The protective effects of estrogen on biliary and liver damage are independent of ER‐β signaling in female Mdr2‐/‐ mice

Abstract

BACKGROUND & AIMCholestatic liver diseases present with ductular reaction, biliary senescence, peribiliary inflammation and bridging fibrosis, and sex‐dependent differences in liver injury exist in different pathologies. The multidrug resistance 2 knockout (Mdr2‐/‐) mouse is a model of toxic bile‐induced cholangitis which show a higher degree of injury in female mice. Estrogen signals through its receptors, ER‐α and ER‐β, that form homodimers and act as transcription factors. Interestingly, ER‐α and ER‐β can have differing downstream responses. In a model of autoimmune cholestasis, inhibition of ER‐β reduces biliary damage, inflammation, immune cell infiltration and liver fibrosis in both male and female mice. We aimed to determine the role of estrogen/ER‐β signaling on biliary and liver damage in female Mdr2‐/‐ mice.METHODSStudies were performed in female wild‐type (WT) and Mdr2‐/‐ mice at 12 wks of age, with a subset of Mdr2‐/‐ mice subjected to ovariectomy (OVX, to reduce estrogen levels) or treated with PHTPP (ER‐β antagonist, 15 mg/kg BW/day by I.P. implanted minipump) for 1 wk. All studies utilized n=4 mice per group. We evaluated liver damage by H&E staining. Ductular reaction was measured by CK‐19 staining and semi‐quantification, and biliary senescence was imaged using p16 co‐staining with CK‐19 (to mark bile ducts). Peribiliary inflammation was evaluated using F4/80 (macrophage marker) staining and semi‐quantification, and qPCR for IL‐6 and TNF‐α in total liver samples. Immune cell infiltration was visualized by staining for CD3 (pan‐T cell marker) and CD20 (pan‐B cell marker). We determined liver fibrosis by (i) Sirius red staining and semi‐quantification, (ii) qPCR for TIMP1, Col1a1 and αSMA in total liver samples and (iii) co‐staining for desmin (hepatic stellate cell marker) with CK‐19. Human female control (avg. age 24.5 yr) and late‐stage, untreated PSC patient (avg. age 51.8 yr) serum samples were evaluated for estrogen levels by EIA.RESULTSOVX exacerbated liver damage, ductular reaction, biliary senescence, inflammation, immune response and liver fibrosis in female Mdr2‐/‐ mice, demonstrating a protective effect of estrogen signaling during cholangitis. Interestingly, female Mdr2‐/‐ mice treated with PHTPP (blocks ER‐β activity) had no change in any parameters of biliary damage or liver injury when compared to controls suggesting that estrogen’s protective effects are not mediated through ER‐β. Serum estrogen levels increased in female PSC samples versus control, potentially as a compensatory response or abnormal dysregulation as a consequence of injury.CONCLUSIONEstrogen may have a protective effect against biliary damage, inflammation, immune response and liver fibrosis during cholestasis. However, the protective effects are not dependent upon ER‐β signaling in female Mdr2‐/‐ mice and future work is ongoing to understand the role or ER‐α in this setting. Alteration of specific estrogen signaling mechanisms may be important for targeting cholestatic liver diseases in female patients.