Emodin Attenuates Acetaminophen-Induced Hepatotoxicity via the cGAS-STING Pathway

Emodin is a natural bioactive compound from traditional Chinese herbs that exerts anti-inflammatory, antioxidant, anticancer, hepatoprotective, and neuroprotective effects. However, the protective effects of emodin in acetaminophen (APAP)-induced hepatotoxicity are not clear. The present study examined the effects of emodin on APAP-induced hepatotoxicity and investigated the potential molecular mechanisms. C57BL/6 mice were pretreated with emodin (15 and 30 mg/kg) for 5 consecutive days and then given APAP (300 mg/kg) to establish an APAP-induced liver injury model. Mice were sacrificed to detect the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and albumin (ALB) and the liver tissue levels of glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD). Histological assessment, Western blotting, and ELISA were performed. Emodin pretreatment significantly reduced the levels of ALT, AST, and ALP; increased the levels of ALB; alleviated hepatocellular damage and apoptosis; attenuated the exhaustion of GSH and SOD and the accumulation of MDA; and increased the expression of antioxidative enzymes, including nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and NAD(P)H quinone dehydrogenase 1 (NQO1). Emodin also inhibited the expression of NLRP3 and reduced the levels of pro-inflammatory factors, including interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α). Emodin inhibited interferon (IFN)-α, cyclic GMP-AMP synthase (cGAS), and its downstream signaling effector stimulator of interferon genes (STING) expression to protect the liver against APAP-induced inflammatory responses and apoptosis. These results suggest that emodin protected hepatocytes from APAP-induced liver injury via the upregulation of the Nrf2-mediated antioxidative stress pathway, the inhibition of the NLRP3 inflammasome, and the downregulation of the cGAS-STING signaling pathway.


INTRODUCTION
Acetaminophen (N-acetyl-p-aminophenol, APAP) is an acetanilide analgesic and antipyretic drug used worldwide that is primarily used against cold-or 1 influenza-induced headache and fever [1,2]. Although APAP is considered highly safe at recommended doses, its intentional or unintentional overdose causes severe nephrotoxicity and hepatotoxicity, which lead to lifethreatening acute kidney injury and liver failure [3,4]. More than 200 million people use APAP annually, and APAP-induced acute hepatic failure results in 200 deaths. However, treatment for APAP poisoning is primarily limited to N-acetyl-l-cysteine (NAC), which is a non-specific antidote that restores endogenous glutathione (GSH) [5]. Therefore, it is of great significance to examine the possible molecular mechanism of APAP-induced liver damage for clinical applications and the development of potential therapeutic drugs against APAP toxicity.
The inflammatory response and oxidative stress are the main mechanisms of APAP-induced liver failure [6,7]. UDP-glucuronidase (UGT) and sulfotransferase (SULT) enzymes in the liver metabolize most ingested APAP into non-toxic compounds that are subsequently excreted in the urine and bile [8]. The CYP450 enzyme metabolizes the remaining APAP into a toxic intermediate metabolite, N-acetyl-p-benzo-quinoneimine (NAPQI), which may lead to the depletion of GSH and the generation of a protein adduct in the liver [9]. The depletion of GSH and NAPQI adducts causes mitochondrial dysfunction and massive reactive oxygen species (ROS) secretion from injured hepatocytes, which lead to hepatocellular apoptosis [10,11]. Intercellular contents released from these damaged cells, called damage-associated molecular patterns (DAMPs), stimulate non-parenchymal cells to produce and release inflammatory mediators and chemokines [12]. These chemokines recruit a variety of immune and inflammatory cells, such as monocytes and neutrophils, into the liver and promote inflammatory responses via the activation of innate immune signal transduction pathways, which results in the necrosis and apoptosis of liver cells [13]. The recognition of DAMPs is essential for defense and triggers signaling cascades that lead to the production of pro-inflammatory cytokines and type I interferons (IFN-α and IFN-β) [14]. Therefore, the blockade of oxidative stress and inhibition of inflammation are important targets for protecting hepatocytes from APAP hepatotoxicity.
The activation of endogenous substances induces inflammation in APAP-induced liver damage. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) pathway plays a role in the activation of the innate immune response. cGAS is a cytosolic sensor of DNA and activates STING, which leads to the phosphorylation of transcription factors, including interferon regulatory factor (IRF)3 and nuclear factor (NF)-κB, and the activation of the transcription of innate immunity-related genes, including type I IFN [15,16]. cGAS-STING signaling was also implicated in several pathogenic processes, such as alcohol intoxication, autoimmune diseases, and kidney injury [17,18]. The activation of the cGAS-STING signaling pathway promotes cell apoptosis, inflammation, and oxidation [19,20], and it likely plays a key role in hepatic injury. Little is known about the role of cGAS-STING signaling in liver injury, and it is valuable to examine the mechanisms to develop treatments. The present study used an APAP-induced liver damage model to study the pathogenic role of cGAS-STING-dependent inflammation in liver damage.
Rheum palmatum L. (RP, Dahuang in Chinese) is one of the most popular plant products in traditional Chinese medicine (TCM). RP-composed formulas have been widely used for the treatment of hematemesis, constipation, enteritis, liver injury, and menorrhagia for many years in China [21,22]. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) (Fig. 1) is a natural anthraquinone derivative, and it is the main active component and quality control index of RP [23,24]. Emodin has various biological activities, such as hepatoprotective, anticancer, antibacterial, neuroprotective, antidiabetic, anti-inflammatory, and antioxidant effects [25,26]. Bhadauria et al. reported that emodin reversed APAPinduced toxicity similarly to silymarin in a rat model [27]. Emodin inhibited the death of HepG2 cells in vitro, attenuated the degeneration of anti-apoptotic proteins, and improved mitochondrial membrane potential [24]. Emodin also promoted the phosphorylation of Yesassociated protein 1 (YAP1), which is the main downstream target of Hippo that mediates oxidative stress. Many studies reported these natural entities and potential benefits to human health. Preliminary results indicated that emodin possessed several biological activities. However, systemic investigations to examine the protective effect on hepatic disorders in vivo are lacking. More studies are needed to determine the precise roles of emodin in hepatic injury and provide more evidence for the clinical application of relevant products.
The present study investigated the protective effects of emodin on APAP-induced liver injury; evaluated its anti-inflammatory, antioxidative stress, and anti-apoptosis effects; and examined the role of the cGAS-STING pathway in the beneficial effects of emodin.

Animal Experiments
Thirty-two male (6-8 weeks) C57BL/6 mice weighing 17-23 g were supplied by Weitonglihua Biotechnology Co., Ltd. (Hangzhou, China) and kept in the Experimental Animal Center of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All animals were fed in the rearing room with a temperature of 22 ± 3 °C, a relative humidity of 55 ± 5%, and a day-night cycle of 12 h. After 7 days of adaptive feeding, the 32 mice were randomly distributed into 4 groups of 8 mice each: a healthy control group (control), APAP group (APAP), emodin low-dose group (Emo-L), and emodin high-dose group (Emo-H).
Mice in the Emo-L and Emo-H groups were orally administered emodin for 5 consecutive days (15 and 30 mg/kg/day, respectively). Emodin was dissolved in 40% polyethylene glycol (PEG). The control and APAP groups received the same volume of vehicle. Two hours after the last emodin administration, APAP was intraperitoneally injected at a concentration of 300 mg/kg body weight to induce acute hepatic injury. APAP was dissolved in saline. The control group received the same volume of vehicle. Twenty-four hours later, all of the mice were euthanized with an overdose of 1% sodium pentobarbital, and specimens were collected immediately. The Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology approved all animal procedures and experiments.

Tissue Collection
Serum samples were obtained from peripheral blood via centrifugation at 3000 rpm for 10 min at 4 ℃. Half of the liver samples from the left lobe were separated and collected. All of these samples were stored at −80 ℃. The remaining liver samples from the left lobe were fixed in 4% paraformaldehyde and embedded in paraffin. All samples were processed on ice as soon as possible to prevent protein degradation.

Hepatic Histological Analysis
Sections of fixed hepatic samples embedded in paraffin were used for routine hematoxylin and eosin (H&E) staining. Sections were observed and photographed using optical microscopy (Olympus, Japan). Injury grades of hepatic samples were evaluated using Suzuki's score based on the H&E staining results [28].

Biochemical Assays
Serum levels of ALT, AST, ALP and ALB, SOD, MDA, and GSH in liver tissues were tested using biochemical kits.

ELISA Analysis
The levels of IL-1β, TNF-α, IL-6, and IL-10 in hepatic tissues and the serum levels of IFN-α were detected using ELISA kits following the protocol provided by the manufacturer.

Protein Extraction and Western Blot Analysis
Total proteins were extracted from hepatic tissues using RIPA buffer. The concentration of total proteins was quantified using the bicinchoninic acid assay. Total proteins were subjected to 10-12% SDS-PAGE electrophoresis, and the proteins were transferred to PVDF membranes (Sigma, MA, USA). PVDF membranes were blocked with 5% non-fat milk for 1.5 h at room temperature. The membranes were incubated with primary antibodies overnight at 4 ℃. PVDFs were incubated with the corresponding secondary antibodies. Grayscale values of bands were analyzed using ImageJ software.

TUNEL Staining Analysis
TUNEL staining was used to detect the apoptosis rate of hepatocytes. Six-micron sections of hepatic tissue were deparaffinized and rehydrated. The sections were treated with proteinase K for 15 min, followed by incubation with TdT at 37 °C for 2 h. The results of TUNEL staining were observed and photographed using optical microscopy. Hepatic TUNEL-positive cell numbers were assessed using Image Picture Pro software.

Statistical Analysis
All experimental data in this study were obtained from at least 3 independent experiments and are shown as the means ± standard deviations (SDs). One-way ANOVA was performed to compare between-group differences. A p ≤ 0.05 was considered statistically significant. Figure 2A shows widespread inflammatory infiltration in hepatic tissue, severe hepatocyte ballooning degeneration, and extensive hepatocyte necrosis in the APAP group, which were not present in the control group. As shown in Fig. 2B, Suzuki's score in the APAP group was increased compared to the control group (p < 0.01). The APAP group showed higher levels of serum ALT, AST, and ALP and lower levels of ALB than the control group (p < 0.01) (Fig. 2C-F). These differences indicated that the APAP-induced liver injury model was successfully established in mice.

Emodin Alleviated APAP-Induced Liver Injury
Compared to the APAP group, serum levels of ALB, ALT, and ALP were decreased, and the levels of ALB were upregulated in the Emo-H group (p < 0.05) (Fig. 2C-F). Suzuki's scores indicated that the administration of high-dose Emo significantly alleviated the hepatic injury (p < 0.05) (Fig. 2B).  6). B Histological changes were graded using Suzuki's score. Serum levels of C ALT, D AST, E ALP, and F ALB were detected using corresponding kits (n = 8). All values are presented as the mean ± SD. ## p < 0.01 vs. the control group; *p < 0.05 vs. the APAP group.

Emodin Inhibited APAP-Induced Oxidative Stress in Liver Tissues
Hepatic damage is associated with the upregulation of oxidative stress. Our results showed that the levels of SOD and GSH in the APAP group were downregulated, and the levels of MDA were upregulated compared to those in the control group (Fig. 3A-C) (p < 0.01). However, the levels of SOD and GSH were increased, and MDA levels were decreased in the Emo-H group, which indicated that emodin inhibited oxidative stress in APAP-mediated liver injury. Previous studies indicated that the transcription factor Nrf2 and its downstream proteins HO-1 and NQO1 exerted antioxidant properties in cells. Therefore, we detected the levels of Nrf2, HO-1, and NQO1 in hepatic tissues. Nrf2, HO-1, and NQO1 were downregulated in the APAP group (Fig. 3D and E) (p < 0.01), which indicated that these proteins failed to fulfill their protective roles in APAP-mediated hepatic injury. However, the expression levels of Nrf2, HO-1, and NQO1 were partially recovered after high-dose Emo intervention (p < 0.05). We also detected the hepatic levels of CYP2E1, which is responsible for the metabolism of APAP to the toxicant NAPQI. The results showed a significant increase in the levels of CYP2E1 in the APAP group compared to the control group, and Emo-H treatment suppressed this increase (Fig. 3D and E) (p < 0.05).

Emodin Suppressed APAP-Induced Hepatic Inflammation
To determine whether emodin inhibited inflammation in APAP-induced hepatic damage, the levels of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α, and an anti-inflammatory cytokine (IL-10) in hepatic tissues were examined. Levels of the proinflammatory factors IL-1β, TNF-α, and IL-6 were upregulated in the APAP group (Fig. 4A-C) (p < 0.01), and the level of the anti-inflammatory factor IL-10 ( Fig. 4D) (p < 0.01) was downregulated, which indicated that APAP induced robust inflammation in the liver. Notably, the pro-inflammatory factors IL-1β, IL-6, and TNF-α were lower and the anti-inflammatory factor IL-10 was higher in the Emo-H group than in the APAP group (p < 0.05). We also detected the expression of NLRP3 inflammasome-associated proteins, including NLRP3, caspase1, and pro-IL-1β. Figure 4E and F show a significant increase in NLRP3 protein levels in the APAP group compared to the control group (p < 0.01).

Emodin Mediated APAP-Induced Hepatocellular Apoptosis
Apoptosis is a programmed cell death process that occurs when cells confront harsh environments or suffer severe destruction. Hepatocytes undergo strict oxidative stress and inflammation during APAP-induced hepatic injury, which may lead to hepatocellular apoptosis. TUNEL staining is an effective method to label fragmented DNA that emerges from cellular apoptosis. Figure 5A and B show that there was more fragmented DNA in the APAP group than in the control group and Emo-H group (p < 0.01).
The levels of Bax in the APAP group were upregulated (p < 0.01) and reduced in the Emo-H group (p < 0.05). The levels of the anti-apoptotic protein Bcl-2 exhibited opposite trends with Bax in the APAP group and Emo-H group (p < 0.05, p < 0.01). These results Fig. 4 Emodin attenuated APAP-induced hepatic inflammation in mice. Hepatic levels of IL-1β A, TNF-α B, IL-6 C, and IL-10 D were detected using ELISA kits (n = 8). E, F Hepatic protein expression of NLRP3, caspase1, and pro-IL-1β was measured using Western blotting (n = 3). All values are presented as the mean ± SD. ## p < 0.01 vs. the control group; *p < 0.05 vs. the APAP group.
showed that APAP-induced hepatic injury also caused severe hepatocellular apoptosis, similar to other acute hepatic injuries, and emodin alleviated apoptosis by rectifying the oxidative stress and inflammation.

Emodin Attenuated the Activity of the cGAS-STING Signaling Pathway
Compared to the control group, the expression of cGAS-STING signaling pathway-related proteins, including P-TBK1, P-IRF3, cGAS, and STING, was significantly increased in the APAP group (p < 0.01) ( Fig. 6A and B). The expression of IFN-α in serum was also significantly increased in the APAP group (p < 0.01) (Fig. 6C). These results suggested that the cGAS-STING signaling pathway was activated in the model mice. The expression of these proteins was significantly reduced (p < 0.05) in the Emo-H group, which indicated that emodin inhibited the APAP-induced hepatocellular injury via the regulation of cGAS-STING signaling pathway activity.

DISCUSSION
APAP is one of the most widely used analgesic and antipyretic drugs worldwide [29]. However, overdoses of APAP may cause liver injury and death [30,31]. The accumulation of NAPQI, one of the intermediate metabolites of APAP in the liver, induces liver injury by promoting oxidative stress and inflammation in hepatic tissues, which ultimately trigger hepatocellular apoptosis [30,32,33]. The present study showed abnormal pathological alterations; an increased Suzuki's score; upregulated expression of AST, ALT, and ALP; and downregulated ALB levels in the APAP group of mice, which indicated successful establishment of the acute hepatic injury model.
Emodin is the major component and one of the quality control indexes of the traditional Chinese herb RP [34][35][36]. Emodin has biological activities and beneficial effects, such as hepatoprotective, anti-inflammatory, antibacterial, antiviral, and neuroprotective effects [26,Fig. 6 Emodin inhibited the cGAS-STING signal pathway. A, B Hepatic protein expression of P-TBK1, P-IRF3, cGAS, and STING was measured using Western blotting (n = 3). All values are presented as the mean ± SD. ## p < 0.01 vs. the control group; *p < 0.05 vs. the APAP group. [37][38][39]. Previous studies demonstrated that emodin protected against APAP-induced hepatic injury via multiple targets, including the cytochrome P450 (CYP) and AMPactivated protein kinase (AMPK)/Yes-associated protein (YAP) signaling pathways [24]. Our study suggested that emodin attenuated APAP-induced hepatic injury via the activation of the Nrf2 antioxidant pathway and the inhibition of NLRP3 by downregulating the cGAS-STING signaling pathway.
Oxidative stress is a landmark event of APAPinduced acute hepatic injury [40]. Routine doses of APAP in experimental rodent models primarily induced glucuronidation and sulfation, and the non-toxic metabolites were excreted in bile and urine [41,42]. However, cytochrome P450 (CYP) oxidizes excessive APAP to NAPQI, which binds with GSH to inhibit toxic responses [43,44]. The accumulation of NAPQI results in the depletion of GSH in the liver, which leads to a decrease in antioxidant enzyme activities and the massive production of ROS [45]. ROS directly causes cytoplasmic vacuolation, hepatocyte apoptosis, and liver failure [46]. SOD, MDA, and GSH are commonly used indexes to measure the levels of intracellular oxidative stress. SOD and GSH are involved in antioxidant processes, and the levels of MDA represent the extent of oxidative injury [47,48]. The levels of SOD and GSH were significantly increased with emodin treatment in our study, and the concentration of MDA was reduced. Antioxidant enzymes, such as HO-1 and NQO1, and the transcription factor Nrf2 are closely related to oxidative-stress-associated cellular damage. The loss of Nrf2 in mice caused severe hepatic injury in a chlorogenic acid-induced acute liver injury model [49]. Nrf2 translocates to the nucleus under ROS stimulation and binds to antioxidant response elements (AREs), which leads to the transcription of antioxidant enzymes, including NQO1 and HO-1 [50,51]. Our results showed that emodin downregulated CYP2E1 expression and upregulated Nrf2, HO-1, and NQO1 expression. These results suggest that emodin alleviates Nrf2-dependent oxidative stress.
Oxidative stress in APAP-induced hepatic injury causes the activation of inflammatory-related signaling pathways, which further aggravates liver injury [52]. NLRP3 is an important pro-inflammatory factor that is activated by oxidative stress [53,54]. NLRP3 is a potential inflammatory mediator in APAP-induced hepatic damage, partially because of the lower levels of NQO1 in the liver [55,56]. Immune cells in the liver are activated by DAMPs, which are associated with mitochondrial DNA (mtDNA), fragmented nuclear DNA, and other proteins that are released from injured cells and may also be involved in hepatic inflammation [40,57]. Inflammation in an APAPinduced damage model is amplified by IL-1β, IL-6, and TNF-α, which are produced by Kupffer cells and hepatic dendritic cells [12]. IL-10 is an anti-inflammatory cytokine that suppresses acute hepatic injury [58]. IL-10-deficient mice show more severe hepatic damage [59]. APAP initiated the activation of the NLRP3 inflammasome in the present study, and treatment with emodin inhibited this activation. These results indicated that emodin inhibited inflammatory responses via the suppression of the NLRP3 inflammasome.
APAP-induced liver damage causes hepatocyte death via necrosis and apoptosis [60]. Bax and Bcl-2 regulate the progression of apoptosis [61]. Excessive APAP adducts promote hepatocellular apoptosis [62]. We found that emodin alleviated APAP-induced hepatocyte necrosis and apoptosis and decreased the Bax/Bcl-2 ratio. These results indicated that emodin inhibited APAP-induced hepatic injury via the regulation of apoptosis.
cGAS is a sensor of DNA that is activated by viral DNA and aberrant intracellular DNA [63]. cGAS recognizes DNA via electrostatic action and hydrogen-bonding interactions [64] and catalyzes the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) from adenosine triphosphate (ATP) and guanosine triphosphate (GTP) after DNA recognition [65]. STING is found on the outer mitochondrial membrane and endoplasmic reticulum in the form of a dimer in an inactivated state [66]. The STING dimer binds with the cGAMP catalyzed by cGAS and subsequently translocates to vesicles around the perinuclear region from the endoplasmic reticulum by the Golgi body [67]. TANKbinding kinase 1 (TBK1) in vesicles phosphorylates and activates STING [16]. Phosphorylated STING phosphorylates the transcription factor IRF3 [16], which is one of the most important downstream transcription factors of the cGAS-STING signaling pathway, and it is closely related to inflammation and apoptosis [68]. Phosphorylated IRF3 enters the nucleus and promotes the transcription of IFN-α [16]. STING is also activated by the second messenger cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) [69].
Notably, the cGAS-STING signaling pathway also participates in multiple types of acute and chronic hepatic injury, including radiation-induced liver injury [70], nonalcoholic fatty liver disease, high-fat diet-associated hepatic injury [71], and hepatitis B virus (HBV) infection-associated liver injury [72]. However, STING is primarily expressed in hepatic non-parenchymal cells, such as intrahepatic macrophages, instead of hepatic parenchymal cells, which results in hepatocellular resistance to HBV infection [72]. STING plays a vital role in APAP-induced hepatic injury in which necrotic hepatocellular cells release generous amounts of mtDNA and fragmented nuclear DNA into the intracellular space, and this DNA may amplify the hepatic injury via DAMPs [73]. The cGAS-STING signaling pathway is associated with the innate immune response and DNA recognition. Araujo et al. found that activation of the cGAS-STING signaling pathway played an important role in APAP-induced hepatic injury [74]. The levels of cGAS and STING were upregulated in hepatic parenchymal cells, and the levels of STING were consistently increased in hepatic non-parenchymal cells. Simultaneously, massive mtDNA accumulated in the extracellular space, which was one of the causes of cGAS-STING signaling pathway activation in hepatocytes. The activated cGAS-STING signaling pathway in hepatic parenchymal cells promotes the inflammation, apoptosis, and necrosis of hepatic tissues [74]. Hepatic non-parenchymal cells with an activated cGAS-STING signaling pathway secrete IFN-α, which also aggravates liver damage. Therefore, the inhibition of the cGAS-STING signaling pathway is a potential therapeutic method for APAP-induced hepatic injury. Our study showed that emodin inhibited the expression of cGAS, STING, P-IRF3, and P-TBK1 in liver tissues. These results suggest that the protective effect of emodin on APAP-induced liver damage is associated with the inhibition of the cGAS-STING signaling pathway. Compared to existing studies, our study provides new evidence of the protective effects of emodin in APAP-induced hepatic injury and a basis for the development of new drugs from natural products. The cGAS-STING signaling pathway provides new perspectives and directions for the study of hepatic injury mechanisms and may play a key role in liver injury. Our results further clarify the mechanisms of APAP-induced injury to promote the development of new targeted drugs and prevent serious complications. There are some shortcomings in this study, including the lack of a comparison of different dosing durations and evaluations of possible side effects of emodin. Our experiments also lack further and deeper mechanistic investigation, including the silencing and overexpression of cGAS-STING signaling using in vivo and in vitro studies. We will perform more studies in this field to obtain a higher level of evidence for clinical application.

CONCLUSION
The results of the present study showed that the administration of emodin attenuated APAP-induced liver injury primarily by alleviating hepatic pathological damage, apoptosis, and oxidative stress and inhibiting the inflammatory response. We also found that emodin suppressed the cGAS-STING signaling pathway in APAP-induced inflammatory responses and apoptosis. This study provides further evidence for the application of emodin and RP. Building on prior research, it is reasonable to suggest emodin as a potential candidate for the prevention and treatment of APAP.