Tea-derived exosome-like nanoparticles prevent irritable bowel syndrome induced by water avoidance stress in rat model

Abstract

Background and aim: Exosome-like nanoparticles (ELNs) have emerged as crucial mediators of intercellular communication, evaluated as potential bioactive nutraceutical biomolecules. We hypothesized that oral ELNs have some therapeutic effect on irritable bowel syndrome (IBS).

Methods: In our study, ELNs from tea (Camellia sinensis) leaves were extracted by differential centrifugation. We investigated the role of ELNs by assessing visceral hypersensitivity, body weight, bowel habits, tight junctions, and corticotropin-releasing hormone (CRH) in rats subjected to water avoidance stress (WAS) to mimic IBS with and without ELNs (1 mg/kg per day) for 10 days.

Results: The average diameter of ELNs from LCC, FD and MZ tea tree were 165 ± 107, 168 ± 94, and 168 ± 108 nm, the concentration of ELNs were 1.2 × 10¹³, 1 × 10¹³, and 1.5 × 10¹³ particles/mL, respectively. ELNs can be taken up by intestinal epithelial cells. In WAS rats, ELNs significantly restored weight, recovered tight junctions, decreased CRH, and CRH receptor 1 expression levels and inhibited abdominal hypersensitivity in comparison to positive control.

Conclusions: Oral tea-derived ELN improves symptoms of IBS by potentially modulating the CRH pathway.

Keywords: abdominal hypersensitivity; biomolecules; corticotropin releasing hormone; exosome‐like nanoparticles; irritable bowel syndrome.

Figure 1

Isolation steps and characterization of TELNs. (a–d) The samples were derived from tea tree (a) and formed juice by grinding with PBS (b). Then TELNs were recovered from juice by using UL (c) and GUL (d). The TELNs are found at the 15/30% interface. (e) TELNs were examined by transmission electron microscopy. Representative TELNs are indicated by white arrows. Magnification: 50 000×. Scale bar: 200 nm. (f) The particle size of TELNs was analyzed by NTA. The sample (TELNs) was diluted 200 000 times of the original sample for NTA detection, and the NTA concentration was the total number obtained by statistics on the number of particles under different nanoparticle sizes. FD, Fuding tea‐derived exosome‐like nanoparticles; GUL, gradient ultracentrifugation; LCC, old Chuancha tea‐derived exosome‐like nanoparticles; MZ, Meizhan tea‐derived exosome‐like nanoparticles; TELNs, tea‐derived exosome‐like nanoparticles; UL, ultracentrifugation.

Figure 2

Cellular uptake of TELNs into epithelial cells. (a) NCM460 cells labeled with DAPI (blue channel) and phalloidin‐FITC (green channel). (b) NCM460 cells incubated with DiI‐LCC and then labeled with DAPI and phalloidin‐FITC. (c) NCM460 cells incubated with DiI‐FD and then labeled with DAPI and phalloidin‐FITC. (d) NCM460 cells incubated with DiI‐MZ and then labeled with DAPI and phalloidin‐FITC. Representative cellular uptake of TELNs is indicated by white arrows. Magnification: 400×. Scale bars: 20 μm. DAPI, 4′,6′‐diamidino‐2‐phenylindole; DiI, 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindocarbocyanine perchlorate; FD, Fuding tea‐derived exosome‐like nanoparticles; FITC, fluorescein isothiocyanate; LCC, old Chuancha tea‐derived exosome‐like nanoparticles; MZ, Meizhan tea‐derived exosome‐like nanoparticles; TELNs, tea‐derived exosome‐like nanoparticles.

Figure 3

TELNs attenuated the water avoidance stress induced change in body weights and defecation. (a) Flow diagram of experimental design employed in these studies. Rats were provided with WAS and/or various TELNs (1 mg protein/kg rat) every day. (b) Variations of body weight over time in (a). , Negative control; , Positive control; , LCC; , FD. , MZ. (c–e). Effect of stress on colonic transit. Fecal pellets were collected during 1 h of WAS, and the number of feces was recorded on day 3 (c), day 7 (d) and day 10 (e). The lowercase letters above bars indicated significant differences by one‐way ANOVA. Data were expressed as the mean ± SD. **P < 0.01 compared to the negative control group; ^## P < 0.01 compared to the positive control group. FD, Fuding tea‐derived exosome‐like nanoparticles; LCC, old Chuancha tea‐derived exosome‐like nanoparticles; MZ, Meizhan tea‐derived exosome‐like nanoparticles; TELNs, tea‐derived exosome‐like nanoparticles; WAS, water avoidance stress.

Figure 4

Water avoidance stress induced EMG in rat was reversed by TELNs treatment. (a,b) Representative recording in rat's EMG representing visceral hypersensitivity reaction treatment without (a) or with (b) balloon distension (BD) (0.4 mL, 10 s) in the different experimental groups. (c,d) Quantification of the area under the curve (AUC) in (a) and (b). The lowercase letters above bars indicated significant differences by one‐way ANOVA. Data were expressed as the mean ± SD. **P < 0.01 compared to the negative control group; ^## P < 0.01 compared to the positive control group. AUC, area under the curve; BD, balloon distension; FD: Fuding tea‐derived exosome‐like nanoparticles; EMG, electromyogram; LCC, old Chuancha tea‐derived exosome‐like nanoparticles; MZ, Meizhan tea‐derived exosome‐like nanoparticles; TELNs, tea‐derived exosome‐like nanoparticles.

Figure 5

Water avoidance stress induced CRH expression in rat brain was reversed by TELNs treatment. (a). Immunofluorescent detection CRH protein in the different experimental groups. Magnification: 400×. Scale bar: 50 μm. (b) The relative fluorescence intensity in (a) was quantified using ImageJ software. The lowercase letters above bars indicated significant differences by one‐way ANOVA. Data were expressed as the mean ± SD. **P < 0.01 compared to the negative control group; ^## P < 0.01 compared to the positive control group. CRH, corticotropin‐releasing hormone; DAPI, 4′,6′‐diamidino‐2‐phenylindole; FD, Fuding tea‐derived exosome‐like nanoparticles; LCC, old Chuancha tea‐derived exosome‐like nanoparticles; MZ, Meizhan tea‐derived exosome‐like nanoparticles; TELNs, tea‐derived exosome‐like nanoparticles.

Figure 6

Water avoidance stress induced CRHR1 expression in rat brain was reversed by TELN treatment. (a) Immunofluorescent detection CRHR1 protein in the different experimental groups. Magnification: 400×. Scale bar: 50 μm. (b) The relative fluorescence intensity in (a) was quantified using ImageJ software. The lowercase letters above bars indicated significant differences by one‐way ANOVA. Data were expressed as the mean ± SD. **P < 0.01 compared to the negative control group; ^## P < 0.01 compared to the positive control group. CRHR1, corticotropin‐releasing hormone receptor 1; DAPI, 4′,6′‐diamidino‐2‐phenylindole; FD, Fuding tea‐derived exosome‐like nanoparticles; LCC, old Chuancha tea‐derived exosome‐like nanoparticles; MZ, Meizhan tea‐derived exosome‐like nanoparticles; TELNs, tea‐derived exosome‐like nanoparticles.

Figure 7

Water avoidance stress induced tight junction protein expression in rat colon was reversed by TELNs treatment. (a,b) Immunofluorescent detection ZO‐1 (a) and occludin (b) protein in the different experimental groups. Magnification: 400×. Scale bar: 50 μm. Representative ZO‐1 or occludin protein expression are indicated by white arrows. (c,d) The relative fluorescence intensity in (a) and (b) was quantified using ImageJ software. The lowercase letters above bars indicated significant differences by one‐way ANOVA. Data were expressed as the mean ± SD. **P < 0.01 compared to the negative control group; ^## P < 0.01 compared to the positive control group. DAPI, 4′,6′‐diamidino‐2‐phenylindole; FD, Fuding tea‐derived exosome‐like nanoparticles; LCC, old Chuancha tea‐derived exosome‐like nanoparticles; MZ, Meizhan tea‐derived exosome‐like nanoparticles; TELNs, tea‐derived exosome‐like nanoparticles; ZO‐1, zonula occluden‐1.