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. 2020 Nov 23:11:573074.
doi: 10.3389/fphar.2020.573074. eCollection 2020.

Physcion Protects Against Ethanol-Induced Liver Injury by Reprogramming of Circadian Clock

Youli Yao  1 , Along Zuo  2 , Qiyu Deng  1 , Shikang Liu  1 , Tianying Zhan  1 , Maolin Wang  1 , Haidong Xu  3 , Junxian Ma  4 , Yingying Zhao  1
Affiliations

Physcion Protects Against Ethanol-Induced Liver Injury by Reprogramming of Circadian Clock

Youli Yao et al. Front Pharmacol. .

Abstract

The circadian clock plays a key role in our daily physiology and metabolism. Alcohol consumption disrupts the circadian rhythm of metabolic genes in the liver; however, the potential contribution of circadian clock modulation to alcoholic liver disease (ALD) is unknown. We identified a novel liver protective agent, physcion, which can alleviate fat accumulation and inflammation in ALD mice via reprogramming the hepatic circadian clock. The model of alcoholic hepatitis was established by intragastrically administering ethanol. In vitro, physcion was investigated by treating HepG2 cells with ethanol. The role of circadian clock in Physcion caused liver protection was tested by knocking down the core circadian gene Bmal1. Physcion application caused reduced lipogenesis and alleviated inflammation in alcohol-induced mice. In alcoholic hepatosteatosis models, physcion upregulated the core circadian genes. And the circadian misalignment triggered by ethanol was efficiently reversed by physcion. Physcion attenuated lipogenesis via reprogramming the circadian clock in HepG2 cells. Suppression of Bmal1 by RNA interference abolished the protective of physcion. In addition, Physcion binds to the active pocket of BMAL1 and promotes its expression. The study identified the novel liver protective effects of physcion on alcohol-induced liver injury, and modulation of the core circadian clock regulators contributes to ALD alleviation. More importantly, strategies targeting the circadian machinery, for example, Bmal1, may prove to be beneficial treatment options for this condition.

Keywords: alcoholic liver steatosis; circadian clock; inflammation; lipogenesis; physcion.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Physcion reduces the symptoms of ethanol-induced hepatic injury. (A) Chemical structure of physcion, (B) animal experimental procedure, (C–E) serum ALT, AST, and TG contents, (F) liver appearance pictures, (G) H&E staining, and (H) Oil Red O staining. Values significantly different from the control are indicated by hash signs (# p < 0.05). Values significantly different from the ethanol are indicated by asterisks (*p < 0.05).
FIGURE 2
FIGURE 2
Physcion effectively reduce the lipogenesis induced by ethanol. (A) The mRNA expression of Srebp1, Fasn, and Pparα. (B) The protein expression of AMPKα, p-AMPKα, PPARα, and SREBP1. (C) Immunofluorescence staining of SREBP1 (×400 original magnification). (D) The phospho-AMPKα staining (green) and nuclei with DAPI (blue) are shown. Values significantly different from the control are indicated by hash signs (# p < 0.05). Values significantly different from the ethanol are indicated by asterisks (*p < 0.05).
FIGURE 3
FIGURE 3
Physcion suppresses inflammasome activation in ethanol-induced acute hepatic injury. (A) The mRNA expression of Nlrp3 and P2x7r. (B) The protein expression levels of P2X7R, and IL-1β. (C) The mRNA expression of Caspase1. (D) Caspase-1 staining (red) and nuclei with DAPI (blue) are shown. (E) The mRNA expression of IL-1β. Values significantly different from the control are indicated by hash signs (# p < 0.05). Values significantly different from the ethanol are indicated by asterisks (*p < 0.05).
FIGURE 4
FIGURE 4
Physcion improves the disorder of the circadian clock pathway in acute alcoholic hepatosteatosis. (A) The protein expression of BMAL1, and CLOCK. (B) Immunofluorescence staining of BMAL1, CLOCK, and PER2. (C) The mRNA expression of Bmal1 and Per2. Values significantly different from the control are indicated by hash signs (# p < 0.05). Values significantly different from the ethanol are indicated by asterisks (*p < 0.05).
FIGURE 5
FIGURE 5
Physcion improves increased inflammasome activation and ameliorates lipid accumulation in HepG2 cells. (A) Cell viability was determined by MTT assay. (B) The protein expression of PPARα, SREBP1, IL1β, and caspase1. (C) Immunofluorescence staining of SREBP1. (D) The protein expression of BMAL1 and CLOCK. (E) Immunofluorescence staining of BMAL1 and CLOCK. Values significantly different from the control are indicated by hash signs (# p < 0.05). Values significantly different from the ethanol are indicated by asterisks (*p < 0.05).
FIGURE 6
FIGURE 6
Effects of physcion on ethanol-induced circadian dysregulation in HepG2 cells. (A–F) The mRNA expression of Bmal1, Clock, Per1, Per2, Cry1, and Cry2 in HepG2 cells.
FIGURE 7
FIGURE 7
Physcion attenuates intracellular lipid accumulation in a Bmal1-dependent way. (A) The protein expression of BMAL1. (B) The mRNA expression of Bmal1. (C) The protein expression of SREBP1, and P2X7R. (D) Immunofluorescence staining of SREBP1 and P2X7R. Values significantly different from the control are indicated by hash signs (# p < 0.05). Values significantly different from the ethanol are indicated by asterisks (*p < 0.05).
FIGURE 8
FIGURE 8
Computer analysis of (A) Bmal1 and Physcion, (B) molecular docking of Bmal1 and Physcion, and (C) interaction of active site and H-bond, 2-D interaction diagram.
FIGURE 9
FIGURE 9
Possible mechanism(s) of suppression of alcoholic hepatosteatosis by physcion. Activation of AMPK-PPARα signaling and inhibition of P2X7R-IL-lβ inflammasome by physcion might be mediated by circadian clock pathway.
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