Celecoxib ameliorates non-alcoholic steatohepatitis in type 2 diabetic rats via suppression of the non-canonical Wnt signaling pathway expression

Abstract

Our aim was to test whether pharmacological inhibition of cycloxygenase-2 (COX-2) reverses non-alcoholic steatohepatitis (NASH) in type 2 diabetes mellitus (T2DM) rats via suppression of the non-canonical Wnt signaling pathway expression. Twenty-four male Sprague-Dawley rats were randomly distributed to two groups and were fed with a high fat and sucrose (HF-HS) diet or a normal chow diet, respectively. After four weeks, rats fed with a HF-HS diet were made diabetic with low-dose streptozotocin. At the 9(th) week the diabetic rats fed with a HF-HS diet or the non-diabetic rats fed with a normal chow diet were further divided into two subgroups treated with vehicle or celecoxib (a selective COX-2 inhibitor, 10 mg/Kg/day, gavage) for the last 4 weeks, respectively. At the end of the 12(th) week, rats were anesthetized. NASH was assessed by histology. Related cytokine expression was measured at both the protein and gene levels through immunohistochemistry (IHC), Western blot and real-time PCR. T2DM rats fed with a HF-HS diet developed steatohepatitis and insulin resistance associated with elevated serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), insulin levels and the non-alcoholic fatty liver disease (NAFLD) activity score (NAS). The expression of Wnt5a, JNK1, NF-κB p65, and COX-2 were all significantly increased in the T2DM-NASH group compared with the control and control-cele group. Hepatic injury was improved by celecoxib in T2DM-NASH-Cele group indicated by reduced serum ALT and AST levels and hepatic inflammation was reduced by celecoxib showed by histology and the NAFLD activity score (NAS). Serum related metabolic parameters, HOMA-IR and insulin sensitivity index were all improved by celecoxib. The expression of Wnt5a, JNK1, NF-κB p65, and COX-2 expression were all suppressed by celecoxib in T2DM-NASH-Cele group. The results of the present study indicated that celecoxib ameliorated NASH in T2DM rats via suppression of the non-canonical Wnt5a/JNK1 signaling pathway expression.

Figure 1. Assessment of insulin resistance in the four groups.

HOMA-IR(A); Insulin sensitivity index(ISI) (B); insulin tolerance test (ITT) (C). ^* P<0.01 vs. the control group and control-cele group; ^** P<0.01 vs. the T2DM-NASH group. White = control; Gray = control-cele; Black = T2DM-NASH; Striped = T2DM-NASH-Cele.

Figure 2. T₂DM rats fed with a HF-HS diet developed severe steatohepatitis.

H&E-stained livers ((A–D; ×200) and (E–H; ×400)). Oil Red-O staining of livers lipid accumulation (I–L; ×200). The results of NAFLD activity score (NAS) (M–P). Quantification results of steatosis (M), lobular inflammation (N), ballooning (O), NAS total scores (P). ^* P<0.01 vs. the control group and the control-cele group;^** P<0.01 vs. the T2DM-NASH group. White = control; Gray = control-cele; Black = T2DM-NASH; Striped = T2DM-NASH-Cele.

Figure 3. Immunohistochemistry(IHC) staining for Wnt5a, JNK1, NF-κB p65, and COX-2.

IHC staining for Wnt5a (A–D; ×400); IHC staining for JNK1 (E–H; ×400); IHC staining for NF-κB p65 (I–L; ×400); IHC staining for COX-2 (M–P; ×400). The Integral Optical Density(IOD) of Wnt5a (Q), JNK1 (R), NF-κB p65 (S); COX-2 (T). ^* P<0.01 vs. the control group and the control-cele group; ^** P<0.01 vs. the T2DM-NASH group. White = control; Gray = control-cele; Black = T2DM-NASH; Striped = T2DM-NASH-Cele.

Figure 4. Western blot analysis of Wnt5a, JNK1, NF-κB p65, and COX-2 protein expression.

Representative western blot images and quantitative analysis of Wnt5a (A), JNK1 p54 and p46 (B), NF-κB p65 (C), and COX-2 (D). GAPDH was used as a loading control. ^* P<0.01 vs. the control group and the control-cele group; ^** P<0.01 vs. the T2DM-NASH group. White = control; Gray = control-cele; Black = T2DM-NASH; Striped = T2DM-NASH-Cele.

Figure 5. Real-time pcr analysis of Wnt5a, NF-κB p65, and COX-2 mRNA expression.

Quantitative analysis of Wnt5a mRNA (A), NF-κB p65 mRNA (B), COX-2 mRNA (C). β-actin was used as a control. ^* P<0.01 vs. the control group and the control-cele group; ^** P<0.01 vs. the T2DM-NASH group. ^# P<0.05 vs. the T2DM-NASH group. White = control; Gray = control-cele; Black = T2DM-NASH; Striped = T2DM-NASH-Cele.