Role of mitofusin 2 in the protective effect of breviscapine against hepatic ischemia/reperfusion injury in rats

Abstract

The purpose of the present study was to investigate the effect of breviscapine injection on hepatic ischemia/reperfusion (I/R) injury in rats. To explore the relevance and discuss the underlying mechanism of mitofusin 2 (Mfn2) in hepatic I/R injury, 40 Sprague-Dawley male rats were randomly and equally divided into five groups (n=8 per group) as follows: Sham, I/R + normal saline 1 (NS1), I/R + breviscapine 1 (Bre1), I/R + NS2 and I/R + Bre2 groups. Groups 1 and 2 represented ischemia for 20 and 60 min, respectively. Breviscapine or normal saline was injected via the tail vein (single dose of 10 mg/kg) 1 h prior to surgery and immediately postoperatively. The classical model of hepatic I/R injury was used in the present study. The blood and liver samples of different groups were collected following reperfusion to observe serum transaminases and histopathological changes. Alterations in Mfn2, cytosolic cytochrome c and cleaved caspase-3 were additionally assessed. The results demonstrated that breviscapine improved liver function, based on histopathological analysis, and decreased levels of the liver enzymes aspartate and alanine aminotransferase in the I/R + Bre groups compared with the I/R + NS group (P<0.05). The expression of Mfn2 was significantly increased in the I/R + Bre groups (P<0.05), whereas the expression of caspase-3 and cytosolic cytochrome c protein was decreased in the I/R + Bre groups (P<0.05) compared with the I/R + NS group. These data provided substantial evidence that breviscapine treatment exerted a protective effect against damage induced by hepatic I/R. This protective effect was possibly due to its ability to inhibit I/R-induced apoptosis and promote the expression of Mfn2.

Keywords: apoptosis; breviscapine; hepatic ischemia/reperfusion; mitofusin 2.

Figure 1.

Breviscapine treatment reduced serum transaminase level following ischemia/reperfusion (I/R) injury. Serum samples were collected 6 h after I/R injury for measuring (A) serum alanine aminotransferase (ALT) and (B) serum aspartate aminotransferase (AST). Data are expressed as the mean ± standard deviation (n=8). *P<0.05, **P<0.01, ***P<0.001 vs. normal saline (NS) groups.

Figure 2.

Breviscapine treatment markedly attenuated ischemia/reperfusion (I/R) injury-induced pathological changes. (A) Histological findings of the liver tissue in the sham, I/R + NS1, I/R + Bre1, I/R + NS2 and I/R + Bre2 groups. Photomicrographs of the liver tissue stained with hematoxylin and eosin (original magnification, ×100). (B) Pathological score following hepatic I/R injury in rats. Data are expressed as the mean ± standard deviation (n=8). *P<0.05, **P<0.01 vs. NS groups. Bre, breviscapine; NS, normal saline.

Figure 3.

Breviscapine treatment markedly reduced ischemia/reperfusion (I/R) injury-induced apoptosis. (A) Cell apoptosis was analyzed using TUNEL staining (original magnification, ×200). (B) The apoptotic index (AI) is quantified as the ratio of TUNEL-positive cells to total number of hepatocytes. Data are expressed as the mean ± standard deviation (n=8). ***P<0.001 vs. Sham group; ⁺⁺P<0.01 vs. normal saline (NS) groups.

Figure 4.

Breviscapine treatment limited the increase in cleaved caspase-3 expression in the hepatic and cytosolic translocation of cytochrome c. (A) The protein levels of cleaved caspase-3 and cytochrome c were determined via western blot analysis. (B) The bar chart demonstrates the relative expression of cleaved caspase-3. (C) The bar chart demonstrates the relative expression of cleaved cytochrome c. Data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs. sham group; ⁺P<0.05, ⁺⁺P<0.01 vs. normal saline (NS) groups.

Figure 5.

Breviscapine treatment limited the downregulation of Mfn2 expression in hepatocytes following ischemia/reperfusion (I/R) injury. (A) The protein levels of Mfn2 were determined via western blot analysis. (B) The bar chart demonstrates the relative expression of Mfn2. (C) The mRNA levels of Mfn2 were determined via reverse transcription-polymerase chain reaction analysis. Data are expressed as the mean ± standard deviation. **P<0.01, ***P<0.001 vs, sham group; ⁺P<0.05, ⁺⁺P<0.01, ⁺⁺⁺P<0.001 vs. NS groups. Mfn2, mitofusin 2; NS, normal saline.