高糖诱导INS-1细胞表达IL-1β及其损伤机制的研究
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摘要
[背景]
随着全球经济的快速发展,人口老龄化及生活方式现代化等变化,2型糖尿病的患病率正在不断增加。其致残率、死亡率仅次于肿瘤和心血管疾病,为第三大非传染性疾病。2型糖尿病严重威胁人类健康并给社会及家庭带来沉重的经济负担。因此,糖尿病的防治成为受到广泛关注的社会卫生问题。
目前普遍认为,胰岛素抵抗和胰岛功能受损是2型糖尿病发病机制的两个关键环节。英国前瞻性糖尿病研究显示,糖尿病患者在被明确诊断时,β细胞功能只剩下正常人的50%,其后胰岛β细胞功能每年以4%～5%的速度下降。以此推算,在糖尿病诊断前10～15年,β细胞功能已开始下降。
既往众多研究认为,在2型糖尿病中糖毒性和脂毒性是造成胰岛β细胞功能受损的主要病理机制。而近年来,研究发现细胞因子介导的炎症损伤在2型糖尿病的发生发展中同样发挥着重要作用。以往认为炎症介质是1型糖尿病的发病关键因素,但不断有研究显示炎症与2型糖尿病之间的同样存在密切联系。有研究报道在2型糖尿病患者的活检中发现胰岛存在纤维化,而纤维化是慢性炎症性疾病的最终阶段。这是支持胰岛局部存在炎症反应的一个有力证据。此外,还有学者指出循环中炎症相关细胞因子和化学因子的表达上调可作为2型糖尿病发病的预测指标。由此可见,炎症因子在2型糖尿病发生发展中的作用,已成为糖尿病发病机制研究领域中的一个倍受关注的热点。
白介素-1β(IL-1β)是在胰岛β细胞损伤研究中受到广泛关注的细胞因子之一。IL-1β是一种主要由单核-巨噬细胞、内皮细胞等分泌的细胞因子,具有广泛的生物学效应。1型糖尿病发生发展中,巨噬细胞产生的IL-1β与肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ等构成细胞因子调控网络诱导胰岛β细胞凋亡。2002年,Maedler首次发现将人胰岛β细胞体外培养于高糖中可诱导其分泌细胞因子IL-1β,利用IL-1β的天然抑制因子白介素1受体拮抗剂(IL-1Ra)能保护高糖环境下的胰岛β细胞。提示,高糖环境下胰岛β细胞分泌细胞因子IL-1β,其分泌的IL-1β可造成胰岛β细胞损伤。体内实验,肥沙鼠以高能量饮食喂养造成2型糖尿病模型,研究发现血糖升高肥沙鼠的胰岛β细胞可表达IL-1β,而血糖正常肥沙鼠的胰岛β细胞无IL-1β表达。随后其他实验室还利用另外两种2型糖尿病动物模型GK大鼠及人胰岛淀粉样多肽转基因大鼠进行研究,同样发现体内高糖环境可诱导胰岛β细胞合成并分泌IL-1β。但同时也有研究报道,长时间的高糖处理未能引起胰岛β细胞分泌IL-1β。
综上所述,细胞因子在2型糖尿病发病过程中的作用受到了越来越多的重视。而关于高糖是否诱导胰岛β细胞表达细胞因子IL-1β,这种IL-1β是否损伤胰岛β细胞,目前存在争议且相关研究报道尚不多见。因此,本课题将以大鼠胰岛细胞瘤株INS-1细胞作为研究模型,探讨高浓度葡萄糖能否诱导INS-1细胞表达IL-1β及其对胰岛β细胞是否具有损伤作用。在以上研究基础上,进一步探讨高糖环境下NF-κB抑制剂及罗格列酮对胰岛β细胞的保护作用及可能机制,为保护胰岛β细胞提供新思路。
第一章高糖诱导INS-1细胞表达IL-1β及其对细胞损伤作用的研究
[目的]
1、探讨高浓度葡萄糖是否诱导INS-1细胞表达细胞因子IL-1β及不同浓度葡萄糖对INS-1细胞IL-1β表达的影响。
2、探讨INS-1细胞表达的IL-1β对细胞胰岛素分泌功能和凋亡的影响。
[方法]
1、实验对象
以大鼠胰岛β细胞瘤株INS-1细胞为研究对象,采用含10%胎牛血清RPMI1640完全培养基培养,置于37℃、5%CO2的培养箱内培养48小时。
2、实验分组
A：不同浓度葡萄糖对INS-1细胞IL-1β表达及细胞活力的影响
(1):11.lmmol/l葡萄糖组(11.1mM组：葡萄糖浓度为11.1 mM／l)；
(2):16.7mmol/l葡萄糖组(16.7mM组：葡萄糖浓度为16.7 mM／l)；
(3):27.8mmol/l葡萄糖组(27.8mM组：葡萄糖浓度为27.8 mM／l)；
(4):33.3mmol/l葡萄糖组(33.3mM组：葡萄糖浓度为33.3 mM／l)；
B：INS-1细胞表达的IL-1β对细胞胰岛素分泌功能和凋亡的影响
(1)：对照组(Control组：葡萄糖浓度为11.1 mM／l)；
(2)：高糖组(HG组：葡萄糖浓度为33.3mM／l)；(3)：高糖+IL-1Ra干预组(HG+IL-1Ra组：葡萄糖浓度为33.3mM的RPMI1640完全培养基+白介素1受体拮抗剂(IL-1Ra 500ng/ml));
3、方法
(1)荧光定量RT-PCR法检测IL-1βmRNA水平
(2)MTT法检测INS-1细胞活力
(3)胰岛素放射免疫试剂盒检测各组胰岛素分泌量
(4) Annexin V-PI-双染法检测各组细胞凋亡率
4、统计学分析
数据应用SPSS13.0统计软件分析。数据以平均数±标准差(x±s)表示。多组间资料比较当满足方差齐性时采用单向方差分析(One-way ANOVA)检验,各组均数多重比较采用LSD法；若不满足方差齐性时分别采用Welch法和Dunnett's T3法。P<0.05认为差异具有统计学意义。
[结果]
1、细胞IL-1βmRNA表达水平11.1mM组、16.7mM组、27.8mM组、33.3mM组RQ值分别是1±0、2.73±0.46、8.57±0.72、114.48±9.75。与11.1mM组相比较,27.8mM组、33.3mM组IL-1βmRNA表达水平分别升高8倍、114倍,差异具有统计学意义(P=0.01、P=0.009)。
2、INS-1细胞活力11.1mM组、16.7mM组、27.8mM组、33.3mM组OD值分别是0.98±0.02、0.95±0.007、0.89±0.003、0.76±0.01。与11.1mM组相比较,27.8mM组、33.3mM组细胞活力显著下降(P=0.05、P=0.002)。
3、相关性分析IL-1βmRNA表达(RQ值)和INS-1细胞活力(OD值)存在显著性负相关(r=-0.862,P<0.01)。
4、INS-1细胞表达的IL-1β对细胞胰岛素分泌功能的影响
(1)基础胰岛素分泌量Control组、HG组、HG+IL-1Ra组细胞BIS分别是16.39±1.78、8.89±1.90、20.85±1.30。与Control组相比较,HG组基础胰岛素分泌量显著降低(P=0.009)。经IL-1Ra处理后,基础胰岛素分泌量显著升高(P=0.001)。
(2)葡萄糖刺激胰岛素分泌量Control组、HG组、HG+IL-1Ra组细胞GSIS分别是29.12±1.78、19.91±1.45、31.81±3.85。与Control组比较,HG组葡萄糖刺激胰岛素分泌量显著降低(P=0.002)。经IL-1Ra处理后,葡萄糖刺激胰岛素分泌量显著升高(P=0.029)。
5、INS-1细胞表达的IL-1β对细胞凋亡的影响Control组、HG组、HG+IL-1Ra组细胞凋亡率分别是3.24±0.78、6.84±0.15、5.76±0.10。与Control组比较,HG组细胞凋亡率显著升高(P=0.000)。经IL-1Ra处理后,细胞凋亡率显著降低(P均=0.000)。
[结论]
1、高浓度葡萄糖可诱导INS-1细胞表达IL-1β,这种IL-1β的表达呈葡萄糖浓度依赖性。
2、高糖诱导INS-1细胞表达的IL-1β可造成β细胞功能障碍与凋亡。
第二章高糖诱导INS-1细胞表达的IL-1β造成细胞损伤可能机制的研究
[目的]
探讨高糖环境下INS-1细胞表达的IL-1β对胰岛β细胞产生损伤作用的可能机制
[方法]
1、实验对象
实验对象及培养方法同第一章。
2、实验分组
(1)：对照组(Control组：葡萄糖浓度为11.1mM／l)；
(2)：高糖组(HG组：葡萄糖浓度为33.3mM mM/l)；
(3)：高糖+IL-1Ra干预组(HG+IL-1Ra组：葡萄糖浓度为33.3 mM／l的RPMI1640完全培养基+白介素1受体拮抗剂(IL-1Ra 500ng/ml))；
(4)：高糖+NF-κB抑制剂干预组(33.3mM组+NF-κB抑制剂组：NF-κB 5umol/l预孵育一个小时后换葡萄糖浓度为33.3mM的RPMI1640完全培养基+NF-κB抑制剂5umol/l共培养)；
(5)：高糖+罗格列酮干预组(HG+罗格列酮组：葡萄糖浓度为33.3mM的RPMI1640完全培养基+罗格列酮5umol/l);
3、方法
(1)免疫细胞荧光定性分析NF-κB蛋白核转位
(2)免疫印迹法(Western blot)检测胞核内NF-κB蛋白表达
(3)荧光定量RT-PCR法检测IKKβmRNA.IL-1βmRNA水平
(4)胰岛素放射免疫试剂盒检测胰岛素分泌量
(5) Annexin V-PI双染法检测细胞凋亡率
4、统计学分析
数据应用SPSS13.0统计软件进行分析。数据以平均数±标准差(x±s)表示。多组间资料比较当满足方差齐性时采用单向方差分析(One-way ANOVA)检验,各组均数多重比较采用LSD法(Least-significant difference test);若不满足方差齐性时分别采用Welch法和Dunnett's T3法。P<0.05认为差异具有统计学意义。
[结果]
1、INS-1细胞免疫荧光Control组图片中细胞呈贴壁状态,触角清晰,红色荧光主要分布在胞浆,提示NF-κB亚基P65蛋白主要在胞浆中表达；HG组胞浆及胞核内均见强红色荧光,提示胞浆与胞核均有P65蛋白的表达,P65蛋白发生核转位；HG+IL-1Ra组、HG+NF-κB抑制剂组、HG+罗格列酮组红色荧光主要分布在胞浆,部分分布细胞胞核内可见弱红色荧光,提示P65蛋白仍主要在胞浆中表达,个别细胞发生P65蛋白核转位。
2、INS-1细胞核内P65蛋白表达Control组、HG组、HG+IL-1Ra组、HG+NF-κB抑制剂组、HG+罗格列酮组胞核内P65蛋白表达量分别是343.47±16.30、835.17±29.63、518.15±15.79、486.9±23.77、386.04±11.10。与Control组相比较,HG组胞核内P65蛋白表达显著增多(P=0.000)。经IL-1Ra、NF-κB抑制剂及罗格列酮处理后,胞核内P65蛋白表达显著减少(P均=0.000)。而与Control组相比较,各干预组胞核内P65蛋白表达量仍是显著增多(P=0.000、0.000、0.029)。
3、INS-1细胞IKKβmRNA表达水平结果由管家基因(GAPDH)扩增效率标准化,经单向方差分析所得。Control组、HG组、HG+IL-IRA组、HG+NF-κB抑制剂组、HG+罗格列酮组RQ值分别是1±0、3.10±0.17、1.21±0.15、1.12±0.15、1.32±0.12。与Control组相比,HG组IKKβmRNA表达水平显著升高,为Control组的3倍(P=0.000)。与HG组相比,HG+IL-IRA组、HG+NF-κB抑制剂组、HG+罗格列酮组IKKβmRNA表达水平显著下降(P均=0.000)。
4、INS-1细胞胰岛素分泌量
(1)基础胰岛素分泌量Control组、HG组、NF-κB抑制剂组、HG+罗格列酮组细胞BIS分别是16.39±1.78、8.89±1.90、21.54±3.42、20.82±4.54。与Control组比较,HG组基础胰岛素分泌量显著降低(P=0.009)。经NF-κB抑制剂及罗格列酮处理后,基础胰岛素分泌量显著升高(P=0.011、0.049)。
(2)葡萄糖刺激胰岛分泌量Control组、HG组、NF-κB抑制剂组、HG+罗格列酮组GSIS分别是29.12±1.78、19.91±1.45、37.63±3.67、42.01±5.66。与Control组比较,HG组葡萄糖刺激胰岛分泌量显著降低(P=0.002)。经NF-κB抑制剂及罗格列酮处理后,葡萄糖刺激胰岛分泌量显著升高(P=0.006、0.017)。
5、INS-1细胞凋亡率Control组、HG组、HG+NF-κB抑制剂组、HG+罗格列酮组细胞凋亡率分别是3.24±0.78、6.84±0.15、5.18±0.13、2.71±0.12。与Control组比较,HG组细胞凋亡率显著升高(P=0.000)。经NF-κB抑制剂及罗格列酮处理后,细胞凋亡率显著降低(P均=0.000)。
6、INS-1细胞IL-1βmRNA表达水平Control组、HG组、HG+IL-1Ra组、HG+NF-κB抑制剂组、HG+罗格列酮组RQ值分别是1±0、124.70±7.22、36.91±5.89、19.86±1.58、17.81±3.11。结果均经过管家基因(GAPDH)扩增效率标准化,由单向方差分析所得。与Control组相比,HG组IL-1βmRNA表达水平升高124倍,差异具有统计学意义(P=0.004)。与HG组相比,HG+IL-1Ra组、HG+NF-κB抑制剂组、HG+罗格列酮组IL-1βmRNA表达水平均显著下降(P值=0.001、0.004、0.002)。
[结论]
1、高糖诱导INS-1细胞表达的IL-1β可能通过激活NF-κB通路造成细胞损伤。
2、罗格列酮通过抑制NF-κB活化,减少胰岛β细胞IL-1β的表达可能是其在高糖环境下保护胰岛β细胞的机制之一。
[Background]
With the rapid development of the global economy, the aging tendency of the population and the modernized lifestyles, the prevalence of type 2 diabetes mellitus is increasing. Nowadays, diabetes has become the third most important chronic non-communicable disease, which is subsequent to cardiovascular and cancer. Diabetes can lead to high rate of disability and mortality in patients. Type 2 diabetes has brought heavy economic burden to the country, society and family. So the prevention and treatment of diabetes have become important social health problems.
Now it is widely accepted that insulin resistance andβ-cell dysfunction are the two main components in the pathogenesis of type 2 diabetes. The United Kingdom Prospective Diabetes Study showed that when a patient was diagnosed diabetes, hisβ-cell function was only 50% of that of a normal person, and then theβ-cell function declined at the rate of 4%-5% every year. On this basis, about ten to fifteen years before being diagnosed with diabetes, the patient'sβ-cell function had begun to fail.
Many previous studies have suggested that glucose toxicity and lipid toxicity are the main pathological mechanisms ofβ-cell dysfunction.β-cell dysfunction and apoptosis caused by high glucose are the important reasons for the onset and development of diabetes. Recent studies have showed that cytokine-mediated inflammation injury also plays an important role in the onset and development of diabetes. In the past, it was generally considered that inflammatory mediators or cytokines are the key factors in the pathogenesis of type 1 diabetes, but recently studies also revealed that inflammation is closely related to type 2 diabetes. Studies indicated that fibrosis could be observed in the pancreatic islets of patients with type 2 diabetes, and fibrosis was the final stage of the chronic inflammatory disease. This is a strong evidence supporting the existence of local inflammation in islets. In addition, some scholars have pointed out that the expression up-regulation of inflammatory cytokines and chemical factors in the blood circulation could be the predictor of type 2 diabetes. Therefore, the harmful role of inflammatory cytokines in the onset and development has been a hot topic in the field of the study on the pathogenesis of the diabetes.
Some studies suggested that pancreaticβcells are more sensitive to IL-1βthan to other inflammatory cytokines. IL-1βis a pro-inflammatory cytokine mainly secreted by monocyte-macrophages, fat cells and endothelial cells, and it has wide biological effects. In the onset and development of type 1 diabetes, such factors as IL-1βproduced by macrophages, tumor necrosis factor (TNF)-α, interferon (IFN)-γand so on constitute a regulatory network of cytokines causing the apoptosis of pancreaticβcells. In 2002, Maedler first reported that high glucose could induce the secretion of pro-inflammatory cytokine interleukin-1β(IL-1β) of human pancreaticβcells and using IL-1 receptor antagonist (IL-1Ra) the natural inhibitor of IL-1βcould block the harmful effects caused by high glucose on the islets, thus offer protection to 0 cells. In vivo, Psammomys were fed by high-energy diet and finally suffered from type 2 diabetes, and researchers found that IL-1βcould be expressed in theβcells of this type 2 diabetes animal model, but IL-1βcould not be expressed in theβcells of non-diabetic Psammomys. From then on, in other two kinds of type 2 diabetes animal model-Goto-Kakizaki (GK) rat and human islet amyloid polypeptid transgenic rat (HIP Rat), also found that in vivo,chronic high glucose could induceβcells to synthesize and secret IL-1β. However, some other studies reported that long-time treatment of high glucose could not induceβcells to secret pro-inflammatory cytokine IL-1β.
In summary, the harmful role of cytokines in the onset and development of type 2-diabetes has gained more and more attention. Whether high glucose could induceβ-cell to express pro-inflammatory cytokine IL-1β? Whether this kind of IL-1βparticipates in the damage process of P-cell? These topics are controversial and related studies are rare. Therefore, in this study, the rat insulinoma-derived cell line INS-1 is used as theβ-cell model, our purpose is to investigate whether high concentrations of glucose could induce INS-1 cells to express IL-1βand whether this kind of IL-1βdoes damage to cells. Based on the above study, we will further explore the protective effect of NF-κB inhibitors and rosiglitazone on the pancreaticβ-cell and related machnisms, thus offer new ideas to studies related to the protection ofβ-cell.
Chapter 1 Study on IL-1βexpression induced by High Glucose and its Injury effects in INS-1 cells
[Objective]
1.To investigate whether high glucose induce INS-1 cells express cytokine IL-1βand effects of different glucose concentrations on the expression of IL-1βin INS-1 cells.
2.To study the effects of the IL-1βthat express by INS-1 cells on cells insulin secretion function and apoptosis.
[Methods]
1.Cell culture:The rat insulinoma-derived INS-1 cells, a widely usedβ-cell surrogate, were cultured in 5% CO2-95% air at 37℃in RPMI-1640 complete medium containing 10% fetal bovine serum(FBS),11.1 mM D-glucose.
2.Experimental group
A:Effect of different glucose concentrations on the expression of IL-1βand Cell viability in INS-1 cells.
(1):11.1mmol/l glucose group (11.1mM Group:glucose concentration was 11.1mM/l);
(2):16.7mmol/l glucose group (16.7mM Group:glucose concentration was 16.7 mM/l);
(3):27.8mmol/l glucose group (27.8mM Group:glucose concentration was 27.8 mM/l);
(4):33.3mmol/l glucose group (33.3mM Group:glucose concentration was 33.3 mM/l)
B:effects of the IL-1βthat express by INS-1 cells on cells insulin secretion function and apoptosis.
(1):The control group (Conctol group, glucose concentration was 11.1mM/l);
(2):The high glucose group (HG group, glucose concentration was 33.3mM/l)
(3):High glucose+IL-1Ra (500ng/ml) group (HG+IL-1Ra group, cells were cultured in RPMI-1640 complete medium containing 33.3 mM glucose+IL-1Ra 500ng/ml for 48 hours);
3.experimental methods
(1) the level of IL-1βmRNA was measured by real-time PCR.
(2) INS-1 cell viability was detected by MTT assay
(3) The apoptosis rate of INS-1 cell were measured by Annexin V-PI apoptosis kit.
(4)basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS) in INS-1 cells were measured by insulin radioimmunoassay kit.
[Results]
1. the levels of IL-1βmRNA:The RQ values of 11.1mM group,16.7 mM group,27.8 mM group,33.3 mM group were 1±0、2.73±0.46、8.57±0.72、114.48±9.75, respectively. The results are standardied by housekeeping gene (GAPDH) amplification efficiency and analysis by one-way ANOVA. In compared with 11.1mM group, the expression levels of IL-1βmRNA in 27.8mM group、33.3mM group were increased significantly, up to 8 fold、114 fold, respectively (P=0.01、P=0.009)
2. INS-1 cell viability:The OD values of 11.1mM group,16.7 mM group, 27.8 mM group,33.3 mM group were 0.98±0.02、0.95±0.007、0.89±0.003、0.76±0.01,respectively. Data analysis by one-way ANOVA. Compared with the 11.1mM group, cell viability in 27.8mM group、33.3mM group were decreased significantly (P=0.05、P=0.002)
3. correlation analysis:IL-1βmRNA expression level (RQ value) and INS-1 cell viability (OD value) showed significant negative.correlation.(r=-0.862,P<0.01).
4. INS-1 cell insulin secretion function
(1)the basal insulin secretion (BIS) in Control group, HG group, HG+IL-1RA group were 16.39±1.78,8.89±1.90,20.85±1.30,respectively. In comparison with Control group, HG group of basal insulin secretion was significantly lower (P=0.009). after IL-1Ra treatment, basal insulin secretion increased significantly (P =0.001).
(2) the glucose-stimulated insulin secretion (GSIS) in Control group, HG group, HG+IL-1Ra group were 29.12±1.78,19.91±1.45,31.81±3.85,respectively. In comparison with Control group, HG group glucose-stimulated insulin secretion was significantly lower (P=0.002). In comparison with HG group, in IL-1Ra group glucose-stimulated insulin secretion were significantly increased (P=0.029).
5.the apoptosis rate of INS-1 cell:apoptosis rates in Control group, HG group, HG+IL-1Ra group were 3.24±0.78,6.84±0.15,5.76±0.10,respectively. In comparison with Control group, the apoptosis rate of INS-1 cell in HG group was significantly higher (P=0.000). In comparison with HG group, after IL-1Ra treatment, the apoptosis rate of INS-1 cells was significantly lower (P=0.000).
[Conclutions]
1. High glucose induced INS-1 cells express IL-1β, the levels of IL-1βexpression dependent on glucose concentrations.
2. IL-1βexpress by INS-1 cells under high glucose environment can injuryβcell function and induceβcell apoptosis.
Chapter2 The damage mechanism of IL-1βexpression induced by High glucose in INS-1 cells
[Objective]
To investigate the mechanism of damage effects by IL-1βthat expressed by INS-1 cells under high glucose environment.
[Methods]
1.Cell culture and experimental grouping were just the same as those of the chapter1.
(1):The control group (Conctol group, glucose concentration was 11.1mM/l);
(2):The high glucose group (HG group, glucose concentration was 33.3mM/l)
(3):High glucose + IL-1Ra (500ng/ml) group (HG+IL-1Ra group, cells were cultured in RPMI-1640 complete medium containing 33.3 mM glucose+IL-1Ra 500ng/ml for 48 hours);
(4):High glucose+NF-κB inhibitors (5umol/l) group (HG+NF-κB inhibitor group, cells were pretreated by NF-κB inhibitor 5umol/l for 1 hours and then exposed in 33.3 mM glucose+NF-κB inhibitor 5umol/l for 48 hours);
(5):High glucose+ Rosiglitazone (5umol/l) group (HG+ rosiglitazone_group, cells were cultured in RPMI-1640 complete medium containing 33.3 mM glucose+ Rosiglitazone 5umol/l for 48 hours);
2.experimental methods
(1) NF-κB protein nuclear translocation of INS-1 cell was analysis by Immunofluorescence.
(2) The expressions of NF-κB protein in cell nucleus were detected by western blot.
(3) The level of IKKβmRNA and IL-1βmRNA was measured by realtime fluorescence quantitative RT-PCR
(4) The apoptosis rate of INS-1 cell were determined by Annexin V-FITC apoptosis kit.
(5) Basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS) in INS-1 cells were determined by insulin radioimmunoassay kit.
[Results]
1. Immunofluorescence:Cells of control group showed a normal attachment state, red fluorescence is mainly distributed in the cytoplasm, suggesting that NF-κB subunit P65 protein mainly expressed in the cytoplasm; in HG group, strong red fluorescencwere seen within the cytoplasm and nucleus of, suggesting that P65 protein happened nuclear translocation; HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+ rosiglitazone group red fluorescence mainly in the cytoplasm, some weak red fluorescence distribution of cell nucleus, indicating P65 protein expression was still mainly in the cytoplasm but individual cells happened P65 protein nuclear translocation.
2. The expression of P65 protein:The expression levels of P65 protein in Control group, HG groups, HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 343.47±16.30,835.17±29.63,518.15±15.79,486.9±23.77,386.04±11.10,respectively. In comparison with C group, HG group nucleus P65 protein was significantly increased (P=0.000). In comparison with HG group,The IL-1RA, NF-KB inhibitor and rosiglitazone group nucleus P65 protein was significantly decreased (P=0.000, respectively). In comparison with C group, the intervention group nuclear P65 protein expression were still significantly increased (P=0.000、0.000、0.029)
3. the expression levels of IKKβmRNA:The RQ values of Control group, HG group, HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+rosiglitazone group were 1±0,3.10±0.17,1.21±0.15,1.12±0.15,1.32±0.12,respectively. In compared with Control group, the expression levels of IKKβmRNA in HG group were increased 3 times (P=0.000). In comparison with the HG group, the expression levels of IKKβmRNA in HG+IL-1Ra group, HG+NF-κB inhibitor group, HG + rosiglitazone group were significantly decreased (P=0.000, respectively)
4.INS-1 cell insulin release function
(1)the basal insulin secretion (BIS) in Control group, HG group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 16.39±1.78,8.89±1.90,21.54± 3.42,20.82±4.54,respectively. In comparison with Control group, HG group of basal insulin secretion was significantly lower (P=0.009). after NF-κB inhibitor and rosiglitazone treatment, basal insulin secretion increased significantly (P=0.011、0.049)
(2) the glucose-stimulated insulin secretion (GSIS) in Control group, HG groups, HG+NF-κB inhibitor group, HG+ rosiglitazone group glucose-stimulated insulin secretion were 29.12±1.78,19.91±1.45,37.63±3.67,42.01±5.66,respectively. In comparison with Control group, HG group glucose-stimulated insulin secretion was-significantly.lower (P=0.002). In comparison-with HG group, in NF-κB inhibitor and rosiglitazone group glucose-stimulated insulin secretion were significantly increased P=0.006、0.017)
5.the apoptosis rate of INS-1 cell:apoptosis rates in Control group, HG group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 3.24±0.78,6.84±0.15, 5.18±0.13,2.71±0.12,respectively. In comparison with Control group, the apoptosis rate of INS-1 cell in HG group was significantly higher (P=0.000). In comparison with HG group, the apoptosis rate after NF-κB inhibitor and rosiglitazone treatment, was significantly lower (P=0.000)
6.the expression levels of IL-1βmRNA:The RQ values of Control group, HG groups, HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 1±0,124.70±7.22,36.91±5.89,19.86±1.58,17.81±3.11,respectively. In compared with Control group, the expression levels of IL-1βmRNA in HG group were increased significantly, up to 124 fold (P=0.004). In comparison with the HG group, the expression levels of IL-1βmRNA in HG+IL-1Ra group, HG+NF-κB inhibitor group, HG + rosiglitazone group were significantly decreased (P=0.001、0.004、0.002, respectively).
[Conclusions]
1.High glucose induced INS-1 cells express IL-1βand its cause cell damage by activated NF-κB pathway.
2.rosiglitazone protect pancreaticβcells by inhibits NF-κB activation, reducedβcell expression of IL-1β.
随着全球经济的快速发展,人口老龄化及生活方式现代化等变化,2型糖尿病的患病率正在不断增加。其致残率、死亡率仅次于肿瘤和心血管疾病,为第三大非传染性疾病。2型糖尿病严重威胁人类健康并给社会及家庭带来沉重的经济负担。因此,糖尿病的防治成为受到广泛关注的社会卫生问题。
目前普遍认为,胰岛素抵抗和胰岛功能受损是2型糖尿病发病机制的两个关键环节。英国前瞻性糖尿病研究显示,糖尿病患者在被明确诊断时,β细胞功能只剩下正常人的50%,其后胰岛β细胞功能每年以4%～5%的速度下降。以此推算,在糖尿病诊断前10～15年,β细胞功能已开始下降。
既往众多研究认为,在2型糖尿病中糖毒性和脂毒性是造成胰岛β细胞功能受损的主要病理机制。而近年来,研究发现细胞因子介导的炎症损伤在2型糖尿病的发生发展中同样发挥着重要作用。以往认为炎症介质是1型糖尿病的发病关键因素,但不断有研究显示炎症与2型糖尿病之间的同样存在密切联系。有研究报道在2型糖尿病患者的活检中发现胰岛存在纤维化,而纤维化是慢性炎症性疾病的最终阶段。这是支持胰岛局部存在炎症反应的一个有力证据。此外,还有学者指出循环中炎症相关细胞因子和化学因子的表达上调可作为2型糖尿病发病的预测指标。由此可见,炎症因子在2型糖尿病发生发展中的作用,已成为糖尿病发病机制研究领域中的一个倍受关注的热点。
白介素-1β(IL-1β)是在胰岛β细胞损伤研究中受到广泛关注的细胞因子之一。IL-1β是一种主要由单核-巨噬细胞、内皮细胞等分泌的细胞因子,具有广泛的生物学效应。1型糖尿病发生发展中,巨噬细胞产生的IL-1β与肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ等构成细胞因子调控网络诱导胰岛β细胞凋亡。2002年,Maedler首次发现将人胰岛β细胞体外培养于高糖中可诱导其分泌细胞因子IL-1β,利用IL-1β的天然抑制因子白介素1受体拮抗剂(IL-1Ra)能保护高糖环境下的胰岛β细胞。提示,高糖环境下胰岛β细胞分泌细胞因子IL-1β,其分泌的IL-1β可造成胰岛β细胞损伤。体内实验,肥沙鼠以高能量饮食喂养造成2型糖尿病模型,研究发现血糖升高肥沙鼠的胰岛β细胞可表达IL-1β,而血糖正常肥沙鼠的胰岛β细胞无IL-1β表达。随后其他实验室还利用另外两种2型糖尿病动物模型GK大鼠及人胰岛淀粉样多肽转基因大鼠进行研究,同样发现体内高糖环境可诱导胰岛β细胞合成并分泌IL-1β。但同时也有研究报道,长时间的高糖处理未能引起胰岛β细胞分泌IL-1β。
综上所述,细胞因子在2型糖尿病发病过程中的作用受到了越来越多的重视。而关于高糖是否诱导胰岛β细胞表达细胞因子IL-1β,这种IL-1β是否损伤胰岛β细胞,目前存在争议且相关研究报道尚不多见。因此,本课题将以大鼠胰岛细胞瘤株INS-1细胞作为研究模型,探讨高浓度葡萄糖能否诱导INS-1细胞表达IL-1β及其对胰岛β细胞是否具有损伤作用。在以上研究基础上,进一步探讨高糖环境下NF-κB抑制剂及罗格列酮对胰岛β细胞的保护作用及可能机制,为保护胰岛β细胞提供新思路。
第一章高糖诱导INS-1细胞表达IL-1β及其对细胞损伤作用的研究
[目的]
1、探讨高浓度葡萄糖是否诱导INS-1细胞表达细胞因子IL-1β及不同浓度葡萄糖对INS-1细胞IL-1β表达的影响。
2、探讨INS-1细胞表达的IL-1β对细胞胰岛素分泌功能和凋亡的影响。
[方法]
1、实验对象
以大鼠胰岛β细胞瘤株INS-1细胞为研究对象,采用含10%胎牛血清RPMI1640完全培养基培养,置于37℃、5%CO2的培养箱内培养48小时。
2、实验分组
A：不同浓度葡萄糖对INS-1细胞IL-1β表达及细胞活力的影响
(1):11.lmmol/l葡萄糖组(11.1mM组：葡萄糖浓度为11.1 mM／l)；
(2):16.7mmol/l葡萄糖组(16.7mM组：葡萄糖浓度为16.7 mM／l)；
(3):27.8mmol/l葡萄糖组(27.8mM组：葡萄糖浓度为27.8 mM／l)；
(4):33.3mmol/l葡萄糖组(33.3mM组：葡萄糖浓度为33.3 mM／l)；
B：INS-1细胞表达的IL-1β对细胞胰岛素分泌功能和凋亡的影响
(1)：对照组(Control组：葡萄糖浓度为11.1 mM／l)；
(2)：高糖组(HG组：葡萄糖浓度为33.3mM／l)；(3)：高糖+IL-1Ra干预组(HG+IL-1Ra组：葡萄糖浓度为33.3mM的RPMI1640完全培养基+白介素1受体拮抗剂(IL-1Ra 500ng/ml));
3、方法
(1)荧光定量RT-PCR法检测IL-1βmRNA水平
(2)MTT法检测INS-1细胞活力
(3)胰岛素放射免疫试剂盒检测各组胰岛素分泌量
(4) Annexin V-PI-双染法检测各组细胞凋亡率
4、统计学分析
数据应用SPSS13.0统计软件分析。数据以平均数±标准差(x±s)表示。多组间资料比较当满足方差齐性时采用单向方差分析(One-way ANOVA)检验,各组均数多重比较采用LSD法；若不满足方差齐性时分别采用Welch法和Dunnett's T3法。P<0.05认为差异具有统计学意义。
[结果]
1、细胞IL-1βmRNA表达水平11.1mM组、16.7mM组、27.8mM组、33.3mM组RQ值分别是1±0、2.73±0.46、8.57±0.72、114.48±9.75。与11.1mM组相比较,27.8mM组、33.3mM组IL-1βmRNA表达水平分别升高8倍、114倍,差异具有统计学意义(P=0.01、P=0.009)。
2、INS-1细胞活力11.1mM组、16.7mM组、27.8mM组、33.3mM组OD值分别是0.98±0.02、0.95±0.007、0.89±0.003、0.76±0.01。与11.1mM组相比较,27.8mM组、33.3mM组细胞活力显著下降(P=0.05、P=0.002)。
3、相关性分析IL-1βmRNA表达(RQ值)和INS-1细胞活力(OD值)存在显著性负相关(r=-0.862,P<0.01)。
4、INS-1细胞表达的IL-1β对细胞胰岛素分泌功能的影响
(1)基础胰岛素分泌量Control组、HG组、HG+IL-1Ra组细胞BIS分别是16.39±1.78、8.89±1.90、20.85±1.30。与Control组相比较,HG组基础胰岛素分泌量显著降低(P=0.009)。经IL-1Ra处理后,基础胰岛素分泌量显著升高(P=0.001)。
(2)葡萄糖刺激胰岛素分泌量Control组、HG组、HG+IL-1Ra组细胞GSIS分别是29.12±1.78、19.91±1.45、31.81±3.85。与Control组比较,HG组葡萄糖刺激胰岛素分泌量显著降低(P=0.002)。经IL-1Ra处理后,葡萄糖刺激胰岛素分泌量显著升高(P=0.029)。
5、INS-1细胞表达的IL-1β对细胞凋亡的影响Control组、HG组、HG+IL-1Ra组细胞凋亡率分别是3.24±0.78、6.84±0.15、5.76±0.10。与Control组比较,HG组细胞凋亡率显著升高(P=0.000)。经IL-1Ra处理后,细胞凋亡率显著降低(P均=0.000)。
[结论]
1、高浓度葡萄糖可诱导INS-1细胞表达IL-1β,这种IL-1β的表达呈葡萄糖浓度依赖性。
2、高糖诱导INS-1细胞表达的IL-1β可造成β细胞功能障碍与凋亡。
第二章高糖诱导INS-1细胞表达的IL-1β造成细胞损伤可能机制的研究
[目的]
探讨高糖环境下INS-1细胞表达的IL-1β对胰岛β细胞产生损伤作用的可能机制
[方法]
1、实验对象
实验对象及培养方法同第一章。
2、实验分组
(1)：对照组(Control组：葡萄糖浓度为11.1mM／l)；
(2)：高糖组(HG组：葡萄糖浓度为33.3mM mM/l)；
(3)：高糖+IL-1Ra干预组(HG+IL-1Ra组：葡萄糖浓度为33.3 mM／l的RPMI1640完全培养基+白介素1受体拮抗剂(IL-1Ra 500ng/ml))；
(4)：高糖+NF-κB抑制剂干预组(33.3mM组+NF-κB抑制剂组：NF-κB 5umol/l预孵育一个小时后换葡萄糖浓度为33.3mM的RPMI1640完全培养基+NF-κB抑制剂5umol/l共培养)；
(5)：高糖+罗格列酮干预组(HG+罗格列酮组：葡萄糖浓度为33.3mM的RPMI1640完全培养基+罗格列酮5umol/l);
3、方法
(1)免疫细胞荧光定性分析NF-κB蛋白核转位
(2)免疫印迹法(Western blot)检测胞核内NF-κB蛋白表达
(3)荧光定量RT-PCR法检测IKKβmRNA.IL-1βmRNA水平
(4)胰岛素放射免疫试剂盒检测胰岛素分泌量
(5) Annexin V-PI双染法检测细胞凋亡率
4、统计学分析
数据应用SPSS13.0统计软件进行分析。数据以平均数±标准差(x±s)表示。多组间资料比较当满足方差齐性时采用单向方差分析(One-way ANOVA)检验,各组均数多重比较采用LSD法(Least-significant difference test);若不满足方差齐性时分别采用Welch法和Dunnett's T3法。P<0.05认为差异具有统计学意义。
[结果]
1、INS-1细胞免疫荧光Control组图片中细胞呈贴壁状态,触角清晰,红色荧光主要分布在胞浆,提示NF-κB亚基P65蛋白主要在胞浆中表达；HG组胞浆及胞核内均见强红色荧光,提示胞浆与胞核均有P65蛋白的表达,P65蛋白发生核转位；HG+IL-1Ra组、HG+NF-κB抑制剂组、HG+罗格列酮组红色荧光主要分布在胞浆,部分分布细胞胞核内可见弱红色荧光,提示P65蛋白仍主要在胞浆中表达,个别细胞发生P65蛋白核转位。
2、INS-1细胞核内P65蛋白表达Control组、HG组、HG+IL-1Ra组、HG+NF-κB抑制剂组、HG+罗格列酮组胞核内P65蛋白表达量分别是343.47±16.30、835.17±29.63、518.15±15.79、486.9±23.77、386.04±11.10。与Control组相比较,HG组胞核内P65蛋白表达显著增多(P=0.000)。经IL-1Ra、NF-κB抑制剂及罗格列酮处理后,胞核内P65蛋白表达显著减少(P均=0.000)。而与Control组相比较,各干预组胞核内P65蛋白表达量仍是显著增多(P=0.000、0.000、0.029)。
3、INS-1细胞IKKβmRNA表达水平结果由管家基因(GAPDH)扩增效率标准化,经单向方差分析所得。Control组、HG组、HG+IL-IRA组、HG+NF-κB抑制剂组、HG+罗格列酮组RQ值分别是1±0、3.10±0.17、1.21±0.15、1.12±0.15、1.32±0.12。与Control组相比,HG组IKKβmRNA表达水平显著升高,为Control组的3倍(P=0.000)。与HG组相比,HG+IL-IRA组、HG+NF-κB抑制剂组、HG+罗格列酮组IKKβmRNA表达水平显著下降(P均=0.000)。
4、INS-1细胞胰岛素分泌量
(1)基础胰岛素分泌量Control组、HG组、NF-κB抑制剂组、HG+罗格列酮组细胞BIS分别是16.39±1.78、8.89±1.90、21.54±3.42、20.82±4.54。与Control组比较,HG组基础胰岛素分泌量显著降低(P=0.009)。经NF-κB抑制剂及罗格列酮处理后,基础胰岛素分泌量显著升高(P=0.011、0.049)。
(2)葡萄糖刺激胰岛分泌量Control组、HG组、NF-κB抑制剂组、HG+罗格列酮组GSIS分别是29.12±1.78、19.91±1.45、37.63±3.67、42.01±5.66。与Control组比较,HG组葡萄糖刺激胰岛分泌量显著降低(P=0.002)。经NF-κB抑制剂及罗格列酮处理后,葡萄糖刺激胰岛分泌量显著升高(P=0.006、0.017)。
5、INS-1细胞凋亡率Control组、HG组、HG+NF-κB抑制剂组、HG+罗格列酮组细胞凋亡率分别是3.24±0.78、6.84±0.15、5.18±0.13、2.71±0.12。与Control组比较,HG组细胞凋亡率显著升高(P=0.000)。经NF-κB抑制剂及罗格列酮处理后,细胞凋亡率显著降低(P均=0.000)。
6、INS-1细胞IL-1βmRNA表达水平Control组、HG组、HG+IL-1Ra组、HG+NF-κB抑制剂组、HG+罗格列酮组RQ值分别是1±0、124.70±7.22、36.91±5.89、19.86±1.58、17.81±3.11。结果均经过管家基因(GAPDH)扩增效率标准化,由单向方差分析所得。与Control组相比,HG组IL-1βmRNA表达水平升高124倍,差异具有统计学意义(P=0.004)。与HG组相比,HG+IL-1Ra组、HG+NF-κB抑制剂组、HG+罗格列酮组IL-1βmRNA表达水平均显著下降(P值=0.001、0.004、0.002)。
[结论]
1、高糖诱导INS-1细胞表达的IL-1β可能通过激活NF-κB通路造成细胞损伤。
2、罗格列酮通过抑制NF-κB活化,减少胰岛β细胞IL-1β的表达可能是其在高糖环境下保护胰岛β细胞的机制之一。
[Background]
With the rapid development of the global economy, the aging tendency of the population and the modernized lifestyles, the prevalence of type 2 diabetes mellitus is increasing. Nowadays, diabetes has become the third most important chronic non-communicable disease, which is subsequent to cardiovascular and cancer. Diabetes can lead to high rate of disability and mortality in patients. Type 2 diabetes has brought heavy economic burden to the country, society and family. So the prevention and treatment of diabetes have become important social health problems.
Now it is widely accepted that insulin resistance andβ-cell dysfunction are the two main components in the pathogenesis of type 2 diabetes. The United Kingdom Prospective Diabetes Study showed that when a patient was diagnosed diabetes, hisβ-cell function was only 50% of that of a normal person, and then theβ-cell function declined at the rate of 4%-5% every year. On this basis, about ten to fifteen years before being diagnosed with diabetes, the patient'sβ-cell function had begun to fail.
Many previous studies have suggested that glucose toxicity and lipid toxicity are the main pathological mechanisms ofβ-cell dysfunction.β-cell dysfunction and apoptosis caused by high glucose are the important reasons for the onset and development of diabetes. Recent studies have showed that cytokine-mediated inflammation injury also plays an important role in the onset and development of diabetes. In the past, it was generally considered that inflammatory mediators or cytokines are the key factors in the pathogenesis of type 1 diabetes, but recently studies also revealed that inflammation is closely related to type 2 diabetes. Studies indicated that fibrosis could be observed in the pancreatic islets of patients with type 2 diabetes, and fibrosis was the final stage of the chronic inflammatory disease. This is a strong evidence supporting the existence of local inflammation in islets. In addition, some scholars have pointed out that the expression up-regulation of inflammatory cytokines and chemical factors in the blood circulation could be the predictor of type 2 diabetes. Therefore, the harmful role of inflammatory cytokines in the onset and development has been a hot topic in the field of the study on the pathogenesis of the diabetes.
Some studies suggested that pancreaticβcells are more sensitive to IL-1βthan to other inflammatory cytokines. IL-1βis a pro-inflammatory cytokine mainly secreted by monocyte-macrophages, fat cells and endothelial cells, and it has wide biological effects. In the onset and development of type 1 diabetes, such factors as IL-1βproduced by macrophages, tumor necrosis factor (TNF)-α, interferon (IFN)-γand so on constitute a regulatory network of cytokines causing the apoptosis of pancreaticβcells. In 2002, Maedler first reported that high glucose could induce the secretion of pro-inflammatory cytokine interleukin-1β(IL-1β) of human pancreaticβcells and using IL-1 receptor antagonist (IL-1Ra) the natural inhibitor of IL-1βcould block the harmful effects caused by high glucose on the islets, thus offer protection to 0 cells. In vivo, Psammomys were fed by high-energy diet and finally suffered from type 2 diabetes, and researchers found that IL-1βcould be expressed in theβcells of this type 2 diabetes animal model, but IL-1βcould not be expressed in theβcells of non-diabetic Psammomys. From then on, in other two kinds of type 2 diabetes animal model-Goto-Kakizaki (GK) rat and human islet amyloid polypeptid transgenic rat (HIP Rat), also found that in vivo,chronic high glucose could induceβcells to synthesize and secret IL-1β. However, some other studies reported that long-time treatment of high glucose could not induceβcells to secret pro-inflammatory cytokine IL-1β.
In summary, the harmful role of cytokines in the onset and development of type 2-diabetes has gained more and more attention. Whether high glucose could induceβ-cell to express pro-inflammatory cytokine IL-1β? Whether this kind of IL-1βparticipates in the damage process of P-cell? These topics are controversial and related studies are rare. Therefore, in this study, the rat insulinoma-derived cell line INS-1 is used as theβ-cell model, our purpose is to investigate whether high concentrations of glucose could induce INS-1 cells to express IL-1βand whether this kind of IL-1βdoes damage to cells. Based on the above study, we will further explore the protective effect of NF-κB inhibitors and rosiglitazone on the pancreaticβ-cell and related machnisms, thus offer new ideas to studies related to the protection ofβ-cell.
Chapter 1 Study on IL-1βexpression induced by High Glucose and its Injury effects in INS-1 cells
[Objective]
1.To investigate whether high glucose induce INS-1 cells express cytokine IL-1βand effects of different glucose concentrations on the expression of IL-1βin INS-1 cells.
2.To study the effects of the IL-1βthat express by INS-1 cells on cells insulin secretion function and apoptosis.
[Methods]
1.Cell culture:The rat insulinoma-derived INS-1 cells, a widely usedβ-cell surrogate, were cultured in 5% CO2-95% air at 37℃in RPMI-1640 complete medium containing 10% fetal bovine serum(FBS),11.1 mM D-glucose.
2.Experimental group
A:Effect of different glucose concentrations on the expression of IL-1βand Cell viability in INS-1 cells.
(1):11.1mmol/l glucose group (11.1mM Group:glucose concentration was 11.1mM/l);
(2):16.7mmol/l glucose group (16.7mM Group:glucose concentration was 16.7 mM/l);
(3):27.8mmol/l glucose group (27.8mM Group:glucose concentration was 27.8 mM/l);
(4):33.3mmol/l glucose group (33.3mM Group:glucose concentration was 33.3 mM/l)
B:effects of the IL-1βthat express by INS-1 cells on cells insulin secretion function and apoptosis.
(1):The control group (Conctol group, glucose concentration was 11.1mM/l);
(2):The high glucose group (HG group, glucose concentration was 33.3mM/l)
(3):High glucose+IL-1Ra (500ng/ml) group (HG+IL-1Ra group, cells were cultured in RPMI-1640 complete medium containing 33.3 mM glucose+IL-1Ra 500ng/ml for 48 hours);
3.experimental methods
(1) the level of IL-1βmRNA was measured by real-time PCR.
(2) INS-1 cell viability was detected by MTT assay
(3) The apoptosis rate of INS-1 cell were measured by Annexin V-PI apoptosis kit.
(4)basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS) in INS-1 cells were measured by insulin radioimmunoassay kit.
[Results]
1. the levels of IL-1βmRNA:The RQ values of 11.1mM group,16.7 mM group,27.8 mM group,33.3 mM group were 1±0、2.73±0.46、8.57±0.72、114.48±9.75, respectively. The results are standardied by housekeeping gene (GAPDH) amplification efficiency and analysis by one-way ANOVA. In compared with 11.1mM group, the expression levels of IL-1βmRNA in 27.8mM group、33.3mM group were increased significantly, up to 8 fold、114 fold, respectively (P=0.01、P=0.009)
2. INS-1 cell viability:The OD values of 11.1mM group,16.7 mM group, 27.8 mM group,33.3 mM group were 0.98±0.02、0.95±0.007、0.89±0.003、0.76±0.01,respectively. Data analysis by one-way ANOVA. Compared with the 11.1mM group, cell viability in 27.8mM group、33.3mM group were decreased significantly (P=0.05、P=0.002)
3. correlation analysis:IL-1βmRNA expression level (RQ value) and INS-1 cell viability (OD value) showed significant negative.correlation.(r=-0.862,P<0.01).
4. INS-1 cell insulin secretion function
(1)the basal insulin secretion (BIS) in Control group, HG group, HG+IL-1RA group were 16.39±1.78,8.89±1.90,20.85±1.30,respectively. In comparison with Control group, HG group of basal insulin secretion was significantly lower (P=0.009). after IL-1Ra treatment, basal insulin secretion increased significantly (P =0.001).
(2) the glucose-stimulated insulin secretion (GSIS) in Control group, HG group, HG+IL-1Ra group were 29.12±1.78,19.91±1.45,31.81±3.85,respectively. In comparison with Control group, HG group glucose-stimulated insulin secretion was significantly lower (P=0.002). In comparison with HG group, in IL-1Ra group glucose-stimulated insulin secretion were significantly increased (P=0.029).
5.the apoptosis rate of INS-1 cell:apoptosis rates in Control group, HG group, HG+IL-1Ra group were 3.24±0.78,6.84±0.15,5.76±0.10,respectively. In comparison with Control group, the apoptosis rate of INS-1 cell in HG group was significantly higher (P=0.000). In comparison with HG group, after IL-1Ra treatment, the apoptosis rate of INS-1 cells was significantly lower (P=0.000).
[Conclutions]
1. High glucose induced INS-1 cells express IL-1β, the levels of IL-1βexpression dependent on glucose concentrations.
2. IL-1βexpress by INS-1 cells under high glucose environment can injuryβcell function and induceβcell apoptosis.
Chapter2 The damage mechanism of IL-1βexpression induced by High glucose in INS-1 cells
[Objective]
To investigate the mechanism of damage effects by IL-1βthat expressed by INS-1 cells under high glucose environment.
[Methods]
1.Cell culture and experimental grouping were just the same as those of the chapter1.
(1):The control group (Conctol group, glucose concentration was 11.1mM/l);
(2):The high glucose group (HG group, glucose concentration was 33.3mM/l)
(3):High glucose + IL-1Ra (500ng/ml) group (HG+IL-1Ra group, cells were cultured in RPMI-1640 complete medium containing 33.3 mM glucose+IL-1Ra 500ng/ml for 48 hours);
(4):High glucose+NF-κB inhibitors (5umol/l) group (HG+NF-κB inhibitor group, cells were pretreated by NF-κB inhibitor 5umol/l for 1 hours and then exposed in 33.3 mM glucose+NF-κB inhibitor 5umol/l for 48 hours);
(5):High glucose+ Rosiglitazone (5umol/l) group (HG+ rosiglitazone_group, cells were cultured in RPMI-1640 complete medium containing 33.3 mM glucose+ Rosiglitazone 5umol/l for 48 hours);
2.experimental methods
(1) NF-κB protein nuclear translocation of INS-1 cell was analysis by Immunofluorescence.
(2) The expressions of NF-κB protein in cell nucleus were detected by western blot.
(3) The level of IKKβmRNA and IL-1βmRNA was measured by realtime fluorescence quantitative RT-PCR
(4) The apoptosis rate of INS-1 cell were determined by Annexin V-FITC apoptosis kit.
(5) Basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS) in INS-1 cells were determined by insulin radioimmunoassay kit.
[Results]
1. Immunofluorescence:Cells of control group showed a normal attachment state, red fluorescence is mainly distributed in the cytoplasm, suggesting that NF-κB subunit P65 protein mainly expressed in the cytoplasm; in HG group, strong red fluorescencwere seen within the cytoplasm and nucleus of, suggesting that P65 protein happened nuclear translocation; HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+ rosiglitazone group red fluorescence mainly in the cytoplasm, some weak red fluorescence distribution of cell nucleus, indicating P65 protein expression was still mainly in the cytoplasm but individual cells happened P65 protein nuclear translocation.
2. The expression of P65 protein:The expression levels of P65 protein in Control group, HG groups, HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 343.47±16.30,835.17±29.63,518.15±15.79,486.9±23.77,386.04±11.10,respectively. In comparison with C group, HG group nucleus P65 protein was significantly increased (P=0.000). In comparison with HG group,The IL-1RA, NF-KB inhibitor and rosiglitazone group nucleus P65 protein was significantly decreased (P=0.000, respectively). In comparison with C group, the intervention group nuclear P65 protein expression were still significantly increased (P=0.000、0.000、0.029)
3. the expression levels of IKKβmRNA:The RQ values of Control group, HG group, HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+rosiglitazone group were 1±0,3.10±0.17,1.21±0.15,1.12±0.15,1.32±0.12,respectively. In compared with Control group, the expression levels of IKKβmRNA in HG group were increased 3 times (P=0.000). In comparison with the HG group, the expression levels of IKKβmRNA in HG+IL-1Ra group, HG+NF-κB inhibitor group, HG + rosiglitazone group were significantly decreased (P=0.000, respectively)
4.INS-1 cell insulin release function
(1)the basal insulin secretion (BIS) in Control group, HG group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 16.39±1.78,8.89±1.90,21.54± 3.42,20.82±4.54,respectively. In comparison with Control group, HG group of basal insulin secretion was significantly lower (P=0.009). after NF-κB inhibitor and rosiglitazone treatment, basal insulin secretion increased significantly (P=0.011、0.049)
(2) the glucose-stimulated insulin secretion (GSIS) in Control group, HG groups, HG+NF-κB inhibitor group, HG+ rosiglitazone group glucose-stimulated insulin secretion were 29.12±1.78,19.91±1.45,37.63±3.67,42.01±5.66,respectively. In comparison with Control group, HG group glucose-stimulated insulin secretion was-significantly.lower (P=0.002). In comparison-with HG group, in NF-κB inhibitor and rosiglitazone group glucose-stimulated insulin secretion were significantly increased P=0.006、0.017)
5.the apoptosis rate of INS-1 cell:apoptosis rates in Control group, HG group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 3.24±0.78,6.84±0.15, 5.18±0.13,2.71±0.12,respectively. In comparison with Control group, the apoptosis rate of INS-1 cell in HG group was significantly higher (P=0.000). In comparison with HG group, the apoptosis rate after NF-κB inhibitor and rosiglitazone treatment, was significantly lower (P=0.000)
6.the expression levels of IL-1βmRNA:The RQ values of Control group, HG groups, HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 1±0,124.70±7.22,36.91±5.89,19.86±1.58,17.81±3.11,respectively. In compared with Control group, the expression levels of IL-1βmRNA in HG group were increased significantly, up to 124 fold (P=0.004). In comparison with the HG group, the expression levels of IL-1βmRNA in HG+IL-1Ra group, HG+NF-κB inhibitor group, HG + rosiglitazone group were significantly decreased (P=0.001、0.004、0.002, respectively).
[Conclusions]
1.High glucose induced INS-1 cells express IL-1βand its cause cell damage by activated NF-κB pathway.
2.rosiglitazone protect pancreaticβcells by inhibits NF-κB activation, reducedβcell expression of IL-1β.
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[2]胡善联,刘国恩,许樟荣,等.我国糖尿病流行病学和疾病经济负担研究现状.中国卫生经济,2008,27(8)：5-8.
[3]Gerasi E.胰岛素生成,胰岛素分泌及2型糖尿病：问题的核心在于p细胞[J].中华内分泌代谢杂志,2005,21(3)：1942198.
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