The bi-functional roles of Ca-Y zeolite in the treatment of ethanol–HCl induced gastric ulcer using a mice model

Abstract

A calcium(II) exchanged zeolite Y (Ca-Y) was prepared and evaluated in ethanol–HCl induced gastric ulcer using a mice model. Benefiting from the high procoagulant activity, good resistance to gastric fluid and anti-acid capability of Ca-Y, a significantly reduced ulcer area percentage from 35.1% ± 4.4% to 11.5% ± 1.9%, was achieved at an oral dosage of 5.0 g kg⁻¹, along with an increased intragastric pH from 2.0 ± 0.5 to 4.5 ± 0.5.

---

Peptic ulcers include gastric and duodenal ulcers and are one of the major human pathologies that affect 10–15% of the world's population.¹ It is a heterogeneous disease of multifactorial etiology, resulting from consumption of nonsteroidal anti-inflammatory drugs (NSAIDs), infection of Helicobacter pylori, excessive alcohol intake as well as irregular dietary manner and suffering from severe stress.² The direct etiological factor is the disorder of the balance between defensive factors (mucus and surface epithelial cells) and offensive factors (hydrochloric acid and pepsin).³ The imbalance of this gastrointestinal self-protective system can cause mucosal damage, which may develop into a gastric ulcer, sometimes with consequent complications such as bleeding, perforation and gastric outlet obstruction.⁴ On account of the adverse environment (hydrochloric acid, pepsin and chyme), the process of hemostasis and tissue repair inside the stomach can face many more obstacles compared to external wound healing. Therefore, additional therapies, with hemostatic-like glue or clotting products have been used more frequently in the endoscopic therapy of gastrointestinal bleeding.⁵

Mineral based hemostatics, such as zeolite and clay, have been widely proven to be effective for controlling external hemorrhage during the last decade.^6,7 Recently, they have also been demonstrated to give promising results towards the therapy of gastric diseases in several animal and clinical trials.^8,9 While the exact mechanism of action remains unclear, their excellent performance have been generally attributed to the special physical and chemical properties of these porous materials. Barkun et al. reported that the Hemospray™ powder demonstrates adhesive properties. It acts as a bandage coupled with local tamponade effect upon contact with moisture and dehydrates tissue through the absorption of water, which concentrates clotting factors at the site of bleeding wherein a subsequent clot forms.¹⁰ Willna and co-workers also found that the gastroprotective benefits of clinoptilolite may be due to its binding to hydrogen ions and biologically active amines/nitrates.⁹ However, limited progress has been made in the fundamental understanding of the interactions between functional proteins (enzymes) or cells with hemostatic materials in this biological process.

Plasma protein adsorption to the surface of nanomaterials has a great influence on their bio-functionality.^11,12 In our previous study, we discovered that in situ generated thrombin in the protein corona of a calcium(II) exchanged zeolite surface displays a calcium(II)-dependent, unusually high (∼3000 NIH U mg⁻¹) procoagulant activity, which is even stable against anti-thrombin deactivation.¹³ Our observations suggest that the thrombin activity can be regulated by the inorganic surface and cations. Most importantly, our discovery indicates the linkage of the biomolecules in the protein corona to the procoagulant activity of the materials, providing a new molecular basis of the procoagulant mechanism for zeolite hemostatics. These results encouraged us to further explore their medical applications in gastrointestinal disorders.

In this study, we explored the in vitro procoagulant activity of a calcium(II) exchanged zeolite Y (Ca-Y) and its composite with hard protein corona (Ca-Y/HPC), which was pre-treated with an artificial gastric fluid. Moreover, we studied its in vivo potency, specifically the gastroprotective effect with an ethanol–HCl induced gastric ulcer using a mice model.

Ca-Y was prepared from commercially available sodium zeolite Y (Na-Y) via a standard ion exchange method (Table 1).¹³ The Si/Al ratio and ion exchange degree with calcium(II) were determined to be 2.57 and 74%, respectively (Table S1, ESI†). SEM images suggest that the Ca-Y has a crystallized particle morphology with a diameter distribution of 2.48 ± 0.93 μm (Fig. 1c, S1 and S2, ESI†). The XRD pattern of Ca-Y confirms that the zeolite sample exhibits a FAU structure (Fig. 1a). The unit cell was cubic (a = 24.7 Å) with Fd3̄m symmetry and has a 3-dimensional pore structure with pores running perpendicular to each other in the x, y and z planes (Fig. 1b). The pore diameter was large (7.4 Å) since the aperture was defined by a 12 membered oxygen ring and leads into a larger cavity with a diameter of 12 Å.

Table 1 The molecular formula and Si/Al ratio of Na-Y and Ca-Y zeolites

Zeolite	Molecular formula	Si/Al^a
a Si/Al: the atomic ratio of Si to Al.
Na-Y	Na0.55(SiO2)1.03(AlO2)0.40·χH2O	2.56
Ca-Y	Ca0.17Na0.12(SiO2)1.08(AlO2)0.42·χH2O	2.57

---

Fig. 1 (a) Powder X-ray diffraction (XRD) pattern of Ca-Y (upper) and standard pattern of zeolite Y (lower). (b) A schematic of zeolite FAU structure where each vertex represents alternating Si and Al atoms and the straight line represents bridging oxygen atoms. (c) Scanning electronic microscopy (SEM) image of Ca-Y.

Ca-Y/HPC was prepared by mixing the Ca-Y and porcine plasma, which was identical to our previous report.¹³ The procoagulant activity was evaluated using an in vitro clotting assay (ESI†). It measures the coagulation response in terms of the clotting time (CT), defined as the time required from activation of the intrinsic pathway of coagulation cascade to the appearance of a firm clot (Fig. S3, ESI†). In this study, the hemostatic agents were evaluated in a mimic gastric environment upon pre-treatment with artificial gastric fluid or deionized water as the reference. A thrombin molecule was used as a control sample since it is a vital clotting factor and has shown effectiveness to control haemorrhage from gastric varices.^14,15

As shown in Fig. 2a, the porcine plasma without the addition of any agent clots slowly in approximately 17 minutes under normal conditions. The introduction of Ca-Y significantly accelerates the coagulation process, resulting in a short CT of 122 ± 3.0 s. Thrombin (20 NIH U) and Ca-Y/HPC both display excellent procoagulant activity with CT of 10.5 ± 2.0 s and 14.5 ± 0.5 s, respectively. However, after treatment with artificial gastric fluid, plasma with/without the addition of thrombin molecules fails to clot within 20 minutes. The thrombin activity assay confirmed that the thrombin molecules completely lost their activity after being treated with artificial gastric fluid, implying that it has been denatured and cannot maintain its basic bio-function under such acidic conditions (Fig. 2b). On the contrary, Ca-Y and Ca-Y/HPC still exhibit effective procoagulant activity with CT of 245 ± 2.0 s and 95 ± 5.0 s, respectively (Fig. 2a).

Fig. 2 (a) Procoagulant activity using an in vitro clotting assay. (b) Thrombin enzyme activity using a thrombin chromogenic assay. The assays were conducted under two different conditions, normal or pre-treatment with gastric fluid.

The moderate procoagulant activity of Ca-Y indicates the stability of the inorganic material against a harsh biological environment. In addition, the significantly reduced clotting time (from 245 s to 95 s) of Ca-Y/HPC can be attributed to the stable in situ generated thrombin in the hard protein corona. Approximately, 68% thrombin activity of Ca-Y/HPC remains after treatment with gastric fluid as determined by the thrombin chromogenic assay, in stark contrast to the free thrombin molecules (Fig. 2b). The remaining activity of thrombin in Ca-Y/HPC is very important due to its crucial role in physiological and pathological coagulation as well as wound healing. The in vitro clotting assay results imply that zeolite based hemostatic agents are a promising option to control hemorrhage and associated diseases in the gastrointestinal tract.

The gastroprotective effect of Ca-Y was further evaluated with an ethanol–HCl induced gastric ulcer using a mice model. Gastric ulcer lesions were induced by the oral administration of 0.2 mL of an HCl/ethanol aqueous solution (200 mM HCl in 50% ethanol). Half an hour before ulcer induction, the Ca-Y suspension was administered at oral doses of 0.5 g kg⁻¹, 2.5 g kg⁻¹, and 5.0 g kg⁻¹. In this study, thrombin (2500 NIH U kg⁻¹) and omeprazole (4.5 mg kg⁻¹) treated groups were included as references, since omeprazole is a proton-pump inhibitor medicine, which has been widely used in gastric disorders and used to suppress gastric acid secretion. One hour after ulcer induction, intense gastric mucosal damage in the form of hemorrhagic streaks and tissue necrosis occurred (Fig. 3a), which was consistent with a previous report.¹⁶ After dissection, the intragastric pH was recorded and the gastric ulcer area percentage was measured and used as an indicator of the degree of ulceration.

Fig. 3 The gastroprotective effect of Ca-Y on the gastric ulcer model induced by ethanol–HCl in mice. The optical images of the stomachs from the mice with a pre-oral administration of (a) saline, (b) thrombin (2500 NIH U kg⁻¹), (c) omeprazole (4.5 mg kg⁻¹) and Ca-Y at (d) (0.5 g kg⁻¹), (e) (2.5 g kg⁻¹) and (f) (5.0 g kg⁻¹). The statistical histogram of the ulcer area percentage (g) in different groups, *p < 0.05, **p < 0.01 vs. control mice, student's t-test and (h) in different dosages of Ca-Y, #p < 0.05, ##p < 0.01 vs. control mice, one-way analysis of variance (ANOVA). n = 6 for each group. Each data are presented as the mean ± standard error (SEM). The intragastric pH was also recorded.

When compared with the control group, the oral administration of Ca-Y (2.5 g kg⁻¹) significantly reduced the gastric ulcer area percentage from 35.1% ± 4.4% to 17.8% ± 2.0% and improved the intragastric pH from 2.0 ± 0.5 to 3.5 ± 0.5, while the administration of thrombin (2500 NIH U kg⁻¹) did not show a statistical discrepancy with 32.9% ± 3.3% ulcer area percentage and an intragastric pH of 2.0 ± 0.5 (Fig. 3g). When combined with the in vitro tests described above, it was reasonable to observe the ineffectiveness of soluble thrombin molecules since it was deactivated in the gastric environment.

Ca-Y shows high gastroprotective potency. As shown in Fig. 3h, the oral treatment of Ca-Y at 0.5 g kg⁻¹, 2.5 g kg⁻¹ and 5.0 g kg⁻¹ can significantly reduce the ulcer area percentage. Moreover, it displays a dose-dependent manner in both ulcer area reduction and increase in intragastric pH (Fig. 3h). This result was in good agreement with a previous report that zeolite is an effective antacid for gastric hyperacidity by proton exchange and hydrolysis of the presented species.¹⁷

It is worth noting that the oral ingestion of Ca-Y in a dosage of 5.0 g kg⁻¹ can effectively increase the intragastric pH to as high as 4.5 ± 0.5, which approximately the same observed with omeprazole at a dosage of 4.5 mg kg⁻¹. However, the ulcer area percentage treated with Ca-Y was much less than the omeprazole group at this dosage level (11.5% ± 1.9% vs. 19.8% ± 1.7%). This implies that in addition to an anti-acid effect, there exist other beneficial factors from Ca-Y participating in treating gastric ulcers. In our in vitro procoagulant activity test, we found that the in situ generated thrombin on the Ca-Y/HPC surface also showed a good resistance to acidity and pepsin, which can be formed rapidly upon contact with the bleeding site or wound. We thus expect that the stable thrombin function in HPC formed on the Ca-Y surface provides a positive influence towards wound healing and the tissue repair process¹⁸ during gastrointestinal injury. Eventually, this synergetic effect enables the Ca-Y zeolite good gastroprotective function and performs well in the in vivo experiments. This may provide an optional therapy for gastrointestinal disorders and more importantly, a different view of the bio-functionality of inorganic materials.

Conclusions

In this study, the in vitro procoagulant activity tests reveal the unusually good resistance of the Ca-Y zeolite to gastric fluid and later, the in vivo experiment of ethanol–HCl induced gastric ulcer using a mice model shows that an oral dosage of 5.0 g kg⁻¹ can significantly reduce the ulcer area percentage from 35.1% ± 4.4% to 11.5% ± 1.9% and improve the intragastric pH from 2.0 ± 0.5 to 4.5 ± 0.5, respectively. This gastroprotective effect was considered to be contributed by a synergist effect via (1) the excellent hemostatic ability and consequently stable highly active thrombin formed on the surface of Ca-Y and (2) the efficient anti-acid properties, which can improve intragastric pH. The results reveal that the linkage of zeolite mediated the thrombin activity to its therapeutic action in gastric disorders, providing a novel aspect for investigating the biological behaviour of inorganic materials in harsh physiological environments, which is crucial to their biological and medical applications.

Acknowledgements

We are grateful for financial support from the NSFC (21222307, 21271153, U1402233 and 21373181) and the Fundamental Research Funds for the Central Universities (2014XZZX003-02). We thank Dr Xiaochun Yang at the Center for Drug Safety Evaluation and Research of ZJU for his the assistance during the in vivo mice experiments.

Notes and references

1. N. D. Paguigan, D. H. Castillo and C. L. Chichioco-Hernandez, Arq. Gastroenterol., 2014, 51, 64–67.
2. K. S. de Lira Mota, G. E. Nunes Dias, M. E. Ferreira Pinto, A. Luiz-Ferreira, A. R. Monteiro Souza-Brito, C. A. Hiruma-Lima, J. M. Barbosa-Filho and L. M. Batista, Molecules, 2009, 14, 979–1012.
3. J. L. Martins, O. R. Rodrigues, S. D. Da, P. M. Galdino, J. R. de Paula, W. Romao, C. H. Da, B. G. Vaz, P. C. Ghedini and E. A. Costa, J. Ethnopharmacol., 2014, 155, 1616–1624.
4. R. J. Leonard, Encyclopedia of Gastroenterology Lower Digestive Tract, Motility and Neurogastroenterology, Science Press, 2008.
5. A. Lanas, Gastroenterol. Hepatol., 2014, 37, 62–70.
6. Y. Ran, E. Hadad, S. Daher, O. Ganor, J. Kohn, Y. Yegorov, C. Bartal, N. Ash and G. Hirschhorn, Prehosp. Disaster Med., 2010, 25, 584–588.
7. S. E. Baker, A. M. Sawvel, N. Zheng and G. D. Stucky, Chem. Mater., 2007, 19, 4390–4392.
8. S. A. Giday, Y. Kim, D. M. Krishnamurty, R. Ducharme, D. B. Liang, E. J. Shin, X. Dray, D. Hutcheon, K. Moskowitz, G. Donatelli, D. Rueben, M. I. Canto, P. I. Okolo and A. N. Kalloo, Endoscopy, 2011, 43, 296–299.
9. W. Potgieter, C. S. Samuels and J. R. Snyman, Clin. Exp. Gastroenterol., 2014, 7, 215–220.
10. A. Barkun, Gastroenterol. Hepatol., 2013, 9, 744–746.
11. M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall and K. A. Dawson, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 14265–14270.
12. S. Tenzer, D. Docter, J. Kuharev, A. Musyanovych, V. Fetz, R. Hecht, F. Schlenk, D. Fischer, K. Kiouptsi, C. Reinhardt, K. Landfester, H. Schild, M. Maskos, S. K. Knauer and R. H. Stauber, Nat. Nanotechnol., 2013, 8, 772–781.
13. Y. Li, X. Liao, X. Zhang, G. Ma, S. Zuo, L. Xiao, G. D. Stucky, Z. Wang, X. Chen, X. Shang and J. Fan, Nano Res., 2014, 7, 1457–1465.
14. N. C. McAvoy, J. N. Plevris and P. C. Hayes, World J. Gastroenterol., 2012, 18, 5912–5917.
15. J. Ramesh, J. K. Limdi, V. Sharma and A. J. Makin, Gastrointest. Endosc., 2008, 68, 877–882.
16. A. Oyagi, K. Ogawa, M. Kakino and H. Hara, BMC Complementary Altern. Med., 2010, 10, 45.
17. G. Rodríguez-Fuentes, A. R. Denis, M. A. Barrios Álvarez and A. I. Colarte, Microporous Mesoporous Mater., 2006, 94, 200–207.
18. Y. Li, H. Li, L. Xiao, L. Zhou, J. Shentu, X. Zhang and J. Fan, Materials, 2012, 5, 2586–2596.

---

Footnote

† Electronic supplementary information (ESI) available: Material synthesis and experimental details. See DOI: 10.1039/c5ra24467f

---