Dynamic observation on capillarization of liver sinusoidal endothelial cells induced by Echinococcus multilocularis infection
Objective To investigate the capillarization of liver sinusoidal endothelial cells(LSECs)and its association with hepatic fibrosis during the development of alveolar echinococcosis,so as to provide the basis for unraveling the mechanisms un-derlying the role of LSEC in the development and prognosis of hepatic injuries and hepatic fibrosis caused by alveolar echinococ-cosis.Methods Forty C57BL/6 mice at ages of 6 to 8 weeks were randomly divided into a control group and 1-,2-and 4-week infection groups,of 10 mice in each group.Each mouse in the infection groups was intraperitoneally injected with 2 000 Echino-coccus multilocularis protoscoleces,while each mouse in the control group was given an equal volume of phosphate-buffered sa-line using the same method.All mice were sacrificed 1,2 and 4 weeks post-infection and mouse livers were collected.The patho-logical changes of livers were observed using hematoxylin-eosin(HE)staining,and hepatic fibrosis was evaluated through semi-quantitative analysis of Masson's trichrome staining-positive areas.The activation of hepatic stellate cells(HSCs)and extracellu-lar matrix(ECM)deposition were examined using immunohistochemical staining of α-smooth muscle actin(α-SMA)and collagen type Ⅰ alpha 1(COL1A1),and the fenestrations on the surface of LSECs were observed using scanning electron microscopy.Pri-mary LSECs were isolated from mouse livers,and the mRNA expression of LSEC marker genes Stabilin-1,Stabilin-2,Ehd3,CD209b,GATA4 and Maf was quantified using real-time fluorescence quantitative PCR(qPCR)assay.Results Destruction of local liver lobular structure was observed in mice 2 weeks post-infection with E.multilocularis protoscoleces,and hydatid cysts,which were surrounded by granulomatous tissues,were found in mouse livers 4 weeks post-infection.Semi-quantitative analysis of Masson's trichrome staining showed a significant difference in the proportion of collagen fiber contents in mouse livers among the four groups(F = 26.060,P<0.001),and a higher proportion of collagen fiber contents was detected in mouse livers in the 4-week infection group[(11.29±2.58)%]than in the control group(P<0.001).Immunohistochemical staining revealed acti-vation of a few HSCs and ECM deposition in mouse livers 1 and 2 weeks post-infection,and abundant brown-yellow stained α-SMA and COL1A1 were deposited in the lesion areas in mouse livers 4 weeks post-infection,which spread to surrounding tis-sues.Semi-quantitative analysis revealed significant differences in α-SMA(F = 7.667,P<0.05)and COL1A1 expression(F = 6.530,P<0.05)in mouse levers among the four groups,with higher α-SMA[(7.13±3.68)%]and COL1A1 expression[(13.18±7.20)%]quantified in mouse livers in the 4-week infection group than in the control group(both P values<0.05).Scanning elec-tron microscopy revealed significant differences in the fenestration frequency(F = 37.730,P<0.001)and porosity(F = 16.010,P<0.001)on the surface of mouse LSECs among the four groups,and reduced fenestration frequency and porosity were observed in the 1-[(1.22±0.48)/μm2 and[(3.05±0.91)%]and 2-week infection groups[(3.47±0.10)/μm2 and(7.57±0.23)%]groups than in the control group(all P values<0.001).There was a significant difference in the average fenestration diameter on the sur-face of mouse LSECs among the four groups(F = 15.330,P<0.001),and larger average fenestration diameters were measured in the 1-[(180.80±16.42)nm]and 2-week infection groups[(161.70±3.85)nm]than in the control group(both P values<0.05).In addition,there were significant differences among the four groups in terms of Stabilin-1(F = 153.100,P<0.001),Stabilin-2(F = 57.010,P<0.001),Ehd3(F = 31.700,P<0.001),CD209b(F = 177.400,P<0.001),GATA4(F = 17.740,P<0.001),and Maf mRNA expression(F = 72.710,P<0.001),and reduced mRNA expression of Stabilin-1,Stabilin-2,Ehd3,CD209b,GATA4 and Maf genes was quantified in three infection groups than in the control group(all P values<0.001).Conclusions E.multi-locularis infections may induce capillarization of LSECs in mice,and result in a reduction in the expression of functional and pheno-typic marker genes of LSECs,and capillarization of LSECs occurs earlier than activation of HSC and development of hepatic fibrosis.
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