Empagliflozin ameliorates liver fibrosis in NASH rat model via targeting hepatic NF-κB/SOX9/OPN signaling and osteocalcin level

Abstract

Non-alcoholic steatohepatitis (NASH) may be associated with tissue fibrotic changes and can be treated via different therapeutic tools which may however either initiate weak or long-term side effects that minimize its use. Empagliflozin (EMPA) is an oral anti-diabetic drug which has characteristic effects during hepatic steatosis regarding lipid accumulation and insulin resistance. In this study, we aimed to investigate an additional mechanism through which EMPA can exert and potentiate its anti-inflammatory and anti-fibrotic effects in NASH rat model. Male Wistar albino rats fed on high fat diet (HFD) and 20% fructose in drinking water for 18 weeks and received EMPA (30 mg/kg/day, orally) starting from week 11. Body and liver weights, homeostatic model assessment of insulin resistance (HOMA-IR), lipid profile, liver function tests, other biochemical and histological parameters were determined. HFD joined with fructose intake significantly increased body and liver weights, HOMA-IR value, hepatic inflammatory and fibrotic markers, liver transaminases, hepatic expression of nuclear factor-kappa B (NF-κB), sex determining region Y box 9 (SOX 9), and osteopontin (OPN) with significant decrease in hepatic osteocalcin (OCN). Intense hepatic lesions with severe microsteatosis and deposition of collagen fibers were clearly observed. Effectively, EMPA restored the normal liver functions, downregulated hepatic inflammatory cytokines, NF-κB, SOX 9, OPN, and increased OCN level. These results highlight another pathway illustrated the anti-fibrotic effects of EMPA against liver fibrosis probably through downregulation of NF-κB/SOX 9/OPN signaling along with upregulation of hepatic OCN which may potentiate the valuable anti-inflammatory and anti-fibrotic effects of EMPA.

Keywords: Empagliflozin; Liver fibrosis; Non-alcoholic steatohepatitis; OPN; Osteocalcin; SOX9.

Fig. 1a

Empagliflozin downregulates hepatic p65 NF-κB protein expression. A) Quantitative analysis of p65 NF-κB protein expression. B) Representative western blots images. NF-κB: nuclear factor –kappa B, RQ: relative quantity, N: normal, NASH: nonalcoholic steatohepatitis, EMPA: empagliflozin (30 mg/kg body weight/day, orally for 8 weeks). Bars and error bars represent mean ± SD. (n=6/group). SD: standard deviation; *P< 0.001 vs. N, #P<0.001 vs. NASH

Fig. 1b

Empagliflozin reduces inflammatory cytokines in hepatic tissues. A) IL-1β: Interleukin-1β, B) IL-6, C) TNF-α: tumor necrosis factor-; N: normal; NASH, non-alcoholic steatohepatitis; EMPA, empagliflozin (30 mg/kg body weight/day; orally for 8 weeks). Bars and error bars represent mean ± SD. (n = 6/group). SD, standard deviation; * P < 0.001 vs. N, ^#P < 0.001 vs. NASH

Fig. 2

Empagliflozin represses hepatic TGF-β1 and SOX 9 gene expression. A TGF-β1, transforming growth factor-β1, B SOX 9, sex determining region Y box 9, N, normal; NASH, non-alcoholic steatohepatitis; EMPA, empagliflozin (30 mg/kg body weight/day, orally for 8 weeks). Bars and error bars represent mean ± SD. (n = 6/group). SD, standard deviation; *P < 0.001 vs. N, ^#P < 0.001 vs. NASH

Fig. 3

Empagliflozin reduces OPN level with significant upregulation of hepatic OCN. A OCN: osteocalcin, B OPN: osteopontin, N, normal; NASH, non-alcoholic steatohepatitis; EMPA, empagliflozin (30 mg/kg body weight/day, orally for 8 weeks). Bars and error bars represent mean ± SD. (n = 6/group). SD, standard deviation; *P < 0.001 vs. N, ^#P < 0.001 vs. NASH

Fig. 4

Representative photomacrographs of liver and photomicrographs of H&E stained sections of hepatic tissues from different experimental groups (H&E staining × 200, scale bar = 100 μm). A Normal control group showing normal hepatic parenchyma. B NASH control group displayed massive microsteatosis (arrow) and fibrosis (arrow head) with mild peripherilobular microsteatosis (arrow). C NASH + EMPA group showed moderate perilobular microsteatosis (arrow) and delicate fibrous strands (arrow head), D NAFLD activity score (NAS) analysis. Bars and error bars represent mean ± SD. (n = 3/group). SD, standard deviation; *P < 0.001 vs. N, ^δP < 0.01 vs. NASH

Fig. 5

Representative photomicrographs of siruis red-stained sections of hepatic tissues from different experimental groups (Siruis red × 200, scale bar = 100 μm). A Normal control group showing no collagen fibers deposits. B NASH control group showing intense collagen fibers stained red in portal areas and interlobular tissue. C NASH + EMPA group showing delicate collagen deposits in portal and interlobular tissue, (n = 3/group)

Fig. 6

Non-parametric Pearson correlation analysis. OCN, osteocalcin; NF-κB, nuclear factor-kappa B; SOX 9, sex determining region Y box 9; OPN, osteopontin; TGF-β1, transforming growth factor- β1