Oxy210 Inhibits Hepatic Expression of Senescence-Associated, Pro-Fibrotic, and Pro-Inflammatory Genes in Mice During Development of MASH and in Hepatocytes In Vitro

Abstract

Background: Senescence, a state of permanent cell cycle arrest, is a complex cellular phenomenon closely affiliated with age-related diseases and pathological fibrosis. Cellular senescence is now recognized as a significant contributor to organ fibrosis, largely driven by transforming growth factor beta (TGF-β) signaling, such as in metabolic dysfunction-associated steatohepatitis (MASH), idiopathic pulmonary fibrosis (IPF), chronic kidney disease (CKD), and myocardial fibrosis, which can lead to heart failure, cystic fibrosis, and fibrosis in pancreatic tumors, to name a few. MASH is a progressive inflammatory and fibrotic liver condition that has reached pandemic proportions, now considered the largest non-viral contributor to the need for liver transplantation.

Methods: We previously studied Oxy210, an anti-fibrotic and anti-inflammatory, orally bioavailable, oxysterol-based drug candidate for MASH, using APOE*3-Leiden.CETP mice, a humanized hyperlipidemic mouse model that closely recapitulates the hallmarks of human MASH. In this model, treatment of mice with Oxy210 for 16 weeks caused significant amelioration of the disease, evidenced by reduced hepatic inflammation, lipid deposition, and fibrosis, atherosclerosis and adipose tissue inflammation.

Results: Here we demonstrate increased hepatic expression of senescence-associated genes and senescence-associated secretory phenotype (SASP), correlated with the expression of pro-fibrotic and pro-inflammatorygenes in these mice during the development of MASH that are significantly inhibited by Oxy210. Using the HepG2 human hepatocyte cell line, we demonstrate the induced expression of senescent-associated genes and SASP by TGF-β and inhibition by Oxy210.

Conclusions: These findings further support the potential therapeutic effects of Oxy210 mediated in part through inhibition of senescence-driven hepatic fibrosis and inflammation in MASH and perhaps in other senescence-associated fibrotic diseases.

Keywords: MASH; Oxy210; fibrosis; oxysterols; senescence.

Figure 1

Sixteen-week time course of hepatic senescence-associated gene expression and inhibition by Oxy210 in APOE*3-Leiden.CETP mice. Expression of p21, p15, p16 and p53 in the livers from control and Oxy210-treated mice was measured by qPCR and normalized to the level of the housekeeping gene Rpl4. Relative gene expression levels are presented as mean ± SD (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Control).

Figure 2

Sixteen-week time course of hepatic SASP gene expression and inhibition by Oxy210 in APOE*3-Leiden.CETP mice. Expression of Pai-1, Chop, and Nox2 in the livers from control and Oxy210-treated mice was measured by qPCR and normalized to the level of the housekeeping gene Rpl4. Relative gene expression levels are presented as mean ± SD (* p < 0.05 and ** p < 0.01 vs. Control).

Figure 3

Sixteen-week time course of hepatic inflammatory gene expression and inhibition by Oxy210 in APOE*3-Leiden.CETP mice. Expression of Ccl2, Il-1b, Il-6, and Tnf-a in the livers from control and Oxy210-treated mice was measured by qPCR and normalized to the level of the housekeeping gene Rpl4. Relative gene expression levels are presented as mean ± SD (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Control).

Figure 4

Sixteen-week time course of hepatic TGF-β target gene expression and inhibition by Oxy210 in APOE*3-Leiden.CETP mice. Expression of Tgf-b1, Col1a1, Ctgf, and Acta2 in the livers from control and Oxy210-treated mice was measured by qPCR and normalized to the level of the housekeeping gene Rpl4. Relative gene expression levels are presented as mean ± SD (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Control).

Figure 5

Sixteen-week time course of hepatic Hedgehog target gene expression and inhibition by Oxy210 in APOE*3-Leiden.CETP mice. Expression of Gli1, Ptch1, and Spp1 in the livers from control and Oxy210-treated mice was measured by qPCR and normalized to the level of the housekeeping gene Rpl4. Relative gene expression levels are presented as mean ± SD (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Control).

Figure 6

Inhibition of TGF-β-induced expression of senescence-associated genes in HepG2 cells by Oxy210. HepG2 cells were treated overnight in EMEM without FBS followed by treatment with Oxy210 at 10μM in EMEM containing 1% FBS for 24 h and then treated with 50 ng/mL of TGF-β in the absence or presence of Oxy210 for 48 h. RNA was extracted and analyzed by Q-RT-PCR for the expression of the genes as indicated and normalized to PMSB4 expression. Data from a representative experiment are reported as the mean of triplicate determinations ± SD (## p < 0.01 vs. TGF-β; # p < 0.05 vs. TGF-b; ** p < 0.01 vs. Control; * p < 0.05 vs. Control).