Endogenous IL-6 of mesenchymal stem cell improves behavioral outcome of hypoxic-ischemic brain damage neonatal rats by supressing apoptosis in astrocyte

Abstract

Mesenchymal stem cell (MSC) transplantation reduces the neurological impairment caused by hypoxic-ischemic brain damage (HIBD) via immunomodulation. In the current study, we found that MSC transplantation improved learning and memory function and enhanced long-term potentiation in neonatal rats subjected to HIBD and the amount of IL-6 released from MSCs was far greater than that of other cytokines. However, the neuroprotective effect of MSCs infected with siIL-6-transduced recombinant lentivirus (siIL-6 MSCs) was significantly weakened in the behavioural tests and electrophysiological analysis. Meanwhile, the hippocampal IL-6 levels were decreased following siIL-6 MSC transplantation. In vitro, the levels of IL-6 release and the levels of IL-6R and STAT3 expression were increased in both primary neurons and astrocytes subjected to oxygen and glucose deprivation (OGD) following MSCs co-culture. The anti-apoptotic protein Bcl-2 was upregulated and the pro-apoptotic protein Bax was downregulated in OGD-injured astrocytes co-cultured with MSCs. However, the siIL-6 MSCs suppressed ratio of Bcl-2/Bax in the injured astrocytes and induced apoptosis number of the injured astrocytes. Taken together, these data suggest that the neuroprotective effect of MSC transplantation in neonatal HIBD rats is partly mediated by IL-6 to enhance anti-apoptosis of injured astrocytes via the IL-6/STAT3 signaling pathway.

Figure 1. MSC transplantation restores learning and memory function in neonatal HIBD rats.

(A) Diagram illustrating the experimental protocols of treatments and tests for rats in the sham, HIBD and HIBD + MSCs groups. (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 sham, HIBD and HIBD + MSCs rats. (D) The escape latencies of each group to locate the visible platform from the 2nd to the 5th day in the Morris water maze test. (E) The number of passes through the former platform region of each group on the final day of the Morris water maze test. n = 15. (F) The discrimination ratio of the exploration time during the test phase of the object-in-place task among the three groups. n = 15. (G) The mean fEPSP slope of the three groups. n = 6. The results are presented as the mean ± SEM. *P < 0.05 vs. the sham group, ^&P < 0.05 vs. the HIBD group.

Figure 2. High level of IL-6 secreted by MSCs and was reduced in MSCs infected with siIL-6-transduced lentivirus.

(A) The concentrations of the cytokines IL-6, IL-1β, IL-8, IL-10, TNFα and IFNβ in the culture medium of primary MSCs. n = 4. (B,C) Primary MSCs infected with lentivirus transduced with an empty vector that expressed GFP or infected with siIL-6-transduced recombinant lentivirus. (D,E) The efficiency of lentivirus infection in MSCs between the siIL-6-transduced recombinant lentivirus and its control. (F,G) The levels of IL-6 mRNA expression and protein release by GFP MSCs and siIL-6 MSCs. n = 3. Scale bar = 200 μm. (H) The concentration of IL-6 in the hippocampus of HIBD + siIL-6 MSCs and HIBD + GFP MSCs rats at 7 and 14 days after HIBD. n = 5. The results are presented as the mean ± SEM. ^#P < 0.05 vs. the GFP MSCs group.

Figure 3. The neuroprotective effect of MSCs is weakened when IL-6 expression is silenced.

(A) Diagram illustrating the experimental protocols of the treatments and tests for rats in the HIBD + siIL-6 MSCs group and HIBD + GFP MSCs group. (B,C) The escape latencies and path lengths to reach the visible platform on the first day of the Morris water maze test for rats in the two groups. n = 15. (D) The escape latencies of each group to locate the visible platform from the 2nd to 5th day in the Morris water maze test. (E) The passing times through the former platform region of the two groups on the final day of the Morris water maze test. (F) The discrimination ratio of the exploration time during the test phase of the object-in-place task between the HIBD + siIL-6 MSCs and HIBD + GFP MSCs groups. n = 15. (G) The standardised fEPSP slope of the two groups. n = 6. The results are presented as the mean ± SEM. ^#P < 0.05 vs. the HIBD + siIL-6 MSCs group.

Figure 4. MSC co-culture activates the IL-6/STAT3 signalling pathway in OGD-injured neurons but has no effect on the ratio of Bcl-2/Bax.

(A) The concentration of IL-6 released in the culture medium of normal neurons and OGD-injured neurons with or without MSC co-culture as determined by ELISA. n = 4. (B–E) The mRNA expression levels of IL-6R, STAT3, Bax and Bcl-2 in the three groups. n = 4. (F) Representative western blot of IL-6R, p-STAT3, STAT3, Bax and Bcl-2 expression in neurons from the control, OGD and OGD + MSCs groups. n = 4. (G) The quantifications of WB signal in F. (H) The ratio between Bcl-2 and Bax protein levels in the three groups. The results are presented as the mean ± SEM. *P < 0.05 vs. the control group, ^&P < 0.05 vs. the OGD group.

Figure 5. Silencing of IL-6 in MSCs suppresses the activation of the IL-6/STAT3 signalling pathway but has no effect on the ratio of Bcl-2/Bax in OGD-injured neurons.

(A) The IL-6 concentration in the culture medium of OGD-injured neurons co-cultured with OGD + GFP MSCs and OGD + siIL-6 MSCs. n = 5. (B) The mRNA expression levels of IL-6R, STAT3, Bax and Bcl-2 in the two groups. n = 4. (C) Representative western blot of IL-6R, p-STAT3, STAT3, Bax and Bcl-2 expression in neurons in the two groups. n = 4. (D) The quantifications of WB signal in C. (E) The ratio of Bcl-2 to Bax protein levels in the two groups. The results are presented as the mean ± SEM. ^#P < 0.05 vs. the OGD + GFP MSCs group.

Figure 6. MSC co-culture activates the IL-6/STAT3 signalling pathway and suppresses apoptosis in OGD-injured astrocytes.

(A) The concentration of IL-6 released in the culture medium of normal astrocytes and OGD-injured astrocytes co-cultured with or without MSCs. n = 5. (B–E) The mRNA expression levels of IL-6R, STAT3, Bax and Bcl-2 in the three groups. n = 4. (F) Representative western blot of IL-6R, p-STAT3, STAT3, Bax and Bcl-2 expression in astrocytes in the three groups. n = 4. (G) The quantifications of WB signal in F. (H) The ratio of Bcl-2 to Bax protein levels among the three groups. The results are presented as the mean ± SEM. *P < 0.05 vs. the control group, ^&P < 0.05 vs. the OGD group.

Figure 7. siIL-6 MSC co-culture impairs the activity of the IL-6/STAT3 signalling pathway and suppresses anti-apoptosis in OGD-injured astrocytes.

(A) The concentrations of IL-6 in the culture medium of astrocytes co-cultured with OGD + GFP MSCs or OGD + siIL-6 MSCs as determined by ELISA. n = 5. (B) The mRNA expression levels of IL-6R, STAT3, Bax and Bcl-2 in the two groups. n = 4. (C) Representative western blot of IL-6R, p-STAT3, STAT3, Bax and Bcl-2 expression in the two groups. n = 4. (D) The quantifications of WB signal in C. (E) The ratio between Bcl-2 and Bax protein levels in the two groups. The results are presented as the mean ± SEM. ^#P < 0.05 vs. the OGD + GFP MSCs group.

Figure 8. siIL-6 MSC co-culture suppresses anti-apoptosis of OGD-injured astrocytes but has no effect on neurons following OGD injury.

(A) Annexin V-FITC/PI double staining of neurons or astrocytes in the OGD + GFP and OGD + siIL-6 MSCs groups. a, d, g and j. Annexin-V-positive cells in neurons or astrocytes in the OGD + GFP group or OGD + siIL-6 MSCs group. b, e, h and k. PI-positive cells in neurons or astrocytes in the OGD + GFP group or OGD + siIL-6 MSCs group. c, f, i and l. Annexin-V-positive cells and PI-positive cells merged with bright-field images of neurons or astrocytes in the OGD + GFP group or OGD + siIL-6 MSCs group. n = 5. (B,C) Annexin-V and PI sum intensity of neurons in the OGD + GFP and OGD + siIL-6 MSCs groups. (D,E) Annexin-V and PI sum intensity of astrocytes in the OGD + GFP and OGD + siIL-6 MSCs groups. Scale bar = 50 μm. The results are presented as the mean ± SEM. ^#P < 0.05 vs. OGD + GFP MSCs.

Figure 9. siIL-6 MSCs transplantation reduces Bcl-2 expression levels in the astrocytes to suppress the anti-apoptotic effect of MSCs but has no effect on neurons following OGD treatment.

(A) Double immunofluorescence staining of NSE or GFAP together with Bcl-2 in the cerebral cortex of rats in the HIBD + GFP MSCs and HIBD + siIL-6 MSCs groups. a and b. Double immunofluorescence staining of NSE together with Bcl-2. The white arrows indicate co-localisation of NSE and Bcl-2. c and d. Double immunofluorescence staining of GFAP together with Bcl-2. The white arrows indicate co-localisation of GFAP and Bcl-2. n = 5. (B) Quantification of the percentage of NSE/Bcl2 or GFAP/Bcl2 double-labelled cells as presented in A. (C) TUNEL staining of the rat cerebral cortex of the HIBD + GFP group and HIBD + siIL-6 MSCs group. a and d. TUNEL-positive cells. b and e. DAPI-stained cells. c and f. Merged images of TUNEL-positive cells and DAPI-stained cells. n = 6. (D) The percentage of TUNEL-positive cells in the HIBD + GFP and HIBD + siIL-6 MSCs groups. Scale bar = 50 μm. The results are presented as the mean ± SEM. ^#P < 0.05 vs. HIBD + GFP MSCs.