Cassia fistula leaves extract profiling and its emphasis on induced ulcerative colitis in male rats through inhibition of caspase 3 and cyclooxygenase-2

2.4.1 DPPH free radical scavenging activity

Measurements were also made of the absorbance of the reference compound ascorbic acid and the DPPH radical without antioxidant (control) (Oboh, 2005). Every calculation was made in three duplicates and averaged. The following formula was used to determine the DPPH radical's percentage inhibition (PI):

The study utilized the 50 % inhibitory concentration (IC₅₀) and graphical representations of the dose–response curve to determine the concentration required to inhibit the (DPPH) radical by 50 %. This approach allowed for the assessment of the effectiveness of the tested compounds in scavenging the DPPH radical.

2.4.2 Ferric reducing antioxidant power (FRAP)

The formula for the (FRAP) of the methanol extract was derived following the method outlined by Nsimba et al. (2008). The reaction progress was tracked by measuring the absorbance change at 593 nm using the Milton Roy Spectronic 1201 spectrophotometer in Houston, Texas, USA. This approach allowed for the quantification of the antioxidant power of the methanol extract.

2.4.3 Total antioxidant capacity (TAC)

The extract TAC was assessed utilizing a phospho-molybdenum test and spectrophotometric analysis. With a few adjustments, the technique was carried out as Saeed et al. (2012) had previously reported. Milton Roy, Spectronic 1201, Houston, TX, USA was used to measure the absorbance using a UV–visible spectrophotometer at 695 nm against a blank sample. Gallic acid equivalents were calculated from the TAC values (mg/g of dry material). The reference substance was butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA).

2.6.1 Experimental and Animal Ethics

Three hundred and fifty male albino rats weighing between one hundred and two hundred grams were acquired at the age of ten weeks from the Egyptian Organisation for Biological Products and Vaccines located in Cairo, Egypt. The rats were acclimatized for ten days under controlled conditions of 25 ± 3 °C, with a regular light/dark cycle, and were provided with unrestricted access to water and laboratory meal pellets during this period. All experimental procedures were approved and performed in compliance with the guide lines of the Research Ethical Committee of the Faculty of Pharmacy, Suez Canal University (Approval No. 202005 M1).

2.6.2 Induction of Ulcerative Colitis and Treated Groups

After a 24-hour fast with unrestricted access to water, the animals' stomach contents were emptied. The procedure that Hagar et al. (2007) had previously outlined was used to produce colitis. An intraperitoneal injection of KX rat cocktail (0.1 mL/100 g rat weight) was used to sedate each rat. includes 9.1 mg/kg of xylazine and 91 mg/kg of ketamine. A 2 mm diameter polyurethane enteral feeding tube was placed to a depth of 4.5 cm, and 2 mL of 3 % AA was injected into the rectum through it. The rats were kept in the Trendelenburg position during reinstallations and for a minute after stellations to stop solution leakage. The rats' diarrhea or rectal bleeding after 24 h suggested that colitis had been induced (Alsharif et al., 2022) C. fistula extract of doses (200 and 400 mg/kg) (Bhakta et al., 1999) were dissolved in 0.1 % DMSO and administered orally to the rats as indicated in the following groups.

Seven groups of five rats each were used in this experimental study, and they were split randomly as follows:

• Group I: For 17 days, 0.1 % dimethyl sulfoxide (DMSO) was administered transrectally to the control group.

• Group II: Colitis group, in which 14 days of 0.1 % DMSO transrectally were administered after 3 days of no therapy to produce ulcerative colitis (UC).

• Group III: After transrectally administering AA for three days straight to induce colitis, 500 mg/kg (Owusu et al., 2020) of sulfasalazine medication for body weight was given for 14 days.

• Group IV: Following UC induction, MLE 200 mg/kg of body weight was administered for 14 days.

• Group V: Following UC induction, MLE 400 mg/kg of body weight was administered for 14 days.

• Group VI: UC was induced after 14 days of MLE 200 mg/kg of body weight being given as a protective dose.

• Group VII: UC was produced after 14 days of MLE 400 mg/kg of body weight being given as a protective dose.

2.6.3 Determination of body weight, and macroscopic scores

The body weight of each animal was measured at the start of the experiment and again after 21 days for every group. The colon's weight after the trial was assessed, as was the colon's weight-to-length ratio.

Macroscopic inflammation scores were assigned based on clinical features of the colon using an arbitrary scale ranging from 0 to 4 as follows: 0 (no macroscopic changes), 1 (mucosal erythema only), 2 (mild mucosal edema, slight bleeding or small erosions), 3 (moderate edema, slight bleeding ulcers or erosions), 4 (severe ulceration, edema, and tissue necrosis) (Salama et al., 2020).

Stool consistency was assessed with the following scoring criteria: 0 for normal, 1 and 2 for loose stool, and 3 and 4 for diarrhea.

2.6.4 Collection of blood and tissue

Blood samples were swiftly collected from the medial retro-orbital venous plexus of starved rats under anesthesia, using capillary tubes (Micro Hematocrit Capillaries, Mucaps) (El-Shenawy et al., 2006). The collected blood was processed to extract serum, which was then centrifuged for 15 min at 4000 rpm to assess liver biomarkers. Additional blood collected in tubes containing EDTA was reserved for hematological analysis.

A suitable colonic slice was completed after adipose tissue was removed and a cool, normal saline rinse was administered. Colon homogenates were extracted from the remaining sections and stored for biochemical research at 80 ⁰C. Colonic segments including the third section were preserved in 10 % neutral buffered formalin for further histological and immunohistochemical examination.

2.6.5 Hematological evaluation

Cell counts were conducted using the Humalog System Analyzer, providing data on white blood cells (WBCs), red blood cells (RBCs), platelets count, hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC). The measurement included the number of WBCs per cubic milliliter of blood, and WBC differentiation was also assessed.

2.6.6 Liver biomarker evaluation

Serum activities of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) were assessed using kits from Sentinel Diagnostics (Milan, Italy). Additionally, total bilirubin and protein levels were determined through spectrophotometry with commercial kits from Boehringer Mannheim GmbH (Mannheim, Germany). In the protein assay, the reaction with copper in the biuret reagent led to an increase in absorbance at an alkaline pH. The resulting rise in absorbance at 550 nm, attributed to the formation of a colored complex, was directly proportional to the concentration of protein in the reaction (Gornall, 1949).

2.6.7 Assessment of oxidative stress and antioxidants

Malondialdehyde (MDA) a marker of lipid peroxidation (LPO) in mucosal tissue induced by reactive oxygen species was measured in serum by the thiobarbituric acid assay (TBA) as previously described by Ahmed and Zaki (2009).

In addition, serum antioxidant defense enzymes that counteract the oxidative stress in colonic tissues were estimated. Superoxide dismutase (SOD) was determined according to Nishikimi et al. (1972). Catalase (CAT) was responsible for the conversion of H₂O₂ into H₂O and was measured as outlined in Aebi (1984). Glutathione peroxidase (GPx) was determined as previously mentioned by Paglia and Valentine (1967). Reduced glutathione (GSH) levels were estimated based on the method of Beutler et al. (1969), who reported that the 5,5′-dithiobis- (2-nitrobenzoic acid) is reduced by the SH group to form 1 mol of 2-nitro-5-mercaptobenzoic acid per mole of SH. Finally, serum total antioxidant capacity was evaluated following the method described (Han et al., 2012).

2.6.8 Histopathological study

The collected colon tissues were preserved for 24 h in 10 % neutral buffered formalin, followed by a tap water wash, dehydration, and xylene clearance. The samples were then regularly prepared to yield paraffin-embedded slices that were 5–7 µm thick, and they were then stained with H&E stain for light microscopy analysis (Nagahashi et al., 2017).

2.6.9 Immunohistochemical (IHC) evaluation of cyclooxygenase-2 (COX-2) and caspase-3

In colonic tissues, the activity of COX-2 (an inflammatory marker) and Caspase-3 (a marker for apoptosis) was determined by IHC. Sections from colon tissues were prepared and stained with anti-caspase 3 (Catalog number GTX30246, GeneTex, Irvine, CA, USA) and anti-COX-2 (Catalog number ab16701, Abcam, Cambridge, UK.) and after preparation according to the methods mentioned (Chang et al., 2019; Eltamany et al., 2021), respectively. The immunoreaction was visualized using 3,3-diaminobenzidine (Power-Stain™ 1.0 Poly HRP DAB Kit for Mouse + Rabbit, GENEMED, South San Francisco, CA, USA) as a chromogen, with Mayer’s hematoxylin used as a counterstain. Selected portions of the colon were captured with a digital camera (Olympus Dp25). Quantification of COX-2 and Caspase-3 reactivity was performed through image analysis software (ImageJ version 1.53 h). The results were expressed as the mean area of immunopositive cells (IHC) per mm2, calculated using a specific equation: $$\%\mathrm{I}\mathrm{H}\mathrm{C}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}=\mathrm{I}\mathrm{H}\mathrm{C}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}/\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}\times 100$$

3.4.1 Rat body weight and colon weight-To-length ratio in AA-induced colitis

Acetic acid (AA) led to a notable decrease in rat body weight within the positive control group. Simultaneously, in the UC (ulcerative colitis) group, AA significantly increased the ratio of wet weight to length, a marker comparable to that of the negative control rats. When compared to the UC group, treatment with sulfasalazine demonstrated a significant decrease in the wet weight/length ratio and showed a notable increase in the percentage change in rat body weight, particularly when administered alongside prevention and treatment involving C. fistula at doses of 200 mg/kg/day and 400 mg/kg (Table 4). The disease activity index of cores of colitis is mentioned in Table 4.

Data presented as mean ± S.E (n = 5). Colon length = 19 cm. I; control, II; acetic acid, III; treated with sulfasalazine drug, IV; protection with a low dose (200 mg/kg), V; protection with a high dose (400 mg/kg), VI; treatment with a low dose and VII; treatment with a high dose. ^a significant difference as compared to control, ^b Significant difference as compared to acetic acid, ^c significant difference as compared to protection with a low dose, ^d significant difference as compared to treatment with a low dose, and ^e significant difference as compared to treatment with sulfasalazine drug (p ≤ 0.05). Macroscopic inflammation scores were assigned based on clinical features of the colon using an arbitrary scale ranging from 0 to 4 as follows: 0 (no macroscopic changes), 1 (mucosal erythema only), 2 (mild mucosal edema, slight bleeding or small erosions), 3 (moderate edema, slight bleeding ulcers or erosions), 4 (severe ulceration, edema, and tissue necrosis)

The phenolic bioactive chemicals, which are present in variable amounts in different extracts of C. fistula can operate either individually or synergistically to give rise to their antioxidant capabilities and pharmacological effects. Motivated by the aforementioned considerations, we have evaluated the therapeutic potential (as a prophylaxis and treatment) of C. fistula against acetic acid-induced UC in rats. In the current study, AA was administered intra-rectally causing a drastic reduction in the body weight of the rats. Weight loss in colitis is brought on by dietary deficiencies due to decreased appetite, food aversion or malabsorption, and rapid loss of bodily fluids owing to diarrhea and colon hemorrhage. These observations are in parallel with a study described by Owusu et al. (2020). The body weight is significantly affected by the standard drug and extract. The protection with the extract is more effective in the body weight than the treatment, especially when using a high dose.

3.4.2 Macroscopic alterations in AA-induced UC

After being longitudinally opened and sanitized with ordinary saline, colons were examined (Fig. 4A-4G). When the colons of the control group were inspected, it was found that the mucosa and serosa were both intact and there were no signs of tissue loss or bleeding (Fig. 4A). Investigation revealed considerable bleeding, extensive ulceration, and tissue edema over a sizable surface area in the acetic acid-induced group (Fig. 4B). Erosions and damage to the mucosal lining were seen (green arrows). Sulfasalazine and treated groups showed the best level of protection against mucosal injury and tissue necrosis, both at high and low dosages, with no obvious bleeding (Fig. 4C, 4E, and 4G). Pretreated groups did not have severe damage to the rat colons or tissue erosions, despite the slight bleeding (Fig. 4D).

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Fig. 4

Macroscopic imaging: A) Shows the colonic section of the control group (Group I), B) The colonic section of acetic-acid-induced colitis (Group II). C) The colonic section of the sulfasalazine-treated group (Group III). D) The colonic section of the pretreated group with a low dose (Group IV). E) The colonic section of the pretreated group with a high dose (Group V). F) The colonic section of the treated group with a low dose (Group VI). G) The colonic section of the treated group with a high dose (Group VII).

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A considerable rise in the rats' colon weight/length ratio was seen in the AA group. This is due to goblet cell hyperplasia, necrosis, severe tissue edema, and inflammatory cell infiltration (El-Abhar et al., 2008). When rats were treated with methanol-leaf extract (MLE), fecal pellets free of obvious blood or mucus stains were observed. This could be a result of the mucus layer not being damaged and the prevention of severe blood loss, both of which are indications that prospective anti-ulcerative medications were successful in preventing the condition (Al-Rejaie et al., 2012; Owusu et al., 2020). It is well known that the mucus layer helps to speed up the healing of chemically caused epithelium injury and also reduces diarrhea and blood loss through feces (Awaad et al., 2017). Therefore, it should come as no surprise that MLE preservation of the mucus layer improved colonic ulceration. Our findings validate that administering MLE to rats before and after AA administration has a significant impact on UC. This is evident through the reduction in the macroscopic examination score, preservation of the intestinal mucosa, and a conspicuous decrease in inflammation of the colon tissue.

3.4.3 Hematological parameters and leukocytes

After acetic acid-induced colitis, the levels of RBCs, HGB, HCT, MCV, and MCH dramatically decreased as compared to the control group, However, the level of PLT significantly increased (Table 5). Sulfasalazine-treated rats had significantly reduced PLT counts and significantly increased RBCS, HGB, and HCT levels when compared to positive control animals. Rats prophylactically administered low and high dosages of crude extract had considerably more RBCs, HGB, HCT, and PLT than UC animals. RBC and HCT levels considerably rose in the low and high treatment groups in comparison to the UC group. The number of RBCs in the low-dosage treatment and MCH and MCHC in the low-dose preventative groups both increased significantly as compared to the sulfasalazine-treated group.

In AA-induced colitis, the WBC count significantly increased while the eosinophil level significantly dropped in comparison to the control group (Table 6). Both the sulfasalazine-treated group and the ones treated with low and high dosages of extract showed a significant decrease in neutrophils and monocytes, and a significant rise in lymphocytes and eosinophils. The number of WBCs and neutrophils significantly dropped in a low dose (200 mg/kg) prophylactic group of C. fistula. The number of RBCs in the low-dosage treatment group and MCH and MCHC in the low-dose preventative group both significantly increased in contrast to the sulfasalazine-treated group.

The WBC count significantly increased in acetic acid-induced colitis (Table 6). In the sulfasalazine-treated group, as well as in the low and high doses of C. fistula-treated groups, neutrophils, and monocytes were dramatically decreased, whereas lymphocytes and eosinophils were significantly increased. When the extract was administered prophylactically at 200 mg/kg, the number of WBCs and neutrophils drastically decreased.

Data presented as mean ± S.E (n = 5). WBCs are white blood cells. I; control, II; acetic acid, III; treated with sulfasalazine drug, IV; protection with a low dose, V; protection with a high dose, VI; treatment with a low dose and VII; treatment with a high dose. ^a significant difference as compared to control, ^b Significant difference as compared to acetic acid, ^c significant difference as compared to protection with a low dose, ^d significant difference as compared to treatment with a low dose, and ^e significant difference as compared to treatment with sulfasalazine drug (p ≤ 0.05).

In current results, different outcomes were found for MLE therapy or protection on the hematological parameters. Reduced RBC characteristics have been associated with anemia brought by the AA treatment as previously described (Alia et al., 2019). The WBCs elevated in the AA group as compared to control animals. According to a report, an increase or reduction in granulocyte cells corresponds to an increase or decrease in inflammatory activity (Alia et al., 2019). Sulfasalazine did not cause any significant change in differential leukocyte parameters that were assessed, these results are in agreement with Owusu et al. (2020).

3.4.4 Effect of C. fistula on liver functions in AA-induced colitis

All liver functions in the acetic acid-induced group showed significant changes in which ALT, AST, total bilirubin, and ALP increased significantly while total protein decreased significantly as compared to control animals (Table 7). ALT, AST, total bilirubin, and ALP were markedly decreased in the sulfasalazine-treated group when compared to the UC group. ALT and AST activities significantly decreased in low and high-dose preventative groups and high-dose treatment animals compared to the sulfasalazine-treated and UC rats.

The ALP activity decreased significantly in all prevention and treatment groups as compared to the UC rats (Table 7). The total protein increased by 1.17-, 1.54-, and 1.28-fold in low and high prevention groups as well as the high treated group as compared to UC animals, respectively (Table 7).

The administration of MLE considerably reduced the rise of ALP in groups treated for colitis; this effect may have been caused by the extract's possible anti-inflammatory properties. The outcomes concur with those of other authors (Sarkar et al., 2015; Hasona and Hussien, 2017) who reported a notable increase in ALP activity in the serum of rats with AA-induced colitis.

3.4.5 Effect of C. fistula on antioxidant and oxidative stress in aa-induced colitis

Examination of all rats' colons revealed the presence of MDA, a marker for LPO. Comparing the UC (ulcerative colitis) group to the control group, a significant elevation in colon MDA levels was observed (Table 8). This is indicated by tissue damage (Vishwakarma et al., 2015). However, rats receiving a high dose for prevention or treatment exhibited a noteworthy decrease in colon MDA levels compared to the UC group. Additionally, the UC group displayed significantly reduced activity of SOD and CAT compared to the negative control group. Conversely, the activity of GPx in UC rats was notably increased compared to the control animals. Furthermore, levels of GSH and TAC declined significantly in the UC group compared to the negative control rats.

Treatment and protection of rats with a high dose (400 mg/kg/day) produced a marked significant decrease in LPO concentration that reached the control value (562.67 ± 11.89 μmol/g and 577.67 ± 8.09, respectively). Interestingly, the activity of SOD and CAT along with the content of GSH showed significant restoration in the groups treated with sulfasalazine, both in the pretreatment and treated groups, compared to the UC (ulcerative colitis) animals (Table 8).

The existence of enzymatic and non-enzymatic antioxidant systems to defend tissues from pro-oxidants is well recognized (Thomas, 1995). GSH is non-enzymatic, whereas SOD, CAT, and GPx are enzymatic endogenous antioxidants (Flora et al., 2012). Another significant conclusion of the current study was that AA therapy causes a drop in the tissue levels of SOD, CAT, and GPx. These results are consistent with the previous study that showed AA-induced colitis to have impaired antioxidant mechanisms and enhanced LPO (Nieto et al., 2000). The TAC level was decreased in AA, while the protection with a high dose has increased significantly, this is consistent with the results of the previous studies (Ermis et al., 2013; Cagin et al., 2016). The results of the current investigation showed that administering the extract orally to rats with AA-induced colitis might lessen the severity of the colonic mucosal injury, stop the rise in MDA levels, and replenish depleted levels of antioxidant enzyme activities. Therefore, the extract is more efficient when given after colitis has been induced in a dose-dependent manner. The findings revealed that extract significantly and dose-dependently reduced both carrageenan-induced hind paw edema and cotton-pellet granuloma (Gobianand et al., 2010).

Numerous plant species, including, Moringa oleifera (Minaiyan et al., 2014), Coriandrum sativum (Heidari et al., 2016), and Helichrysum oligocephalum (Popov et al., 2006), have had protective effects on colonic tissues against UC examined. However, there aren’t many publications examining the impact of C. fistula. The pathological analysis supported the biochemical changes. The detected histological alternation, such as clogged blood arteries, leukocytic infiltration, and various degrees of cell degeneration, were consistent with prior studies of UC by other authors (Tanide et al., 2014; 2016).

The present investigation is in parallel with the study of Zabihi et al. (2024) that revealed the extract from the C. fistula plant has beneficial effects on the healing process of ulcerative colitis, demonstrating a reduction in colitis symptoms. Particularly, the composition of the aqueous extract proved effective in diminishing the activity of the myeloperoxidase (MPO) enzyme when compared to the control group. These findings suggest a potential therapeutic role for C. fistula in managing ulcerative colitis by mitigating inflammation and influencing specific enzyme activity involved in the condition as well as controlling the oxidative/antioxidant balance. Zabihi et al. (2024) used high doses with short time; 600 and 800 mg/kg once daily for 5 days.

3.4.6 Histological evaluation

Colonic mucosa from the control group was shown by H&E-stained sections to be made up of densely aggregated simple tubular glands (Lieberkuhn crypts), which extended down to the muscularis mucosa and were embedded in the lamina propria. The crypts' lining was made up mostly of numerous goblet cells and surface epithelial columnar absorptive cells with an apical brush border and basal oval nuclei. Basal nuclei and vacuolated cytoplasm were visible in goblet cells. A small number of intraepithelial lymphocytes were also visible. Undifferentiated cells with basal oval vesicular nuclei were present in the basal regions of the crypts (Fig. 5A). In the positive control group, staining revealed focal areas of severe mucosal deformation, exfoliation, and ulceration with loss of the whole crypt lining in the acetic acid-induced UC group. Some crypts displayed dilatation along with a notable reduction in goblet cells or their disappearance. Transmural necrosis, edoema, and diffuse inflammatory cell infiltration in the mucosa were histological features of untreated rats. Desquamated regions, loss of the epithelium, and focal ulceration of the colonic mucosa that penetrated the muscularis mucosa were present. Hyperplasia of the crypt cells and shorter crypt cell lengths are signs of malabsorption. Tubular adenomas are present along with hyperplastic colorectal polyps (hyperplastic polyposis) in (Fig. 5B).

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Fig. 5

(A-G) Light micrographs of colonic mucosa. A) Control rats showing, normal architecture, and arrangement of the lining layers; mucosa (M), muscularis mucosa (MM), submucosa (S), and musculosa (MS). Apical parts of the mucosa; absorptive crypt cells I, numerous Goblet cells (GC), and obvious lamina propria (LPB). B) colonic mucosa was observed 17 days after induction of colitis and oral treatment with saline, with some generation in the acetic acid group. Showing, IC: invasion of some inflammatory cells, separation of the epithelium (SE), and damaging crypts (DC). C) Colonic mucosa 17 days after induction of colitis and oral treatment with sulfasalazine. Showing, maintained the regular arrangement of cell structure, epithelium remained intact and Goblet cells and crypts were both normally distributed. D) Colonic mucosa observed 17 days after pretreatment with a low dose extract to induce colitis; showing alteration in mucosal, submucosal, and musculosal layers, with damaged crypts. E) colonic mucosa observed 17 days after pretreatment with a high dose to induced colitis: minimal inflammatory reaction in the mucosa and submucosa without any feature of tissue destruction. Note also, that Crypts had a normal architecture with intact vacuolated goblet cells. F) colonic mucosa observed 17 days after induction of colitis and oral treatment with a low dose; more connective tissue cells (CT) and increased number of crypts. G) Colonic mucosa 17 days after induction of colitis and oral treatment with a high dose; the treatment ameliorates.

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The degree and severity of the histological signs of cell damage were considerably decreased in the sulfasalazine-treated group. The lamina propria had a few mildly inflammatory cells, but the epithelium was undamaged. However, sulfasalazine reduced microscopic harm and preserved the normal alignment of cell structure. Lieberkühn's goblet cells and crypts were both discernible and well-spaced. Reduced leukocytosis was seen (Fig. 5C). The prophylactic low dose (200 mg/kg/day) group's histological characteristics included necrosis of a few crypts and a moderate inflammatory response in the mucosa and submucosa (Fig. 5D). Fig. 5E showed pseudo-Peyer's patches without any discernible polyps. The mucosa deteriorated and infiltrated with fewer inflammatory cells in the preventative group, which received a high dose (400 mg/kg/day), than in the protection group which received a low dose. With the growth of multiple goblet cells, crypt cells grew larger. Polyps cannot be seen. little inflammation without any signs of tissue damage in the mucosa and submucosa. Additionally, crypts displayed a typical architectural design with complete vacuolated goblet cells.

The treatment groups showed desquamated regions and epithelium loss after receiving a lower dose (200 mg/kg/day) of C. fistula. With many increases in the quantity and total degeneration of goblet cells, the crypt architecture was also altered. Inflammatory cells had invaded the lamina propia with no evidence of polyps (Fig. 5F). However, higher-dose therapy (400 mg/kg/day) improved microscopic damage and preserved the regular organization of cell structure. Lieberkühn's goblet cells and crypts were both distinct and evenly spaced. Infiltration and necrosis were diminished (Fig. 5G).

Rats given a high dose of MLE showed normal epithelium on their surface and only a small amount of inflammatory cells were infused. Animals administered the extract as treatment were protected against the AA's effects of inflammation, congestion, ulceration, erosion, necrosis, and hyperplasia. The complete colonic mucosa's cytoarchitecture was also preserved. This demonstrates the extract's capacity to safeguard the animals and slow the spread of the illness. The treated animals had better healing than the protective male rats.

3.4.7 Immunohistochemically evaluation

As seen in the figures, several cells in the negative control group had a mildly positive cytoplasmic COX2 immuno-histochemical reaction, which was manifested as a brownish color (Fig. 6A). However, sections taken from group II showed many cells with a high positive cytoplasmic COX2 reaction (Fig. 6B). The section from group III, on the other hand, had several cells that had a positive cytoplasmic COX2 reaction (Fig. 6C). Sections from groups IV and VI, however, revealed a few cells with a slightly positive COX2 reaction in the cytoplasm (Fig. 6D and 6E). Sections from groups V and VII displayed a COX2 reaction that was almost normal (Fig. 6F and 6G). Meanwhile, immuno-stained sections from the control group showed a few cells with a weakly positive cytoplasmic caspase-3 immuno-histochemical reaction, shown as a brownish coloring (Fig. 7A). However, the group II section showed several cells with a markedly positive caspase-3 reaction (Fig. 7B). Additionally, a few cells in the group III section had a weakly positive caspase-3 reaction (Fig. 7C). A mildly positive caspase-3 reaction was seen in several cells in sections from groups IV and VI (Fig. 7D and 7E). Sections from groups V and VII, on the other hand, displayed a nearly typical caspase-3 response (Fig. 7F and 7G).

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Fig. 6

COX-2 immunostaining. A) Shows control COX-2 expression (Group I), brown color indicates specific immunostaining of cleaved COX-2 and light blue color indicates nuclear hematoxylin staining. B) The colonic section of acetic-acid-induced colitis (Group II) depicts numerous cells with a strong positive mainly cytoplasmic COX-2 reaction. C) The colonic section of the sulfasalazine-treated group (Group III) shows a near-control COX-2 expression. D) The colonic section of the pretreated group with a low dose (Group IV); shows multiple cells with a moderately positive cytoplasmic COX-2. E) The colonic section of the pretreated group with a high dose (Group V); reaction reveals few cells with a weak positive cytoplasmic COX-2 reaction. F) The colonic section of the treated group with a low dose (Group VI); shows multiple cells with a moderately positive cytoplasmic COX-2. G) The colonic section of the treated group with a high dose (Group VII); shows a near-control COX-2 expression. [Original magnification: 40x].

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Fig. 7

Caspase-3 immunostaining. A) Shows control caspase-3 expression (Group I), brown color indicates specific immunostaining of cleaved caspase-3 and light blue color indicates nuclear hematoxylin staining. B) The colonic section of acetic-acid-induced colitis (Group II) depicts numerous cells with a strong positive mainly nuclear caspase-3 reaction. C) The colonic section of the sulfasalazine-treated group (Group III) shows a near-control caspase-3 expression. D) The colonic section of the pretreated group with a low dose (Group IV); shows multiple cells with a moderately positive cytoplasmic caspase-3. E) The colonic section of the pretreated group with a high dose (Group V); reaction reveals few cells with a weak positive cytoplasmic caspase-3 reaction. F) The colonic section of the treated group with a low dose (Group VI); shows multiple cells with a moderately positive cytoplasmic caspase-3. G) The colonic section of the treated group with a high dose (Group VII); shows a near-control caspase-3 expression.[Original magnification: 40x].

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Image J software (version 1.53 h) was used for morphometric analysis to semi-quantify COX-2 and Caspase −3 reactivity in the analyzed tissues. Following Fig. 8, when compared to the control group, group II showed a highly significant rise in the mean percentage area of positive COX-2 reactivity. In contrast to the AA-induced colitis, sulfasalazine, and low C. fistula-treated groups, group VII showed a considerable decline. When compared to the AA-induced group, a substantial reduction was also seen in groups III, IV, V, and VI. On the other hand, group II showed a highly significant increase in the mean percentage of positive caspase-3 reaction when compared to the control group. Group VII among the II, III, and VI groups showed a notable improvement in the mean percentage area. Comparing Groups III, IV, and V to the AA-induced UC group, there was a noticeable decline. Group VI and the AA-induced UC rats, however, showed no statistically significant difference (Fig. 9).

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Fig. 8

Effect of C. fistula on COX-2 positively stained area percentage in colon rats. Data presented as mean ± S.E (n = 5). I; control, II; acetic acid, III; treated with sulfasalazine drug, IV; protection with a low dose, V; protection with a high dose, VI; treatment with a low dose and VII; treatment with a high dose. ^a significant difference as compared to control, ^b Significant difference as compared to acetic acid, ^c significant difference as compared to protection with a low dose, ^d significant difference as compared to treatment with a low dose, and ^e significant difference as compared to treatment with sulfasalazine drug (p ≤ 0.05).

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Fig. 9

Effect of C. fistula on caspase-3 positively stained area percentage in colon rats. Data presented as mean ± S.E (n = 5). I; control, II; acetic acid, III; treated with sulfasalazine drug, IV; protection with a low dose, V; protection with a high dose, VI; treatment with a low dose and VII; treatment with a high dose. ^a significant difference as compared to control, ^b Significant difference as compared to acetic acid, ^c significant difference as compared to protection with a low dose, ^d significant difference as compared to treatment with a low dose, and ^e significant difference as compared to treatment with sulfasalazine drug (p ≤ 0.05).

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The colonic caspase-3 and COX-2 activity of the AA group was higher than that of the control group concerning the inflammatory response. A highly substantial rise in group AA when compared to the control group was revealed by morphometrical analysis of the mean percentage of positive caspase-3 and COX-2 reaction, which confirmed this. Improvement in the mean percentage area was remarkable in the treated group with a high dose of MLE amongst the AA, sulfasalazine-treated, and treated group with a low dose.