| BackgroundDM (Diabetes mellitus) is a systemic disease caused by genetic andenvironmental factors, and its effect on cardiovascular system is a very serioushazard. AGEs (Advanced glycation end products) play a great influence on thedevelopment of diabetes and its complications. AGEs and diabetes mellitus aresignificantly related. To prevent AGEs generation can reduce or mitigate thedevelopment of diabetes and its complications. Notch signaling pathway isclosely associated with the occurrence of certain organs and diseases. γ-secretase inhibitor (DAPT) is a specific inhibitor of Notch signaling pathway.Studies have shown that the Notch signaling pathway is involved inangiogenesis and other important physiological processes.This study is designed to investigate the effects of γ-secretase inhibitor(DAPT) under AGEs on cardiac microvascular endothelial cells (CMECs) andthe mechanism of Notch signaling pathway. Methods:Part I: To get rat ventricle in a sterile environment, using enzymedigestion and isolated CMECs. After being centrifugal, using complete culturemedium (containing20%FCS) resuspended and then placing at37℃,5%CO2incubator, observing cell morphology. BSA and glucose in the dark conditions,constant temperature at37℃were incubated for80days, and prepared the BSA(using the same conditions) for the control group. Successfully prepared, usingthe fluorescence spectrophotometer to identify and calculate the protein content.Part II: Take the2nd generation of the above cultured cells and add200mg/L AGEs-BSA to CMECs. Then ues different concentrations of DAPT(μmol/L:0,0.25,0.5,1.0,5.0,10.0) intervention for1day, or DAPT (5μmol/L),intervention at different times (0h,24h,48h,72h,96h). MTT assay was used todetect cell proliferation capacity. Transwell method was used for the detectionof cell migration. Use the capillary tube-like structure formation experiment toobserve the DAPT on cell angiogenesis.Part III: Experiment was divided into the BSA group, AGEs-BSA groupand (AGEs-BSA+DAPT) group, respectively being observed for1day,2days,3days,4days. The cell proliferation capacity was measured by MTT assay. Thecell angiogenesis was observed by tube-like structure formation assay. The cellapoptosis was measured by TUNEL assay. Notch protein was measured byWestern blot.Results1. CMECs were isolated and cultured in vitro successfully.2. Successfully prepared out of AGEs-BSA. After being identified, the contentof AGEs was98.6kU/g, the content of control group was2.2kU/g. 3. DAPT can significantly inhibit the cells survival. With the higherconcentration and the longer intervention time, the inhibitory effect wasmore obvious (P<0.05).4. Compared with the other two groups, AGEs-BSA group absorbance values at1d,2d,3d were significantly higher (P<0.01). The length of tube-likestructures and Notch protein expression increased significantly (P<0.05).After4d, in AGEs-BSA group, the length of tube-like structures and Notchprotein expression decreased significantly (P<0.01). Compared withAGEs-BSA group,(AGEs-BSA+DAPT) group absorbance values and thelength of tube-like structures decreased significantly (P<0.05), while theapoptotic index was significantly increased (P<0.01).Conclusions1. By blocking Notch signaling pathway, DAPT can inhibit the effects of AGEson cardiac microvascular endothelial cells (CMECs), such as proliferation,migration and angiogenesis, and have some correlations with concentrationand time.2．AGEs can significantly enhance proliferation of CMECs and tube-likestructure-forming ability in the early days, and its mechanism may be relatedto Notch signaling pathway.3．In the late days, AGEs can increase the cells apoptosis and make the tube-likestructures collapse. These phenomena may be associated with reducedexpression of Notch. |
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