The Role Of Mfn2 On Breviscapine Against Hepatic Ischemia/Reperfusion Injury In Rats

ObjectivesPartial hepatic ischemia/reperfusion(I/R) injury model in rat was established to investigate the effect of Breviscapine(BS) preconditioning in hepatic I/R injury. To analyze the relationship between Mfn2 expression and mitochondrial damage on the molecular level and ultrastructure and discuss its possible mechanism. The study provides a theoretical basis for protecting liver after I/R injury in clinic.Methods40 Sprague-Dawley(SD) male rats were randomly and equally divided into five groups(n=8):sham group, control groups(I/R+NS1, I/R+NS2), breviscapine groups(I/R+BS1, I/R+BS2).Group 1 and group 2 represented ischemia time for 20 min and 60 min individually, and duration time of reperfusion was 6 h. Sham and control groups were pretreated with 2ml normal saline and by intraperitoneal injection for continuous 7days. Breviscapine groups were pretreated with5mg/kg in the same condition. The classical model of hepatic I/R injury was used in this study.The liver and venous blood samples of different groups were collected 6 hours after reperfusion.Subsequently, the serum levels of ALT and AST were detected; the hepatic content of MDA and SOD were determined by colorimetry; gross and microscopic morphologic changes of liver tissues were observed; the morphology ultrastructure change of hepatic cellular mitochondrion was investigated under a transmission electron microscope; the expression levels of Mfn2 m RNA and protein were detected by real-time PCR and western blot.Results1. 6 h after reperfusion, the levels of ALT and AST in control group were higher than Sham group, especially in I/R+NS2 group(P<0.01), whereas the levels of ALT and AST in experimental group were reduced when compared to control group(P<0.05).2. Compared with sham group, the content of MDA increased and SOD activity decreased incontrol group, especially in I/R+NS2 group(P<0.01); compared with control group,experimental group significantly reduced MDA content and increased activity of SOD(P<0.01).3. In the control group, HE staining indicated that diffuse perfusion abnormality in lobule,necrotic tissue, local necrosis, extensive hepatocyte swelling, and partial vacuolar degeneration, hepatic cord derangement, accompanied with a large number of inflammatory cell infiltration. However, breviscapine group significantly alleviated liver damage with a few part of neutrophil infiltration.4. Swelling, even vacuolar degeneration and decreased of hepatic mitochondria can been seen in the control group, and followed reduction of matrix density and mitochondrial crest and incomplete mitochondrial membrane, of which I/R+NS2 group is the most severe. In the contrast, there was no apparent mitochondria swelling in breviscapine group, less mitochondrial morphological changes with normal shape and membrane integrity, and cristae of the mitochondria were arranged regularly, no obvious changes of matrix.5. Compared with sham group, the expression levels of Mfn2 m RNA and protein were significantly decreased in control group(P<0.05); whereas expression levels of Mfn2 m RNA and protein were elevated as compared to corresponding control group(P<0.05).Conclusions1. Breviscapine preconditioning had the protective effects on mitochondrial morphology and function in hepatic cell after I/R injury.2. Breviscapine may promote mitochondria fusion and recovery of mitochondrial structure and function induced by hepatic ischemia/reperfusion injury by promoting the expression levels of Mfn2 m RNA and protein, and reduce oxidative stress, thereby reducing hepatic ischemia/reperfusion injury.