CXCR2 Activated JAK3/STAT3 Signaling Pathway Exacerbating Hepatotoxicity Associated with Tacrolimus

Abstract

Purpose: Tacrolimus could induce hepatotoxicity during clinical use, and the mechanism was still unclear, which posed new challenge for the prevention and treatment of tacrolimus-induced hepatotoxicity. The aim of this study was to investigate the mechanism of tacrolimus-induced hepatotoxicity and provide reference for drug development target.

Methods: In this study, biochemical analysis, pathological staining, immunofluorescent staining, immunohistochemical staining, transcriptomic analysis, Western blotting was used to investigate the mechanism of tacrolimus-induced hepatotoxicity in gene knockout mice and Wistar rats.

Results: In gene knockout mice, compared to wild-type mice, CXCR2-deficiency alleviated tacrolimus-induced hepatotoxicity (P < 0.05 or P < 0.01). In Wistar rats, compared to control group, CXCL2-CXCR2, JAK3/STAT3 signaling pathway (phosphorylation of JAK3 and STAT3) were up-regulated, the expression of CIS was lowered and the expression of PIM1 was raised, inducing liver pathological change (P < 0.05 or P < 0.01); Inversely, blocking CXCR2 could reverse the expression of p-JAK3/p-STAT3 and tacrolimus-induced hepatotoxicity (P < 0.05 or P < 0.01).

Conclusion: CXCR2 activated JAK3/STAT3 signaling pathway (phosphorylation of JAK3 and STAT3) exacerbating hepatotoxicity associated with tacrolimus, meanwhile the expression of CIS was down-regulated, the expression of PIM1 was up-regulated. Blocking CXCR2 could reverse the expression of p-JAK3/p-STAT3, CIS, PIM1, and tacrolimus-induced hepatotoxicity.

Keywords: CIS; CXCR2; JAK3/STAT3 signaling pathway; PIM1; hepatotoxicity; tacrolimus.

Graphical abstract

Figure 1

CXCR2-deficiency alleviated tacrolimus-induced hepatotoxicity. (A) Flow diagram. (B) ALT. (C) AST. (D) TBIL. (E) SOD. (F) MDA. (G) GSH-Px. (H) Masson staining and E-cadherin/α-SMA fluorescent staining. (I) Mean of IOD from Masson. (J) Mean of IOD from E-cadherin. (K) Mean of from α-SMA. *P < 0.05, **P < 0.01 vs N-WT, ^#P < 0.05, ^##P < 0.01 vs T-WT. WT, wild-type mice. CXCR2-KO, CXCR2 knockout mice. N, control group. T, tacrolimus intervention group. IOD, integral optical density. Scale bar, 100 µm.

Figure 2

CXCL2-CXCR2 was up-regulated in tacrolimus-induced hepatotoxicity. (A) Flow diagram. (B) Fluorescent staining of CXCL2/CXCR2. (C) Western blotting of CXCL2 and CXCR2. (D) The expression of CXCL2. (E) The expression of CXCR2. *P < 0.05, **P < 0.01 vs N. N, control group. T, tacrolimus intervention group. Scale bar, 200 µm.

Figure 3

Pathological change of tacrolimus-induced hepatotoxicity. (A) Masson staining, sirius red staining and immunohistochemical staining from vimentin, COL-1, COL-3, FN. (B) E-cadherin/α-SMA fluorescent staining. (C) Western blotting of vimentin, E-cadherin and α-SMA. (D) The expression of vimentin. (E) The expression of E-cadherin. (F) The expression of α-SMA. *P < 0.05, **P < 0.01 vs N. N, control group. T, tacrolimus intervention group. Scale bar, 200 µm.

Figure 4

Transcriptomic analysis of tacrolimus-induced hepatotoxicity. (A) Density map. (B) Violin plot. (C) Pearson correlation between samples. (D) Differential gene cluster map. (E) Volcano map of differential gene expression distribution. (F) Statistical histogram of differentially expressed genes. (G) GSEA enrichment analysis. (H) KEGG pathways enrichment. (I) JAK-STAT signaling pathway. (J) Interaction network map of differentially expressed genes.

Figure 5

JAK3/STAT3 signaling pathway was activated in tacrolimus-induced hepatotoxicity. (A) Western blotting of p-JAK3, p-STAT3, and p21. (B) The expression of p-JAK3. (C) The expression of p-STAT3. (D) The expression of p21. (E) Western blotting of PIM1, CIS, and slug. (F) The expression of PIM1. (G) The expression of CIS. (H) The expression of slug. (I) The hypothesis of CXCR2 activating JAK3/STAT3 signaling pathway in tacrolimus-induced hepatotoxicity. The red and green arrows represented the expression of proteins after tacrolimus intervention, where the red arrows facing up meant the expression of proteins were up-regulated, the green arrows facing down meant the expression of proteins were down-regulated. JAK3 and STAT3 referred to their phosphorylated forms (p-JAK3 and p-STAT3). *P < 0.05, **P < 0.01 vs N. N, control group. T, tacrolimus intervention group.

Figure 6

CXCR2 antagonist alleviated tacrolimus-induced hepatotoxicity. (A) Flow diagram. (B) ALT. (C) AST. (D) TBIL. (E) SOD. (F) MDA. (G) GSH-Px. (H) Western blotting of E-cadherin, α-SMA, and vimentin. (I) The expression of E-cadherin. (J) The expression of α-SMA. (K) The expression of vimentin. *P < 0.05, **P < 0.01 vs N; ^#P < 0.05, ^##P < 0.01 vs T; N, control group. T, tacrolimus intervention group. C, CXCR2 antagonist group.

Figure 7

CXCR2 antagonist down-regulated JAK3/STAT3 signaling pathway in tacrolimus-induced hepatotoxicity. (A) Western blotting of p-JAK3, p-STAT3, and p21. (B) Western blotting of PIM1, CIS, and slug. (C) The expression of p-JAK3. (D) The expression of p-STAT3. (E) The expression of p21. (F) The expression of PIM1. (G) The expression of CIS. (H) The expression of slug. (I) Fluorescent staining of p-JAK3, p-STAT3, p21, slug, PIM1 and immunohistochemical staining of CIS. *P < 0.05, **P < 0.01 vs N; ^#P < 0.05, ^##P < 0.01 vs T; N, control group. T, tacrolimus intervention group. C, CXCR2 antagonist group. Scale bar, 200 µm.

Figure 8

CXCR2 activated JAK3/STAT3 signaling pathway exacerbating hepatotoxicity associated with tacrolimus. The red and green arrows represented the expression of proteins after tacrolimus intervention, where the red arrows facing up meant the expression of proteins were up-regulated, the green arrows facing down meant the expression of proteins were down-regulated. The brown arrows represented the expression of proteins after CXCR2 antagonist intervention, where the arrows facing up meant the expression of proteins were up-regulated, the arrows facing down meant the expression of proteins were down-regulated. JAK3 and STAT3 referred to their phosphorylated forms (p-JAK3 and p-STAT3).