Structural changes and expression of hepatic fibrosis-related proteins in coculture of Echinococcus multilocularis protoscoleces and human hepatic stellate cells

Abstract

Background: Echinococcus multilocularis is the causative agent of human hepatic alveolar echinococcosis (AE). AE can cause damage to several organs, primarily the liver, and have severe outcomes, such as hepatic failure and encephalopathy. The main purpose of this study was to explore the interactions between hepatic stellate cells (HSCs) and E. multilocularis protoscoleces (PSCs). The results of this study provide an experimental basis for further examination of the pathogenesis of hepatic fibrosis due to AE infection.

Methods: We investigated the role of Echinococcus multilocularis (Echinococcus genus) PSCs in hepatic fibrosis by examining structural changes and measuring hepatic fibrosis-related protein levels in cocultures of PSCs and human HSCs. Structural changes were detected by transmission electron microscopy (TEM), and levels of the hepatic fibrosis-related proteins collagen I (Col-I), alpha-smooth muscle actin (α-SMA) and osteopontin (OPN) were measured by western blotting and enzyme-linked immunosorbent assay (ELISA).

Results: Under coculture (1) both PSCs and HSCs exhibited morphological changes, as observed by TEM; (2) Col-I, α-SMA, and OPN expression levels, which were determined by western blotting and ELISA, significantly increased after 3 days of incubation.

Conclusions: The results of this study provide insights into the molecular mechanisms of AE-induced hepatic fibrosis.

Keywords: Alpha-smooth muscle actin; Collagen-I; Echinococcus multilocularis; Hepatic fibrosis; Hepatic stellate cell; Osteopontin; Protoscoleces.

Fig. 1

a–f Transmission electron microscopy (TEM, a–c: 30 000×; d–f: 10 000×) images of the internal structure of Echinococcus multilocularis protoscoleces (PSCs). a Normal cell structure of E. multilocularis PSC on co-culture 0 day. b Cell structure of E. multilocularis PSC on co-culture the day 4. vacuoles appearing in the cytoplasm. c Cell structure of E. multilocularis PSC on co-culture the day 4. the perinuclear space widening and mitochondria swelling and vacuoles appearing in the cytoplasm. d the microcilia on wall of E. multilocularis PSC were clearly visible on the 0 day (arrow). The microcilia on wall of E. multilocularis PSC become shorter and cell structure disappearing on the day 3 (e; arrow). The microcilia on wall of E. multilocularis PSC are disappeared and cell structure dissolved on the day 4 (f; arrow)

Fig. 2

Structural characteristics of HSCs (TEM, 10 000 ×) (HSCs exposed to PSCs). a Lipid droplets are commonly seen in the cytoplasm in HSC on the 0 day (arrow). b Microvilli on the HSC clearly visible on the day 3(arrow). c Lipid droplets decreasing in cytoplasm; mitochondrial election density increasing and lysosomes increasing in cytoplasm in HSC (arrow). d The nuclei are malformed and the mitochondria are swelling on the day 3 (arrow). e The nuclei are irregular and microvilli on the cell membrane are increasing on the 4th day

Fig. 3

Structural characteristics of HSC (TEM, 30 000×) (HSCs exposed to PSCs). a The oval lipid droplets clearly visible in HSCs on 0 day. (arrow). b, d The mitochondrial cristae space widening and the microfilaments increasing in cytoplasm on the day 3 (arrow). c The lysosomes increasing and mitochondria swelling occurred in HSCs (arrow) on the day 3. e microvilli on the HSC cell membrane (arrow)

Fig. 4

a–f Collagen I (Col-I) expression profile measured by western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). a Expression of Col-I assessed by WB. b At a PSC:HSC-LX2 ratio of 1:200, Col-I expression had significantly increased by days 2 and 3 but not by day 1. c Col-I expression at a PSC:HSC-LX2 ratio of 2:200 was significantly higher on days 1, 2 and 3 compared to the control. d At a PSC:HSC-LX2 ratio of 3:200, Col-I expression had increased by days 1 and 2, but decreased by day 3. e Col-I expression increased in HSCs cocultured with PSCs for 48 h. f By day 3, Col-I expression had increased at a PSC:HSC-LX2 ratio of 1:200 but had started to decrease at PSC:HSC-LX2 ratios of 2:200 and 3:200. For other abbreviations, see Fig. 1

Fig. 5

a–g Alpha-smooth muscle actin (α-SMA) expression profile measured by WB and ELISA. a α-SMA expression assessed by WB. b α-SMA expression was significantly higher on days 2 and 3 days, but was not on day 1 for the PSC:HSC-LX2 ratio of 1:200. c α-SMA expression was significantly higher on days 1, 2 and 3 for the PSC:HSC-LX2 ratio of 2:200. d For the PSC:HSC-LX2 ratio of 3:200, α-SMA expression had increased by days 1 and 2 but decreased by day 3. e The expression of α-SMA in HSCs cocultured with PSCs for 24 h increased. f By day 2, α-SMA expression had increased at all ratios of PSC:HSC-LX2 (1:200, 2:200 and 3:200). g By day 3, α-SMA expression had increased at a PSC:HSC-LX2 ratio of 1:200 but had started to decrease at ratios of 2:200 and 3:200. For other abbreviations, see Fig. 1

Fig. 6

a–d Osteopontin (OPN) expression profile measured by WB and ELISA. a Expression of OPN assessed by WB. b OPN expression was significantly higher on days 2, 3 and 4, but not on day 1 day for the PSC:HSC-LX2 ratio of 1:200. c OPN expression was significantly higher on days 1, 2 and 3 at a PSC:HSC-LX2 ratio of 2:200. d At a PSC:HSC-LX2 ratio of 3:200, OPN expression had increased by days 1 and 2, but decreased by days 3 and 4. For other abbreviations, see Fig. 1