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Abstract
The present study was conducted in the light of the continuing medical unachieved combating of the gastric ulcer disease owing to disease recurrence after cessation of medication with either prostaglandins or eradication of the implicated bacterial infection. This indicates presence of molecular defect(s) that was not corrected properly with such approaches. In this concern, there were attempts to investigate new avenues to controlling the disease by usage of organic copper complexes. This laboratory had pioneered the studying of the molecular mechanisms of the gastric antiulcerogenic abilities of copper nicotinate and glycinate complexes. Previous study revealed accomplished abilities for each of these two complexes in prevention of fundic ulceration in the Shay Rat experimental model of the disease. The model is composed basically of 3 days fasting followed by ligation of the pylorus, installation of the solution of the complexes and abdominal wall closure and fasting for a further of 19 hr. The rats received saline subcutaneously to guard against dehydration. Rats were scarified under light diethyl ether anesthesia for gastric juice, blood plasma and fundic gastric wall collection for subsequent histopathological and biochemical investigations. The model in such format was horrendous because of the massive stress leading to perforating ulceration and frequent death before completion of the experiment complicated by drenching pneumonia due to accumulation of uncontainable amount of gastric juice due to the fluid therapy. The used dose of the nicotinate seemed lipid peroxidative.
Dependent on such experience, the model was modified to be more humane by abstinence form fluid therapy and usage of deep anesthesia by Ketamine and diazepam that gave the rats a deep sedation and analgesia for at least 6 hr post-operation - conducted in optimized surgical conditions in the Unit of Experimental Surgery, Faculty of Medicine, Assiut University. The modified model prevent animal death due to absence of perforating ulcer with low severity ulcers formation reaching 50% of the original model confined to the fundic region of the stomach. Also the dosage of the nicotinate complex was reduced to be similar to that of the glycinate that resulted in close copper content for both in the final doses used (3 mg/kg body weight of the used Wister Albino rats ranging from 200 – 250 gm). The two complexes were synthesized according to published methods and their elemental analysis was conducted in the Unit for Elemental Analysis, Department of Chemistry, Faculty of Science, Assiut University. The experiments were conducted in DROP-through cages to prevent coprophagy.
The initial hypothesis of the present study was investigating a more commonly shared mechanism(s) for the two complexes antiulcerogenic action based on the fact that in the present model ulceration had happened due to specific molecular defect(s). Such molecular targets need identification and be corrected by the complexes for the seek of ulceration prevention. The previous study revealed that two complexes worked differentially on the antiulcerogenic PGE2, the ulcerogenic TXA2 and lipid peroxidation through induction of SOD activity. The present study hypothesized that the pathogenetic change include inflammation and inflammatory cells infiltration particularly the neutrophils with subsequent secretion of acute inflammatory cytokines particularly IL-6 that result in apoptosis of the gastric wall cells and ulcer lesion formation – where they also cause peroxidative damage of the microcirculatory endothelium leading to starvation of mucosal cells of nutrients and O2. The later would be complicated with deficiency of VEGF the major endothelial surveillance angiogenic growth factor and the TGF-α necessary for the integrity of the mucosal cells and that is also angiogenic. All of these disturbances are expected to precipitate a state of oxidative stress locally and possibly systemically as reflected on total peroxides and antioxidants contents – preferentially used over individual oxidative stress biomarkers.
The histopathological investigations of the lesion areas of the gastric fundic wall on section stained by each of H&E and toluidine blue were conducted.
Total juice volume and its mucin content; juice and plasma contents of total peroxides and total antioxidants; juice total DNA and % of its fragmentation as apoptotic markers confirmed by measurement of caspase 3 activity in the fundic wall homogenate were all measured by colorimetric published methods; and each of IL-6, VEGF and TGF-α were measured by quantitative commercially available ELISA assays; and juice acid content was measured by titration against NaOH with phenolphthalein as end point indicator. Rats were divided into 4 groups - 16 each; Group I was ulcerated untreated control, Group II was ulcerated nicotinate treated, Group II was ulcerated glycinate treated and Group IV was ulcerated therapeutic control omeprazole treated. The later is one of the most efficient therapeutic for combating gastric ulceration by inhibiting the H+ pomp and preventing acid secretion and was used at 20 mg/kg body weight dissolved in DMSO and subcutaneously injected 1 hr before surgery as previously published.
The results revealed the outstanding antiulcerogenic ability of the complexes better than the therapeutic control drug used. Associating the suppression of ulceration there was lower microcirculatory failure seen as congestion of the blood capillaries and reduced inflammatory cells infiltration and the histopathological apoptotic changes (condensation of nuclei and their fragmentation within acidic cytoplasm surrounded by clear spaces). This was reflected on reduced level of IL-6 equipotently. Medications prevented oxidative stress in juice and plasma by increasing total antioxidants and decreasing total peroxides with superior ability for the complexes over omeprazole despite its outstanding ability. They also reduced total juice volume and increased its gastroprotectant mucin content best with glycinate followed by nicotinate then omeprazole; same for acid content but with nicotinate the best. They also reduced the apoptotic markers; total juice DNA content and % fragmented DNA equipotently and also reduced caspase 3 activity with omeprazole the weakest. They induced the basal level of TGF-α with omeprazole the most potent followed by nicotinate then glycinate particularly in juice. The very low basal level of VEGF was also induced with the complexes only.
The gastro-destructive biomarkers (ulcer index, total peroxides, oxidative stress index, DNA content and % fragmented, caspase 3 activity, acid content, juice volume and IL-6 content) correlated amongst themselves positively and they correlated negatively with the gastro-protective biomarkers (mucin content, total antioxidants, VEGF and TGF-α contents) that in turn correlated positively amongst themselves. However, the magnitude of changes in the measured investigations at juice level comparing treated vs. untreated rats was massive compared to such difference at the plasma level. This confirms a local rather than a system affliction in the present model that - of course - was reflected although modestly systemically.
The results showed surprising new mechanistic venues for the omeprazole action at IL-6 and TGF-α levels. And, also showed a commonly shared mechanism for the two complexes evident from their almost equipotent antiulcerogenesis, effect on microcirculatory failure, inflammation and inflammatory cells infiltration, oxidative stress, apoptosis, IL-6 and VEGF; although they were mildly distinct at TGF-α level. Therefore, these changes possible have an upper hand in the pathogenesis of the present model of gastric ulceration. Continuation in exploring these mechanisms necessitates further investigations using radiolabeled complexes for studying their kinetics and dynamics and locating in vivo targets and compounds derived from them.