Elsevier

Free Radical Biology and Medicine

Volume 160, 20 November 2020, Pages 163-177
Free Radical Biology and Medicine

Baicalin promotes liver regeneration after acetaminophen-induced liver injury by inducing NLRP3 inflammasome activation

https://doi.org/10.1016/j.freeradbiomed.2020.05.012Get rights and content

Highlights

  • Baicalin improved liver regeneration after APAP-induced acute liver injury in mice.

  • Baicalin induced NLRP3 inflammasome activation and the subsequent IL-18 produce.

  • IL-18 promoted the proliferation of hepatocytes.

  • IL-18 was critical for the baicalin-provided promotion on liver regeneration after APAP-induced liver injury.

  • Baicalin increased NLRP3 inflammasome assembly by inducing Nrf2 accumulation in cytoplasm.

Abstract

Liver regeneration has become a new hotspot in the study of drug-induced liver injury (DILI). Baicalin has already been reported to alleviate acetaminophen (APAP)-induced acute liver injury in our previous study. This study aims to observe whether baicalin also promotes liver regeneration after APAP-induced liver injury and to elucidate its engaged mechanism. Baicalin alleviated APAP-induced hepatic parenchymal cells injury and enhanced the number of mitotic and proliferating cell nuclear antigen (PCNA)-positive hepatocytes in APAP-intoxicated mice. Baicalin increased hepatic PCNA and cyclinD1 expression in APAP-intoxicated mice. Baicalin induced the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome, leading to the increased hepatic expression of interleukin-18 (IL-18) and IL-1β in APAP-intoxicated mice. The results in vitro demonstrated that IL-18 promoted the proliferation of human normal liver L-02 cells. Moreover, the baicalin-provided promotion on liver regeneration in APAP-intoxicated mice was diminished after the application of NLRP3 inhibitor MCC950 and the recombinant mouse IL-18 binding protein (rmIL-18BP). Baicalin induced the cytosolic accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), and increased the interaction between Nrf2 with Nlrp3, ASC and pro-caspase-1 in livers from APAP-intoxicated mice. Furthermore, the baicalin-provided NLRP3 inflammasome activation and promotion on liver regeneration after APAP-induced liver injury in wild-type mice were diminished in Nrf2 knockout mice. In conclusion, baicalin promoted liver regeneration after APAP-induced acute liver injury in mice via inducing Nrf2 accumulation in cytoplasm that led to NLRP3 inflammasome activation, and then caused the increased expression of IL-18, which induced hepatocytes proliferation.

Introduction

Drug-induced liver injury (DILI) is a serious clinical problem around the world and is responsible for over 50% of reported cases of acute liver failure in the United States [1,2]. Acute liver injury induced by acetaminophen (APAP) overdose is reported to be the main cause for DILI in many western countries [[3], [4], [5]]. Liver has a high regeneration and repair capacity. It is the only visceral organ that has the ability to regenerate in a time-limited manner with the restoration of its original size [6,7]. It has been widely reported that liver regeneration occurs after any insult that induces liver inflammation or hepatocytes necrotic death during a variety of liver diseases including DILI [6].

Previous studies mainly focused on elucidating the engaged mechanism of necrotic death of hepatocytes during APAP-induced liver injury [8,9]. Recently, liver regeneration is reported to be crucial for the recovery of liver after APAP-induced acute liver injury [10]. Schisandrol B and Liuweiwuling tablet are reported to alleviate APAP-induced hepatotoxicity via promoting liver regeneration [11,12]. Thus, promoting liver regeneration has the huge potential to be developed as a novel therapeutic strategy for the detoxification of APAP-induced hepatotoxicity. However, the concrete mechanism of liver regeneration is mostly studied in experimental hepatectomy, which is considerably different from the APAP-induced hepatotoxicity.

The NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome consists of Nlrp3 (NACHT, LRR and PYD domain-containing protein 3), ASC (apoptosis-associated speck-like protein containing a CARD) and the inactive caspase-1 (cysteine-dependent aspartate-directed protease 1) [13,14]. The major function of NLRP3 inflammasome is to recognize a wide variety of danger signals including pathogen-related molecular patterns (PAMPs) and damage-related molecular patterns (DAMPs), and thus leads to the activation of caspase-1, which further conducts the cleavage of pro-interleukin-1β (pro-IL-β) and pro-interleukin-18 (pro-IL-18) into mature IL-1β and IL-18 [13,14]. A previous study showed that NLRP3 inflammasome activation contributed to the APAP-induced acute liver injury, which may be due to the IL-1β-initiated liver inflammatory injury [15]. However, a later study showed that NLRP3 inflammasome activation had little impact on APAP-induced acute liver injury [16]. Therefore, it can be seen that the critical role of NLRP3 inflammasome involved in APAP-induced acute liver injury is still conflicting. Moreover, there is still no report about whether NLRP3 inflammasome activation is also involved in liver regeneration after APAP-induced acute liver injury.

Baicalin is the main compound isolated from Chinese herbal medicine Scutellaria baicalensis Georgi. Baicalin is reported to alleviate a variety of inflammatory disorders [17]. Previous studies have shown the protection of baicalin from APAP-induced liver injury via reducing liver inflammation [18,19]. Moreover, our previous study has shown that baicalin attenuated APAP-induced acute liver injury via inducing Nrf2 activation [20]. This study aims to investigate the promotion of baicalin on liver regeneration after APAP-induced acute liver injury and to further explore whether NLRP3 inflammasome is involved in this process.

Section snippets

Chemicals reagents and antibodies

Baicalin (≥98.0%) was purchased from Shanghai Yuanye Biological Technology Co., Ltd (Shanghai, China). Alanine/aspartate aminotransferases (ALT/AST) activity and myeloperoxidase (MPO) activity assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Adenosine triphosphate (ATP) assay kits were purchased from Beyotime Institute of Biotechnology (Shanghai, China). 5-Ethynyl-2′-deoxyuridine (EdU) assay kits were purchased from KeyGEN BioTECH (Nanjing, China).

Baicalin alleviated hepatocellular damage and promoted liver regeneration in APAP-intoxicated mice

As shown in Fig. 1A-a, serum ALT/AST activities in mice were increased at 24 h, 48 h or 72 h after APAP were orally given to mice. When baicalin was given to mice at 6 h, 12 h or 18 h after APAP administration, the elevated transaminase activities were significantly decreased in mice at 24 h after APAP administration as compared to mice without baicalin administration. However, when baicalin was given to mice at 12 h, 36 h and 60 h after APAP administration, baicalin had no obvious inhibition

Discussion

The replacement of necrotic hepatocytes and restoration of liver normal function after acute liver injury can be acquired through promoting liver regeneration, and enhancing liver regeneration after APAP-induced acute liver injury has already been reported to improve the final outcome [10,24]. Generally, it is reported that the most serve time point of APAP-induced acute liver injury in C57BL/6 mice is at about 6 h after APAP administration, and liver regeneration occurred at about 12 h after

Funding

This work was financially supported by the leadership in Science and Technology innovation of the third batch of national “Ten Thousand People Plan” for Lili Ji, National Natural Science Foundation of China (81573679, 81960748) and National Key R&D Program of China (2018YFC1707302).

Declaration of competing interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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