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Histopathological evidence of protective action of garlic against collagen and arachidonic acid toxicity in rabbits
Prostaglandins Leukotrienes and Essential Fatty Acids (1992) 46, 301-306 Q Longman Group UK Ltd 199’2
Histopathological Evidence of Protective Action of Garlic Against Collagen and Arachidonic Acid Toxicity in Rabbits M. A. Alnaqeeb*.
M. Ali, M. Thomson,
S. H. Khater, S. A. Gomes and J. M. Al-Hassan
Departments of *Zoology and Biochemistq, Faculty of Science. Kuwait University, P.O.Box 5969,13060-Safat, Kuwait (Reprint requests to M. Ali) ABSTRACT. Soluble rat tail tendon collagen produced respiratory distress, agitation, convulsions and finally death in rabbits when infused intravenously (i.v.) in lethal doses. Similar observations were noted when a lethal dose of arachidonic acid (unsaturated essential fatty acid) was infused. These agents caused thrombocytopenia, indicative of in vivo platelet aggregation, hypotension and increased levels of thromboxane (TX) B, (a stable metabolite of TXA,) in the plasma. Histopathological examination of lung, heart and liver tissue indicated that the lungs and livers of treated animals were adversely affected, while heart tissues appeared to be normal. Histopathological examination of lung and liver tissues of animals pretreated with garlic, then treated with a lethal dose of collagen or arachidonic acid showed a significant reduction in the damage observed compared to animals not pretreated with garlic.
INTRODUCTION
antibacterial, antifungal, antiviral, antitumor, hypoglycemic, hypolipidemic and antiartherosclerotic properties (8, 9). It has been shown that aqueous extracts of garlic inhibit in vitro and in vivo platelet aggregation and thromboxane formation ( 1O- 12). We have recently observed that intravenous injection of lethal doses of collagen or arachidonic acid into rabbits causes convulsions, agitation, labored breathing and death within 2 min (12). Analysis of plasma of these animals showed increased levels of enzymes, such as lactate dehydrogenase, glutamate-pyruvate transaminase and glutamate-oxalate transaminase, indicative of liver and cardiac damage (13). Increased plasma levels of thromboxane BZ (TXB,) and 6-keto-prostaglandin F,, (6-keto-PGF,,), a stable metabolite of prostaglandin I, (PGI,), were also observed after i.v. injection of soluble collagen or arachidonic acid in rabbits (12). These results suggested that the deaths of animals injected with collagen or arachidonic acid may be related to prostaglandin formation and increased plasma levels of enzymes due to damage of organs. In the present study, we wished to study the effect of garlic as a potential antithrombotic agent, and also to assess its ability to prevent tissue damage in rabbits injected i.v. with a lethal dose of collagen or arachidonic acid.
It is well known that collagen and arachidonic acid both in vitro and in vivo cause platelet aggregation (1, 2) with the subsequent release of serotonin, ADP and other platelet contents (3). These effects are not seen when platelets are pretreated with non-steroidal antiinflammatory drugs (NSAIDs), such as aspirin and indomethacin (4). The collagen in blood vessels is involved in the initial events of hemostasis when the endothelial cell lining is injured or damaged. The collagen containing subendothelium is exposed and platelets undergo adhesion and aggregation (5). It has been shown that collagen or arachidonic acid, when injected into the ear vein of rabbits, causes sudden death, associated with the occlusion of the microcirculation in the lungs by platelet aggregates (6). These platelet thrombi were not seen in the lungs of animals pretreated with NSAIDs (7). This suggests that arachidonic acid may be a key compound in hemostasis. Injection of arachidonic acid directly, or stimulation of its release by collagen from phospholipids can thus trigger platelet aggregation and formation of lethal prostaglandins. Garlic (Allhm sativum) is used as a common food item all over the world. During the past 10 years, there has been great interest in the potential medicinal uses of garlic, which has been shown to possess insecticidal,
MATERIALS AND METHODS Sample preparation
Date received 13 December 1991 Date accepted 7 January 1992
Sodium arachidonate 301
was prepared by dissolving
10 mg
302
Prostaglandins
Leukotrienes
and Essential Fatty Acids
of arachidonic acid (Sigma Chemical Co, USA) in about 50 ~1 of ethanol, followed by conversion into the sodium salt by the addition of equimolar sodium carbonate, and dilution with saline to reach the desired concentration. Lyophilized collagen was dissolved at a concentration of 4 mg/ml in 0.5 M acetic acid followed by dialysis against 0.05 M potassium phosphate buffer, pH 7.6, and then 0.4 M NaCl before injection into rabbits. Aqueous extracts of garlic were prepared from locally available fresh garlic cloves by the method of Ali and Mohammed ( 10). Treatment of animals Female, mixed breed healthy rabbits (2-2.5 kg) were used in all experiments. Rabbits were kept under observation for at least 10 days before use to allow for screening. A 21-gauge butterfly cannula was inserted into the marginal ear vein and used for the administration of sodium thiopentol (30 mg/kg) for anesthesia, garlic, collagen or arachidonate. The animals were divided into 6 groups. The animals in the first 2 groups served as controls and received either saline or garlic (500 mg/kg). Two test groups of animals received either a lethal dose of arachidonate (1.4 mg/kg) or a lethal dose of rat tail collagen (2 mg/ kg), respectively. The animals in the remaining 2 test groups were treated intravenously with an aqueous extract of garlic (500 mg/kg), and after 10 min were injected with the same lethal dose of arachidonate or collagen. Test animals not pretreated with garlic were sacrificed as soon as death seemed imminent. Those pretreated with garlic were observed for 10 min before sacrifice. Rabbits were sacrificed by exsanguination and organs were removed and processed for histological examination. Histological preparations Liver, lung and heart samples were removed for histological examination. The left lung lobe was slowly infused in situ via the trachea with 10% formylsaline. The left lobe was then ligated, excised and immersed
in formylsaline for 7 days. Segments of liver and heart samples were also removed and immersed in 10% formlysaline for 7 days. The fixative on the samples was changed daily. After the completion of fixation, the samples were dehydrated in a series of alcohols, cleared m toluene and then embedded in Paraplast. Blocks were cut at 5-7 microns. Sections were dewaxed, rehydrated in a series of alcohols. stained in Harris hematoxylin for 6 min and counterstained in 1% aqueous eosin for 1 min. Sections were then dehydrated in alcohol, cleared in xylene and mounted in DePeX. Stained sections were examined and photographed on an Opton Photomicroscope III.
RESULTS Lungs and livers of saline treated animals appeared to be normal (Figs 1A & 2A). Lungs of the garlic treated control rabbits appeared intact with thin alveolar walls and small numbers of RBCs within the alveoli (Fig. 1B). Some capillaries in the walls of alveoli appeared swollen. The RBCs in the alveoli may have originated from such capillaries. Livers of garlic treated control animals showed signs of bleeding (Fig. 2B) and pockets of intercellular RBCs were observed occasionally. Injection of a lethal dose of arachidonate or collagen into the marginal ear vein of rabbits caused rapid, shallow respiration, strong convulsions and finally death within 2 min. When animals were pretreated with garlic and then injected with a lethal dose of arachidonate or collagen, no obvious symptoms were observed. Garlic provided complete protection against the toxic effects of these two thrombogenic agents. No apparent symptoms were observed in animals receiving garlic only. These observations are summarized in the Table. Superficial examination of rabbit organs revealed clotting in the lungs of rabbits injected with collagen or arachidonate. These clots were not observed in the garlic pretreated animals. Livers of collagen or arachidonate treated rabbits were darker in appearance than livers of rabbits pretreated with garlic. Some granulation on the surface of livers of arachidonate or collagen treated
Table Prevention of lethal effects of collagen and arachidonate injection of lethal doses of collagen or arachidonic acid
by garlic. Response to intravenous
Pretreatment
Infusion
No. of animals
Mortality
Symptoms
None None None
Saline Garlic” Collagenb
10 8 9
o/10 O/8 919
Garlic None
Collagen ArachidonateC
9 11
*1/9 1 l/l 1
Garlic
Arachidonate
8
None None Rapid respiration, strong convulsion, death within 2 min None Same symptoms observed with collagen None
*l/8
* Animal died within 10 min after infusion of lethal dose of collagen or arachidonic B Garlic concentration was 500 mg/kg. b Collagen concentration was 2 t&kg. c Arachidonate concentration was 1.4 mg/kg.
acid.
Protective Action of Garlic Against Collagen and AA Toxicity
rabbits. which was absent in garlic pretreated rabbit livers, was noticed. No obvious changes in appearance were apparent in the hearts of treated animals. The above observations were confirmed upon histopathological examination of these organs. Lungs of rabbits injected with a lethal dose of arachidonate or collagen showed massive disruptions (Fig. 1C & E ). Alveolar walls were
303
thickened, more so in arachidonate treated animals, capillaries were dilated, and aggregated red blood cells (RBCs) were found blocking the lumen of capillaries. Significant edema within alveoli was also evident. Except for the more severe effects of arachidonate, no significant differences in the effects of these two thrombogenie agents on the lungs were noted. Lungs of animals
Fig. 1 Effect of garlic pretreatment followed by arachidonic acid or collagen treatment on rabbit lung. (A) Control saline injected rabbit: (B) Rabbits treated with 500 mg/kg garlic showed a small number of red blood cells within the alveoli (+): (C) Rabbits treated with 2 mg/kg collagen showed slight thickening of alveolar walls, capillaries in walls of alveoli were congested with red blood cells; (D) Rabbits pretreated with 500 mg/ kg garlic and then treated with collagen showed little disruption; (E) Rabbits treated with 1.4 mg/kg arachidonic acid showed massive disruptions, alveolar walls were thickened, capillaries were dilated and blocked with red blood cells: (F) Rabbits pretreated with 500 mgikg garlic and then treated with arachidonic acid were similar to garlic control. Size of bar = 100 pm.
304
Prostaglandins
Leukotrienes
and Essential Fatty Acids
pretreated with garlic and then injected with a lethal dose of collagen or arachidonate showed little disruption (Fig. ID & F). Alveolar walls were thin, and little bleeding and edema were noticed. No clumping or aggregation of RBCs was noted in the microcirculation of the lung. Livers of rabbits treated with arachidonate or collagen showed significant numbers of RBCs appearing inbetween cells (Fig. 2C & E). The aggregation of these RBCs was also evident in blood vessels. Hepatic cells
showed vacuolation in the cytoplasm. Again, except for the more severe effect of arachidonate, no differences between the effects of collagen or arachidonate were found. Livers from animals pretreated with garlic contained very little intercellular RBCs (Fig. 2D & F). Cytoplasmic vacuolation was observed occasionally in arachidonate, but not collagen treated animals. No clumping or aggregation of RBCs in capillaries was noted in either group of garlic pretreated animals.
Fig. 2 Effect of garlic pretreatment followed by arachidonic acid or collagen treatment on rabbit liver. (A) Control saline injected rabbit; (B) Rabbits treated with 500 mgikg garlic showed a small number of red blood cells in-between cells (+): (C) Rabbits treated with 2 mg/kg collagen showed necrosis with a large number of vacuoles of varying size appearing in the cytoplasm of hepatocytes (-1). red blood cells were also evident intercellularly; (D) Rabbits pretreated with 500 mgikg garlic and then treated with collagen showed little disruption; (E) Rabbits treated with 1.4 mgikg arachidonic acid also showed vacuolation and intercellular aggregates of red blood cells (+): (F) Rabbits pretreated with 500 mg/kg garlic and then treated with arachidonic acid were similar to garlic controls. Size of bar = 50 ,nm.
Protective Action of Garlic Against Collagen and
DISCUSSION Histopathological results clearly indicate that intravenous injection of toxic collagen or arachidonic acid causes extensive damage to the lungs and liver, which may have been a major factor leading to death in the rabbits. Massive platelet thrombi present in the vessels of the microcirculation of the lung were probably the major cause of death in animals injected with the thrombogenic agents, collagen and arachidonic acid. This agrees with earlier observations that i.v. injection of a lethal dose of arachidonic acid caused sudden death in rabbits (7). Garlic pretreated rabbits were protected from the toxic effects of collagen and arachidonic acid. Very few thrombi were observed in the vessels of the microcirculation of the lungs of garlic pretreated animals. Similar observations were made previously in aspirin pretreated animals receiving a lethal dose of arachidonic acid intravenously (7). The histopathological study of livers confirmed our previous observations that the levels of plasma enzymes in rabbits injected with different preparations of collagen were elevated indicating liver damage ( 13). The livers of rabbits injected i.v. with collagen or arachidonic acid exhibited significant numbers of RBCs appearing inbetween cells. The aggregation of RBCs was also evident in the liver blood vessels. Hepatic cells showed vacuolation in the cytoplasm. In garlic pretreated animals, almost complete protection against the effects of collagen or arachidonic acid were observed. These results suggest that the cause of death in these animals was due to the blockage of the microcirculation of the lungs by platelet aggregates. Such aggregates could be partially responsible for the tissue damage by blocking blood flow and causing bursting of blood vessels. However, other factors may have been involved, such as bronchoconstriction caused by the production of thromboxane AZ (TXA?) by either platelets (14) or lung tissue (II?), perhaps as a result of the disruptive influence of clusters of platelet aggregates (16). The present data also support our previous observation that collagen and arachidonic acid caused thrombocytopenia, hypotension and elevation of plasma TXB, in animals receiving a lethal dose of either arachidonic acid or collagen intravenously (6, 17). In the same studies, it was also observed that non-steroidal antiinflammatory drugs inhibited the thrombocytopenia and blocked the elevation of TXB?. Similar observations were made in a recent study when an aqueous extract of garlic was used instead of NSAIDs ( 12). These observations provide evidence that the toxic effects observed are mediated, at least in part, through the formation of toxic TX. These results show that arachidonic acid may play an important role in hemostasis and thrombosis, since arachidonic acid and collagen can cause platelet aggregation and TX formation in vitro and in vivo. Perhaps collagen and other aggregating agents such as adenosine diphosphate and thrombin may be responsible for triggering phos-
pholipase
activity
and causing
release
AA Toxicity 305 of arachidonic
acid, which, in turn, becomes converted into lethal TXs and PGs. Since garlic was given i.v., some effects, such as slight edema and some intercellular RBCs, were observed in the lungs of garlic control animals. These effects may have been avoided if the garlic was administered orally or intraperitoneally. More work is being conducted to investigate these possibilities. In summary, our results suggest that garlic may be a potent antithrombotic agent in rabbits, since it can prevent the lethal effects of the thrombogenic agents, collagen and arachidonic acid. Thus, the present study contributes to a better understanding of the biochemical mechanisms of thrombosis, and may lead to a more effective preventive treatment for thrombosis.
Acknowledgements This study was supported by Kuwait University which the authors are grateful.
Grant No. SBO 26, for
References R L, Remiers H K, Mustard J F. 1. Kinlough-Rathbone Packham M A. Sodium arachidonate can induce platelet shape change and aggregation which are independent of the release reaction. Science 192: 1011, 1976. 2. Ali M, McDonald J W D. Effects of sulfinpyrazone on platelets prostaglandin synthesis and platelet release of serotonin. .I. Lab. C/in. Med. 89: 868, 1977. 3. Hamberg M, Svensson J. Sammuelson B. Thromboxanes, a new group of biological active compounds derived from prostaglandin endoperoxides. Proc. Natl. Acad. Sci. b’.S.A. 72: 2994, 1978. 4. Ali M. McDonald J W D. Reversible and irreversible inhibition of platelet cyclooxygenase and serotonin release by non-steroidal antiinflammatory drugs. Thromh. Res. 13: 1057, 1978. 5. Harker L A, Ross R. Glomset J A. The role of endothelial cell injury and platelet response in atherogenesis. Thromh. Res. 39: 312. 1978. 6. Cerskus A L, Ali M, McDonald J W D. TXBz and 6-ketoPGF,, during infusion of collagen and arachidonic acid in rabbits. Inhibition by aspirin and sulfinpyrazone. Thromh. Rrs. 18: 693, 1980. 7. Silver M J, Hoch W, Kocsis J J, Ingerman C M, Smith J B. Arachidonic acid causes death in rabbits. Scieflc,e 183: 1085. 1974. 8. Lau B H S. Adetumbi M A, Sanchez A. Allium sari\wm (garlic) and atherosclerosis: A review. Nurr. Res. 3: I 19. 1983. 9. Adetumbi M A. Lau B H S. Album sativum (garlic) - A natural antibiotic. Med. Hypotheses 12: 227. 1983. 10. Ali M. Mohammad S Y. Selective suppression of platelet thromboxane formation with sparing of vascular prostacyclin synthesis by aqueous extract of garlic in rabbits. PI-osra~qlandins, Leukotrienes and Medicine 25: 139, 1986. 11. Srlvastava K C. Effects of aqueous extract of onion, garlic and ginger on platelet aggregation and metabolism of arachidonic acid in blood vascular systems. In vitro study. Prostucglandins. Leukotr-ienes and Medicine 11: 177. 1984. 12. Ali M. Thomson M, Alnaqeeb M A. Al-Hassan J M. Khater S H. Gomes S A. Antithrombotic activity of garlic: Its inhibition of the synthesis of TXB? during infusion of arachidonic acid and collagen in rabbits. Prosraglandins, Lcukotrienes and Essenfial Fatty Acids 41: 95. 1990.
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Prostaglandins
Leukotrienes
and Essential Fatty Acids
13. Gomes S A. Thomson M, Ali M, Alnaqeeb M A, Khater S H. Collagen toxicity in rabbits. To,ricol. Left. 56: 229. 1991. 14. Ali M, Cerskus A L, Zamecnik J. McDonald J W D. Synthesis of prostaglandin D? and thromboxane B? by human platelets. Thromb. Res. 11: 485, 1977. 15. McDonald J W D, Ali M, Morgan E. Townsend E R, Cooper J D. Thromboxane synthesis by sources other than platelets in association with complement-induced
pulmonary leukostasis and pulmonary hypertension in sheep. Circul. Res. 51: 1, 1983. 16. Piper P. Vane J. The release of prostaglandins from lung and other tissues. Atm. N. Y. Acad. Sci. 180: 362. 1971. 17. Cerskus A L, Ali M, Zamecnik J, McDonald J W D. Effects of indomethacin and sulfinpyrazone on in vivo formation of TXB, and PGD, during arachidonate infusion in rabbits. Thromb. Res. 12: 549, 1978.
Histopathological Evidence of Protective Action of Garlic Against Collagen and Arachidonic Acid Toxicity in Rabbits M. A. Alnaqeeb*.
M. Ali, M. Thomson,
S. H. Khater, S. A. Gomes and J. M. Al-Hassan
Departments of *Zoology and Biochemistq, Faculty of Science. Kuwait University, P.O.Box 5969,13060-Safat, Kuwait (Reprint requests to M. Ali) ABSTRACT. Soluble rat tail tendon collagen produced respiratory distress, agitation, convulsions and finally death in rabbits when infused intravenously (i.v.) in lethal doses. Similar observations were noted when a lethal dose of arachidonic acid (unsaturated essential fatty acid) was infused. These agents caused thrombocytopenia, indicative of in vivo platelet aggregation, hypotension and increased levels of thromboxane (TX) B, (a stable metabolite of TXA,) in the plasma. Histopathological examination of lung, heart and liver tissue indicated that the lungs and livers of treated animals were adversely affected, while heart tissues appeared to be normal. Histopathological examination of lung and liver tissues of animals pretreated with garlic, then treated with a lethal dose of collagen or arachidonic acid showed a significant reduction in the damage observed compared to animals not pretreated with garlic.
INTRODUCTION
antibacterial, antifungal, antiviral, antitumor, hypoglycemic, hypolipidemic and antiartherosclerotic properties (8, 9). It has been shown that aqueous extracts of garlic inhibit in vitro and in vivo platelet aggregation and thromboxane formation ( 1O- 12). We have recently observed that intravenous injection of lethal doses of collagen or arachidonic acid into rabbits causes convulsions, agitation, labored breathing and death within 2 min (12). Analysis of plasma of these animals showed increased levels of enzymes, such as lactate dehydrogenase, glutamate-pyruvate transaminase and glutamate-oxalate transaminase, indicative of liver and cardiac damage (13). Increased plasma levels of thromboxane BZ (TXB,) and 6-keto-prostaglandin F,, (6-keto-PGF,,), a stable metabolite of prostaglandin I, (PGI,), were also observed after i.v. injection of soluble collagen or arachidonic acid in rabbits (12). These results suggested that the deaths of animals injected with collagen or arachidonic acid may be related to prostaglandin formation and increased plasma levels of enzymes due to damage of organs. In the present study, we wished to study the effect of garlic as a potential antithrombotic agent, and also to assess its ability to prevent tissue damage in rabbits injected i.v. with a lethal dose of collagen or arachidonic acid.
It is well known that collagen and arachidonic acid both in vitro and in vivo cause platelet aggregation (1, 2) with the subsequent release of serotonin, ADP and other platelet contents (3). These effects are not seen when platelets are pretreated with non-steroidal antiinflammatory drugs (NSAIDs), such as aspirin and indomethacin (4). The collagen in blood vessels is involved in the initial events of hemostasis when the endothelial cell lining is injured or damaged. The collagen containing subendothelium is exposed and platelets undergo adhesion and aggregation (5). It has been shown that collagen or arachidonic acid, when injected into the ear vein of rabbits, causes sudden death, associated with the occlusion of the microcirculation in the lungs by platelet aggregates (6). These platelet thrombi were not seen in the lungs of animals pretreated with NSAIDs (7). This suggests that arachidonic acid may be a key compound in hemostasis. Injection of arachidonic acid directly, or stimulation of its release by collagen from phospholipids can thus trigger platelet aggregation and formation of lethal prostaglandins. Garlic (Allhm sativum) is used as a common food item all over the world. During the past 10 years, there has been great interest in the potential medicinal uses of garlic, which has been shown to possess insecticidal,
MATERIALS AND METHODS Sample preparation
Date received 13 December 1991 Date accepted 7 January 1992
Sodium arachidonate 301
was prepared by dissolving
10 mg
302
Prostaglandins
Leukotrienes
and Essential Fatty Acids
of arachidonic acid (Sigma Chemical Co, USA) in about 50 ~1 of ethanol, followed by conversion into the sodium salt by the addition of equimolar sodium carbonate, and dilution with saline to reach the desired concentration. Lyophilized collagen was dissolved at a concentration of 4 mg/ml in 0.5 M acetic acid followed by dialysis against 0.05 M potassium phosphate buffer, pH 7.6, and then 0.4 M NaCl before injection into rabbits. Aqueous extracts of garlic were prepared from locally available fresh garlic cloves by the method of Ali and Mohammed ( 10). Treatment of animals Female, mixed breed healthy rabbits (2-2.5 kg) were used in all experiments. Rabbits were kept under observation for at least 10 days before use to allow for screening. A 21-gauge butterfly cannula was inserted into the marginal ear vein and used for the administration of sodium thiopentol (30 mg/kg) for anesthesia, garlic, collagen or arachidonate. The animals were divided into 6 groups. The animals in the first 2 groups served as controls and received either saline or garlic (500 mg/kg). Two test groups of animals received either a lethal dose of arachidonate (1.4 mg/kg) or a lethal dose of rat tail collagen (2 mg/ kg), respectively. The animals in the remaining 2 test groups were treated intravenously with an aqueous extract of garlic (500 mg/kg), and after 10 min were injected with the same lethal dose of arachidonate or collagen. Test animals not pretreated with garlic were sacrificed as soon as death seemed imminent. Those pretreated with garlic were observed for 10 min before sacrifice. Rabbits were sacrificed by exsanguination and organs were removed and processed for histological examination. Histological preparations Liver, lung and heart samples were removed for histological examination. The left lung lobe was slowly infused in situ via the trachea with 10% formylsaline. The left lobe was then ligated, excised and immersed
in formylsaline for 7 days. Segments of liver and heart samples were also removed and immersed in 10% formlysaline for 7 days. The fixative on the samples was changed daily. After the completion of fixation, the samples were dehydrated in a series of alcohols, cleared m toluene and then embedded in Paraplast. Blocks were cut at 5-7 microns. Sections were dewaxed, rehydrated in a series of alcohols. stained in Harris hematoxylin for 6 min and counterstained in 1% aqueous eosin for 1 min. Sections were then dehydrated in alcohol, cleared in xylene and mounted in DePeX. Stained sections were examined and photographed on an Opton Photomicroscope III.
RESULTS Lungs and livers of saline treated animals appeared to be normal (Figs 1A & 2A). Lungs of the garlic treated control rabbits appeared intact with thin alveolar walls and small numbers of RBCs within the alveoli (Fig. 1B). Some capillaries in the walls of alveoli appeared swollen. The RBCs in the alveoli may have originated from such capillaries. Livers of garlic treated control animals showed signs of bleeding (Fig. 2B) and pockets of intercellular RBCs were observed occasionally. Injection of a lethal dose of arachidonate or collagen into the marginal ear vein of rabbits caused rapid, shallow respiration, strong convulsions and finally death within 2 min. When animals were pretreated with garlic and then injected with a lethal dose of arachidonate or collagen, no obvious symptoms were observed. Garlic provided complete protection against the toxic effects of these two thrombogenic agents. No apparent symptoms were observed in animals receiving garlic only. These observations are summarized in the Table. Superficial examination of rabbit organs revealed clotting in the lungs of rabbits injected with collagen or arachidonate. These clots were not observed in the garlic pretreated animals. Livers of collagen or arachidonate treated rabbits were darker in appearance than livers of rabbits pretreated with garlic. Some granulation on the surface of livers of arachidonate or collagen treated
Table Prevention of lethal effects of collagen and arachidonate injection of lethal doses of collagen or arachidonic acid
by garlic. Response to intravenous
Pretreatment
Infusion
No. of animals
Mortality
Symptoms
None None None
Saline Garlic” Collagenb
10 8 9
o/10 O/8 919
Garlic None
Collagen ArachidonateC
9 11
*1/9 1 l/l 1
Garlic
Arachidonate
8
None None Rapid respiration, strong convulsion, death within 2 min None Same symptoms observed with collagen None
*l/8
* Animal died within 10 min after infusion of lethal dose of collagen or arachidonic B Garlic concentration was 500 mg/kg. b Collagen concentration was 2 t&kg. c Arachidonate concentration was 1.4 mg/kg.
acid.
Protective Action of Garlic Against Collagen and AA Toxicity
rabbits. which was absent in garlic pretreated rabbit livers, was noticed. No obvious changes in appearance were apparent in the hearts of treated animals. The above observations were confirmed upon histopathological examination of these organs. Lungs of rabbits injected with a lethal dose of arachidonate or collagen showed massive disruptions (Fig. 1C & E ). Alveolar walls were
303
thickened, more so in arachidonate treated animals, capillaries were dilated, and aggregated red blood cells (RBCs) were found blocking the lumen of capillaries. Significant edema within alveoli was also evident. Except for the more severe effects of arachidonate, no significant differences in the effects of these two thrombogenie agents on the lungs were noted. Lungs of animals
Fig. 1 Effect of garlic pretreatment followed by arachidonic acid or collagen treatment on rabbit lung. (A) Control saline injected rabbit: (B) Rabbits treated with 500 mg/kg garlic showed a small number of red blood cells within the alveoli (+): (C) Rabbits treated with 2 mg/kg collagen showed slight thickening of alveolar walls, capillaries in walls of alveoli were congested with red blood cells; (D) Rabbits pretreated with 500 mg/ kg garlic and then treated with collagen showed little disruption; (E) Rabbits treated with 1.4 mg/kg arachidonic acid showed massive disruptions, alveolar walls were thickened, capillaries were dilated and blocked with red blood cells: (F) Rabbits pretreated with 500 mgikg garlic and then treated with arachidonic acid were similar to garlic control. Size of bar = 100 pm.
304
Prostaglandins
Leukotrienes
and Essential Fatty Acids
pretreated with garlic and then injected with a lethal dose of collagen or arachidonate showed little disruption (Fig. ID & F). Alveolar walls were thin, and little bleeding and edema were noticed. No clumping or aggregation of RBCs was noted in the microcirculation of the lung. Livers of rabbits treated with arachidonate or collagen showed significant numbers of RBCs appearing inbetween cells (Fig. 2C & E). The aggregation of these RBCs was also evident in blood vessels. Hepatic cells
showed vacuolation in the cytoplasm. Again, except for the more severe effect of arachidonate, no differences between the effects of collagen or arachidonate were found. Livers from animals pretreated with garlic contained very little intercellular RBCs (Fig. 2D & F). Cytoplasmic vacuolation was observed occasionally in arachidonate, but not collagen treated animals. No clumping or aggregation of RBCs in capillaries was noted in either group of garlic pretreated animals.
Fig. 2 Effect of garlic pretreatment followed by arachidonic acid or collagen treatment on rabbit liver. (A) Control saline injected rabbit; (B) Rabbits treated with 500 mgikg garlic showed a small number of red blood cells in-between cells (+): (C) Rabbits treated with 2 mg/kg collagen showed necrosis with a large number of vacuoles of varying size appearing in the cytoplasm of hepatocytes (-1). red blood cells were also evident intercellularly; (D) Rabbits pretreated with 500 mgikg garlic and then treated with collagen showed little disruption; (E) Rabbits treated with 1.4 mgikg arachidonic acid also showed vacuolation and intercellular aggregates of red blood cells (+): (F) Rabbits pretreated with 500 mg/kg garlic and then treated with arachidonic acid were similar to garlic controls. Size of bar = 50 ,nm.
Protective Action of Garlic Against Collagen and
DISCUSSION Histopathological results clearly indicate that intravenous injection of toxic collagen or arachidonic acid causes extensive damage to the lungs and liver, which may have been a major factor leading to death in the rabbits. Massive platelet thrombi present in the vessels of the microcirculation of the lung were probably the major cause of death in animals injected with the thrombogenic agents, collagen and arachidonic acid. This agrees with earlier observations that i.v. injection of a lethal dose of arachidonic acid caused sudden death in rabbits (7). Garlic pretreated rabbits were protected from the toxic effects of collagen and arachidonic acid. Very few thrombi were observed in the vessels of the microcirculation of the lungs of garlic pretreated animals. Similar observations were made previously in aspirin pretreated animals receiving a lethal dose of arachidonic acid intravenously (7). The histopathological study of livers confirmed our previous observations that the levels of plasma enzymes in rabbits injected with different preparations of collagen were elevated indicating liver damage ( 13). The livers of rabbits injected i.v. with collagen or arachidonic acid exhibited significant numbers of RBCs appearing inbetween cells. The aggregation of RBCs was also evident in the liver blood vessels. Hepatic cells showed vacuolation in the cytoplasm. In garlic pretreated animals, almost complete protection against the effects of collagen or arachidonic acid were observed. These results suggest that the cause of death in these animals was due to the blockage of the microcirculation of the lungs by platelet aggregates. Such aggregates could be partially responsible for the tissue damage by blocking blood flow and causing bursting of blood vessels. However, other factors may have been involved, such as bronchoconstriction caused by the production of thromboxane AZ (TXA?) by either platelets (14) or lung tissue (II?), perhaps as a result of the disruptive influence of clusters of platelet aggregates (16). The present data also support our previous observation that collagen and arachidonic acid caused thrombocytopenia, hypotension and elevation of plasma TXB, in animals receiving a lethal dose of either arachidonic acid or collagen intravenously (6, 17). In the same studies, it was also observed that non-steroidal antiinflammatory drugs inhibited the thrombocytopenia and blocked the elevation of TXB?. Similar observations were made in a recent study when an aqueous extract of garlic was used instead of NSAIDs ( 12). These observations provide evidence that the toxic effects observed are mediated, at least in part, through the formation of toxic TX. These results show that arachidonic acid may play an important role in hemostasis and thrombosis, since arachidonic acid and collagen can cause platelet aggregation and TX formation in vitro and in vivo. Perhaps collagen and other aggregating agents such as adenosine diphosphate and thrombin may be responsible for triggering phos-
pholipase
activity
and causing
release
AA Toxicity 305 of arachidonic
acid, which, in turn, becomes converted into lethal TXs and PGs. Since garlic was given i.v., some effects, such as slight edema and some intercellular RBCs, were observed in the lungs of garlic control animals. These effects may have been avoided if the garlic was administered orally or intraperitoneally. More work is being conducted to investigate these possibilities. In summary, our results suggest that garlic may be a potent antithrombotic agent in rabbits, since it can prevent the lethal effects of the thrombogenic agents, collagen and arachidonic acid. Thus, the present study contributes to a better understanding of the biochemical mechanisms of thrombosis, and may lead to a more effective preventive treatment for thrombosis.
Acknowledgements This study was supported by Kuwait University which the authors are grateful.
Grant No. SBO 26, for
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