Design of Antioxidant Nanoparticle, which Selectively Locates and Scavenges Reactive Oxygen Species in the Gastrointestinal Tract, Increasing The Running Time of Mice

Abstract

Excess reactive oxygen species (ROS) produced during strong or unfamiliar exercise cause exercise-induced gastrointestinal syndrome (EIGS), leading to poor health and decreased exercise performance. The application of conventional antioxidants can neither ameliorate EIGS nor improve exercise performance because of their rapid elimination and severe side effects on the mitochondria. Hence, a self-assembling nanoparticle-type antioxidant (RNP^O ) that is selectively located in the gastrointestinal (GI) tract for an extended time after oral administration is developed. Interestingly, orally administered RNP^O significantly enhances the running time until exhaustion in mice with increasing dosage, whereas conventional antioxidants (TEMPOL) tends to reduce the running time with increasing dosage. The running (control) and TEMPOL groups show severe damage in the GI tract and increased plasma lipopolysaccharide (LPS) levels after 80 min of running, resulting in fewer red blood cells (RBCs) and severe damage to the skeletal muscles and liver. However, the RNP^O group is protected against GI tract damage and elevation of plasma LPS levels, similar to the nonrunning (sedentary) group, which prevents damage to the whole body, unlike in the control and TEMPOL groups. Based on these results, it is concluded that continuous scavenging of excessive intestinal ROS protects against gut damage and further improves exercise performance.

Keywords: exercise-induced gastrointestinal syndrome; high-intensity running; leaky gut; polymeric nanoparticle antioxidants; reactive oxygen species.

Scheme 1

Schematic illustration of RNP^O.

Figure 1

Results of the all‐out running test. A) All‐out time for each mouse. The number in parentheses denotes the dose of TEMPO in 10⁻² mmol kg⁻¹ body weight (Black: Control, 0 mmol nitroxide radicals kg⁻¹ body weight, Blue: TEMPOL, Green: RNP^O). B) Effect of dose dependency on the average all‐out time for the obtained data in Figure 1A. Mice were forced to run until they reach their limit and were no longer able to move forward. TEMPOL was administered at 0.39–0.69 mmol kg⁻¹. RNP^O was administered at the same dose as the TEMPOL group (0.39–0.69 mmol‐TEMPO kg⁻¹, 200–400 mg‐RNP^O kg⁻¹). Data are expressed as the mean ± standard error of the mean (SEM) (n = 7–9). *p < 0.05 versus Control (0 mmol nitroxide radicals kg⁻¹ body weight in the graph), ##p < 0.01 versus within same concentration between the TEMPOL and RNP^O groups.

Figure 2

Suppression of high‐intensity running‐induced GI tract damage by RNP^O. A) Representative histopathological sections of duodenum tissues stained with H&E, scale bar = 100 µm. B) Damage score in the duodenum after rest or high‐intensity running on 80 min (n = 16–17). C) Villi height (n = 103–131), D) Crypt depth (n = 103–131), E) The villus height/crypt depth ratio after rest or high‐intensity running for 80 min. F‐H) Gene expression of inflammatory cytokines in the small intestine after rest or high‐intensity running for 80 min, F) Interleukin‐6 (IL‐6) (n = 8–9), G) Tumor necrosis factor alpha (TNF‐α (n = 7–8), H) Interferon‐gamma (IFN‐γ (n = 8–9). I‐L) Protein expression of inflammatory cytokines in the small intestine after rest or high‐intensity running for 80 min, I) Immunoblots of β‐actin, IL‐6, TNF‐α, and IFN‐γ, J) IL‐6 (n = 8–9), K) TNF‐α (n = 8–9), L) IFN‐γ (n = 8–9). M, N) Oxidative stress levels in the small intestine after rest or high‐intensity running for 80 min, M) Protein carbonyl (n = 8), N) Malondialdehyde (MDA) (n = 8–9). TEMPOL was administered at 0.69 mmol kg⁻¹. RNP^O was administered at the same dose as the TEMPOL group (0.69 mmol‐TEMPO kg⁻¹, 400 mg‐RNP^O kg⁻¹). Data are expressed as the mean ± SEM (*p < 0.05, **p < 0.01).

Figure 3

Protection against high‐intensity running‐induced RBC damage by RNP^O. A) The number of RBCs after rest or high‐intensity running for 80 min (n = 8–9). B, C) Oxidative stress levels after rest or high‐intensity running for 80 min, B) Protein carbonyl (n = 8–9), C) MDA (n = 8–9). D, E) Osmotic fragility of the RBCs collected after rest or high‐intensity running for 80 min, D) Hemolyzed fraction of RBCs in response to NaCl concentration, E) Half‐hemolytic concentrations of NaCl (n = 8–9). TEMPOL was administered at 0.69 mmol kg⁻¹. RNP^O was administered at the same dose as that in the TEMPOL group (0.69 mmol‐TEMPO kg⁻¹, 400 mg‐RNP^O kg⁻¹). Data are expressed as the mean ± SEM (*p < 0.05, **p < 0.01).

Figure 4

Suppression of high‐intensity running induced‐leaky gut by scavenging ROS in the GI tract. LPS levels in plasma after rest or high‐intensity running for 80 min (n = 8–9). TEMPOL was administered at 0.69 mmol kg⁻¹. RNP^O was administered at the same dose as the TEMPOL group (0.69 mmol‐TEMPO kg⁻¹, 400 mg‐RNP^O kg⁻¹). Data are expressed as the mean ± SEM (*p < 0.05, **p < 0.01).

Figure 5

Prevention of high‐intensity running‐induced skeletal muscle damage by suppression of leakiness of gut wall (leaky gut) by oral RNP^O administration. A, B) Skeletal muscle damage marker in plasma after rest or high‐intensity running for 80 min, A) LDH (n = 16–17), B) CK (n = 15–17). C‐E) Gene expression of inflammatory cytokines in the muscle after rest or high‐intensity running for 80 min, C) IL‐6 (n = 8–9), D) TNF‐α (n = 7–9), E) IFN‐γ (n = 8–9). F‐I) Protein expression of inflammatory cytokines in the muscle after rest or high‐intensity running for 80 min, F) Immunoblots of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), IL‐6, TNF‐α, and IFN‐γ, G) IL‐6 (n = 8–9), H) TNF‐α (n = 8–9), I) IFN‐γ (n = 8–9). J, K) Oxidative stress levels in the muscle after rest or high‐intensity running for 80 min, J) Protein carbonyl (n = 8–9), K) MDA (n = 8–9). TEMPOL was administered at a 0.69 mmol kg⁻¹. RNP^O was administered at the same dose as the TEMPOL group (0.69 mmol‐TEMPO kg⁻¹, 400 mg‐RNP^O kg⁻¹). Data are expressed as mean ± SEM (*p < 0.05, **p < 0.01).

Figure 6

Suppression of high‐intensity running‐induced liver injury caused by EIGS. A, B) Liver damage marker in plasma after rest or high‐intensity running for 80 min, A) ALT (n = 15–17), B) AST (n = 16–17). C‐E) Gene expression of inflammatory cytokines in the liver after rest or high‐intensity running for 80 min, C) IL‐6 (n = 7–9), D) TNF‐α (n = 8–9), E) IFN‐γ (n = 8–9). F‐I) Protein expression of inflammatory cytokines in the liver after rest or high‐intensity running for 80 min, F) Immunoblots of GAPDH, IL‐6, TNF‐α, and IFN‐γ, G) IL‐6 (n = 8–9), H) TNF‐α (n = 8–9), I) IFN‐γ (n = 8–9). J, K) Oxidative stress levels in the liver after rest or high‐intensity running for 80 min, J) Protein carbonyl (n = 8–9), K) MDA (n = 8–9). TEMPOL was administered at 0.69 mmol kg⁻¹. RNP^O was administered at the same dose as the TEMPOL group (0.69 mmol‐TEMPO kg⁻¹, 400 mg‐RNP^O kg⁻¹). Data are expressed as the mean ± SEM (*p < 0.05, **p < 0.01).

Figure 7

Gastrointestinal ROS impairs systemic function whereas RNP^O improves exercise performance.