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2 By Hussam A.S. Murad and Khaled A. Mahmoud Department of Pharmacology and Therapeutics Faculty of Medicine, Ain Shams University

3 Systemic modest inflammation is defined as 2-4 fold increase in circulating levels of proinflammatory mediators & acute-phase proteins (as C- reactive protein) and minor increase in neutrophils & natural killer cells. It accompanies aging, smoking, obesity and some chronic disorders as CV, DM type 2, colorectal cancer, stroke, COPD and Alzheimer’s disease. New more sensitive assays for proinflammatory mediators have demonstrated an increased risk of mortality in persons who were previously thought to have plasma levels within the normal range Bacterial infection causes systemic endotoxemia, however, modest endotoxemia also occurs due to absorption of toxins produced by Gram-negative intestinal flora. This modest endotoxemia is enhanced by alterations in diet, alcohol consumption, GI disease, liver disease, anesthesia and surgery and this may initiate a modest inflammation in tissues. Ranitidine is H 2-receptor antagonist used in treatment of peptic ulcer. It causes sinus bradycardia through block of H 2-receptor in heart and may cause idiosyncratic liver injury in human. The present work was designed to investigate whether bradycardia and hepatic injury induced by ranitidine are exaggerated in a rat model of modest inflammation or not.

4 Induction of modest inflammation and effects of ranitidine: The endotoxin produced by Gram-negative bacteria contains lipopolysaccharide (LPS) as its main active component. LPS is used experimentally to induce inflammation. The rats were subjected to recording of ECG and measurement of serum ALT (alanine aminotransferase) & AST (aspartate aminotransferase ) and GGT (γ-glutamyltransferase). Only rats with normal findings were included in the study. 4 groups (6/group) were fasted for 24 hours and given the followings intravenously: Group I (Control group) : 0.5 ml saline. Group II (LPS group): Lipopolysaccharide (44.4 x 106 units/kg, a non-hepatotoxic dose) to induce a modest inflammation. Group III (RAN group): Ranitidine (30 mg/kg, a non-hepatotoxic dose). Group IV(LPS+RAN group): LPS & after 2 hours RAN in the previous doses. The rats in all groups were subjected to : (I) Determination of ECG changes :   The rat's limbs were connected to electrodes of Nihon Kohden Cardiofax by needles.   The apparatus was switched on lead II and run at a paper speed of 25 mm/ second. (II) Measurement of serum ALT & AST (markers of hepatocellular necrosis i.e. hepatic parenchymal cell injury) and GGT (a marker of hepatic cholestasis i.e. biliary injury ):   By the enzymatic methods after 24 hours of receiving the previous treatments

5 (I) Effects of lipopolysaccharide (LPS), ranitidine (RAN) and the combination of both (LPS+RAN) on ECG in rats : LPS produced a non-significant change in heart rate in contrast to both RAN and the combination groups which produced significant reductions compared with the control group. Both RAN and the combination groups produced significant reductions compared with LPS group. The combination group produced a non- significant change compared with RAN group. (II) Effects of lipopolysaccharide (LPS), ranitidine (RAN) and the combination of both (LPS+RAN) on serum ALT, AST and GGT in rats : LPS and RAN produced non-significant changes while the combination produced significant increases compared with the control group. On the other hand, ranitidine produced non- significant changes while the combination produced significant increases compared with LPS group. The combination produced significant increases compared with ranitidine.

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7 Systemic modest inflammation Some patients may suffer idiosyncratic drug reactions after the first or second administration of the drug, while others may suffer after weeks or months of therapy. Mild inflammation during the course of therapy may render tissues especially liver more susceptible to idiosyncratic reactions. Liver is the most vulnerable because it contains most (80–90%) of the body's fixed macrophages (i.e. Kupffer cells), which are highly sensitive to activation by inflammatory and xenobiotic agents. Also liver remove these agents from the circulation protecting other organs.Moreover, the liver is the first organ exposed to larger concentrations of agents absorbed from the intestine. Ranitidine is given intravenously in resistant ulcer, patients unable to take oral medication, in prophylaxis of recurrent hemorrhage or in prophylaxis of upper GI hemorrhage from stress ulcer in seriously ill patients in ICU.

8 LPS-induced modest inflammation exaggerates hepatic injury induced by ranitidine in rats due to many causes. LPS/RAN-induces expression of several genes controlling the inflammatory mediators (as COX-II products, IFN-γ, IL 1&6, PAF, reactive oxygen species and toxic proteases). Expression of these genes is confirmed by real-time PCR. The coagulation system and neutrophils are mediators of LPS/RAN-induced hepatic injury. Heparin reduces this injury indicating a link between hemostasis-induced gene expression and inflammation in the genesis of LPS/RAN-hepatotoxic injury. LPS/RAN-induced hepatotoxicity is accompanied by a marked neutrophil infiltration & fibrin deposition and it is inhibited by antineutrophil antibodies and heparin. This suggests that LPS- potentiated toxicity involves a combination of fibrin deposit-induced hypoxia and neutrophil-mediated cell damage. Receptors for LPS and other inflammagens have been identified on inflammatory cells leading to activation of these cells, synthesis and release of proinflammatory mediators which are essential in defense against pathogens but also alter the homeostasis of host cells.

9 Gut-derived LPS plays a role in the pathogenesis of liver diseases like fibrosis. Alkaline phosphatase in the intestinal wall and liver dephosphorylates LPS. LPS enhances alkaline phosphatase expression in hepatocytes leading to an attenuation of LPS-induced responses in vivo. Thus gut-derived alkaline phosphatase and its upregulation within hepatocytes by LPS may be a protective mechanism. Ranitidine causes sinus bradycardia through block of H 2-receptor in heart. Patients with ranitidine-induced bradycardia have experienced it with a single injection of ranitidine or with oral administration. Modest inflammation induced by a small non-hepatotoxic dose of LPS exaggerates the idiosyncratic hepatic injury but not bradycardia induced by a non-toxic dose of ranitidine in rats. Clinically, this should be taken into consideration in case of use of ranitidine in conditions associated with systemic low-level inflammation as aging, smoking, obesity and some chronic medical disorders as cardiovascular diseases, type 2 diabetes, colorectal cancer, stroke,chronic obstructive pulmonary disease (COPD) and Alzheimer’s disease. Additional work is recommended to study effects on other organs like kidney and lung.

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