Modulating effect of luteolin on ethanol and cerulein induced inflammation— In silico and in vivo studies using rat model of pancreatitis

NLRP3 inflammasomes are induced in pancreas during inflammatory insult, which contain caspase activation and recruitment domain (CARD) and pyrin domain (PYD) of apoptosis associated speck-like protein containing a CARD (ASC). It is known that NLRP3-ASC interaction via PYD domain recruits and activate caspase-1. Through CARD, ASC brings monomers of procaspase-1 into close proximity and activate them which in turn activate pro-inflammatory cytokines. The assembly of inflammasome and activation of procaspase-1 is thought to be the molecular target for anti-inflammatory drugs. Here, we investigated the molecular interactions of luteolin, a potent anti-inflammatory agent with procaspase-1, ASC-CARD and ASC-PYD using Acceryls Discovery Studio Visualizer. To assess the pancreato-protective effect of luteolin, rats were administered with ethanol (36% of total calories for five weeks) and cerulein (20 µg/kg body wt. i.p., weekly thrice for last three weeks). In addition, a group of rats also received 2 mg luteolin/kg body wt. orally from third week till the experimental period. Co-administration of luteolin reduced the levels of pancreatic and inflammatory marker enzymes in serum and maintained the antioxidant status in pancreas. Immunohistochemical analysis confirmed the presence of ASC protein in excess in the pancreas of ethanol-cerulein administered rats than in rats co-administered with luteolin. Luteolin satisfies Lipinski’s rule of five to determine the drug likeness property. The energy value and the hydrophobic interactions obtained in the present docking study confirmed that there was a strong binding affinity of luteolin towards procaspase-1, ASC-CARD, ASC-PYD that might interfere with the molecular assembly of NLRP3 inflammasome and the production of pro-inflammatory cytokines.

Hydroalcoholic extract of licorice (Glycyrrhiza glabra L.) root attenuates ethanol and cerulein induced pancreatitis in rats

Objective: To evaluate the therapeutic potential of hydroalcoholic extract of licorice root against ethanol and cerulein induced chronic pancreatitis in rats. Methods: The phytochemical profile of hydroalcoholic extract of licorice root was determined by high performance liquid chromatography (HPLC) and gas chromatography coupled to mass spectrometry (GC-MS). Chronic pancreatitis was induced in male albino Wistar rats by feeding them a diet containing ethanol (0%-36% of total calories) for 4 weeks and cerulein (20 μg/kg b.wt, i.p.) thrice a week for 3 weeks. Lipase and amylase in serum, lipid peroxides and antioxidants including reduced glutathione, glutathione peroxidase, superoxide dismutase and catalase in pancreas were determined. Inflammatory response was measured by myeloperoxidase in the pancreas, caspase-1 and the concentrations of IL-1 β and IL-18 in serum. Moreover, histological evaluation of the pancreas and liver was carried out. Results: Different flavonoids and saponins were identified in the hydroalcoholic extract of licorice root through HPLC and GC-MS. A marked increase in the levels of serum lipase, amylase, lipid peroxides, caspase-1, myeloperoxidase, IL-1 β, and IL-18 and a marked decrease in the levels of antioxidants were observed after ethanol and cerulein administration. Treatment with hydroalcoholic extract of licorice root attenuated these changes. In addition, histological observation confirmed the protective effect of the extract in the pancreas and liver against inflammatory changes induced by ethanol and cerulein. Conclusions: The licorice root extract attenuates ethanol and cerulein induced pancreatitis in rats probably due to its antioxidant phytonutrients since ethanol and cerulein-induced production of reactive oxygen species contributes to severe inflammation in the pancreas.

Effect of Pithecellobium dulce (Roxb.) Benth. fruit extract on cysteamine induced duodenal ulcer in rats.

The edible fruits of Pithecellobium dulce (Roxb.) Benth. are traditionally used for various gastric complications in India. Here, we investigated the antiulcer activity of hydroalcoholic fruit extract of P. dulce (HAEPD) by applying cysteamine induced duodenal ulcer model in rats. Duodenal ulcer was induced in male albino Wistar rats by oral administration of cysteamine @ 420 mg/kg body wt. as a single dose. The rats were pre-administered orally with HAEPD @ 200 mg/kg body wt. for 30 days prior to ulcer induction. Rats pre-administered with ranitidine @ 30 mg/kg body wt. served as reference drug control. Ulcer score, thiobarbituric acid reactive substances (TBARS), glycoproteins, superoxide dismutase, catalase and glutathione peroxidase and reduced glutathione levels were measured in the duodenum. Rats pre-administered with the HAEPD showed significantly reduced ulcer score comparable to that of ranitidine pretreated rats. The co-administration of HAEPD lowered the TBARS level and also restored the levels of glycoproteins, enzymatic and non-enzymatic antioxidants. Histopathological observations confirmed the presence of inflammation, necrosis and hemorrhagic spots in the duodenum of ulcer control rats which were significantly reduced due to HAEPD treatment. No abnormal alterations were observed in normal rats treated with HAEPD at the dosage studied. The results demonstrated antioxidant and cytoprotective nature of P. dulce, and thereby its significant anti ulcer property.

Effect of thymoquinone on ethanol and high fat diet induced chronic pancreatitis—a dose response study in rats.

A significant increase in serum lipase, amylase, capase-1 and myeloperoxidase activities, oxidative stress index (OSI), IL-1β and IL-18 was observed in rats receiving ethanol (EtOH) and high fat diet (HFD). Thymoquinone (TQ) supplementation along with EtOH and HFD significantly decreased the levels of serum lipase, amylase, capase-1, myeloperoxidase, OSI and maintained the antioxidant status when compared to untreated EtOH and HFD fed rats. Among the 4 doses, 100 mg of TQ/kg body weight was found to provide optimum protective effect on pancreas against EtOH and HFD induced abnormal changes. Histological observations added more evidence for the anti-inflammatory effect of TQ.

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis.

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.

Effect of ursodeoxycholic acid on hydrogen peroxide induced lipid peroxidation in sheep liver mitochondria.

The effect of various concentrations of ursodeoxycholic acid (UDCA), a potent hepatoprotective agent on hydrogen peroxide-induced mitochondrial swelling was evaluated in vitro to find out the mechanism of action of the drug. Aliquots of sheep liver mitochondria were pre-incubated with various concentrations of UDCA [0-600 micrograms] and swelling was induced by hydrogen peroxide [1 mM]. Swelling was assessed at various time intervals and lipid peroxide, reduced glutathione status were also evaluated simultaneously. UDCA minimized hydrogen peroxide-induced swelling in a dose-dependent manner. Time-dependent elevation in the level of lipid peroxides was noted in mitochondria treated with hydrogen peroxide and this elevation was minimized in UDCA pre-treatment. UDCA also maintains the reduced glutathione level in mitochondria. UDCA acts against the oxidative stress imposed in liver mitochondria. It reduces lipid peroxidation-induced abnormalities such as swelling and thiol group depletion and the anti lipid peroxidative efficacy of the drug may be related to its hydrophilic nature which might protect the hydrophobic regions of the mitochondrial membranes which are prone for free radical-mediated reactions.