In Vivo Gastroprotective Upshots of the Novel Schiff Base CdCl2 (C14H21N3O2) Compound by Bax/HSP-70 Signaling and Inflammatory Cytokines

Abstract

Background: Synthesis of the original Schiff base CdCl₂ (C₁₄H₂₁N₃O₂) compound (Schiff base complex) displays an extensive range of bioactivities and was predictably utilized to treat several syndromes.

Purpose: The goal of the existing experiment is to evaluate the gastroprotective effects of a novel Schiff base CdCl₂ (C14H21N3O2) compound in alcohol-induced gastric ulcers in rats by examining its antioxidant activity, anti-inflammatory effects, and modulation of key molecular markers, including heat shock protein-70 (HSP-70) and Bcl-2-associated X protein (Bax) proteins.

Methods: Five groups of rats were utilized in the current study. Control and model groups were orally administered 10% Tween 20. The treated groups were orally administered 20 mg/kg omeprazole or Schiff base compound (25 or 50 mg/kg). One hour later, only the control group received oral 10% Tween 20, and the treated groups received oral (5 ml/cage) absolute alcohol. During the second hour, all rats were sacrificed.

Results: All treated rats presented considerable improvement in alcohol-induced gastric injury recognized by decreasing ulcer index and raising % of ulcer inhibition. Increased mucus and gastric pH content and decreased ulcerated portion, reduced or non-appearance of edema, and leucocytes penetrated the subcutaneous layer. In stomach epithelium homogenate, the Schiff base compound obtainable significant upsurge superoxide dismutase (SOD), catalase (CAT) activities, considerable declining malondialdehyde (MDA) quantity. Moreover, the Schiff base compound raised the intensity of periodic acid-Schiff (PAS) stains gastric epithelium. Furthermore, the Schiff base compound formed up-regulated HSP-70 and down-regulated Bax proteins gastric epithelium Schiff base compound, reduced the level of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and improved the quantity of IL-10. Administering a high dose of 500 mg/kg Schiff base compound revealed the nontoxic nature of the compound in rats.

Conclusion: Schiff base compound exhibited gastroprotective effects attributed to its antioxidant nature, its capability to enhance mucus excretion, SOD and CAT, reduce MDA amount, up-regulate HSP-70 protein, down-regulate Bax protein, and inflammatory cytokines.

Keywords: bax; gastric ulcer; histopathology; hsp-70; il-10; il-6; immunohistochemistry; novel schiff base cdcl2 (c14h21n3o2) compound; tnf-α.

Figure 1. Histology slices of the liver (A, C, and E) and kidney (B, D, and F) for acute toxicity assessment.

Rats administered 5 ml/kg 10% Tween 20 (A, B); animals administered Schiff base compound (250 mg/kg) (C, D); and rats administered Schiff base compound (500 mg/kg) (E, F). There are no substantial changes detected in the constructions of the liver and kidney (hematoxylin and eosin (H&E) stains, amplification ×40).

Figure 2. Macroscopic presence of gastric epithelium (A-E) and statistical analysis of ulcer area (F).

(A) G1: Normal control showed undamaged stomach epithelium; (B) G2: Ulcer control had extensively damaged stomach mucosa (black arrows), absolute alcohol formed widespread noticeable hemorrhage to damage stomach mucosa; (C) G3: Omeprazole (20 mg/kg) damages stomach epithelium with very minor injuries (black arrows) compared to the ulcer control group; (D) G4: Schiff base compound (25 mg/kg) decreases the development of stomach lacerations induced by absolute alcohol (black arrows); (E) G5: Schiff base compound (50 mg/kg) minor damages stomach epithelial mucosa (black arrows). The statistical results indicated that Schiff base compound at dosages of 25 and 50 mg/kg, respectively, produced significantly different inhibition effects in preventing the formation of stomach ulcers to that of ulcer control (p<0.01) (G2) (F). ns, non-significant. ****p<0.0001.

Figure 3. Effect of Schiff base compound on the histological study of the absolute alcohol-induced stomach mucosal injury.

(A) G1: The normal group displayed intact stomach epithelia. (B) G2: Ulcer group pre-fed with 10% Tween 20. Extensive disturbance superficial mucosa dead lacerations infiltrate intensely mucosa (red arrows) widespread edema of submucosa coating inflammatory cells permeation existing (black arrows). (C) G3: Rats nourished omeprazole (20 mg/kg). Slight interruption of outward mucosa exists (black arrow); however, profound epithelial injury was absent. Decreasing of submucosal edema and white blood cell penetration (black arrow). (D) G4: Schiff base compound (25 mg/kg), minor disturbance surface epithelium existing then unfathomable mucosal impairment absent. Decrease submucosal edema inflammatory permeation (black arrow). (E) G5: Schiff base compound (50 mg/kg), minor disturbance surface epithelium existing nonetheless profound mucosal injuries is inattentive. Decline submucosal edema and inflammatory penetration (black arrow) (hematoxylin and eosin (H&E) stain, magnification 20×).

Figure 4. Influence of the Schiff base compound on stomach material glycoprotein-PAS stained (A-E) and statistical analysis (F).

(A) G1: The control presented undamaged stomach epithelium. (B) G2: Ulcer control exhibited sever stomach tissue damage with very appearance of magenta PAS stain, (C) G3: Omeprazole group (20 mg/kg) had non-significant stomach tissue disruptions with a high PAS staining concentration. (D) G4: Schiff base compound 25 mg/kg mild to moderate damage of gastric mucosa with mild PAS stain of gastric mucosa. (E) G5: Schiff base compound 50 mg/kg mild damage of gastric mucosa and with moderate PAS stain of gastric mucosa. Magenta color apical epithelial cells of Schiff base compound administered group’s demonstrations regularly increase mucosal excretion stomach glands (black arrow). The powerful excretion of mucus stomach glands confirmed (E). The statistical analysis indicated that the Schiff base compound of dose (50 mg/kg), and omeprazole group showed the highest PAS stained percentage as significant different to that of the ulcer control group (G2) (p<0.01) (F). The arrow points to the glycoprotein gathering (PAS stain, magnification 20×). PAS: periodic acid-Schiff; ns: non-significant. ****p< 0.0001.

Figure 5. Influence of Schiff base compound on HSP-70 protein expression of alcohol-induced stomach epithelial grazes in diverse groups of animals (A-E) and quantitative analysis (F).

G1: Normal control exhibited complete stomach mucosa and very weak HSP-70 protein expression in the gastric mucosa layer (A, F), G2: Ulcer control group presented severe injury of the gastric mucosa and down-regulation of HSP-70 protein expression in alcohol-induced gastric ulcers in rats compared to the other treated groups (B, F). G3: Omeprazole group exhibited mild damage of the gastric mucosa and up-regulation of HSP-70 protein expression of gastric mucosa in alcohol-induced gastric ulcers in rats (blue arrow) (C, F), G4: Rats administered Schiff base compound 25 mg/kg had moderate stomach lesions (blue arrow) with increased representations of HSP-70 in their stomach tissues (D, F). G5: Rats administered Schiff base compound 50 mg/kg showed a mild stomach tissue injury (blue arrow) with increased representations of HSP-70 protein, compared to the ulcer-controlled rats (E, F). The antigen place seemed brown (HSP-70 stain magnification 20x). Values expressed as means ± SEM. The antigen area is observed in a brown color. The significant difference in the HSP-70% protein expressions are presented as ns. ns: non-significant;  HSP-70: heat shock protein-70. *p < 0.05; ****p < 0.0001.

Figure 6. Influence of Schiff base compound on Bax protein expression alcohol-induced stomach epithelial lesions in different groups of rats (A-E) and statistical analysis (F).

G1: The normal control displayed unharmed stomach mucosa and very weak expression of Bax protein in the gastric mucosa (A, F). G2: The ulcer control group presented severe damage to the gastric mucosa and up-regulation of Bax protein expression in alcohol-induced gastric ulcers in rats (B, F). G3: The omeprazole group showed mild damage of the gastric mucosa and down-regulation of Bax protein expression of the gastric mucosa  (orange arrow) compared to the ulcer control group in alcohol-induced gastric ulcers in rats (C, F). G4: Schiff base compound 25 mg/kg showed moderate injury of the gastric mucosa and down-regulation of Bax protein expression of the gastric mucosa (orange arrow) compared to the ulcer control group in alcohol-induced gastric ulcers in rats (D, F). G5: Schiff base compound 50 mg/kg displayed mild damage of the gastric mucosa and down-regulation of Bax protein expression of the gastric mucosa (orange arrow) compared to the ulcer control group in alcohol-induced gastric ulcers in rats (E, F). The antigen position looks brown (Bax stain magnification 20x). There was significant differentiation between rat groups in Bax protein expressions with the highest expressions recorded for the G2 rat group. ns: non-significant; Bax: Bcl-2-associated X protein. ***p<0.001. ****p<0.0001.

Figure 7. The Effect of Schiff base compound on SOD, CAT, and MDA in absolute alcohol prompted stomach ulcers.

Pre-treatment groups: G1: normal control (10% Tween 20). G2 ulcer control (10% Tween 20), G3 Omeprazole 20 mg/kg, G4 Schiff base compound 25 mg/kg. G5 Schiff base compound 50 mg/kg. Data are expressed as mean ± SEM. Means among groups (n = 6 rate/group) show a significant difference. ns, non-significant, SOD: superoxide dismutase, CAT: catalase, MDA: malondialdehyde. *** p < 0.001. **** p < 0.0001.

Figure 8. Influence of Schiff base compound on TNF-α, IL-6, and IL-10 in alcohol-induced stomach ulcers.

G1: normal control (10% Tween 20). G2: ulcer control (10% Tween 20), G3 Omeprazole 20 mg/kg, G4 Schiff base compound 25 mg/kg. G5 Schiff base compound 50 mg/kg. Data are expressed as mean ± SEM. Means among groups (n = 6 rate/group) show a significant difference. ns, non-significant. *p < 0.05. **p < 0.01. ****p < 0.0001. TNF-α: tumor necrosis factor-alpha; IL-6: interleukin-6.

Figure 9. Graphical abstract: in vivo gastroprotective upshots of novel Schiff base CdCl2 (C14H21N3O2) compound.

PAS: periodic acid-Schiff; TNF-α: tumor necrosis factor-alpha; IL-6: interleukin-6; HSP-70: heat shock protein-70; Bax: Bcl-2-associated X protein; MDA: malondialdehyde.