PROSTAGLANDINSLEUKOTRIEVES ANDEWZITIALFATTYACIDS

The Effect of Iloprost and NDGA in Ischemia Reperfusion Injury in Rat Liver

N. Okboy. C. Yegen. A. 0. Aktan, H. H. Dosluoglu, A. Sav*, R. Yalin and S. Ercan’

Marmara University School of Medicine. Departments of General Surgery and *Pathology, Marmara universite Hastanesi, Gene1 Cerrahi Bbhtmii, Altwkade, 81190 Istanbul, Turkey, ‘Gazi University School of Medicine, Department of Pharmacology (Reprint requests to AOA}

ABSTRACT. In this study, the effects of iloprost (ZK 36374) and NDGA on warm ischemia and reperfusion injury in rat liver were investigated. Rats were given isotonic saline (control group), iloprost 25,ug/kg i.v. (group II) just before warm ischemia or NDGA lOpg/kg i.v. (group III) 5 min before reperfusion or the same drugs were given together (group IV). Serum SGOT, SGPT, and LDH values and tissue malonedialdehyde (MDA), gluthathione (GSH), prostaglandin (PG)E,, and leukotriene (LT)C, levels were determined after ischemia- reperfusion injury. Histopathologic examination of the liver was carried out under the light microscope. The serum SGOT, SGPT and LDH levels improved significantly in groups II, III, and IV when compared with the control group (p c 0.05). There was a significant decrease (p < 0.05) in tissue MDA levels and significant increase (p c 0.05) in tissue GSH levels in group I, when compared with group IV and the control groups. The values did not differ significantly in group IV when compared to controls. The LTCJPGE, ratio was low and histologic findings were worse in group III. In conclusion, iloprost was found to be beneficial in preventing the ischemia-reperfusion injury in the rat livers. NDGA, either by direct toxic effect or by shifting the arachidonic acid metabolism to the cyclooxygenase route, was not found to be as effective. Iloprost and NDGA did not exert a synergistic effect.

INTRODUCTION

Reperfusion injury of the liver is a common problem en-

countered in widely performed liver transplantations in

our days (1). Ischemia and reperfusion (IR) injury of the

liver can also expected during surgery of extensive he-

patic trauma or tumors as well as after low perfusion

states (2). Various studies have demonstrated the ben-

eficial effects of different agents affecting the arachi-

donic acid (AA) pathway and metabolites (3-5) on different organ systems after IR injury. Besides other

agents such as superoxide radical scavenger enzymes

(6-9). antioxidants (10. 1 I) and xanthine oxidase (X0) inhibitors (9. 12). a synthetic stable analogue of prosta-

cyclin, iloprost (ZK 36374) (3,4) has also been found to

have beneficial effects on reperfusion injury. Lypoxy- genase inhibitors, by reducing the leukotriene (LT) for-

mation, are also effective in preventing the IR injury (13, 14).

This study was undertaken to investigate the effects of

Dare received 8 Ma) 1992 Date nccep~ecl IO July I997

prostacyclin analogue iloprost and lypoxygenase inhibi-

tor NDGA on reperfusion injury in the liver.

MATERIAL AND METHODS

Wistar Albino rats weighing 18&3_5Og were fasted 24 h

before surgery but allowed water ad libitum. The ani-

mals were anesthetized with ether and the right internal

jugular vein was cannulated for drug administration and

fluid replacement. Midline laparatomy was performed

and 100~ of heparin administered. Under the operative

microscope, the liver hilus was exposed and the blood

vessels to the left and median lobes were occluded with

a microvascular clamp as previously described (15). The

abdominal incision was temporarily closed with 3-O silk sutures and the animals were reanesthetized after 1 h of

warm ischemia. The clamp was removed and the liver reperfused. The abdomen was closed with 3-O silk su- tures after the observation of complete reperfusion.

Animals were divided into 4 groups. Rats in group I (control group n= 15) received 2cc isotonic saline before ischemia and 0.2~~ isotonic saline 5 min before reperfu- sion (i.v.). Rats in group II (iloprost treated group n=15)

292 Prostaglandins Leukotrienes and Essential Fatty Acids

were given 25,ugikg iloprost in 2cc isotonic saline and 0.2~~ isotonic saline 5 min before reperfusion iv. Rats in

group III (NDGA treated group n=l5) were given 2cc

isotonic saline before ischemia and 10 pm/kg NDGA in

0.2~~ saline 5 min before reperfusion i.v. Group IV rats

(iloprost+NDGA treated group n= 15) received 2 pug/kg

iloprost in 2cc isotonic saline before ischemia and

10 pug/kg NDGA in 0.2~~ isotonic saline i.v. Sham oper-

ated rats (group v n=4) were prepared as in group I but

the vessels were not occluded. 6 min after reperfusion,

liver biopsies from the ischemia area were obtained and

immediately immersed into liquid nitrogen to determine

LTC4 (16, 17), prostaglandin (PG) E? (16, 18), glu-

tathione (GSH) (19) and malondialdehyde (MDA) (20)

levels, as described in the literature. Liver biopsies were

also taken for histologic evaluation and stored in 4%

formaldehyde. The blood samples were obtained on the

6th min, 2nd h, 2nd day and 7th day. Serum glutamic

oxaloacetate transaminase (SGOT), serum glutamic

pyruvic transaminase (SGPT) and serum lactate dehy-

drogenase (LDH) levels were determined from these

blood samples. Light microscopic review was performed

by a single pathologist in a blinded fashion and necrosis,

picnosis, vacuolization, granulation and congestion were

rated as mild, moderate and severe. Student’s t-test was

used for statistical analysis.

RESULTS

SGOT 1)

While the SGOT were 129 + 41 u is-

1 Serum glutamic oxaloacetate transaminase (SGOT) levels. *, Significantly lower compared to control, NDGA vs iloprost + NDGA groups (p< 0.001) g, Significantly higher compared to all other groups (p < 0.001).

Fig. 2 Serum glutamic pyruvic transaminase (SGPT) levels. *, Significantly lower compared to control, NDGA vs iloprost + NDGA groups (p < 0.001) 8, Significantly higher compared to all other groups (p < 0.001).

SGPT (Fig. 2)

SGFT values were found to be 87 f 37 u before is-

chemia. SGPT values were found to have increased in all

groups when compared with the preischemic levels on

the 6th min and 2nd h. On the 6th min, SGPT values in

group II (425 k 218 u) were significantly lower than

group I (1644 -t 152 u), group III (1252 & 535 u), and

group IV (1716 f 331 u) (p < 0.001). On the 2nd h, in all treatment groups, SGPT values were found to be lower

than the control group. The difference was statistically

significant with the control group but no differences

were noted among the treatment groups. In all groups

SGPT levels returned to the preischemic values on the

2nd and 7th day.

LDH (Fig. 3)

LDH levels in the treatment groups and in the control

group were found to be significantly increased when

compared with group V on the 6th min after reperfusion.

LDH levels in group III were significantly higher than

groups I, II, and IV (p < 0.05). On the 2nd h, the values

were significantly decreased in the treatment groups

when compared with the control group (p < 0.05) but

there were no statistically significant differences among

the treatment groups. On the 2nd day, levels were sig-

nificantly lower in group II when compared with group

III and IV (p < 0.001). There were no differences on the

7th day among the groups,

Fig. 3 Serum lactate dehydrogenase (LDH) levels. *, Significantly higher compared to iloprost group (p < 0.05). 5, Significantly higher compared to all drug-treated groups (p < 0.05).

The Effect of lloprost and NDGA in lschemia Reperfusion Injury in Rat Liver 293

Fig. 4 MDA levels in liver tissue. *. Significantly lower compared to control. NDGA vs NDGA + iloprost treated groups (p < 0.001~.

MDA (Fig. 4) Histologic findings (Table 2)

MDA levels on the 6th min after reperfusion were found

to be elevated in group I when compared with the sham

operated group (p < 0.001). There were significantly

lower levels of lipid peroxidation in the iloprost treated

group but no significant differences were observed

among groups III and IV and the control group.

Severe injury was noted in the control group when com-

pared with the sham group. No marked histologic differ-

ences were observed among groups I, II, and IV but

in group III ischemic signs were more severe than the

others on the 6th min and 2nd day.

GSH (Fig. 5)

On the 6th min after reperfusion, GSH levels were found

to be decreased in the control group when compared

with the sham group. There were significantly higher

levels of GSH only in group II when compared with the control group. GSH levels were not statistically different

in groups III and IV when compared with the control

group.

LTC, and PGEz (Table 1)

There are no statistically significant differences among

the values. But decreased LTCJPGE, ratios were ob- served in NDGA treated group on the 2nd and 7th day.

On the 2nd day, the same ratio was high in the iloprost

treated group.

-

Fig. 5 The reduced GSH levels in liver tissue. *, Significantly higher compared to all other groups (p < 0.05). 8. Significantly higher compared to all other groups except for the iloprost group (p < 0.05).

DISCUSSION

The role of superoxide radicals in IR injury of different

organ systems has been demonstrated in numerous

studies (21-27). In this study, high levels of lipid

peroxidation and low GSH levels in the tissue samples

obtained 6 min after reperfusion of the liver in group I have also supported this fact. PGI,, by inhibiting throm-

bocyte aggregation, dilating the coronary vessels, sta- bilizing the lysozomal and cytoplasmic membranes,

increasing the blood flow to the splanchnic area, and

preventing tromboxane (TX) A, synthesis in trombo-

cytes, has been found to be useful in shock and ischemia

(3, 27). The cytoprotective effect of PG12 is indepen-

dent from these properties (28). It is thought that this

effect is mediated by maintaining high ATP and cyclic

nucleotide levels together with low intracellular calcium

concentrations (3).

Table 1 LTQPGE, tissue levels (nmol/g tissue) and LTCJPGE, ratios

6th min 2nd day 7th day

Sham LTC, PGE, LTCJ’GEL

Control LTC, PGE, LTCJ’GE?

Ilosprost LTC, PGEL LTCJPGE:

NDGA LTC, PGEz LTCJPGE,

LTC, Iloprost + NDGA PGEz

LTCJPGE2

I.55 k 0.23 I .47 k 0.56 3.95 k 0.57 3. 40 f 0.56

0.39 0.43

1.20 * 0.90 1.80 * 0.64 3.32 k 2.07 3.85 IO.25

0.36 0.46

1.81 kO.72 I .03 + 0.75 3.86 i I .26 1.85 + 1.19

0.46 0.55

I .74 * 0.9-J I .30 f 0.88 3.88 5 1.50 3.93 + 2.28

0.44 0.33

I .30 + 0.86 I .24 + 0.47 3.24 k 1.62 3.26 k 0.93

0.40 0.3R

1.56 i 0.84 3. 2Ok 1.36

0.48

1.12 * 0.71 2.63 + 1.34

0.42

0.95 + 1.01 2.22 + 2.30

0.42

0.72 k 0.63 1.65 + 1.47

0.43

0.73 + 0.49 2.45 k 0.98

0.29

294 Prostaglandins Leukotrienes and Essential Fatty Acids

Table 2 Degree of histological injury (<% 15: absent, %15-35: mild, %35-55:moderate. >%55: severe)

6th min 2nd day 7th day

Sham absent 2 1 I mild _ _ _

moderate _ _ _

severe _ _

Control absent 2 3 mild 3 3 2 moderate 2 _

severe _ _

Ilosprot absent I _ mild 1 _ 3 moderate 3 4 3 severe _ 1 _

NDGA absent _

mild 2 1 3 moderate 2 2 2 severe 1 2 _

lloprost + NDGA absent 1 1 _

mild 3 4 moderate 4 1 1 severe _ _ _

In this study, iloprost prevented lipid peroxidation and maintained GSH levels after reperfusion. This indicates

that the protective effect of iloprost in IR injury is also related with oxygen free radicals (OFR). Tanaka et al

demonstrated that PGI, in IR injury is not protective

alone but when infused together with superoxide dismu-

tase (SOD) and catalase, it increased the effects of these agents (4). On the other hand, Ontell et al reported the

superiority of SRI 63441, a potent platelet activating fac-

tor antagonist, to SOD and cyclooxygenase inhibitor

ibuprofen, and they emphasized the importance of inhib-

iting the trombocyte aggregation in IR injury (29). It is reported that PGIJTXA, ratio alterations are

responsible for the development of many pathologic

conditions (30). Besides the cytoprotective effect of

prostacyclins, increasing the PGI2/TXA2 ratio may lead

to decreased levels of intracellular calcium and thus pre-

vents the synthesis of OFR. Granger et al hypothesized

that decreased levels of ATP in ischemic tissue may in- crease OFR synthesis by activating the xanthine oxidase

(X0) system (8, 25). Iloprost may also have exerted its

protective effect by maintaining the ATP and CAMP

levels. Marabayashi et al state that the activity of X0 is

different in different tissues and they showed that lipid peroxidation is high when the liver is exposed to 90- 120 min of ischemia, whereas the activity of X0 in liver

tissue after this period was only 15% of the total X0 activity (3 1). This indicates that system or systems other than X0 may also be responsible for the synthesis of

OFR. It is reported that neutrophyl based myeloperoxi- dase system may also generate OFRs (2, 3, 32). Iloprost may have also affected this system.

The high LTC,/PGE: ratio in the iloprost treated group on the 2nd day is probably related to uninhibited

lypoxygenase pathway and inhibited endogen PG syn- thesis by this prostacyclin analogue. In the NDGA

treated group. liver enzymes were elevated on the 6th

min. The histologic findings were also worse in this

group. On the 2nd h, the enzyme and histologic findings

were better but still worse than the iloprost treated

group. Although the absolute levels of LTC, and PGE?

were not different, decreased levels or LTCflGE? ratio have been observed in NDGA treated group on the 2nd

and 7th day. This finding suggests that LTC, generation

has been prevented and/or PGE? increased. The inhibi-

tion of the lypoxygenase pathway shifts the AA metabo-

lism to the cyclooxygenase pathway and thus together

with increased PGE?, the increased TXA, prevents the

expected beneficial effect of NDGA. NDGA at this dos-

age may also have a direct toxic effect on the liver as

reflected by the histologic changes on the 6th min. To

differentiate the TXA, effect from the direct toxic effect

of NDGA on liver tissue. further studies with TXAl

synthetase inhibitors will be helpful. The unchanged LTCl levels in the control group and increased LTC,/

PGE, ratio in the iloprost group suggest the non-relation-

ship of LTC3 to IR injury to the liver. LTs and pro-

stanoids have been shown to have increased in many

pathologies and can exhibit synergistic effects (33). The

expected synergistic effect was not seen in this study and, furthermore, NDGA appears to have a negative

effect on the protective iloprost effect. Theoretically

NDGA should have reduced the LT formation and thus

have a protective effect on IR injury in the liver. LTs in

this study seemed to have no effect on IR injury of the

liver. LTCq was found to be elevated in the IR injury of

the brain (34) and this result may be another example of

different organ responses to IR injury. The minimal

change in PGE, and LTC, values with iloprost and

NDGA implies an interaction of lipoxygenase and

cyclooxygenase pathways. In conclusion. the beneficial effect of iloprost on the

IR injury of the liver seems to be related to the increased

PGI,nXA, ratio. The histologically demonstrated toxic

effect of NDGA may be related to its direct toxic effect

or increased TXA, levels due to the shift of the AA pathway to the cyclooxygenase route. This study also

supports the hyphothesis that the cyclooxygenase and lipoxygenase pathways are not two independent path-

ways but are interrelated.

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