丁酸钠对肥胖孕鼠代谢和胰岛β细胞功能的影响和其分子机制

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非涉密论文□论文作者签名：日期：导师签名：日期：丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制中文摘要丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制中文摘要随着饮食习惯及生活方式的改变，肥胖、糖尿病的发病率逐年攀升。

高龄、肥胖孕妇的增多，妊娠期糖尿病（gestational diabetes millus, GDM）的发病率也不断增加，GDM产后2型糖尿病(Type 2 diabetes mellitus,T2DM)及子代肥胖发病率都明显增加；GDM的发病与胰岛β细胞增殖不足密切相关,研究者已把目光投向了胰岛β细胞增殖这一焦点，且认为无论是GDM还是糖尿病患者，寻找可以有效保护修复胰岛β细胞，恢复胰岛功能，抑制胰岛β细胞凋亡的药物是当今治疗糖尿病发展的重要方向。

研究显示组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitors, HDACIs）可以改善胰岛β细胞功能，促进胰岛素释放与合成；减轻炎症反应对胰岛β细胞的损伤，还可以改善外周组织的胰岛素抵抗，调节全身免疫对T1DM和T2DM致病的影响。

而丁酸钠(CH3CH2CH2COONa)作为HDACIs一员，存在于抗性淀粉，奶酪等食物中，大肠中的菌群酵解膳食纤维也可合成，合成后立即被结肠细胞作为能量吸收和利用，其能量占到了人体基本能量需求的5-10%，因而受到研究者的广泛关注。

本研究将丁酸钠添加于肥胖孕鼠饮食中，探讨其对代谢及胰岛β细胞相关功能及胰岛微环境相关影响。

1.肥胖孕鼠模型的建立及丁酸钠对代谢表型的影响本研究将SPF级4W龄C57BL/6J雌鼠60只适应性喂养一周后，根据饲料成分不同随机分为3组分别进行喂养：高脂饲料组（40%脂肪，20%蛋白质, 40%碳水化合物），高脂加丁酸钠组（95%高脂料+5%丁酸钠），普通饲料组（14%脂肪，26%蛋白质，60%碳水化合物）。

测体重每周1次，空腹血糖每两周1次（禁食12h后断尾法），喂养至14W，禁食12h球后静脉丛取血测定空腹胰岛素水平，且行腹腔葡萄糖耐量试验（Intraperitoneal glucose tolerance test, IPGTT）及腹腔胰岛素耐量试验（Intraperitoneal Insulin Tolerance Test, ITGTT）实验，测定三组小鼠胰岛素抵抗情况及对葡萄糖及胰岛素的反应性。

结果发现此时高脂组小鼠与其他两组比较体重出现明显差异；高脂组空腹血糖偏高，出现明显的胰岛素抵抗；高脂组孕前胰岛素水平为1.580±0.23ng/ml ，中文摘要丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制与其他各组比较均有明显差异(P<0.001)，高脂加丁酸钠组为0.690±0.120ng/ml与对照组比较有差异(P<0.05)。

小鼠休息1W后与雄鼠合笼，结合小鼠的肥胖和妊娠孕14.5d状态，此模型是目前最接近人类GDM发病机理的模型。

至孕14.5d，处死后心脏取血，分别分析血清甘油三酯，总胆固醇，胰岛素，TNF-α和IL-1β水平，测定丁酸钠对肥胖孕鼠代谢的影响。

结果发现：至孕14.5d高脂组小鼠平均体重增长了24g,高脂加丁酸钠组增长了14.8g，对照组增长了11g(各组间P<0.001)。

在高脂饮食添加的第10W后小鼠血糖增长较迅速(P<0.001), 对照组血糖在整个实验中波动不明显，高脂加丁酸钠组血糖一直高于对照组，却也低于高脂组(p>0.05)。

高脂组的清除葡萄糖的及胰岛素刺激下清除葡萄糖的能力在IPGTT及IPITT实验中各时间点均明显减低，丁酸钠显示了对血糖调节能力的改善；高脂组甘油三酯235.21±29.08 mg/dl，而对照组140.45 ±18.02 mg/dl（P<0.001）；总胆固醇水平：高脂组351.08 ±59.07 mg/dl；对照组：236.8 ±41.8 mg/dl (P<0.001)，而高脂加丁酸钠组甘油三酯仅176.6±13.92 mg/dl；及总胆固醇244.42±25.17 mg/dl，丁酸钠明显降低甘油三酯及总胆固醇水平（P<0.001）；与高脂组相比，丁酸钠明显降低外周血IL-1β（P<0.001）及TNF-α水平(P<0.05)。

2.丁酸钠对胰岛β细胞胰岛素合成及增殖凋亡功能的影响第一部分研究发现丁酸钠可改善肥胖孕鼠糖脂代谢，那么为了进一步探讨丁酸钠对胰岛β细胞是否有影响，本研究在此肥胖GDM模型基础上，在胰岛质量峰值期－－孕14.5d，胰岛增殖及抗凋亡高度活跃的状态下研究胰岛β细胞如何适应这双重的胰岛素抵抗状态，丁酸钠对胰岛β细胞增殖及凋亡的影响在这一特殊时期更容易显现。

在孕14.5d，处死孕鼠后迅速分离胰腺组织，一部分OCT中包埋，冰冻切片后-70°C 保存，用胰岛素抗体标记胰岛β细胞，利用免疫荧光组织化学方法测定胰岛β胰岛素表达及质量变化，PCNA及Bcl-2分别作为增殖及抗凋亡的标记分子太探讨丁酸钠对胰岛β细胞增殖及抗凋亡功能的影响。

使用Image-Pro Plus software软件测定各组胰岛素表达灰度值，同时测定各组胰岛β细胞面积，胰岛β细胞质量，及各组胰岛β细胞中PCNA, Bcl-2表达比率。

结果发现：胰岛素荧光强度灰度值测定下的结果为对照组0.17±0.03,高脂组0.037± 0.007，高脂加丁酸钠组灰度值为0.09±0.01（P<0.001)。

高脂组小鼠的β细胞体积增长最大（112.1 ± 7.18um2）（P<0.001），而高脂加丁酸钠组及对照组分别为94.5 ± 4.21um2、90.6 ± 6.73 um2, 高脂加丁酸钠组与对照组比较无丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制中文摘要统计学意义。

高脂组小鼠胰岛β细胞质量2.67±0.17mg, 高脂加丁酸钠组为2.18± 0.19 mg，对照组为1.94 ± 0.11 mg，高脂组与另两组有明显统计学差异(P<0.001)，高脂加丁酸钠与对照比较，差异有统计学意义(P<0.05)。

胰岛β细胞的PCNA的阳性比率：高脂组（1.70±0.27）%，高脂加丁酸钠组（1.45±0. 15）%，对照组为（1.4±0.11）%，在对照组及高脂加丁酸钠组间的结果没有差异，但高脂组的PCNA表达水平较高(P<0.05)。

另外高脂组抗凋亡因子Bcl-2的表达水平较另两组较低（P<0.001），高脂组（1.26±0.49）%，高脂加丁酸钠组（4.32±0.81）%，对照组为（4.46±0.49）%，同样Bcl-2水平高脂加丁酸钠与对照组比较无统计学差异。

3.丁酸钠对胰岛β细胞微环境中炎症反应及孕鼠仔鼠发育影响越来越多的研究证实胰岛内较高的炎症因子的释放，影响胰岛β细胞的功能及存亡。

GDM与胰岛炎症反应也密切相关，它们的病理过程也可以通过抑制炎症反应得到缓解或治疗。

丁酸钠在众多研究中发现可以抑制自身免疫性疾病的炎症反应水平。

为进一步探讨丁酸钠改善肥胖孕鼠代谢的机理，在此模型的基础上我们也选取了部分胰腺组织，同时研究选取了经典的NF-κB及其磷酸化NF-κB分子，利用免疫组织化学方法测定其在胰岛中的不同表达，探讨丁酸钠对胰岛炎症反应的影响。

以上各组在孕14.5d，在处死孕鼠分离的胰腺组织后，第二部分实验中的另一部分在福尔马林中固定24h后，石蜡包埋切片，测定三组间胰腺胰岛中NF-κB及pNF-κB表达，使用Image-Pro Plus software软件测定胰岛中阳性着色的细胞数目。

结果发现：NF-κB 表达水平高脂组: (6.48 ±2.39)%，高脂加丁酸钠组：(2.92±0.73)%，对照组：(0.56±0.15)%，高脂加丁酸钠组与对照组比较有统计学意义(P<0.05)；pNF-κB高脂组: (2.07±0.78)%，高脂加丁酸钠组: (0.78±0.15) %, 对照组: (0.286± 0.08), P<0.001) %；高脂加丁酸钠组胰岛中NF-κB及pNF-κB阳性细胞数目，及pNF-κB/NF-ΚB比值明显低于高脂组（P<0.001），高脂加丁酸钠组pNF-κB及pNF-κB/NF-κB与对照组间无统计学差异（p>0.05）。

实验中每组孕鼠都留取3只待其自然分娩，在整个丁酸钠喂养的周期中，高脂加丁酸钠组小鼠无一例死亡。

14.5d仔鼠未发现发育的异常及畸形，母鼠的孕期也无明显改变。

综上所述，本研究首先建立肥胖孕鼠模型，孕鼠出现明显的体重差异、糖耐量受损及胰岛素抵抗即为GDM建模成功。

在此模型的基础上，系统地研究了丁酸钠对糖脂代谢的改善,同时发现其增强胰岛β细胞胰岛素表达，抑制β细胞凋亡,抑制胰岛炎中文摘要丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制症反应,同时发现丁酸钠对孕鼠及仔鼠无明显毒性。

故丁酸钠有可能为GDM、及T1DM、T2DM等代谢性疾病的预防及治疗开辟崭新的途径。

关键字：丁酸钠；肥胖；β 细胞；炎症反应；糖尿病；作者：陈轩指导教师：李华萍丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制英文摘要Effects and Mechanisms of Butyrate on Metabolism and Pancreatic β-Cell Function in Obesity Pregnancy MiceAbstractAs diet and lifestyle changes, the incidence of obesity and diabetes increase year by year. And with the number of aged and obesity women growing, the incidence of gestational diabetes millus (GDM) is also arising quickly with an serious results of more Type 2 diabetes mellitus (T2DM) women and obesity children after pregnancy; GDM is closely related to the deficiency expansion of pancreatic β cells, so many researchers pay their attentions on the expansion of β cells in pregnancy, and they think it’s important to look for a drug that can prevent and prom ote β cells from damage, restoreβ cells function, and inhibit their apoptosis function, which may be an important direction to deal with GDM and other diabetes .Histone deacetylase inhibitors (HDACIs) show promising properties, which can promote β cells function, enhance insulin production and secretion, prevent β cells from inflammatory damage, improve peripheral insulin resistance, keep immune system balance in pathologic process of T1DM and T2DM. Sodium butyrate (CH3CH2CH2COONa) is one member of HDACI s, it’s also a fatty acid derivative found in foods, such as resistant starch and cheese, and it can produced physiologically in large amounts from fermentation of dietary fiber in the large intestine, it can be used as substrates energy production for intestinal and ruminal epithelial cells, which can meet 5-10% basic energy need of human. So in this study, we explored whether sodium butyrate has an influence on metabolism, pancreatic β-cell function and microenvironment in islet in mice model of obesity pregnancy.1. Established obesity pregnancy animal model and explored whether high fat diet with butyrate can improve metabolic impairments SPF C57BL/6J 60 female mouse (4 weeks old) were randomly divided into three groups with three different diets after 1W quarantine, diets were the high fat food (HF) (40% fat, 20% protein,40% carbohydrate); High-fat diet with sodium butyrate food (HSF) was fed the above high-fat diet with sodium butyrate at 5% wt/wt; control food (CF)(14% fat, 26% protein, 60% carbohydrate) was fed a control food. Body weight was measured英文摘要丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制once a week; Blood glucose was determined by glucometer at indicated time points from cutting tails after overnight fasted 12 h every 2 weeks; Insulin of blood samples were collected by retro orbital sinus puncture after overnight fasted 12h after 14 weeks dietary intervention for insulin analysis. And after 14 weeks dietary intervention, conventional intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal Insulin tolerance test (IPITT )were performed after fasting for 12h and 4h respectively; Results showed the body weight had great difference in groups, HF showed a higher blood glucose and with a significant insulin resistance state: insulin level of HF was 1.580±0.23 ng/ml (P<0.001), as while HSF was at a 0.690±0.120 ng/ml (P<0.05), CF was at 0.343±0.06ng/ml. Then after 1 week rest, mouse were mating with male mouse, combined with the obesity and GD14.5 status, the establishment of obesity mouse model is the most close to the pathogenesis of human GDM. To explore whether high fat diet with butyrate can improve metabolic impairments, dams of pregnant mouse of groups were sacrificed at GD14.5, maternal serum was analyzed for triglyceride, total cholesterol, insulin, TNF-α, and IL-1β levels respectively. In our results: at GD14.5, the body weight of HF gained 24g, HSF gained 14.8g, CF gained 11g (in groups P<0.001). Blood glucose levels showed a progressive increase in 10th W of HF feeding(P<0.001), but not showed in CF or HSF groups, glucose levels in HSF were elevated compared to CF, but were still lower compared to HF(p>0.05). IPGTT demonstrated that glucose clearance is significantly retarded in HF mice, and IPITT of HF also exhibited much worse glucose clearance response to insulin stimulus at all time points compared to HSF and CF. HSF had improved insulin resistance someway. At GD14.5, Groups of fast insulin were all a little increase compared before pregnancy, but the statistical differences between groups were not changed. Our results showed sodium butyrate can clear away triglyceride and total cholesterol（P<0.001）: HF triglyceride (235.21±29.08 mg/dl); CF(140.45 ±18.02 mg/dl); total cholesterol: HF 351.08 ±59.07 mg/dl, CF 236.8 ±41.8 mg/dl (P<0.001), triglyceride and total cholesterol in HSF : 176.6±13.92 mg/dl and 244.42±25.17 ; and compared to HF, HSF has a lower IL-1β (P<0.001）and TNF-α level (P<0.05).2. Butyrate influenced β cells insulin expression and anti-apoptosis function in obesity pregnancy miceIn part I, butyrate showed an important regulation on metabolic impairments of obesity, we wonder if butyrate can influenceβ cells, so based on th is obesity pregnant mouse model, at the GD14.5, in which time β cell has the peak mass with much excited丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制英文摘要proliferation and anti-apoptosis functions during pregnancy, and wonder how β cell to meet these double (obesity and pregnancy) insulin resistance stat e, the influence on β cells which butyrate played at this time can be easily found. Dams of pregnant mouse were sacrificed at gestational 14.5d, entire pancreata were removed and weighed quickly, one part of tissues snapped frozen in OCT, and stored in -70°C after frozen section. Insulin expression as a marker of the β cell and β cell mass was examined by immunohistochemistry, PCNA and Bcl-2 were chosen to be marker protein of proliferation and anti-apoptosis respectively to incubation with insulin to assess the influence of sodium butyrate on β cells. Then morphometrical analysis was explored by Image-Pro Plus software, the insulin expressions were measured by determining the average pixel value of staining per islet, and the size of β cell, the area of β c ells in pancreases, the rate of PCNA and Bcl-2 in β cells were all measured and counted. The results showed that the average pixel value of insulin of CF was 0.17±0.03, and HF was 0.037±0.007, HSF 0.09±0.01, (P<0.001). The area of β each cell was biggest in HF 112.1 ±7.18um2, as HSF 94.5 ±4.21um2 and CF: 90.6 ± 6.73 um2, but there was no statistically difference between last two groups. The β cell mass in HF was 2.67±0.17mg, HSF: 2.18± 0.19 mg，CF:1.94 ± 0.11, β cell mass of HF was significantly different from HSF and CF (P<0.001), and there only a litter difference between HSF and CF (P<0.05). The percentage of PCNA positive β cells, with no difference between CF and HSF groups, PCNA levels were higher in HF （1.70±0.27）%compared to HSF（1.45±0. 15）%,(P<0.05) , CF（1.4±0.11）%, there was no significant difference between HSF and CF. On the other hand, the number of Bcl-2 positive β cells was obviously lower in the HF group compared to HSF and CF (HF: 1.26±0.49 vs. HSF: 4.32±0.81 and CF: 4.46±0.49, P<0.001), and there was still no significant difference between HSF and CF.3. The influence of sodium butyrate on pancreatal islet inflammatory response and mother and infant mouse development.And more and more studies have confirmed that the microenvironment in islet is very important, higher release of inflammatory cytokines in islets can affect the function and survival of β cells. GDM is also closely related with islet inflammation. The pathological process can be relieved or treated by inhibiting the inflammatory reaction. Besides above functions of regulate metabolic disorder and enhance β cells functions, sodium butyrate had found that can suppress autoimmune diseases in numerous studies.To futher exploer the influence on metabolism, based on the model, we also chose英文摘要丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制part pancreatic tissue to explore the expression of NF-κB, pNF-κB to evaluate islet inflammatory of groups, since the activation of NF-κB is an important and classical marker of inflammatory response. Dams of pregnant mouse of groups were sacrificed at GD14.5, the other part pancreata left from the part II experiment was fixed in 4% paraformaldehyde for 24 h, and then laid flat for paraffin section, then detected the expression of NF-κB, pNF-κB by immunohistochemistry method. The assessment was performed by determining the number of positive cells per islet section by Image-Pro Plus software. Results in our study showed: NF-κB positive cells in islet of HF were greater than that of HSF and CF (HF: 6.48 ±2.39 VS. HSF 2.92±0.73 and CF 0.56±0.15, p<0.001), and inflammatory response in HSF islet was greater than that in CF (P<0.05), as measuring pNF-ΚB positive cells in islet, HF still showed increased inflammatory response compared to HSF and CF (HF: 2.07 ±0.78 vs. HSF: 0.78 ±0.15, P<0.005; HF vs. CF: 0.286± 0.08, P<0.001); the ratio of pNF-ΚB to NF-ΚB positive cells in HF was higher than that of other groups (HF: 0.35 ±0.052 VS. HSF:0.27 ±0.03, P< 0.001; HF VS.CF:0.2666 ±0.04, P<0.001), but there was no statistically significant difference between CF and HSF. 3 pregnant mouse of each group were let them born without human intervention, as litters were born from mothers of HSF, it did not show any visible deformity to the naked eye were not found and any change of pregnancy duration.Therefore, in our research we established obesity pregnant mouse model, when the obesity mouse has significant difference on body weight, glucose glucose tolerance impaired and insulin resistance, with above symptoms GDM models were thought to be successfully established. And based on this model, we found sodium butyrate has an effect on metabolism, and β cells biological behaviors such as anti-apoptosis, and inhibited islet inflammatory response without any toxicity found in pregnancy and new born mouse, so butyrate was presented itself as a new therapeutic target for GDM, T1DM, T2DM and other metabolic diseases.Keywords: Sodium butyrate; Obesity; β cell; Inflammatory response;Diabetes;Written by: Chen XuanSupervised by: Li hua-ping目录序言 (1)第一部分肥胖孕鼠模型的建立及丁酸钠对代谢表型的影响实验方法 (5)结果 (9)讨论 (11)第二部分丁酸钠对胰岛β细胞胰岛素合成及增殖凋亡功能的影响实验方法 (15)结果 (19)讨论 (22)第三部分丁酸钠对胰岛β细胞微环境中炎症反应及孕鼠仔鼠发育影响实验方法 (25)结果 (28)讨论 (30)结论 (33)参考文献 (34)综述组蛋白去乙酰化酶抑制剂在糖尿病治疗中的作用研究进展 (39)攻读硕士学位期间发表的文章 (44)致谢 (45)附录 (46)丁酸钠对肥胖孕鼠代谢及胰岛β细胞功能的影响及其分子机制序言序言肥胖是现代工业社会的一种流行病，随着饮食习惯及生活方式的改变，肥胖的发病率逐年攀升。