3.1. Bioinformatic analysis of AA-interacted genes indicated perturbation of metabolism-related pathways.
A total of 7408 genes interacted with AA were collected from the Comparative Toxicogenomics Database (CTD), among which the top 3 genes were the key metabolizing enzymes of AA including NQO1, CYP1A1, and CYP1A2 (Fig. S1A). The 7408 genes were significantly enriched into endogenous metabolic pathways and exogenous compound metabolic pathways through CTD relevantly functional modules (Fig. S1B), suggesting that AA exposure is closely related to disturbances of various metabolic processes regardless of the target organs.
Next, a total of 1134 genes known to be associated with Chemical-induced liver injury (CILI) in CTD were identified from the above 7408 AA-interacted genes (Fig.S1C). Acquired genes were then analyzed using Qiangen Ingenuity Pathway Analysis (IPA) software, with the selection of “Homo Sapien” and “hepatic” as the setting of organisms and source, respectively. Nuclear receptors-mediated pathways were significantly enriched, including Aryl Hydrocarbon Receptor (AHR) signaling, NRF2-mediated oxidative stress response (NRF2) signaling, Peroxisome proliferators-activated receptor alpha (PPARα) signaling, Liver X receptors (LXR) signaling, Farnesoid X receptor (FXR) signaling, Constitutive androgenic receptor (CAR) signal, et al. (Fig. S1D). A correlation analysis investigating AA and the downstream genes governed by respective nuclear receptors again confirmed the significant perturbance of the nuclear receptor-mediated metabolizing pathways (Fig. S1E).
3.2. 28-day exposure of AA-induced hepatotoxicity and triglycine increase in rats.
A subacute liver injury model was established by oral gavage using SD rats as previously published[27]. As shown in Fig. 1A&B, the body weight of the 28-day treatment of 20 mg/kg AA was significantly decreased comparing to the control group (P < 0.05), while the liver-to-body weight ratio was significantly higher than the control group (P < 0.05). Also as described in our previous study, histological evaluation and serum biochemical measurement confirmed AA-induced liver injury (Fig. 1C&D). In addition to the published index, increases of triglycine in both serum and liver tissues were observed as well, indicating a disorder of lipogenesis in the liver (Fig. 1E).
3.3 LXR-, FXR-, NRF2-, CAR-, and AHR-mediated pathways were enriched in the injured liver of the AA-exposed rat model.
Transcriptomic profiling of the AA-exposed rat model was conducted, and a total of 2186 differently expressed genes (p < 0.05) were analyzed using IPA software. The top 20 pathways ranked by -log (p-value) are shown in Fig. 2A, including metabolism-related pathways mediated by LXR, FXR, NRF2, AHR, and CAR, which is basically consistent with the results of the above bioinformatics analysis. PPARα-mediated pathway did not show up in the top pathway analysis and was therefore, not included in the following investigation. Besides, CAR was widely acknowledged as mediating rodent liver tumor formation rather than hepatotoxicity; more seriously, such a mode of action lacks human relevance[17, 28] and was excluded from further investigation in this study as well.
Four nuclear receptors, LXR, FXR, NRF2, and AHR were to be further studied. As shown in Fig. 2B&C, the mRNA and protein levels of these nuclear receptors were examined: LXR and FXR were significantly decreased in AA-exposed rat liver suggesting inhibition of corresponding pathways, while NRF2 and AHR were significantly increased in this model suggesting activation of corresponding pathways, all of which were consistent with the RNA-sequencing and pathway analysis. In addition, as numerous studies have established that FXR inhibition or LXR activation can lead to hepatic TG accumulation[5, 29, 30], we inferred that the increase of hepatic TG in the AA-exposed rats was due to the inhibition of FXR pathways rather than the activation of the LXR pathways; that’s to say, the FXR mediated AA-induced lipogenesis disorder in AA-exposed rats.
3.4 AAI exposure decreased cell viability, induced cytotoxicity, and activated nuclear translocation of AHR, NRF2, and FXR in HepG2 cells.
In order to investigate the toxicological mechanism, the most potent component of AA, AAI was used in the cell experiments. The human hepatocellular carcinoma cell line HepG2 was subject to a series of concentrations of AAI ranging from 25–150 µM. Cell viability detected by CCK-8 assay after 24 h declined in a concentration-dependent manner, whose decline was significant (P < 0.05) at 50 µM of AAI and above with IC50 at 144.2 µM (Fig. 3A), while the cellular release of LDH, an indicator of injured cell membrane structure, after 24 h was significantly increased (P < 0.05) in a dose-dependent manner (Fig. 3B). Above results showed a cytotoxic effect of AAI on HepG2 cells, and therefore, the concentration of 100 µM AAI was selected for subsequent steps.
It is well-established that nuclear receptors, as a class of ligand-activated transcription factors, function by translocating into the cell nucleus, binding to the promoter of DNA with its DNA-binding domain, so as to regulate the expression of downstream genes[31]. Therefore, observation of their nuclear translocation in response to exogenous stress gives a good indication of their activation. To investigate whether AAI exposure activates AHR, FXR, and NRF2 proteins, an immunofluorescence assay was first performed on HepG2 cells after 24h treatment of 100 µM AAI, and the results showed that all of the three nuclear receptors were in the cytoplasm under untreated condition while translocated into the nucleus upon AAI exposure (Fig. 3C). Such observation demonstrated that the HepG2 cell line is an ideal model to investigate the nuclear receptor-mediated mode of action and that AHR, FXR, and NRF2 were all activated upon AAI exposure. Next, to explore the regulatory effects among these proteins and corresponding pathways, the translocation of these nuclear receptors upon 100 µM of AAI at 1h and 6 h were investigated. As shown in Fig. 3D, AHR, FXR, and NRF2 all enter from the cytoplasm into the nucleus within 1h after exposure, and the translocation of these proteins was more obvious at 6h. The above results showed that the translocation of AHR, NRF2, and FXR, and the consequent regulations of downstream genes, respectively, were all early molecular events rather than one being an effective event that was governed by another nuclear receptor, in the case of AAI exposure.
3.5. AA-induced intracellular TG accumulation by inhibiting FXR
To identify the role of FXR in AA-induced TG accumulation, GW4064, a FXR agonist, and guggulsterone (GUGG), a FXR antagonist, were used separately or together, along with AAI treatment on HepG2 cells. As shown in Fig. 4A, GW4064 significantly decreased intracellular TG level, and GUGG significantly increased TG accumulation while GUGG with GW4064 significantly inhibited such accumulation as compared with GUGG alone. Alike, AAI exposure at concentrations of 50 µM and 100 µM led to significant intracellular TG accumulation as compared with control, while such accumulation was significantly inhibited in AAI treatment with GW4064 groups when compared with AAI alone groups at respective concentrations. The above results showed the inhibition of FXR mediated AA-induced TG accumulation.
Next, FXR downstream effectors were investigated for their role in the toxicological processes. It is known that FXR can regulate the expression of SHP[32, 33], which has an inhibitory role on lipogenesis pathways involving SREBP1[34, 35], FASN[35], SCD1[36], etc. In our study, similar to GUGG, AAI at 25, 50, and 100 µM significantly down-regulated the mRNA expression of FXR, and that of SHP at 50 and 100 µM of AAI as compared with control (Fig. 4B). In addition, such inhibition on the expression of FXR and SHP was all significantly relieved in AAI treatment with GW4064 groups when compared with AAI alone at respective concentrations. SREBP1 encodes for sterol regulatory element-binding protein-1c, a major regulator of fatty acids synthesis, FASN encodes a key lipogenic enzyme fatty acid synthase, and SCD1 decodes a key enzyme converting saturated fatty acids to monounsaturated fatty acids during lipogenesis. In our study, similar to GUGG, AAI at any treatment concentration significantly up-regulated the mRNA expression of SREBP1, FASN, and SCD1 as compared with control (Fig. 4B), whereas such up-regulation was all significantly blocked in AAI treatment with GW4064 groups (except for 25 µM of AAI with GW4064) when compared with AAI alone at respective concentrations. Together, all the results in Fig. 4B suggested that AA-induced lipogenesis disorder was induced by the inhibition of FXR, which inhibited the expression of SHP and consequently led to the over-expression of lipogenesis-related genes.
3.6. AA-induced hepatotoxicity by activating AHR but not NRF2
To investigate the mechanism underlying AA-induced hepatotoxicity, benzo(a)pyrene (BaP), an AHR agonist, and α-naphthoflavone (ANF), an AHR antagonist, and likewise, NK252, a NRF2 agonist, and ML385, a NRF2 antagonist, were used separately or in combination, along with AAI treatment on HepG2 cells. As shown in Fig. 5A, the treatment of BaP induced significant ALT elevation in HepG2 cells, so as the treatment of 100 µM AAI. Such elevation was significantly inhibited by 100 µM AAI combined with ANF which abolished AHR activation. Similarly, such elevation was inhibited by 100 µM AAI combined with ML385 which abolished NRF2 activation, although the inhibition of ML385 was not statistically significant. The above results suggested that the activation of AHR and NRF2 may mediate ALT elevation in AAI -exposed HepG2 cells. Similarly, Fig. 5B also suggested that the activation of AHR and NRF2 may both mediate ROS generation that was induced by any concentration of AAI treatment to HepG2 cells.
Next, downstream effectors belonging to AHR and NRF2 pathways, respectively, were investigated to further reveal the mechanism. AHR nuclear translocation activated the transcription of a battery of xenobiotics metabolizing enzymes, including the cytochrome P450 subfamilies CYP1[37], the phase II metabolizing enzymes NQO1, as well as pro-apoptotic proteins, including Bcl-2 member BAX[38, 39] and TNF-receptor member FAS[40], etc. As shown in Fig. 5C, both AAI treatment (at any concentration) and AHR agonist, BaP, could significantly up-regulate the mRNA expression of AHR-responsive genes, while a combination of AAI with AHR antagonist, ANF, could significantly inhibit such up-regulation. It is worth noticing that both BaP and AAI significantly decreased the mRNA expression of AHR itself. In sustained AHR activity, after transcriptional activation of target genes, the activated AHR undergoes rapid receptor degradation due to proteolytic degradation by the 26S proteasome[41], which phenomenon further demonstrated that AAI-induced AHR activation and induced expression of responsive genes involved in chemical bioactivation and cell apoptosis, and consequently led to hepatocellular injury.
The downstream effectors of NRF2 pathways also include metabolizing enzymes NQO1 and GST, in addition to antioxidant proteins-encoding SOD and CAT. As shown in Fig. 5D, 24h treatment of 100 µM AAI did not up-regulate NRF2 downstream target genes as NRF2 agonist NK252 did (except for NQO1); instead, the mRNA expression of GST, CAT, and SOD was significantly down-regulated upon AAI exposure, which was further inhibited by the addition of ML385 that was supposed to abolish NRF2 activation. These results in Fig. 5D suggested that AA did not induce hepatotoxicity by the function of NRF2 and its downstream effectors; rather, the activation of NRF2 and perturbation of downstream genes were possibly a result of ROS generation and crosstalk with other pathways, rather than a causal mechanism of hepatocellular injury.