Protective Effects Of α 7nAChR On High Glucose Injury In Rat Myocardial Microvascular Endothelial Cell And It’s Mechanism

BackgroundBy 2012, the morbidity of Diabetes(Diabetes mellitus, DM) in Britain, the United States, China and Arab countries has reached 8%, 11.5%, 16% and 18% respectively1. Meanwhile, the prevalence of diabetes is increasing in China, there are more than 92.5 million people with diabetes, and about 151 million people with pre-diabetes2. Currently diabetic cardiovascular complications is the "first killer" in diabetes patients, 74%- 81% of diabetes patients were died of acute cardiovascular accidents. Recently years, the researches of pathogenesis of atherosclerosis(AS) and recanalization treatment have been well performed, as well as the study focused on the mechanism of coronary artery vascular lesions caused by diabetes, however, the mechanism of diabetic Cardiac Microvascular Endothelial Cells(CMEC) injury remains unknown 3. Treatment for diabetes mainly includes controling blood glucose, regulating energy metabolism, and application of calcium channel blockers, etc4,5, but unfortunately, the morbidity and motality of cardiovascular accidents in patients with diabetes remains high, prevention and control of task is still very difficult.One of the important factors affecting the mortality and cardiovascular accidents is myocardial microcirculation level, it direct impacts on myocardial perfusion and coronary reserve, and play key role in maintaining physiological function in normal myocardial cells, N 2007 Engl J Med article pointed out, the prognosis of patients with diabetic heart disease is closely related to myocardial microcirculation; and forecast myocardial micro vessel may be an important therapeutic target in heart disease in diabetes mellitus. However, so far, the mechanism of diabetic myocardial microvascular injury is still unclear.The latest study shows diabetic myocardial injury is caused by chronic inflammation, but the inflammatory factors in myocardial tissue are not secreted by myocardial cells6. Under the support of the National Natural Science Foundation of China(No.81070126, No.81300078), our laboratory has determined the mechanism of diabetic myocardial damage, the results showed: in the condition of diabetes, the density of myocardial microvascular decreased, its barrier function damaged, CMEC produced a large number of ROS and TNF- alpha, IL-1 beta, IL-2 inflammatory cytokines which induced local inflammatory reaction,(Inflamm Res. 2011; PLo S ONE. 2012; Basic Res Cardiol. 2014), meanwhile, the results also showed that the ability of CMEC to mediate angiogenesis was decreased(Microvasc Res. 2010; Apoptosis. 2010). Then, diabetic myocardial microvascular endothelial cells also declined to enhance local inflammation and angiogenesis of the phenomenon, whether it is just a coincidence? Or is connected behind? It is not clear7,8.Study shows, alpha 7 nicotinic acetylcholine receptor(a7n ACh R) is the key effector molecules of cholinergic anti-inflammatory pathway(Nature, 2003). It also showed that after interfering a7 n ACh R generation or knockout a7 n ACh R, whether using electrical stimulation or using a7 n ACh R agonist, could not inhibit the release of inflammatory factors such as TNF alpha. In the author’s laboratory, it found that nicotine acetylcholine receptor(n ACh Rs) could regulate stem cell biological activity and to stimulate myocardial angiogenesis(Plos one, 2009). Other research has shown that, in the n ACh Rs family, the one who mediates angiogenesis is the subtypes of alpha 7-n ACh R(J Cell Biochem, 2009). At the same time, the author’s laboratory also found that in view of the important role of alpha 7n ACh R in mediating angiogenesis, the targeted molecular imaging has the hope to be used for early disease early angiogenesis of non-invasive diagnosis(NSFC No.81000141). Since a7 n ACh R play such an important role in the inflammatory pathway and angiogenesis pathways, does it has any effect on the diabetic cardiac injury? In February 4, 2012, we searched “a7n ACh R, diabetes, heart” as keywords on the Pub Med, the retrieval result is zero, which means that the role of a7 n ACh R, which plays an important role in anti-inflammation and pro-angiogenesis, is still blank in the research field of the diabetic heart damage.Based on aboved, this study intends to explore the role of a7 n ACh R high glucose induced CMEC injury, and whether it’s the common important reason behind diabetic myocardial inflammation of CMEC and the decreasing of angiogenesis ability. If so, to intervene a7 n ACh R in the diabetic heart tissue, there will be hope to achieve at the same time, reduce the inflammatory response in diabetic myocardial ischemic injury and promoting angiogenesis treatment, and it is expected to provide a new idea of double protection and target for the prevention and treatment of diabetes and ischemic heart disease.Objective:1 Identify the role of α 7n ACh R in myocardial microvascular endothelial cell damage caused by high glucose.2. Preliminary explore the mechanisms of α 7n ACh R’s protective effects and possible mechanism on myocardial microvascular endothelial cell injury by high glucose.Methods:1.Experiment(all the experiments were repeated with 3-times):1.1 Culturing and identification the myocardial microvascular endothelial cells in different groups:normal or high glucose(25mmol/L) culture, at the same time according to the grouping add the α 7n ACh R agonist GTS-21 dihydrochloride(DMBX- A)(10μM) or antagonist(Methyllycaconitine)of α 7n ACh R.1.2 Experimental groups: control group(Con), high glucose medium(HG), Con+DMBX-A(DMBX-A), Con+ Methyllycaconitine(Methyllycaconitine), high glucose medium + DMBX-A group(HG+DMBX-A), high glucose medium+ Methyllycaconitine group(HG+ Methyllycaconitine).1.3 Using the Tunel/DAPI immunofluorescence method to detected CMECs’ apoptosis, Transwell Chambers detected CMECs’ migration ability, the In vitro vascularpermeability assy kit to detected CMECs’ permeability.1.4 Flow cytometry instrument detected the level of reactive oxygen species(ROS).1.5 Enzyme Linked Immunosorbent Assay detected the nitric oxide(NO), nitric oxide synthase(NOS), superoxide dismutase(SOD).1.6 Tube-like structure and angiogenesis were tested by Matrigel matrix culture.2.Experiment 2(all the experiment are repeated 3 times) :2.1 The primary culture and identification of myocardial microvascular endothelial cells, the method with grouping experiment 1.1.2.2 The nucleus and the cell membrane were separated via Subcellular Protein Fractionation kit for Cultured Cells.2.3 α 7n ACh R/JAK2 / AKT/NFk B expressional level were analyzed by the Western blot.2.4 The expression of i NOS/e NOS were detected by RT-PCR(reverse transcription-polymerase chain reaction).Results:1. The experiment 1:1.1 CMECs were identified by Ac LDL staning.1.2 Compared with the Con, The apoptosis index of CMECs in the HG group increased, DMBX-A significantly reduced the cell apoptosis, and Methyllycaconitine has increased the index significantly.1.3 The migration ability is abate in HG group compared with the Con group, and recovery with DMBX-A, in addition significantly reduced by Methyllycaconitine.1.4 DMBX-A attenuated the permeability change caused by high glucose. Whereas, the effect was apparently decreased in the Methyllycaconitine group.1.5 Compared with Con,a higher ROS index was obtained in HG group. DMBX-A had normalized the ROS index and Methyllycaconitine had significantly increased this index.1.6 Compared with the Con, NOS activity in the HG was rised, the level of NO increased, and the vitality of SOD reduced at the same time. DMBX-A could reduce the trend, and Methyllycaconitine trained increased the NOS activity and NO production and significantly reduced the SOD’s vitality.1.7 The ability of tube formation is more higher in the CMECs treated with DMBX-A than Con and HG, meanwhile the little tube structures were found in the cells treated with Methyllycaconitine.2.The Experiment 2:2.1 Western blot results show that compared with normal group, the HG induced CMECs’ JAK-2 / p-AKT expression and up-regulate the expression of NF kappa B predominately.DMBX-A has eased the above phenomenon, and this phenomenon is more obvious in Methyllycaconitine group.2.2 In HG group,a apparent increase of i NOS and decrease of e NOS were demonstrated, furthermore, the Methyllycaconitine significantly aggravate the imbalance of i NOS/e NOS. However, the DMBX-A recovered this balance of i NOS/e NOS.Conclusions:1. Our study represents that activation of α 7n ACh R plays a important roll in the protective effect of injured CMECs cause by high glucose, and is closely related to the pathological and physiological changes in the myocardial microvascular endothelial cells. The α 7n ACh R activated by DMBX-A can improve CMECs’ pathological changes: reduce the apoptosis, increase the ability of migration and tube formation, reduce the cell permeability, lower the ROS generation.2. Our research also indicates the mechanism of the protecion effect in activation α7n ACh R: Via JAK2-p AKT-NFk B-i NOS/e NOS pathway, α 7n ACh R is helpful to adjust the balance of i NOS/e NOS, and improve CMECs’ funtion and angiogenesis...