Luteolin alleviates inorganic mercury-induced liver injury in quails by resisting oxidative stress and promoting mercury ion excretion

Background Inorganic mercury is a well-known toxic substance that can cause oxidative stress and liver damage. Luteolin (Lut) is a kind of natural antioxidant, which is widely found in plants. Therefore, we focused on exploring the alleviative effect of Lut on liver injury induced by mercuric chloride (HgCl2), and the potential molecular mechanism of eliminating mercury ions in quails. Methods and results Twenty-one-day-old male quails were randomly split into four groups: control group, Lut group, HgCl2 group, and HgCl2 + Lut group. The test period was 12 weeks. The results showed that Lut could significantly ameliorate oxidative stress, the release of inflammatory factors, and liver damage caused by HgCl2, and reduce the accumulation of Hg2+ in quail liver. Furthermore, Lut evidently increased the levels of protein kinase C α (PKCα), nuclear factor-erythroid-2-related factor 2 (Nrf2), and its downstream proteins, and inhibited nuclear factor-kappaB (NF-κB) production in the liver of quails treated by HgCl2. Conclusions To sum up, our results suggest that Lut not only reduces the levels of oxidative stress and inflammation, but also promotes the excretion of Hg2+ by promoting the PKCα/Nrf2 signaling pathway to alleviate HgCl2-induced liver injury in quails.


Introduction
Mercury (Hg) is a heavy metal, which widely exists in rocks, soil, water, and air [1]. Due to its unique properties, mercury has recently been utilized extensively in non-ferrous metals, electronic devices, cement, insecticides, chemical fertilizers, and other sectors, resulting in a significant amount of inorganic mercury being discharged into the environment [2]. However, with the increasing understanding of the toxicity of mercury, mercury has been considered one of the most dangerous elements [3]. In addition, due to the severe toxicity of inorganic mercury, it has adverse effects on the immune system, digestive system, nervous system, and reproductive system [4]. Studies have shown that mercury induces diseases in livestock and poultry and accumulates in their bodies [5]. Therefore, animal-derived foods with excessive mercury content pose a great threat to human health [5]. The liver is not only an important metabolic organ of organisms, but also the main target organ of toxic chemicals. Chronic poisoning tests have shown that inorganic mercury can cause particularly severe liver damage, which is manifested in liver morphological changes, liver cell apoptosis, and liver function injury [6]. The acute mercury poisoning test has suggested that inorganic mercury can cause acute liver injury by inducing oxidative stress, lipid peroxidation, and other mechanisms [7]. Notably, oxidative stress is one of the important ways that inorganic mercury causes damage to animal tissues and organs [8,9]. Therefore, the use of natural antioxidants may treat the liver injury in quails by inhibiting oxidative stress.
Luteolin (Lut) is a kind of natural flavonoid with health care function and widely exists in traditional Chinese medicinal plants [10]. Furthermore, the results of some experiments indicate that Lut has a wide range of pharmacological effects, such as anti-tumor, anti-oxidation, anti-fibrosis, antiviral, and so on [11]. It is worth noting that Lut protects tissues and organs by improving antioxidant capacity and promoting toxin excretion. However, whether Lut has a therapeutic effect on liver injury induced by mercuric chloride (HgCl 2 ) in quail remains unclear.
Flavonoids can regulate oxidative stress by activating the Protein Kinase C (PKC) pathway [12]. PKCα is a typical subtype of PKC that plays a key role with downstream nuclear factor-erythroid-2-related factor 2 (Nrf2) during antioxidant stress [13]. Notably, Nrf2 can alleviate body poisoning by up-regulating a variety of antioxidant enzymes and detoxifying enzymes [14]. Nrf2 initiates the transcription of antioxidant enzymes and adenosine triphosphate (ATP)-binding cassette (ABC) transporters [15]. The ATPbinding cassette (ABC) transporters superfamily is one of the largest transporters families present in organisms and regulated by Nrf2, consisting of seven subfamilies (from A to G), which can effectively remove endogenous substances and exogenous compounds in cells and play a key role in the field of toxicology [16]. Among them, ATP-binding cassette subfamily G member 2 (ABCG2) is widely expressed in various normal tissues, and relevant experimental studies have shown that it is involved in the excretion process of Hg 2+ in vivo [17]. Thus, we discussed the related role of Lut in HgCl 2 -induced liver injury in quails and the molecular mechanism of activation of the PKCα/Nrf2 signal pathway.

Reagents and antibodies
Mercuric chloride used in the test was produced in Beijing Chemical Plant (Beijing, China). Lut was purchased from Xi'an Four Seasons Biotech Corporation (Purity = 98%, Shanxi, China). Superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and hemoglobin (Hb) were indicators of clinically applied oxidative stress, and the related detection kits were supplied by Nanjing Jiancheng Institute of Biotechnology (Jiangsu, China). Trizol was obtained from Ambion (Foster City, CA, USA). SYBR Green RT-qPCR SuperMix and cDNA synthesis kits were from Vazyme Biotech Co., Ltd (Nanjing, China), and 2 × PCR Taq Plus Master Mix with dye was provided by Applied Biological Materials (Vancouver, Canada). Nuclear extraction kits were bought from Beyotime Biotechnology (Shanghai, China). DNA marker was supplied by Tian gen Biotech Co., Ltd (Beijing, China). All the kits for protein extraction came from the Beyotime Institute of Biotechnology (Jiangsu, China). Primary antibodies to Nrf2, nuclear factor-kappa B (NF-κB), and tumor necrosis factor-α (TNF-α) were from Bioss (Beijing, China). The antibody of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was supplied by Goodhere Biotechnology Company (Hangzhou, China). Secondary antibodies were purchased from ZSGB-BIO (Beijing, China).

Animals and treatments
The animal experiment protocol was performed under the approval of the Ethical Committee for Animal Experiments (Northeast Agricultural University, Grant Number: 20210317). All experiments were carried out according to the requirements of the Animal Experimental Ethics Committee of Northeast Agricultural University. We acquired 40 healthy male quails (21 days old, weighing 90-105 g) from Wanjia poultry farm (Heilongjiang, China), and randomly split them into 4 groups (n = 10 per group): control group, Lut group, Hg group, and Hg + Lut group. Before starting the experiment, all quails were fed in standard animal feeding laboratory cages (24-27 °C, humidity 50-60%, free access to water and food) with 12 h light/dark cycle for 7 days. The control group was given normal saline (NS) by intragastric administration and fed with normal feed. The Lut group was given NS and mixed Lut into the feed (800 mg/kg, proportion of Lut and feed) [18,19]. The Hg group was given HgCl 2 solution (6 mg/kg body weight, dissolved in NS) and normal feed [20]. The Hg + Lut group was given HgCl 2 solution and mixed Lut into the feed (800 mg/ kg, proportion of Lut and feed). The experimental period was 12 weeks. The general activity, body weight, food and water intake of quails were recorded. After the last administration, quails were fasted and given water for 24 h. Fasting is to exclude the effect of food on the animal's liver function indicators. It was also to avoid food backflow after anesthesia, which would affect the experimental operation and test results, and to avoid unnecessary suffering of animals during the experiment. In addition, to exclude the effect of fasting on our study, we set up the control group to observe and compared with other experimental groups under the same experimental conditions. All the quails were anesthetized with ether and sacrificed after blood was collected from the inferior wing vein for subsequent blood index detection and biochemical analysis. The liver was immediately removed and frozen at − 80 °C for subsequent use.

Hepatic histopathological analysis
The liver tissues of quails were fixed in 4% formaldehyde for 24 h, then dehydrated with alcohol, and embedded in paraffin, finally tissues were carefully sectioned into 3 μm sections. The sections were first unfolded flat in hot water and dried overnight in a thermostat. The dried tissue sections were dewaxed in xylene and then dehydrated in alcohol. The sections from the previous step were stained with hematoxylin and eosin (HE) [21]. Under the optical microscope, observed the pathological changes in the section (BX-FM, Olympus Corp, Tokyo, Japan).

Blood testing
The number of red blood cells (RBC) and white blood cells (WBC) in the blood was measured by the blood cell counter, and hemoglobin (Hb) levels were measured by the kit [22].

Biochemical analysis
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) belong to clinically applied liver function indexes, and they were measured by a Beckman DXC 800 biochemical analyzer (Beckman-Coulter, Brea, CA, USA) from the Laboratory department of Heilongjiang Electric Power Hospital [23].

Measurement of biomarkers oxidative stress
Liver tissues (0.1 g) and 0.9 mL phosphate-buffered saline (PBS) were added to the homogenizer (S10, Scientz Corporation, Ningbo, China) for homogenization. Tissue homogenates were centrifuged (2500 rpm, 10 min, 4 °C). Collecting the supernatant to test SOD activity, MDA level, and GSH concentration according to the instructions of corresponding commercial kits [24].

Hg content
The Hg content in the liver was detected by atomic fluorescence absorption spectrometry. After the Hg standard working curve was prepared, the liver tissues were accurately weighed at 0.5 g. After homogenization, a small amount of HNO 3 -H 2 SO 4 solution was added and heated on low heat without drying the pot until the residue was free of charcoal particles. After cooling, add hydrochloric acid to make up to 10 mL, well mixed, and finally dilute to 50 mL with deionized water. The value of the prepared solution was measured by atomic fluorescence spectrometry (AFS-930, Jitian Instrument Co., Ltd., Beijing, China), and the Hg content was calculated according to the standard curve.

Quantitative real-time PCR analysis
In this experiment, we extracted total RNA from liver tissue samples of quails using Trizol reagent. Then, we used a High Capacity cDNA Reverse Transcription kit to synthesize cDNA. Reverse transcription was performed at 37 °C for 20 min, 98 °C for 5 min, and finally stored at 4 °C. The quantitative real-time PCR (qRT-PCR) was conducted after adding SYBR Green RT-qPCR SuperMix and all primers. As listed in Online Resource 1, all primer sequences were designed by Sangon Biotech (Shanghai, China). For the 20 μL system used, add 2 μL template DNA, 0.4 μL upstream primer, 0.4 μL downstream primer, and 10 μL 2 × SYBR Green Mix respectively, and finally make up to 20 μL with Nuclease-free-H 2 O and mix thoroughly. Subsequently, all mRNA levels were detected with a Bio-Rad CFX96 Touch Quantitative PCR Instrument (Hercules, CA, USA), and the final data were gained by the standard method (2 −ΔΔCT ) [25].

Western blot analysis
The total protein was extracted from the liver of quails with the assistance of the related protein extraction kit. On the basis of the manufacturer's direction, the concentration of protein was determined by bicinchoninic acid (BCA) kit. Equal amounts of the diluted protein samples were aspirated and placed on a polyacrylamide gel, afterwards, all the target proteins were converted onto polyvinylidene fluoride (PVDF) membranes.

Statistical analysis
The test data were sorted out in Excel and processed by SPSS 20.0 statistical software (SPSS, Chicago, IL, USA). The obtained data are expressed as mean ± standard error of the mean (SEM). One-way ANOVA was used in the comparison between the experimental groups following Dunnett's post-hoc test. P < 0.05 was deemed to be statistically significant.

Effects of Lut on the clinical symptom
Except for the Hg group, the mental, physical, and behavioral conditions of quails were normal in the control group, Lut group, and Hg + Lut group. In contrast, the quails in the Hg group were characterized by different degrees of disheveled and lusterless feathers, drooping of both wings, slow response to external stimuli, lethargy, loss of appetite, and reduced locomotion, so we concluded that quails in the Hg group were mentally depressed. Nevertheless, the administration of Lut increased the appetite, and improved the mental state and motor function of quails in the Hg + Lut group.

Effects of Lut on the change of liver organ index
Compared with the data results obtained by the control group, the liver index of the Hg group increased significantly. Nonetheless, the liver index of the Hg + Lut group was distinctly lower than that of the Hg group. There was no marked difference in liver index between the Hg + Lut group and the control group (Fig. 1A).

Effects of Lut on hematological change in liver induced by HgCl 2
As indicated by the content displayed in Fig. 1B and C application of Lut alone did not increase RBC and Hb levels, but remarkably restored the decrease in RBC and Hb levels caused by HgCl 2 .
The amount of WBC was increased after Hg treatment, but Lut lightened this change (Fig. 1D).

Effects of Lut on histopathological change of liver
The presence of inflammatory cells can be observed by HE staining, because inflammatory cells have their characteristics, usually with large nuclei and large nucleoplasmic ratios. After HE staining, the nuclei were stained with hematoxylin, a distinct blue color, and the cytoplasm was stained with eosin, showing varying shades of pink. Thus, in HE staining at 200 × magnification, a predominantly blue-dotted inflammatory cell (mainly neutrophils, lymphocytes, and monocytes) can be seen, while other cells are larger, with more cytoplasm pink and smaller, blue nuclei. Therefore, we evaluated the intensity of the inflammatory response mainly by the size of the inflammatory cell infiltration area in HE staining results. As shown in Fig. 2A, the liver tissue structure of the control group and Lut group was normal, and the cell structure was clear and complete and arranged neatly. In the Hg group, the liver structure was disordered, with damage to the cell structure, a large number of fat vacuoles, and inflammatory cell aggregation. The sinusoids were hemorrhagic. Compared with the Hg group, hepatic cell damage, the number of inflammatory cells, and the sinusoids hemorrhage were significantly reduced in the Hg + Lut group.

Effects of Lut on total mercury concentration in liver tissue
Under the condition of comparison with the control group, the content of Hg in the liver of Hg group increased strikingly, while the content of Hg in liver of Hg + Lut group decreased evidently compared with that of Hg group (Fig. 2B).

Effects of Lut on liver biochemical indexes induced by HgCl 2
The liver dysfunction was clinically evaluated with AST and ALT activities. Compared with the control group, AST (Fig. 2C) and ALT (Fig. 2D) activities were strikingly ascended in the Hg group. Whereas, the pharmacological action of Lut dramatically reversed the effects of HgCl 2 on serum AST and ALT activities.

Effects of Lut on oxidative stress in liver tissue
Compared with the control group, the SOD activity (Fig. 3A) and GSH (Fig. 3B) concentration were significantly decreased, and the MDA level (Fig. 3C) was significantly increased in the Hg group. Meanwhile, the Hg + Lut group significantly attenuated these changes in the Hg group.

Effects of Lut on liver inflammation induced by HgCl 2
The results of qRT-PCR exhibited that compared with the control group, the mRNA levels of NF-κB, IL-1β, and IL-6 in the Hg group increased distinctly, but Lut treatment restored these mRNA levels (Fig. 4A). Western blot results Fig. 3 Effects of Lut on liver oxidative stress in quails. The SOD activity (A), GSH concentration (B), and MDA level (C) were determined. Values are mean ± SEM (n = 10). * indicates observable difference when compared with the control group (p < 0.05). # indicates observable difference when compared with the Hg group (p < 0.05) Fig. 4 Effects of Lut on liver inflammation and PKCα/Nrf2 pathway proteins in liver injury induced by HgCl 2 in quails. A The mRNA expression levels of NF-κB, IL-6, and IL-1β (n = 7). B The protein levels of NF-κB and TNF-α (n = 4). C The mRNA expression levels of PKCα, Nrf2, HO-1, NQO1, and ABCG2 (n = 7). (D) The protein expression level of Nrf2 (n = 4). Values are mean ± SEM. * indicates observable difference when compared with the control group (p < 0.05). # indicates observable difference when compared with the Hg group (p < 0.05) manifested that the changes in NF-κB and TNF-α protein levels were similar to those of mRNA mentioned above (Fig. 4B).

Effects of Lut on the PKCα/Nrf2 pathway in liver induced by HgCl 2
Lut treatment significantly upregulated PKCα, Nrf2, HO-1, NQO1, and ABCG2 gene expressions compared with HgCl 2 treatment (Fig. 4C). Furthermore, the protein level of Nrf2 was observably reduced in the Hg group as detected by western blot, but the Hg group restored Nrf2 protein levels under the pharmacological action of Lut (Fig. 4D).

Discussion
With the acceleration of industrialization, mercury exposure from both the natural environment and human activities are increasing significantly [2]. Human exposure to various forms of mercury can cause many pathological changes in organ systems, such as inflammation of the organ systems [27]. Prolonged exposure to mercury, even in small amounts, can cause hepatotoxicity [5]. Lut is a natural flavonoid compound with a wide source and no overt toxic or side effects. It has many characteristics such as anti-inflammation, antioxidation, and regulation of metabolism [28]. In this study, we found that Lut can significantly alleviate the liver damage caused by HgCl 2 by enhancing the ability of antioxidation and Hg 2+ efflux in quails.
The activities of ALT and AST are commonly used in the clinical diagnosis of liver pathological changes [29]. Our results showed that Lut could reduce the increase in serum AST and ALT activities caused by HgCl 2 and relieve liver injury. In addition, the results of HE staining also provided strong evidence for the protective effect of Lut on the liver. All in all, these results confirm the alleviative effect of Lut on HgCl 2 -induced liver injury.
Studies have demonstrated deposits of inorganic mercury cause oxidative stress and tissue damage in organisms [7]. Moreover, the appearance of reactive oxygen species (ROS) initiates a series of pathological events, including inflammation, fibrosis, genotoxicity, and lipid peroxidation [30]. MDA, SOD, and GSH are all considered biomarkers of oxidative stress in organisms [31,32]. Our research results indicated that HgCl 2 treatment decreased the GSH concentration and SOD activity as well as increased the MDA level in the liver, and these effects were diminished after Lut treatment. Subsequently, we deduce that Lut mitigates liver injury by way of reducing levels of oxidative stress induced by HgCl 2 .
NF-κB is a key transcription factor of inflammation [33]. NF-κB is localized to the cytoplasm in the resting state, while during oxidative stress, the activated NF-κB undergoes nuclear translocation [34], thereby upregulating inflammatory cytokines such as TNF-α, interleukin-6 (IL-6), and interleukin1β (IL-1β), thus resulting in an inflammatory reaction [33,35]. Our hematological examination showed that HgCl 2 caused a significant increase in WBC levels in the blood, suggesting that HgCl 2 induced inflammatory reactions in the body. Results from other tests showed that the mRNA levels of NF-κB and related inflammatory factors as well as the protein levels of NF-κB and TNF-α in the liver of quails in the Hg group were significantly increased, while the liver inflammation of poisoned quails treated with Lut was mitigated. Therefore, the results of this study reveal that Lut attenuates HgCl 2 -induced hepatic inflammation in the quail.
As a transcription factor against oxidative stress, Nrf2 is now considered to alleviate many aspects of stress including xenobiotics, inflammation, etc. [36]. Under oxidative stress or exogenous attack, Nrf2 in the cytoplasm is rapidly released from the combination with Kelch-like ECH-associated protein 1 (Keap1) and transferred to the nucleus [37]. In the nucleus, activated Nrf2 interacts with other transcription factors and auxiliary factors to adjust the transcription of its target genes, and the target genes encode protein associated with antioxidants and drug detoxification [38,39]. Heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1) are the downstream antioxidant proteins of Nrf2, which play a pivotal role in preventing oxidative stressinduced cell damage [23,40]. The ABCG2 gene downstream of Nrf2 encodes breast cancer resistance protein (BCRP), a significant transporter in the body, which is a relevant tool for removing potentially harmful exogenous substances from cells and thereby reducing their toxicity [16,41]. The BCRP is positioned in the cell membrane and highly expresses in the hepatic tissue of the animal body [42]. Furthermore, we also found that the use of Lut reduced the accumulation of mercury in the liver. In our study, it was verified from the perspective of mRNA and protein that Lut up-regulated the expression of ABCG2 through the Nrf2 signaling pathway. Taken together, Lut may intensify antioxidant defense and increase Hg 2+ excretion by promoting Nrf2, thereby alleviating HgCl 2 -induced liver injury.
PKC, a superfamily of serine-threonine kinase, which is widely expressed in tissues [15,43], plays a vital role in a variety of illnesses [44], and it is also a key link in a range of cellular signaling pathways [45]. The activation of PKC mediates the Nrf2 response to oxidative stress [46]. Stimulation of the PKC/Nrf2 pathway has been reported to be critical to the maintenance of heart resistance to injury by significantly increasing the antioxidant defense system [47]. PKCα is an important member of PKC family, which is of great significance in the process of antioxidant stress. It contains 10 serine/threonine kinases encoded by 9 different genes [48]. Antioxidants further induce PKCα phosphorylation and Nrf2 nuclear translocation by opening the PKCα/Nrf2 signaling pathway, and thus participate in the antioxidant reactions [13,49]. In the present study, Lut can alleviate the decrease of PKCα and Nrf2 levels induced by HgCl 2 . Therefore, our results indicate that stimulation of PKCα/Nrf2 signaling pathway participates in the alleviative effect of Lut on quail liver injury induced by HgCl 2 . Thus, the study provides convincing evidence that dietary Lut protects HgCl 2 -induced hepatic damage by stimulating the PKCα/Nrf2 signaling pathway (Fig. 5).

Conclusion
To sum up, the experimental results of this study demonstrate that Lut protects against oxidative stress and promotes mercury excretion by stimulating the PKCα/ Nrf2 pathway to attenuate HgCl 2 -induced hepatic damage. Consequently, Lut may be used as a feed additive to prevent mercury accumulation and poisoning in livestock and poultry in the future.  5 The mechanism of luteolin on the mitigation of liver injury induced by Hg 2+ via the PKCα/Nrf2 signaling pathway