Abstract: Objective:To explore the effect of heme oxygenase1 (HO-1)on expressions of hypoxia inducing factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF)and regeneration of hepatic vascular plexus after orthotopic liver transplantation ischemiareperfusion injury in rats.
Methods:The experimental study was conducted. According to the random number table, 240 SD rats were divided into the 3 groups, 80 rats in each group. Empty virus group: rats were transfected with the empty virus. Induced group: rats were transfected with HO-1 overexpression adenovirus. Inhibited group: rats were transfected with HO-1 RNAi adenovirus. Rats were made pairs (1:1) and established rat liver transplantation model according to “two cuffs method”. Rats with less weight and with heavier weight were respectively chosen as donor rats and recipient rats, and then recieved tail intravenous injection of adenovirus at 24 hours before operation. (1) Detection of transfection efficiency of adenovirus before operation: HO-1 expression of liver tissue of rats in each group was detected by Western blot at 12 and 24 hours after injection of adenovirus. (2) Liver function test of recipient rats after liver transplantation: liver functions of recipient rats ［alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gammaglutamyl transferase (GGT)］ were detected at 1, 3, 7 and 14 days postoperatively. (3) Pathological histology of liver tissue and injury scores of recipient rats in the 3 groups after liver transplantation: paraffin sections of recipient rats in the 3 groups at postoperative 1 and 14 days were stained by HE staining and observed by light microscope, and were evaluated by Suzuki damage score standard. (4) Relative expressions of HIF-1α, VEGF and HO-1 in liver tissue of recipient rats were detected by Western blot. (5) Von Willebrand factor (vWF) in liver tissue of recipient rats at 14 days postoperatively was detected by immunofluorescence staining and small vessels were counted. Measurement data with normal distribution were represented as ±s. Comparison between groups was analyzed by the independentsample t test, comparison among groups was done using oneway ANOVA, and pairwise comparison was analyzed by the LSD test.
Results:(1) Detection of transfection efficiency of adenovirus before operation: the relative expression of HO-1 of liver tissue of rats at 12 and 24 hours preoperatively after injection of adenovirus was 1.08±0.16 and 1.08±0.26 in the empty virus group, 1.18±0.21 and 1.39±0.19 in the induced group, 0.87±0.26 and 0.57±0.12 in the inhibited group, respectively, with statistically significant differences in different time points (F=4.232, 36.513, P<0.05). (2) Liver function test of recipient rats after liver transplantation: level of ALT at 3 days postoperatively in the empty virus group, induced group and inhibited group was (504±67)U/L, (438±47)U/L and (490±39)U/L, with a statistically significant difference (F=3.517, P<0.05). Levels of ALT, AST and ALP at 7 days postoperatively were (443±49)U/L, (430±34)U/L, (455±38)U/L in the empty virus group and (382±49)U/L, (372±50)U/L, (394±25)U/L in the induced group and (493±44)U/L, (455±62)U/L, (470±72)U/L in the inhibited group, respectively, with statistically significant differences (F=10.950, 5.667, 5.398, P<0.05). Levels of ALT, AST, ALP and GGT at 14 days postoperatively were (394±46)U/L, (361±68)U/L, (417± 17)U/L, (4.5±1.1)U/L in the empty virus group and (283±47)U/L, (288±60)U/L, (332±46)U/L, (2.5±0.5)U/L in the induced group and (446±43)U/L, (422±51)U/L, (423±63)U/L, (4.3±1.3)U/L in the inhibited group, respectively, with statistically significant differences (F=26.906, 9.924, 8.013, 9.279, P<0.05). (3) Pathological histology of liver tissue and injury scores of recipient rats in the 3 groups after liver transplantation: liver cell swelling, loose cytoplasm and a varying quantity of inflammatory cell infiltration in the portal regions in the liver tissue of 3 groups were observed at 1 day postoperatively. A few inflammatory cell infiltrations in the portal regions, basically normal liver cell arrangement and a slightly swelling of liver cell were found in the empty virus group at 14 days postoperatively. Reduced liver cell swelling and basically normal structure of liver lobule were observed in the induced group. There were small patchy or focal necrosis of liver cell, masses of inflammatory cell infiltration in the portal regions and damage of bile duct in the inhibited group. Suzuki score at 1 day postoperatively in the empty virus group, induced group and inhibited group were respectively 6.7±1.7, 6.1±1.2 and 7.6±1.3, with no statistically significant difference (F=2.257, P>0.05). Suzuki score at 14 day postoperatively in the empty virus group, induced group and inhibited group were respectively 4.0±0.8, 2.9±0.8 and 5.1±1.4, with a statistically significant difference (F=9.776, P<0.05). (4) Western blot results: the relative expressions of HIF-1α and VEGF (43 KD) in liver tissue of recipient rats at 1 day postoperatively were 0.21±0.10, 0.30±0.12 in the empty virus group and 0.23±0.09, 0.34±0.14 in the induced group and 0.17±0.06, 0.29±0.11 in the inhibited group, respectively, with no statistically significant difference (F=0.902, 0.410, P>0.05). The relative expressions of VEGF (24 KD) and HO-1 in liver tissue of recipient rats at 1 day postoperatively were 1.21±0.25, 0.55±0.12 in the empty virus group and 2.13±0.40, 0.72±0.12 in the induced group and 0.91±0.22, 0.26±0.07 in the inhibited group, respectively, with statistically significant differences (F=35.158, 39.082, P<0.05). The relative expressions of HIF-1α, VEGF (43 KD), VEGF (24 KD) and HO-1 in liver tissue of recipient rats at 7 days postoperatively were 0.49±0.22, 0.46±0.13, 0.98±0.37, 0.98±0.37 in the empty virus group and 0.83±0.26, 0.63±0.19, 1.60±0.33, 1.49±0.46 in the induced group and 0.24±0.09, 0.30±0.12, 0.64±0.18, 0.75±0.26 in the inhibited group, respectively, with statistically significant differences (F=16.853, 10.021, 20.756, 8.156, P<0.05). (5) Immunofluorescence staining results: number of small vessels at 14 days postoperatively in the empty virus group, induced group and inhibited group was respectively 7.9±2.0, 10.6±1.9 and 7.6 ±1.9, with a statistically significant difference (F=5.921, P<0.05).
Conclusion:HO-1 could promote expressions of HIF-1α and VEGF in liver tissue after liver transplantation ischemiareperfusion injury and regeneration of intrahepatic vascular plexus, and it also alleviate bile duct ischemiareperfusion injury after liver transplantation.