| Background:With the improvement of people’s living standard and the changes of life style, the morbidity of type2diabetes (T2DM) presents a tendency of rapid increasing in our country. According to the latest data in2009, the morbidity of T2DM has reached9.7%in our country, and the total number of patients was92.40million which has climbed atop the first in the world. Therefore, to explore the mechanisms of diabetes mellitus has been a hotspot and focus in the field of diabetes. The effect of incretin is an important mechanism for regulating and controlling postprandial insulin secretion and postprandial glucose, which is a breakthrough for the pathogenesis and treatment of T2DM. Glucagon-like peptide-1(GLP-1) is produced in L cells with the functions of glucose-dependent insulin secretion and improving the proliferation and apoptosis of p-cells. Many studies suggest that the effect of incretin is significantly impaired in patients with T2DM, favoring the hyposecretion of GLP-1. Therefore we propose that the early damage and apoptosis of intestinal L cells may play a central role in the hyposecretion of GLP-1and the development of T2DM.Present studies mostly focus on the regulating effect of different medicine in secretion of GLP-1and its possible pathway, whereas few studies aim at the early damage and apoptosis of intestinal L cells, especially the effects of advanced glycation end-products (AGEs) in controlling the function of L cells. The pathway of AGEs and the receptor for advanced glycation end products (RAGE) is an important mechanism in the damage of various histocyte with diabetes. So to explore whether AGEs-RAGE result in the damage of L cells and its mechanism plays a central role in understanding and improving the pathogenesis of T2DM.AGEs are toxic and irreversible compounds that are formed by the glycosylation between long-term hyperglycemia and various proteins. The production and accumulation of massive irreversible AGEs is an important mechanism in cellular damage and chronic progress of diabetes. AGEs combining with RAGE can up-regulate the inflammatory response and lead to the destruction of histiocyte, so AGEs may be a pre-inflammatory mediator that can activate cells and promote the response to oxidative stress. Many studies suggest that the pathway of AGEs-RAGE not only mediates damage of various cells but also leads to apoptosis and dysfunction in islet cells. The similar results are also obtained by the latest study in2013:high concentration of AGEs can obviously up-regulate the expression of RAGE on the surface of islet cells. Moreover, the combination between AGEs and RAGE activates NADPH oxidase to increase the production of intracellular reactive oxygen species (ROS), then promotes the expression of nuclear factor kappa (NF-κB) and up-regulates the ratio of Bax/Bcl-2, which finally obviously increases the apoptosis rates of islet cells. Therefore, using the inhibitor of NADPH oxidase can improve the apoptosis of islet cells induced by AGEs. It follows that the pathway of AGEs-RAGE that activates NADPH oxidase to increase the production of intracellular ROS, then promotes the expression of NF-κB and up-regulates the ratio of Bax/Bcl-2is an important mechanism for the apoptosis of inlet cells and the reduced secretion of insulin. Interestingly, our colleagues conducted a study about the effect of lipopolysaccharide (LPS) in the apoptpsis and secretion punction of L cells, and confirmed that lipopolysaccharide (LPS) could induced the apoptpsis of L cells and reduced the secretion of GLP-1through promoting the expression of NF-κB and the downregulation of Bcl-2. This damage was a time-dependent and dose-dependent effect. Moreover, LPS, as well as AGEs, could induced the damage of cells through RAGE and its related pathway. It indicated that the apoptosis of L cells was a cause for reduction of GLP-1, and RAGE played an important role in it.Therefore, this program focus on L cell line (GLUTag cells), and discuss from the following aspects:1. we observe the effect of AGEs in the early damage and apoptosis of GLUTag cells and the secretion of GLP-1.2. we observe the effect of AGEs-RAGE and its related mechanism with monoclonal antibody interfering RAGE in the damage and apoptosis of GLUTag cells. The study will further explain the cause why GLP-1decrease secretion in patients with T2DM and provide new foundation to explore the new therapy in T2DM.Part1The effects of AGEs on the early damage and apoptosis of GLUTag and the secretion of GLP-1Objective:To study the effect of AGEs in the early damage and apoptosis of GLUTag cells and the secretion of GLP-1.Methods:(1)Preparation of AGEs-BSA in vitro Briefly, BSA was dissolved in phosphate-buffered saline (PBS; pH=7.4) with D-glucose, and incubated for12weeks in the dark. AGE-BSA was identified using a fluorescence spectrophotometer.(2)Culture of GLUTag cellsGLUTag cells were subcultured with low glucose DMEM containing10%fetal bovine serum,100IM/ml penicillin and100IM/ml streptomycin.(3)Groups:The effect of different concentration of AGEs in L cells:The blank control group was cultured with no intervention factor. The BSA control group was cultured with BSA as the positive control. The100μg/ml AGEs group was cultured with100μg/ml AGEs. The200μg/ml AGEs group was cultured with200μg/ml AGEs. The300μg/ml AGEs group was cultured with300μg/ml AGEs.The effect of AGEs in L cells for different times:200μg/ml AGEs incubated with cells for0,12,24,48h.(4) The effects of AGEs in the apoptosis of GLUTag cellsThe cells were divided into5groups as above. The morphological change of apoptosis were detected with Hoechst33258fluorescence staining.The apoptosis rate was detected with AnnexinⅤ-FITC/PI method.(5) The effects of AGEs in the proliferation of GLUTag cellsThe cells were divided into5groups as above. The proliferation of GLUTag cells was tested with CCK-8method.(6)The effects of AGEs in the secretion of GLP-1in GLUTag cellsThe cells were divided into5groups as above and the supernate of cells were collected. The secretion level of GLP-1in GLUTag cells was tested with ELISA method.Statistical Analysis: All analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Apoptotic rate was determined by the factorial design ANOVA. Statistical significance was defined as two-sided p<0.05.Results:(1)The effect of different concentration of AGEs in the morphological change of GLUTag cellsAll the groups were observed with fluorescence microscope by Hoechst33258staining after intervention for24h. The apoptotic cells in100μg/ml AGEs group showed little morphological change. The apoptotic cells in200ug/ml AGEs group and300μg/ml AGEs group showed topic morphological changes. Apoptotic cells were observed as intact round nuclei and fragmented (or condensed) nuclei.(2)The effect of200μg/ml AGEs in the morphological change of GLUTag cells for different timeThe apoptotic cells in12h group showed little morphological change. The apoptotic cells in24h group and48h group showed topic morphological changes were observed with Hoechst33258staining. Apoptptic cells were observed as intact round nuclei and fragmented (or condensed) nuclei.(3) The effect of different concentration of AGEs in the apoptosis of GLUTag cellsThe results obtained from FCM showed that the apoptotic rate in100μg/ml AGEs group was (15.30±2.89)%, which significantly higher than that of control group (6.43±1.03)%and BSA group (7.37±1.50)%(P=0.000). Apoptptic rate in200μg/ml AGEs group was (24.10±2.95)%, which significantly higher than that of control group, BSA group and100μg/ml AGEs group (P=0.000). Apoptptic rate in300μg/ml AGEs group was (35.19±3.76)%, which significantly higher than that of control group, BSA group and100μg/ml AGEs group (P=0.000), and higher than 200μg/ml AGEs group (P<0.05).(4) The effect of200μg/ml AGEs in the apoptpsis of GLUTag cells for different timeThe results obtained from FCM showed that the apoptotic rate in12h group was (12.61±1.94)%, which significantly higher than that of control group (6.30±2.23)%(P=0.000). Apoptptic rate in24h group was (21.77±3.41)%, which significantly higher than that of control group and12h group (P=0.000). Apoptptic rate in48h group was (29.52±4.42)%, which significantly higher than that of control group and12h group (P=0.000), and higher than24h group (P<0.05).(5) The effect of different concentration of AGEs in the activity of GLUTag cellsAfter treatment with100,200,300μg/mL AGEs for24h, the OD value were0.86±0.05,0.66±0.05,0.46±0.06, respectively, and significantly lower than control group (1.21±0.05) and the BSA group (1.19±0.03)(P=0.000). Moreover, the OD value of200μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000). The OD value of300μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).(6) The effect of200μg/ml AGEs in the activity of GLUTag cells for different timeAfter treatment with200μg/mL AGEs for12h, the OD value was0.87±0.05, which significantly lower than control group (Oh,1.110±0.05)(P=0.000). The OD value of24h group was0.66±0.04, which significantly lower than control group and12h group (P=0.000). The OD value of48h group was0.39±0.04, which significantly lower than control group and12h group (P=0.000), and also significantly lower than24h group (P<0.05).(7) The effect of different concentration of AGEs in GLP-1secretion of GLUTag cellsAfter treatment with100,200,300μg/mL AGEs for24h, the concentration of GLP-1from the GLUTag cells were69.97±4.30,55.27±6.20,37.13±5.55, respectively, and significantly lower than control group (91.27±5.23) and the BSA group (86.63±6.71)(P=0.000). Moreover, the concentration of GLP-1in200μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000). The concentration of GLP-1in300μg/mL AGEs group significantly lower than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).(8) The effect of200μg/ml AGEs in the GLP-1secretion of GLUTag cells for different timeAfter treatment with200μg/mL AGEs for12h, the concentration of GLP-1was71.10±4.75, which significantly lower than control group (Oh,83.97±7.28)(P=0.000). The concentration of GLP-1in24h group was55.27±6.20, which significantly lower than control group and12h group (P=0.000). The concentration of GLP-1in48h group was33.93±6.01, which significantly lower than control group and12h group (P=0.000), and also significantly lower than24h group (P<0.05).Conclusions:The apoptosis of GLUTag cells became severer with both the concentration of AGEs increasing for the same aging time and the longer time of exposure to AGEs in the same does. We found for the first time that the apoptpsis of L cells were induced by AGEs in a dose, time-dependent manner and the GLP-1secretion from L cells decreased.Part2The effect of AGEs-RAGE and its related mechanism in the damage and apoptosis of GLUTag cellsSection1The effect of AGEs-RAGE in the apoptosis of GLUTag cells and its related machanismObjective:To investigate the effect of AGEs-RAGE and its related pathway in the apoptosis of GLUTag cells induced by AGEsMethods:(1) GroupsThe effect of different concentration of AGEs in the expression of RAGE of GLUTag cells:The blank control group was cultured with no intervention factor. The BSA control group was cultured with BSA as the positive control. The100μg/ml AGEs group was cultured with100μg/ml AGEs. The200μg/ml AGEs group was cultured with200μg/ml AGEs. The300μg/ml AGEs group was cultured with300μg/ml AGEs. All the groups were incubated for24h. The effect of AGEs in the expression of RAGE and its related pathway:The blank control group was cultured with no intervention factor. The BSA control group was cultured with BSA as the positive control. The AGEs group was cultured with200μg/ml AGEs. The AGEs+siRNA-RAGE group was cultured with AGEs after the transfection of siRNA-RAGE. The AGEs+apocynin group was cultured with AGEs and apocynin.(2) The effect of AGEs in the expression of RAGE of GLUTag cellsGLUTag cells were subcultured (the method as part1), and AGEs-BSA was prepared. The concentration and time of AGEs were adopted the identified one. The expression of RAGE mRNA was detected with RT-PCR. The expression of protein for RAGE was detected with Western blot.(3) The effects of AGEs in the level of ROS and the expression of NADPH oxidase in GLUTag cells The cells were divided into5groups as above. The expression of gp22phox and p47phox was detected with Western blot.The change of intracellular ROX was tested with fluorescent probe method.(4) The effects of AGEs in the expression of P53and Bax in GLUTag cellsThe cells were divided into5groups as above.The change of P53and Bax was detected with Western bolt.(5) The effects of AGEs in the activity of caspase-3,-9in GLUTag cellsThe cells were divided into5groups as above. The activity of caspase-3,-9was detected with ELISA method.Statistical Analysis:All analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p<0.05.Results:(1) The effect of different concentration of AGEs in the expression of RAGE mRNA in GLUTag cellsAfter treatment with100,200,300μg/mL AGEs for24h, the expression of RAGE mRNA were1.45±0.13,2.14±0.010,2.45±0.32, respectively, and significantly higher than control group (1.01±0.04) and the BSA group (1.09±0.03)(P=0.000). Moreover, the expression of RAGE mRNA in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The expression of RAGE mRNA in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).(2) The effect of different concentration of AGEs in the protein level of RAGE in GLUTag cellsAfter treatment with100,200,300μg/mL AGEs for24h, the protein level of RAGE were0.36±0.04,0.56±0.009,0.72±0.06, respectively, and significantly higher than control group (0.14±0.04) and the BSA group (0.18±0.03)(P=0.000). Moreover, the protein level of RAGE in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The protein level of RAGE in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000) and200μg/mL AGEs group (P<0.05).(3) The effect of AGEs in the expression of NADPH oxidase subunit in GLUTag cellsCells in groups were treated with indicated treatment for24h. The expressions of gp22phox and p47phox in200μg/mL AGEs group were0.66±0.08and0.76±0.09, which significantly higher than that of the BSA group (0.27±0.06and0.30±0.06)(P<0.05). The expressions of gp22phox and p47phox in AGEs+apocynin group were0.40±0.08and0.47±0.09, which significantly higher than that of the BSA group (P<0.05) and lower than that of the200μg/mL AGEs group (P<0.05). The expressions of gp22phox and p47phox in AGEs+siRNA-RAGE group were0.32±0.04and0.34±0.056, which significantly lower than that of the200μg/mL AGEs group (P<0.05).(4)The effect of AGEs in the activity of ROS in GLUTag cellsCells in groups were treated with indicated treatment for24h. The fluorescence intnesity of200μg/mL AGEs group was57.58±4.69, which significantly higher than that of the BSA group (23.16±3.47)(P<0.05). The fluorescence intnesity of AGEs+apocynin group was34.97±0.08, which significantly higher than that of the BSA group (P<0.05) and lower than that of the200μg/mL AGEs group (P<0.05). The fluorescence intnesity of AGEs+siRNA-RAGE group was29.44±5.27, which significantly lower than that of the200μg/mL AGEs group (P<0.05).(6) The effect of AGEs in the expression of p53and Bax in GLUTag cells Cells in groups were treated with indicated treatment for24h. The expression of p53in200μg/mL AGEs group was0.42±0.05, which significantly lower than that of the BSA group (0.73±0.05)(P<0.05), wherase the expression of Bax in this group was0.66±0.08, which significantly higher than that of the BSA group (0.27±0.08)(P<0.05). The expression of p53in AGEs+apocynin group and AGEs+siRNA-RAGE group were0.64±0.05and0.71±0.06, which significantly higher than that of the200μg/mL AGEs group (P<0.05), wherase the expression of Bax in these group were0.40±0.08and0.32±0.04, which significantly lower than that of the200μg/mL AGEs group (P<0.05).(7) The effect of AGEs in the activity of caspase-3and-9in GLUTag cellsCells in groups were treated with indicated treatment for24h. The activity of caspase-3and-9in200μg/mL AGEs group were1.03±0.10and1.22±0.05, which significantly higher than that of the BSA group (0.43±0.05and0.47±0.02)(P<0.05). The activity of caspase-3and-9in AGEs+apocynin group were0.65±0.07and0.68±0.09, which significantly higher than that of the BSA group (P<0.05) and lower than that of the200μg/mL AGEs group (P<0.05). The activity of caspase-3and-9in AGEs+siRNA-RAGE group were0.55±0.08and0.57±0.07, which significantly lower than that of the200μg/mL AGEs group (P<0.05).Conclusions:The expression of RAGE mRNA and the protein level of RAGE in GLUTag cells increased with the concentration of AGEs increasing for the same aging time, which suggested AGEs could enhance the oxidative stress of GLUTag cell via the increasing the expression of RAGE. After indicated treatment for24h, the expression of gp22phox and p47phox and the activity of ROS increased, resulting to the downregulation of p53and the upregulation of Bax, as well as increasing the activity of caspase-3and-9, which finally increased the apoptpsis of L cells. That meant that NADPH oxidase, ROS, p53/Bax and caspase-3,-9were the majoy pathway of the apoptosis in GLUTag cells induced by AGEs-RAGE.Section2The effect of AGEs-RAGE in the inflammation of GLUTag cells and its related machanismObjective:To observe the effect of AGEs on the inflammation of GLUTag cells and the pathway of p38MAPK/NF-κBMethods:(1) The effect of AGEs in the phosphorylation of p38MAPK in GLUTag cellsGLUTag cells were subcultured (the method as part1), and AGEs-BSA was prepared. The time of AGEs were adopted the identified one.Groups:The blank control group was cultured with no intervention factor;The BSA control group was cultured with BSA as the positive control;The100g/ml AGEs group was cultured with100μg/ml AGEs;The200μg/ml AGEs group was cultured with200μg/ml AGEs;The300μg/ml AGEs group was cultured with300μg/ml AGEs;The AGEs+apocynin group was cultured with AGEs and apocynin.All the groups were accepted the following tests. The phosphorylation of p38MAPK was detected with Western blot.(2) The effect of AGEs in the activity of NF-κB in GLUTag cellsThe cells were divided into6groups as above.The activity of NF-κB was detected with Western blot.(3) The effect of AGEs in the inflammatory factor of GLUTag cellsThe cells were divided into6groups as above. The change of inflammatory factor TNF-α、IL-1、IL-6were detected with ELISA method.Statistical Analysis:All analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p<0.05.Results:(1) The effect of AGEs in the phosphorylation of p38MAPK in GLUTag cellsAfter treatment with100,200,300μg/mL AGEs for24h, the expression of p-p38MAPK were0.41±0.05,0.61±0.05,0.82±0.04, respectively, and significantly higher than control group (0.22±0.03) and the BSA group (0.24±0.04)(P=0.000). Moreover, the expression of p-p38MAPK in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The expression of p-p38MAPK in300μg/mL AGEs group significantly higher than that of1000μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The expression of p-p38MAPK in AGEs+apocynin group was0.28±0.03, which significantly lower than that of200μg/mL AGEs group (P=0.000).(2) The effect of AGEs in the expression of NF-κB in GLUTag cellsAfter treatment with100,200,300μg/mL AGEs for24h, the expression of NF-κB were0.36±0.04,0.55±0.08,0.67±0.05, respectively, and significantly higher than control group (0.18±0.02) and the BSA group (0.20±0.02)(P=0.000). Moreover, the expression of NF-κB in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The expression of NF-κB in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The expression of NF-κB in AGEs+apocynin group was0.22±0.03, which significantly lower than that of200μg/mL AGEs group (P=0.000).(3)The effect of AGEs in the inflammatory factor of GLUTag cells After treatment with100,200,300μg/mL AGEs for24h, the content of TNF-a were27.23±4.25,38.93±3.62,54.47±5.85, respectively, and significantly higher than control group (15.30±1.71) and the BSA group (16.10±2.15)(P=0.000). Moreover, the content of TNF-α in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The content of TNF-α in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The content of TNF-α in AGEs+apocynin group was18.40±3.75, which significantly lower than that of200μg/mL AGEs group (P=0.000).After treatment with100,200,300μg/mL AGEs for24h, the content of IL-1were136.50±13.05,191.17±12.01,287.7±13.75, respectively, and significantly higher than control group (50.67±4.99) and the BSA group (55.33±6.06)(P=0.000). Moreover, the content of IL-1in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The content of IL-1in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The content of IL-1in AGEs+apocynin group was68.17±8.72, which significantly lower than that of200μg/mL AGEs group (P=0.000).After treatment with100,200,300μg/mL AGEs for24h, the content of IL-6were95.93±12.29,177.93±9.26,234.27±19.74, respectively, and significantly higher than control group (43.80±5.94) and the BSA group (50.40±5.64)(P=0.000). Moreover, the content of IL-6in200μg/mL AGEs group significantly higher than that of100μg/mL AGEs group (P=0.000). The content of IL-6in300μg/mL AGEs group significantly higher than that of100μg/mL AGEs group and200μg/mL AGEs group (P=0.000). The content of IL-6in AGEs+apocynin group was63.50±7.62, which significantly lower than that of200μg/mL AGEs group (P=0.000).Conclusions:The phosphorylation of p38MAPK and the expression of NF-κB, TNF-α, IL-1, IL-6increased with the concentration of AGEs increasing for the same aging time. The NADPH oxidase inhibitor apocynin could regulate the expression of p38MAPK, NF-κB, TNF-α, IL-1, IL-6in GLUTag cells induced by AGEs. This part suggested that AGEs could induce the inflammatory injury of L cells through the pathway of p38MAPK/NF-κB. |
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