间歇性冷刺激对高脂饮食小鼠的血清脂质相关指标、肝脏组织形态及肝脏脂质合成相关基因和蛋白表达的影响

Effects of Intermittent Cold Stimulation on Serum Lipid⁃Related Indexes,Liver Tissue Morphology and Expression of Genes and Proteins Related to Liver Lipid Synthesis in Mice Fed High Fat Diets

为了探究间歇性冷刺激对高脂饮食小鼠血清脂质相关指标、肝脏组织形态以及肝脏脂质合成的影响,试验选择5周龄的雄性C57BL/6小鼠48只,随机分为常温对照组、常温高脂组、冷刺激组和冷刺激高脂组,每组3个重复,每个重复4只。常温组小鼠饲养温度为(26±2)℃,冷刺激组小鼠饲养温度为4℃,试验期30 d(每天6 h)。处理结束后采集各组小鼠的肝脏组织和血清样本,通过苏木素-伊红(HE)染色、蛋白质免疫印迹(Western blot)、实时荧光定量PCR(qRT-PCR)等方法,对小鼠肝脏形态结构和脂质合成相关指标进行检测。结果显示:1)饲喂高脂饲粮的小鼠在14周时,体重极显著高于常温对照组(P<0.01)。2)HE结果表明,常温对照组小鼠肝脏肝小叶结构清晰,肝索排列规则有序,未见水肿变性和坏死,未见炎细胞;冷刺激组小鼠的肝脏组织出现颗粒变性,炎性细胞浸润,并观察到充血和出血现象;常温高脂组小鼠肝脏可见大小不一的圆形空泡,发生大泡性脂肪变;冷刺激高脂组小鼠肝脏肝小叶排列紊乱,肝细胞水肿可见空泡变性,炎性细胞浸润。3)小鼠高脂造模后,常温高脂组小鼠血清中血脂相关指标甘油三酯(TG)、游离脂肪酸(NEFA)、总胆固醇(T-CHO)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的含量均极显著升高(P<0.01)。4)冷刺激组小鼠血清中TG、NEFA、T-CHO、HDL-C、LDL-C的含量极显著低于对照组(P<0.01)。5)常温高脂组小鼠在冷刺激后血清中TG、NEFA、T-CHO、HDL-C、LDL-C的含量极显著低于高脂组(P<0.01)。6)小鼠高脂造模后,常温高脂组小鼠肝脏脂质合成相关[脂肪酸合酶(FASN)、乙酰辅酶A羧化酶1(ACC1)、固醇元件结合蛋白1c(SREBP1c)、ATP-柠檬酸裂解酶(ACLY)]基因和蛋白表达水平极显著升高(P<0.01);小鼠间歇性冷刺激后,冷刺激组小鼠肝脏ACC1蛋白表达水平极显著升高(P<0.01),FASN蛋白表达水平极显著降低(P<0.01);与冷刺激组相比,冷刺激高脂组小鼠肝脏SREBP1c、ACC1、FASN和ACLY蛋白表达水平显著降低(P<0.05)。综上所述,间歇性冷刺激可使高脂饮食小鼠肝脏脂肪组织变得致密并对其血脂异常现象具有一定的缓解作用,并抑制肝脏的脂质合成。

To investigate the effects of intermittent cold stimulation on liver tissue morphology,serum lipid in-dexes and liver lipid synthesis in obese mice,48 male C57BL/6 mice at 5 weeks of age were randomly divided into room temperature control group,room temperature high fat group,cold stimulation group,and cold stimu-lation high fat group.There were three replicates in each group,and there were four mice in each replicate.The feeding temperature of mice in the room temperature group was(26±2)℃,and the feeding temperature of the mice in the cold stimulation group was 4℃for 30 days(6 h per day).After the end of treatment,liver tissue and serum samples of each group of mice were collected,and the liver morphological structure and lipid syn-thesis related indexes of mice were detected by hematoxylin-eosin(HE)staining,Western blot,and real-time PCR(qRT-PCR).The results showed as follows:1)the weight of mice fed high fat diet was significantly higher than that in the control group at 14 weeks(P<0.01).2)The results of HE showed that the liver lobules in the room temperature control group had a clear structure,regular and orderly arrangement of liver cords,no edema degeneration and necrosis,and no inflammatory cells;the liver tissue of mice in the cold stimulation group showed granular degeneration,inflammatory cell infiltration,and hyperemia and bleeding were ob-served;round vacuoles of different sizes and bullous steatosis occurred in the livers of mice in the room temper-ature high fat group;cold stimulation high fat group mice with disordered hepatic lobular arrangement,hepato-cyte edema showed vacuolar degeneration,inflammatory cell infiltration.3)After high-fat modeling,the ser-um lipid-related indexes of triglyceride(TG),non-esterified fatty acid(NEFA),total cholesterol(T-CHO),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C)contents was sig-nificantly increased in high fat group(P<0.01).4)The contents of TG,NEFA,T-CHO,HDL-C and LDL-C in serum of mice in the cold stimulation group were significantly lower than those in the room temperature control group(P<0.01).5)The contents of TG,NEFA,T-CHO,HDL-C,LDL-C and LDL-C in serum of mice in the high fat group after cold stimulation were significantly lower than those in the room temperature high fat group(P<0.01).6)After high-fat modeling,liver lipid synthesis genes and proteins[fatty acid syn-thase(FASN),acety-CoA carboxylase(ACC1),sterol regulatory element binding protein1c(SREBP1c),ATP-citrate lyase(ACLY)]expression levels were significantly elevated in high fat group(P<0.01);after in-termittent cold stimulation,the expression level of ACC1 protein in the liver of mice in the cold stimulation group was significantly increased(P<0.01),and the expression level of FASN protein was significantly re-duced(P<0.01);compared with the cold stimulation group,the expression levels of SREBP1c,ACC1,FASN and ACLY proteins in the liver of cold stimulation high-lipid mice were significantly reduced(P<0.05).In summary,intermittent cold stimulation can densify the liver adipose tissue of high fat diet mice and have a certain alleviating effect on their dyslipidemia,and inhibit lipid synthesis in the liver.