SERP1 reduces inchoate acute hepatic injury through regulation of endoplasmic reticulum stress via the GSK3β/β‑catenin/TCF/LEF signaling pathway

Abstract

The liver is a crucial digestive organ of humans and in charge of detoxification. Acute hepatic injury is an aggressive type of hepatic disease and its harmful effect cannot be ignored. The present study examined the role and mechanism of stress‑associated endoplasmic reticulum protein 1 (SERP1) in acute hepatic injury. Mice were injected intraperitoneally with D‑galactosamine/lipopolysaccharide (LPS) and rat hepatocytes were induced by LPS to establish an acute hepatic injury model. Tissue lesions were observed by H&E staining, and biomarkers of hepatic injury in the serum were examined. Western blotting, immunohistochemistry and reverse transcription‑quantitative PCR were performed to assess SERP1 expression in tissues and hepatocytes. A SERP1 overexpression plasmid was constructed to evaluate the role of SERP1 in inflammation, apoptosis, endoplasmic reticulum stress (ERS) and the GSK3β/β‑catenin/T‑cell factor (TCF)/lymphoid enhancing factor (LEF) signaling pathway. In addition, a GSK3β overexpression plasmid was constructed to investigate the role of GSK3β/β‑catenin signal activation. Additionally, the present study investigated whether SERP1 regulated the endoplasmic reticulum via this pathway. In the present study, reliable animal and cellular hepatic injury models were established and verified. SERP1 overexpression reduced the expression of inflammatory factors, apoptosis‑related proteins and ERS‑related proteins, as well as the expression of proteins related to GSK3β/β‑catenin/TCF/LEF signaling pathways. A GSK3β overexpression plasmid was constructed and it was revealed that GSK3β overexpression could reverse the effects of SERP1 overexpression in aforementioned aspects. This suggested that the activation of the GSK3β/β‑catenin/TCF/LEF signaling pathway may be required for the regulation of SERP1. In conclusion, SERP1 regulated ERS via the GSK3β/β‑catenin/TCF/LEF signaling pathway, thereby reducing inchoate acute hepatic injury.

Keywords: D‑galactosamine/lipopolysaccharide; GSK3β; endoplasmic reticulum stress; hepatic injury; stress‑associated endoplasmic reticulum protein 1.

Figure 1.

Establishment of an inchoate acute hepatic injury model and detection of ERS-related proteins. (A) Pathological changes of tissues of mice in the model group detected by H&E staining. (B) Levels of alanine aminotransferase and aspartate aminotransferase in the serum. (C) ERS-related protein expression detected by western blotting. SERP1 expression in acute hepatic injury tissues detected by (D) immunohistochemistry and (E) western blotting. (F) SERP1 expression in acute hepatic injury cells detected by western blotting and reverse transcription-quantitative PCR. *P<0.05, **P<0.01 and ***P<0.001 vs. the control; n≥3. ERS, endoplasmic reticulum stress; SERP1, stress-associated endoplasmic reticulum protein 1; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LPS, lipopolysaccharide; D-GalN, D-galactosamine; GRP78, glucose-regulated protein 78; GRP94, glucose-regulated protein 94.

Figure 2.

Effect of SERP1 overexpression on the expression of inflammatory factors in hepatocytes. Expression levels of SERP1 following transfection with SERP1 plasmid detected by (A) western blotting and (B) reverse transcription-quantitative PCR. **P<0.01, ***P<0.001 vs. OE-NC. (C) Expression levels of inflammatory factors (TNF-α, IL-18, IL-6 and IL-1β) detected by ELISA. (D) NLRP3 inflammasome (NLRP3, ASC and caspase-1) expression detected by western blotting. ***P<0.001 vs. the control; ^#P<0.05, ^###P<0.001 vs. LPS + NC; ^ΔP<0.05, ^ΔΔP<0.01 vs. LPS + SERP1; n≥3. SERP1, stress-associated endoplasmic reticulum protein 1; OE, overexpression; NC, negative control; NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein containing a CARD; LPS, lipopolysaccharide.

Figure 3.

Effect of SERP1 overexpression on apoptosis of hepatocytes. (A) A TUNEL assay was employed to detect cell apoptosis. (B) Apoptosis-related protein expression was detected by western blotting. ***P<0.001 vs. the control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. LPS + NC; ^ΔP<0.05 vs. LPS + SERP1; n≥3. SERP1, stress-associated endoplasmic reticulum protein 1; LPS, lipopolysaccharide; NC, negative control.

Figure 4.

Effect of SERP1 overexpression on endoplasmic reticulum stress-related protein expression and GSK3β/β-catenin/TCF/LEF signaling in hepatocytes. (A) Expression levels of GRP78, GRP94 and CHOP in hepatocytes detected by western blotting. (B) TOP Flash/FOP Flash fluorescent gene reporter assay to detect TCF/LEF activity. (C) GSK3β/β-catenin expression was detected by western blotting. ***P<0.001 vs. the control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. LPS + NC; ^ΔΔΔP<0.001 vs. LPS + SERP1; n≥3. SERP1, stress-associated endoplasmic reticulum protein 1; GRP78, glucose-regulated protein 78; GRP94, glucose-regulated protein 94; TCF, T-cell factor; LEF, lymphoid enhancing factor; LPS, lipopolysaccharide; NC, negative control.

Figure 5.

GSK3β/β-catenin signaling activation reverses the effect of SERP1 overexpression on ERS and cell apoptosis. (A) Overexpression was verified by reverse transcription-quantitative PCR and western blotting. ***P<0.001 vs. OE-NC; n≥3. (B) TCF/LEF activity was detected using a luciferase assay. (C) GSK3β/β-catenin expression was detected by western blotting. (D) Western blotting was applied to detect ERS-related protein expression. (E) A TUNEL assay was employed to detect cell apoptosis. (F) Apoptosis-related protein expression was detected by western blotting. *P<0.05, **P<0.01, ***P<0.001 vs. LPS; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. LPS + SERP1 + NC; n≥3. SERP1, stress-associated endoplasmic reticulum protein 1; ERS, endoplasmic reticulum stress; OE, overexpression; TCF, T-cell factor; LEF, lymphoid enhancing factor; LPS, lipopolysaccharide; NC, negative control.