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. 2024 Jun;28(12):e18458.
doi: 10.1111/jcmm.18458.

ATF3 is involved in rSjP40-mediated inhibition of HSCs activation in Schistosoma japonicum-infected mice

Jing Li  1   2 , Jiali Zhang  1   3 , Bei Zhang  1 , Guo Chen  1 , Min Huang  1 , Boyin Xu  4 , Dandan Zhu  1 , Jinling Chen  1 , Yinong Duan  1 , Wenxi Gao  1   5
Affiliations

ATF3 is involved in rSjP40-mediated inhibition of HSCs activation in Schistosoma japonicum-infected mice

Jing Li et al. J Cell Mol Med. 2024 Jun.

Abstract

Schistosomiasis is a parasitic disease characterized by liver fibrosis, a process driven by the activation of hepatic stellate cells (HSCs) and subsequent collagen production. Previous studies from our laboratory have demonstrated the ability of Schistosoma japonicum protein P40 (SjP40) to inhibit HSCs activation and exert an antifibrotic effect. In this study, we aimed to elucidate the molecular mechanism underlying the inhibitory effect of recombinant SjP40 (rSjP40) on HSCs activation. Using a cell model in which rSjP40 inhibited LX-2 cell activation, we performed RNA-seq analyses and identified ATF3 as the most significantly altered gene. Further investigation revealed that rSjP40 inhibited HSCs activation partly by suppressing ATF3 activation. Knockdown of ATF3 in mouse liver significantly alleviated S. japonicum-induced liver fibrosis. Moreover, our results indicate that ATF3 is a direct target of microRNA-494-3p, a microRNA associated with anti-liver fibrosis effects. rSjP40 was found to downregulate ATF3 expression by upregulating microRNA-494-3p in LX-2 cells. This downregulation led to the inhibition of the expression of liver fibrosis proteins α-SMA and COL1A1, ultimately alleviating liver fibrosis caused by S. japonicum.

Keywords: Schistosoma japonicum; ATF3; Schistosoma japonicum protein P40; hepatic stellate cell; microRNA‐494‐3p.

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Conflict of interest statement

The authors confirm that there are no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Atf3 mRNA expression in the liver of S. japonicum‐infected mice. (A) Atf3 mRNA expression at different weeks post S. japonicum infection, as detected by RT‐qPCR. (B) Liver tissue from an S. japonicum‐infected mouse model. (C) HE staining of liver tissue from S. japonicum‐infected mice at different weeks. (D) Immunohistochemical staining of liver tissue from S. japonicum‐infected mice at different weeks. ***p < 0.001 compared with the normal group.
FIGURE 2
FIGURE 2
rSjP40 inhibits α‐SMA expression in LX‐2 cell by down‐regulating ATF3. (A) Infection of LX‐2 cells with LV‐ATF3 lentivirus, observed for GFP fluorescence intensity. (B) Detection of ATF3 overexpression efficiency and the effect of rSjP40 treatment on ATF3 protein expression by Western blot. (C) Detection of the effect of rSjP40 on α‐SMA expression in LX‐2 cells stably overexpressing ATF3 by Western blot. (D) Quantitative statistical analysis of Western blot results. (E) Immunofluorescence showing ATF3 expression in the nucleus after rSjP40 treatment. (Red: ATF3 staining; Blue: DAPI staining for nucleus). **p < 0.01 compared to LV‐NC group, ***p < 0.001 compared to LV‐NC group, #p < 0.05 compared to LV‐NC + rSjP40 group, ## p < 0.01 compared to LV‐NC + rSjP40 group.
FIGURE 3
FIGURE 3
Effect of ATF3 overexpression on rSjP40 inhibition of LX‐2 cell migration. (A) Cell scratch assay after LX‐2 cells were infected with LV‐ATF3 and LV‐NC Lentivirus (green fluorescence represents virus‐infected positive cells). (B) Transwell assay after LX‐2 cells were infected with LV‐ATF3 and LV‐NC lentivirus. (C) Quantitative statistical analysis of cell scratch and transwell assays. *p < 0.05 compared to LV‐NC group; ***p < 0.001 compared to LV‐NC group; ### p < 0.001 compared to LV‐NC + rSjP40 group.
FIGURE 4
FIGURE 4
Reducing ATF3 expression ameliorates S. japonicum‐induced liver fibrosis. (A) Detection of ATF3 protein expression in mouse liver tissues by Western blot (WT: wild‐type mice, KD: ATF3 knockdown mice). (B) Quantitative statistical analysis of ATF3 knockdown efficiency in mouse liver tissue by Western blot. (C) Detection of Atf3, Acta2 and Col1a1 mRNA expression in mouse liver tissue by RT‐qPCR. (D) Evaluation of liver fibrosis by macroscopic examination, H&E, and Masson staining. (E) Immunohistochemical staining for ATF3, α‐SMA, and COL1A1 (scale: 100 μm). **p < 0.01 compared to shNC group.
FIGURE 5
FIGURE 5
TLR‐4 contributes to down‐regulation of ATF3 expression induced by rSjP40. (A) Detection of ATF3 expression in control group, LPS group, or LPS + rSjP40 group by Western blot. (B) Quantitative statistical analysis of ATF3 expression in each group by Western blot. (C) Detection of TLR4 expression in control group, LPS group, or LPS + rSjP40 group by RT‐qPCR. (D) Transwell assay to determine the migration ability of LX‐2 cells in control group, LPS group, or LPS + rSjP40 group. (E) Quantitative statistical analysis of transwell assays. **p < 0.01 compared to NC group; ## p < 0.01 compared to LPS group.
FIGURE 6
FIGURE 6
MicroRNA‐494‐3p targets ATF3. (A) Prediction of upstream microRNAs of ATF3 using three databases (Targetscan, miRDB, and miRtarBase). (B) Potential binding site between microRNA‐494‐3p and ATF3. (C) Detection of the effect of rSjP40 on microRNA‐494‐3p expression in LX‐2 cells by RT‐qPCR. (D) Detection of the combination of microRNA‐494‐3p and ATF3 3’ UTR by double luciferase reporter assay. **p < 0.01 compared to NC group.
FIGURE 7
FIGURE 7
MicroRNA‐494‐3p negatively regulates ATF3, α‐SMA and COL1A1 expression. (A) Detection of inhibitory efficiency of microRNA‐494‐3p inhibitor by RT‐qPCR. (B, C) Changes in ATF3, α‐SMA, and COL1A1 protein levels upon silenced expression of microRNA‐494‐3p. (D) Changes in ATF3, ACTA2, and COL1A1 mRNA levels upon silenced expression of microRNA‐494‐3p. **p < 0.01 compared to NC group; ## p < 0.01 compared to rSjP40 + NC group.
FIGURE 8
FIGURE 8
Effects of microRNA‐494‐3p inhibitor on rSjP40 inhibition of LX‐2 cell migration. (A) Impact of microRNA‐494‐3p inhibitor on LX‐2 cell migration assayed by cell scratch assay. (B) Impact of microRNA‐494‐3p inhibitor on LX‐2 cell migration assayed by transwell assay. (C) Quantitative statistical analysis of cell scratch and transwell assays. **p < 0.01 compared to NC group; ## p < 0.01 compared to rSjP40 + NC group.
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