الفهرس 
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Abstract
The main goal of this work to study the treatment of stomach ulcer of male albino rats using prepared gold nanoparticles with zinc for the drug delivery.
Gastric ulcer disease (GUD) is common problem of the gastrointestinal tract with increasing incidence and prevalence attributed to loss of balance between aggressive and protective factors (Nartey et al., 2012). Gastric lesion results as a consequence of impaired balance between gastro-destructive substances (Tytgat et al., 2011). Gastro-destructive substances include several endogenous aggressive factors such as hydrochloric acid, pepsin, refluxed bile, leukotriene’s, reactive oxygen species (ROS) and several exogenous factors including non- steroidal anti-inflammatory drugs (NSAIDs), stress, alcohols and Helicobacter pylori infection which are major causative agents for gastric mucosal damage and ulceration (Rang et al., 2003; Nartey et al., 2012).
Photothermal effect from the absorption of green solid state laser beam with AuNPs at localized gastric area was considered advanced proved to have many effects mostly tend to be to the being treated.
Nano-technology has different applications in the field of biomedical research. Nanoparticles materials have emerged as novel ”treatment agents” due to their high surface area to volume ratio and their unique physio-chemical properties. Recent reports have demonstrated that the use of metallic nanoparticles, especially gold nanoparticles, provides an opportunity for novel therapy for cancer but lack it information on peptic ulcer. Recently, metal nanomaterials have been developed to be used as drug deliver, such as gold nanoparticles delivered with zinc. Here in this study, laser radiation has been used to induce photothermic effect of prepared gold nanoparticles that had been showed to have a promising application for stomach ulcers in albino rat’s therapy.
The AuNPs with an average size of 13 and 42 nm were synthesized via chemical ionotropic method and characterized by electron microscopy.
Low-level laser power (LLL) exposure, using green solid state laser at wave length 532nm has advanced method that has been used with various power densities. It was found that laser exposed has a positive stimulating effect on ulcerated stomach tissue in male albino rats. The results indicated that:-
1) Genetic results:-
A: - cytogenetic results:-
Induction of ulceration through administration of single dose of indomethacin(IND) (30mg/kg b.wt.) followed by treatment with AuNPs of 13 and 42 nm with zinc and laser exposure for 10 and 15 mints significantly increased the sperm abnormalites in comparison with normal group 0.6%. In addition, groups of Zn+ IND, GNS+ Zn, GNL+ Zn, GNS+Zn+L10, GNL+Zn+L10, GNS+Zn+L15 and GNL+Zn+L15 groups showed non-significant changes in the sperm morphology as compared to IND group (ulceration model group) 1.16%.
B:-molecular results:-
Quantitative real-time PCR:
The expression levels of Bax-mRNA in rat’s stomach were showed in figs. 2 &3. Its level was very low in control group. However, this level was found to be significantly (p≤0.05) upregulated in ulcerated group (IND group) and that treated with indomethacin and zinc and AuNPs. The higher expression level was observed in ulcerated group. However, the expressions of Bax were blocked in ulcerated groups treated with AuNPs and zinc with later exposure to laser at 10 and 15 min.
As showed in figs. 4&5 the expression levels of Caspase3-mRNA in rat’s stomach were significantly (p≤0.05) increased in all group as compared to control group. However higher levels were found in ulcerated group which received indomethacin. After treatment with AuNPs and zinc with exposure to laser blocking effect were observed through down regulation of caspase-3 expression level to nearly like that of control group especially with AuNPs (42 nm) and laser for 15 min.
The BCL2-mRNA expression levels were detected in rat’s stomach figs. 6 &7. It was significantly (p≤0.05) increased in all group as compared to control group. However, the expression levels were upregulated gradually after treatment with AuNPs and exposure to laser for 10 and 15 min respectively.
DNA Fragmentation Assay.DNA fragmentation was done to investigate the effect of AuNPs in the treatment of gastric ulcer, stability and DNA replication. The purity and concentration of extracted DNA was determined. The A260/A280 ratios of DNA were ranged from 1.8 to 2.0 that indicated highly purified DNA a generally acceptable DNA purity. This gave us the ability to determine the volume which contains the same concentration amount of DNA in nano grams (ng). The final concentration of the extracted DNA in all tested sampled showed fluctuation between tested groups.
Fragmented level of DNA detected by gel electrophoresis clearly showed that the biosynthesized GNPs at GNS+Zn and GNL+Zn groups appeared as a mild DNA smear which, however, no DNA changes were observed at Control, GNS+Zn+L10, GNL+Zn+L10, GNS+Zn+L15 and GNL+Zn+L15 groups.
Also DNA fragmentation analysis clearly demonstrated that DNA bands of stomach tissue treated with gold nanoparticles and zinc with or without exposure of laser with a concentration of 250 μg/ml after incubation for 7 days was intact, and nearly with no fragmentation, as same as the (control) band. But, bands of DNA extracted after gold nanoparticles and with or without exposure of laser demonstrated that the fragmented the DNA characteristic necrotic smear of degenerative DNA.
These data showed that the intact genomic DNA of control group appeared nearly non damage in comparison with the maximum damage with high level of random DNA degraded and strong DNA smears, in the gastric ulceration model group (IND). On the other hand, a mild smear observed in the with small size gold nanoparticles (13nm) and large size gold nanoparticles (42nm) only furthermore, the smear decreased significantly especially in group of gold nanoparticles with zinc and exposure of laser doses which recorded the lowest level of random DNA degradation detected as slight dese smear.
In the after with gold nanoparticles (13 and 42 nm) with zinc and exposure of laser doses 15 min. The smears were just a little less than that in the IND group. While, in the post-treated combination group gold nanoparticles (13 and 42 nm) with zinc the smearing decreased in comparison with IND group and other post-treatment groups.
2) Biochemical studies:
i. There was significant increase of gastric MDA level in IND group (ulceration model group) as compared to control. The GNS+Zn+L10, GNL+Zn+L10, GNS+Zn+L15 and GNL+Zn+L15 (2.70±0.03b, c, 3.07±0.02b, 2.64±0.01b and 2.68±0.01b respectively) showed significant decreased of MDA levels as compared to IND group (ulceration model group, 7.28±0.38). In addition, there was changes in MDA level between IND group (ulceration model group) and (GNL+Zn+L15, 2.64±0.01).
ii. There was significant decrease of gastric GST level in IND group (ulceration model group) as compared to control. The level of Gastric GST concentration at IND group (453.00±4.16) is significantly lower than GNS+Zn+L10, GNL+Zn+L10, GNS+Zn+L15 and GNL+Zn+L15 (364.39±3.00c, 375.72±1.61c, 363.70±2.53b and 329.69±9.60c respectively).
iii. A significant decrease was recorded in the gastric CAT concentration of rats administered single dose of IND (30mg/kg b.w.) compared to control group. The GNS+Zn+L10, GNL+Zn+L10, GNS+Zn+L15 and GNL+Zn+L15 groups showed significant increase in gastric CAT levels compared to IND group (ulceration model group, 72.83±1.25). Whereas, the Zn+ IND showed non-significant change to in CAT level as compared to IND group. On the other hand, GNS+ Zn group and IND group (ulceration model group) showed changes to in CAT level compared to GNS+Zn+L15, 1594.67±27.14).
iv. The level of gastric SOD decreased significantly in the IND group (ulceration model group) as compared to the control. The GNS+ Zn, GNL+ Zn, GNS+Zn+L10 and GNL+Zn+L10 groups (26.87±0.15d, 26.33±0.19d, 34.53±0.47e and 33.00±0.07f respectively) showed significant increase in SOD levels as compared to IND group14.97±0.37. While GNS+Zn+L10 group showed non- significant change in SOD level compared to the GNL+Zn+L10 group. On the other hand, the levels of gastric SOD in the GNS+Zn+L15 and GNL+Zn+L15 (34.53±0.47 were significantly increased as compared to the IND group (ulceration model group).
v. There was significant increase of gastric NO level in IND group (ulceration model group) as compared to control group, 101.70±2.15a). The GNS+Zn+L10, GNL+Zn+L10, GNS+Zn+L15 and GNL+Zn+L15 (203.40±1.03b, 223.67±5.10c, 106.53±1.46a and 109.22±1.06a respectively) showed significant decreased of NO levels as compared to IND group (ulceration model group, 426.27±6.04g).
3) Histopathology of the stomach rats:
Administration of IND (30mg/kg b.wt.) caused maximum damage and high level of focal ulceration in the mucosa layer associated with inflammatory cells infiltration in the underlying lamina propria layer, which assayed by histopathology in gastric IND group (ulceration model group) as compared to normal group. On the other hand, Zn+ IND group were showed, necrosis and inflammatory cells infiltration in the mucosal layer with few others in the submucosa, muscular is and serosa with edema in the later. While, the GNS+ Zn group exhibited histological intact in mucosal layer while the underlying submucosa showed odema with inflammatory cells infiltration. There was thickening in the serosal layer by oedema but GNL+ Zn group presented the lining mucosal and glandular epithelium degenerative change. GNL+Zn+L10 group degeneration was observed in the mucosal and glandular lining epithelium associated with congestion in the submucosal blood vessels. The GNS+Zn+L10 group showed inflammatory cells infiltration was detected in the base of the mucosal layer. The GNS+Zn+L15 group recorded fibroblast cells replacement ulcer and GNL+Zn+L15 seen no histopathological alteration in the mucosa but muscular layer and serosa were detected few inflammatory cells infiltration was in the submucosa.
4) Administration of single dose of IND (30mg/kg b.wt., 124.33 ±1.48a) with AuNPs of 13 and 42 nm with zinc and laser for 10 and 15 mints significantly decreased the body weight in comparison with normal group, 150.67 ± 1.83e). A increase in body weight was recognized in IND with GNS+Zn+L10, GNL+Zn+L10, GNL+Zn+L15 and GNS+Zn+L15 groups ( 141.67 ± 0.76c,d, 140.50 ±1.48b,c,d, 144.83 ±0.65d,e and 145.17 ±1.01d,e) as compared to ulcerated group.
5) The means value of mortality rate of rats showed an increase after 7 days in ulcer group as compared to control group. The values of mortality rate of GNS+Zn+L10, GNL+Zn+L10, GNL+Zn+L15 and GNS+Zn+L15 groups Compared to IND group after 7 days in these groups was decreased as . In relation to the control animals, a significant increase (P ≤ 0.05) was indicated in the IND alone and gold nanoparticles with zinc. The gold nanoparticles with zinc and laser for 10 and 15 mints for 10 and 15 min group’s treatment for 7 days showed increase level than IND and Zn+ IND.
6) Administration of single dose of IND (30mg/kg b.wt.) 1.35±0.01a with AuNPs of 13 and 42 nm with zinc and laser for 10 and 15 mints significantly decreased the stomach weight in comparison with (normal group, 1.74±0.02e) .A increase in stomach weight was recognized in GNS+Zn+L10, GNL+Zn+L10, GNL+Zn+L15 and GNS+Zn+L15 groups (1.67±0.01d, 1.60±0.02c, 1.74±0.01e and 1.78±0.02 respectively) comparison with (IND group, 1.35±0.01a).
7) Administration of single dose of IND (30mg/kg b.wt.) 1.03
with AuNPs of 13 and 42 nm with zinc and laser for 10 and 15 mints significantly decreased the relative stomach weight in comparison with (normal group, 1.5) .A increase in stomach weight was recognized in GNS+Zn+L10, GNL+Zn+L10, GNL+Zn+L15 and GNS+Zn+L15 groups (1.19, 1.11, 1.20 and 1.40 respectively comparison with (IND group, 1.03.
8) Elisa :
I. Gastric PGE were significantly decreased after administration of single dose of IND (30mg/kg b.wt.),(11.77±0.38a) with AuNPs of 13 and 42 nm with zinc and laser for 10 and 15 mints in comparison with (control group,50.67±0.17g). were gastric PGE significantly decreased in comparison with the control at (p<0.05). While, the gastric PGE content was significantly increased in all treated groups as compared with the IND group. Moreover, the GNS+Zn+L10, GNL+Zn+L10 , GNS+Zn+L15 and GNL+Zn+L15 groups( 42.52±1.63e, 38.63±0.23d, 48.11±1.45g and 45.45±1.34f respectively) exhibited a significant increase in gastric PGE contents as compared with the Zn +IND, group at (p<0.05).
II. Administration of single dose of IND (30mg/kg b.wt.) with AuNPs of 13 and 42 nm with zinc and laser for 10 and 15 mints showed that all the treated groups were significantly increased in comparison with the control at (p<0.05). While, the gastric TNF-content was significantly decreased in all treated groups as compared with the IND group. Moreover, the GNS+Zn+L10, GNL+Zn+L10, GNS+Zn+L15 and GNL+Zn+L15 groups exhibited a significant decrease in gastric TNF-α as compared with the Zn+IND, group at (p<0.05).