【作者】 王琦；

【导师】 吕正梅；

【作者基本信息】 安徽医科大学 ， 人体解剖与组织胚胎学， 2015， 硕士

【摘要】 背景现代社会久坐、能量摄入过多等不健康生活方式导致肥胖发病逐年增加。肥胖能够引起包括性腺机能减退在内的多种病理状态。目前研究表明肥胖引起的慢性炎症是导致肥胖相关疾病损害如胰岛素抵抗的重要机制。白藜芦醇具有抗炎、抗氧化应激作用,对于肥胖相关代谢性疾病的治疗价值是当前研究热点。目的探讨白藜芦醇对肥胖相关脂肪组织炎症及氧化应激的抑制机制,以及对睾丸内分泌功能的保护作用。方法30只6周龄的C57BL/6J雄性小鼠,随机分为四组:标准饮食组(Ctrl1组,n=7),高脂组(HCD组,n=8),高脂加药组(HCD+Res组,n=8),标准饮食加药组(Ctrl2组,n=7)。自然的光照节律,每两天换一次垫料,并提供充足的水分和食物。HCD组给予高糖高胆固醇饮食(high-calorie and high-cholesterol diet,配方:59%的维持料、10%猪油、10%蛋黄粉、1%胆固醇、20%蔗糖);HCD+Res组给予高糖高胆固醇饮食,并灌胃量为400mg/(kg·d)的白藜芦醇;Ctrl1组给予普通维持料饮食;Ctrl2组是在普通维持料的基础上喂饲与HCD+Res组等量的白藜芦醇。造模结束后,摘除眼球取血进行处死,然后:①测量小鼠体重和附睾脂肪组织的重量;②采用放射免疫法测小鼠血清睾酮的浓度;③取附睾脂肪组织做石蜡切片,HE染色观察脂肪细胞形态学的改变;巨噬细胞特异性抗体F4/80进行免疫组织化学染色观察巨噬细胞的浸润情况;④Western blotting分别检测NF-κB、IκB-α和抗氧化物Sirt1、Mn SOD的蛋白表达情况;⑤RT-PCR检测附睾脂肪组织中Sirt1、Rel A、TNF-α、IL-1β和Mn SOD的m RNA的表达情况。结果1、小鼠称重及血清睾酮水平:HCD组小鼠的体重和附睾脂肪组织的重量比Ctrl组显著增高(p<0.05),HCD+Res组则较HCD组明显下降(p<0.05)。HCD组血清T的浓度较Ctrl组明显降低(p<0.05);白藜芦醇能够改善肥胖导致的T浓度的降低(p<0.05)。2、脂肪组织形态学:HE染色显示,HCD组脂肪细胞平均面积较Ctrl组明显增大(p<0.01),而HCD+Res组较HCD组明显减小(p<0.01)。3、白藜芦醇抑制附睾脂肪组织的炎症反应:我们用脂肪组织中的冠状结构(Crown-like structure,CLS)的密度来表示巨噬细胞的浸润情况,免疫组织化学染色显示,HCD组CLS的密度明显高于Ctrl组(p<0.01),而HCD+Res组中CLS的密度则明显低于HCD组(p<0.01)。我们检测白藜芦醇对NF-κB信号通路的作用。RT-PCR结果显示,Rel A的m RNA水平在HCD组较Ctrl组显著增高,HCD+Res组较HCD组显著降低(p<0.01);这种变化也表现在NF-κB及其抑制物IκB-α的蛋白水平上。Western blotting结果显示:NF-κB蛋白表达HCD组较Ctrl组显著增高,HCD+Res组则较HCD组明显降低(p<0.01);与Ctrl组比较,IκB-α的蛋白水平在HCD组明显下降,白藜芦醇的使用则升高了HCD组IκB-α的蛋白水平。和NF-κB变化一致的是,其下游靶标炎症因子TNF-α和IL-1βm RNA的表达在HCD组显著高于Ctrl组,HCD+Res组TNF-α和IL-1β的水平则显著低于HCD组(p<0.01)。4、白藜芦醇促进Sirt1以及Mn SOD表达:Sirt1、Mn SOD的表达在各组之间有显著差异:RT-PCR结果显示,HCD组Sirt1和Mn SOD的m RNA水平较Ctrl组显著降低(p<0.01),HCD+Res组则较HCD组显著升高(p<0.01)。Western blotting结果显示相似的变化,HCD组Sirt1和Mn SOD的蛋白表达较Ctrl组显著降低(p<0.01),HCD+Res组则较HCD组显著升高(p<0.05)。结论我们的结果表明白藜芦醇能够抑制肥胖相关的附睾脂肪组织中的炎症反应和氧化应激,提高睾酮水平,对肥胖相关的性腺功能减退具有潜在的治疗价值。更多还原

【Abstract】 Background In modern society, unhealthy lifestyles such as sedentary, excessive intake of energy can lead to an increasing incidence of obesity year by year. Obesity can induce a variety of pathological conditions, including hypogonadism. The present study suggests that chronic inflammation induced by obesity is an important mechanism of obesity-related diseases such as insulin resistance. Resveratrol have properties of anti-inflammatory and antioxidant stress, and its value in the treatment of obesity-related metabolic diseases is a hot spot of current research.Object To investigate the mechanism of resveratrol on obesity-related inflammatory and oxidative stress, and the protection on testicular endocrine function.Methods Thirty 6-week-old C57BL/6J male mice were randomly divided into four group: high-calorie and high-cholesterol diet group(59.5% maintaining materials, 0.5% cholesterol, 10% yolk powder, 20% cane sugar and 10% lard)(HCD, n=8), high-calorie and high-cholesterol diet with resveratrol supplementation(given by intragastric administration at a dose of 400mg/kg/day)(HCD+Res, n=8); standard-diet-fed control group(maintaining materials same to HCD group)(Ctrl1, n=7); standard-diet-fed with resveratrol supplementation(Ctrl2, n=7). Diet and water were provided ad libitum for approximately 18 weeks. Mice were sacrificed by the removal of the eye ball. Then ①Epididymal adipose tissue was harvested and weighed. ②measure the concentration of serum testosterone by radioimmunoassay. ③Epididymal adipose tissue was embedded in paraffin, on the one hand, hematoxylin and eosin staining was used for morphological measurement. on the other hand, immunohistochemistry with macrophages special antibody F4/80 to observe the infiltration of macrophages. ④Western blotting to detect the protein expression of NF-κB and IκB-α, and antioxidants Sirt1, Mn SOD. ⑤RT-PCR were used for detecting the m RNA expression of Sirt1、Rel A、TNF-α、IL-1β and Mn SOD in epididymal adipose tissue.Results 1、 weight of mice and the level of serum TBody weight exited significant increased when compared with Ctrl group(p<0.05); however, HCD+Res group showed significant decreased when compared with HCD group(p<0.01). The weight of epididymal adipose tissue was consistent with body weight: HCD group showed a significant increase when compared with Ctrl group(p<0.05), resveratrol supplementation decreased the weight in HCD group(p<0.05). The concentration of serum T showed significant decreases in HCD group when compared with Ctrl group(p<0.05); Resveratrol supplementation increased the concentration of serum T in HCD+Res group when compared with HCD group(p<0.05).2、Morphology of adipose tissue HE staining showed that mean adipocyte area in HCD group were larger than Ctrl group; resveratrol supplementation decreased the area fed by HCD(p<0.01).3、Resveratrol decreased obesity-induced infiltration We used CLS from adipose tissue to stand for the infiltration of macrophages. Dates showed that CLS was more numerous in HCD group than standard-diet-fed control group(p<0.01), however, its density was dramatically decreased in HCD+Res group when compared with HCD group(p<0.01).We used RT-PCR and western blotting to analysis the effect of resveratrol on NF-κB signaling pathway. HCD group showed significantly elevated m RNA level of Rel A(p<0.01), as compared to standard-diet-fed control group. However, after treated with additional resveratrol, the m RNA level of Rel A showed significantly down-regulated(p<0.05). Changes in m RNA level of Rel A can also be confirmed by the protein expression levels of NF-κB and IκB-α. In accordance with m RNA level of Rel A, the protein expression level of NF-κB was significantly increased in HCD group and dramatically decreased with additional resveratrol supplementation(p<0.01). However, The protein expression of IκB-α showed an opposite tendency with the protein level of NF-κB. It decreased significantly in HCD group when compared to standard-diet-fed control group(p<0.01), and increased significantly with resveratrol supplementation in HCD+Res group(p<0.01). Dates showed that the relative m RNA expressions of two classic inflammatory cytokines TNF-α and IL-1β were elevated in HCD group, however, obviously decreased were showed in HCD+Res group as compared to HCD group(p<0.01).4、Resveratrol increased the expression of Sirt1 and Mn SOD The m RNA level of Sirt1 and Mn SOD were significantly decreased in HCD group when compared with standard-diet-fed control group(p<0.01). However, this HCD-induced decrease were markedly improved by resveratrol supplementation(p<0.01). Western blot analysis demonstrated that the similarly reduced translation of Sirt1 and Mn SOD protein existed in HCD mice when compared to standard-diet-fed control group(p<0.01), and this decrease was significantly improved by resveratrol supplementation(p<0.05).Conclusion Resveratrol can increase the concentration of serum T, alleviate obesity-related inflammation and oxidative stress in HCD mice. The findings suggest that resveratrol may have a potent therapeutic value for obesity-related hypogonadism.更多还原