Targeted delivery of Nitric Oxide triggered by α-Glucosidase to Ameliorate NSAIDs-induced Enteropathy

Abstract

Nonsteroidal anti-inflammatory drugs (NSAIDs) increase risks of severe small intestinal injuries. Development of effective therapeutic strategies to overcome this issue remains challenging. Nitric oxide (NO) as a gaseous mediator plays a protective role in small intestinal injuries. However, small intestine-specific delivery systems for NO have not been reported yet. In this study, we reported a small intestine-targeted polymeric NO donor (CS-NO) which was synthesized by covalent grafting of α-glucosidase-activated NO donor onto chitosan. In vitro and in vivo experiments demonstrated that CS-NO could be activated by intestinal α-glucosidase to release NO in the small intestine. Pre-treatment of mice with CS-NO significantly alleviated small intestinal damage induced by indomethacin, as demonstrated by down-regulation of the levels of pro-inflammatory cytokines and chemokines CXCL1/KC. Moreover, CS-NO also attenuated indomethacin-induced gut barrier dysfunction as evidenced by up-regulation of the levels of tight junction proteins and restoration of the levels of goblet cells and MUC2 production. Meanwhile, CS-NO effectively restored the defense function of Paneth cells against pathogens in small intestine. Our present study paves the way to develop NO-based therapeutic strategy for NSAIDs-induced small intestinal injuries.

Keywords: Anti-inflammation; Nitric oxide donor; Small intestinal injury; Small intestine-targeting; α-Glucosidase.

Graphical abstract

Fig. 1

Alpha-glucosidase-induced NO-releasing mechanism from CS-NO.

Scheme 1

The synthetic route for preparation of CS-NO.

Fig. 2

Characterization of synthesized CS-NO. (a) ¹H NMR spectrum of CS-NO; (b) Elemental analysis of CS-NO; (c) TEM images of CS-NO nanoparticles. Scale bar: 1.0 μm; d) size distribution and hydrodynamic diameter from DLS.

Fig. 3

In vitro NO release from CS-NO. a) NO release from CS-NO (0.5 mg/mL) was measured by Griess assay in phosphate buffer solution (PB, pH 5.0, 6.0 and 7.4) containing α-glucosidase (0.01 mg/mL) at 37 °C; data was expressed at mean ± s.e.m, n = 3. b) The NO levels released from CS-NO (0.5 mg/mL)were measured by spin trapping technique in PB (pH 5.0, 6.0, 7.4) containing α-glucosidase(0.01 mg/mL); c) The NO levels released from CS-NO (3.5 mg/mL)were measured by spin trapping technique in PBS (pH 7.4, 20 mM) containing mice ileum homogenate (1 mg/mL); data was expressed at mean ± s.e.m, n = 3; d) EPR spectra of the NO adduct (i.e., MGD-Fe-NO) at different incubation times which were generated from the mixture of CS-NO (3.5 mg/mL), mice ileum homogenate (1 mg/mL) and MGD-Fe (1 mM) in PBS (pH 7.4, 20 mM).

Fig. 4

In vitro and in vivo NO release from CS-NO. a) EPR spectra showing the NO release from CS-NO in duodenum and ileum after intragastric administration of 100 μL of 50% CS-NO solution (20 mg/mL). b) The amount of the NO adducts (DETC)₂Fe–NO) measured in duodenum and ileum; data was expressed at mean ± s.e.m, n = 5; *p < 0.05, ***P < 0.001. c) In vivo NO generation from CS-NO was detected by fluorescence imaging. CS group was administered intragastrically with 100 μL of 50% CS (20 mg/mL) and 20 μL of DAC-S (0.4 mM), while CS-NO group was administered with 100 μL of CS-NO (20 mg/mL) and DAC-S (0.4 mM, 20 μL). d) Total radiant efficiency from the mice administrated with CS and CS-NO respectively. Mean ± s.e.m; n = 3; *P < 0.05. Statistical analyses were performed with a two-tailed student's test. e) The sensing mechanism of DAC-S for NO.

Fig. 5

Suppression of indomethacin-induced body weight loss, intestinal injuries and the intestinal low-grade inflammation in mice by CS-NO. a) Time-course percentage changes of body weights after indomethacin administration. b) Histological scores from the small intestine (Left) and the complete length of small intestine (Right). c) Mucosal bleeding of the distal small intestine. d) H&E staining of the distal small intestine. Scale bars: 1000 μm (Left), 100 μm (Right). e) Expression of inflammatory cytokines including TNF-α, IL-1β, IL-6 and KC were quantitatively analysed by Realtime PCR. Total RNA was extracted from the distal small intestine. CS-NO: n = 10, Chitosan: n = 7, PBS: n = 8. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6

Protection of the intestinal barrier against indomethacin-induced small intestinal injury by CS-NO. a) The relative expressions of the intestinal tight junction proteins including Claudin3, Occludin, ZO-1, and Goblet cell product MUC2 were measured by Realtime PCR. Total RNA was extracted from the distal small intestine. b) Periodic acid Schiff staining for Goblet cells and MUC2 staining in the distal small intestine (Left) and the number of positive staining cells in each villus (Right). Scale bars: 50 μm. c) The morphology of Paneth granules was presented by H&E staining. The morphology of Paneth granules could be classified into four categories by the expression of lysozyme (represented in red, white represents areas that exclude lysozyme): normal (D0), disordered (D1), depleted (D2), and diffuse (D3). Percentage of Paneth cells showed normal (D0) and abnormal (D1-D3) patterns of lysozyme expression. One hundred Paneth cells for each mouse were scored. Scale bars: 100 μm (Left), 20 μm (Right).CS-NO: n = 10, Chitosan: n = 7, PBS: n = 8. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)