内质网应激介导高脂及棕榈酸致骨骼肌胰岛素抵抗及非诺贝特的干预机制初探

发布时间：2018-06-14 06:06

本文选题：骨骼肌 + 胰岛素抵抗　；参考：《安徽医科大学》2015年硕士论文

【摘要】：目的:观察高脂饮食(high-fat diet,HFD)对雌性SD大鼠骨骼肌的影响,探讨非诺贝特(fenofibrate,FF)对高脂及棕榈酸导致的骨骼肌胰岛素抵抗的保护作用是否与内质网应激(endoplasmic reticulum stress,ERS)有关。方法:雌性SD大鼠随机分为三组:标准饮食组(standard control diet,SCD)、高脂饮食组(HFD)及治疗组(HFD+FF)。高脂组和治疗组大鼠先予以高脂饮食20wk后,高脂组大鼠继续予以高脂饮食8wk,治疗组大鼠予以非诺贝特(30mg·kg-1·d-1)灌胃治疗8wk。每周监测大鼠体重,第28周检测血清生化指标TC、TG、LDL-C、HDL-C,葡萄糖耐量试验(GTT)和胰岛素耐量试验(ITT)测定胰岛素抵抗(IR),Real-time RT-PCR测定骨骼肌组织葡萄糖调节蛋白78(GRP78)、转录因子GADD153(CHOP)、肌醇酶1α(IRE1α)、真核起始因2的α亚基(e IF2α)和过氧化物酶体增殖物激活受体α(PPARα)基因表达,Western-blot法检测骨骼肌组织GRP78蛋白的表达水平。将骨骼肌细胞C2C12分组:正常对照组(normal control group,NC)、模型组(棕榈酸组,palmitic acid,PA)、阳性对照药组(衣霉素组,tunicamycin,TM)、治疗组(棕榈酸+非诺贝特酸,PA+FA),RT-PCR测定GRP78、CHOP、IRE1α和e IF2αm RNA的表达水平,Western-blot检测GRP78、Akt和p-Akt蛋白表达水平,免疫荧光检测CHOP蛋白表达水平。结果:(1)GTT实验提示HFD组大鼠给予葡萄糖30 min后血糖值达高峰,且较SCD组、HFD+FF组血糖峰值升高,此后缓慢下降;与HFD组大鼠相比,HFD+FF组大鼠空腹血糖降低,给予葡萄糖后30 min时峰值降低,血糖波动较小。ITT实验显示大鼠注射胰岛素后,SCD组血糖迅速下降,30 min时达最低值后血糖缓慢上升;HFD组血糖缓慢下降,且始终维持在较高水平,90 min时达最低值后血糖缓慢上升;HFD+FF组大鼠各点血糖值介于HCD和SCD组之间,注射胰岛素后血糖下降至60 min时达最低值,随后缓慢上升。(2)与SCD组大鼠相比,HFD组大鼠体重显著增加(P0.05),血清TG、TC水平明显升高,HDL-C水平显著下降(P0.05);与HFD组大鼠相比,非诺贝特干预后能够显著减少大鼠体重、改善肥胖,降低血清TC、TG水平,增加血清HDL-C水平(P0.05)。(3)与SCD组大鼠相比,HFD组大鼠GRP78、CHOP、IRE1α和e IF2α基因表达水平均明显上调,与HFD组大鼠相比,非诺贝特治疗后PPARα表达水平明显增加,GRP78、CHOP、IRE1α和e IF2α基因表达水平下调;(4)与NC组相比,PA组骨骼肌细胞GRP78、CHOP、IRE1α和e IF2α基因表达水平显著增加,p-Akt蛋白表达水平明显下调;与PA组相比,PA+FA组骨骼肌细胞GRP78、CHOP、IRE1α和e IF2α基因表达显著降低,p-Akt蛋白表达水平明显上调。结论:高脂饮食成功构建雌性高脂血症伴IR大鼠模型,非诺贝特干预可显著控制大鼠体重,调节血脂紊乱,改善胰岛素抵抗,其作用机制可能与显著抑制ERS标志物CHOP、GRP78、IRE1α和e IF2α的表达水平有关。
[Abstract]:Aim: to observe the effect of high-fat HFDs on skeletal muscle of female SD rats and to investigate whether the protective effect of fenofibrate FFF on insulin resistance induced by hyperlipidemia and palmitic acid is related to endoplasmic reticulum stress (endoplasmic reticulum stress). Methods: female SD rats were randomly divided into three groups: standard diet group, high fat diet group and treatment group. Rats in hyperlipidemia group and treatment group were treated with high-fat diet (20wk). Rats in high-fat group were given high-fat diet for 8 wk, and rats in treatment group were treated with fenofibrate 30mg kg-1 d-1 (gavage) for 8 wk. The body weight of rats was monitored weekly. Determination of Serum biochemical Indexes TCX TGN LDL-Con HDL-C (glucose tolerance Test GTT) and Insulin tolerance Test (ITT) in skeletal muscle tissue by Real-time RT-PCR for determination of Glucose-regulated protein 78GRP78, transcription Factor GADD153, Inositase 1 伪 -IRE1 伪, Eukaryotic initiation Factor 2 伪 Subunit e IF2 伪) and peroxisome proliferator activated receptor 伪 PPAR 伪) were used to detect the expression of GRP78 protein in skeletal muscle by Western-blot. C2C12 skeletal muscle cells were divided into normal control group (normal control group), model group (palmitic acidopyridine), positive control group (tunicamycin TMN), treatment group (fenofibrate palmitate PA FAFA), RT-PCR for the detection of GRP78 CHOPIRE1 伪 and e IF2 伪 mRNA expression levels by Western-blot. The expression level of GRP78, Akt and p-Akt protein, The expression of chop protein was detected by immunofluorescence. Results the blood glucose level of HFD group reached the peak after 30 min glucose administration, which was higher than that of SCD group, and then decreased slowly, and the fasting blood glucose in HFD FF group was lower than that in HFD group. The peak value of glucose decreased at 30 min after glucose administration, and the blood glucose fluctuated slightly. ITT test showed that the blood glucose of the SCD group decreased rapidly after insulin injection and reached the lowest level at 30 min. The blood glucose of the HFD group slowly increased after 30 min, and the blood glucose of the HFD group decreased slowly. The blood glucose level of HFDFF group was between HCD group and SCD group, and the blood glucose level decreased to 60 min after insulin injection. Compared with the rats of SCD group, the weight of HFD group increased significantly (P 0.05), serum TGN TC level increased significantly and HDL-C level decreased significantly compared with HFD group, fenofibrate could significantly reduce body weight and improve obesity. Compared with SCD group, the expression of GRP78 CHOPIRE1 伪 and e IF2 伪 gene in HFD group was significantly up-regulated, compared with that in HFD group, and that in HFD group was significantly higher than that in HFD group, and that in HFD group was significantly higher than that in HFD group, and the expression of IRE1 伪 and e IF2 伪 in HFD group was significantly higher than that in HFD group. After fenofibrate treatment, the expression of PPAR 伪 increased significantly (P < 0.05). The expression levels of GRP78-CHOPOP-IRE1 伪 and eIF2 伪 decreased significantly (P < 0.01). Compared with NC group, the expression levels of GRP78-CHOPON-IRE1 伪 and eIF2 伪 in skeletal muscle cells of PA group increased significantly, and the expression levels of p-Akt protein decreased significantly. Compared with PA group, the expression of GRP78 CHOPIRE1 伪 and eIF2 伪 gene in skeletal muscle cells in PA FA group was significantly lower than that in PA group. Conclusion: female hyperlipidemia with IR rat model was successfully constructed by high-fat diet. Fenofibrate intervention can significantly control the weight of rats, regulate the disorder of blood lipids, and improve insulin resistance. The mechanism may be related to the significant inhibition of the expression levels of ERS markers CHOP-GRP78, IRE1 伪 and eIF2 伪.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589

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