PDZK1对成石喂养小鼠肝脏SRB1调节作用的研究

Regulation of hepatic SRB1 expression by PDZK1 in mice fed with lithogenic diet

目的 :研究成石饲料对小鼠肝脏脂质代谢相关基因表达的影响,以及PDZK1(PDZ domain-containing 1)的表达对B族Ⅰ型清道夫受体(scavenger receptor B type 1,SRB1)的调节作用。方法:以成石饲料喂养C57BL/6J雄性小鼠,观察肝脏基因表达的影响。分别构建小鼠PDZK1基因过表达慢病毒载体和Si RNA干扰腺病毒载体,并以成石饲料喂养C57BL/6J小鼠。分别采用实时定量PCR检测小鼠肝脏脂质代谢相关基因的m RNA表达,Western印迹法检测肝脏PDZK1及SRB1的蛋白质表达。结果:成石饲料喂养4周后,小鼠肝脏胆固醇合成关键酶3-羟基-3甲基戊二酰辅酶A还原酶基因表达显著增加(P<0.01)、胆汁酸合成关键酶胆固醇7α-羟化酶基因表达显著下降(P<0.001);PDZK1基因的表达显著抑制,SRB1基因表达相应降低(P<0.001)。PDZK1过表达慢病毒载体经腹腔注射成石饲料喂养的小鼠,4周后肝脏PDZK1蛋白表达增加37%(P<0.01),SRB1在总蛋白的表达量未改变,但在膜蛋白中表达增加136%(P<0.05)。将PDZK1的Si RNA腺病毒载体经腹腔注射成石饲料喂养的小鼠,2周后肝脏PDZK1蛋白的表达量降低49%(P<0.05),SRB1在总蛋白中表达增加60%(P<0.05),但在膜蛋白中的表达则下降14%。结论:成石饲料影响小鼠肝脏脂质代谢相关基因的表达。SRB1表达降低可能与PDZK1的抑制有关。干预小鼠肝脏PDZK1的表达会影响SRB1在肝细胞膜上的表达。

Objective To investigate the expression of genes involving in hepatic lipid metabolism in mice fed with lithogenic diet and the regulation of hepatic scavenger receptor B type 1 （SRB1） by PDZ domain-containing 1 （PDZK1） protein. Methods Male C57BL/6J mice were fed with lithogenic diet and the differences in the expression of hepatic genes were studied. PDZKl-overexpressing lentivirus vector and small interfering RNA adenovirus vector were constructed and were used to infect C57BL/6J mice fed with lithogenic diet. mRNA expression of the genes involved in lipid metabolism in liver was quantified by real-time PCR. The protein levels of PDZK1 and SRB1 were determined by Western blot. Results The mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key enzyme in cholesterol synthesis, in- creased （P〈0.01） in the mice fed with lithogenic diet for 4 weeks. However the expression of cholesterol 7-α hydroxylase, the rate-limiting enzyme of bile acid synthesis, decreased significantly （P〈0.001）. Both the mRNA expression of SRB1 and PDZK1 decreased under lithogenic diet （P〈0.001）. Intraperitoneal injection to mice with PDZKl-overexpressing lentivirus increased the hepatic PDZK1 protein 37% （P〈0.01） after 4 weeks fed with lithogenic diet. The increased expression of SRB1 protein was found in the liver plasma membrane （＋136%, P〈0.05） but not in the whole liver lysates. On contrary, the intraperitoneal injection to the mice with SiPDZK1 adnovirus after feeding with lithogenic diet 2 weeks reduced PDZK1 protein 49% （P〈0.05）. In accompany, the total SRB1 protein of liver increased 60% （P〈0.05） and SRB1 protein in hepatic membrane decreased 14%. Conclusions Lithogenic diet changed the expression of hepatic genes in lipids metabolism in mice. The down-regulation of SRB1 might be due to the suppression of PDZK1 expression. Intervention of hepatic PDZK1 expression could affect the expression of SRB1 protein in the membrane of hepatocytes.