ω-3多不饱和脂肪酸缓解非酒精性脂肪性肝病炎性损伤的研究

发布时间：2019-06-04 09:13

【摘要】：目的：非酒精性脂肪性肝病(non-alcoholic fatty liver diseases，NAFLD)是临床上最为常见的的慢性肝病之一，其炎性反应贯穿了整个病变过程。近年来发现ω-3系多不饱和脂肪酸(ω-3polyunsaturated fatty acid，ω-3PUFA)对NAFLD炎性损伤有正向干预作用，但具体作用机制尚不明确，所以本课题旨在初步探讨ω-3PUFA缓解NAFLD炎性损伤的可能作用机制。 方法：采用高脂饲喂建立NAFLD动物模型。选用体重160~180g的雄性SD大鼠，随机分为4组，每组48只：正常饲料饲喂的对照组（CON组）、高脂（20%猪油）饲喂的NAFLD模型组（HFD组）、高脂饲料基础上加鱼油（10%猪油+10%鱼油）的鱼油干预组（FOH组）、高脂饲料基础上加紫苏油(14.83%猪油+5.17%紫苏油)干预的紫苏油干预组（POH组），分别于实验的4w、8w、12w和16w时间点断头取血处死（每时间点12只/组），称取体重、肝重，计算肝指数。采用酶法检测血清肝功（ALT、AST）、血脂（TCTG、HDL-C、LDL-C）、SOD、GST和FFA变化情况；HE染色观察肝细胞气球样变、脂肪变性程度和炎性反应程度；Masson染色观察肝组织纤维化分期；免疫组织化学技术检测巨噬细胞特异性抗体CD68蛋白的表达；使用荧光实时定量PCR技术检测肝组织中IL-6、IL-1β、TNF-、MCP-1、NF-κB P65的mRNA的表达水平。 结果：（1）HFD组较CON组8w时大鼠肝指数明显升高，肝细胞气球样变明显，呈现出大小泡混合型肝细胞脂肪变性，小叶内和汇管区炎性细胞浸润，纤维化明显，即发生了典型的NASH病变。另外，8w时血清ALT、TCH、FFA水平均显著性升高，HDL-C显著性降低。至12w时AST、TG、LDL-C也显著性升高，SOD显著性降低，16w时GST显著性升高。炎性细胞因子（IL-6、IL-1β、TNF-）、趋化因子MCP-1的mRNA表达均在8w都显著性升高，到12w时NF-κB P65的mRNA表达也显著性升高。蛋白水平，16w时CD68阳性表达明显增多，巨噬细胞大片聚集。 （2）鱼油干预后，肝细胞气球样变，脂肪变性，小叶内和汇管区炎性细胞浸润，纤维化均有不同程度的改善。并且4w时血清ALT、TCH、FFA较HFD组显著性降低；到8w时LDL-C、GST较HFD组显著性降低；12w时AST、TG较HFD组显著性降低，SOD较HFD组显著性升高。RNA水平上，炎性细胞因子IL-6在4w时开始显著性降低，TNF-在8w时开始显著性降低，IL-1β在12w时开始显著性降低；趋化因子MCP-1在8w时开始显著性降低；NF-κB P65在12w时开始显著性降低。蛋白水平，16w时CD68阳性表达明显减少。 （3）紫苏油干预后，肝细胞气球样变，脂肪变性，小叶内和汇管区炎性细胞浸润，纤维化也呈现不同程度的改善。4w时血清ALT较HFD组显著性降低；到8w时TCH、LDL-C、FFA、GST较HFD组显著性降低；12w时AST、TG较HFD组显著性降低，SOD较HFD组显著性升高。RNA水平，炎性细胞因子IL-6有降低但没有显著性变化， TNF-、IL-1β在12w时开始显著性降低；趋化因子MCP-1在12w时开始显著性降低；NF-κB P65在16w时开始显著性降低。蛋白水平，16w时CD68阳性表达明显减少。 结论：(1)高脂饮食8w时，大鼠肝脏进展到了NASH病程，鱼油、紫苏油干预不同程度缓解了NAFLD炎性损伤。 (2) ω-3PUFA可能通过NF-κB信号通路，抑制炎性细胞因子的产生，，最终缓解了NAFLD炎性进程。
[Abstract]:Objective: Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases. In recent years, it has been found that the trans-3 PUFA has a positive effect on the inflammatory injury of NAFLD, but the mechanism of specific action is not clear. Methods: The animal model of NAFLD was established by high-fat feeding. Type: Male SD rats weighing 160-180 g were randomly divided into 4 groups,48 rats in each group: control group (CON group) fed with normal feed, and NAFLD model group (HFD group) fed with high fat (20% lard). (10% lard + 10% fish oil) on the basis of high-fat diet (FOH group), and perilla oil (14.83% lard + 5.17% perilla oil) was added to the base of high-fat diet to intervene with Perilla oil intervention group (POH group). At the time point of w, 12w, and 16w, the blood was killed (12/ group at each time point), and the weight and liver weight were weighed, and the liver finger was calculated. The changes of serum liver function (ALT, AST), blood lipid (TCTG, HDL-C, LDL-C), SOD, GST and FFA in serum were detected by enzyme method. The expression of IL-6, IL-1, TNF-, MCP-1, NF-CDB P65 in liver tissue was detected by fluorescent real-time quantitative PCR (RT-PCR). Results: (1) The liver index of the rats was significantly higher in the HFD group than that of the CON group, and the balloon of the liver cells was obvious, presenting a large-size mixed type of hepatocyte fat degeneration, infiltration of the inflammatory cells in the lobular and foreign exchange tubes, and the obvious fibrosis, that is, the typical NAS H. In addition, the levels of ALT, TCH and FFA in serum increased significantly at 8w, and HDL-C was significant. The results showed that AST, TG, LDL-C in AST, TG and LDL-C increased significantly at 12w. The expression of IL-6, IL-1, TNF-and the expression of MCP-1 in the expression of MCP-1 increased significantly at 8w. The expression of CD68 was significantly increased at the level of protein and 16w, and the macrophage was large. (2) After the intervention of fish oil, the cell infiltration and fibrosis of the liver cells were different after the intervention of the fish oil. The levels of serum ALT, TCH and FFA were significantly lower than those in the HFD group at the time of 4w. The levels of LDL-C and GST were significantly lower at 8w, and the levels of AST and TG in the lower group were significantly lower than those in the HFD group, and the SOD was lower than that of the HFD group. There was a significant increase in the level of the RNA, the level of IL-6 of the inflammatory cytokines decreased significantly at 4w, the level of TNF-1 was significantly lower at 8w, the level of IL-1 was significantly lower at 12 w, and the expression of the chemokine MCP-1 was significantly lower at 8w; and the NF-EMAB P65 started at 12w Significant decrease. Protein level, CD68 positive table at 16 w (3) After the intervention of Perilla oil, the cell infiltration and fibrosis of the liver cell balloon and the inflammatory cells of the interlobular area and the sink area were improved, and the level of the serum ALT was significantly decreased at the time of 4w. The levels of TCH, LDL-C, FFA and GST in the group were lower than those of the HFD group at the time of 8w. At 12w AST, TG was significantly lower than that in the HFD group, and the SOD was higher than that of the HFD group. There was a significant increase in the level of the D group. The level of the RNA, the IL-6 of the inflammatory cytokine, but no significant change, the TNF-, IL-1 level began to decrease significantly at 12w; the chemokines MCP-1 began to significantly decrease at 12w; and the NF-EMAB P65 at 16 w Start significant reduction. Protein level, CD68 male at 16 w Conclusion: (1) When the high-fat diet is 8w, the liver of the rat has progressed to the course of NASH, fish oil and Perilla oil. NAFLD inflammatory injury. (2) The 3-3 PUFA may pass through the NF-B signal pathway to inhibit the production of inflammatory cytokines and eventually slow down
【学位授予单位】：武汉轻工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5

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