EVALUATION OF ANTI-ULCERATIVE COLITIS ACTIVITY OF ORAL AND RECTAL DOSED TEST FORMULATIONS IN RAT MODEL OF DEXTRAN SULFATE SODIUM (DSS)-INDUCED ULCERATIVE COLITIS

 EVALUATION OF ANTI-ULCERATIVE COLITIS ACTIVITY OF TEST FORMULATIONS IN RAT MODEL OF DEXTRAN SULFATE SODIUM (DSS)-INDUCED ULCERATIVE COLITIS 

1.0  TEST SYSTEM DETAILS:

Species                   : Rattus norvegicus (Rat)

Age                        : 6-8 weeks

Sex                        : Male/Female

No. of animals        : 08 /Group for Study 1 and Study 2 each

Total animals         : 104 (56 animals for Study 1 and 48 animals for Study 2) 

2.0   ALLOCATION OF GROUPS:

Study 1:

Group No.	Group Description	Disease-Induction agent administered	Treatment administered	Dose Volume and Route
G1	Normal Control	Normal drinking water	0.5% MC, b.i.d.	5 mL/kg, p.o.
G2	Disease Control	4 % w/v DSS in autoclaved drinking water for 8 consecutive days	0.5% MC, b.i.d.
G3	Treated with Sulfasalazine		100 mg/kg, q.d.
G4	Treated with low dose of HF1		HF1-X1 mg/kg, b.i.d. in 0.5% MC
G5	Treated with intermediate dose 1 of  HF1		HF1-X2 mg/kg, b.i.d. in 0.5% MC
G6	Treated with  intermediate dose 2 of  HF1		HF1-X3 mg/kg, b.i.d. in 0.5% MC
G7	Treated with high dose of  HF1		HF1-X4 mg/kg,  b.i.d. in 0.5% MC

Abbreviations: MC-Methyl Cellulose, p.o.-per os; q.d.: quaque die, bid: bis in die. X1, X2, X3, X4  are defined as the incremental doses of the Ayurvedic formulations. The dose range will be from 10 mg/kg to 1000 mg/kg, b.i.d.

 

Study 2:

Group No.	Group Description	Disease- Induction agent administered	Treatment administered	Dose Volume and Route
G1	Normal Control	Normal drinking water	Water for injection	0.3 mL, i.r.
G2	Disease Control	4 % w/v DSS in autoclaved drinking water for 8 consecutive days	Water for injection
G3	Treated with low dose of HF2		HF2-X1mg/kg, q.d. formulated in sterile water
G4	Treated with intermediate dose 1 of  HF2		HF2-X2 mg/kg, q.d. in water  sterile water
G5	Treated with  intermediate dose 2 of  HF2		HF2-X3 mg/kg, q.d. in water  sterile water
G6	Treated with high dose of  HF2		HF2-X4 mg/kg,  q.d. in water  sterile water

 

Abbreviations: i.r. – intra-rectal, q.d. – quaque die. X1, X2, X3, X4 are defined as the incremental doses of the respective Ayurvedic formulations. The dose range will be from 20 mg/kg to 2000 mg/kg q.d.

 

3.0  METHOD:

·         After completion of quarantine, healthy animals will be selected for the study. Subsequently, they will be randomized based on body weight and allocated into 7 groups for Study 1 and 6 groups for study 2, consisting of 8 animals each.

·         Post-randomization, animals will be acclimatized for 5 days in an experimental room earmarked for the experiment.

·         For the induction of ulcerative colitis, animals allocated to groups G2-G7 for Study 1 and G2-G6 for Study 2 will be administered with freshly prepared DSS (4% w/v) in autoclaved drinking water for 8 consecutive days. The animals assigned to the normal-control groups in both the studies (i.e. G1) will receive normal drinking water throughout the study period.

·         Treatments to be administered:

v Study 1

Ø  Animals of the Group G1 and G2, designated as normal-control and disease-control respectively, will be administered 0.5% MC, p.o., b.i.d.

Ø  Animals of group G3 will be treated with reference drug, Sulphasalazine at the dose of 100 mg/kg, p.o., q.d.

Ø  Animals of group G4-G7 will be treated with HF-1, orally at different dose levels ranging from 10-1000 mg/kg, b.i.d.  

v Study 2

Ø  Animals of the Group G1 and G2, designated as normal-control and disease-control respectively, will be administered 0.3 mL of water for injection, q.d. by intra-rectal route.

Ø  Animals of group G3-G6 will be treated with HF2, at dose levels ranging from 20 to 2000 mg/kg, i.r., q.d.

·         The animals will be monitored daily for appearance of abnormal clinical signs and loss in body weight and scored for their stool consistency (0 = well-formed stool pellet, 2 = semi-formed stool, 4 = liquid stool that adhere to anal region) and hematochezia (0 = no blood, 2 = blood trace in the stool clearly visible, 4 = gross rectal bleeding).

·         On day 9, the animals will be sacrificed under overdose of thiopentone anaesthesia. After suitable anaesthesia but before the animal dies, blood will be collected and serum will be separated for estimation of cytokines.

·         After completion of the procedure, the colon of the sacrificed animals will be excised and length and weight will be recorded. Further, one portion of the colon will be fixed in 10% neutral buffered formalin for the histopathological analysis, whereas the other portion will be snap frozen in liquid nitrogen and stored at -80°C for biochemical and gene expression analysis.

 

4.0  PARAMETERS TO BE EVALUATED:

·         Clinical signs

·         Body weight

·         Disease Activity Index (DAI): Stool consistency and hematochezia score

·         Serum/tissue levels of TNF-α, IL-6 and IL-1β

·         Colon Length

·         Colon Weight

·         Colonic myeloperoxidase (MPO) assay

·         Estimation of protein expression of TNF, IL-6 and COX-2 in the colon

·         Histopathology of colon

 

5.0  REFERENCES:

1.        Liu, Y. et al. Pulsatilla chinensis Saponins Ameliorate Inflammation and DSS-Induced Ulcerative Colitis in Rats by Regulating the Composition and Diversity of Intestinal Flora. Front. Cell. Infect. Microbiol. 11, 1–11 (2021).

2.        Zhu, L., Gu, P. Q. & Shen, H. Protective effects of berberine hydrochloride on DSS-induced ulcerative colitis in rats. Int. Immunopharmacol. 68, 242–251 (2019).

3.        Saber, S. et al. BBG enhances OLT1177-induced NLRP3 inflammasome inactivation by targeting P2X7R/NLRP3 and MyD88/NF-κB signaling in DSS-induced colitis in rats. Life Sci. 270, 119123 (2021).

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