APAP is one of the most widely used analgesic and antipyretic drugs around the world [21]. However, it has been reported that overdosage of APAP could cause liver injury even death [22,23]. Accumulation of NAPQI, one of the intermediate metabolites of APAP in liver, could actuate the liver injury by promoting oxidative stress and inflammation of hepatic tissues, which finally triggers the hepatocellular apoptosis [22,24,25]. In the present study, abnormal pathologic alternations, increased Suzuki’s score, upregulated expression of AST, ALT and ALP, as well as downregulated ALB level in APAP group of mice indicated that the modelling of acute hepatic injury had been successfully established.
Emodin is the major component and one of quality control indexes of RP, a traditional Chinese herb [26–28]. Emodin has been reported that it has biologic activities and beneficial effects, such as hepatoprotective, anti-inflammatory responses, antibacterial, antivirus, as well as neuroprotective [19,29–31]. Previous studies demonstrated that emodin could protect against APAP-induced hepatic injury via multiple targets, including cytochrome P450 (CYP), and AMP-activated protein kinase (AMPK)/ Yes-associated protein (YAP) signaling pathway [17]. Our study suggested that emodin attenuated APAP-induced hepatic injury by activating Nrf2 anti-oxidant pathway and inhibiting NLRP3 via downregulating cGAS-STING signaling pathway.
Oxidative stress is one of landmark events of APAP-induced acute hepatic injury [32]. In experiments of rodent model, routine dose of APAP were mainly involved in glucuronidation and sulfation, and the non-toxic metabolites were then excreted through vile and urine [33,34]. However, when excessive APAP is oxidated to NAPQI by cytochrome P450 (CYP), which binds with GSH to inhibit toxic responses [35,36]. The accumulation of NAPQI result in the depletion of GSH in liver, leading to the decrease of anti-oxidant enzyme activities, and the massive production of ROS [37]. ROS directly cause cytoplasmic vacuolation, hepatocyte apoptosis, and liver failure [38]. SOD, MDA and GSH are the commonly used indexes to measure the levels of intracellular oxidative stress, and SOD and GSH are involved in the anti-oxidant processes, the levels of MDA represent the extents of oxidative injury [39,40]. In our study, the levels of SOD and GSH were significantly increased with the treatment of emodin, the concentration of MDA was reduced. Anti-oxidant enzymes like HO-1 and NQO1, transcription factor Nrf2 is closely related to oxidative stress-associated cellular damage. The loss of Nrf2 in mice caused severe hepatic injury in chlorogenic acid induced acute liver injury [41]. Nrf2 can translocate to nucleus under the stimulation of ROS, and binds to anti-oxidant response element (ARE), leading to the transcription of anti-oxidant enzymes, including NQO1 and HO-1 [42,43]. Our results shown that emodin downregulated CYP2E1 expression and upregulated Nrf2, HO-1, NQO1 expression.
In APAP-induced hepatic injury, oxidative stress causes the activation of inflammatory related-signaling pathways, which further aggravates liver injury [44]. NLRP3 has been considered as an important pro-inflammatory factor activated by oxidative stress [45,46]. It has been reported that NLRP3 is one of potential inflammatory mediators in APAP-induced hepatic damage, partly because of lower levels of NQO1 in the liver [47,48]. Besides, immune cells in hepatic activated by damage-associated molecular patterns (DAMPs), which was conducted with mitochondrial DNA (mtDNA) and fragmented nucleus DNA as well as other proteins released from injured cell, could also be involved in hepatic inflammation [32,49]. Inflammation in APAP-induced damage model is thought to be amplified by IL-1β, IL-6, and TNF-α, produced by Kupffer cells and hepatic dendric cells [12]. IL-10 is known as an anti-inflammatory cytokine and could suppress the acute hepatic injury, moreover, IL-10 deficiency mice showed more severe hepatic damage. In the present study, APAP was found to initiated the activation of NLRP3 inflammasome, which was inhibited by treatment with emodin.
APAP-induced liver damage causes hepatocyte death via necrosis and apoptosis [50]. Bax and Bcl-2 can regulate the of progression apoptosis[51]. The excessive APAP-adducts have been proved to promote hepatocellular apoptosis [52]. We found that emodin alleviated hepatocyte necrosis and apoptosis induced by APAP, as well as inhibited Bax/Bcl-2 ratio. Above results indicated that emodin could inhibit APAP-induced hepatic injury via multiple processes.
Cyclic GMP-AMP synthase (cGAS), a sensor of DNA, which could be activated by virus DNA or aberrant intracellular DNA [53]. cGAS can recognize DNA via electrostatic action and hydrogen-bonding interaction [54], and catalyze the synthesis of Cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) from adenosine triphosphate (ATP) and guanosine triphosphate (GTP) after recognizing DNA [55]. Stimulator of interferon genes (STING) is found on the outer mitochondrial membrane and endoplasmic reticulum in the form of a dimer in inactivated state [56]. Dimer STING can bind with cGAMP catalyzed by cGAS and subsequently translocated to the vesicles around perinuclear region from endoplasmic reticulum by Golgi body [57]. TANK-binding kinase 1 (TBK1) were enrolled into the vesicles to phosphorylated and activated STING [58]. Furthermore, phosphorylated STING can phosphorylate transcription factor interferon regulatory factor 3 (IRF3) [58], one of the most important downstream transcription factors of cGAS-STING signaling pathway, which is closely related to inflammation and apoptosis [59]. Finally, phosphorylated IRF3 enter the nucleic and promote the transcription of IFN-α [58]. In addition to cGAMP, STING can also be activated by second messenger cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) [60].
It is noteworthy that cGAS-STING signaling pathway also participate in multiple kinds of acute and chronic hepatic injury, including radiation-induced liver injury [61], non-alcoholic fatty liver disease (NAFLD) and high fat diet-associated hepatic injury [62], and hepatitis B virus (HBV) infection-associated liver injury [63]. However, it is interesting that STING mainly expressed in hepatic nonparenchymal cells like intrahepatic macrophages instead of hepatic parenchymal cells, which resulted that the resistance to HBV infection is failed in hepatocellular [63]. It plays vital role in APAP-induced hepatic injury that necrosis hepatocellular released generous mtDNA and fragmented nucleus DNA to intracellular space, and the DNA might amplify the hepatic injury as DAMPs [64]. The cGAS-STING signaling pathway, is associated with innate immune response and DNA recognition. Araujo et al. found that the activation of cGAS-STING signaling pathway plays an important role in APAP-induced hepatic injury [65]. The levels of cGAS and STING were upregulated in hepatic parenchymal cells, and the levels of STING were also consistently increased in hepatic nonparenchymal cells. Simultaneously, massive mtDNA accumulated in extracellular space, which is one of the causes of the activation of cGAS-SITNG signaling pathway in hepatocellular. Activated cGAS-STING signaling pathway in hepatic parenchymal cells promotes inflammation, apoptosis, and necrosis of hepatic tissues [65]. In addition, hepatic nonparenchymal cells with activated cGAS-STING signaling pathway secret IFN-α, which also aggravates the liver damage. Therefore, inhibiting the cGAS-STING signaling pathway is a potential therapeutic method of APAP-induced hepatic injury. Our study shown that emodin could inhibit the expression of cGAS, STING, P-IRF3, and P-TBK1 in liver tissues. These results suggested that the protective effect of emodin on APAP-induced liver damage might be associated with the inhibition of the cGAS-STING signaling pathway.