慢性酒精对肝胰岛素信号通路的影响及其机制

发布时间：2018-06-16 15:35

本文选题：肝脏 + 酒精　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景随着物质生活水平的提高,各种酒精饮料越加广泛地成为人们生活的一部分。随之,酒精相关的疾病也在增加。例如,大量饮酒可导致肝脏脂肪性变,形成酒精性肝病,进一步可发展为酒精性肝硬化,甚至发展到酒精性肝癌。肝脏脂肪性病变后可导致多种病理结果,其中一个重要方面则是导致胰岛素抵抗,即机体对胰岛素生理作用的反应性降低或敏感性降低。而胰岛素抵抗与其它多种疾病(如糖尿病、肥胖、脂代谢紊乱和高血压等)的发生和发展密切关联[1-2]。 PI3K/AKT和MAPK/ERK是胰岛素调节细胞代谢或生长的两条重要通路。正常情况下,胰岛素与胞膜上的受体结合后,引起胰岛素受体自身磷酸化,从而激活酪氨酸激酶和胰岛素受体底物(Insulin Receptor Substrate, IRS)。在PI3K/AKT信号通路中,活化的IRS进而激活下游的3-磷酸肌醇激酶(phosphoinositide-3-kinase, PI3K)的活性。P13K的激活可使磷脂酰肌醇4,5二磷酸(phosphatidylinositol-4,5-bisphosphate, PI(4,5)P2)转化为磷脂酰肌醇3,4,5三磷酸(phosphatidylinositol-4,5-triphosphate, PI(3,4,5)P3),后者可进一步激活蛋白丝氨酸-苏氨酸蛋白激酶B(serine-threonine protein kinase B, AKT),进而引起葡萄糖转运体易位[3-4]。在MAPK/ERK通路中,Ras和Raf结合,将Raf从细胞浆转移到细胞膜,在细胞膜上Raf丝／苏氨酸发生磷酸化而被激活。激活后的Raf以其C端催化区与MEK结合,并使其催化区Ser磷酸化,从而使MEK激活。活化的MEK进一步激活下游的ERK。抑癌基因第10号染色体同源性磷酸酶-张力蛋白基因(phosphatase and tensin homologue, PTEN)是Insulin/PI3K/AKT信号通路的主要负调控分子,其行为的变化直接影响胰岛素信号通路的稳态[5-6]。因为Insulin信号通路接受许多分子的调控,并进而介导许多的下游分子事件,因而,该通路的调节异常也与细胞的许多病变相联系。例如癌症、糖尿病、胰岛素抵抗。慢性酒精刺激可以导致胰岛素抵抗。然而,酒精刺激导致胰岛素抵抗的分子机制不甚清楚,有待深入的研究。本课题是国家基金慢性酒精对肝脏胰岛素抵抗形成的影响的一部分。该基金旨在探讨抑癌基因PTEN在慢性酒精刺激损害Insulin/PI3K/AKT信号通路的分子机制。而本课题,主要是该基金的前期工作,即研究酒精小鼠的模型建立。我们使用液体喂养模型,采用Lieber-DeCarli液体饲料。本课题开展了体外细胞的培养,以印证体内试验的结果。目的慢性酒精刺激对肝脏胰岛素信号通路的研究影响及其机制。方法一、体外实验部分 1、本基金用肝癌细胞系HepG2细胞为研究对象。细胞常规培养(10%胎牛血清的DMEM培养液培养,温度37℃、5%二氧化碳恒温箱)。 1) HepG2细胞胰岛素刺激下PI3K/AKT信号通路的激活。细胞分为对照组和胰岛素刺激组。无血清饥饿12小时后,胰岛素刺激10min,对照组用PBS刺激。裂解细胞,提取蛋白。测量蛋白浓度后,经过western blot(制胶、电泳、电转移、封闭、一抗孵育、二抗孵育、漂洗PVDF膜、胶片曝光)蛋白分析法检测对照组和胰岛素刺激组P-AKT蛋白表达水平。 2)慢性酒精效应细胞分为酒精刺激组和对照组。无血清饥饿12小时后,无水乙醇刺激(100mM/L)72小时,对照组为无血清DMEM培养液。血清饥饿12小时后,胰岛素刺激10min,裂解细胞,提取蛋白。测量蛋白浓度后,P-AKT、P-ERK蛋白表达水平的检测见上节1。 2、凝胶图像分析。将胶片进行扫描,利用Image J软件分析信号灰度值,以GAPDH作为内参,P-AKT以总AKT作为参照,P-ERK以总ERK作为参照,以相应蛋白条带与GAPDH或AKT或ERK的比值计算目的蛋白的相对表达量。二、体内实验部分 1、实验设计：SPF级Balb/c小鼠购自南方医科大学动物中心,南方医院动物房按清洁级标准饲养。周龄为6至8周,雄性小鼠,14只,随机分为2组：对照组和慢性酒精组。根据Lieber-DeCarli液体饲料配方配制含酒精和不含酒精而热卡相同的液体饲料。予小鼠每日定量喂养：对照组喂对照液体饲料(热量比例：脂肪35%,碳水化合物47%,蛋白质18%,不含酒精),慢性酒精组喂酒精液体饲料(与对照液体饲料相同,但用酒精代替其中的一部分碳水化合物,酒精占36%,酒精体积分数为5%),两组小鼠最终热卡摄入量基本一致。整个造模过程分成过渡期和造模期,小鼠共饲养56天。在整个造模过程中,每7天给小鼠测一次空腹体重。处死当天,再次测量小鼠空腹体重。56天后,予腹腔注射戊巴比妥钠溶液,待小鼠麻醉充分后打开腹腔,采集肝脏组织,取一小部分肝脏组织用4%甲醛溶液固定,剩余部分立即放于液氮速冻,再移至-80℃冰箱长期保存。 2、小鼠肝脏组织的制片与观察。肝脏组织在4%甲醛溶液中固定。然后组织块经过脱水、透明、浸蜡、包埋、切片、烤片、脱蜡、复水、染色、脱水、透明、封片,制成病理切片,显微镜观察对照组和慢性酒精组两组肝脏组织的结构变化。 3、Western blot检测对照组和慢性酒精组肝脏组织中P-AKT、P-ERK和PTEN的表达量。-80℃碎冰上剪取肝脏组织,并于普通冰上裂解研磨肝脏组织(RIPA buffer液),提取蛋白,蛋白样品用western blot(制胶、电泳、电转移、封闭、一抗孵育、二抗孵育、漂洗PVDF膜、胶片曝光)检测P-AKT、P-ERK和PTEN表达量。 4、凝胶图像分析。将胶片进行扫描,利用Image J软件分析信号灰度值,以GAPDH作为内参,P-AKT以总AKT作为参照,P-ERK以总ERK作为参照,以相应蛋白条带与GAPDH或AKT或ERK的比值计算目的蛋白的相对表达量。三、统计分析：所有结果经Exce12007与SPSS13.0软件统计分析,结果以“算术平均值±标准差(arithmetic mean±standard deviation,M±SD)"表示。两组间比较采用两独立样本t检验(Independent-Samples T Test)或wilcoxon秩和检验(Mann-Whitney U检验),P值0.05定义为有显著性差异。实验结果一、体外实验结果 1、Western blot检测结果显示,相比对照组,胰岛素刺激组在胰岛素刺激10min后,P-AKT水平显著提高(t=-12.758,P=0.009),两组灰度值比分别为0.313±0.156及2.765±0.222。 2、我们的检测结果中,酒精刺激组较对照组P-AKT蛋白表达水平下降(t=2.645,P=0.048),两组灰度值比分别为0.454±0.085及0.681±±0.150,AKT蛋白无显著差异,两组灰度值比值为0.588±±0.213及0.705±±0.052(t=-1.063,P=0.329)。 3、酒精刺激组较对照组P-ERK蛋白表达水平下降(t=2.911,P=0.027),两组灰度值比分别为0.058±0.031及0.125±±0.034,ERK蛋白无显著差异,两组灰度值比值为1.725±±0.539及1.727±0.196(t=0.008,P=0.994)。二、体内实验结果 1、酒精喂养前,各组小鼠精神状态好,食欲佳,毛发色泽光亮。实验过程中,对照组小鼠与之前对比,变化不大；慢性酒精组小鼠与之前对比,精神差,食欲差,毛发色泽较晦暗,且有2只小鼠死亡。 2、体重经过56天慢性酒精刺激后,对照组实验结束时平均体重(31.943±±1.381)较实验前重(18.300±1.253),差异有统计学意义(P0.01)。慢性酒精组实验结束时平均体重(19.160±1.539)与实验前(18.900±0.860)对比,变化不大,差异无统计学意义(P0.05)。对照组体重的增加(13.643±2.343)显著多于酒精组小鼠的体重增加(0.420±0.550),差异有统计学意义(P0.01)。 3、HE染色结果显示,对照组的肝脏病理切片基本正常,肝小叶结构较清晰,肝细胞呈多角形,由中央静脉向四周呈放射性排列。慢性酒精组肝小叶结构轮廓基本消失,肝细胞排列不规则,细胞形态不规则,胞质中可见大小不等的空泡,为脂肪滴,部分细胞呈印戒状,即以酒精性脂肪肝病理表现为主。 4、Western blot检测结果 1)慢性酒精刺激降低小鼠肝脏内P-AKT的表达水平,慢性酒精组P-AKT水平(0.268±0.208)显著低于对照组(0.646±0.309)(t=3.216,P=0.005),但肝脏内总AKT蛋白水平无明显差异,灰度值比值为慢性酒精组(1.735±1.003)及对照组(1.644±0.618)(t=-0.255,P=0.801),western blot以GAPDH作为内参。 2)慢性酒精刺激降低小鼠肝脏内P-ERK的表达水平,慢性酒精组P-ERK水平(0.072±0.028)显著低于对照组(0.178±0.019)(t=6.322,P=0.001),但肝脏内总ERK蛋白水平无明显差异,灰度值比值为慢性酒精组(1.938±0.320)及对照组(1.582±0.218)(t=-1.842,P=0.115),western blot以GAPDH作为内参。 3)慢性酒精刺激增加小鼠肝脏内PTEN的表达水平,慢性酒精组PTEN水平(1.173±0.536)显著高于对照组(0.725±0.304)(t=-2.516,P=0.020)。结论 1、慢性酒精组小鼠体重实验前后没有增长,对比对照组有明显差异,这可能表明工业酒精对小鼠有一定的毒性,或小鼠对工业酒精饲料有一定抵制。从而,实验表明,目前广泛应用的工业酒精可能不适宜小鼠此类试验的研究。从而提示,下一步的研究,将选择不同的食用酒精。 2、通过显微镜观察对照组和慢性酒精组的病理切片,慢性酒精刺激可导致正常小鼠肝脏脂肪性变,形成酒精性肝病。 3、体外实验中,胰岛素刺激激活PI3K/AKT信号通路,上调P-AKT的表达水平。 4、体外体内实验western blot检测,慢性酒精组P-AK1、P-ERK的表达水平较对照组低,慢性酒精刺激降低小鼠肝脏内P-AKT、P-ERK的表达水平,形成肝内胰岛素抵抗。 5、体内western blot检测,慢性酒精组PTEN的表达水平较对照组高,慢性酒精刺激增加小鼠肝脏内PTEN的表达水平。从而可知,在健康小鼠中,慢性酒精刺激可能通过上调PTEN的表达量进而诱导其P-AKT的表达水平下降,形成肝内胰岛素抵抗。然而,PTEN表达水平的提高,是因为可能的工业酒精的潜在毒性,还是因为酒精本身效应的因素,有待于进一步研究。该问题的理清对酒精肝胰岛素抵抗的机制具有重要的作用。
[Abstract]:Research background
With the improvement of the living standard of material, alcoholic beverages have become more and more widely used as part of people's life. Accordingly, alcohol related diseases are also increasing. For example, a large amount of alcohol can lead to fatty changes in the liver, form alcoholic liver disease, further develop into alcoholic liver harden, and even develop to alcoholic liver cancer. Fatty disease of the liver. A variety of pathological results can lead to a variety of pathological results, one of which is the cause of insulin resistance, that is, the body's response to insulin physiology is reduced or reduced, and insulin resistance is closely associated with the development and development of many other diseases, such as diabetes, obesity, lipid metabolic disorders, and hypertension, [1-2].
PI3K/AKT and MAPK/ERK are two important pathways for insulin to regulate cell metabolism or growth. Under normal conditions, insulin receptor binding to the receptor on the membrane causes the phosphorylation of the insulin receptor itself, which activates the tyrosine kinase and the insulin receptor substrate (Insulin Receptor Substrate, IRS). In the PI3K/AKT signaling pathway, the activated IRS Activation of the active.P13K in the downstream 3- phosphoric acid inositol kinase (phosphoinositide-3-kinase, PI3K) can convert phosphatidylinositol 4,5 two phosphoric acid (phosphatidylinositol-4,5-bisphosphate, PI (4,5) P2) into phosphatidyl inositol 3,4,5 three phosphoric acid (phosphatidylinositol-4,5-triphosphate, PI), which can further activate the eggs. B (serine-threonine protein kinase B, AKT), resulting in a glucose transporter translocation [3-4]. in the MAPK/ERK pathway, Ras and Raf binding, and Raf from cytoplasm to cell membrane, activated by phosphorylation of Raf filament / threonine on the cell membrane. The active MEK further activates the ERK. suppressor gene tenth homologous phosphatase - tension protein gene (phosphatase and tensin homologue, PTEN), the main negative regulator of the Insulin/PI3K/AKT signaling pathway, which directly affects the insulin signal. The steady-state [5-6]. of the path
Because the Insulin signaling pathway is regulated by many molecules and thus mediates many downstream molecular events, the regulation of the pathway is associated with many lesions of the cell, such as cancer, diabetes, insulin resistance. Chronic alcohol stimulation can lead to insulin resistance. However, alcohol stimulates the molecules that cause insulin resistance. The study is part of the impact of the national fund chronic alcohol on the formation of insulin resistance in the liver. The fund aims to explore the molecular mechanism of the tumor suppressor gene PTEN in the chronic alcohol irritation damage Insulin/PI3K/AKT signaling pathway. This subject is mainly the early work of the fund, that is, to study alcohol The mice model was established. We used the liquid feeding model and used the Lieber-DeCarli liquid feed. The culture of the cells in vitro was carried out in this subject to verify the results of the test in vivo.
objective
Effects of chronic ethanol stimulation on hepatic insulin signaling pathway and its mechanism.
Method
First, in vitro experimental section
1, the fund used the hepatoma cell line HepG2 cells as the research object. Cell culture (the DMEM culture medium of 10% fetal bovine serum, temperature 37, 5% carbon dioxide thermostat).
1) activation of PI3K/AKT signaling pathway stimulated by insulin in HepG2 cells.
The cells were divided into the control group and the insulin stimulation group. After 12 hours of serum free starvation, insulin stimulated 10min, and the control group was stimulated by PBS. The protein concentration was measured. After the measurement of protein concentration, the control group was detected by Western blot (glue, electrophoresis, electrical transfer, closure, incubation, two anti incubation, rinsing PVDF membrane, film exposure) protein analysis. The level of P-AKT protein expression in the insulin stimulation group.
2) chronic alcohol effect
The cells were divided into the alcohol stimulation group and the control group. After 12 hours without serum starvation, anhydrous ethanol stimulation (100mM/L) was 72 hours, and the control group was serum-free DMEM culture. After 12 hours of serum starvation, insulin stimulated 10min, lysis cells and protein extraction. After measuring protein concentration, the level of P-AKT and P-ERK protein expression was detected in the previous section 1..
2, gel image analysis. The film is scanned and the Image J software is used to analyze the gray value of the signal. GAPDH is used as the internal reference. P-AKT is used as the reference for the total AKT. P-ERK is used as the reference of the total ERK. The relative expression of the target protein is calculated by the ratio of the corresponding protein strip to GAPDH or AKT or ERK.
Two, the experimental part of the body
1, experimental design: SPF class Balb/c mice were bought from the animal center of Southern Medical University, and the animal room of the southern hospital was kept in accordance with the standard of clean grade. The week age was 6 to 8 weeks, male mice and 14 rats were randomly divided into 2 groups: the control group and the chronic alcohol group. The same liquid diet containing alcohol and non alcohol was made according to the formula of Lieber-DeCarli liquid feed. Feed. Give the mice daily quantitative feeding: the control group was fed the control liquid feed (the proportion of heat: fat 35%, carbohydrate 47%, protein 18%, no alcohol), and the chronic alcohol group was fed with alcohol liquid feed (the same as the control liquid feed, but alcohol replaced part of the carbohydrate, alcohol accounted for 36%, the alcohol volume fraction was 5%), two groups. The final intake of heat card was basically the same. The whole process was divided into the transition period and the mold making period. The mice were fed for 56 days. During the whole process, the mice were given an empty abdominal body weight every 7 days. The day of death, the abdominal body weight of.56 days after the abdominal injection of pentobarbital sodium was given, and the abdominal cavity was opened after the mice were fully anesthetized. Liver tissue was collected and a small amount of liver tissue was fixed with 4% Formaldehyde Solution. The rest was immediately frozen in liquid nitrogen and then moved to -80 C refrigerator for long-term preservation.
2, the production and observation of liver tissue in mice. The liver tissue was fixed in 4% Formaldehyde Solution. Then the tissue block was dehydrated, transparent, impregnated, embedded, sliced, roasted, dewaxed, rehydrated, dyed, dehydrated, transparent, and made into pathological sections, and the structural changes of two groups of liver tissues in the control group and the chronic alcohol group were observed by microscope.
3, Western blot detected the expression of P-AKT, P-ERK and PTEN in the liver tissues of the control group and the chronic alcohol group at.-80 centigrade ice to cut the liver tissue, and lysed and lapping the liver tissue (RIPA buffer liquid) on the ordinary ice. The protein samples were extracted with Western blot (glue, electrostroke, electrotransfer, sealing, one anti incubation, two anti incubation, and rinse PVDF membrane, " The expression of P-AKT, P-ERK and PTEN was detected by film exposure.
4, gel image analysis. The film is scanned and the Image J software is used to analyze the gray value of the signal. GAPDH is used as the internal reference. P-AKT is used as the reference for the total AKT. P-ERK is used as the reference of the total ERK. The relative expression of the target protein is calculated by the ratio of the corresponding protein strip to GAPDH or AKT or ERK.
Three, statistical analysis: all results were analyzed by Exce12007 and SPSS13.0 software, and the results were "arithmetic mean + standard deviation (M + SD)". The two groups were compared with two independent sample t test (Independent-Samples T Test) or rank sum test (0.05). Significant differences.
experimental result
First, in vitro experimental results
1, the results of Western blot detection showed that compared with the control group, the insulin stimulation group increased the level of P-AKT significantly (t=-12.758, P=0.009) after insulin stimulation of 10min, and the ratio of the two groups was 0.313 + 0.156 and 2.765 + 0.222., respectively.
2, in our test results, the expression level of P-AKT protein in the alcohol stimulation group was lower than that in the control group (t=2.645, P=0.048). The ratio of gray value of the two groups was 0.454 + 0.085 and 0.681 + 0.150 respectively, and the AKT protein was not significantly different. The ratio of the gray value of the two groups was 0.588 + + + + 0.213 and 0.705 + 0.052 (t=-1.063, P=0.329).
3, the expression level of P-ERK protein in the alcohol stimulation group was lower than that in the control group (t=2.911, P=0.027). The ratio of gray value of the two groups was 0.058 + 0.031 and 0.125 + 0.034 respectively. There was no significant difference in the ERK protein. The ratio of the gray value of the two groups was 1.725 + + + 0.539 and 1.727 + 0.196 (t=0.008, P=0.994).
Two, experimental results in vivo
1, before the alcohol was fed, the mice in each group had good mental state, good appetite, and bright hair color. In the experiment, the control group had little change, and the mice in the chronic alcohol group were compared with the previous, poor spirit, poor appetite, dark hair and dark color, and 2 mice died.
2, after 56 days of chronic alcohol stimulation, the average weight of the control group was 31.943 + 1.381 (18.300 + 1.253) at the end of the experiment (18.300 + 1.253), and the difference was statistically significant (P0.01). The average weight (19.160 + 1.539) at the end of the chronic alcohol group was compared with the pre experiment (18.900 + 0.860), and the difference was not significant (P0.05). Body weight gain (13.643 + 2.343) was significantly higher than that of alcohol group (0.420 + 0.550), and the difference was statistically significant (P0.01).
3, the results of HE staining showed that the pathological sections of the liver of the control group were basically normal, the structure of hepatic lobule was clearer, the liver cells were polygonal, and the central vein was arranged radially from the central vein to the surrounding area. The structure of the hepatic lobule in the chronic alcohol group disappeared basically, the liver cells were irregular, the cell shape was irregular, and the vacuoles of different sizes were visible in the cytoplasm, and the fat was fat. Some of the cells showed a signet ring, which was mainly alcoholic fatty liver.
4, Western blot detection results
1) chronic alcohol stimulation reduced the expression level of P-AKT in the liver of mice. The P-AKT level of chronic alcohol group (0.268 + 0.208) was significantly lower than that of the control group (0.646 + 0.309) (t=3.216, P=0.005), but there was no significant difference in the total AKT protein level in the liver, the ratio of gray value was (1.735 + 1.003) and the control group (1.644 + 0.618) (t=-0.255, P=0.801), Wester N blot takes GAPDH as the internal reference.
2) chronic alcohol stimulation reduced the expression level of P-ERK in the liver of mice. The P-ERK level of chronic alcohol group (0.072 + 0.028) was significantly lower than that of the control group (0.178 + 0.019) (t=6.322, P=0.001), but there was no significant difference in the total ERK protein level in the liver, the ratio of gray value was (1.938 + 0.320) and the control group (1.582 + 0.218) (t=-1.842, P=0.115), Wester N blot takes GAPDH as the internal reference.
3) chronic alcohol stimulation increased the expression level of PTEN in the liver of mice, and the level of PTEN in chronic alcohol group (1.173 + 0.536) was significantly higher than that of the control group (0.725 + 0.304) (t=-2.516, P=0.020).
1, the mice in the chronic alcohol group did not increase before and after the weight test, and there was a significant difference between the control group and the control group. This may indicate that the industrial alcohol has some toxicity to the mice, or the mice have a certain resistance to the industrial alcohol feed. One step of the study will choose different edible alcohol.
2, the pathological sections of the control group and chronic alcohol group were observed by microscope. Chronic alcohol stimulation could cause fatty liver in normal mice to form alcoholic liver disease.
3, in vitro, insulin stimulates the activation of PI3K/AKT signaling pathway and upregulates the expression level of P-AKT.
4, in vitro test of Western blot, the expression level of P-AK1 and P-ERK in chronic alcohol group was lower than that of the control group. Chronic alcohol stimulation reduced the expression level of P-AKT and P-ERK in the liver of mice, and formed the insulin resistance in the liver.
5, in vivo Western blot detection, the expression level of PTEN in chronic alcohol group is higher than that of the control group. Chronic alcohol stimulation increases the expression level of PTEN in the liver of mice. Thus, in healthy mice, chronic alcohol stimulation may induce the decrease of the expression level of P-AKT by up regulating the expression of PTEN and forming the insulin resistance in the liver. However, PT The increase in the expression level of EN is due to the potential toxicity of industrial alcohol, or the factors of alcohol itself, which need to be further studied. It is important to clarify the mechanism of insulin resistance to alcohol and liver.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5

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