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

Abstract

Abstract:

Objective To investigate the effect of hepatic stellate cell (HSC) membrane receptor regulation by heat shock protein 47 (HSP47)-shRNA 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-6240BD multi-channel physiological signals were used to measure the portal vein pressure of mice, flow cytometry was used to detect 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/3T3 cells and mouse model of Schistosoma japonicum-induced liver fibrosis (n = 12) were transfected with HSP47-shRNA 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/3T3 cells at 0, 24 and 48 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 before 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/3T3 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-shRNA 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-shRNA transfected group were 4 249 ± 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-shRNA 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.

Key words: Heat shock protein 47, Hepatic fibrosis, Schistosoma japonicum, Short hairpin RNA interference, Endothelin receptors

CLC Number:

R383.24

Ju-hong DENG, Ran TAO, Qi-qin SONG, Hong-yan KONG, Yun-tao JIAO, Jia-quan HUANG. Regulation of hepatic stellate cell membrane receptors by Hsp47-shRNA affects hepatic fibrosis in mice with Schistosoma japonicum infection[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2018, 36(4): 317-324.

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