Schiff base metal derivatives enhance the expression of HSP70 and suppress BAX proteins in prevention of acute gastric lesion.

Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP) 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg), the positive control (Tween 20 5% v/v, 5 mL/kg), and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg). After 1 h, all of the groups received ethanol 95% (5 mL/kg) but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg). The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E), immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

1. Introduction

Wide spectrum applications of the Schiff base in biological systems have opened a new horizon in pharmaceutical researches. Schiff bases are usually synthesized through the condensation process of primary amines and active carbonyl groups. Furthermore, indole derivatives have critical roles in variety of biological activities. In novel therapeutics, the development of hybrid molecules consisting different pharmacophores in one frame may lead to remarkable pharmacological effects. The coadministration of the moieties, acting by different mechanisms, may have a synergistic effect with higher activities [1]. The pharmacological activity of Schiff bases metal complexes is solely attributed to the metal, its ligands, or both. Two important factors such as maximum thermodynamic stability and large degree of selectivity are crucial in the design of Schiff base metal complexes or ligands for pharmaceutical application. Various metal complexes have been introduced for their potential therapeutic applications. Possessing various biological activities, some of the first row transition metals are important in metallo-proteins. The chemistry of the metal complexes of Schiff bases containing nitrogen and other donors is well described by Tarafder and colleagues [2]. Various Schiff base complexes have shown variety of properties such as anticancer [3-5], antimicrobial, antifungal [6, 7], antiviral [8] and antioxidant, and anti-inflammatory activity [9]; also see [10]. It is believed that some complexes are more effective in metal complexes than free ligands [11]. Recent studies showed that Schiff base complexes also have remarkable effects against peptic ulcer [12-17].

Peptic ulcer is a result of imbalance between aggressive factors and protective factors. Ethanol has been widely used to induce acute hemorrhagic gastric lesions in animal model studies. Ethanol as an aggressive factor can induce apoptosis in the gastric mucosa. In both prokaryotic and eukaryotic cells, these proteins are classified according to their size [18]. Small heat shock proteins with molecular mass between 15 and 30 kDa act as molecular chaperones in the unfolding and refolding reactions [19, 20]. Heat shock proteins also are important in the translocation of polypeptides across mitochondria membranes [21], nucleus [22], and endoplasmic reticulum [23]. Upregulation of HSPs is a protective mechanism in many biological systems. 70 kDa heat shock protein (HSP70) mediates its biological roles through an interaction with other proteins [24]. Several studies have shown that HSP70 is a type of protein in variety of biological systems. This protein could inhibit apoptosis [25], maybe through its chaperone function stress-induced apoptosis independent from the immune response [26]. This protein is also important in proteins translocation across the nucleus [22]. Heat shock proteins play a critical role in gastroprotection against acute hemorrhagic lesion of gastric mucosa. Studies showed that gastric tissues produce HSP70 as a protective mechanism against ethanolic induced gastric lesions [16, 27–29]. Tsukimi and Okabe reviewed that enhance in expression of HSP70 is an important protective mechanism in mucosal defence against ethanolic-induced lesions [30]. Several studies introduced variety of synthetic compounds and natural products that enhanced the expression of HSP70 in protection of gastric tissue [16, 28, 29, 31]. BAX, a member of Bcl-2 family, is a proapoptotic protein which induces apoptosis [32] to maintain homeostasis. The cytosol in a cell normally contains BAX protein but during apoptosis it migrates to the mitochondria [33]. Ethanol produces reactive oxygen species that increases the expression of BAX protein and causes acute hemorrhagic gastric lesions [16, 27–29]. In our previously published work, copper Schiff base complexes caused a remarkable protection against ethanolic-induced gastric ulcer [16]. The present article was to evaluate whether Schiff bases metal derivatives enhance the expression of heat shock proteins and suppress the expression of BAX proteins in prevention of acute gastric lesion. The gastroprotective properties of each Schiff base derivative complexes were not the main focus in this work.

2. Materials and Methods

2.1. Preparation of Schiff Bases

Condensation between carbonyl group and amines is possible in different reaction conditions and in different solvents [34]. The presence of dehydrating agents normally favours the formation of Schiff bases. Acid salts (usually MgSO4 or Na2SO4) are commonly employed as a dehydrating agent. Primary alcohols such as ethanol are the main solvent for the preparation of Schiff bases. Schiff bases have been purified by crystallization methods. Chromatography of Schiff bases on silica gel might cause decomposition of the Schiff bases, through hydrolysis [35]. Stirring the crude reaction mixture in hexane or cyclohexane can purify the Schiff base in these solvents [36]. Moderate polar solvent, such as diethyl ether and dichloromethane, might enhance the purity [37]. In general, Schiff bases are stable solids and can be stored without many precautions [35]. This type of compound is known as an imine or Schiff base. Figure 1 illustrates the general structure of Schiff base derivative. In this study, four Schiff base derivatives with different metal groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 were tested to assess the expression levels of HSP70 and BAX proteins during their gastroprotective activity.

Figure 1

Reaction pathway for the Schiff base derivative.

2.2. Animals

Sprague Dawley (8–10 weeks old, weighted 210–230 g) rats were obtained from the Experimental Animal House, Faculty of Medicine, University of Malaya. Animal care and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institute of Health) with approval from the committee for animal experimentation, Faculty of Medicine, University of Malaya (ethic no. (ISB/30/05/2012/SG (R))). They were maintained at ~24°C with free access to standard laboratory pellet and water. 12 h before the experiment, the animals' access for food was restricted. Their access to water was also restricted from 2 h before the experiment.

2.3. Animals Experiment

In this experiment, in addition to two control groups, named the normal group (n = 6) and the positive group (n = 6), the animals were assigned to four Schiff base groups (Schiff_1, Schiff_2, Schiff_3, and Schiff_4) of 6 rats. The experimental diagram shows specifications for each group (Table 1). In brief, the experiment was divided two sections (Sections 1 and 2). An hour interval was introduced between the sections. In the first section, the normal group and the positive group received 5 mL/kg Tween 20 (5% v/v) while the other groups received 25 mg/kg of their respective Schiff base complexes (Schiff_1 to Schiff_4) dissolved in Tween 20 (5% v/v). The animals received a volume of 5 mL/kg of their respective complexes. The animals received 5 mL/kg of ethanol (5% v/v) after 1 h but the normal group received 5 mL/kg of Tween 20 (5% v/v). The animals were euthanized after 1 h and their stomachs were dissected for further analysis.

Table 1

Specifications of groups for first and second section of the experiment.

Groups	First section	Second section
Normal group	Tween 20 (5% v/v), 5 mL/kg	Tween 20 (5% v/v), 5 mL/kg
Positive group	Tween 20 (5% v/v), 5 mL/kg	Ethanol (95% v/v), 5 mL/kg
Schiff_1	25 mg/kg*	Ethanol (95% v/v), 5 mL/kg
Schiff_2	25 mg/kg*	Ethanol (95% v/v), 5 mL/kg
Schiff_3	25 mg/kg*	Ethanol (95% v/v), 5 mL/kg
Schiff_4	25 mg/kg*	Ethanol (95% v/v), 5 mL/kg

 *Dissolved in Tween 20 (5% v/v) and orally given at the dose of 5 mL/kg.

The gastric lesion indexing was performed as described by several works [38-41]. In brief, a dissecting microscope of the lesion area (mm2) was measured. The index was calculated as inhibition percentage, accordingly [29, 31].

2.4. Histological Observation

Gastric tissues were fixed in buffered formalin (10% v/v) for 18 h and processed by a tissue-processing machine (Leica, Germany). Sections of the stomach were prepared on proper histological slides at a thickness of 5 μm and proceeded to hematoxylin and eosin (H&E) staining and immunohistochemical staining.

Immunohistochemistry staining was performed according to the manufacturer's protocol (DakoCytomation, USA). Tissue sections were incubated with HSP70 (1 : 500) and BAX (1 : 200) biotinylated primary antibodies. Streptavidin-HRP (streptavidin conjugated to horseradish peroxidase) was incubated as a secondary antibody. Under light microscope, the secondary antibody appeared as brown stains.

2.5. Western Blotting

Stomachs of each rat were washed and tissue homogenates were prepared with phosphate buffer (50 mM, pH 7 at ~4–8°C). The homogenates were centrifuged at 4000 rpm for 20 min (4°C) and the supernatant was subjected for protein extraction using the lysis buffer (50 mM Tris-HCL pH 8.0, 120 mM NaCl, 0.5% NP-40, 1 mM PMSF), Protein extract (25 g) was separated by SDS-PAGE (10%), then transferred to a polyvinylidenedifluoride membrane (Bio-Rad), and blocked with 5% nonfat milk in TBS-Tween buffer 7 (0.12 M Tris-base, 1.5 M NaCl, 0.1% Tween 20). The membrane was incubated with the antibodies (HSP70 (1 : 1000), BAX (1 : 1000), and β-actin 1 : 10,000; Cruz Biotechnology, USA) following incubation with horseradish peroxidase conjugated secondary antibody. The bound antibody was detected with peroxidase conjugated anti-rabbit antibody (1 : 10,000) or anti-mouse antibody (1 : 10,000) followed by chemiluminescence (ECL System) and exposed by autoradiography [42].

3. Results

3.1. Lesion Indexing

Gross appearance of luminal surface of the stomach for each group that received the Schiff base complexes showed significant protection when compared with the positive group. However, the protection indexes varied among the rats that received Schiff base complexes. The protection index for Schiff_1 was 73.6% ± 5.50%; Schiff_2 68.06% ± 6.28%; Schiff_3 83.37% ± 6.85%; Schiff_4 79.83% ± 6.47%.

3.2. Histological Observation

Figure 2 shows the H&E staining of gastric tissue for each group. Histological evaluation of gastric tissues showed that ethanol imposed extensive acute haemorrhagic damages into gastric tissue of the positive group. The mucosal and submucosal tissues were disrupted by lesions. Edema was typically observable at the submucosal layer with neutrophil migration. In the experimental groups that received the Schiff base complexes, the damages were significantly reduced when compared with the positive control group. The Schiff base complexes protected the mucosal and submucosal tissues. The histology slides showed that these groups possessed moderate lesion and disruption of the histological layers. Furthermore, the edema was remarkably declined and neutrophil migration was negligible (Figure 2, left column).

Figure 2

Microscopic study of gastric mucosa in rats (20x). Histological H&E staining of the gastric tissues (left column) and immunohistochemistry (IHC) of HSP70 and BAX proteins (mid column and right column, resp.) are shown for different experimental groups. Normal control group (1st row) shows intact gastric tissue with moderate presence of HSP70 and low presence of BAX. The ulcer control group (2nd row) shows epithelial disruption with acute hemorrhagic lesions into the mucosa. Remarkable submucosa edema and leucocyte penetration are noticeable. The presence of HSP70 is apparently suppressed while the BAX protein is considerably overwhelmed. The Schiff base derivative complexes (3rd–6th rows) show considerable protection against the mucoepithelial disruption caused by ethanol. The edema is not significant. The IHC of HSP70 and BAX shows that these proteins are meaningfully regulated by the Schiff base derivative complexes. The presence of HSP70 in considerably increased when compared to either of the control groups while that of BAX proteins is dramatically declined.

3.3. Immunohistochemistry

Immunohistochemistry study showed the expression of HSP70 and BAX proteins in tissue sections of the gastric tissues among the groups. Figure 2 shows the expression of HSP70 and BAX proteins (mid and right columns, resp.) in different groups. Microscopic evaluation of the gastric sections showed that acute haemorrhagic gastric lesions induced by ethanol caused an increase in the expression of BAX protein (Figure 2(f)) and decline in the expression of HSP70 protein (Figure 2(e)). Oral administration of the Schiff base complexes enhanced the expression of HSP70 protein (Figures 2(h), 2(k), 2(m), and 2(q)) and suppressed the expression of BAX protein (Figures 2(i), 2(l), 2(o), and 2(r)). In Figure 2, the expression of these two proteins is characterized by the brown colour staining of the cytoplasm.

3.4. Western Blotting

Throughout the western blot analysis, we conducted a comparable analysis of these proteins in the tissue homogenates of each sample where the β-actin protein was considered the loading control. The result showed that HSP70 was significantly induced in those rats that received the Schiff base complexes as compared with the positive group (Figure 3). On the other hand, the expression of BAX protein was suppressed when the animals received the Schiff based complexes (Figure 3). These results confirmed the role of Schiff base complexes in the regulation of certain proteins as a protective mechanism.

Figure 3

Western blot assay against HSP70 and BAX mouse monoclonal antibody. Corresponding β-actin blots are considered as the loading control. Nor, the normal control group, Pos, the ulcer control group.

4. Discussion

Ethanol intoxication caused extensive and acute haemorrhagic lesions on the luminal surface of the gastric tissue and damaged the gastric layers. Previous studies showed that such disruption caused decline in antioxidant properties of the stomach and increased in neutrophil infiltration [16, 28, 29, 31]. These studies also confirmed that ethanol reduced the expression of HSP70 protein but enhanced the regulation of BAX protein. Antiulcer properties of several agents have proven the importance of upregulation of HSP70 protein for gastroprotection. For instance, Polygonum chinense aqueous leaf extract and Mucuna pruriens at the dose of 250 mg/kg and 500 mg/kg, respectively, showed the highest expression level of HSP70 [28, 31]. Our previous study also confirmed that Schiff base derived copper (II) significantly enhanced the expression of HSP70 [16]. In all of these studies, the enhancement of HSP70 protein was parallel with the suppression of BAX protein.

In this study we assessed the protein expression levels of HSP70 and BAX proteins when the animals were administered with different Schiff base complexes. Based on our pilot study, the dose of 25 mg/kg was the optimal dose for its preventive properties. Moreover, to evaluate the biological effect of Schiff base derivative complexes, all of the Schiff base groups received the same dose (25 mg/kg) of their respective Schiff base complexes. The microscopic evaluation of the stomach for each group showed that Schiff base complexes remarkably increased the gastric protection against ethanol-induced ulcers in rats. Although the protection level of each Schiff base complexes did not appear to be similar, they showed protective property for Schiff base derivative complexes. This work mainly focussed on the expression level of HSP70 and BAX proteins in the protection against acute haemorrhagic gastric lesions. Histological examination with H&E staining confirmed the protective effect of the Schiff base complexes, accordingly. This study revealed that the Schiff base complexes inhibited neutrophil infiltration to the submucosal layer. Moreover, immunohistochemical staining against HSP70 and BAX proteins showed the effect of the Schiff base complexes in enhancement of HSP70 protein and suppression of BAX protein while the animals received their respective administration of Schiff base complexes. Western blot assay showed that the expression level of HSP70 was enhanced, in the tissue homogenates, when the rats received the respective administration of Schiff base complexes in the first section of the experiment. Expectedly, the expression level of BAX protein was suppressed upon receiving Schiff base complexes.

5. Conclusion

Schiff base derivative complexes are among the promising compounds with several biological properties. This study was conducted to evaluate the expression level of HSP70 and BAX proteins in acute haemorrhagic gastric lesions in rats administrated with Schiff base derivative complexes. The Schiff base complexes showed direct relationship with the expression level of HSP70 protein. They also showed that they might suppress the expression of BAX protein as a protective mechanism. Each Schiff base complexes showed a different level of protection but all of them had the same regulatory properties on the expression of HSP70 and BAX proteins at the dose of 25 mg/kg. Consistent with several studies on gastroprotective complexes, Schiff base derivative complexes showed prosperous protective potential through protein regulation pathways. The immunohistochemistry evaluation of gastric tissues showed the protein regulation in situ which was confirmed by western blot assay accordingly.