Mesenchymal stem cells suppress neuronal apoptosis and decrease IL-10 release via the TLR2/NFκB pathway in rats with hypoxic-ischemic brain damage

Abstract

Background: Hypoxic-ischemic brain damage (HIBD) is a major cause of infant mortality and neurological disability in children. Many studies have demonstrated that mesenchymal stem cell (MSC) transplantation facilitates the restoration of the biological function of injured tissue following HIBD via immunomodulation. This study aimed to elucidate the mechanisms by which MSCs mediate immunomodulation via the key effectors Toll-like receptor 2 (TLR2) and interleukin-10 (IL-10).

Results: We showed that TLR2 expression in the brain of HIBD rats was upregulated following HIBD and that MSC transplantation suppressed the expression of TLR2 and the release of IL-10, thereby alleviating the learning-memory deficits of HIBD rats. Following treatment with the specific TLR2 agonist Pam3CSK4 to activate TLR2, learning-memory function became further impaired, and the levels of nuclear factor kappa B (NFκB) and Bax expression and IL-10 release were significantly increased compared with those in HIBD rats that did not receive Pam3CSK4. In vitro, we found that MSC co-culture downregulated TLR2/NFκB signaling and repressed Bax expression and IL-10 secretion in oxygen and glucose deprivation (OGD)-injured adrenal pheochromocytoma (PC12) cells. Furthermore, NFκB and Bax expression and IL-10 release were enhanced following Pam3CSK4 treatment and were decreased following siTLR2 treatment in OGD-injured PC12 cells in the presence or absence of MSCs.

Conclusions: Our data indicate that TLR2 is involved in HIBD and that MSCs decrease apoptosis and improve learning-memory function in HIBD rats by suppressing the TLR2/NFκB signaling pathway via a feedback mechanism that reduces IL-10 release. These findings strongly suggest that MSC transplantation improves HIBD via the inhibition of the TLR2/NFκB pathway.

Fig. 1

MSC transplantation improved the learning-memory function and reduced the TLR2/IL-10 expression levels of HIBD rats. a Diagram illustrating the experimental protocols of the treatments and tests used in the rats. b-c The escape latencies and path lengths to reach the visible platform on the first day of the Morris water maze test for the control, HIBD and MSCs rats. d From the 2^nd to the 5^th day of the Morris water maze test, the escape latencies to locate the visible platform gradually decreased in all three groups. e The duration spent in the former platform quadrant by each of the three groups on the final day of the Morris water maze test. n = 20 in each group. f-g The TLR2 mRNA and protein expression levels in the rat brains on the 3^rd, 7^th and 14^th days following HIBD. n = 5 in each group. h The quantifications of WB signal in g. i The changes in the IL-10 secretion levels in the brains at the three time points after HIBD in all three groups. n = 6 in each group. ^# P < 0.05 vs. the control group; &P < 0.05 vs. the HIBD group

Fig. 2

Pam3CSK4 exacerbated learning-memory dysfunction and stimulated the TLR2/NFκB signaling pathway and IL-10 secretion in HIBD rats. a Diagram illustrating the experimental protocols of treatments and tests used in the rats. b-c The escape latencies and path lengths to reach the visible platform on the first day of the Morris water maze test for the HIBD and Pam3CSK4 groups. d The escape latencies to reach the visible platform on the 2^nd through the 5^th days of the Morris water maze test for the Pam3CSK4-treated and HIBD groups. e The duration spent in the hidden platform quadrant by the Pam3CSK4-treated and HIBD groups. n = 20 in each group. f-g The Pam3CSK4-induced mRNA and protein expression levels of TLR2, NFκB P65 and Bax in the brains of 4-week-old rats. n = 5 in each group. h The quantifications of WB signal in G. i IL-10 release in the brains of 4-week-old rats in HIBD and Pam3CSK4 groups. n = 6 in each group. ^# P < 0.05 vs. the HIBD group

Fig. 3

MSC transplantation decreased the expression of TLR2 in neurons and suppressed apoptosis in the cerebral cortex of HIBD rats. a Double immunofluorescence staining of NSE or GFAP or Iba1 (green) together with TLR2 nred) in the cerebral cortex of rats of the control, HIBD and MSCs groups. a, d and g. Double immunofluorescence staining of NSE together with TLR2. The white arrows indicate co-localization of NSE and TLR2. b, e and h. Double immunofluorescence staining of GFAP together with TLR2. c, f and i. Double immunofluorescence staining of Iba1 together with TLR2. Scale bar = 50 μm. n = 4 in each group. b TUNEL staining in the rat cerebral cortex of the control, HIBD and MSCs groups. a, d and g. TUNEL-positive cells. b, e and h. DAPI-stained cells. c, f and i. Merged images of TUNEL-positive cells and DAPI-stained cells. The white arrows indicate TUNEL-positive cells that were merged with DAPI-stained cells. Scale bar = 50 μm. c The percentage of TUNEL-positive cells in the three groups of control, HIBD and MSCs. n = 5 in each group. The results are presented as the mean ± SEM. ^# P < 0.05 vs. the control group; &P < 0.05 vs. the HIBD group

Fig. 4

Co-culturing OGD-injured PC12 cells with MSCs decreased the expression of TLR2, NFκB, Bax and IL-10. a-b The mRNA and protein expression levels of TLR2 in OGD-injured PC12 cells in the presence or absence of MSCs. n = 6 in each group. c The quantifications of WB signal in b. d-e The mRNA and protein expression levels of NFκB P65 in OGD-injured PC12 cells in the presence or absence of MSCs. n = 6 in each group. f The quantifications of WB signal in e. g-h The mRNA and protein expression levels of Bax in OGD-injured PC12 cells in the presence or absence of MSCs. n = 6 in each group. i The quantifications of WB signal in h. j The concentrations of IL-10 released into the culture media were measured in the control, OGD and OGD + MSCs groups via ELISA. n = 6 in each group. ^# P < 0.05 vs. the control PC12 cells; &P < 0.05 vs. the OGD-injured PC12 cells

Fig. 5

MSC co-culture suppressed apoptosis in OGD-injured PC12 cells. a Annexin V-FITC/PI double staining of PC12 cells in the control, OGD and OGD + MSCs groups. a, d and g. Annexin-V-positive PC12 cells in the control, OGD and OGD + MSCs groups. b, e and h. PI-positive PC12 cells in the control, OGD and OGD + MSCs groups. c, f and i. Annexin-V-positive cells and PI-positive cells merged with bright-field images of PC12 cells in the control, OGD and OGD + MSCs groups. Scale bar = 100 μm. b-c Annexin-V and PI sum intensity of PC12 cells in the control, OGD and OGD + MSCs groups. n = 5 in each group. The results are presented as the mean ± SEM. ^# P < 0.05 vs. control PC12 cells; &P < 0.05 vs. OGD-injured PC12 cells

Fig. 6

Pam3CSK4 effectively activated and Ad-siTLR2 specifically blocked the TLR2/NFκB signaling pathway in PC12 cells. a-b The mRNA and protein expression levels of TLR2 in PC12 cells following Pam3CSK4 treatment or Ad-siTLR2 infection. c The quantifications of WB signal in b. n = 6 in each group. d-e The mRNA and protein expression levels of NFκB in PC12 cells following Pam3CSK4 treatment or Ad-siTLR2 infection. f The quantifications of WB signal in e. n = 6 in each group. ^# P < 0.05 vs. untreated PC12 cells; &P < 0.05 vs. Ad-RFP-infected PC12 cells

Fig. 7

MSCs modulated the TLR2/NFκB signaling pathway and suppressed the apoptosis of OGD-injured PC12 cells and the secretion of IL-10. a Diagram illustrating the experimental protocols of the treatments and tests for PC12 cells. b-c The TLR2 mRNA and protein expression levels in co-cultures of MSCs with OGD-injured PC12 cells that received Pam3CSK4 or Ad-siTLR2 treatment. n = 6 in each group. d The quantifications of WB signal in c. e-f The mRNA and protein expression levels of NFκB P65 in co-cultures of MSCs with OGD-injured PC12 cells that were treated with Pam3CSK4 or Ad-siTLR2. n = 6 in each group. g The quantifications of WB signal in f. h-i The changes in the mRNA and protein expression levels of Bax in co-cultures of MSCs with OGD-injured PC12 cells that were treated with Pam3CSK4 or Ad-siTLR2. n = 6 in each group. j The quantifications of WB signal in i. k The changes in IL-10 release in co-cultures of MSCs with OGD-injured PC12 cells that received Pam3CSK4 or Ad-siTLR2 treatment. n = 6 in each group. ^# P < 0.05 vs. OGD-injured PC12 cells that were treated with Pam3CSK4 or Ad-siTLR2