TL1A promotes the postoperative cognitive dysfunction in mice through NLRP3-mediated A1 differentiation of astrocytes

Abstract

Aim: We investigated the mechanism, whereby tumor necrosis factor-like ligand 1A (TL1A) mediates the A1 differentiation of astrocytes in postoperative cognitive dysfunction (POCD).

Methods: The cognitive and behavioral abilities of mice were assessed by Morris water maze and open field tests, while the levels of key A1 and A2 astrocyte factors were detected by RT-qPCR. Immunohistochemical (IHC) staining was used to examine the expression of GFAP, western blot was used to assay the levels of related proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory cytokines.

Results: The results showed that TL1A could promote the progression of cognitive dysfunction in mice. Astrocytes differentiated into A1 phenotype, while unobvious changes were noted in astrocyte A2 biomarkers. Knockout of NLRP3 or intervention with NLRP3 inhibitor could inhibit the effect of TL1A, improving the cognitive dysfunction and suppressing the A1 differentiation.

Conclusion: Our results demonstrate that TL1A plays an important role in POCD in mice, which promotes the A1 differentiation of astrocytes through NLRP3, thereby exacerbating the progression of cognitive dysfunction.

Keywords: POCD; TL1A; astrocytes; cognitive dysfunction.

FIGURE 1

TL1A promoted the progression of POCD. (A–E) Morris water maze (n = 10), the Normal mice exhibited shorter escape latency, longer total movement distance and navigation time, as well as a higher number of platform crossings compared to the POCD mice. TL1A could exacerbate POCD, as manifested by the significantly longer escape latency, shorter total movement distance and navigation time, as well as less platform crossings compared to the POCD group, showing obvious differences over time. *p < 0.05 versus Normal; ^# p < 0.05 versus POCD. (F, G) OFT (n = 10), the movement distance was significantly longer in the Normal group than in the POCD group, while TL1A could shorten the movement distance to become shorter than that in the POCD group. *p < 0.05 versus Normal; ^# p < 0.05 versus. POCD. (H) IHC (n = 5), the expression of GFAP, a RAs‐A1 marker, was low in Normal, while was upregulated in POCD, leading to increased number of positive cells. TL1A could elevate the positive cell counts. (I–K) ELISA (n = 10), TNF‐α, IL‐6, and IL‐1β were significantly upregulated in POCD than in Normal, while TL1A could further promote the expression of inflammatory cytokines.

FIGURE 2

Effects of TL1A on the RAs‐A1 differentiation. (A–F) A1 RASmRNA (n = 10), the mRNA levels of A1 biomarkers in POCD were significantly higher than those in Normal. TL1A could further upregulate these mRNA levels. **p < 0.0 inter‐group comparisons. (G–L) A2 RASmRNA (n = 10), the mRNA levels of A2 biomarkers did not change significantly, showing absence of inter‐group differences. (M, N) Relative protein expressions (n = 3), the POCD group exhibited higher levels of NLRP3, C3 compared to the Normal group. TL1A could further elevate the levels of NLRP3, C3. *p < 0.05 versus Normal; ^# p < 0.05 versus POCD.

FIGURE 3

NLRP3 knockout could antagonize the action of TL1A and ameliorate the POCD in mice. (A–E) Morris (n = 10), obvious cognitive dysfunction appeared in TL1A + WT‐POCD and TL1A + KO‐POCD mice. Compared to the TL1A + WT‐POCD group, the TL1A + KO‐POCD group exhibited shorter escape latency, longer total movement distance and navigation time, as well as higher number of platform crossings. *p < 0.05 versus WT; ^# p < 0.05 versus TL1A + WT‐POCD. (F, G) OFT (n = 10), the total movement distance was longer in the TL1A + KO‐POCD group than in the TL1A + WT‐POCD group. *p < 0.05 versus WT; ^# p < 0.05 versus TL1A + WT‐POCD. (H) IHC (n = 5), no distinctly positive cells were found in WT and KO mice, while the number of positive cells in TL1A + KO‐POCD was significantly lower than that in TL1A + WT‐POCD. (I–K) ELISA (n = 10), the levels of inflammatory cytokines in TL1A + KO‐POCD were significantly lower than those in TL1A + WT‐POCD. **p < 0.0 inter‐group comparisons.

FIGURE 4

Effects of NLRP3 knockout on the TL1A‐induced RAs‐A1 differentiation. (A–F) A1 RA mRNA (n = 10), the mRNA levels of Serping1, Ggta1, C3, Psmb8, Srgn, and Amigo2 were lower in the TL1A + KO‐POCD group than in the TL1A + WT‐POCD group. **p < 0.0 inter‐group comparisons. (G–L) A2 RA mRNA (n = 10), the mRNA levels of A2 RA biomarkers changed insignificantly, showing absence of inter‐group differences. (M, N) Relative protein expressions (n = 3), compared to the TL1A + WT‐POCD group, the expressions of C3 were significantly lowered in the TL1A + KO‐POCD group. *p < 0.05 versus WT; ^# p < 0.05 versus TL1A + WT‐POCD.

FIGURE 5

MCC950 could inhibit the action of TL1A and reduce the RAs‐A1 differentiation. (A–F) A1 RA mRNA (n = 10), MCC950 could inhibit the RAs‐A1 differentiation, as manifested by significantly declined mRNA levels of A1 RA biomarkers in the POCD‐TL1A + MCC950 group than in the POCD‐TL1A group. **p < 0.0 inter‐group comparisons. (G–L) A2 RA mRNA (n = 10), the mRNA levels of A2 RA biomarkers changed insignificantly, showing absence of inter‐group differences. (M) IHC (n = 5), GFAP assay revealed significantly lower number of positive cells in the POCD‐TL1A + MCC950 group than in the POCD‐TL1A group. (N–P) ELISA (n = 10), ELISA assay also revealed significantly declined levels of inflammatory cytokines in the POCD‐TL1A + MCC950 group than in the POCD‐TL1A group. **p < 0.0 inter‐group comparisons. (Q, R) Relative protein expressions (n = 3), MCC950 could lower the levels of NLRP3, C3, showing significant differences from the POCD‐TL1A group. *p < 0.05 versus Normal; ^# p < 0.05 versus POCD.

FIGURE 6

TL1A promoted the primary RAs‐A1 differentiation in vitro. (A–F) A1 RA mRNA (n = 5), TL1A could promote the RAs‐A1 differentiation and elevate the mRNA levels of A1 RA biomarkers. **p < 0.0 inter‐group comparisons. (G–L) A2 RA mRNA (n = 5), the mRNA levels of A2 RA biomarkers changed insignificantly, showing the absence of inter‐group differences. (M–O) ELISA (n = 5), TL1A could promote the secretion of inflammatory cytokines by RAs, as manifested by significantly upregulated cytokine levels in the A1 + TL1A group than in the A1 group. **p < 0.0 inter‐group comparisons. (P, Q) Relative protein expressions (n = 3), TL1A could promote the expressions of NlRP3, C3. *p < 0.05 versus Control; ^# p < 0.05 versus A1.

FIGURE 7

NLRP3 knockout could inhibit the action of TL1A in vitro. (A–F) A1 RAs mRNA (n = 5), the A1 differentiation was inhibited in the NLRP3 ^−/− ‐A1 + TL1A group, where the mRNA levels of A1 biomarkers were significantly lower than those in the A1 + TL1A group. **p < 0.0 inter‐group comparisons. (G–L) A2 RAs mRNA (n = 5), the mRNA levels of A2 biomarkers changed insignificantly, showing absence of inter‐group differences. (M–O) ELISA (n = 5), the inflammatory cytokine levels were significantly lower in NLRP3 ^−/− ‐A1 + TL1A than in A1 + TL1A. **p < 0.0 inter‐group comparisons. (P, Q) Relative protein expressions (n = 3), the inter‐group differences in C3 were insignificant after knockout of NLRP3. *p < 0.05 versus Control; ^# p < 0.05 versus A1.