The protective mechanism of Yisheng Injection against hepatic ischemia reperfusion injury in mice

Abstract

Aim: Hepatic ischemia/reperfusion injury may cause acute inflammatory, significant organ damage or dysfunction, and remains an important problem for liver transplantation. Our previous in vivo and in vitro studies demonstrated that Yisheng injection (YS), a traditional Chinese medicine, had protective effect on ischemia/reperfusion injury. In this study, we examined whether YS had protective effect for hepatic ischemia/reperfusion injury and explored its protective mechanism.

Methods: Hepatic warm ischemia/reperfusion was induced in mice. YS at different doses (5, 10, 20 mg/kg) was injected intraperitoneally 24 h and 1 h before ischemia and a third dose was injected intravenously just before reperfusion. The hepatocellular injury, oxidative stress, neutrophil recruitment, proinflammatory mediators and adhesion molecules associated with hepatic ischemia/reperfusion injury were assayed by enzyme-linked immunosorbent assay (ELISA), immunohistochemical assay and reverse transcription polymerase chain reaction (RT-PCR).

Results: Undergoing 90 min of ischemia and 6 h of reperfusion caused dramatical injuries in mouse livers. Administration of YS at doses of 5, 10 and 20 mg/kg effectively reduced serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), from 3 670+/-463 U/L, 2 362+/-323 U/L and 12 752+/-1 455 U/L in I/R group to 1 172+/-257 U/L, 845+/-193 U/L and 2 866+/-427 U/L in YS (20 mg/kg) treated group, respectively (P<0.01). The liver myeloperoxidase (MPO) and malondialdehyde (MDA) contents were decreased from 1.1+/-0.2 (U/mg protein) and 9.1+/-0.7 (nmol/mg protein) in I/R group to 0.4+/-0.1 (U/mg protein) and 5.5+/-0.9 (nmol/mg protein) in YS (20 mg/kg) treated group, respectively (P<0.01). Moreover, the serum levels of tumor necrosis factor-alpha (TNF-alpha) were reduced from 55+/-9.9 (pg/mL) in I/R group to 16+/-4.2 (pg/mL) (P<0.01). Furthermore, the over-expressions of TNF-alpha and intercellular adhesion molecule-1 (ICAM-1) were suppressed by YS treatment in a dose-dependent manner.

Conclusion: YS attenuates hepatic warm ischemia/reperfusion injury by reducing oxidative stress and suppressing the over-expression of proinflammatory mediators and adhesion molecules.

Figure 1

Effects of YS on hepatocellular injury induced by hepatic ischemia and reperfusion. After 90 min of ischemia and 6 h of reperfusion, serum levels of ALT, AST (A) and LDH (B) were determined. YS at different doses (5, 10, 20 mg/kg) was injected intraperitoneally 24 h and 1 h before ischemia and a third dose was injected intravenously just before reperfusion. For all groups, n = 8. ^bP < 0.01 compared with sham group; ^dP < 0.01 compared with I/R group.

Figure 2

Effects of YS on neutrophil recruitment (A) and liver edema (B) induced by 90 min of ischemia and 6 h of reperfusion. MPO contents in liver tissues were analyzed as the index of neutrophil recruitment. Liver edema was determined by tissue wet-to-dry weight ratio. YS at different doses (5, 10, 20 mg/kg) was injected intraperitoneally 24 h and 1 h before ischemia and a third dose was injected intravenously just before reperfusion. For all groups, n = 8. ^bP < 0.01 compared with sham group; ^cP < 0.05, ^dP < 0.01 compared with I/R group.

Figure 3

Effects of YS on SOD (A) and MDA (B) levels in liver tissues. After 90 min of ischemia and 6 h of reperfusion, liver tissues were homogenized and assayed for SOD and MDA levels as the index of hepatic oxidative stress. YS with different dose (5, 10, 20 mg/kg) was injected intraperitoneally 24 h and 1 h before ischemia and a third dose was injected intravenously just before reperfusion. For all groups, n = 8. ^bP < 0.01 compared with sham group; ^cP < 0.05, ^dP < 0.01 compared with I/R group.

Figure 4

Effects of YS on serum levels of TNF-α analyzed by enzyme-linked immunosorbent assay. Mice were subjected to 90 min of ischemia and 6 h of reperfusion. Saline or YS with differ-ent dose (5, 10, 20 mg/kg) was injected intraperitoneally 24 h and 1 h before ischemia and a third dose was injected intravenously just before reperfusion. For all groups, n = 8. ^bP < 0.01 compared with sham group; ^cP < 0.05, ^dP < 0.01 compared with I/R group.

Figure 5

Immunohistochemical assay of TNF-α on liver tissue. Compared with sham control (A), the expression of TNF-α was highly up-regulated by 90 min of ischemia and 6 h of reperfusion (B), and the up-regulation of TNF-α expression was markedly inhibited by YS (20 mg/kg) (C). Original magnification: × 400.

Figure 6

Immunohistochemical assay of ICAM-1 on liver tissue. Compared with sham control (A), the expression of ICAM-1 was highly up-regulated by 90 min of ischemia and 6 h of reperfusion (B), and the increase of ICAM-1 expression was dramatically inhibited by YS (20 mg/kg) (C). Original magnification: × 400.

Figure 7

RT-PCR analysis of TNF-α (A) and ICAM-1 (B) mRNA expression in the liver tissues. β-actin was used as control. Mice were subjected to 90 min of ischemia and 6 h of reperfusion. Saline or YS with different dose (5, 10, 20 mg/kg) was injected intraperitoneally 24 h and 1 h before ischemia and a third dose was injected intravenously just before reperfusion. Liver RNA extracts were analyzed by reverse transcription PCR and stained with SYBR green dye. M: marker; Lain 1: sham control; Lain 2: I/R group; Lain 3-5: 5, 10 and 20 mg/kg of YS treated groups. The band intensities of TNF-α (C) and ICAM-1 (D) in sham, I/R and YS-treated groups were compared. One representative experiment from three performed is shown. ^bP < 0.01 compared with sham group; ^cP < 0.05, ^dP < 0.01 compared with I/R group.