罗格列酮对OLETF大鼠肝脏组织病理变化及IL-17、Bcl-xl表达的影响
发布时间：2018-08-28 06:59
【摘要】：目的糖尿病(diabetes mellitus DM)是一组代谢性疾病,由于胰岛素分泌缺陷和/或障碍引起的以慢性高血糖为主的疾病。长期持续的高血糖和代谢紊乱,可导致全身组织器官损伤和功能障碍。肝脏在2型糖尿病(T2DM)的致病中有着重要的作用,作为一个靶器官,肝脏在糖尿病状态下也会受到损害。持续的高血糖使得晚期糖基化产物增加,肝小叶结构破坏,纤维化增加,肝细胞结构和功能发生变化,肝细胞炎症增加,细胞凋亡加剧等一系列变化。本实验以与OLETF大鼠其同系的LETO鼠作为对照组,OLETF大鼠以及经罗格列酮治疗大鼠均为实验组,观察各组大鼠肝脏组织的病理改变。并测定肝脏细胞炎症因子IL-17以及凋亡因子Bcl-xl的表达变化。进一步来探讨罗格列酮对2型糖尿病OLETF大鼠肝脏组织的影响。方法大鼠单笼饲养,标准饲料喂养,12/12h循环光照。LETO为正常组12只,给予普通饲料喂养,仅注射缓冲溶液。饲养一个月后,每两周做一次糖耐量实验,大鼠禁食16h后灌胃给予单剂的糖(25%葡萄糖溶液1mL/100g)2.5g/kg作为进食,在给药前及给药后30、60、120min分别从尾静脉取血,以血糖仪测得血糖浓度。以血糖峰值16.7mmol/L和负荷后120min血糖11.1mmol/L诊为糖尿病,若只具备其中一个条件则为糖耐量减低。到30周时,共有16只OLETF大鼠作为实验组模型。之后随机分为两组(n=8),RGZ组灌以罗格列酮稀释液,剂量为3mg/kg·d。DM组和NC组灌以等量蒸馏水,每日1次灌胃12周。三组大鼠分别采集血液进行测量,之后三组大鼠同时处死,分别取肝脏组织。放血后以无菌器械迅速取肝脏组织3块,其中2块分别放入DEPC处理过的冻存管中,丢入液氮保存备用,1块置于10%的福尔马林中,行石蜡包埋切片。之后进行组织学染色,包括:HE染色、AO染色、Schiff实验、Masson三色染色、氯化金染色和免疫组织化学染色。观察大鼠肝脏组织学变化、测定肝肝巨噬细胞和肝储脂细胞数量的变化、观察肝脏组织细胞RNA变化、胶原纤维的变化以及IL-17和Bcl-x L的阳性表达改变。另外,用置于-70℃的肝脏组织制成组织匀浆来,羟脯氨酸法测定胶原蛋白的含量。所有数据均采用SPSS13.0软件进行处理。结果1.大鼠一般状况比较:与leto大鼠相比,oletf大鼠体型较为肥胖,神经较为敏感易激怒,懒动畏寒,精神萎靡、焦虑,且肝脏体积明显变大。2.生化指标的改变:处死大鼠之前分别测得oletf大鼠餐后两小时血糖,甘油三酯、谷丙转氨酶和谷草转氨酶以及肝指数。与nc组相比,dm组餐后两小时血糖,甘油三酯、谷丙转氨酶、天冬氨酸转移酶和肝指数指标均有明显升高,rzg组与dm组相比,dm组餐后两小时血糖,甘油三酯、谷丙转氨酶有显著降低,但谷草转氨酶无明显差异。3.肝脏组织学变化:nc组大鼠肝小叶规则,肝细胞索呈放射状整齐排列,肝血窦结构清晰,肝细胞核圆居中,胞质均匀,没有炎性细胞浸润。dm组肝小叶失去正常结构,肝细胞索变窄,肝血窦界限不清,肝细胞肿胀、脂肪变,坏死,偶见点状炎性灶。rgz肝细胞结构尚清晰,肝细胞索结构排列尚整齐,虽然也存在脂性空泡和炎性细胞,但较糖尿病对照组明显减少4.肝脏肝巨噬细胞的比较:通过观察he染色结果,计数各组织中肝巨噬细胞的数量,nc组肝巨噬细胞计数结果为17.05±0.89,dm组肝巨噬细胞数量为25.31±1.23,rgz组结果为38.61±4.02。结果显示dm组较nc组相对增多,但rgz组肝巨噬细胞结果较nc组和dm组都明显增加(p0.01)。5.肝脏肝细胞糖原含量的变化:nc组肝糖原定位于胞浆,分布均匀密集,糖原颗粒染色均一呈粉红色。dm组肝细胞肿胀、拥挤,糖原分布疏松,糖原颗粒不明显。rgz组肝糖原较糖尿病组密布,颗粒相对均一。测定肝细胞糖原颗粒灰度,nc组肝细胞糖原的灰度值为62.38±10.16,dm组(109.54±11.18)与nc组相比明显增高,具有统计学意义(p0.01),rgz组(79.36±9.98)与nc组相比无统计学意义,与dm组相比明显降低(p0.05)。6.肝脏组织rna含量变化:吖啶橙荧光染色使正常细胞核dna呈绿色或黄绿色均匀荧光,细胞质和核仁的rna染为桔黄(红)色荧光,测定胞质中rna荧光染色灰度,与nc组(47.21±8.72)相比,dm组(89.84±10.71)和rgz组(61.25±8.13)灰度值增高,差异均有统计学意义(p0.01),与dm组相比,rgz组灰度值降低(p0.05)。7.肝脏储脂细胞(hsc)的观察:肝脏储脂细胞位于狄氏间隙和肝细胞之间,经氯化金染色使得肝储脂细胞呈紫褐色,背景淡灰色。通过高倍镜下储脂细胞计数,与NC组肝储脂细胞(2.75±0.21)相比DM组(7.19±0.45)升高(P0.01),与DM组相比RGZ组储脂细胞数值(4.93±0.48)降低(P0.01)。8.胶原纤维的变化:NC组大鼠肝小叶中央静脉管壁薄,门管区有少量结缔组织,其所占面积的百分比为[(3.19±0.75)%],与NC组相比,DM组[(15.87±0.63)%]肝小叶中央静脉管壁增厚,门管区结缔组织增多,胶原沉积(P0.01),与DM组相比,RGZ组[(10.06±0.54)%]在门管区可见纤维间隔,较DM组胶原纤维减少(P0.05)。9.胶原蛋白含量的比较:高效液相色谱法测定每克肝脏组织中的胶原蛋白的含量,与NC组(64.52±21.51 mg/g)相比,DM组(134.54±18.78 mg/g)胶原蛋白含量明显增加(P0.01),RGZ组(91.59±10.75 mg/g)低于DM组(P0.01),有统计学意义。10.肝脏组织IL-17表达变化:与NC组成肝脏IL-17阳性表达吸光度值(0.129±0.045)相比,DM组(0.520±0.021)升高(P0.01),而RGZ组(0.235±0.034)低于DM组(P0.01)。11.肝脏组织Bcl-xl表达变化:与NC组肝脏组织Bcl-x L阳性表达吸光度值(0.613±0.014),DM组(0.242±0.041)降低(P0.05),RGZ组肝脏组织Bcl-x L阳性表达(0.369±0.048)较DM组升高(P0.01)。结论1.糖尿病大鼠肝小叶结构紊乱,肝细胞肿胀,胶原纤维沉积,胶原蛋白含量明显增加。2.糖尿病组HSC细胞活化增生,导致肝胶原纤维含量增加。3.糖尿病组肝小叶和门管区炎性细胞增多,可能与肝巨噬细胞数量增加有关;过多肝巨噬细胞加重糖尿病肝组织损伤。4.糖尿病组IL-17阳性表达增加,Bcl-xl表达降低,且以中央静脉为中心向周围发射状分布,可能与血液供给有关。5.罗格列酮对OLETF 2型糖尿病大鼠肝脏组织的有一定的保护作用,能够减轻糖尿病对肝脏组织损害程度。
[Abstract]:Objective Diabetes mellitus (DM) is a group of metabolic diseases, mainly caused by chronic hyperglycemia due to insulin secretion deficiencies and/or disorders. Long-term persistent hyperglycemia and metabolic disorders can lead to systemic tissue and organ damage and dysfunction. The liver plays an important role in the pathogenesis of type 2 diabetes mellitus (T2DM). As a target organ, the liver can also be damaged in diabetic state. Persistent hyperglycemia leads to the increase of advanced glycosylation products, the destruction of hepatic lobules, the increase of fibrosis, the change of hepatocyte structure and function, the increase of hepatocyte inflammation and the increase of apoptosis. As control group, OLETF rats and rosiglitazone-treated rats were both experimental groups. The pathological changes of liver tissues were observed and the expression of IL-17 and Bcl-xl were measured. The effect of rosiglitazone on liver tissues of OLETF rats with type 2 diabetes mellitus was further investigated. Methods Rats were fed in single cage. The rats were fed with standard diet for 12/12 hours and were fed with normal diet and injected with buffer solution only. At 30 weeks, 16 OLETF rats were randomly divided into two groups (n=8). The RGZ group was given rosiglitazone dilution, and the RGZ group was given rosiglitazone. The rats in the three groups were collected blood for measurement, and then the liver tissues were taken. After bleeding, three pieces of liver tissues were quickly taken out with aseptic instruments. Two of them were put into DEPC-treated cryopreserve tubes and stored in liquid nitrogen for reserve. The rats were placed in 10% formalin and paraffin-embedded sections, then stained histologically, including HE staining, AO staining, Schiff test, Masson trichrome staining, gold chloride staining and immunohistochemical staining. Changes of collagen fibers and positive expression of IL-17 and Bcl-x L were observed. In addition, liver tissue homogenate was prepared at - 70 C and the content of collagen was determined by hydroxyproline method. All data were processed by SPSS 13.0 software. Results 1. Comparing with Leto rats, the body type of OLETF rats was more than that of Leto rats. Obesity, nervous sensitivity, irritability, laziness, chills, depression, anxiety, and liver volume significantly increased. 2. Biochemical indicators: before the execution of OLETF rats were measured two hours postprandial blood glucose, triglyceride, alanine aminotransferase and glutamic oxaloacetic transaminase and liver index. Compared with DM group, blood glucose, triglyceride and alanine aminotransferase in DM group were significantly decreased, but there was no significant difference in glutamic oxaloacetic aminotransferase. 3. histological changes of liver: the hepatic lobules of NC rats were regular, the hepatic cords were arranged in a radial fashion, and the hepatic sinusoids were in good order. In DM group, hepatic lobules lost normal structure, hepatic cords narrowed, hepatic sinusoids were unclear, hepatocytes swelled, steatosis, necrosis, occasional punctate inflammatory foci. RGz hepatocyte structure was still clear, hepatic cords were arranged regularly, although there were also lipid vacuoles and inflammation The number of hepatic macrophages in the NC group was 17.05 (+ 0.89), the number of hepatic macrophages in the DM group was 25.31 (+ 1.23) and that in the RGz group was 38.61 (+ 4.02). The content of glycogen in hepatocytes of RGz group was more than that of NC group and DM group (p0.01). 5. the content of glycogen in hepatocytes of RGz group was higher than that of NC group and DM group. The gray level of hepatocyte glycogen granules in NC group was 62.38 (+ 10.16), while that in DM group (109.54 (+ 11.18) was significantly higher than that in NC group (p0.01), and that in RGz group (79.36 (+ 9.98) was significantly lower than that in NC group (p0.05). Acridine orange fluorescence staining made the normal nuclear DNA show green or yellow-green uniform fluorescence, cytoplasm and nucleolus RNA stained orange (red) fluorescence, and measured the gray level of cytoplasmic RNA fluorescence staining, compared with NC group (47.21 + 8.72), DM group (89.84 + 10.71) and RGz group (61.25 + 8.13) gray level increased, the difference was statistically significant (p0.01), compared with DM group (61.25 + 8.13). The observation of hepatic fat storage cells (hsc) in the dieldrin space and between the hepatocytes, the liver fat storage cells were stained with gold chloride to appear purple brown, and the background was light gray. The number of fat storage cells in the high power microscope was higher in the DM group (7.19 + 0.45) than that in the NC group (2.75 + 0.21). Compared with DM group, the number of fat storage cells in RGZ group decreased (P 0.01). 8. Changes of collagen fibers: the wall of hepatic lobule central vein was thin and there were a few connective tissues in portal area in NC group. The percentage of connective tissue in portal area was [(3.19 [0.75)%]. Compared with NC group, the wall of hepatic lobule central vein was thickened and the connective tissue in portal area was thickened in DM group [(15.87 [(0.63)%)]. Collagen deposition increased (P 0.01). Compared with DM group, fibrous septum was observed in RGZ group [(10.06.54)%] and decreased in DM group ((P 0.05). 9. Comparison of collagen content: Collagen content in every gram of liver tissue was determined by high performance liquid chromatography, and collagen content in DM group ((134.54 18.78 mg/g) was compared with NC group (64.52 21.51 mg/g). The expression of IL-17 in liver tissue was significantly higher in DM group than in DM group (P 0.01). The expression of Bcl-xl in RGZ group was significantly lower than that in DM group (P 0.01). Changes: Compared with NC group, the absorbance value of Bcl-x L positive expression in liver tissue (0.613+0.014), DM group (0.242+0.041) decreased (P 0.05), RGZ group liver tissue Bcl-x L positive expression (0.369+0.048) increased (P 0.01). Conclusion 1. Diabetic rats liver lobule structure disorder, hepatocyte swelling, collagen fibril deposition, collagen content increased significantly.2. Activation and proliferation of HSC cells in diabetic group lead to the increase of hepatic collagen fiber content. 3. Increase of inflammatory cells in hepatic lobules and portal tract may be related to the increase of hepatic macrophages; excessive hepatic macrophages aggravate liver injury in diabetic group. 4. Increase of IL-17 positive expression and decrease of Bcl-xl expression in diabetic group, and the central vein as the center. Peripheral emission distribution may be related to blood supply. 5. Rosiglitazone has a protective effect on liver tissue of OLETF type 2 diabetic rats, and can reduce the degree of liver damage caused by diabetes mellitus.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1
本文编号：2208638
[Abstract]:Objective Diabetes mellitus (DM) is a group of metabolic diseases, mainly caused by chronic hyperglycemia due to insulin secretion deficiencies and/or disorders. Long-term persistent hyperglycemia and metabolic disorders can lead to systemic tissue and organ damage and dysfunction. The liver plays an important role in the pathogenesis of type 2 diabetes mellitus (T2DM). As a target organ, the liver can also be damaged in diabetic state. Persistent hyperglycemia leads to the increase of advanced glycosylation products, the destruction of hepatic lobules, the increase of fibrosis, the change of hepatocyte structure and function, the increase of hepatocyte inflammation and the increase of apoptosis. As control group, OLETF rats and rosiglitazone-treated rats were both experimental groups. The pathological changes of liver tissues were observed and the expression of IL-17 and Bcl-xl were measured. The effect of rosiglitazone on liver tissues of OLETF rats with type 2 diabetes mellitus was further investigated. Methods Rats were fed in single cage. The rats were fed with standard diet for 12/12 hours and were fed with normal diet and injected with buffer solution only. At 30 weeks, 16 OLETF rats were randomly divided into two groups (n=8). The RGZ group was given rosiglitazone dilution, and the RGZ group was given rosiglitazone. The rats in the three groups were collected blood for measurement, and then the liver tissues were taken. After bleeding, three pieces of liver tissues were quickly taken out with aseptic instruments. Two of them were put into DEPC-treated cryopreserve tubes and stored in liquid nitrogen for reserve. The rats were placed in 10% formalin and paraffin-embedded sections, then stained histologically, including HE staining, AO staining, Schiff test, Masson trichrome staining, gold chloride staining and immunohistochemical staining. Changes of collagen fibers and positive expression of IL-17 and Bcl-x L were observed. In addition, liver tissue homogenate was prepared at - 70 C and the content of collagen was determined by hydroxyproline method. All data were processed by SPSS 13.0 software. Results 1. Comparing with Leto rats, the body type of OLETF rats was more than that of Leto rats. Obesity, nervous sensitivity, irritability, laziness, chills, depression, anxiety, and liver volume significantly increased. 2. Biochemical indicators: before the execution of OLETF rats were measured two hours postprandial blood glucose, triglyceride, alanine aminotransferase and glutamic oxaloacetic transaminase and liver index. Compared with DM group, blood glucose, triglyceride and alanine aminotransferase in DM group were significantly decreased, but there was no significant difference in glutamic oxaloacetic aminotransferase. 3. histological changes of liver: the hepatic lobules of NC rats were regular, the hepatic cords were arranged in a radial fashion, and the hepatic sinusoids were in good order. In DM group, hepatic lobules lost normal structure, hepatic cords narrowed, hepatic sinusoids were unclear, hepatocytes swelled, steatosis, necrosis, occasional punctate inflammatory foci. RGz hepatocyte structure was still clear, hepatic cords were arranged regularly, although there were also lipid vacuoles and inflammation The number of hepatic macrophages in the NC group was 17.05 (+ 0.89), the number of hepatic macrophages in the DM group was 25.31 (+ 1.23) and that in the RGz group was 38.61 (+ 4.02). The content of glycogen in hepatocytes of RGz group was more than that of NC group and DM group (p0.01). 5. the content of glycogen in hepatocytes of RGz group was higher than that of NC group and DM group. The gray level of hepatocyte glycogen granules in NC group was 62.38 (+ 10.16), while that in DM group (109.54 (+ 11.18) was significantly higher than that in NC group (p0.01), and that in RGz group (79.36 (+ 9.98) was significantly lower than that in NC group (p0.05). Acridine orange fluorescence staining made the normal nuclear DNA show green or yellow-green uniform fluorescence, cytoplasm and nucleolus RNA stained orange (red) fluorescence, and measured the gray level of cytoplasmic RNA fluorescence staining, compared with NC group (47.21 + 8.72), DM group (89.84 + 10.71) and RGz group (61.25 + 8.13) gray level increased, the difference was statistically significant (p0.01), compared with DM group (61.25 + 8.13). The observation of hepatic fat storage cells (hsc) in the dieldrin space and between the hepatocytes, the liver fat storage cells were stained with gold chloride to appear purple brown, and the background was light gray. The number of fat storage cells in the high power microscope was higher in the DM group (7.19 + 0.45) than that in the NC group (2.75 + 0.21). Compared with DM group, the number of fat storage cells in RGZ group decreased (P 0.01). 8. Changes of collagen fibers: the wall of hepatic lobule central vein was thin and there were a few connective tissues in portal area in NC group. The percentage of connective tissue in portal area was [(3.19 [0.75)%]. Compared with NC group, the wall of hepatic lobule central vein was thickened and the connective tissue in portal area was thickened in DM group [(15.87 [(0.63)%)]. Collagen deposition increased (P 0.01). Compared with DM group, fibrous septum was observed in RGZ group [(10.06.54)%] and decreased in DM group ((P 0.05). 9. Comparison of collagen content: Collagen content in every gram of liver tissue was determined by high performance liquid chromatography, and collagen content in DM group ((134.54 18.78 mg/g) was compared with NC group (64.52 21.51 mg/g). The expression of IL-17 in liver tissue was significantly higher in DM group than in DM group (P 0.01). The expression of Bcl-xl in RGZ group was significantly lower than that in DM group (P 0.01). Changes: Compared with NC group, the absorbance value of Bcl-x L positive expression in liver tissue (0.613+0.014), DM group (0.242+0.041) decreased (P 0.05), RGZ group liver tissue Bcl-x L positive expression (0.369+0.048) increased (P 0.01). Conclusion 1. Diabetic rats liver lobule structure disorder, hepatocyte swelling, collagen fibril deposition, collagen content increased significantly.2. Activation and proliferation of HSC cells in diabetic group lead to the increase of hepatic collagen fiber content. 3. Increase of inflammatory cells in hepatic lobules and portal tract may be related to the increase of hepatic macrophages; excessive hepatic macrophages aggravate liver injury in diabetic group. 4. Increase of IL-17 positive expression and decrease of Bcl-xl expression in diabetic group, and the central vein as the center. Peripheral emission distribution may be related to blood supply. 5. Rosiglitazone has a protective effect on liver tissue of OLETF type 2 diabetic rats, and can reduce the degree of liver damage caused by diabetes mellitus.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1
【参考文献】
相关期刊论文 前2条
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2 Wen-Shan Lv;Rui-Xia Sun;Yan-Yan Gao;Jun-Ping Wen;Rong-Fang Pan;Li Li;Jing Wang;Yu-Xin Xian;Cai-Xia Cao;Ming Zheng;;Nonalcoholic fatty liver disease and microvascular complications in type 2 diabetes[J];World Journal of Gastroenterology;2013年20期
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