Prior transfusion of umbilical cord mesenchymal stem cells can effectively alleviate symptoms of motion sickness in mice through interleukin 10 secretion

Abstract

Background: Motion sickness (MS) is a disease that occurs during unbalanced movement, characterized by gastrointestinal symptoms and autonomic nervous system activation. Current clinical treatments for MS are limited. Recent evidence indicates that the levels of pro-inflammatory cytokines increase during MS and are associated with an inner ear immune imbalance. In the present study, mesenchymal stem cells (MSCs) have been shown to exert strong immuno-suppressive effects.

Aim: To explore whether umbilical cord-derived mesenchymal stem cells (UC-MSCs) can prevent the occurrence of MS, and the underlying mechanism regulated by MSCs in a mouse model of MS.

Methods: A total of 144 (equal numbers of males and females) 5wkold BALB/c mice were randomly divided into five groups: Normal group (n = 16), MS group (n = 32), MSCs group (n = 32), MS + MSCs group (n = 32), and MS + AS101/MSCs group (n = 32). The MSCs group (n = 32), MS + MSCs group (n = 32), and MS + AS101/MSCs group (n = 32) were preventively transplanted with UC-MSCs or AS101-treated UC-MSCs (1 × 10⁶ cells/mouse). Mice in the MS (n = 32), MS + MSCs, and MS + AS101/MSCs groups were subjected to rotation on a centrifuge for 10 min at 8 × g/min for MS model establishment on days 3, 5, 8, and 10 after UC-MSCs injection. The Morris water maze (MWM) test was used to observe the symptom of dizziness. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect the levels of inflammatory cytokines in mice peripheral blood and the petrous part of the temporal bone samples. Western blot analysis was performed to analyze the JAK2/STAT3 signaling pathway in the cochlear tissues. Histological examination was performed by hematoxylin and eosin (HE) staining for conventional morphological evaluation in the petrous part of temporal bone samples.

Results: The MWM test demonstrated that UC-MSCs improved the symptoms of MS. The MS + MSCs group was faster than the MS group on days 3 and 5 (P = 0.036 and P = 0.002, respectively). ELISA and RT-qPCR showed that the serum and mRNA levels of interleukin-10 (IL-10) in the cochlear tissues were increased after transplantation with UC-MSCs (MS + MSCs group vs MS group at 3 and 5 d, P = 0.002 and ^c P < 0.001, respectively). RT-qPCR results confirmed a significant increase in IL-10 levels at four time points (MS + MSCs group vs MS group, P = 0.009, P = 0.009, P = 0.048, and P = 0.049, respectively). This suggested that UC-MSCs reduced the sensitivity of the vestibular microenvironment by secreting IL-10. Moreover, Western blot analysis showed that the MSCs activated the JAK2/STAT3 signaling pathway in the cochlear tissues. The levels of IL-10, IL-10RA, JAK2, STAT3, and phosphorylated JAK2 and STAT3 in the MS + MSCs group were increased compared to those of the MS group (P < 0.05). The morphological changes in the four groups showed no significant differences. The role of IL-10 secretion on the ability of UC-MSCs to successfully improve the symptoms of MS was confirmed by the diminished therapeutic effects associated with treatment with the IL-10 inhibitor ammonium trichloro (dioxoethylene-o,o') tellurate (AS101).

Conclusion: Prophylactic transplantation of UC-MSCs can alleviate the clinical symptoms of MS in mice, particularly at 3-5 d after preventive transplantation. The mechanism for UC-MSCs to reduce the sensitivity of vestibular cortex imbalance may be the secretion of IL-10. The next step is to demonstrate the possibility of curing MS in the vestibular environment by intermittent transplantation of MSCs. Above all, MSCs are expected to become a new method for the clinical prevention and treatment of MS.

Keywords: Immune microenvironment; Inflammation; Interleukin-10; JAK2/STAT3; Mesenchymal stem cell; Motion sickness.

Figure 1

Immunophenotypes of umbilical cord-derived mesenchymal stem cells determined by flow cytometry. Images illustrate the expression of positive stem markers CD29, CD44, CD73, CD90, and CD105 and negative stem markers CD31, CD45, and CD271.

Figure 2

AS101 inhibits spontaneous interleukin-10 secretion by umbilical cord-derived mesenchymal stem cells. A: Secreted interleukin-10 (IL-10) levels in the supernatant under exposure to different AS101 concentrations, as measured by enzyme-linked immunosorbent assay. The IL-10 level decreased with 1.5-3.0 μg/mL of AS101; B and C: Flow cytometry analysis of the viability of umbilical cord-derived mesenchymal stem cells (UC-MSCs) after co-culture with AS101. There was no significant difference in the proportion of UC-MSCs according to AS101 concentration. IL-10: Interleukin-10.

Figure 3

Motion sickness model evaluation and effects of umbilical cord-derived mesenchymal stem cells treatment on performance of mice in Morris water maze tests. A: Motion sickness (MS) index score reflecting the degree of MS symptoms in mice; B: Serum glucose levels in the three groups before and after exposure to rotation (^cP < 0.001); C-F: Time taken for the mice to reach the platform after different treatments. The time of the MS + mesenchymal stem cells (MS + MSCs) group was faster than that of the MS group at 3 d and 5 d (^aP < 0.05; ^bP < 0.01); G: The 5-day group had the shortest time among the four MS + MSCs subgroups (^aP < 0.05); H: Swimming trajectories in the MSCs, MS, MS + MSCs, and MS + AS101/MSCs groups on day 5; the platform is shown as a small circle. In addition to the MSCs group, the path of mice in the MS + MSCs group was clearer. MWM: Morris water maze; MS: Motion sickness; MSCs: Mesenchymal stem cells.

Figure 4

The levels of interleukin-10 and interleukin-10RA in the five groups. A: Statistical analyses of interleukin-10 (IL-10) levels in the peripheral blood in the five groups (^aP < 0.05; ^bP < 0.01; ^cP < 0.001; n = 8); B: Statistical analyses of IL-10 mRNA levels in the petrous part of temporal bone tissues of mice at four time points, normalized to mouse Gapdh (^aP < 0.05; ^bP < 0.01; n = 3); C-E: Western blot analysis showing upregulated IL-10 and IL-10RA expression in the petrous part of the temporal bone in mice of the motion sickness + mesenchymal stem cells (MS + MSCs) group compared to the MS group, and downregulated expression in the MS + AS101/MSCs group on days 3 and 5 (^aP < 0.05; ^bP < 0.01; ^cP < 0.001; n = 3). These experiments were performed three times with similar results. IL: Interleukin; MS: Motion sickness; MSCs: Mesenchymal stem cells; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Western blot analysis of JAK2/ pJAK2 and STAT3/pSTAT3 expression. JAK2/STAT3 and pJAK2/pSTAT3 protein expression in the petrous part of the temporal bone was significantly upregulated in the motion sickness + mesenchymal stem cells (MS + MSCs) group compared to the MS group, and was downregulated in the MS + AS101/MSCs group on days 3 and 5 (^aP < 0.05; ^bP < 0.01; ^cP < 0.001; n = 3). A-C: JAK2/STAT3; D-F: pJAK2/pSTAT3. These experiments were performed three times with similar results. MS: Motion sickness; MSCs: Mesenchymal stem cells; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

Representative histology images (hematoxylin and eosin staining) of the cochleae in the four groups. Black arrows indicate the organ of Corti, blue arrows indicate vascular striate cells, and orange arrows indicate spiral ganglion cells. The dotted boxes are featured in the magnified inserts. Images of the four groups are shown in high-power fields (bar, 100 μm). MS: Motion sickness; MSCs: Mesenchymal stem cells.

Figure 7

Proposed mechanism of action of mesenchymal stem cells in the vestibular microenvironment. Mice were first prophylactically infused with mesenchymal stem cells (MSCs). After that, the motion sickness (MS) model was established at specified time points. At this time, pro-inflammatory cytokines in the vestibular environment of mice were released. MSCs were stimulated by these cytokines to secrete interleukin-10 (IL-10). Then, IL-10 receptors on fibrocytes in the cochlear lateral wall were triggered to activate the JAK2/STAT3 signaling pathway. Finally, the sensitivity of the vestibular cortex was reduced. IL: Interleukin; TNF-α: Tumor necrosis factor-alpha; MSCs: Mesenchymal stem cells; SOCS: Suppressor of cytokine signalling; Myc: Myelocytomatosis oncogene; CTFs: C-terminal fragments.