reversible, known collectively as the early glycosylation. In the late stage of the reaction, Amadori products become highly active carbonyl compounds after further dehydration and rearrangement, which can react with free amino groups to form AGEs. Glycosylation reactions usually occur in the longevity protein, such as matrix proteins, collagen, elastin. AGEs have the following physicochemical properties:(1) AGEs show brownish yellow;(2) AGEs have a special absorption spectra and fluorescence characteristics;(3) irreversible;(4) It can form physical crosslinking between AGEs and amino acid-based groups. Even excluding sugar moiety, AGEs and other AGEs can still form macromolecules in a way of the side chain cross-linked, and the other macromolecules such as proteins, fats, nucleic acids can form the larger macromolecules in a way of glycosylation.(5)AGEs are not easy to be degraded within protein-degrading enzyme.(6)AGEs-modified proteins,loss of biological activity, binded with specific AGEs receptor on the monocyte-macrophage cell, were decomposed the low molecular weight AGEs peptides (the LWM-AGE) by phagocytosis or extracellular proteolytic system, which was released into the blood circulation and cleared by the kidneys. In the normal human body, the glycosylation reaction occurs slowly, but the rate of glycosylation reaction will accelerate markedly in the condition of high blood sugar and in the state of oxidative stress and so on. The levels of AGEs will also increase with increasing age.The study has confirmed that the vascular endothelial cells can express Human Receptor for advanced glycation end products (Human Receptor for advanced glycation end products, RAGE). After combining with endothelial cell surface RAGE, AGEs will play the following role:①Accelerate synthesis of endothelin-1and lead to vasoconstriction through nuclear factor kappa B pathway (NF-κB)[3].②Inhibit the formation of nitric oxide (NO) from endothelial cells and reduce the vasodilative effect induced by nitric oxide.③Activate the expression of two kinds of Ca2+-related genes in endothelial cells-phospholipase C (PLC) and calcium/calmodulin-dependent protein kinase IV (CAMK IV), then activate the expression of nuclear factor kappa B gene (NF-kappa B) and induce endothelial cells to produce inflammatory factors;④Increase the content of the oxygen family (ROS) and Ca2+directly and induce the apoptosis of endothelium cells. Now there are less drugs in clinical practice which can inhibit the formation of AGEs, such as the antioxidant drug aminoguanidine, lymphokines such as IL-10,immune agents such as soluble receptor of AGEs, the fracture of AGEs cross-linking agent such as hydrochloric horses Magee, antipyretic and analgesic drugs such as aspirin. From the above-mentioned study, we found that Ca2+may play an important role in endothelial cell injury induced by AGEs. Ca2+is widely considered the second messenger, and the increase of intracellular Ca2+content can activate some transcription factors, also has an impact on cell proliferation and differentiation. From the present study,we found AGEs combined with vascular endothelial cells could increase the content of intracellular Ca2+and induce apoptosis of endothelial cells, also induce the inflammatory response of endothelial cells through Ca2+-NF-kappa B signal pathway.Studies have confirmed the second-generation dihydropyridine calcium antagonist (calcium channel blocker, CCB), nifedipine can inhibit the generation of reactive oxygen species from endothelial cells and decrease the expression of RAGE on endothelial cell surface. The third-generation long-acting dihydropyridine CCB, L amlodipine (L-amlodipine) is a pharmacologically active part of amlodipine which was removed toxic right-hand part of lacking pharmacological activity, whose efficacy is2times higher than amlodipine’s and whoes security in clinical application is higher than amlodipine’s. Recently, as Levamlodipine was widely used in clinic practice, we found that it had a variety of other effects, such as improving endothelial function, reversing left ventricular hypertrophy,inhibiting the deposition of AGEs in the vascular smooth muscle cells, in addition to the direct dilation of blood vessels and blood pressure control. This study aims to investigate whether Levamlodipine can inhibit the inflammatory response of endothelial cell induced by AGEs, which will help to explain further why Levamlodipine will be helpful in the treatment of cardiovascular diseases, which also provides the theory basis for Levamlodipinei used in patients with essential hypertension, especially in patients with hypertension and type2diabetes.Objectives:1. Learn to prepare common solutions and master useful cell culture techniques2. To investigate the concentration of MCP-1and the expression of NF-κB gene in endothelial cells treated with advanced glycosylation protein (AGE) and make it clear whether the expression of MCP-1and NF-κB p65protein can be induced by the AGE.3. To observe whether Levamlodipine besylate decreaeses the level of mononuclear chemoattractant protein1(MCP-1) secreted by endothelial cells treated with advanced glycosylation protein (AGE) by down-regulating the expression of NF-κB gene. Methods1. Isolation, identification,culture and subculture of endothelial cellsUnder sterile conditions, fresh umbilical cord from healthy mothers after cesarean section was cleaned with phosphate buffer solution (PBS) and digested with type I collagenases, inoculated in M199medium supplemented with20%fetal bovine serum (FBS) and incubated under the condition of5%CO2,37℃. Medium was changed after24hours. They were digested with0.125%trypsin and passaged when cells grew to cover80%-90%of bottom. Fourth to fifth generation of good cells were used in experiments after we observed cell morphology under an inverted microscope. Endothelial cells were identified with methods of morphology or testing Ⅷ related antigen of endothelial cells. They were passaged when the primary cells grew to cover95%or more of bottom and gather into a single layer as paving stones under the microscope after4-5days. We removed the old medium, washed endothelial cells2-3times with PBS (preheated for20minutes at37degrees), digested them20s with1ml of0.125%trypsin (preheated for20minutes at37degrees) at room temperature, removed the trypsin when cells became rounding or shrinking that observed by microscope, then added the M199complete medium to terminate the digestion, isolated cells gentlely to a uniform cell suspension and divided them into2groups randomly.2. Frozen storage and recovery of endothelial cells3. GroupsHUVECs were randomly divided into blank control group, the BSA control group, AGEs group and SHD group. Blank control group was cultured with equal M199medium supplemented with2%fetal bovine serum (FBS), the BSA control group was cultured with50mg/L BSA, AGEs group was cultured with AGEs (25,50,100mg/L) for24h. HUVECs were treated with SHD (2.5mg/L,5mg/L,10mg/L)for1hour, then 100mg/L AGE-BSA(select the best quality concentration from results of AGEs induced group) was added to co-culture for24h. Enzyme-linked immuno sorbent assay (ELISA) was used to measure MCP-1concentration; RIPA cell lysates were used to extract whole-cell protein samples; The method of BCA was used to determine protein concentration; Western Blot was performed to detect the level of NF-κB p65protein after predetermined time.4. ELISAEnzyme-linked immuno sorbent assay (ELISA) was used to measure MCP-1concentration.5. Western BlotHuman umbilical vein endothelial cells were extracted from the6-well plates; RIPA cell lysates were used to extract whole-cell protein samples; The method of BCA was used to determine protein concentration; Then protein samples were used to SDS-PAGE electrophoresis, were transferred to PVDF membrane, were made produce an immune response and were made light chemically. Then developing, fixing and make an analysis of gel image at last.6. Data analysisSPSS13.0statistical software was used to analyze data. Data are presented as Mean±S.D. One-way ANOVA test was used to single comparison between different groups of data; LSD was used to a multiple comparisons among different groups of data. P value<0.05was considered to be significant.Results1. The concentration of MCP-1secreted by human umbilical vein endothelial cells was increased with the increasing concentration of AGEs, while the difference between AGE group and blank control group or BSA control group was statistically significant (p=0.000). 2. The concentration of MCP-1secreted by human umbilical vein endothelial cells treated with AGEs was decreased with the increasing concentration of Levamlodipine besylate, while the difference between SHD group(5.0、10.0mg/L) and100mg/L AGE group was statistically significant (p=0.000).3. The level of NF-κB p65protein was increased with the increasing concentration of AGEs in umbilical vein endothelial cells, compared with blank control group or BSA control group. The level of NF-κB p65protein in AGEs(25、50、100mg/L) group was higher than blank control group(1.24times、3.10times、4.05times), and the difference between AGEs groups(5.0,10.0mg/L) and control group was statistically significant(p=0.000).4. The level of NF-κB p65protein was decreased with the increasing concentration of SHD in umbilical vein endothelial cells treated with100mg/L AGEs, compared with100mg/L AGEs group. The level of NF-κB p65protein in SHD groups(2.5、5.0.10.0mg/L) was lower than100mg/L AGEs group (1.08times、1.74times、2.94times), and the difference between SHD groups(5.0、10.0mg/L) and AGEs group was statistically significant(p=0.000).5. The concentration of MCP-1and the level of NF-κB p65protein were reduced with increasing concentrations of Levamlodipine besylate (2.5、5、10mg/L), compared with the cells treated with1OOmg/L AGE group.ConclusionThe anti-inflammatory properties of Levamlodipine besylate in HUVECs treated with AGE may partly be attributed to downregulation of NF-κB signal pathway. |
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