热休克蛋白47-shRNA调节肝星状细胞膜受体对日本血吸虫鼠肝纤维化的影响

Regulation of hepatic stellate cell membrane receptors by Hsp47-shRNA affects hepatic fibrosis in mice with Schistosoma japonicum infection

目的了解热休克蛋白47-短发夹RNA(HSP47-sh RNA)调节肝星状细胞(HSCs)膜受体对日本血吸虫鼠肝纤维化的影响。方法建立日本血吸虫鼠肝纤维化模型,感染前(健康对照组)和感染后第4、9、14周,分别随机取5只小鼠,提取肝脏组织总RNA,采用RT-PCR检测HSP47 mRNA相对表达水平,蛋白质印迹(Western blotting)检测HSP47蛋白表达情况,用RM-6240BD型多道生理信号系统动态检测模型鼠门静脉压力,流式细胞术检测HSCs膜内皮素受体(ETAR和ETBR)的平均荧光强度(MFI),免疫组织化学法检测感染前和感染后14周的模型鼠肝组织膜受体表达情况,采用线性相关分析方法分析各时间点的HSP47 mRNA相对表达水平与门静脉压力、ETAR和ETBR相对表达量的关系。用HSP47-sh RNA质粒转染小鼠成纤维细胞NIH/3T3细胞和血吸虫肝纤维化模型鼠,并设阴性对照组(空质粒)和空白对照组(PBS),每组12只血吸虫感染模型鼠。采用RT-PCR检测NIH/3T3细胞转染0、24、48 h及模型鼠干预至第14周的HSP47 mRNA、Ⅰ型和Ⅲ型胶原mRNA相对表达水平,Western blotting检测对应蛋白的表达情况,流式细胞术检测膜受体(ET、TGF-β及PDGF的受体)的MFI。两组间均数的比较采用Student-t检验,多组间采用单因素方差分析。结果模型鼠感染日本血吸虫后第4、9、14周,RT-PCR结果显示,HSP47 mRNA相对表达水平分别为0.592±1.031、1.253±2.101和0.651±1.532,较感染前(0.253±0.120)均升高(P<0.01);Western blotting结果显示,HSP47蛋白表达情况与mRNA变化一致;门静脉压力分别为(3.010±0.250)、(8.850±0.63)和(12.390±0.830)mm Hg,感染后第14周与感染前的(2.350±0.180)mm Hg比较,差异有统计学意义(P<0.01)。流式细胞术检测结果显示,ETAR的MFI分别为3 245±186、6 108±213和8 784±257,高于感染前的2 104±232(P<0.01);ETBR的MFI分别为3 812±158、4 524±197和5 554±156,高于感染前的2 091±237(P<0.01)。相关性分析结果显示,HSP47 mRNA相对表达水平与门静脉压力、ETAR和ETBR的MFI均呈正相关(r=0.750、0.750、0.508,P<0.05)。免疫组织化学结果显示,感染后第14周,ETB、TGF-β和PDGF的受体在肝纤维聚集部位呈强阳性表达。HSP47-sh RNA转染NIH/3T3细胞48 h后,RT-PCR结果显示,转染组HSP47 mRNA相对表达水平为0.254±2.358,低于阴性对照组的0.911±1.391(P<0.01);Western blotting结果显示,HSP47蛋白表达情况与mRNA变化一致;Ⅰ型和Ⅲ型前胶原mRNA相对表达水平分别为0.656±0.061、1.330±0.155,较阴性对照组的1.521±0.314、2.243±0.142均下调(P<0.01);流式细胞术检测结果显示,ETBR的MFI为53.433±5.243,高于转染前的30.825±5.460(P<0.05),TGF-β及PDGF受体转染前后差异无统计学意义(P>0.05)。转染组小鼠体内干预至第14周后,RT-PCR结果显示,HSP47 mRNA相对表达水平为2.686±0.711,低于阴性对照组的6.001±0.458(P<0.01);Western blotting结果显示,HSP47蛋白表达情况与mRNA变化一致;Ⅰ型、Ⅲ型前胶原mRNA相对表达水平分别为10.956±2.079和14.071±1.915,均较空白对照组的22.687±1.478和29.065±2.537下降(P<0.01);流式细胞术检测结果显示,转染组ETAR表达阳性率为(51.48±4.27)%,低于空白对照组的(81.60±6.07)%(P<0.05);转染组小鼠HSC膜受体ETAR、ETBR的MFI分别为4 249±344和3 706±194,均低于空白对照组的8 538±494和5 052±213(P<0.05),TGF-β和PDGF受体转染前后差异无统计学意义(P>0.05)。结论HSP47-sh RNA可干预活化的HSC和血吸虫肝纤维化鼠,ETR变化为影响肝纤维化尤其门静脉高压的因素。

Objective To investigate the effect of hepatic stellate cell（HSC） membrane receptor regulation by heat shock protein 47（HSP47）-sh RNA on the Schistosoma japonicum-induced liver fibrosis in mice. Methods The mouse model of Schistosoma japonicum-induced liver fibrosis was established. Total RNA was extracted from liver tissues before infection and at weeks 4, 9 and 14 after infection（ n = 5 mice in each group）. The mRNA expression and protein levels of HSP47 were assessed by RT-PCR and Western blotting, respectively. The RM-6240 BD multichannel physiological signals were used to measure the portal vein pressure of mice, flow cytometry was used to de-tect the mean fluorescence intensity（MFI） of endothelin receptor（ETAR and ETBR） in HSC membranes, and immunohistochemistry was used to detect the expression of membrane receptors in liver before and at week 14 after infection. The relationships between the expression of HSP47 and portal vein pressure as well as expression of ETAR and ETBR in HSC were analyzed using the linear correlation analysis method. The NIH/3 T3 cells and mouse model of Schistosoma japonicum-induced liver fibrosis（n = 12） were transfected with HSP47-sh RNA expression plasmid, accompanied by a negative control group（control plasmid） and a blank control group（PBS group）. RT-PCR was used to detect the mRNA expression of HSP47, type Ⅰ collagen and type Ⅲ collagen in NIH/3 T3 cells at 0, 24 and48 h after transfection and in the mouse model at week 14. Their protein levels were determined by Western blotting. MFI of the membrane receptors for ET, TGF-β and PDGF was detected by flow cytometry. Data were analyzed by student＇s t-test for comparisons between groups, and by ANOVA for comparisons among groups. Results RT-PCT results showed that the relative expression levels of HSP47 mRNA were 0.592 ± 1.031, 1.253 ± 2.101 and 0.651 ± 1.532,respectively, in the mouse model at weeks 4, 9 and 14 after infection, all significantly increased compared with that be-fore infection（0.253 ± 0.120）（P〈0.01）. Western blotting revealed consistent changes of HSP47 protein level. The portal vein pressures were（3.010 ± 0.250）,（8.850 ± 0.630）, and（12.390 ± 0.830） mm Hg, respectively, significantly higher than that before infection [（2.350 ± 0.18） mm Hg; P〈0.01]. Flow cytometry showed that the MFI levels of ETAR were 3 245 ±186, 6 108 ± 213 and 8 784 ± 257, all higher than that before infection（2 104 ± 232; P〈0.01）. Similarly, the MFI levels of ETBR were also significantly higher at weeks 4, 9 and 14 after infection（3 812 ± 158, 4 524 ± 197 and 5 554 ± 156）than that before infection（2 091 ± 237）（P〈0.01）. The relative expression of HSP47 mRNA was positively correlated with the portal vein pressure, relative MFI of ETAR, and relative MFI of ETBR（r = 0.750, 0.750 and 0.508, P〈0.05）. Immunohistochemical results showed that at week 14 after infection, receptors for ETBR, TGF-β and PDGF were strongly expressed in the hepatic sites of fiber aggregation. RT-PCT results showed that the relative expression of HSP47 mRNA in the transfected NIH/3 T3 cells 0.254 ± 2.358 was significantly lower than that in the negative control group（0.911 ±1.391）（P〈0.01） at 48 h after transfection, and consistent protein changes were revealed by Western blotting. The relative expression levels of type Ⅰ and type Ⅲ procollagen mRNA were 0.656 ± 0.061 and 1.330 ± 0.155, significantly downregulated compared with the negative control group（1.521 ± 0.314 and 2.243 ± 0.142, P〈0.01）. The relative MFI of ETBR measured by flow cytometry was 53.433 ± 5.243, significantly higher than that before transfection（30.825 ± 5.460, P〈0.05）. There was no significant difference in TGF-β and PDGF receptors between cells before and after transfection（P〉0.05）. RT-PCT results showed that the relative expression level of HSP47 mRNA was 2.686 ± 0.711, significantly lower than that of the negative control group（6.001 ± 0.458, P〈0.01） at week 14 after transfection. Western blotting showed consistent changes of the protein level. The mRNA expression of procollagentype Ⅰ and Ⅲ in the Hsp47-sh RNA transfected group（10.956 ± 2.079, 14.071 ± 1.915） were both decreased compared with those in the blank control group（22.687 ± 1.478, 29.065 ± 2.537）（P〈0.01）. Flow cytometry showed that the rate of ETAR positive expression in the transfected group [（51.48 ± 4.27）% ] was significantly lower than that in the blank control group [（81.60 ± 6.07）%,P〈0.05]. The MFI values of ETAR and ETBR on the membrane of HSCs in the Hsp47-sh RNA transfected group were4 249 ± 344 and 3 706 ± 194, respectively, both lower than that of the blank control group（8 538 ± 494, 5 052 ±213; P〈0.05）. There was no significant difference in TGF-β or PDGF receptor between the transfection group and blank control group（P〉0.05） as measured by flow cytometry. Conclusion HSP47-sh RNA has an effect on activated HSCs and in mice with Schistosoma japonicum-induced liver fibrosis. The change of ETR is one of the possible mechanisms of live fibrosis, especially portal hypertension.