Gomphrena globosa L. extract alleviates carbon tetrachoride-induced liver injury in mice by activating antioxidant signaling pathways and promoting autophagy

Carbon tetrachloride (CCl4) is highly toxic to animal liver and is a major contributor to liver injury. Gomphrena globosa L. (GgL) is an edible plant with anti-inflammation and antioxidation properties. The aim of this study was to investigate the potential therapeutic effects of GgL on liver injury. A model of chronic liver injury in mice was established by intraperitoneal injection of CCl4 (0.4 mL/kg) for 3 weeks, and the mice were treated intraperitoneally with different concentrations of GgL crude extract (GgCE; 100, 200, 300 mg/kg) or Bifendatatum (Bif; 20 mg/kg) in the last 2 weeks. The results showed that GgCE treatment alleviated the liver injury, improved the pathological changes caused by CCl4 on the mice liver, and enhance the antioxidant capacity. We also found that GgCE increased the expression of antioxidant stress related proteins, decreased the phosphorylation levels of autophagy related proteins PI3K and mTOR, and decreased the expression of LC3 II and P62 proteins. These results suggest that GgCE alleviated CCl4-induced chronic liver injury in mice by activating antioxidant signaling pathways and promoting autophagy, indicating a potential therapeutic effect of GgCE on liver injury.


Introduction
Liver injury has become a common health hazard in clinical practice. Chronic liver injury occurs as a result of abnormal liver function caused by long-term repeated damage to hepatocytes, and chronic liver dysfunction can lead to liver fibrosis or cirrhosis [1]. CCl 4 is a colorless, toxic liquid organic compound. At high concentrations, CCl 4 can Yunfei Wei, Wenxi Tan, and Haiyan Qin have equally contributed to this work. cause severe damage to the liver, leading to lipid peroxidation and inflammatory responses in liver tissue, resulting in hepatocyte damage and necrosis, and in severe cases, even liver fibrosis and cirrhosis. CCl 4 is widely used to establish models of chronic liver injury in animals [2]. CCl 4 stimulation induces liver parenchyma damage, causing aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities are increased [3]. However, no effective drugs have been identified for the treatment of CCl 4 -induced chronic liver injury.
Many liver diseases are associated with the occurrence and development of reactive oxygen species (ROS), and excess ROS has been found to be a direct cause of oxidative stress [4]. Formation of ROS is one of the manifestations of CCl 4 -induced liver injury in mice [5], and importantly, excessive accumulation of ROS can promote lipid peroxidation and produce lipid peroxidation products, such as malondialdehyde [6]. Glutathion (GSH), glutathion peroxidase (GSH-Px), malondialdehyde (MDA) and superoxide dismutase (SOD) are common indicators used to evaluate resistance to oxidative stress [7].
Nrf2, a key factor in the cellular response to oxidative stress, is regulated by Keap1, a binding protein for Nrf2 in the cytoplasm, bound to actin [8]. There is evidence that modification of cysteine residues of Keap1 allows Nrf2 to accumulate in the nucleus and express Nrf2 target genes [9]. Nrf2 in hepatocytes plays an important role in blocking CCl 4 -induced liver injury by regulating oxidative and inflammatory responses [10]. Conversely, Nrf2 silencing can aggravate the process of CCl 4 -induced liver injury in mice [11]. Indeed, chemically induced Nrf2 expression has been shown to protect the liver from various hepatotoxic agents [12]. Similarly, Nrf2-activated downstream antioxidant proteins have antioxidant effects. Glutamate cysteine ligase (GCL) consists of a catalytic subunit (GCLC) and a modifing subunit (GCLM) [13]. As a downstream molecule of Nrf2, it catalyzes the synthesis of glutathione (GSH), which is an important antioxidant and cofactor for glutathione S-transferase binding [14]. HO-1 and NQO1 are also downstream molecules of Nrf2 signaling pathway [15]. Under normal conditions, Nrf2 is inhibited. However, when free radicals attack the body, Nrf2 enters the nucleus and activates HO-1 and NQO1 to scavenge free radicals and increase the antioxidant capacity of the body to combat liver injury [16]. Therefore, activation of Nrf2 and its downstream target proteins is an important research direction for the treatment of CCl 4 -induced liver injury.
Autophagy is an important phenomenon in the removal of metabolic waste by the body [17]. And PI3K/AKT signaling pathway, which can regulate downstream mTOR, inhibits autophagy by activating mTOR is an important upstream signaling pathway of autophagy [18]. Numerous studies have shown that autophagy can be inhibited when liver injury occurs. In fact, enhanced autophagy can prevent the progression of a variety of liver diseases, including liver injury [19]. When autophagy is enhanced, LC3 I can be converted to LC3 II and translocated to the autophagosome membrane, facilitating the formation of double-membrane autophagosome [20]. P62 interacts with LC3 and acts as a cargo adapter to transport damaged mitochondria to the autophagosome [21]. It is generally accepted that accumulation of P62 indicates impaired autophagy, while reduction of P62 indicates enhanced autophagy [22]. In conclusion, dysregulation of autophagy is associated with the occurrence of liver injury, and regulation of autophagy homeostasis is considered as a potential therapeutic strategy for liver injury.
Chinese herbal medicine has a long history of application in China, due to their abundant resources and low toxic side effects. A large number of studies have proved that Chinese herbals and their active ingredients can significantly improve liver function, reduce inflammatory response and apoptosis, thus alleviating liver injury caused by various factors, and have a promising application [23]. Gomphrena globosa L. (GgL) is an edible plant from the amaranthaceae families [24]. In recent years, many studies have found that GgL contains polysaccharides, volatile oil, flavonoids and their glycosides, saponins and other active ingredients [25]. Its monomer has been studied for anti-inflammation and anti-oxidation purposes. However, it has not been reported whether GgL crude extract (GgCE) can protect liver injury by anti-oxidation and promoting autophagy. Our experiments were conducted to investigate the protective effect of GgCE against CCl 4 -induced chronic liver injury in mice. It provides a theoretical basis for the treatment of liver injury with comestible plant.

Plant material and extracts preparation
The GgL was purchased from a medicinal plants distributor (Morais e Costa & C.ª Lda, Portugal). The crude extract of GgL (GgCE) was obtained by boiling 10 g GgL with 500 mL water for 5 min, extracting it by filtration through a Buchner funnel, and lyophilizing the filtrate. The active ingredients can be found in Silva's research [26].

Experimental animals
Male C57BL/6 (20 ± 2g ) mice were offered by the Liaoning Changsheng Biotechnology (produc-tion license: SCXK2010-0001; Liaoning, China). All mice studies conformed to the U.S. National Institutes of Health (NIH), the Guide for the Care and Use of Laboratory Animals, and were authorized by the related ethnical regulations of Jilin University (Number of permit: SY202110003). Animals were housed at a room temperature of 24 ± 1 ℃ (Relative humidity: 40-80%) with a 12 h light/dark cycle and had free access to food and water. Mice were randomly divided into 7 groups with 5 mice in each group, including control group, CCl 4 (0.4 mL/kg) group, GgCE (300 mg/kg) group, CCl 4 + GgCE (100 mg/kg) group, CCl 4 + GgCE (200 mg/kg) group, CCl 4 + GgCE (300 mg/kg) group, and CCl 4 + Bifendatatum (Bif; 20 mg/kg) group. CCl 4 was dissolved in olive oil, and Bif was dissolved in saline. Mice were injected intraperitoneally with CCl 4 twice a week for 3 weeks to establish a CCl 4 -induced chronic liver injury model in mice. Starting from the second week, mice were intragastric with different concentrations of GgCE for the last 2 weeks. After the last intragastric administration, mice fasted without water, and blood was collected from the eye pit 12 h later. Then the liver of mice was taken for index determination or stored at −80 ℃.

Measurement of AST, ALT, ROS and SOD in mice serum
The mice blood was allowed to stand at room temperature for 8 h and then centrifuged at 3000 rpm/min for 10 min. Serum was taken, and serum AST and ALT levels were determined. The specific experimental steps were performed according to the instructions of the commercial kit (Nanjing Jiancheng Bioengineeering Institute, Jiangsu, China).

Measurement of MPO, TP, MDA, GSH, and GSH-Px in mice liver
Mice liver tissues were collected, homogenized according to the kit instructions and hepatic ROS (FEIYA BIOTECH-NOLOGY, Jiangsu, China) levels, GSH, GSH-Px (Solarbio, Beijing, China) activity, MDA content, myeloperoxidase (MPO), and SOD (Nanjing Jiancheng Bioengineeering Institute, Jiangsu, China) activity were measured. The specific test steps were performed according to the commercial kit instructions.

Histopathological studies
Mice livers were preserved in 10% formalin solution and embedded in paraffin. They were cut into 5 μm sections and stained with hematoxylin and eosin (H&E). The pathological changes of liver were observed under a light microscope.

Protein expressions studies by Western blot
Fresh liver tissue was cleaved with RIPA lysate, phosphatase inhibitor and PMSF at 4 °C, supernatant was collected by centrifugation, and the protein weight was unified to 20 ug by adding PBS and loading buff. It was injected into 12% SDS polyacrylamide gel, electrophoresis and then transferred to a PVDF membrane, sealed with 5% skim milk made with TBST, and then Nrf2, β-actin, LC3 I/II (from Proteintech (Boston, MA, USA)), Keap1, GCLC, GCLM, P62 (from Abcam (Cambridge, MA, USA)), HO-1, NQO1, p-PI3K, PI3K, p-mTOR and mTOR (from Cell Signal Technology (Beverly, MA, USA)) primary antibody incubated at 4 °C overnight. After 4 washes with TBST for 5 min each, the membrane was incubated with HRP (Boster, CA, USA) coupled secondary antibody (1: 5000) at room temperature for 1 h, and washed 4 times with TBST for 5 min each. Finally, the membranes were visualized with Omni-ECL Pico Light Chemiluminescence Kit (YAMEI, Shanghai, China), and the images were obtained by using the Tanon 4200 Luminous Imaging System (YPHBIO, Beijing, China). Image J software was used for Western blot bands quantitative analysis.

Statistical analysis
All experiments were repeated more than three times. All data are presented as mean ± SD. Data were analyzed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Differences between groups were analyzed by Ordinary one-way ANOVA test. P > 0.05 indicated no significant difference, 0.01 < P < 0.05 indicated significant difference, P < 0.01 was considered to be a very significant difference. n.s. indicates a nonsignificant difference.

GgCE alleviated CCl 4 -induced liver injury in mice liver
To test the protective effect of GgCE on CCl 4 -induced liver injury, AST, ALT, MPO and TP kits were used for detection. The results showed that CCl 4 significantly increased serum AST and ALT levels and hepatic MPO activity compared with control group (Fig. 1). Different concentrations of GgCE (100, 200, 300 mg/kg) could reduce serum AST, ALT levels and hepatic MPO activity in CCl 4 -induced chronic liver injury, with extremely significant differences compared to CCl 4 group. GgCE reduced serum AST, ALT levels and hepatic MPO activity, and there was no significant difference between the treatment effect of GgCE (300 mg/kg) and Bif. CCl 4 could observably reduce hepatic TP content ( Fig. 1. D), GgCE treatment group could markedly improve hepatic TP content reduction compared with CCl 4 group. These results demonstrated that GgCE alleviated CCl 4 -induced liver injury in mice.

GgCE improved CCl 4 -induced oxidative damage in mice liver
To further explore the effects of GgCE on CCl 4 -induced liver oxidative damage, we detected the changes of serum ROS levels, hepatic MDA content, GSH and GSH-Px activity and serum SOD activity. As shown in Fig. 2, CCl 4 notably increased serum ROS levels and hepatic MDA content compared with the control group. Different concentrations of GgCE treatment group could reduce the cumulative serum ROS levels and alleviate the increased hepatic MDA content caused by CCl 4 . Compared with control group, CCl 4 could also markedly reduce hepatic GSH, GSH-Px activity and serum SOD activity, and signally increased that after treatment with different concentrations of GgCE. In addition, we also compared the treatment effect of GgCE (300 mg/kg) with that of Bif. We found that they were not significantly different in biochemical indexes serum ROS, hepatic MDA, GSH and GSH-px, but the treatment effect of GgCE (300 mg/kg) was better than that of Bif in serum SOD. These results suggested that GgCE improved CCl 4 -induced oxidative damage in mice.

GgCE improved CCl 4 -induced pathological changes in mice liver
For purpose of observe the effect of GgCE on CCl 4 -induced liver injury in mice in more detail, we made tissue sections of the large lobe of the liver and stained them with H&E. In Fig. 3, under microscope, the liver lobule structure of the control group was normal. However, in the CCl 4 group, the liver tissue structure was disorganized, the hepatocytes were swollen and degenerated, the central vein showed obvious bleeding symptoms, with congestion and inflammatory cell infiltration, and the tube wall was blurred and unclear. GgCE could improve liver injury in levels. C Hepatic MPO activity. D Hepatic TP content. All data are presented as mean ± SD (three independent experiments). ## P < 0.01, # P < 0.05 versus control group; **P < 0.01, *P < 0.05 versus CCl 4 group. n.s. versus CCl 4 + GgCE (300 mg/kg) group/CCl 4 + Bifendatatum group a dose-dependent manner, especially under the action of high dose (300 mg/mL) GgCE, the liver tissue structure was orderly, the central vein had no bleeding and inflammatory cell infiltration, and the overall status of the liver was close to normal. These results indicated that GgCE improved CCl 4 -induced pathological changes in mice liver.

GgCE activated antioxidant signaling pathways in CCl 4 -induced chronic liver injury
The above results showed that GgCE could resist oxidative damage of mice liver induced by CCl 4 . In order to explore the effect of GgCE on CCl 4 -induced liver oxidative damage B Hepatic MDA content. C Hepatic GSH activity. D Hepatic GSH-Px activity. E Serum SOD activity. All data are presented as mean ± SD (three independent experiments). ## P < 0.01, # P < 0.05 versus control group; **P < 0.01, *P < 0.05 versus CCl 4 group. n.s., && P < 0.05 versus CCl 4 + GgCE (300 mg/kg) group/CCl 4 + Bifendatatum group from the mechanism, we used Western blot assay to detect the expression of related proteins (Fig. 4). The results showed that the expressions of Nrf2, GCLC, GCLM, HO-1 and NQO1 proteins in mice liver tissue were inhibited by CCl 4 , while GgCE treatment activated the expression of Nrf2 protein and downregulated the CCl 4 -induced increase in Keap1 protein levels in mice liver. In addition, the expression of GCLC, GCLM, HO-1 and NQO1 proteins, the downstream of Nrf2, was activated. Here we also found that GgCE (300 mg/kg) had a significantly stronger effect on antioxidant protein activation than Bif. These consequences suggest that GgCE alleviated oxidative damage in CCl 4 -induced chronic liver injury by activating antioxidant signaling pathways.

GgCE promoted autophagy in CCl 4 -induced chronic liver injury
The above study found that GgCE had a protective effect on CCl 4 -induced liver injury in mice. In order to investigate whether autophagy is the further reason for GgCE to protect liver injury, we used Western blot to detect the expression of related autophagy proteins (Fig. 5). By analyzing the protein content, it was found that CCl 4 promoted the phosphorylation of PI3K and mTOR proteins and promoted the expression of P62, and inhibited the expression of LC3 II protein. GgCE treatment effectively reduced the phosphorylation PI3K and mTOR proteins, inhibit the expression of P62 protein, and activate the expression of LC3 II protein. In additional, we combined the results with those of autophagyrelated proteins, and we found that GgCE (300 mg/kg) promoted autophagy significantly more than Bif. The results showed that GgCE alleviated CCl 4 -induced chronic liver injury by promoting autophagy.

Potential mechanism of GgCE to alleviate CCl 4 -induced chronic liver injury
CCl 4 stimulation in mice caused oxidative damage and impaired autophagic pathway, whereas GgCE treatment could activate antioxidant signaling pathways against oxidative damage and promote autophagy to alleviate liver injury in mice (Fig. 6).

Discussion
Liver injury seriously affects liver function and endangers health, so we need to find more effective drugs to treat liver injury. From the study of Luís R et al. [26], we can analyze that GgCE contains 24 phenolic compounds, includes free phenolic acids, flavonoid glycosides and their aglycones, and 8 betacyanins. These bioactive components have been All data are presented as mean ± SD (three independent experiments). ## P < 0.01 versus control group; **P < 0.01 versus CCl 4 group. n.s., && P < 0.05 versus CCl 4 + GgCE (300 mg/kg) group/ CCl 4 + Bifendatatum group studied in the field of antioxidants, but no reports have been seen on the treatment of liver injury, and we can get the content of these components from Luís R's study.
In our study, we investigated whether GgCE has a protective effect on CCl 4 -induced liver injury. To demonstrate the therapeutic effect of GgCE, we used biochemical kits, H&E staining and Western blot. Our experimental results showed that GgCE alleviated CCl 4 -induced chronic liver injury in mice by activating antioxidant signaling pathways and promoting autophagy.
CCl 4 can lead to the occurrence of liver injury, which can cause elevation of ALT and AST, hepatocyte inflammation, necrosis, steatosis and other changes [27]. AST and ALT mainly exist in hepatocyte, and when the liver is damaged, transaminases in hepatocyte are transferred to the blood, resulting in an increase in the content of transaminase  Potential mechanism of GgCE to alleviate CCl 4 -induced liver injury. The mechanism of GgCE is opposite to that of CCl 4 , GgGE alleviated CCl 4 -induced chronic liver injury by activating antioxidant signaling pathways and promoting autophagy in the blood. ALT is more sensitive to the damage of liver cells than AST [28]. The results of this study showed that injection of CCl 4 directly increased the serum AST and ALT levels in mice, which decreased after GgCE treatment, suggesting that GgCE can resist the effects of CCl 4 . MPO activity can be used as a measure of neutrophil count, which tends to increase and exacerbate inflammatory responses in models of liver injury [29]. We found a significant increase in hepatic MPO activity after CCl 4 treatment, indicating that CCl 4 can cause increased an inflammatory response in mice liver, and GgCE treatment can significantly downregulate hepatic MPO activity, suggesting that GgCE alleviated inflammatory response in mice liver caused by CCl 4 . TP is an important test item for clinical liver biochemical indexes. TP maintains the normal colloid osmotic pressure and PH of blood, and it has the functions of transporting various metabolites, regulating the physiological effects of transported substances, relieving toxicity, immunity and nutrition. TP is usually used to detect the nutritional status of the body and also to identify and diagnose liver diseases [30]. We found that GgCE treatment significantly increased TP levels in mice liver and improved liver dysfunction caused by CCl 4 .
In this study, we found that CCl 4 could induce the increase of serum ROS in mice. It is well known that excessive ROS accumulation can lead to oxidative stress and lipid peroxidation, which can aggravate the liver damage process [31]. MDA is a metabolite of lipid peroxidation that accumulates in the body after liver injury. MDA can effectively indicate the degree of liver oxidative damage [32]. Similarly, we found that CCl 4 can induce MDA production and aggravate liver oxidative damage. It was found that GSH is an important superoxide anion scavenger that scavenges ROS and protects the liver from oxidative stress, and that loss and depletion of GSH leads to the accumulation of ROS [33]. Unfortunately, we found that CCl 4 inhibited the expression of GSH. The body can protect itself from oxidative damage through enzyme-induced and non-enzyme-induced antioxidant pathways [34]. The regulation of GSH-Px and SOD is the main mechanism of enzymatic antioxidant activity in vivo. SOD catalyzes the degradation of superoxide free radicals and remove free radicals. GSH-Px catalyzes H 2 O 2 reduction to prevent cell damage. However, CCl 4 can reduce the activities of GSH-Px and SOD, and hinder the body's antioxidant reaction [35]. The results showed that GgCE can reduce ROS and MDA to normal levels, and increase hepatic GSH, GSH-Px and serum SOD activities, suggesting that GgCE can alleviate CCl 4 -induced liver oxidative damage by enhancing the body's ability to resist oxidative stress.
When liver injury occurs, liver tissue is disorganized, the cells are swollen and degenerated, and there is significant hemorrhage and inflammatory cell infiltration in the central vein. Similarly, we found the above-mentioned pathological changes in CCl 4 -induced liver injury. Interestingly, the overall state of the mice liver was near to normal after the treatment of CCl 4 -induced liver injury with GgCE, indicating that GgCE can effectively improve chronic liver injury in mice induced by CCl 4 .
We found that CCl 4 not only promoted the increase of ROS, but also inhibited the expression of Nrf2 and its downstream proteins. The Nrf2-ARE signaling pathway was found to be a key pathway of cells to resist oxidative stress [36]. Moreover, activating Nrf2 can induce the expression of downstream genes such as GCLC, GCLM, HO-1 and NQO1 to resist oxidative stress injury caused by various stimuli [37, [38]. Chun's study found that berberine could alleviate CCl 4 -induced liver injury in rats by regulating Nrf2-ARE signaling pathway [39], which is similar to our findings that GgCE could counteract CCl 4 -induced oxidative stress injury by activating Nrf2 and other antioxidant proteins.
It is well known that liver injury can inhibit autophagy. In autophagy, mTOR, a protein kinase that regulates cell growth and metabolism, is the upstream negative regulator of autophagy and the best characterized regulator of autophagy [40]. Activation of PI3K can increase mTOR activity, leading to down-regulation of autophagy [41]. LC3 II and P62 are markers of detecting autophagy. LC3 II exists only in mature autophagosomes, whereas substrate protein P62 recognizes ubiquitinated protein aggregates and directly bind to LC3 II specific motifs [42]. Several studies have shown that in the treatment of CCl 4 -induced liver injury by promoting autophagy, LC3 II protein content will decrease and P62 protein content will increase [43]. We also found that CCl 4 has an inhibitory effect on autophagy. However, these effects were reversed by GgCE. This suggests that GgCE may alleviate CCl 4 -induced liver injury by promoting autophagy.
As a positive drug control, BIf did improve CCl 4 -induced chronic liver injury in mice in our study. Our GgCE was not significantly different from Bif in the biochemical index assay related to liver injury and anti-oxidative stress, but GgCE (300 mg/kg) was significantly better than Bif in activating antioxidant proteins and promoting autophagy process. This difference may be caused by the different mechanism of action of Bif on CCl 4 -induced liver injury in mice. However, our GgCE is more affordable and convenient to obtain than Bif, and GgCE is extracted from pure natural herbs, which is less toxic. In summary, we believe that GgCE may have a greater advantage over Bif in the treatment of liver injury.
In conclusion, our study suggests that GgCE alleviated CCl 4 -induced chronic liver injury in mice, possibly by activating antioxidant signaling pathways and promoting autophagy (Fig. 6.). Currently, the therapeutic effect of