Effect of leflunomide on liver regeneration after partial hepatectomy in rats.

PURPOSE: Partial hepatectomy (PH) can be an inevitable surgical therapy in some conditions, such as hepatic malignancies, trauma or partial liver transplantation. Its capacity for regeneration distinguishes the liver from other essential organs. Regeneration is a complex process involving growth factors, cytokines, transcription factors, hormones, and oxidative stress products. In the event of ineffective or total absent liver regeneration, the life threatening picture of acute liver failure may supervene. In the present research, we studied the effect of leflunomide, a novel immunosuppressive and antiinflammatory agent against autoimmune disease, on hepatic regeneration after PH in Wistar Albino rats. METHODS: Thirty-five Wistar albino rats were divided into five groups: group 1, control; group 2, sham; group 3, drug control (was treated with leflunomide 10 mg/kg/d/i.g.); group 4, PH; group 5, PH + leflunomide. As for PH, approximately 70% of the rat liver was surgically removed under general anesthesia. On postoperative day 3, all rats were humanely killed. Catalase (CAT), superooxide dismutase (SOD) and myeloperoxidase (MPO) activities with malondialdehyde (MDA), nitric oxide and protein carbonyl (PC) levels were determined in remnant liver tissue. Inflammatory process and liver regeneration were evaluated with H&E and KI67, respectively. RESULTS: The tissue levels of MDA, PC and MPO were lower in group 5 than levels in group 1. PH significantly decreased the enzymatic activity of CAT (p < 0.05) and SOD. This reduction was significantly improved by the treatment with leflunomide. Histopathologically the enhancement of the liver parenchymal regeneration in the group 5 was significantly greater than the group 4. CONCLUSION: The findings imply that oxidative stress products play a preventive role in liver regeneration after PH and leflunomide ameliorates the regeneration probably by the radical scavenging and antioxidant activities.

Polyenylphosphatidylcholine pretreatment protects rat liver from ischemia/reperfusion injury.

BACKGROUND: Hepatic injury induced by ischemia/reperfusion following surgery, transplantation, or circulatory shock combined with resuscitation is a major clinical problem. Polyenylphosphatidylcholine (PPC) has strong antioxidant, cytoprotective and anti-inflammatory effects. AIM: In this study, the influence of PPC pretreatment on ischemia-reperfusion (I/R) injury of the liver was examined in rats. METHODS: The animals were divided into three groups: control (n=10), I/R (n=15) and I/R+PPC (n=15). PPC was given 100mg/day for 7 days before experiment. Several parameters of hepatic damage, oxidative stress, neutrophil infiltration and nuclear factor kappa beta (NF-kappaB) expression were measured as well as microscopic examination. RESULTS: We observed that a significant reduction in AST and ALT values in the PPC treated group when compared with the ischemic group. The increases in hepatic total NO(2)+NO(3) and MDA, and decreases in SOD and GSH levels after reperfusion were partially, but significantly, inhibited by PPC pretreatment. I/R induced increase in hepatic myeloperoxidase content and NF-kappaB expression were also lowered by PPC pretreatment. Animals pretreated with PPC presented minimal hemorrhage and reduced signs of liver injury. CONCLUSION: PPC pretretament provided significant protection againts I/R injury to the liver. This treatment could be therapeutic in liver transplantation and other conditions associated with I/R injury.

Attenuation of ureteral obstruction-induced renal injury by polyenylphosphatidylcholine.

BACKGROUND: The cytoprotective, antioxidant and antifibrotic effects of polyenylphosphatidylcholine (lecithin, PPC) have been demonstrated both experimentally and clinically. The present study investigated whether PPC treatment has any beneficial effect on renal injury in unilateral partial ureteral obstruction (UUO) in rats. METHODS: Forty Wistar-Albino rats were split into three groups (sham-operated controls, untreated and treated rats). Rats of the untreated and treated groups (n = 15) underwent UUO with two-thirds of the left ureter embedded in the psoas muscle. In group 3, PPC was given orally at a dose of 100 mg/day for 30 days. At the end of the 30th day of the experimental period, obstructed kidneys and blood samples were harvested. To investigate the therapeutic efficacy of PPC treatment in UUO kidneys, oxidant and antioxidant enzyme levels, lipid peroxidation, proinflammatory cytokines (interleukin-1, interleukin-6, tumor necrosis factor alpha), transforming growth factor beta-1 (TGFbeta-1), alpha smooth muscle actin (alpha-SMA) and nuclear factor kappa beta (NF-kappabeta) expression, leukocyte infiltration (ED1, ED2, CD4 and CD8 immunohistochemistry), and tubulointerstitial damage in the obstructed kidneys were studied. RESULTS: Oxidative stress, neutrophil infiltration, release of cytotoxic mediators, TGFbeta-1 levels, tubulointerstitial damage, alpha-SMA and NF-KB expressions in kidney tissue were significantly increased in the UUO rats. PPC treatment attenuated oxidative stress, leukocyte infiltration, cytotoxic mediator, and TGFbeta-1 levels and also decreased expressions of alpha-SMA and NF-kappabeta. It was associated with decreased tubulointerstitial damage, compared with UUO alone. CONCLUSIONS: These results indicate that PPC treatment protects against UUO-induced renal injury in rats possibly through its antioxidant, anti-inflammatory and antifibrotic actions.

Fluvastatin reduced liver injury in rat model of extrahepatic cholestasis.

Inhibitors of 3-hydroxy-3methylglutarly coenzyme A, reductase, namely statins, exert pleiotropic actions beyond lipid-lowering effects. In ex vivo and in vitro studies, statins have antioxidative and antiinflammatory effects. Herein, we sought to determine whether treatment with fluvastatin (FV) would be beneficial in a rat model of common bile duct ligation (BDL)-induced liver injury. Female rats were subjected to a sham (n=10) or BDL (n=20). Obstructive jaundice was induced in rats by the ligation and division of the common bile duct. Three days after operation, rats subjected to CBDL were randomized to receive treatment with either FV (10 mg/kg) or saline every day over a 10 days experimental period. High levels of alanine aminotransferase, aspartate aminotransferase, and gamma glutamyltransferase decreased significantly (P<0.05) in animals treated with FV with compared to saline-administrated BDL animals. Compared with sham-operated rats, CBDL rats showed significantly higher levels of total nitrite and nitrate, malondihaldehyde, tumor necrosis factor alpha, myeloperoxidase, and lower concentrations of glutathione, superoxide dismutase, and catalase in the liver tissue (P<0.001). All of these changes were significantly attenuated (P<0.05) by treatment with FV after CBDL. CBDL was associated with increased apoptosis and nuclear factor kappa beta expression in saline-treated rats. Treatment with FV also decreased these parameters. These data support the view that FV ameliorates hepatic inflammation, lipid peroxidation, and tissue injury in rats subjected to CDBL. FV warrants further evaluation as an adjunctive treatment to ameliorate liver injury from extrahepatic biliary obstruction.

Sulfasalazine reduces inflammatory renal injury in unilateral ureteral obstruction.

The purpose of this study was to test whether sulfasalazine has a protective action against interstitial inflammation and the development of renal fibrosis in obstructive nephropathy. Female rats were subjected to a sham (n = 10) or unilateral ureteral obstruction (UUO, n = 30). UUO was induced in rats by ligating the left ureter. Three days after operation, rats subjected to UUO were randomized to receive tretment with either sulfasalazine (100 mg/kg) or vehicle every day for the last 7 days of the experiment. At 10 days following UUO, the obstructed kidney exhibited tubulointerstitial injury and leukocyte infiltration (mainly monocytes) that were associated with high levels of reactive oxygen species, cytokines, transforming growth factor (TGF)-beta1, myeloperoxidase (MPO), and lipid peroxidation. Ten days after UUO, the obstructed kidney was also associated with increased nuclear factor kappa beta (NF-kappabeta) expression in saline-treated rats. Compared with sham-operated rats, UUO rat kidneys showed lower concentrations of antioxidant enzymes in the obstructed kidney tissue. All of these changes were significantly attenuated by treatment with sulfasalazine in the obstructed kidney. Sulfasalazine protected against the renal interstitial inflammation and tissue damage elicited by ureteral occlusion. Inhibition of the NF-kappabeta-dependent pathway and inflammatory response and oxidative stress inhibition is likely to be involved in the beneficial effects of sulfasalazine.

Protective effects of leflunomide against ischemia-reperfusion injury of the rat liver.

Hepatic ischemia-reperfusion (I/R) injury may be developed in some conditions, such as trauma, major hepatic resection, hemorrhagic shock or liver transplantation. I/R injury of the liver causes hepatocellular damage that may lead to hepatic failure. A considerable body of evidence indicates that reactive oxygen species (ROS) and inflammation may contribute to hepatocellular injury in liver I/R. Leflunomide is an isoxazole derivative, and a unique immunomodulatory agent. In the present study, we examined the effects of leflunomide on the neutrophil activation with oxidative stress and some antioxidant enzymes in the reperfusion following I/R in the rat liver. Thirty-two rats divided into four groups: group 1 (control); was given leflunomide 10 mg/kg, i.g.; group 2 (SHAM), animals were only laparotomized; group 3 (liver I/R), and group 4 (liver I/R + Leflunomide). In group 4, rats were pretreated with leflunomide (10 mg/kg, i.g.) two doses prior to experiment. In groups 3 and 4, occluding the hepatic pedicel for 60 min induced ischemia and reperfusion was allowed thereafter for 60 min. At the end of the reperfusion period, rats were sacrificed. superoxide dismutase, catalase, nitric oxide, xanthine oxidase, malondialdehyde, protein carbonyl and myeloperoxidase levels were determined in hepatic tissue as well as histological examination with H and E staining. Group 3 animals demonstrated severe deterioration of liver morphology and a significant liver oxidative stress. Pretreatment of animals with leflunomide markedly attenuated morphological alterations and neutrophil activation, reduced elevated oxidative stress products levels and restored the depleted hepatic antioxidant enzyme. The findings imply that ROS play a causal role in I/R-induced hepatic injury, and leflunomide exerts hepatoprotective effects probably by the anti-inflammatory effect with radical scavenging and antioxidant activities.

Prevention of renal ischemia/reperfusion-induced injury in rats by leflunomide.

OBJECTIVE: There is increasing evidence to suggest that toxic oxygen radicals play an essential role in the pathogenesis of ischemia/reperfusion (I/R) injury in the kidney. This study was designed to investigate the effects of leflunomide, an isoxazole derivative and a unique immunomodulatory agent, in I/R-induced renal injury in rats. METHODS: Forty female Sprague-Dawley rats were divided equally into four groups: (I) control (only leflunomide 10 mg/kg, intragastrically treated); (II) sham operated (only unilateral nephrectomy); (III) I/R; and (IV) leflunomide (10 mg/kg for two doses prior to experiment) plus I/R groups. In groups III and IV, after unilateral nephrectomy, the rats were subjected to 60 min of left renal pedicle occlusion, followed by 6 h of reperfusion. At the end of the reperfusion period, rats were killed and kidneys and blood were removed. Catalase, myeloperoxidase and xanthine oxidase activities, and malondialdehyde, nitric oxide and protein carbonyl levels were determined in renal tissue. Serum creatinine, blood urea nitrogen and aspartate aminotransferase were measured for the evaluation of renal function. In histopathological examination, renal damage was scored 0-3. RESULTS: Group III animals demonstrated severe deterioration of renal function, renal morphology and a significant renal oxidative stress. Pretreatment of animals with leflunomide markedly attenuated renal dysfunction, morphological alterations, reduced elevated oxidative stress products levels and restored the depleted renal antioxidant enzyme. CONCLUSION: The findings imply that oxygen radicals play a causal role in I/R-induced renal injury, and leflunomide exerts renoprotective effects probably by the radical scavenging and antioxidant activities with immunomodulatory effect.

Hepatic damage in biliary-obstructed rats is ameliorated by leflunomide treatment.

Cholestasis, or impaired bile flow, occurs in a wide variety of liver diseases and causes hepatic damage by retention and accumulation of toxic hydrophobic bile salts inducing persistent inflammation and oxidative stress. In the present research, we studied the effect of leflunomide, a novel immunosuppressive and anti-inflammatory agent against autoimmune disease, on hepatic damage produced by double ligature of the extrahepatic biliary duct in Wistar Albino rats. Cholestasis was done by double ligature and section of the extrahepatic biliary duct (BDL). Leflunomide was given i.g. 10 mg/kg/day. The severity of cholestasis and hepatic injury was determined by changes in the plasma enzyme activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and levels of direct bilirubin. Malondialdehyde (MDA), protein carbonyl (PC), nitric oxide (NO), catalase (CAT) and superoxide dismutase (SOD) were determined to the oxidative status in the liver tissue. Myeloperoxidase (MPO) activity and levels of tissue hydroxyproline (HPR) were determined to neutrophil activation and collagen accumulation, respectively. Further, histological changes were studied. Treatment with leflunomide markedly reduced serum transaminase activities as compared to BDL rats. At the same time leflunomide significantly inhibited increases in liver MDA, PC and NO levels and also attenuated the depletion of CAT and SOD in the liver after bile duct ligation. Similarly, increase in tissue MPO activity and HPR due to BDL was also attenuated by leflunomide treatment. These findings were supported by histopathological findings. These findings suggested that leflunomide can attenuate hepatic damage in extrahepatic cholestasis by prevention of oxidative stress and inflammatory process.

The NF-kappaB inhibitors attenuate hepatic injury in bile duct ligated rats.

Cholestasis-induced liver injury during bile duct obstruction causes an inflammatory response and this inflammatory process may be an important source of tissue injury. We hypothesized that NF-kappaB inhibition would decrease liver injury in a rat model of extrahepatic biliary obstruction. A total of 40 female rats of Sprague-Dawley strain were allocated to four groups. First group was sham operated control. The second group underwent common bile duct ligation (BDL) and was monitored for 10 days. Third group of rats underwent BDL and received pyrrolidine dithiocarbomate (PDTC) at a dose of 100 mg/kg/day intraperitoneally. Fourth group underwent BDL and received sulfasalazine at a dose of 100 mg/kg b.w. Both inhibitors were administered once a day throughout last 7 days of the experimental period. Rats were terminated 10 days after sham operation or BDL. Aspartate aminotransferase, alanine aminotransferase, gamma-glutamil transpeptidase, and tumor necrosis factor-alpha levels were elevated in the BDL group as compared to the control group, while this increase was significantly decreased by treatment with PDTC and sulfasalazine (P < 0.05). Hepatic GSH, SOD and catalase levels were significantly depressed by BDL, but were elevated back to control levels in NF-kappaB inhibitor-treated BDL groups. Increases in tissue free radical and MDA levels and MPO activity due to BDL were reduced back to control levels by NF-kappaB inhibitor treatment (P < 0.05). Similarly histological damage in the BDL rats was reduced by treatments. These results indicate that inhibitors of NF-kappaB activity such as PDTB and sulfasalazine exert a therapeutic effect on cholestatic liver injury in rats with BDL through anti-inflammatory and antioxidant actions.

Polyenylphosphatidylcholine pretreatment ameliorates ischemic acute renal injury in rats.

AIM: Polyenylphosphatidycholine has been demonstrated to have antioxidant, cytoprotective and anti-inflammatory effects. Whether polyenylphosphatidycholine pretreatment affects ischemia/reperfusion-induced renal damage in vivo is not known and was investigated here in rats. METHODS: Forty female Sprague-Dawley rats were divided into three groups. Group 1 (n = 10) was given saline (control, sham operated). Group 2 (n = 15) were given saline, and Group 3 (n = 15) were given polyenylphosphatidycholine (100 mg/day for 10 days prior to experiment). Groups 2 and 3 were subjected to bilateral renal ischemia (60 min) followed by reperfusion (6 h). After the reperfusion period, the rats were sacrificed and kidney tissue superoxide dismutase, glutathione, total nitrite and nitrate, malondialdehyde and myeloperoxidase levels, plasma aspartate aminotransferase, blood urea nitrogen and creatinine concentrations, and nuclear factor kappa beta expression were determined. RESULTS: Serum levels of aspartate aminotransferase, blood urea nitrogen and creatinine were significantly decreased (P < 0.05) in the treatment group compared to those in the ischemic group. There were significant differences between treatment and ischemic groups regarding the tissue superoxide dismutase, glutathione, total nitrite and nitrate, malondialdehyde, and myeloperoxidase levels (P < 0.05). In addition, polyenylphosphatidycholine pretreatment reduced nuclear factor kappa beta expression in ischemic kidney tissue. Kidneys obtained from rats pretreated with polyenylphosphatidycholine demonstrated marked reduction of the histological features of renal injury compared to kidneys obtained from Group 2 rats, including a little vacuolization, pyknosis and necrosis. CONCLUSIONS: Polyenylphosphatidycholine pretreatment provided significant protection against ischemia/reperfusion injury to the kidney. This treatment could be therapeutic in kidney transplantation and other conditions associated with ischemia/reperfusion injury to the kidney.

Glial cell line-derived neurotrophic factor and synaptophysin expression in pelviureteral junction obstruction.

OBJECTIVES: To examine the expression of neuronal markers in congenital pelviureteral junction (PUJ) obstruction as a causative factor. The findings from some investigations have suggested that defective neuronal innervation may play an important role in the pathogenesis of PUJ obstruction. METHODS: Using specific antibodies, we studied the neuronal markers of specimens from 12 cases of PUJ obstruction and 10 normal PUJs by immunohistochemistry using glial cell line-derived neurotrophic factor (GDNF), synaptophysin, S-100, and neurofilament. RESULTS: In the PUJ obstruction specimens, staining with hematoxylin-eosin and Masson's trichrome revealed muscular hypertrophy and an increase in collagen tissue and fibrosis in the lamina propria and tunica muscularis. The most striking finding on immunohistochemistry was the marked nuclear staining of cells with synaptophysin in all layers of the PUJ obstruction specimens that was totally absent in the normal PUJ specimens. In addition, significantly less intense staining for GDNF was found in the PUJ obstruction specimens compared with the normal PUJ specimens. The underexpression of GDNF in PUJ obstruction specimens was localized in the muscular layer especially. Immunohistochemical staining for S-100 and neurofilament showed no differences in the expression level of these neuronal markers in normal and PUJ obstruction specimens. CONCLUSIONS: Because GDNF is a survival factor for central and peripheral neurons, defective expression of GDNF could play an important role in the defective neuronal innervation of PUJ obstruction. Intense nuclear expression of synaptophysin in all layers of obstructed PUJ specimens suggested that obstructed PUJs have a serious structural abnormality.

Delayed gastric emptying in gastroesophageal reflux disease: the role of malrotation.

The association between gastroesophageal reflux (GER) and intestinal malrotation (IM) has been well described. Delayed or impaired gastric emptying in IM is thought to be a contributing factor in the development of gastroesophageal reflux disease (GERD). The current study assessed the role of malrotation in delayed gastric emptying in children with GERD. We also evaluated the interactions between GERD, malrotation, gastric pH abnormalities, and gastric dysmotility. Sixty-seven patients between 1 and 5 years of age (mean 3.08+/-1.2) and with symptoms of GER, such as emesis, reactive or recurrent lung disease, and/or growth retardation, were studied in 2001-2005. Upper and lower gastrointestinal contrast studies were performed for the diagnosis of malrotation. Gastric motility was evaluated with a liquid gastric emptying protocol. GER was documented by upper gastrointestinal studies, scintigraphy, and/or 24-h pH monitoring. In our series of 44 children with GERD, there was an unexpectedly high incidence of IM: 54.5% (24/44). IM has previously been known to occur in 25% of patients with GERD. GERD was found in 24 (82.7%) of 29 patients with IM. Mean nuclear gastric emptying (MNGE) was 51.6+/-8.04 min in patients with isolated GERD and 96.6+/-20.5 min in children with IM and GERD. There was a statistically significant difference in MNGE time (p<0.05) between children with primary GERD and in those with GERD and IM. Esophageal pH monitoring showed that mean fraction time below pH 4 was 7.06+/-1.1% in patients with isolated GERD and 14.7+/-4.1% in patients with IM and GERD. GERD is common in children between 1 and 5 years old. Using gastric emptying studies and esophageal pH monitoring, we have shown that gastric dysmotility and esophageal pH abnormalities are highly prevalent, especially in children with malrotation compared with children with isolated GERD. These findings suggest that malrotation is an important factor responsible for delayed gastric emptying in GERD. Hence, we recommend that all infants and children with GERD and delayed gastric emptying undergo careful evaluation for malrotation.