何首乌肝损伤模型大鼠胆汁淤积现象及相关蛋白表达研究

Study on cholestasis and related protein expression in rats with liver injury induced by Polygonum multiflorum

目的观察经脂多糖(LPS)诱导后连续给予何首乌醇提液(AEP)7 d大鼠的肝损伤程度及胆汁淤积相关指标的变化。方法将雄性SD大鼠随机分为5组:对照组、LPS组、LPS+对乙酰氨基酚(APAP)组、AEP组、LPS+AEP组,LPS、LPS+APAP、LPS+AEP组按大鼠体质量分别尾iv给予4 mg/kg LPS,对照组与AEP组给予等体积生理盐水;2 h后LPS+APAP组ig给予625 mg/kg APAP,AEP组与LPS+AEP组ig给予12 g/kg,每天1次,连续给药7 d,建立特异质肝损伤炎症模型。观察体质量变化,并分别于造模后2 h、14 h、5 d、8 d检测血清生化中肝功能相关指标的变化,同时收集胆汁,计算胆汁流速与密度,并检测胆汁中主要成分变化。进行肝脏系数及组织病理学检查,并对胆汁淤积相关蛋白胆盐输出泵转运蛋白(BSEP)、多药耐药蛋白2(MRP2)及多药耐药蛋白3(MRP3)进行Real-Time PCR检测。结果经过LPS诱导2 d后,LPS+AEP组与对照组、AEP组比较体质量明显下降,5、8 d肝脏系数显著增加(P<0.05);血清生化指标分析结果显示,与对照组比较,LPS+AEP组丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)水平无明显变化;造模后8 d的总胆红素(TBIL)明显降低(P<0.05),ALP明显升高(P<0.05);可观察到胆汁密度和胆汁流速明显降低(P<0.05),对胆汁成分分析显示,与对照组比较,LPS+AEP组总胆固醇(TCHO)显著升高、TBIL显著降低(P<0.05);病理结果显示,AEP组出现轻微肝细胞变性,而LPS+AEP组可见严重局灶坏死;转录水平结果发现,LPS诱导后可使得BSEP、MRP2在14 h时出现短期抑制(P<0.05),单独给予AEP可使BSEP、MRP2和MRP3的表达水平短时显著升高(P<0.05、0.01),而LPS+AEP给药第8 d对大鼠肝脏BSEP、MRP2无显著性影响,可使MRP3转录水平升高(P<0.05)。结论经LPS诱导的AEP可明显损伤大鼠肝细胞并干扰胆汁分泌功能,引起胆汁成分相关生化指标的改变,对BSEP与MRP2水平无显著影响,MRP3出现代偿性升高,提示确实存在胆汁淤积症状,但可能是存在其他机制。

Objective To observe the rats with liver damage induced by lipopolysaccharide （LPS） after administration of alcohol extract of Polygonum multiflorum （AEP） for 7 d,and study the change of related indicators of cholestasis.Methods Male SD rats were randomly divided into five groups：control group,LPS group,LPS ＋ acetaminophen （APAP） group,AEP group,and LPS＋AEP group.A dosage of 4 mg/kg LPS was injected to the caudal vein of rats in the LPS and the LPS＋AEP groups.Two hours later,rats wereig administered with AEP （12 g/kg,equivalent to 22 times the dose of clinical medication） respectively for 7 d,once daily.Since the liver-injury model of inflammation was successfully established,the general conditions of rats such as body weight and clinical observation were observed.Liver function-related indicators were detected and bile was collected to determine flow rate,density and changes of major compositions 2 h,14 h,5 d,and 8 d after modeling.After getting the organ to body weight ratio for each rat,liver samples were treated for histopathological observation and the mRNA expression of hepatic BSEP,MRP2,and MRP3 was detected by real-time PCR.Results Compared with the rats in control group and AEP group,rats treated with LPS＋AEP performed weight losing 2 d after modeling,and organ to body weight ratio was significantly increased on day 5 and 8.The results of liver function-related indicators showed that LPS＋AEP did not change the contents of ALT and AST in serum,but increased the content of ALP （P 〈 0.05） and decreased the content of TBIL in serum （P 〈 0.05）.The bile flow and density were decreased by LPS＋AEP,while bile composition was obviously changed,the content of TCHO was increased and the content of TBIL was decreased （P 〈0.05）.Histopathological examination showed that a small amount of hepatocyte degeneration in AEP group was observed,but livers were damaged and grown necrosis partially in the LPS＋AEP group.After 14 h,mRNA expression of BSEP and MRP2 in LPS group showed transitory inhibition,but AEP group had a significant up-regulation of mRNA expression of B SEP,MRP2,and MRP3 （P 〈 0.05）.The following two time points,compared with the control group,mRNA expression of BSEP and MRP2 appeared no obvious change in LPS＋AEP group,while mRNA expression of MRP3 was significantly increased on day 8 （P 〈0.05）.Conclusion AEP induced by LPS performs obvious hepatocyte damage,showed a certain effect on expression of bile acid synthesis and changed bile compositions and liver function-related indicators.The mRNA expression of BSEP and MRP2 was not changed significantly,while mRNA expression of MRP3 is compensatory increased,suggesting that cholestatic symptoms exist,but there may be other mechanisms.