Effect of ischemic preconditioning on P-selectin expression in hepatocytes of rats with cirrhotic ischemia-reperfusion injury

Abstract

AIM: To investigate the effects and mechanisms of ischemic preconditioning (IPC) on the ischemia/reperfusion (I/R) injury of liver cirrhosis in rats and the effect of IPC on P-selectin expression in hepatocytes.

METHODS: Forty male SD rats with liver cirrhosis were randomly divided into sham operation group (SO group), ischemia/reperfusion group (I/R group), ischemic preconditioning group (IPC group), L-Arginine preconditioning group (APC group), L-NAME preconditioning group (NPC group), eight rats in each group. Hepatocellular viability was assessed by hepatic adenine nucleotide level and energy charge (EC) determined by HPLC, ALT, AST and LDH in serum measured by auto- biochemical analyzer and bile output. The expression of P-selectin in the liver tissue was analyzed by immunohistochemical technique. Leukocyte count in ischemic hepatic lobe was calculated.

RESULTS: At 120 min after reperfusion, the level of ATP and EC in IPC and APC groups was higher than that in I/R group significantly. The increases in AST, ALT and LDH were prevented in IPC and APC groups. The livers produced more bile in IPC group than in I/R group during 120 min after reperfusion (0.101 ± 0.027 vs 0.066 ± 0.027 mL/g liver, P = 0.002). There was a significant difference between APC and I/R groups, (P = 0.001). The leukocyte count in liver tissues significantly increased in I/R group as compared with SO group (P < 0.05). The increase in the leukocyte count was prevented in IPC group. Administration of L-arginine resulted in the same effects as in IPC group. However, inhibition of NO synthesis (NPC group) held back the beneficial effects of preconditioning. Significant promotion of P-selectin expression in hepatocytes in the I/R group was observed compared with the SO group (P < 0.01). IPC or L-arginine attenuated P-selectin expression remarkably (P < 0.01). However, inhibition of NO synthesis enhanced P-selectin expression (P < 0.01). The degree of P-selectin expression was positively correlated with the leukocyte counts infiltrating in liver (r = 0.602, P = 0.000).

CONCLUSION: IPC can attenuate the damage induced by I/R in cirrhotic liver and increase the ischemic tolerance of the rats with liver cirrhosis. IPC can abolish I/R induced leukocyte adhesion and infiltration by preventing post-ischemic P-selectin expression in the rats with liver cirrhosis via a NO-initiated pathway.

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INTRODUCTION

Ischemic preconditioning (IPC) refers to a phenomenon in which a tissue is rendered resistant to the deleterious effects of prolonged ischemia and reperfusion (I/R) by prior exposure to a short period of vascular occlusion. This phenomenon was first demonstrated in the heart a decade ago[1] and has been the subject of intensive investigation ever since. Although it is clear that activation of adenosine receptors and protein kinase C (PKC) is critical to the development of the beneficial action of IPC, the downstream effectors in the signaling cascade initiated by IPC are uncertain. Akimisn et al[2] and Kubes et al[3] have demonstrated that IPC prevents intestinal and skeletal muscle I/R injury by inhibiting postischemic leukocyte-endothelial cell interaction. However, identification of the end effectors of the ant adhesive effects of IPC remains unclear. A likely candidate effector molecule that may be targeted by the signaling cascade initiated by IPC is P-selectin, because post-ischemic leukocyte rolling (and thus subsequent stationary adhesion and emigration) is critically dependent on the expression of P-selectin on venular endothelium[4]. IPC has been commonly studied in the heart, but few studies have been performed on cirrhotic liver IPC. This study was aimed to determine the effects and mechanisms of IPC on the I/R injury rats with liver cirrhosis and the effect of IPC on the expression of P-selectin.

MATERIALS AND METHODS

Reproduction of rat cirrhotic liver model

Sprague-Dawley (SD) Male rats initially weighing 200 ± 20 g were used.

Subcutaneous injection of 60% CCl₄ (0.3 mg/kg) was made once every 4 d for 8 weeks and 5% ethanol was allowed for 60 d[5].

Operative procedure

At first, ligamentous attachments around the liver were dissected. The common bile duct was then cannulated and bile output was measured. Ischemia was induced in the median and left lateral hepatic lobes by clamping the corresponding hepatic arterial and portal vein, while the blood flowing to the other lobe was left intact. When the assigned period of warm ischemia was completed, the clamp was removed and the pedicles to the non-ischemic lobe were ligated[6].

Grouping of animals

Forty male SD rats with liver cirrhosis were divided into 5 groups randomly, eight rats in each group. Animals in sham Operation group (SO group) were subjected to anesthesia and laparotomy. Animals in ischemia/reperfusion group (I/R group) were subjected to 30 min of left and middle lobe hepatic ischemia, followed by 120 min of reperfusion. Animals in ischemic preconditioning group (IPC group) were same as I/R group, but subjected to 10 min of ischemia and 5 min of reperfusion prior to I/R. Animals in L-arginine preconditioning group (APC group) were same as IPC group, but treated with a continuous intravenous infusion of L-Arginine (10 mg/kg, portal vein) for 5 min before preconditioning. Animals in L-NAME preconditioning group (NPC group) were same as IPC group, but treated with a continuous intravenous infusion of L-NAME (10 mg/kg, portal vein) for 5 min before preconditioning.

The animals were killed after blood samples were collected from the inferior vena cava after 120 min of reperfusion. Liver samples were excised from the anterior edge of the median lobe before ischemia, after the induction of ischemia and 120 min after reperfusion respectively. The specimens were immersed in liquid nitrogen immediately after sampling to measure the tissue concentration of adenine nucleotides, adenosine 5’-triphosphate (ATP), adenosine 5’-diphosphate (ADP), adenosine 5’-monophosphate (AMP) and total adenosine nucleotide. At 120 min after reperfusion, livers were perfused and fixed in situ with 4% para formaldehyde.

Energy metabolism

ATP and its metabolites, ADP and AMP in the liver tissue were assayed as follows. After the frozen tissue with a mortar and pestle was ground, the powder was mixed with 1 mL 6% perchloric acid at 4 °C. The mixture was centrifuged at 3000 rpm for 10 min and the supernatant was stored at 4 °C, then 0.5 mL 6% perchloric acid was added to the precipitate and centrifuged in the same manner. The supernatant was neutralized with 3 mol/L potassium carbonate and centrifuged at 3000 rpm for 10 min. The final supernatant was used as a sample for ATP and its metabolites. The tissue concentration of ATP and its metabolites was determined by high-performance liquid chromatography ( HPLC). Energy charge (EC) was equal to (ATP + 1/2ADP)/(ATP + ADP + AMP)[7].

Measurement of serum cytosolic enzymes

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured at 4 °C using commercially available kits (Horizon, American) by an auto-biochemistry analyzer.

Measurement of bile output

Bile output from the ischemic liver was measured through a choledochotomy tube placed in the common bile duct.

P-selectin expression in liver tissues

Immunohistochemical staining for P-selectin protein was performed using SP technique[8]. The immunostaining of P-selectin was visually classified into four groups: no staining present in any tumor cells (-), slight staining in most of the hepatocytes (+), most of the hepatocytes with moderate staining (++), and strong staining in most of the hepatocytes (+++). Two senior pathologists who did not know the clinicopathological data did the classification.

Histological examination

Liver samples were excised from the anterior edge of the median lobe 120 min after reperfusion. Small portions (0.5 cm × 0.5 cm) were fixed immediately in 4% buffered para formaldehyde (pH7.2) and embedded in paraffin. These portions were cut into 4 μm thick sections and stained with hematoxylin and eosin (H & E). Leukocyte count in ischemic hepatic lobe could be calculated randomly under microscopy(× 400).

Statistical analysis

The results were expressed as x^-±s. The one-way NOVA and H test were used for statistical significance of differences between groups. Correlation analysis between two factors was made by Spearman method. P < 0.05 was considered significant.

RESULTS

Change of ATP, ADP, AMP and EC levels in liver after ischemia and reperfusion

At 30 min of hepatic inflow occlusion, the ATP and EC levels in liver tissues were significantly decreased in I/R, IPC, APC and NPC groups (P < 0.05). At 120 min after reperfusion, the ATP and EC levels in IPC and APC groups were significantly higher than those in I/R group (P = 0.000, P = 0.001). But there was no significant difference between NPC and I/R groups (P > 0.05) (Table 1).

Table 1 ATP, ADP, AMP and EC levels in liver after ischemia and reperfusion (U/L).

Groups	n	After ischemia				After reperfusion
		ATP	ADP	AMP	EC	ATP	ADP	AMP	EC
SO	8	5.4 ± 1.3	3.1 ± 0.8	1.0 ± 0.2	0.7 ± 0.0	5.5 ± 0.8	3.2 ± 1.0	1.0 ± 0.1	0.7 ± 0.0
I/R	8	0.5 ± 0.2b	2.3 ± 0.6a	3.5 ± 1.0b	0.3 ± 0.0b	1.5 ± 0.6b	2.3 ± 1.2	2.6 ± 1.3a	0.4 ± 0.1b
IPC	8	0.5 ± 0.1b	2.1 ± 0.5a	3.6 ± 1.5b	0.3 ± 0.1b	4.1 ± 1.6ac	2.3 ± 0.8	1.9 ± 0.9	0.6 ± 0.1ac
APC	8	0.5 ± 0.1b	2.2 ± 0.5a	3.4 ± 0.7b	0.3 ± 0.0b	4.0 ± 1.6ac	2.5 ± 1.1	2.2 ± 1.2a	0.6 ± 0.1ac
NPC	8	0.5 ± 0.2b	2.0 ± 0.7a	3.3 ± 0.6b	0.3 ± 0.0b	2.3 ± 1.6b	2.2 ± 0.9a	3.2 ± 1.1b	0.4 ± 0.1b

^aP < 0.05,

^bP < 0.01, vs SO group;

^cP < 0.01, vs I/R group.

Change of ALT, AST and LDH in serum

Significant increases of ALT, AST and LDH levels in serum were observed in the group subjected to ischemia and reperfusion (I/R group) in comparison with the control group (SO group). When ischemia was preceded by 10 min of ischemia and 5 min of reperfusion (IPC), the increases of AST, ALT and LDH in serum were prevented (P = 0.000). Administration of L-Arginine (APC group) resulted in the same effects on ALT, AST and LDH as above (P = 0.001). However, infusion of L-NAME (NPC group) inhibited the beneficial effects of preconditioning (Table 2).

Table 2 Results of ALT, AST and LDH in serum after reperfusion (U/L).

Groups	Cases	ALT	AST	LDH
SO	8	300.5 ± 159.2	551.1 ± 84.7	4612.3 ± 1042.8
I/R	8	2218.8 ± 825.3a	3043.8 ± 1198.9a	13762.5 ± 5371.9a
IPC	8	568.8 ± 214.6b	1315.0 ± 958.9b	6266.3 ± 2425.5b
APC	8	508.8 ± 142.8b	1108.8 ± 637.2b	5355.0 ± 1237.9b
NPC	8	2091.3 ± 684.6a	3083.8 ± 844.5a	11361.3 ± 4211.8a

^aP < 0.01, vs SO group;

^bP < 0.01, vs I/R group.

Results of bile output and leukocyte count in ischemic hepatic lobe

The livers produced more bile in IPC group than in I/R group during 120 min after reperfusion (0.101 ± 0.027 vs 0.066 ± 0.027 mL/g liver, P = 0.002). There was a significant difference between APC and I/R, NPC and SO groups (P = 0.001, P = 0.000) respectively. However, there was no significant difference between NPC and I/R groups (P > 0.05). The leukocyte counts in liver tissue showed a more significant increase in I/R group than in SO group (P = 0.000). The increase in leukocytes count was inhibited in IPC group (P = 0.028). Administration of L-arginine resulted in the same effects as in IPC group (P = 0.020). However, inhibition of NO synthesis (NPC group) prevented the beneficial effects of preconditioning (P > 0.05) (Table 3).

Table 3 Results of bile output and leukocyte count in ischemic hepatic lobe.

Groups	Cases	Bile output (mL/g liver)	Leukocyte count (piece/HP)
SO	8	0.15 ± 0.02	181.38 ± 59.23
I/R	8	0.07 ± 0.03a	442.38 ± 94.10a
IPC	8	0.10 ± 0.03ab	353.00 ± 84.11ba
APC	8	0.10 ± 0.02ab	347.75 ± 51.53ba
NPC	8	0.07 ± 0.01a	407.88 ± 90.40a

^aP < 0.01, vs SO group;

^bP < 0.05, vs I/R group.

Results of P-selectin expression in liver tissues

Significant promotion of P-selectin expression in hepatocytes in the I/R group was observed in comparison with the SO group (P = 0.000). IPC or L-arginine attenuated P-selectin expression significantly (P = 0.005). However, inhibition of NO synthesis enhanced the expression of P-selectin (P = 0.001) (Table 4).

Table 4 Expression of P-selectin in liver tissues.

Groups	Cases	Grade			Average rank
		(+)	(++)	(+++)
SO	8	7	1	0	10.69
I/R	8	0	5	3	30.50a
IPC	8	5	3	0	15.06b
APC	8	4	4	0	17.25b
NPC	8	0	6	2	29.00a

^aP < 0.01, vs SO group;

^bP < 0.05, vs I/R group.

Correlation between leukocytes infiltration and P-selectin expression in liver tissues

Leukocytes infiltration was significantly correlated with P-selectin expression in liver tissues. The degree of P-selectin expression was positively correlated with the counts of leukocyte infiltration in liver (r = 0.602, P = 0.000).

DISCUSSION

IPC is a unique phenomenon which attenuates organ injury caused by I/R. This is accomplished through a brief preceding episode of vascular occlusion which renders these tissues resistant to the deleterious effects of prolonged ischemia and reperfusion. The protective effects of IPC have been well documented in the previous studies involving different tissues and organs. These included cardicmuscle[1,9], skeletal muscle[2], small intestines[10] and more recently the liver[11]. Although the mechanism of IPC is still unclear up to now, several potential mediators (nitrogen monoxide, adenosine, oxide radical, bradykinin and so on) have been found to play different roles in different organs[11-14]. Adenosine and protein kinase C (PKC) were critical to the beneficial actions of IPC in the heart[15]. IPC-induced adenosine A₁-receptor stimulation during the period of preconditioning ischemia increased phospholipose C (PLC) activity, an event that is coupled by pertussis toxin-sensitive G proteins[12,13]. Activation of PLC induced the formation of diacylglycerol, which in turn promotes the translocation and activation of PKC. Activation of PKC stimulated the activation of ATP-sensitive potassium (Katp) channels, and the beneficial actions of IPC in the heart were induced[15], while adenosine stimulated NO production in IPC to protect against the injury associated with I/R in liver[16]. In the case of the cirrhotic liver, our work revealed that the ATP and EC levels in IPC group were higher than those in I/R group. There was significantly more bile produced by the livers in IPC group too. However, the increase of AST, ALT and LDH release was attenuated, when IPC was performed before ischemia. This fact shows the protective effect of IPC on preventing ischemia-reperfusion damage of cirrhotic liver. In addition, we found that L-arginine administration in hepatic ischemia reperfusion attenuated the injury in a manner similar to that of IPC. Accordingly, inhibition of NO synthesis abolished the beneficial effects of IPC. Thus, our data suggest that NO is one of the potential mediators of the protective effects of IPC.

Akimisn.[2] and Kubes[3] demonstrated that IPC prevented intestinal and skeletal muscle I/R injury by inhibiting postischemic leukocyte-endothelial cell interactions. These observations are important because they indicate that in addition to protecting against the deleterious effects of ischemia and reperfusion, IPC could induce cellular changes that also prevent leukocyte recruitment to ischemic tissues. This might limit the reperfusion component of I/R injury, which was primarily leukocyte dependent in the small intestine and other organs[17,18]. Thus, in addition to K⁺-ATP channels, IPC appears to target effector molecules that modulate the inflammatory response to I/R. A likely candidate effector molecule that may be targeted by the signaling cascade initiated by IPC is P-selectin, for several researches have shown that I/R injury is a leukocyte-mediated event resulted from a cascade of acute phase reactants causing leukocyte-endothelial cell interactions. The interactions progressed from rolling to saltation to firm adhesion with subsequent tissue infiltration and organ injury[19-24]. The cascade of acute phase reactants was critically dependent on the expression of P-selectin on venular endothelium[4,25]. In our study, significant promotion of the expression of P-selectin in hepatocytes in the I/R group was observed in comparison with the SO group. IPC or L-arginine attenuated P-selectin expression significantly. However, inhibition of NO synthesis enhanced the expression of P-selectin. The increase in the leukocytes count was prevented in IPC group. Administration of L-arginine resulted in the same effects as in IPC group. In the mean time, there was a significant correlation between leukocytes infiltration and P-selectin expression in liver tissues. The degree of P-selectin expression was positively correlated with the counts of leukocyte infiltration in liver.

In summary, IPC can abolish I/R induced leukocyte adhesion and infiltration by preventing postischemic P-selectin expression in rats with liver cirrhosis via a NO-initiated pathway.