Cardiovascular disease (CVD) based on atherosclerosis and microangiopathy is a major complication of diabetes and a major cause of disability and death in diabetes. Damage and dysfunction of endothelial cells (ECs) are the initiating factors in the development and progression of diabetic vascular complications. Therefore, studying the mechanism of endothelial injury and finding effective ways to repair the structure and function of damaged endothelium has become a hot research topic.
Endothelial progenitor cells (EPCs) are bone marrow-derived cells that can expand and further differentiate into ECs in vitro and participate in the repair and revascularization of damaged endothelium, and both animal experiments and clinical studies have confirmed that 25% of ECs in neovascularization are differentiated from EPCS, so EPCs are considered to be important biochemical factors for the treatment of various cardiovascular diseases and damaged vessels. Endothelial nitric oxide synthase (eNOS) is an important substance that ensures the migratory capacity of EPCs.
In a high glucose environment in vivo, oxidative stress can trigger the appearance of oxidative stress, which in turn can lead to impaired function of EPCs and reduced production of nitric oxide (NO), while the application of superoxide dismutase (SOD) intervention in EPCs can lead to increased NO secretion and functional recovery. Antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), etc. Non-enzymatic antioxidant systems, including vitamin C, vitamin E, glutathione, melatonin, alpha-lipoic acid (ALA), carotenoids, trace elements copper, zinc, selenium (Se), etc.
In this study, we applied high concentration of glucose to incubate EPCs to induce oxidative stress, measured malondialdehyde (MDA) and nitric oxide (NO) levels in culture supernatants, and also observed the mRNA expression of GPx-1 and eNOS by real-time fluorescence quantitative RTPCR, determined the protein expression of GPx-1 and eNOS by western blot, and then applied the antioxidant ALA for Then, the antioxidant ALA was applied to intervene to investigate the mechanism of glucose-induced EPCs damage and therapeutic measures, and to provide a basis for the prevention and treatment of diabetic vascular complications.
1. Materials and methods
1.1 Materials
1.1.1 Experimental animals: 15 clean grade healthy male Wistar rats (weight 180-200 g), provided by the Experimental Animal Center of Hebei Medical University, animal certificate number: 902035.
1.1.2 Reagents MDA and NO assay kit (Nanjing Jiancheng Institute of Biological Engineering), 50% glucose injection (Huarui Pharmaceutical Co., Ltd.), EGM-2MV medium (LONZA, USA), rat lymphocyte isolate (Tianjin Hao Yang Co., Ltd.);
Human fibronectin (Solexpro Technology Co., Ltd.), trypsin (sigma, USA), RNA extraction reagent Trizol Reagent (Invitrogen, USA), reverse transcriptase (M-MLV ), random primers were produced by Promega, rabbit anti-human polyclonal GPx-1 antibody (ABCAM, UK), rabbit anti-human polyclonal eNOS antibody (Santa, USA), ALA (Wizai Pharmaceutical Co., Ltd., Japan).
1.1.3 Experimental instruments UV spectrophotometer (756MC) (Shanghai Precision Instrument Science Co.
), ABI 7300 real-time fluorescence quantitative PCR system (PE, USA), UVP gel scanning system (UVP, USA), DYZ22A double constant-time electrophoresis instrument and DYY-III bridge electrophoresis instrument (Beijing Liuyi Instrument Factory), inverted microscope (Olympus, Japan) ultra-clean table (HFsafe1200) (Likang Development Co., Ltd.), carbon dioxide incubator (MCO-15AC) (Heraeus, Germany), cell culture plates and culture bottles (Corning, USA)
1.2 Experimental methods
1.2.1 Rat bone marrow EPCs culture
The lower limb of the rat was removed with surgical scissors, the skin of the lower limb was cut off, the lower limb was soaked in 75% alcohol for 30 min, the femur was separated from the tibia along the knee joint in an ultra-clean table, the muscle of the lower limb was removed, the lower limb bone was exposed under aseptic conditions, immersed in a culture dish containing PBS, the lower limb bone was cut off from both ends, and the bone marrow cavity was repeatedly rinsed with PBS in an ultra-clean table;
Then the rinsing solution was slowly superimposed on the rat lymphocyte isolate, centrifuged at 2000 rpm for 20 min, the liquid in the tube was divided into three layers after centrifugation, the middle white cloudy layer was aspirated out, 4 times the volume of PBS was added and mixed, centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and then the washing was repeated once. After the final centrifugation, discard the supernatant and add EGM-2MV to resuspend the cells.
Fn was spread into the well plates according to 5ug/cm2, and the cell suspension was inoculated into six-well plates and 24-well plates at a density of 2×106 cells/mL. The first full volume of fluid was changed after 48 h, and then the fluid was changed every day, and every other day after 7 days. Cell growth was observed daily under an inverted microscope.
1.2.2 Identification of 2EPCs
Morphological identification: Observe cell morphology and growth under an inverted microscope every day.
Experiment of EPCs uptake of DIL-Ac-LDL, combined with FITC-Lectin-UEA-1: Cells on the 10th day of culture were incubated with medium containing DIL-Ac-LDL (2.5ug/mL) for 4h, washed 3 times with PBS, fixed with 2% paraformaldehyde for 20min, and then FITC-Lectin-UEA-1 ( 10ug/mL), incubate for 1h, observe under laser confocal microscope, double-stained as orange-yellow cells as EPCs, and count double-stained cells.
1.2.3 Experimental grouping
EPCs cultured for 4 days were digested with 0.25% trypsin/0.02% EDTA and grouped for 24 h after apposition, respectively
The EPCs were incubated with glucose 5 mmol/L as normal control group (NC), 30 mmol/l glucose as high glucose group (HS), and glucose (30 mmol/l) + α lipoic acid (40µg/l) as ALA group for 48 hours, and the indexes were detected.
1.2.4 NO and MDA were measured by chemical colorimetric method, Western blot method to determine the expression of GPx-1 and eNOS in EPCs, and real-time fluorescence quantitative RT-PCR method to measure the expression of GPx-1 mRNA in EPCs.
1.3 Statistical methods
All data were analyzed using the SPSS 13.0 statistical analysis package. All measurement data were expressed as mean ± standard deviation ( ±s). Multiple sample means were compared using one-way ANOVA with a completely randomized design, and significant differences were considered at P<0.05.
2, Results
2.1 Effect of different interventions on MDA secretion by EPCs (Table 3)
The culture supernatant MDA level was higher than that of the normal control group after 48 h of high glucose intervention (P<0.05); the MDA level decreased after the application of ALA intervention compared with the high glucose group (P<0.05).
2.2 Effects of different interventions on GPx-1 and eNOS expression in EPCs (Fig1-4, Table 1)
The expression of GPx-1 and eNOS of EPCs was significantly lower than that of the control group after 48 hours of high glucose intervention, while the expression of GPx-1 and eNOS of EPCs was significantly increased after ALA intervention compared with the high glucose group (both P<0.05).
2.3 Effects of different interventions on GPx-1 and eNOS mRNA expression of EPCs (Fig5-8, Table 2)
The expression levels of GPx-1 and eNOS mRNA in EPCs were significantly lower than those in the control group after 48 hours of high glucose intervention, while the expression of GPx-1 and eNOS mRNA in EPCs increased significantly after ALA intervention compared with the high glucose group (both P<0.05).
2.4 Effect of different interventions on the level of secreted NO in EPCs (Fig9, Table 3)
After 48 hours of high sugar intervention EPCs secreted NO levels were lower than normal control group (P < 0.05), while after ALA intervention EPCs eNOS expression was significantly increased compared to high sugar group (P < 0.05), and culture supernatant NO levels were increased.
3, Discussion
In 2004, at the annual meeting of the American Diabetes Association (ADA), Brownlee, a Banting Award winner, gave a lecture: both macrovascular and microvascular complications are caused by the same pathogenesis, i.e., oxidative stress, which is the common basis of high glucose damage; in the same year, the European Association for the Study of Diabetes (EADS) gave a lecture on the importance of the same pathogenesis, i.e., oxidative stress. In the same year, Ceriello, winner of the top prize at the European Association for the Study of Diabetes (EASD) annual meeting, gave a lecture on oxidative stress as a common mechanism of insulin resistance, diabetes and cardiovascular disease – the common soil theory.
EPCs are precursor cells present in circulating peripheral blood capable of differentiating into vascular endothelial cells (ECs), which are derived from bone marrow, umbilical cord blood or fetal liver. In the case of vascular injury or tissue ischemia, EPCs present in bone marrow can proliferate in response to locally released growth factors and cytokines, differentiate into mature endothelial cells, and mobilize to the peripheral circulation, where they specifically nest at the site of injury or ischemia to further proliferate and differentiate into mature endothelial cells that participate in vascular neogenesis and re-endothelialization.
Once the number and function of EPCs are impaired, the balance between endothelial damage and repair is disrupted and the integrity of the endothelial layer is damaged, resulting in atheromatous lesions. Therefore, endothelial progenitor cells play an important role in regulating the apoptosis/regeneration balance of endothelial cells and maintaining the integrity of the endothelial layer. Therefore, the function of EPCs is closely related to the development and progression of diabetic vascular complications and is a predictor of cardiovascular disease.
Dernbach et al [6] examined the oxidative sensitivity of healthy adult EPCs and found that the intracellular ROS levels and oxidant-mediated apoptosis were lower in healthy adult EPCs than in umbilical vein endothelial cells, and the expression of antioxidants such as manganese superoxide dismutase (MnSOD), GSH-Px and CAT on the surface of EPCs was increased compared to umbilical vein endothelial cells, suggesting that healthy human EPCs have a stronger antioxidant capacity than endothelial cells;
This promotes their viability, but it is not sufficient to counteract the effects of risk factors on the endogenous antioxidant system of EPCs, which impairs their function under high levels of oxidative stress by imbalancing the redox status within EPCs, leading to apoptosis and senescence.
Elevated blood glucose can activate intracellular NADPH through the PKC pathway, generating large amounts of reactive oxygen species (ROS) and damaging cells, causing a decrease in normal cellular bioactivity, disruption of energy metabolism, cell signaling and other functions, which means that oxidative stress can be induced in a high glucose state. It has also been shown that high glucose can affect the bone marrow mobilization and survival of EPCs through oxidative stress and the P38MAPK pathway.
Diabetic and glucose-treated EPCs cause decreased bioavailability of NO or decreased tetrahydrobiopterin (BH4), the cofactor of eNOS, which results in uncoupling of eNOS, generating superoxide negative ions (O-) and leading to accumulation of reactive oxygen species clusters (ROS), further reducing the number and migratory capacity of EPCs. Oxidative stress in cells mediated by oxLDL plays a critical role in the pathogenesis of atherosclerosis.
OxLDL affects the function of EPCs by promoting their senescence through its inhibitory effect on EPCs eNOS. In recent studies, ox-LDL in diabetic patients leads to EPC dysfunction through P53-mediated signaling pathways, in contrast to HDL which is considered as a protective factor for vascular function due to its antioxidant and anti-inflammatory properties.
In this study, we applied high glucose to incubate rat bone marrow-derived EPCs for 48 h. The results showed that MDA levels in culture supernatants were significantly increased, indicating that high glucose can lead to oxidative stress in EPCs. Quagliaro et al. also suggested that high glucose concentrations, especially fluctuating high glucose, can activate protein kinase C (PKC) channels through the overexpression of diacylglycerol and mitochondrial superoxide anion, altering The activation of the mitogen-activated protein kinase pathway contributes to apoptosis and oxidative DNA damage.
GPx-1 is a key redox protein that plays an important role in protecting the endothelium from oxidative stress and thus maintaining a stable vascular environment. Studies have shown that in plaques of isolated carotid arteries GPx-1 activity is decreased. It has also been shown that GPx-1 overexpression maintains normal vascular endothelial function in endothelial cells cultured with high concentrations of cysteine.
In addition, it has also been shown that lack of GPx-1 not only results in vascular endothelial dysfunction but also in the presence of cardiac and vascular structural abnormalities. In addition, Ac-LDL+ Lectin+ VEGFR-2+ eNOS+ cells isolated from GPx-1 knockout mice are functionally defective in promoting angiogenesis and have increased sensitivity to oxidative stress both in vivo and in vitro [21].C-reactive protein (CRP) intervention in EPCs leads to an upregulation of their glycosylation end-product receptor expression, resulting in a downregulation of GPx-1 and other antioxidant enzymes expression down-regulated, while leading to senescence and dysfunction of EPCs in vivo, further leading to increased apoptosis.
eNOS is a cytochrome P450 reductase-like enzyme that catalyzes the flavin-mediated electron transfer from the electron donor NADPH to the subtilisin cofactor. eNOS cofactor BH4 is close to the subtilisin group and can transfer electrons to the guanidine nitrogen of L arginine to produce NO. In the absence of L arginine or BH4, eNOS produces O2- and H2O2, a phenomenon known as This phenomenon is called eNOS uncoupling.
eNOS plays a critical role in the mobilization and functional regulation of EPCs. eNOS uncoupling causes an increase in the level of ROS, which results in impaired function of EPCs. In diabetic patients, eNOS uncoupling reduces the number of EPCs in diabetic patients, decreasing their function and ultimately leading to the development of vascular disease.
It has been shown that eNOS is an important substance to ensure the migratory capacity of EPCs, and eNOS knockout mice can lead to reduced migratory capacity of EPCs [25], and ensuring adequate expression of eNOS, normal secretion of NO and normal biological activity are important factors to maintain the normal biological function of EPCs. In this study, we applied high concentration of glucose to intervene in EPCs for 48 h and found that The GPx-1 protein expression and mRNA expression levels of EPCs decreased, while there was a decrease in eNOS protein expression mRNA expression and down-regulation and NO secretion;
Therefore, the results of this study indicate that high glucose can cause oxidative stress in EPCs in addition to downregulating the expression of their antioxidant enzymes, thus impairing their antioxidant capacity and affecting their function.
ALA is a natural antioxidant, which can scavenge various free radicals generated during the physiological and pathological processes of the body and reduce the oxidative stress of the body. Studies have shown that ALA can improve vascular endothelial function in rats by increasing NO activity, reducing ROS production [26] and inhibiting NF-κB expression, and can also reduce the level of oxidative stress in vivo by activating the AMPK pathway in endothelial cells and reversing the mitochondrial membrane hyperpolarization induced by high glucose.
In this study, by applying ALA intervention to EPCs incubated with high glucose, it was found that GPx-1 and eNOS expression were upregulated in EPCs, NO secretion was increased and MDA secretion was decreased in EPCs, indicating that ALA treatment could reduce the oxidative stress produced by EPCs induced by high glucose and restore their antioxidant capacity, thus protecting their functions.
In conclusion, high glucose concentration can induce oxidative stress in EPCs on the one hand, and reduce the production and expression of their antioxidant enzymes and impair their antioxidant capacity on the other hand, while antioxidant stress treatment can improve the antioxidant capacity and NO secretion capacity of EPCs. This suggests to us that antioxidant stress treatment has some significance in the prevention and treatment of the occurrence and development of diabetic vascular complications.