Protective Effect of Daidzein on TNBS Induced Acute Ulcerative Colitis on Rats

The present study aimed at investigating the potential protective effect of Daidzein on 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced acute ulcerative colitis in rats. Animals were treatment with TNBS 20 mg, TNBS dissolved in 50% ethanol by single intra-colonic application into the descending colon, Daidzein 40 and 80 mg/kg per oral, and standard sulfasalazine (SSZ) 360 mg/kg. for Two weeks. Colon was removed, length (CL), weight (CW), microscopic index (MI) processed for histopathological evaluation and estimation of oxidative colonmarker contents of active lipid peroxidation (MDA)myeloperoxidase (MPO), reduced glutathione (GSH). Enzymatic activity of superoxide dismutase (SOD) and serum nitrate levels were assessed. TNBS induced signi icant (p < 0.001), increase in CW, MI, oxidative marker MDA, MPO, and serum nitrate content, TNBS induced a signi icantly decrease in CL, SOD, and GSH content. Treatment of Daidzein 40 and 80mg with TNBS decreased preserved colon parameter and histology close to normal, increased (P<0.001) SOD, CAT, GSH and TNFα IL-6 and IL-8 down-regulate the levels to compare SSZ and Daidzein. Daidzein 40and 80mg restored TNBS-induced colon injury via inhibition of oxidative stress. Daidzein found to protect the TNBS Induced Acute Ulcerative Colitis in rats.

IBD, Daidzein, TNBS, MPO, MDA, SOD, TNFα IL-6 A The present study aimed at investigating the potential protective effect of Daidzein on 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced acute ulcerative colitis in rats. Animals were treatment with TNBS 20 mg, TNBS dissolved in 50% ethanol by single intra-colonic application into the descending colon, Daidzein 40 and 80 mg/kg per oral, and standard sulfasalazine (SSZ) 360 mg/kg. for Two weeks. Colon was removed, length (CL), weight (CW), microscopic index (MI) processed for histopathological evaluation and estimation of oxidative colon marker contents of active lipid peroxidation (MDA) myeloperoxidase (MPO), reduced glutathione (GSH). Enzymatic activity of superoxide dismutase (SOD) and serum nitrate levels were assessed. TNBS induced signi icant (p < 0.001), increase in CW, MI, oxidative marker MDA, MPO, and serum nitrate content, TNBS induced a signi icantly decrease in CL, SOD, and GSH content. Treatment of Daidzein 40 and 80mg with TNBS decreased preserved colon parameter and histology close to normal, increased (P<0.001) SOD, CAT, GSH and TNFα IL-6 and IL-8 down-regulate the levels to compare SSZ and Daidzein. Daidzein 40and 80mg restored TNBS-induced colon injury via inhibition of oxidative stress. Daidzein found to protect the TNBS Induced Acute Ulcerative Colitis in rats.

INTRODUCTION
In lammatory bowel diseases (IBD) are inveterate diseases of the intestine with obscure aetiology. Hereditarily vulnerable individuals are thought to have a dysregulated mucosal immune response to commensal gut lora, which comes about in bowel in lammation (Abraham and Cho, 2009). The vari-ous types of IBD are Crohn's disease (CD), Ulcerative colitis (UC) and, IBD unspeci ied (IBDU). Differences in hereditary inclination, clinical, endoscopic, and histological characteristics are used to differentiate these types (Ordás et al., 2012). Environmental factors are one of the key roles in intervening the risk of IBD, although no single environmental factor has been con irmed to have an unequivocal causative function (Bernstein, 2012).
An inappropriate and continuing in lammatory response to gut microbiota on a background of hereditary susceptibility causes ulcerative colitis (UC), a chronic idiopathic in lammatory condition of the gastrointestinal tract. UC is accelerated by a complex interaction of environmental, hereditary, and immunoregulatory components (Tazneem and Khan, 2018).
The main objective of the present study was to scrutinize Daidzein exerted anti-in lammatory effects in a 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced acute ulcerative colitis in rats.

Chemicals
TNBS Purchase from Sigma Aldrich USA, Daidzein purchase from Cyman chemical USA, sulfasalazine gift sample from Valens Molecule Private Limited, Hyderabad, all additional reagents were used AR grade.

Experimental animals
Adult wistar rats both male and female (weigh 150-180 g) procure from Sanzyme bio-analytical laboratory, Gaganphad, Hyderabad were used. All the animals were maintained at standard laboratory conditions (20 ± 2 0 C, 45-55 %RH and alternating light/ dark cycle). The animals caged in polycarbonate cage were allowed access to rat chow and water ad libitum under strict hygienic conditions. The study conducted as per guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). (IAEC/1292/09/31/A)(21-01-2109).

Induction of Colitis
UC was induced following the procedure given by (Morris et al., 1989), animals mildly anesthetized using anesthetic ether, fasted for 24h .20 mg TNBS solubilised in 50% ethyl alcohol (0.25mL) and inserted 8 cm into anus using poly urethane catheter, aided with glycerin for lubrication. Post instillation of the hapten, the rats placed in headdown position for about a minute to avert leakage. The control group received enema of physiological saline, Daidzein 40 and 80mg/mg p. and Sulfasalazine 360 mg/kg p.o as standard. Control group received vehicle in equivalent volume. The rats were inspected every day for behavioral and stool constancy. The animals forfeited after 14 days by administrating over dose of anesthetic agent (Jurjus et al., 2004;Tazneem and Razdan, 2010;da Silva et al., 2010).

Assessment of Ulcerative Colitis
Daidzein at the doses of 40 and 80 mg/kg were administered orally to the test group animals with TNBS effected colitis. Whereas, Sulfasalazine 360 mg/kg was administered to group V animal served as standard. Both Daidzein and Sulfasalazine were administered for 14 days' post TNBS inducted colitis. On 14 th day post induction of TNBS-colitis, the rats sacri iced, abdomens opened, the portion of distal colon excised, cleaned with 0.9% saline. 5 cm of segment weighed to determine the colon oedema. The results presented with reference to increased colon weight (g)/5 cm ratio, in comparison to control group.

Assessment of Colitis
8 cm of colon starting from rectum is removed, opened longitudinally, cleaned for any feacals using 0.9% saline, dried completely and weight noted (Galvez et al., 2001).

Macroscopic Assessment
Macroscopic scores for in lammations are assigned according to morphological characters of the colon according to the criteria (Prakash et al., 2008).
Qualitative analysis of TNFα, IL-6 and IL-8 by RT-PCR mRNA Extraction mRNA from each tissue sample was extracted by using GITC method (Chomzynski, 1987), cDNA was prepared using reverse transcriptase enzyme (MMuLVRT) and mRNA speci ic primer.
Constructed cDNAs were stored at -80 • C for further experimental purposes.

mRNA expression analysis
Selected in lammatory genes (TNF-α, IL6 and IL8) expression levels were quanti ied (Before treatment and after treatment with various doses) by using real time-quantitative polymerase chain reaction (ABI 7200, Applied Biosystems, Singapore) based on SYBR-Green miR relative quanti ication assay. The amounts of expression level of all the genes were calculated relative to the amount of GAPDH (used as internal control) in the same sample. Reactions were carried out in triplicates and mean Ct values were taken for to calculate the fold difference. The changes in expression of all selected genes at before and after the treatment were normalized to GAPDH (Livak and Schmittgen, 2001;Pfaf l, 2001).

Statistical Analysis
The data analyzed using statistical package of social version 17.0 (SPSS) software. Descriptive statistics used to present data in terms of mean ± SEM employing ANOVA, followed by Tukey's Multiple Comparison Test post hoc test. For in lammatory markers data, statistical analysis carried out using primer statistical software. The clinical analyzed adopting paired t test, Wilcox on test, Mann Whitney U test, Kruskal Wallis test and ANOVA. Differences were considered signi icant whenever the P value are reported as mean ± Score data expressed in terms of mean ± S.E.M, n=6. (ANOVA) followed by Tukey Multiple Comparison Test a P<0.001 vs Normal group, b P<0.01, c P<0.001 vs TNBS group.

RESULTS AND DISCUSSION
Physical variations in animals were obvious 24 hr post intra colonic instillation of TNBS. Colon length, Colon weight and macroscopic examination of cecum, colon, and rectum of TNBS-induced animals displayed indication of severe colonic mucosal damage, with edema, deep ulceration and hemorrhage The macroscopic variations in the distal colon also examined TNBS-induced animals displaced increase in colon weight/length ratio and Microscopic index (MI) indicative of in lammation extent and severity of colonic injury, as re lected in the lower macroscopic scores TNBS-treated rats as shown in [Figures 2 and 5 A-E]. Treatment of animals with Daidzein signi icantly increased colon length in a dose-dependent manner. Initially during in lammatory process taking place in TNBS -IC (induced colitis), the damage to tissues was caused by ROS stimulate in iltration of MPO-positive neutrophils signi icantly increased14 days after instillation of TNBS in comparison to control group, rats treated with Daidzein considerably decreased MPO activity in dosage-dependent pattern. GSH undergoes oxidation in presence of toxic lipids/ H 2 O 2 by action of GST. Rats with TNBS-IC have decrease in GSH level in colon in comparison to normal control, the GSH level was reinstated by treating animals with Daidzein signi icantly increased GSH level in dosage-dependent pattern. MDA these increased levels in TNBS-treated rat colons are indicative of lipid peroxidation, treatment with Daidzein signi icantly decreased MDA level in a dosage-dependent pattern. In in lammation, higher levels of ROS can upset SOD antioxidant enzymes and reduce SOD enzymatic activity, SOD level restore by treating rats with Daidzein signi icantly increased SOD level in dosge-dependent pattern.
The NO level in the colon of rat induced with TNBS appreciably augmented compared to normal group and Treating the animals with Daidzein consider-

Figure 2: Effect of Daidzeinon colonic injury in rats with TNBS-IC. [A] Colonic tissues in Control group [B] TNBS treatment group [C] TNBS and Daidzein40 mg/kg [D] TNBS and Daidzein 80 mg/kg and [E] TNBS and SSZ 360 mg/kg.
ably reduced inhibited nitrite generation in a dosedependent manner. A signi icantly lower level of the tissue concentration of levels was observed in rats administered with standard SSZ when compared to TNBS induced colitis group as shown in [Table 1 and Expression of TNF-α, IL-6, and IL-8 was found to be increased in TNBS-induced colitis compare to control. After treatment with Daidzein and SSZ, the expression of TNF-α IL-6 and IL-8 signi icantly decreased. Could down-regulate the levels of these pro-in lammatory factors in a dose-dependent manner as shown in [ Figure 4 A-C and Figure 6].
Differences were considered signi icant whenever the P value are reported as mean ± Score data expressed in terms of mean ± S.E.M, n=6.

Figure 6: Qualitative analysis TNF-α expression of Daidzein in TNBS induced colitis.
colonic in lammation in rat and this serves an established colitis model. Investigations conducted on animal models of ulcerative colitis (UC) suggested a signi icant role in understanding the disease with underlying mechanisms involved and has led to identifying different therapeutic agents, especially those with the natural origin (Jurjus et al., 2004). TNBS induced colitis model has many similarities resembled the human features of UC especially with the immunological perspective (Kawada, 2007). The ethanol carrier used in this model causes the disruption of epithal layers and the exposure of underlying layers to both TNBS agents and the bacterial component present in the colon (Kawada, 2007).
This colitis model was based on the fact that, ethanol as a vehicle damages the colonic epithelium and thereby facilitates the permeation of TNBS into the propria and behaves as an antigen by binding to the tissue (Wallace et al., 1995). In lammation produced by TNBS model causes signi icant thickening of colonic wall and incorporation of cellular in iltration and ulceration persisting for a longer period (Prakash et al., 2008). In the present study, there was actual damage to colonic mucosa and submucosa characterized by ulceration and in iltration of the in lammatory cell after TNBS administration. Increase in colon weight /length ratio and macroscopic index of colonic tissue in TNBS administered and Daidzein40, 80mg treated group was signi icantly reduced compared to the group administered with TNBS alone. Histopathology images suggest the similarities in the healing process in the Daidzein treated and SSZ treated groups of animals.
Myeloperoxidase enzyme found in neutrophils, it is an indicator of in lammatory injury to the tissues (Stein et al., 1998). The neutrophils secrete myeloperoxidase enzymes during in lammation. Hence, neutrophils counts are directly co-related with myeloperoxidase activity. Neutrophils play an important role in the oxidative process in in lammation (Zheng, 2000). Lessening in the concentration of myeloperoxidase enzyme is inferred as an anti-in lammatory effect of a drug (Stein et al., 1998). In the present study increase in MPO enzyme was found after TNBS administration. There was a signi icant reduction in the activity of MPO in Daidzein 40mg and 80mg treatment. Histopathological studies established the reduction in the MPO activity since the leukocyte in iltration level of the colonic mucosa was lower in the animals receiving the Daidzein 40, 80mg when equated with TNBS induced colitis group. This outcome could be due to the anti-in lammatory potential of Daidzein. Free radical chain reactions are potentiated by lipid peroxidation process associated with the oxidative stress. These reactions activate the in lammatory mediators thereby disrupting the integrity of the intestinal mucosal barrier system. Studies have demonstrated an increase in colonic MDA contents and a decrease in colonic SOD levels in human and experimental animal involving IBD studies (Ek et al., 2008). The levels of MDA are often used as an indicator of oxidative damage and as a marker for free radical-induced lipid peroxidation. There was a substantial reduction in the malondialdehyde levels in the Daidzein treated animals compared to TNBS effected colitis group, this may be attributed to the inhibition of lipid peroxidation (Ferhan Girgin, 2000).
Sustained in lammation in IBD is usually associated with NO. The tissue damage is weakened by targeted inhibition of inducible NO synthase (NOS) (Kolios et al., 2004). Literature indicates that an increased nitrate levels appear to be secondary to the extent of in lammation. Serum nitrous oxide levels in TNBS induced colitis group were signi icantly increased compared to the normal control group. However, in Daidzein and SSZ treated groups, NO levels were signi icantly decreased. Previous studies have suggested that increased NO level dilates vasculature, enhances vascular permeability, and inactivates the anti-oxidase activity of SOD, CAT, and GSH by reacting with hydro sul ide group (-SH) in the enzymes (Kolios et al., 2004;Lundberg et al., 1997). In the present study, Daidzein treated group showed a signi icant increase in antioxidant enzymes SOD, CAT, and GSH compared to TNBS induced colitis group, suggesting its antioxidant activity. Being the vital cytokine in "in lammation cascade" of UC, TNFα involved in stimulating the synthesis of oxygen free radicals and IL-6, IL-8, NO besides the other mediators of in lammation. Also, TNFα triggered the leucocytes promoted migration of in lammatory cells in the inter cellular matrix, thus initiating the in lammatory response by stimulating a cascade of immune cells (Neurath et al., 1997).
Ulcerative colitis is associated with a radical imbalance in the initiation of pro in lammatory and anti-in lammatory signaling pathways in the gut (de Jesus and Isidro, 2016;Zhang et al., 2016;Ramli et al., 2016). Daidzein signi icantly inhibited TNF-a, IL-6, and IL-8, thereby indicating its potential anti-in lammatory properties. The present study explains the scope of using Daidzein for treating UC with in lammation and its protective mechanism. Colo rectal cancer is associated with chronic in lammation. Therefore, it is important to counter the in lammatory mediators such as TNFα, a cytokine, and a vital in lammatory mediator that plays a signi icant role in the malignant cellular proliferation, angiogenesis, tissue invasion, and metastasis (Banday et al., 2016). The results of this study revealed that Daidzein could down-regulate the levels of pro in lammatory factors TNF-a, IL-1b, IL-6, and IL-8 in a dose-dependent manner. Results showed that Daidzein acts by countering the in lammation and oxidation and by lowering lipid peroxidation effects. Hence, Daidzein is found to be effective in experimentally induced ulcerative colitis in rats.

CONCLUSIONS
The present study's indings suggest that Daidzein possesses a protective effect against IBD induced by TNBS in rats. Daidzein's protective effect was equivalent with the standard drug, sulfasalazine, and supported by the antioxidant assays and the histopathological studies. The therapeutic effect of Daidzein evident the possible use as an alternative therapy to treat IBD. Extended study is needed to explore the actual mechanism of action, safety, and ef icacy of Daidzein in treating patients with IBD and the development of herbal remedy for the treatment of IBD.