Mesenchymal stem cells' exosomes alleviate chronic visceral pain through Nrf-2-mediated oxidative stress pathway in rats

Abstract

Introduction: Irritable bowel syndrome (IBS) is a prevalent chronic functional gastrointestinal disorder characterized by visceral hypersensitivity (VH), affecting over 10% of the global population. Current treatments for IBS have notable limitations, highlighting the need for novel therapeutic strategies to address chronic visceral pain and associated comorbidities. Objectives: This study aimed to investigate whether bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) can alleviate chronic visceral pain in IBS and to explore the underlying molecular mechanisms, with a specific focus on the Nrf-2/HO-1 oxidative stress pathway.

Methods: High-quality BMSC-Exos were isolated and characterized. In vitro, their neuroprotective effects were assessed in oxidatively stressed neurons. In vivo, IBS model rats received intrathecal injections of BMSC-Exos, and their effects on visceral pain sensitivity and anxiety-like behaviors were evaluated. Spinal cord tissues were analyzed to determine modulation of the Nrf-2/HO-1 pathway.

Results: In vitro studies demonstrated that BMSC-Exos effectively rescued oxidative stress-induced neuronal damage. In IBS rats, intrathecally administered BMSC-Exos were internalized by spinal cord neurons, significantly reducing visceral hypersensitivity and anxiety-like behaviors. Mechanistically, BMSC-Exos upregulated the Nrf-2/HO-1 antioxidant pathway, mitigating oxidative stress-induced neuronal damage.

Conclusion: These findings demonstrate that BMSC-Exos alleviate chronic visceral pain and comorbid anxiety in IBS rats, likely through Nrf-2/HO-1-mediated oxidative stress reduction in spinal neurons. These results highlight BMSC-Exos as a promising acellular therapeutic strategy for IBS, offering potential applications for this debilitating disorder.

Keywords: Anxiety mood; Bone marrow mesenchymal stem cell–derived exosomes; Chronic visceral hyperalgesia; Nrf-2/HO-1.

Figure 1.

Neonatal rats exposed to CRD developed chronic visceral hyperalgesia and anxiety-like behaviors in adulthood. (A) Experimental design. (B) Hematoxylin and eosin (HE) staining of the descending colon, magnification ×100. (C and D) Representative EMG recordings in control and IBS rats under 40 and 60 mm Hg CRD stimulation, with statistical analysis of EMG amplitudes (n = 10, 2-way ANOVA, F = 52.730, ***P < 0.001). (E–H) Representative trajectories in the OFT, statistical graphs of total distance moved, time spent in the central zone, and distance traveled in the central zone in control and IBS rats (n = 7, t test, t = 3.868, t = 5.260, **P < 0.01, ***P < 0.001). (I–L) Representative trajectories in the EPM, statistical graphs of percentage of distance traveled in the open arms, percentage of time spent in the open arms, and percentage of open arm entries in control rats and IBS rats (n = 7, t test, t = 3.762, t = 2.230, t = 2.267, *P < 0.05, **P < 0.01). CRD, colorectal distension; EMG, electromyography; EPM, elevated plus maze; IBS, irritable bowel syndrome; OFT, open field test.

Figure 2.

Imbalance of the Nrf-2/HO-1 signaling pathway in spinal cord neurons of IBS rats. (A) Immunofluorescence (IF) showing predominant Nrf-2 expression (red) in neurons (NeuN) in the spinal cord, with no apparent co-localization with astrocytes (GFAP) or microglia (Iba-1). DAPI was used for nuclear staining (blue). Scale bar: 100 μm (log-mag) and 20 μm (high-mag). (B and C) Representative IF images and quantitative analysis of Nrf2 in the spinal cord of control and IBS rats (n = 4, t test, t = 4.646, **P < 0.01; scale bar: 50 μm). (D–F) Representative images and statistical graphs of Nrf-2 and HO-1 protein expression in the spinal cord of control and IBS rats (n = 8, t test, t = 5.198, t = 4.723, ***P < 0.001). IBS, irritable bowel syndrome.

Figure 3.

Characteristics of BMSCs and their derived exosomes. (A) Morphological characteristics of rat bone marrow mesenchymal stem cells under the microscopy, magnification ×100. (B–F) Flow cytometry histograms showing fluorescence intensity with antibodies against surface markers (red area) compared with isotype controls (blue area). CD73 (99.7%), CD90 (99.8%), and CD105 (99.8%) were positive, whereas CD34 (0.013%) and CD45 (0.013%) were negative. (G) Representative TEM image of exosomes (arrowheads). Scale bar, 100 nm. (H) Size distribution of isolated BMSC-Exos. (I) Western blot analysis of protein expression in BMSC-Exos with 293T cells as control. BMSC, bone marrow mesenchymal stem cell; TEM, transmission electron microscopy.

Figure 4.

BMSC-Exos reversed oxidative stress-induced neuronal damage in vitro. (A) IF showing co-localization of different doses of PKH26-labeled BMSC-Exos (red) with PC12 cells. DAPI was used for nuclear staining (blue). Scale bar: 100 μm (log-mag) and 20 μm (high-mag). (B) Quantification of NSE expression in PC12 cells by ELISA (n = 6, t test, t = 3.758, **P < 0.01). (C) Efficiency ranking of candidate Nrf-2 siRNA at 10 nM (n = 4, 1-way ANOVA, F = 23.312, *P < 0.05). (D and E) Statistical graphs of Nrf-2 and HO-1 mRNA expression in PC12 cells across groups (n = 5, 1-way ANOVA, F = 18.886, *P < 0.05, **P < 0.01). (F–H) Statistical graphs of MDA, SOD, and CAT levels in PC12 cells in the si-NC + Exos and si-Nrf-2 + Exos groups (n = 6, t test, t = 3.118, t = 2.243, t = 3.346, *P < 0.05, **P < 0.01). BMSC, bone marrow mesenchymal stem cell; CAT, catalase; IF, immunofluorescence; MDA, malondialdehyde; NSE, neuron-specific enolase; SOD, superoxide dismutase; TEM, transmission electron microscopy.

Figure 5.

BMSC-Exos administration improved visceral hypersensitivity and anxiety-like behaviors in IBS rats. (A) Experimental design. (B) IF showing predominant localization of BMSC-Exos (red) in neurons (NeuN) of the spinal cord, with minimal co-localization in astrocytes (GFAP) or microglia (Iba-1), DAPI was used for nuclear staining (blue). Scale bar: 100 μm (log-mag) and 10 μm (high-mag). (C and D) Representative EMG recordings and statistical analysis of EMG amplitudes under 40 and 60 mm Hg CRD in rats treated with different doses (n = 6, 2-way ANOVA, *P < 0.05). (E–H) Representative OFT trajectories and statistical graphs of total distance moved, time spent in the central zone, and distance traveled in the central zone in control and exosome-injected rats (n = 6, t test, t = 2.90, t = 3.957, *P < 0.05, **P < 0.01). (I–L) Representative EPM trajectories and statistical graphs of percentage of distance traveled, time spent, and entries into the open arms in control rats and IBS rats treated with exosomes (n = 6, t test, t = 2.830, t = 2.287, t = 2.494, *P < 0.05). BMSC, bone marrow mesenchymal stem cell; CRD, colorectal distension; EMG, electromyography; EPM, elevated plus maze; IBS, irritable bowel syndrome; IF, immunofluorescence; OFT, open field test.

Figure 6.

The antioxidative effect of BMSC-Exos is dependent on the Nrf-2/HO-1 pathway. (A–C) Representative images and statistical graphs of Nrf-2 and HO-1 protein expression in the spinal cord of the Model group (IBS + Vehicle) and Treatment group (IBS + BMSC-Exos) (n = 8, t test, t = 3.686, t = 3.867, **P < 0.01). (D–F) Representative images and statistical graphs of Nrf-2 and HO-1 protein expression in the Treatment group (IBS + Exos + Vehicle) and Antagonist group (IBS + Exos + ML385) (n = 8, t test, t = 3.655, t = 5.044, **P < 0.01, ***P < 0.001). (G–H) Representative EMG recordings and statistical graphs of rats in the Treatment group (IBS + Vehicle + Exos) and Antagonist group (IBS + ML385 + Exos) under 40 and 60 mm Hg CRD (n = 8, 2-way ANOVA, F = 44.790, **P < 0.01). (I–L) Representative OFT trajectories and statistical graphs of total distance moved, time spent in the central zone, and distance traveled in the central zone by rats (n = 6, t test, t = 0.682, t = 2.854, t = 2.555, *P < 0.05). (M–P) Representative EPM trajectories and statistical graphs of percentage of entries, distance traveled, and time spent in the open arms (n = 6, t test, t = 2.736, t = 5.085, t = 4.729, *P < 0.05, ***P < 0.001). (Q–S) Statistical graphs of MDA content and SOD and CAT activities in spinal cord tissues of the Blank group (Control + Vehicle), Model group (IBS + Vehicle), Treatment group (IBS + Exos), and Antagonist group (IBS + Exos + ML385) (n = 6, t test, *P < 0.05, **P < 0.01, ***P < 0.001). BMSC, bone marrow mesenchymal stem cell; CAT, catalase; CRD, colorectal distension; IBS, irritable bowel syndrome; MDA, malondialdehyde; OFT, open field test; SOD, superoxide dismutase.