EVALUATION OF EFFICACY OF TEST FORMULATIONS IN RAT MODEL OF L-ARGININE-INDUCED ACUTE PANCREATITIS

EVALUATION OF EFFICACY OF TEST FORMULATIONS IN RAT MODEL OF L-ARGININE-INDUCED ACUTE PANCREATITIS

1.0  TEST SYSTEM DETAILS:

Species                   : Rattus norvegicus (Rats)

Strain                     : Sprague Dawley or Wistar

Age                        : 6-7 weeks

Sex                        : Male

No. of animals        : 8/Group

Total animals         : 88

 

2.0  TEST ARTICLES DETAILS

HF1: Rheum emodi-based herbal formulation 

 

3.0  VEHICLE DETAILS

The test articles will be formulated by utilizing 0.5% methylcellulose as the vehicle.

4.0   ALLOCATION OF GROUPS:

 

Group No.	Group Description	Disease Induction procedure	Treatment administered	Dose Volume and Route
G1	Normal Control	Normal Saline administered by intraperitoneal route (i.p.), twice at 1-hour interval	0.5% MC, p.o., b.i.d.	5 ml/kg, p.o.
G2	Disease Control	L-Arginine-3.2 g/kg (dissolved in Normal Saline), i.p., twice at an interval of 1 hour	0.5% MC, p.o., b.i.d.
G3	Reference Control		Carvedilol-5 mg/kg, p.o., b.i.d.
G4	Treated with low dose of  HF1		HF1: 5-15 mg/kg, b.i.d.
G5	Treated with intermediate dose 1 of HF1		HF1: 15-50 mg/kg, b.i.d.
G6	Treated with intermediate dose 2 of HF1		HF1: 50-150 mg/kg,   b.i.d.
G7	Treated with high dose of HF1		HF1: 150-500 mg/kg,  b.i.d.

Abbreviations: MC: Methylcellulose, p.o.-per os. q.d.: quaque die; bid: bis in die.

5.0  METHOD:

·         Healthy animals will be selected for the study, randomized based on body weight, and will be assigned to 11 groups consisting of 8 animals each.

·         Animals of Group G1 will be designated as normal-control and administered 0.5% MC, p.o., b.i.d., two weeks prior to disease induction and will be continued till the end of the experiment.

·         Disease control animals (assigned to group G2) will receive 0.5% MC, p.o., b.i.d., two weeks prior to disease induction and will be continued till the end of the experiment.

·         Animals of group G3 will be treated with reference drug carvedilol at the dose of 5 mg/kg, p.o., b.i.d., two weeks prior to disease induction and will be continued till the end of the experiment.

·         Animals of group G4-G7 will be treated with HF1, at different incremental dose levels as outlined in Section 4.0 of Annexure-I, twice daily, two weeks prior to disease induction, and will be continued till the end of the experiment.

·     Normal control group (G1) will be administered two injections of Normal Saline by intraperitoneal route, whereas animals allocated to groups G2 – G7 will be administered two injections of 20% L-Arginine solution (3.2 g/kg, i.p.) dissolved in Normal Saline.

·         20% L-Arginine solution will be prepared in Normal Saline, its pH adjusted to 7.0 and filtered through syringe filter into a sterile tube in a properly sterilised environment.

·         Three days after administration of L-Arginine they will be sacrificed under overdose of thiopentone anaesthesia. After suitable anaesthesia but before the animal dies, blood will be collected from the retro-orbital plexus for the estimation of biochemical parameters. Immediately after the animal dies, pancreas will be weighed and one half will be fixed in 10% neutral buffered formalin for histopathology whereas the other half will be stored at -80°C for various biochemical and molecular evaluations.

 

6.0  PARAMETERS TO BE EVALUATED:

·         Body weight: Twice a week.

·         Pancreatic weight and its relative organ weight.

·         Serum biochemistry parameters: Lipase, Amylase, LDH, Total Proteins.

·         Cytokine estimation in serum: IL-10, IL-1β, TNF-α, IL-6, IL-22 (anti-inflammatory)

·      Oxidative stress parameters in the pancreas: Reduced and oxidized Glutathione, Malondialdehyde, Catalase, Total nitrate, Total anti-oxidant capacity (TAC).

·         Gene expression analysis by Real Time PCR: Pancreatitis associated protein (PAP), Nrf2, Sirt1, TNF-α, Caspase-3, iNOS, TGF-β1

·         Pancreatic amylase and Total protein estimation

·         Histopathological analysis of pancreas (Hematoxylin & Eosin-stained).

·         Immunofluorescence staining of the pancreatic tissue

 

7.0  REFERENCE(S):

1.        El Morsy, E. M. & Ahmed, M. A. E. Carvedilol attenuates L-arginine induced acute pancreatitis in rats through modulation of oxidative stress and inflammatory mediators. Chem. Biol. Interact. 327, 109181 (2020).

2.        Abdelzaher, W. Y. et al. Vinpocetine ameliorates L-arginine induced acute pancreatitis via Sirt1/Nrf2/TNF pathway and inhibition of oxidative stress, inflammation, and apoptosis. Biomed. Pharmacother. 133, 110976 (2021).

3.        Sidhu S. et al. Beneficial effects of Emblica officinalis in L-arginine-induced acute pancreatitis in rats. J Med Food. 2011 Jan-Feb;14(1-2):147-55. doi: 10.1089/jmf.2010.1108.

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