J Neurocrit Care.  2024 Dec;17(2):49-56. 10.18700/jnc.240041.

Direct co-culture with human neural stem cells suppresses hemolysate-induced inflammation in RAW 264.7 macrophages through the extracellular signal-regulated kinase pathway

• Kim TJ
• Sun J
• Kang L
• Kim YJ
• Ko SB
• Yoon BW

• KMID: 2563021
• DOI: http://doi.org/10.18700/jnc.240041

Abstract

Background
Inflammation following stroke is associated with poor outcomes, and the anti-inflammatory effects of neural stem cells (NSCs) have been reported. However, the direct immunomodulatory effects of NSCs in hemorrhagic stroke remain unclear. In the present study, we investigated the anti-inflammatory mechanism of direct co-culture with NSCs on RAW 264.7 cells stimulated by hemolysate.
Methods
RAW 264.7 cells were stimulated with the hemolysate for 4 hours to induce hemorrhagic inflammation in vitro. Regarding direct co-culture, RAW 264.7 cells were cultured with HB1.F3 cells for 24 hours in normal medium and stimulated with hemolysate for 4 hours. Inflammatory cell signaling molecules, including cycloxygenase-2 (COX-2), interleukin-1β (IL-1β), and extracellular signal-regulated kinase (ERK), as well as tumor necrosis factor-α (TNF-α), were evaluated.
Results
After stimulation with the hemolysate, levels of the inflammatory markers COX-2, IL-1β, and TNF-α were increased in RAW264.7 cells. Inflammatory marker production was reduced in the group subjected to direct co-culture with HB1.F3 in comparison to that in the RAW264.7 group stimulated by the hemolysate. In addition, direct co-culture with HB1.F3 significantly suppressed the phosphorylation of ERK 1/2 in hemolysate-stimulated RAW 264.7 cells. Moreover, treatment of the ERK inhibitor (U0126) suppressed the expression levels of inflammatory markers in hemolysate-stimulated RAW246.7 cells.
Conclusion
These results demonstrate that direct co-culture with HB1.F3 suppresses inflammation by attenuating the ERK pathway. These findings suggest that direct NSC treatment modulates the inflammatory response in hemorrhagic stroke.

Keyword

Neural stem cell; Hemolysate-induced inflammation; Raw 264.7 cell; Direct co-culture; ERK1/2 pathway; Hemorrhagic stroke

Figure

• 

  Fig. 1. Hemolysate activates cycloxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) expression in RAW 264.7 cells. (A) Western blot analyses of inflammatory markers. COX-2 levels in RAW 264.7 cells increased and reached a peak level after 1 hour, and IL-1β and TNF-α reached peak levels after 4 hours. The increased inflammatory mediators (COX-2 shown in Fig. 1B, IL1-β in Fig. 1C, and TNF-α in Fig. 1D) decreased gradually. The band intensity was quantified using scanning densitometry and standardized with respect to α-tubulin. Values are presented as mean±standard deviation of three independent experiments.

• 

  Fig. 2. Direct co-culture with HB1.F3 cells reduced the hemolysate-stimulated expression of cycloxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in RAW 264.7 cells. The cells were divided into four groups. One was the control group, which was labeled R. The second group comprised RAW 264.7 cells stimulated with hemolysate for 4 hours and labeled RH. The third group was RAW 264.7 cells co-cultured with HB1.F3 cells and stimulated with hemolysate for 4 hours and labeled RHF. The fourth group was RAW 264.7 cells co-cultured with HB1.F3 cells for 24 hours and labeled RF. (A) Band quantification was performed using scanning densitometry against standardized levels of α-tubulin. (B) Direct co-culture with HB1.F3 cells suppressed COX-2 level compared with the levels in hemolysate-stimulated RAW 264.7 cells alone after 4 hours. (C) IL-1β expression decreased by direct co-culture with HB1.F3 cells after 4 hours compared with the expression in hemolysate-stimulated RAW 264.7 cells alone. (D) Direct co-culture with HB1.F3 cells inhibited TNF-α the hemolysate-stimulated RAW 264.7 cells after 4 hours. These results were obtained using enzyme-linked immunosorbent assay. Values are presented as mean±standard deviation of three independent experiments. NS, not significant. *P<0.05, **P<0.01, and ***P<0.001 compared to the control.

• 

  Fig. 3. Direct co-culture with HB1.F3 cells downregulated the hemolysate-activated phosphorylation of extracellular signal-regulated kinase (ERK) in RAW 264.7 cells. The cells were divided into four groups. One was the control group, which was labeled R. The second group comprised RAW 264.7 cells stimulated with hemolysate for 4 hours and labeled RH. The third group was RAW 264.7 cells co-cultured with HB1.F3 cells and stimulated with hemolysate for 4 hours and labeled RHF. The fourth group was RAW 264.7 cells co-cultured with HB1.F3 cells for 24 hours and labeled RF. (A) Band quantification was performed using scanning densitometry against standardized levels of α-tubulin. (B) ERK phosphorylation increased significantly after hemolysate stimulation for 4 hours. However, ERK pathway activation in hemolysate-induced RAW 264.7 cells was markedly suppressed by direct co-culture with HB1.F3. Moreover, the ERK1/2 pathway in RAW 264.7 cells was not activated by HB1.F3. Values are presented as mean±standard deviation of three independent experiments. p-ERK, phosphorylated ERK; NS, not significant. **P<0.01, ***P<0.001 compared to the control.

• 

  Fig. 4. The phosphorylation of extracellular signal-regulated kinase (ERK) participates in hemolysate-stimulated expression of cycloxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in RAW 264.7 cells. The cells were divided into three groups. One was the control group, which was labelled R. The second group was activated with the hemolysate for 4 hours and labeled RH. The third group was treated with U0126 (an ERK inhibitor) for 1 hour, incubated with hemolysate, and labeled RHU. (A) Band quantification was performed using scanning densitometry against standardized levels of α-tubulin. COX-2 (B), IL-β (C), and TNF-α (D) expressions were significantly suppressed by treatment with U0126 in hemolysate-stimulated RAW 264.7 cells. Values are presented as mean±standard deviation of three independent experiments. *P<0.05, ***P<0.001 compared to the control.

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