HSC-LX2 promoted the growth of PSCs
PSCs and HSC-LX2 were cocultured in DMEM for 3 days to examine their interaction. The PSCs became highly motile, developed rapidly and evaginated in the presence of HSC-LX2. TEM was used to observe the structural changes on day 3 in the PSCs cocultured with HSC-LX2. The micrographs showed a slight change in the walls of the PSCs cocultured with HSC-LX2, as the walls of the PSCs cocultured with HSC-LX2, as the microcilia on the walls of E. multilocularis PSCs become shorter and cell structure disappearing and dissolved on day 3 compared with those of the PSC control (Fig. 1a, d, e, f). Vacuoles appeared and the perinuclear space widening and mitochondria swelling in the cellss of PSCs occurred during co-culture on day 4 (Fig. 1b, c).
Structural changes of HSC-LX2 in coculture with PSCs
HSC-LX2 were activated at PSC:HSC-LX2 ratios of 1:200 and 2:200 for 3 days, but were inhibited at a PSC:HSC-LX2 ratio of 3:200. On day 3 of the coculture, structural changes were analysed by TEM. The lipid droplets that were detected by TEM in control HSC-LX2 on day 0 were present until day 3 (Fig. 2a; Fig. 3a). Lipid droplets were not observed in all the cells, but where they were observed in HSC-LX2 exposed to PSCs they had degenerated by day 3. This indicated that fibrogenesis had occurred (Fig. 2d; Fig. 3b). Furthermore, the lysosomes increasing and mitochondria swelling occurred in HSCs on the day 3 than normal HSC (Fig. 3c). Some changes in the cytoplasm were observed, such as the mitochondrial cristae space widening and the microfilaments increasing in HSC cytoplasm (Fig. 3d, e). The nuclei of HSCs cocultured wth PSCs also exhibited structural changes in comparison with those of normal control HSCs, as they are malformed or irregular, and microvilli on the cell membrane are increasing and the mitochondria are swelling on day 3 and 4 (Fig. 2d, e).
Col-I expression in HSC-LX2 exposed to PSCs
Col-I plays a significant role in liver fibrosis. Therefore, the expression of Col-I was examined in HSC-LX2 exposed to PSCs. The expression of Col-I at different ratios of PSC:HSC-LX2 (1:200, 2:200, and 3:200) was measured on days 1, 2 and 3 by western blotting (WB) (Fig. 4a). The Col-I level in the coculture supernatant was measured by ELISA. At a PSC:HSC-LX2 ratio of 1:200, the expression of Col-I had risen dramatically by days 2 (Dunnett’s various comparisons test, the following data processing by this statistical method, q = 3.83, p < 0.05) and 3 (q = 5.39, p < 0.01) (Fig. 4b, ANOVA: F(3, 8) = 14.30, p = 0.0014). At a PSC:HSC-LX2 ratio of 2:200, the level of Col-I had significantly increased by days 1 (q = 3.37, p < 0.05), 2 (q =10.04, p < 0.001) and 3 (q = 7.18, p < 0.01) for the 2:200 ratio (Fig. 4c, ANOVA: F(3, 8) = 38.49, p < 0.0001). At a PSC:HSC-LX2 ratio of 3:200, the elevation of Col-I was higher on day 2 (q = 5.15, p < 0.001) than on days 1 (q = 7.08, p < 0.01) and 3 (q = 4.66, p < 0.05) (Fig. 4d, ANOVA: F(3, 8) = 42.01, p < 0.0001). Col-I expression levels had increased gradually by days 1 and 2 days at PSC:HSC-LX2 ratios of 1:200 and 2:200 (Fig. 4e, ANOVA: F(10, 22) = 34.85, p < 0.0001). However, by day 3, at a ratio of 1:200, Col-I had continued to rise, but at ratios of 2:200 and 3:200 showed a decline (Fig. 4f, ANOVA: F(3, 8) = 35.15, p < 0.0001).
α-SMA expression in HSC-LX2 exposed to PSCs
PSCs have been reported to play an essential role in the activation and proliferation of HSCs in coculture accompanied by the expression of α-SMA. α-SMA expression in HSCs exposed to PSCs was measured by WB (Fig. 5a) and confirmed by ELISA (Fig. 5b–g). α-SMA expression increased significantly at a PSC:HSC-LX2 ratio of 1:200 on the day 2 (q = 7.54, p < 0.01) and 3 day (q = 9.69, p < 0.01) (Fig. 5b, ANOVA: F(3, 8) = 47.12, p < 0.0001). A significant increase in α-SMA expression was observed for the 2:200 ratio on the days 1, 2 and 3 (q = 6.35, p < 0.05; q = 22.9, p < 0.01; and q = 13.12, p < 0.05, respectively) (Fig. 5c, ANOVA: F(3, 8) = 188.0, p < 0.0001). Additionally, a significant elevation in α-SMA expression was observed for 3:200 on days 1, 2 and 3 (q = 8.29, p < 0.01; q = 16.67, p < 0.001 and q = 12.17, p < 0.05, respectively) (Fig. 5d, ANOVA: F(3, 8) = 119.0, p < 0.0001). α-SMA expression on day 1 was significantly higher at the PSC:HSC-LX2 ratios of 2:200 and 3:200 but not at 1:200 (Fig. 5e, ANOVA: F(3, 8) = 93.85, p < 0.0001). The expression of α–SMA on day 2 was significantly higher at a ratio of 2:200 followed by 1:200 and then 3:200 (Fig. 5f, ANOVA: F(3, 8) = 73.37, p < 0.0001). On day 3, α-SMA expression was moderate at a PSC:HSC-LX2 ratio of 1:200 and had decreased at ratios of 2:200 and 3:200 (Fig. 5g, ANOVA: F(3, 8) = 48.42, p < 0.0001).
OPN Expression in HSC-LX2 exposed to PSCs
In addition to Col-I and α-SMA, OPN is also a major ECM protein produced during fibrosis. HSCs were incubated under coculture to identify the role of PSCs in OPN expression, which was measured by WB (Fig. 6a). OPN expression increased in the coculture of HSC-LX2 with PSCs during incubation for 4 days (Figs. 6b–d). The relationship at a PSC:HSC-LX2 ratio of 1:200 was significant on days 2 (q = 14.16, p < 0.01), 3 and 4 (q = 21.78, p < 0.001) (Fig. 6b, ANOVA: F(4, 10) = 116.4, p < 0.0001); for a ratio of 2:200, the results were significant on days 1 (q = 8.11, p < 0.05), 3 (q = 13.83, p < 0.001), 2 (q = 13.43, p < 0.01) and 4 (q = 12.91, p < 0.01) (Fig. 6c, ANOVA: F(4, 10) = 68.95, p < 0.0001). At a PSC:HSC-LX2 ratio of 3:200, the relationship was significant on days 1 (q = 12.29, p < 0.01), 2 (q = 18.20, p < 0.001) and 3 (q=13.47, p<0.001) and 4 (q = 11.48, p < 0.001) (Fig. 6d, ANOVA: F(4, 10) = 90.42, p < 0.0001); however, OPN expression had increased by days 1 and 2, but decreased by days 3 and 4.