Elsevier

Life Sciences

Volume 276, 1 July 2021, 119367
Life Sciences

Estrogen cholestasis induces gut and liver injury in rats involving in activating PI3K/Akt and MAPK signaling pathways

https://doi.org/10.1016/j.lfs.2021.119367Get rights and content

Abstract

Backgrounds

Estrogen and its metabolites often lead to intrahepatic cholestasis in susceptible women with pregnancy, administration of oral contraceptives and postmenopausal hormone replacement therapy. Recently, dysfunction of the gut-liver axis has been suggested to play a pivotal role in the progression of cholestasis, but details about estrogen cholestasis (EC)-induced gut and liver injury are still largely unknown. This study aims to gain insight into EC-induced gut and liver injury and cell signaling implicated.

Methods

Male rats were exposed to 5 and 10 mg/kg of 17α-ethinylestradiol via subcutaneous injection for 5 successive days to simulate human EC.

Results

By detection of these estrogen cholestatic rats, we found that EC induced inflammation in the liver but not in the intestine through activating NF-κB signaling pathway. EC strongly induced oxidative stress in both the liver and intestine, and activated the hepatic Nrf2/Gclm/Gclc pathway and the intestinal Nrf2/Ho-1 pathway, respectively, for adaptively regulating oxidative stress. EC increased cell apoptosis in both the liver and intestine. Additionally, EC elevated phosphorylation of Akt, ERK1/2, and p38 in the liver and increased phosphorylation of p38 in the intestine.

Conclusions

EC induces liver inflammation, both gut and liver oxidative stress and apoptosis, involving in activating PI3K/Akt and MAPK signaling pathways. Investigation of EC-induced gut and liver injury contributes to the development of new potential therapeutic strategies.

Introduction

Intrahepatic cholestasis results from an impairment of bile secretion and leads to intrahepatic retention of toxic bile constituents, which is a common manifestation of several liver diseases. The etiologies and pathogenesis of intrahepatic cholestasis are complicated, including infection, heredity, environment, pregnancy, and diet [1]. In recent years, however, studies have indicated that intestinal damages are observed in patients with cholestatic liver diseases, such as obstructive jaundice, primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) [2]. It has been reported that approximately 70%–80% of PSC patients represent extraintestinal manifestations of inflammatory bowel disease [3]. Besides, intestinal damages are also observed in bile duct ligation (BDL)- and CCl4-induced cholestatic animal models [4]. The close association between liver and intestine highlights the importance of the gut-liver axis in the progression of cholestasis.

Estrogen is a female sex hormone, which plays a critical role in the menstrual cycle and reproductive system. Furthermore, estrogen is also a major component in many drugs, such as oral contraceptives and hormone replacement medications [5]. However, estrogen and its metabolites are causally associated with intrahepatic cholestasis, especially in women who are pregnant (intrahepatic cholestasis of pregnancy, ICP), receiving oral contraceptive administration and postmenopausal hormone replacement therapy [6]. It has been demonstrated that estrogen can repress bile acid secretion through the ERα/FXR/BSEP pathway, which results in excessive accumulation of toxic bile acid in hepatocytes and leads to estrogen cholestasis (EC) [7]. EC is mainly characterized by high estrogen level and hepatic toxic bile acid accumulation, which induce liver injury through activating multiple cell signaling pathways. A recent study has reported that some ICP patients displayed unexplained gastrointestinal discomfort and intestinal leakage [8]. Moreover, women with a history of ICP during pregnancy are more likely to die from intestinal diseases later in life [9]. We have recently provided preliminary evidence suggesting that intestinal damages present in estrogen-induced cholestatic rats [10]. However, details about pathological conditions of EC-induced gut and liver injury are largely unknown and need to be confirmed.

Considering human relevance, experimental cholestasis induced by 17α-ethynylestradiol (EE) administration has been widely used to investigate the molecular mechanisms involved in EC [6]. In this work, we aim to gain insight into EC-induced gut and liver injury and the cell signaling implicated. Overall, our data indicate that EC induces liver inflammation, both gut and liver oxidative stress and apoptosis, and the activation of PI3K/Akt and MAPK signaling pathways represents important mechanisms underlying EC-induced gut and liver injury.

Section snippets

Materials

EE was purchased from Sigma (St. Louis, MO, USA). Antibodies against NF-κB, p-NF-κB, Iκbα p-Iκbα, Bax, Caspsae3, Cleaved Caspsae3, Akt, p-Akt, ERK1/2, p-ERK1/2, JNK, p-JNK, p38, and p-p38 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibody against Bcl-2 was obtained from R&D Systems (Minneapolis, MN, USA). RIPA lysis buffer and protease inhibitor cocktail were obtained from Beyotime Biotechnology (Jiangsu, China). All other reagents were of analytical grade.

Animal experiments

Male

The rat model of EC as well as EC-induced gut and liver injury

The body weight of rats treated with EE was dose-dependent decreased compared with the control group (Fig. 1A). Liver weight and liver/body weight were significantly increased after EE treatment (Fig. 1A). ALP, γ-GT, and TBA are biomarkers to indicate cholestasis, and all of them were dramatically increased after EE treatment (Fig. 1B). These results suggest that we have successfully established an estrogen cholestatic model which can be used for further investigation.

Histological abnormalities

Discussion

Intrahepatic cholestasis derives from the accumulation of hepatic bile acids and disruption of bile secretion caused by various factors, gradually progressed to liver injury, fibrosis, cirrhosis, and even liver cancer [1]. In addition to liver damage, recent studies have suggested the intestine is also implicated [3]. Although the gut-liver injury in partial types of cholestasis is confirmed, however, the pathological conditions of EC-induced gut and liver injury remain largely unknown. Our

Funding

This work was supported by the National Natural Science Foundation of China (NO.81803798, NO.82073939, and NO.81573788) and the Tongji Hospital Fund (2019A08).

Declaration of conflicting interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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    These authors contributed equally to this study.

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