Inhibition of cysteine-serine-rich nuclear protein 1 ameliorates ischemia-reperfusion injury during liver transplantation in an MAPK-dependent manner

Abstract

Hepatic ischemia-reperfusion injury (HIRI) is a critical pathophysiological process during liver transplantation (LT). Multiple genes and signal pathways are dysregulated during HIRI. This study aims to identify genes as potential therapeutic targets for ameliorating HIRI. Datasets containing samples from the human donor liver (GSE151648) and mouse HIRI model (GSE117066) were analyzed to determine differentially expressed genes (DEGs). The selected DEGs were confirmed by real-time PCR and western blot in the hepatocyte hypoxia-reoxygenation (HR) model, mouse HIRI model, and human liver samples after transplantation. Genetic inhibition was used to further clarify the underlying mechanism of the gene in vitro and in vivo. Among the DEGs, CSRNP1 was significantly upregulated (|log FC|= 2.08, P < 0.001), and was positively correlated with the MAPK signal pathway (R = 0.67, P < 0.001). CSRNP1 inhibition by siRNA significantly suppressed apoptosis in the AML-12 cell line after HR (mean Annexin⁺ ratio = 60.62% vs 42.47%, P = 0.0019), but the protective effect was eliminated with an additional MAPK activator. Knocking down CSRNP1 gene expression by intravenous injection of AAV-shRNA markedly reduced liver injury in mouse HIRI model (ALT: AAV-NC vs AAV-shCsrnp1 = 26,673.5 ± 2761.2 vs 3839.7 ± 1432.8, P < 0.001; AST: AAV-NC vs AAV-shCsrnp1 = 8640.5 ± 1450.3 vs 1786.8 ± 518.3, P < 0.001). Liver-targeted delivery of siRNA by nanoparticles effectively inhibited intra-hepatic genetic expression of Csrnp1 and alleviated IRI by reducing tissue inflammation and hepatocyte apoptosis. Furthermore, CSRNP1 inhibition was associated with reduced activation of the MAPK pathway both in vitro and in vivo. In conclusion, our results demonstrated that CSRNP1 could be a potential therapeutic target to ameliorate HIRI in an MAPK-dependent manner.

Keywords: Cysteine-serine-rich nuclear protein-1 (CSRNP1); Hepatic ischemia–reperfusion injury (HIRI); Liver transplantation; Mitogen-activated protein kinases; Nanoparticles.

Fig. 1

Identification of DEGs in the GSE151648 and GSE117066 cohort. a Heatmap displaying some of the DEGs between pre-LT and post-LT samples in the GSE151648 dataset. b Heatmap displaying some of the DEGs between HIRI samples and negative controls in the GSE117066 dataset. c Volcano plot displaying all the DEGs between pre-LT and post-LT samples in the GSE151648 dataset. d Volcano plot displaying all the DEGs between HIRI samples and negative controls in the GSE117066 dataset. e Venn plot showing the commonly upregulated genes in both the GSE151648 and GSE117066 cohort

Fig. 2

KEGG and GO analysis of the commonly upregulated genes in different species. a Chordal graph showing the enriched GO pathways in humans. b Chordal graph showing the enriched GO pathways in mice. c Bubble plot showing the enriched KEGG pathways in humans. d Bubble plot showing the enriched KEGG pathways in mice

Fig. 3

Identification of CSRNP1 as a potential therapeutic target in the datasets. a MAPK scores of pre-transplant and post-transplant samples in the GSE151648 dataset. b Comparison of the MAPK score before and after LT in the GSE151648 dataset. c Correlation heatmap displaying the Pearson correlation coefficient between the MAPK score and relevant genes. d Correlation plot displaying the Pearson correlation coefficient between the MAPK score and CSRNP1 expression. ***P < 0.001 vs pre-transplant group. LT: liver transplantation

Fig. 4

Identification of CSRNP1 upregulation in vitro and in vivo. a Representative photographs of the cell HR model. b Apoptosis of AML-12 cells was assessed by flow cytometry analysis n = 6). c Relative mRNA levels of CSRNP1 assessed by qRT-PCR in AML-12 cells of the sham and different HR groups (H: hypoxia; R: reoxygenation) (n = 6). d Western blot showing the levels of Bax, cleaved caspase 3, SAPK, phosphorylated SAPK, p38, phosphorylated p38, and CSRNP1 in AML-12 cells of the sham and HR group. e Serum ALT and AST levels in mice after sham surgery or HIRI (n = 6). f Representative H&E staining images and necrotic areas of mouse liver lobes after HIRI (scale bar = 200 μm). g Relative mRNA levels of CSRNP1 were assessed by qRT-PCR in liver tissues from sham and HIRI mice (n = 6). h Western blot showing the levels of CSRNP1 in liver tissues of sham and HIRI groups. i Western blot showing the levels of Bax and cleaved caspase 3 in liver tissues of sham and HIRI groups. j Relative mRNA levels of CSRNP1 were assessed by qRT-PCR in liver tissues before and after LT (n = 32). k Representative images of IHC for CSRNP1 protein in human liver samples before and after LT (scale bar = 100 μm). l Representative H&E staining images of human liver samples before and after LT (scale bar = 100 μm) (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IRI: ischemia–reperfusion injury; HR: hypoxia-reoxygenation; LT: liver transplantation

Fig. 5

CSRNP1 knockdown exerted a protective effect on hepatocytes after HR via MAPK signaling pathway in vitro. a Schematic of the workflow (Created with BioRender.com, Agreement number: DU26KZDY58). b Relative mRNA levels of CSRNP1 were assessed by qRT-PCR in AML-12 cells of the sham and HR group with or without transfection of CSRNP1-siRNA (n = 6). c Apoptosis of AML-12 cells after HR was assessed by flow cytometry (n = 6). d Quantification charts of flow cytometry in Fig. 5c (n = 6). e Representative blots showing the protein level of CSRNP1; apoptosis related Bax, Bcl2 and cleaved caspase3; MAPK related SAPK, phosphorylated SAPK, P38 MAPK, and phosphorylated P38 MAPK; and internal control protein β-actin. **P < 0.01, ***P < 0.001, ****P < 0.0001. HR: hypoxia-reoxygenation; si: CSRNP1-siRNA; B: Blank; NC: negative control; A: Anisomycin (JNK activator)

Fig. 6

CSRNP1 Knockdown Ameliorates Mouse HIRI via MAPK Pathway. a Schematic of the workflow (Created with BioRender.com, Agreement number: VB26NQ5MCI). b Relative mRNA levels assessed by qRT-PCR in liver tissue of the sham (n = 5), AAV-NC (n = 6) and AAV-shCsrnp1 (n = 6) group. c Serum ALT and AST levels in mice after sham surgery or HIRI. d Western blot showing the levels of marker proteins related to apoptosis, MAPK signal pathway, and corresponding phosphorylated forms. e Representative H&E staining images (scale bar = 200 μm), IHC for CSRNP1, cleaved caspase3 and phosphorylated SAPK (scale bar = 100 μm). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

Delivering siNP attenuates mouse HIRI in vivo. a Schematic of workflow for construction of siNP (Created with BioRender.com, Agreement number: EX26KZEGAI). b The optimized size distribution of siNP is determined by dynamic light scattering (DLS). c Representative zeta potential of siNP determined by DLS. d Representative image of siNP under the transmission electron microscope (TEM). e RNase protection assay. f Schematic of workflow for treatment of siNP in mouse HIRI model (Created with BioRender.com, Agreement number: DB26NP7AXT). g Relative mRNA levels of CSRNP1 were assessed by qRT-PCR in liver tissue of the Sham (n = 5), IRI-NS (n = 6), IIR-NP (n = 6), and IRI-siNP (n = 6) groups. h Serum ALT and AST levels in mice after sham surgery or HIRI. i Relative mRNA levels of Il-1β, Tnf-α, and Sod1 assessed by qRT-PCR in mouse liver. j Representative H&E staining images (scale bar = 100 μm) and corresponding Suzuki score quantification. k Western blot showing the levels of marker proteins related to apoptosis, and MAPK signaling pathway. l Representative images of TUNEL staining among liver tissues from different groups (scale bar = 100 μm). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

Targeting CSRNP1 to ameliorate ischemia–reperfusion injury during liver transplantation. This graph illustrates the potential therapeutic strategies targeting CSRNP1, including viral and nanomedical delivery of genetic interfering tools, to potentially alleviate graft injury via inhibiting MAPK-dependant hepatocyte apoptosis. (Created with BioRender.com, Agreement number: IT26KZELCN)