Wistar rats (male, 8 wk old, weight 200-250 g) were randomly divided into control group, GaCl₃ group, SMCo group, GaCl₃ + SMCo group (n = 15 each).

Gadolinium chloride hexahydrate (Wako pure medicine, Japan) was dissolved in physiological saline (2 g·L^-1), which was injected into tail vein at a dose of 7 mg·kg^-1 on d 1 and d 2 in GaCl₃ group. Salvia miltiorrhiza compound (First Shanghai pharmacy) was injected into hind limb muscle at a dose of 2 mL·kg^-1 on d 1 and d 2 in SMCo group. The GaCl₃ + SMCo group received both GaCl₃ and SMCo injection. The control group received saline only on d 1 and d 2.

On d 3, after 12 h fasting, each rat was anesthetized with sodium thiopental at a dose of 50 mg·kg^-1 (im). A midline incision was made to expose the liver vessels. The arteries to middle and left lobes of the liver were dissected and the blood flow was blocked with a pair of vessel clamps. The abdomen was then closed. The blood flow was restored by releasing the clamps after 2 h. The blood reperfusion in each group lasted for 2 h, 6 h and 18 h respectively (n = 5 for each time point).

The blood sample was taken from heart 2 h, 6 h and 18 h after reperfusion for ALT assay.

The liver was excised and washed with saline solution, cleaned with filter paper and then homogenized with 0.25 g·L^-1 saccharose-EDTA solution. The homogenized solution was centrifugated for 10 min in 1000 g of centrifugal force, and the supernatant was taken out and centrifugated again for 15 min in 7000 g. The deposit after second centrifugation, which contained mitochondria, was reserved. All approaches were performed at 4 °C and each procedure was repeated three times to make mitochondria pure.

The separated mitochondria was fixed by entering 2 mL of 25 g·L^-1-tris phosphoric acid glutaradehyde for 2 h and then dehydrated bleach routinely. After embedded with EPSONB for 48 h, an extreme thin slice was made and lead acetate-uranium double staining was performed. The ultrastructure of mitochondria was observed under electronic microscopy.

The separated mitochondria were mixed with 0.125 mol·L^-1 kalium chloride up to 1 mL. The content of MDA in hepatocellular mitochondria was determined with biuret reaction at the 525 nm of wave.

The hepatic tissue fixed with formaldehyde was embedded into paraffine, slices were made and stained with HE. Pathological changes were observed under optical microscopy.

Analysis of variance and Student-newman-Keuls test were used to determine the difference between groups. The results were considered statistically significant when P < 0.05.

In control group, ALT became significantly activated 6 h after reperfusion. In GaCl₃, SMCo and GaCl₃ + SMCo treated groups, ALT levels were all significantly inhibited compared with control group (Table 1).

The dynamic changes of MDA were identical to the pattern of ALT (Table 2).

In the control group, the major change of hepatocytes after 2 h of I/R was swelling without necrosis. The swelling became more and more severe with some scattered and focal necrosis and deposition of fibrin in central lobular vein after 6 h of I/R. In addition to obvious swelling, denaturalization and necrosis were seen after 18 h of I/R, which were associated with destroyed hepatic sinuses and infiltration of numerous white blood cells. In GaCl₃, SMCo and GaCl₃ + SMCo groups, there was no necrosis 2 h and 6 h after I/R, and the hepatocyte swelling and denaturalization were much less than those in control group. In treated groups, the hepatic structure was maintained with less necrosis and infiltration of leucocyte 18 h after I/R.

The I/R injury in hepatocellular mitochondria became more severe with the prolongation of I/R time in control group. It was obvious that swelling, rupture of inner membrane ridge, damage or even breakdown of mitochondrial structure with outline only (Figure 1, Figure 2, Figure 3). The changes in GaCl₃ and SMCo group were less severe than those in control group. Except for slight swelling, the structure of mitochondria was unchanged. The protective effects of GaCl₃ and SMCo against I/R damage on mitochondria were seen not only 2 h, 6 h, and 18 h after I/R (Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9). The effect of GaCl₃ + SMCo combination on mitochondria was similar to that of either GaCl₃ or SMCo alone.

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Figure 1 Hepatocellular mitochondria in I/R 2 h in control group, × 10 K.

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Figure 2 Hepatocellular mitochondria in I/R 6 h in control group, × 10 K.

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Figure 3 Hepatocellular mitochondria in I/R 18 h in control group, × 10 K.

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Figure 4 Hepatocellular mitochondria in I/R 2 h in GdCl₃ group, × 10 K.

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Figure 5 Hepatocellular mitochondria in I/R 6 h in GdCl₃ group, × 10 K.

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Figure 6 Hepatocellular mitochondria in I/R 18 h in GdCl₃ group, × 10 K.

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Figure 7 Hepatocellular mitochondria in I/R 2 h in SMCo group, × 10 K.

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Figure 8 Hepatocellular mitochondria in I/R 6 h in SMCo group, × 10 K.

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Figure 9 Hepatocellular mitochondria in I/R 18 h in SMCo group, × 10 K.

The experimental results from the present study showed that ALT increased after 2 h of I/R, and reached the highest value after 6 h of I/R in control group, indicating that I/R injury in hepatic tissues is maximized 6 h after I/R. Previous studies have suggested that activation of Kupffer cells is involved in the injury process at the early stage of liver I/R. GaCl₃ can effectively blockade the function of Kupffer cells and reduce the secretion of cytokines and reactive oxygen species from activated Kupffer cells, so that MDA in hepatocellular mitochondria is inhibited[11]. The fact that both ALT and MDA in mitochondria in GaCl₃ group were markedly decreased because of the use of GaCl₃ suggests that inhibition of Kupffer cells can protect the hepatic function[12]. This study also showed that there was no significant decrease in ALT and MDA 18 h after I/R, which was in agreement with the hypothesis that inhibition of Kupffer cells could only decrease the injury in the liver at the early stage of I/R. These biochemistry alterations are in parallel to the pathohistological changes. In control group, slight but detectable structural change was seen after 2 h, severe changes including necrosis and denaturalization were seen after 6 h, and more severe changes were seen after 18 h following I/R. In the mean time, by inhibiting Kupffer cells, GaCl₃ remarkably ameliorated those detrimental changes, in particular, 6 h after I/R. It should be pointed out that the role played by Kupffer cells is only one of the factors that is involved I/R injury in the liver. In addition to GaCl₃, other endogenous and exogenous compounds have also shown protective effects. For example, hepatocyte growth factor could reduce the impairment of both hepatocytes and sinusoidal endothelial cells[13]. Administration of epoprostenol could decrease hepatocellular I/R injury in transplanted liver[14], and nonselective endothelin-1 receptor antagonists could improve hepatic microcirculatory impairment[15]. Special neutrophil eastase inhibitor[16], tissue factor pathway inhibitor[17], adenosine[18], precondition of low-dose TNF-α[19] or ischemic precondition[20,21], etc, could alleviate I/R injury. All of these suggest that I/R is a complicated process. Although the potential role of Kupffer cells in I/R injury is still controversial[22,23], our data provide the strongest evidence in favor of its important role because blockade of Kupffer cells greatly reduces I/R injury in hepatocellular mitochondria.

Our data showed that SMCo treatment decreased the content of MDA in mitochondria in SMCo group. As a scavenger of oxygen free radicls, the SMCo can effectively inhibit lipid peroxidation in hepatocellular mitochondria[24,25], apoptosis of cells[26], and adhesion of neutrophil[27], etc, and accordingly protect hepatocellular mitochondria. Whereas simple inhibition of lipid peroxidation is not enough to prevent entirely the occurrence of I/R injury since a lot of factors are involved. Our results imply that SMCo can protect the function and structure of mitochondria, maintain energy metabolism of hepatocytes, and thereby reduce significantly I/R injury in the liver. Because a lot of factors are involved in I/R injury, it is understandable that inhibition of lipid peroxidation by SMCo can improve, but not prevent I/R injury.

Although both GaCl₃ and SMCo are able to alleviate I/R injury in hepatocellular mitochondria when used separately, there is no additive effect when used in combination. We speculate that the two compounds act at different level in a same mechanism. While GaCl₃ inhibits mainly the functions of Kupffer cells, decreases the production of oxygen free radicls, SMCo is mainly to promote microcirculation and to restrain lipid peroxidation. Therefore, the effect of SMCo will become insignificant when oxygen free radicls are reduced when GaCl₃ is used.

In conclusion, both GaCl₃ and SMCo can ameliorate I/R injury in hepatocellular mitochondria at some extent.