Scutellarin Ameliorated Carbon Tetrachloride-Induced Chronic Liver Injury in Mice

Background: Erigeron breviscapus (Vant.) Hand. -Mazz. is an edible and traditional medical herb and its extract scutellarin (SCU) is a widely used avonoid showing anti-oxidant and anti-inammatory activities. The purpose of this study was to evaluate the hepatoprotective effect of SCU on carbon tetrachloride (CCl 4 )-induced chronic liver injury in mice and reveal the underlying mechanisms. Methods: Chronic liver injury in mice was induced by intraperitoneal injection of 1 ml/kg CCl 4 every three days. SCU (15 mg/kg, 30 mg/kg and 60 mg/kg) was administered through gavage every day. Bifendate (120 mg/kg) serves as a positive drug to validate the effectiveness of SCU. Results: The hepatoprotective effect of SCU was conrmed by liver function analysis, histological analysis and TUNEL assay. Administration of SCU recovered the activities of superoxide dismutase (SOD) and reduced the production of malondialdehyde (MDA). Additionally, treatment with SCU signicantly decreased the mRNA levels of pro-inammatory cytokines including IL-6, IL-1β and TNF-α. Moreover, SCU treatment suppressed the activation of NF-κB by decreasing the degradation of IκBα and inhibited the expression of CYP2E1. The 16S rRNA sequencing demonstrated that intake of SCU signicantly remodeled gut microbiota, especially enriching the following: Lactobacillus, Coprobacillus, Akkermansia, Bidobacterium, Parabacteroides. Conclusion: Our ndings showed that SCU effectively ameliorated CCl 4 -induced chronic liver injury. This hepatoprotective effects might be attributed to inhibition of CCl 4 -induced NF-κB and CYP2E1 activation and enrichment of benecial microbial community.


Background
The liver is a central organ and performs numerous vital functions including regulation of glycogen storage and detoxi cation of metabolites to maintain homeostasis of the body. Due to its multiple metabolic effects, the liver is vulnerable to get injured by long-term drug therapy (such as rifampicin, isoniazid and phenytoin), virus and alcohol, and eventually form chronic liver injury. Repeated hepatic damage leads to the detriment of the hepatic architecture, resulting in functional impairments and jaundice of hepatocytes [1]. As of now, the clinical application of hepatoprotective drugs is limited due to various side effects. Thus, it is of critical importance to develop a hepatoprotective drug with high e cacy and few side effects.
Chronic liver injury is de ned by the chronic in ammatory insult to the hepatic parenchyma which results in aggravating brosis with ultimate progression to end-stage liver disease over time. The cycle of hepatocyte damage and regeneration not only leads to persistent in ammation but also disrupts the regulation of an intricately balanced relationship between the gut and the liver [2]. The commensal bacteria such as Lactobacillus [3], Bi dobacterium [4] that have been widely reported to have antiin ammatory properties and produce bene cial metabolites (such as indoles, propionic acid and secondary bile acid), and play a vital role in hepatic disease and in preserving the balance of the gut-liver axis [5,6]. Thus, the focus on them as a therapeutic target to treat chronic liver injury has been growing.
Erigeron breviscapus (Vant.) Hand. -Mazz., a well-known ethnomedicine, has been traditionally used to treat cerebral thrombosis, cerebral hemorrhage and cerebral embolism in Yunnan. Its main extract is scutellarin (SCU), an herbal avonoid. In recent years, bioactive phytonutrients such as avonoids and polyphenols derived from plants and foods have attracted considerable attention for hepatoprotection due to their unique bioavailability, multiple targets and low adverse effects [7,8]. SCU (Fig. 1) possesses multiple bene cial effects including anti-in ammation and anti-oxidation. It has been indicated that SCU administration is safe to various normal cell types [9][10][11]. Recently, mounting evidence suggests a potential hepatoprotective effect of SCU but the critical mechanism for SCU that protects against liver injury remains elusive [9,10]. Carbon tetrachloride-induced liver injury in rodent models is widely used to clarify potential for natural compounds to protect the liver against damage due to its similarity with druginduced liver injury in humans [12]. CCl 4 is a well-known hepatotoxin, mainly metabolized by the hepatic CYP2E1 to yield toxic trichloromethyl-free radical and peroxyl radical. The CCl 4 -derived free radicals could attack hepatocyte membranes, resulting in lipid peroxidation, oxidative stress and in ammatory response [13]. NF-κB corresponded to the regulation of pro-in ammatory mediators expression and could signi cantly induce the expression of various pro-in ammatory genes after translation to the nucleus, thus resulting in adverse consequences [14,15]. Therefore, CYP2E1 and NF-κB may be potential therapeutic targets of SCU.
In the present study, the therapeutic effect of SCU on CCl 4 -induced chronic liver injury was investigated and the underlying mechanisms were explored to develop a potential candidate for chronic liver injury treatment.

Animal Model
Male BALB/c mice (6-8 week and 18-22 g) were obtained from Tianqin biotechnology Co., Ltd with a certi cate of quality No.SCXK-2019-0004 (Changsha, Hunan, China) and acclimatized for 7 days. All animal experiments were performed in accordance with the guidelines of the Care and Use of Laboratory Animals of the Laboratory Animal Ethical Commission of Dali University and all efforts were taken to minimize animals suffering. Chronic liver injury in mice was induced by intraperitoneal injection of 1 ml/kg CCl 4 (diluted 1:9 in olive oil, Sinoreagent, Shanghai, China) every three days. SCU (PubChem ID 185617) (15 mg/kg, 30 mg/kg, 60 mg/kg, Yunnan Plant Pharmaceutical Co., Ltd, Kunming, Yunnan, China) was given by gavage every day. The groups were treated as follows (n = 10 each group): (1) Normal group (N) was given an equal volume of solvent (0.5% CMC-Na) and an equal volume of olive oil.

Liver Function and Oxidative Stress Assessment
Blood samples were centrifuged at 2000 rpm and 4℃ for 7 min to obtain serum. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and albumin (ALB) were measured according to the manufacture's instructions (Nanjing jiancheng bioengineering institute, Nanjing, Jiangsu, China). Liver tissues were homogenized for 1 min in physiological saline, the homogenates were centrifuged at 12000 rpm and 4℃ for 30 min and the supernatant was collected.

Histological Analysis
Liver samples were xed in 4% paraformaldehyde and embedded in para n wax. Embedded tissues were cut into 4 µm thick sections and stained with hematoxylin-eosin (H&E) for the histological analyses. The histological score of sections was assessed by two pathology experts (Table S3).

TUNEL Assay
Cell apoptosis in the liver tissue was detected by DeadEndTM Fluorometric TUNEL System according to the manufacture's protocol (Promega, Wisconsin, USA). Para n sections were brie y digested by 20 µg/mL of proteinase K solution for 7 min and then equilibrated with equilibration buffer for 10 min at room temperature. The sections were incubated with TdT reaction mix for 60 min at 37℃ in a humidi ed chamber before being transferred to 2 × SSC buffer for 15 min to stop the reaction. After immersing in propidium iodide solution (PI) for 15 min at room temperature in the dark, the sections were analyzed under a uorescence microscope. Areas of apoptosis were quanti ed by Image J (National Institute of Health, Bethesda, MA, USA).

Immunohistochemistry (IHC)
Immunohistochemistry for CYP2E1 was performed with the para n-embeded liver sections. Antigen retrieval was carried out by incubation in EDTA buffer after the slides were depara nized. Endogenous peroxidase in the section was blocked with 0.3% hydrogen peroxide solution in the dark for 10 min. The sections were incubated with primary antibody against mouse CYP2E1 (1:500, Abcam, Cambridge, UK) at 4℃ for 60 min and then with HRP-conjugated secondary antibodies at room temperature for 15 min. Finally, the sections were stained with DAB substrate and counterstained with hematoxylin. Areas of CYP2E1 expression were quanti ed by Image J (National Institute of Health, Bethesda, MA, USA).

Real-Time Quantitative PCR (RT-qPCR)
Total RNA was extracted from liver tissues with TRIzol reagent (Invitrogen, Thermo Fisher Scienti c, NY, USA). 3 µg of total RNA was reverse-transcribed into complementary DNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c, NY, USA) following the supplier's protocol. The PCR primer sequences are listed in Table 1. RT-qPCR was performed in a StepOnePlus™ Real-Time PCR system (Thermo Fisher Scienti c, NY, USA) in combination with TB Green® Premix Ex Taq™ II (Takara Bio, Inc., Shiga, Japan). The reaction was as follows: a precycling stage at 95℃ for 30 s, 40 cycles of denaturation at 95℃ for 5 s and annealing at 60℃ for 30 s. The expression levels, which were normalized to GAPDH, were calculated using the 2 −ΔΔCt method.

Western Blot
The total protein from liver tissues was extracted by RIPA lysis buffer (Solarbio, Beijing, China) on ice and then quanti ed using BCA protein assay kit (Solarbio, Beijing, China). The protein was resolved through SDS-PAGE gel separation with a Bio-Rad electrophoresis system and transferred to polyvinylidene di uoride membranes (Millipore, MA, USA). The membranes were blocked with 5% skim milk (BD, USA) for 1. . The sequencing data analysis was carried out as previously described [16]. The Chao1, Shannon and Pielou indices were calculated for α-diversity evaluation. UniFrac-based principal coordinates analysis (PCoA) was employed to assess β-diversity. The different abundant bacteria among all groups were performed by hierarchical clustering heatmap. The correlation between gut microbiota and liver injury indicators was analyzed using spearman index. All gures were performed by Personalbio genescloud (https://www.genescloud.cn/chart/). The raw data were deposited into NCBI Sequence Read Archive (SRA) database and accession numbers are SRR12278403-SRR12278407.

Statistical analysis
Statistical analysis was performed with SPSS 22.0 (IBM Corp., NY, USA). All data are presented as the mean ± SD (n = 5). Comparisons between groups were assessed by a one-way analysis of variance (AVONA), and Dunnett's test was employed as a post hoc test. Statistical signi cance was set at P < 0.05.

Effects of SCU on serum parameters
In order to assess the degree of liver injury, we detected the levels of ALT, AST, TBIL and ALB in serum. We also used bifendate (BIF) as a positive control, which is widely prescribed to treat hepatic injury. As presented in Fig. 2, the serum ALT, AST, TBIL levels in the M group were elevated obviously compared to those in the N group. In contrast, SCU treatment signi cantly blunted the serum ALT, AST, TBIL levels in the CCl 4 -exposed mice. As for ALB, no signi cant difference was observed among the ve groups. These results suggest that SCU harbors hepatoprotective properties against CCl 4 -induced chronic liver injury.

Effects of SCU on Histopathologic Changes
H&E staining was performed to evaluate the protective effects of SCU on CCl 4 -induced chronic liver injury.
As shown in Fig. 3, in the N group, the hepatic lobule structure was clear, the hepatic cells were arranged in neat row and there were no hepatocyte necrosis, hepatocyte swelling and in ammatory cell in ltration.

Effects of SCU on CCl4-Induced Hepatocyte Apoptosis
As shown in Fig. 4, TUNEL assay demonstrated that compared with the N group, there was a large amount of hepatocyte apoptosis that occurred in the liver tissue of the M group, while in SCU-treatment groups, hepatocyte apoptosis was signi cantly decreased in a dose-dependent fashion. These results imply that SCU treatment effectively attenuates CCl 4 -induced hepatocyte apoptosis.

Effects of SCU on Liver Oxidative Stress
In order to quantify oxidative liver injury, we measured the levels of SOD and MDA in the liver. As shown in Fig. 5, compared with the N group, SOD activity was decreased signi cantly, and MDA level was increased signi cantly in the M group. SCU treatment signi cantly inhibited MDA level and increased SOD activity. These data demonstrate that SCU exerts hepatoprotective effects by alleviating oxidative stress.

Effects of SCU on the Pro-in ammatory Cytokines Production
In ammatory response is an important factor that leads to liver injury, we then assessed the IL-6, IL-1β and TNF-α mRNA levels that are the key in ammatory cytokines in the liver tissues by RT-qPCR. In the M group, the IL-6, IL-1β and TNF-α mRNA levels were rapidly increased but oral administration of SCU resulted in signi cant attenuation of these pro-in ammatory cytokines in the liver (Fig. 6). These data hint that the hepatoprotective effects of SCU are achieved by suppressing in ammatory response.

Effects of SCU on NF-κB Activation
We further explored the underlying anti-in ammatory mechanism of SCU against CCl 4 -induced chronic liver injury. IκBα and NF-κB play a critical role in the production of in ammatory cytokines such as IL-6, IL-1β and TNF-α, thus we detected the changes in the protein levels of IκBα and NF-κB. As shown in Fig. 7, after CCl 4 intoxication, the protein level of IκBα was decreased signi cantly and the protein level of NF-κB was increased signi cantly. Compared with the M group, the protein levels of IκBα were increased and the protein levels of NF-κB were decreased in the SCU treatment group. These data suggest that SCU suppresses in ammatory response through inhibiting the activation of NF-κB by decreasing the degradation of IκBα.

Effects of SCU on CYP2E1 Expression
CYP2E1 have been deemed as a marker of chemical liver injury, playing a pivotal role in CCl 4 -induced liver injury. We then examined the expression of CYP2E1 mRNA and protein in the liver tissue through IHC, RT-qPCR and western blot, respectively. As shown in Fig. 8, the expression of CYP2E1 mRNA and protein were dramatically up-regulated in the M group, which were then dose-dependently inhibited by SCU treatment. These data suggest that the hepatoprotective effects of SCU are achieved by suppressing CYP2E1 expression.

Effects of SCU on gut microbiota diversity and composition
Gut microbiota plays a pathogenic role in the development of many disease and modulation of the microbiome is a potential therapeutic approach for prevention of liver injury [16,17]. Moreover, in our previous work, we proved that SCU markedly modulated the gut microbiota (Table S1, Figure S1). Therefore, we hypothesized the SCU exert its hepatoprotective effect through modulation of the gut microbiota. In order to verify the conjecture, we analyzed the effects of SCU on the diversity and composition of gut microbial community in CCl 4 -exposed mice by 16S rRNA sequencing. Repeated injection of CCl 4 signi cantly raised the α-diversity of the gut microbiota as indicated by the Chao1, Shannon and Pielou indices that represent the richness, diversity and uniformity of gut microbiota, respectively. Treatment of SCU signi cantly reduced richness, diversity and uniformity with lower Chao1, Shannon and Pielou indices compared with those of the M group (Fig. 9A). β-diversity was used to compare the similarity of the overall community structure, which employed an unsupervised multivariate statistical assessment such as PCoA. PCoA displayed a marked structure shift in the M group in contrast to that of N group, while after daily SCU treatment, gut microbiota was restored to be similar with that of N group (Fig. 9B). Furthermore, a taxon-based analysis was used to clarify the alteration of gut microbiota in each group at phylum level. Overall, a total of twenty phyla were shared from all ve groups (Fig. 9C). Bacteroidetes and Firmicutes were selected due to their quantitative dominance in the microbial community. CCl 4 injection signi cantly raised the relative abundance of Bacteroidetes and reduced the relative abundance of Firmicutes compared to the N group. SCU treatment signi cantly raised the relative abundance of Bacteroidetes compared to the M group (Fig. 9D). Altogether, SCU administration had a substantial effect on remodeling the gut microbiota in response to CCl 4 .

Effect of SCU on key phylotypes of gut microbiota
To further investigate the differences of various bacteria in ve groups, we used a hierarchical clustering heatmap based on genus level, and the major genera ranking the top forty from each group were selected (Fig. 10A, Table S2). Although LefSe analysis indicated that no genus is speci c for any group, there were ve genus exhibited obvious differences. As shown in Fig. 10B, Lactobacillus, Coprobacillus, Akkermansia, Bi dobacterium, Parabacteroides were evidently higher in SCU treatment groups relative to the M group. To investigate the relationship between gut microbiota and liver injury indicators, the top forty genera in all samples and AST, ALT, TBIL, ALB, SOD, MDA, IL-6, TNF-α, IκBα, NF-κB, CYP2E1 were analyzed using the spearman index. As shown in Fig. 10C, Lactobacillus, Coprobacillus, Akkermansia, Bi dobacterium, Parabacteroides had bene cial effects on the liver because they are negatively related with AST, ALT, TBIL, MDA, IL-6, TNF-α, NF-κB, CYP2E1 and positively related with ALB, SOD, IκBα. These data suggest Lactobacillus, Coprobacillus, Akkermansia, Bi dobacterium, Parabacteroides may be the speci c genera that are responsible for the hepatoprotective effects of SCU.

Discussion
Chronic liver injury induced by long-term drug therapy, virus and alcohol occurs frequently in clinic, which brings a considerable burden on the healthcare system and the pharmaceutical industry. Natural products such as silymarin and oleanolic acid act as hepatoprotective drugs due to high e ciency and few adverse effects, making the development of hepatoprotective agents based on natural products popular worldwide [18,19]. SCU is an herbal avonoid, possessing various bene cial effects and widely used to treat cardiovascular and cerebrovascular diseases in China. Recently, emerging studies have suggested that SCU has the potential for liver protection but its mechanisms of hepatoprotective effect are still unclear. In this study, we investigated the impacts of SCU on CCl 4 -induced chronic liver injury as well as revealed potential mechanisms underlying the hepatoprotective effect of SCU. CCl 4 -induced hepatocellular damage and the subsequent rupture of the plasma membrane cause the release of intracellular enzymes such as ALT and AST into the extracellular space. The released enzymes eventually enter the circulation, thereby increasing serum ALT and AST levels. Serum ALT and AST levels re ect hepatocyte damage and are considered to be a quite speci c clinical biomarker of hepatotoxicity [20]. In our study, we clearly observed that CCl 4 intraperitoneal injection dramatically increased the levels of serum ALT and AST compared to the N group. Most importantly, our results demonstrated that serum ALT and AST levels were alleviated by SCU in a dose-dependent manner. The concentration of bilirubin in the serum is determined by the balance between bilirubin production and clearance by hepatocytes. Elevated serum bilirubin levels may be caused by release of unconjugated or conjugated bilirubin from injured hepatocytes. TBIL is another speci c biomarker of liver injury. The results showed that SCU treatment dose-dependently reduced serum TBIL level. The liver is the exclusive site of synthesis of ALB. Thus, serum ALB serves as true test of hepatic synthetic function. However, serum ALB has a very long half-life, which may not be affected in the short-term experimental liver injury model and low serum albumin may be a sign of advanced liver disease [21]. Our results con rmed that Both CCl 4 intoxication or SCU treatment did not produce any apparent effects to ALB. A great many studies demonstrate that lipid peroxidation and oxidative stress are vital factors for hepatic dysfunction in the CCl 4 -induced mice [22]. CCl 4 -induced lipid peroxidation and oxidative stress can bring injury to the lipids, proteins and nucleic acids, ultimately inducing liver injury. MDA, the nal product of lipid peroxidation, can be detected to estimate the severity of CCl 4 -induced lipid peroxidation. SOD, an antioxidant enzyme with high endogenous expression in the liver, is the major defense mechanism against reactive oxygen species [23]. In our study, ingestion of SCU signi cantly repressed the increase of MDA in the liver induced by CCl 4 and markedly enhanced SOD activity. An excessive in ammatory response can worsen injured hepatic tissue and hinder repair processes [24]. In ammatory response can be activated at the local sites of injury after hepatic damage, followed by elevated expression of proin ammatory cytokines including IL-6, IL-1β and TNF-α, which can subsequently damage endothelial cells to aggravate liver injury [14]. In ammatory systems operate through various signaling pathways, among which the most important pathway, NF-κB has been long studied as a major target for anti-in ammatory treatments [25]. CCl 4 stimulation leads to the degradation of IκBα, resulting in the free pass and translocation of NF-κB from cytoplasm to nucleus [26]. The activation of NF-κB leads to release of proin ammatory cytokines and aggravation of liver injury [27]. Therefore, NF-κB might be a potential target for the treatment of chronic liver injury. The results demonstrated that SCU treatment signi cantly reduced IL-6, IL-1β and TNF-α mRNA levels, and restored IκBα degradation as well as suppressed NF-κB activation. Histological analysis can directly re ect the physiological and pathological process of tissues; the results showed that intake of SCU signi cantly reduced necrosis and in ammatory cell in ltration induced by CCl 4 . After TUNEL assay examination, it was con rmed that CCl 4 -induced hepatocyte apoptosis was weakened by SCU treatment. Taken together, SCU shows the hepatoprotective effect on CCl 4 -induced chronic liver injury and the hepatoprotective properties of SCU may be associated with its anti-oxidative and anti-in ammatory capacities.
It is well-documented that CCl 4 mediated by CYP2E1 to generate trichloromethyl free radical that could attack hepatocyte membranes, causing lipid peroxidation, oxidative stress and in ammatory response [28]. At the same time, highly destructive reactive radicals can also signi cantly elevate the activity of CYP2E1, concentrating the activation of the positive feedback and nally causing severe hepatic damage [29]. Our results showed that SCU signi cantly down-regulated the CYP2E1 expression levels in the liver of mice exposure to CCl 4 , indicating the bene cial roles of SCU on CCl 4 -induced chronic liver injury may be mediated by down-regulating the expression of CYP2E1.
Compelling evidence supports that gut microbiota is related to various liver diseases, such as nonalcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (AFLD) and hepatocellular carcinoma (HCC) [2,30,31]. In this study, the results of the 16S rRNA sequencing demonstrated that parenterally administered (intraperitoneal injection) CCl 4 signi cantly modulated the composition of gut ora, as shown by the result of α-, β-diversity indices. Oral administered SCU signi cantly remodeled the gut microbiota, making it similar to the N group. At the genus level, intake of SCU had a selective increase in the enrichment of Lactobacillus, Coprobacillus, Akkermansia, Bi dobacterium, Parabacteroides. Lactobacillus and Bi dobacterium are widely known as probiotics for their potential pathogens inhibition, useful metabolites production (butyrate, succinic acid) and intestinal barrier protection [3,4]. Coprobacillus has been found to play bene cial role in maintaining intestinal stability and liver function [32]. Akkermansia is also a butyrate-producing genus and has been con rmed to improve immunological disorders and the gut barrier to protect against immune-mediated liver injury [33]. Parabacteroides can produce succinic acid, secondary cholic acid and activate different signaling pathways and has been deemed as a potential and new type of probiotics against metabolic syndrome and liver injury [34]. Moreover, their metabolites such as short chain fatty acids can maintain gut mucosal permeability and function, preventing the translocation of microbiota-derived toxin (such as LPS) to the liver to induce in ammation[35]. Short chain fatty acids can also activate hepatic GPCRs via the portal vein and/or systemic circulation, leading to the suppression of pro-in ammatory mediators such as IL-6, IL-1β and TNF-α [4].Correlation analysis also demonstrated that Lactobacillus, Coprobacillus, Akkermansia, Bi dobacterium, Parabacteroides showed bene cial effect to the liver. Therefore, the bene cial roles of SCU on CCl 4 -induced chronic liver injury may be through raising the abundance of Lactobacillus, Coprobacillus, Akkermansia, Bi dobacterium, Parabacteroides.