Catechin hydrate

The involvement of Nrf2 antioxidant signaling pathway in the protection of monocrotaline-induced hepatic sinusoidal obstruction syndrome in rats by (+)-catechin hydrate

Xiaoqi Jing, Jiaqi Zhang, Zhenlin Huang, Yuchen Sheng, Lili Ji
A The MOE Key Laboratory for Standardization of Chinese Medicines and Shanghai Key Laboratory of Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
B Center for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

ABSTRACT
Hepatic sinusoidal obstruction syndrome (HSOS) is a rare and life-threatening liver disease. (+)-Catechin is a natural dietary flavonol with high antioxidant capacity. This study aims to investigate the involvement of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant signaling pathway in the protection of (+)-catechin hydrate (CAT) against monocrotaline (MCT)-induced HSOS. Results of serum alanine/aspartate aminotransferases (ALT/AST) activities, total bilirubin (TBil) and bile acids (TBA) amounts, liver histological observation, scanning electron microscope evaluation and hepatic metalloproteinase-9 (MMP-9) expression all demonstrated the protection of CAT against MCT-induced HSOS in rats. CAT attenuated MCT-induced liver oxidative injury in ratsand the formation of cellular reactive oxygen species (ROS) in human hepatic sinusoidal endothelial cells (HHSECs). CAT enhanced Nrf2 nuclear translocation in livers from MCT-treated rats and in HHSECs treated with MCT, and further increased the expression of Nrf2-dependent genes including catalytic or modify subunit of glutamate-cysteine ligase (GCLC/GCLM), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). Moreover, GCL inhibitor L-buthionine-(S, R)-sulfoximine (BSO), NQO1 inhibitor diminutol (Dim) and HO-1 inhibitor zinc protoporphyrin (ZnPP) all abrogated CAT-provided the protection against MCT-induced cytotoxicity in HHSECs. The results of molecular docking analysis indicated the potential interaction of CAT with the Nrf2 binding site in kelch- like ECH-associated protein-1 (Keap1) protein. In summary, this study demonstrated the critical involvement of Nrf2 antioxidant signaling pathway in CAT-provided the protection against MCT-induced HSOS.

Introduction
Catechins are polyphenolic phytochemicals and belong to flavanols, which is part of the chemical family of flavonoids. Catechins are widely distributed in food and medicinal plants, and they are the main ingredients in all kinds of tea including green tea, white tea and black tea that are all popular consumed and functional drinks in the world [1,2]. Catechins are found to have a variety of biological functions including antioxidant, anti-inflammation, anti-hypertension, anti-cancer, anti-fibrosis, hepato-protection, body weight control and neuro-protection [1-5]. A growing number of studies have shown that the intake of catechins-rich foods is helpful for the prevention of various human chronic diseases including cardiovascular diseases, inflammatory bowel disease, liver diseases, cancer, metabolic syndromes and neurodegenerative diseases [1-7].
Hepatic sinusoidal obstruction syndrome (HSOS), also known as hepatic veno- occlusive disease (HVOD), is the most frequent complication in patients in early phase following hematopoietic stem-cell transplantation [8]. The severe HSOS is commonly associated with multi-organ failure with a mortality rate of 84.3% [9]. HSOS was also associated with the ingestion of tea or herbal medicines containing hepatotoxic pyrrolizidine alkaloids (HPAs), and about thousands of HSOS clinical cases due to the ingestion of HPAs have been reported in the world since 1920 [10,11]. Besides, some other drugs that cause bone marrow suppression are also found to be related with HSOS development, such as 6-thioguanine, cyclophosphamide, oxaliplatin and azathioprine [12].
The damage on hepatic sinusoidal endothelium has been reported as the key and primary event of HSOS [13]. However, the mechanism of the development of HSOS is very complicate and still not clear, which greatly hinders the finding of drugs for HSOS treatment in clinic. Currently, pharmacologic options for HSOS are very limited, and defibrotide is the only drug approved for treatment of serious HSOS in European Union [14]. However, under the recommended safe and efficacious dose (25 mg/kg/day) of defibrotide in the treatment of HSOS, the survival rate was just 54% at day 100 [15]. Thus, it is urgent to find more efficacious and safe drugs for HSOS treatment in clinic.
(+)-Catechin is one of the most common isomers of catechins. (+)-Catechin and its gallic acid conjugates are widely found in various fruits, tea, cacao bean and chocolate [16,17], and they are also distributed in some traditional herbal medicines such as Paeonialactiflora, Uncariarhynchophylla, Ephedra sinica Stapf, Rhodiolakirilowii (Regel) Maxim. and Loranthusparasiticus Merr [18-22]. (+)- Catechin and (+)-catechin hydrate (CAT) has been reported to have anti- neurodegeneration, antioxidant, anti-cancer, anti-inflammatory and anti-viral activities [22-25]. However, there is still no report about whether (+)-catechin or CAT has protection against HSOS. In this study, the attenuation of CAT against HSOS induced by monocrotaline (MCT) and the involvement of Nrf2 antioxidant signaling pathway were observed.

Materials and methods
Chemical compounds and reagents
MCT was purchased from Sigma Chemical Co. (St. Louis, MO). CAT with purity ≥98.0% was purchased from Beyotime Institute of Biotechnology (Haimeng, China). Kits for detecting alanine/aspartate aminotransferases (ALT/AST) activities, total bilirubin (TBil) and bile acids (TBA) contents, malondialdehyde (MDA), protein carbonylation, reduced glutathione (GSH) and oxidized glutathione (GSSG) amounts, and glutathione-S-transferase (GST) activity were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Trizol, fetal bovine serum (FBS) and 2’-7’-dichlorodihydrofluorescein diacetate (H2DCFDA) were bought from Life Technology (Carlsbad, CA). Reagents for determining prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB) amount were obtained from Dade Behring Inc. (Deerfield, IL). Antibodies for MMP-9, Lamin B1 and β-actin were all purchased from Cell Signaling Technology (Danvers, MA). Antibodies for Nrf2, GCLC, GCLM, Keap1, HO-1, NQO1 and p62 were all obtained from Santa Cruz (Santa Cruz, CA). Peroxidase-conjugated goat anti-Rabbit IgG (H+L) and anti-Mouse IgG (H+L) were purchased from Jackson ImmunoResearch (West Grove, PA). Whole cell protein extraction kit and enhanced chemiluminescence kit were obtained from Millipore (Darmstadt, Germany). NE- PER nuclear and cytoplasmic extraction reagents and BCA Protein Assay Kits were purchased from ThermoFisher Scientific (Waltham, MA). PrimeScript Master Mix and SYBR Premix Ex Taq were purchased from Takara (Shiga, Japan). Other reagents unless indicated were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals
Specific pathogen free male Sprague-Dawley rats (weight: 200-240 g) were bought from Shanghai Laboratory Animal Center of Chinese Academy of Science (Shanghai, China). Rats were fed with a standard laboratory diet, given free access to tap water, living in a controlled room temperature (22 ± 1 °C), humidity (65 ± 5%) with a 12:12 h light/dark cycle. All rats have received humane care in compliance with the institutional animal care guidelines approved by the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine.

Animal treatment
Rats were divided into four groups as following: (1) Vehicle control (n=9), (2) MCT (90 mg/kg) (n=11), (3) MCT (90 mg/kg) + CAT (40 mg/kg) (n=10), (4) CAT (40mg/kg) (n=9). Rats were orally administered with MCT (90 mg/kg,intragastrical administration) or vehicle for once, and then were given with CAT (40 mg/kg, intragastrical administration) twice at 6 h and 30 h after MCT administration. Rats were sacrificed 48 h after MCT administration, and blood and livers were collected.

Serum biochemistry analysis
Fresh blood was obtained and kept at room temperature for 1 h. Serum was then collected after centrifugation at 840 × g for 15 min. Serum ALT/AST activity, TBil and TBA amounts were determined with an automatic biochemical analyzer (HITACHI 7080, Japan) by using commercial kits.

Blood cell analysis
Fresh blood was collected from rats of each group by using anticoagulant solution. Blood cells were analyzed by using BAYER ADVIA-120 (German).

Liver histological evaluation
A piece of the liver was fixed in 10% phosphate buffered saline (PBS)-formalin solution and embedded in paraffin.Samples were subsequently sectioned (5 μM) and stained with hematoxylin-eosin, and then observed under a microscope (Olympus, Japan) to evaluate liver injury.

Scanning electron microscope observation
Three rats in each group were firstly perfused with PBS through the abdominal aorta, and then perfused with a fixative solution containing 2.5 % glutaraldehyde. The livers were further cut into small pieces (approximately 5 mm3), and each sample was ion- sputter-coated and observed with a scanning electron microscopy (Hitachi S-4700).

Plasma coagulation assay
The whole blood was freshly obtained from abdominal aorta of rats and placed in a test-tube pre-coated with 3.8% sodium citrate. After being centrifuged at 1000 rpm for 10 min at room temperature, the plasma (platelet-rich plasma) was subjected to detecting PT, APTT, TT and FIB amount according to the manufacturer’s protocols. The experiments were performed using a SysmexCA-1500 plasma coagulation analyzer supplied by Sysmex Corporation (Kobe, Japan) [26].

Measurement of liver lipid peroxidation (LPO)
Liver tissues were collected in cold PBS. MDA, formed as the product of LPO and served as an index for LPO, was determined by using commercial kits.The MDA amount was expressed as nmol/mg protein based on liver protein concentration.

Analysis of liver GSH/GSSG
Liver GSH and GSSG amounts were determined by using commercial kits, and the ratio of GSH/GSSG was calculated.

Analysis of liver protein carbonylation
Liver protein carbonylation was detected by using commercial kits. The amount of protein carbonylation was expressed as nmol/mg protein based on liver protein concentration.

Analysis of liver GST activity
Liver GST activity was determined by using commercial kits. The GST activity was expressed as U/mg protein based on liver protein concentration.

Cell culture
PrimaryHuman Hepatic Sinusoidal Endothelial Cell (HHSEC) was bought from ScienCell (Carlsbad, CA). HHSECs were cultured in ECM supplemented with 5% [v/ v] FBS and 1% ECGS, 100 U/ml penicillin and 100 mg/ml streptomycin. Experiments were performed on HHSECs from passage 4 to 10.

Cell viability assay
HHSECs were pre-incubated with or without various inhibitors for 15 min, and then incubated with or without CAT for 15 min, and then incubated with MCT for another 72 h. After treatments, cells were incubated with 500 μg/ml 3-(4,5-dimethyl- thiazol-2-yl) 2,5-diphenyltetra-zolium bromide (MTT) for 4 h. The formed blue formazan was dissolved in 10% SDS-5% iso-butanol-0.01 M HCl, and the optical density was measured at 570 nm with 630 nm as a reference. Cell viability was normalized as the percentage of control.

Measurement of cellular and hepatic ROS
HHSECs were pre-incubated with H2DCFDA (20 μM) and CAT for 15 min, and then incubated with MCT for 72 h. Cellular ROS were measured as described in our previous published paper [27]. As for measuring hepatic ROS level, cold liver homogenate were centrifuged at 10,000 g (4 °C). The supernatants were incubated with 10 μM H2DCFDA for 1 h, and then fluorescence was detected at excitation 485 ± 20 nm, emission 525 ± 20 nm by using a spectrophotometer (BioTek Synergy H4, Winooski, VT). Protein concentrations in supernatants were detected by using BCA kits. The results were calculated as units of fluorescence per microgram of protein and presented as the percentage of control.

Real-time PCR analysis
The whole RNA was extracted from cells and livers by using TRIZOL reagent, and cDNA was further synthesized according to the manufacturer’s instruction. Real- time PCR was performed using a SYBR green premix according to the instruction. Relative expressions of target genes were standardized to Actin or GAPDH, evaluated by 2-ΔΔCt method and given as ratio compared with the control. The primer sequences used in this study are shown in Table 1.

Protein extraction
HHSECs were seeded into dishes. After attachment, cells were pre-incubated with or without CAT for 15 min, and then incubated with MCT for 72 h. After treatment, cellular proteins were isolated by using whole cell protein extraction kits. Cytosolic and nuclear proteins in cells or liver tissues were isolated as described in kits.
The protein concentration of samples was detected by BCA Protein Assay Kit, and all the samples in the same experiment were normalized to the equal protein concentration.

Western-blot analysis
Protein samples were separated by SDS-PAGE and blots were probed with appropriate combination of primary and horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized by enhanced chemiluminescence kits. The gray densities of the protein bands were normalized by using β-actin or Lamin B1 density as internal controls, and the results were further normalized to control.

Molecular docking analysis
Molecular docking analysis was performed as described in our previous study [28]. The confirmation of CAT is generated using Comformational Search (MMFF94X force field) in MOE2015.

Statistical analysis
Data were expressed as means ±standard error of the mean (SEM). The significance of differences between groups was evaluated by one-way ANOVA with LSD post hoc test, and P<0.05 was considered as statistically significant differences. Results CAT attenuated MCT-induced SOS in rats As shown in Figs.1A-C, CAT (40 mg/kg) obviously decreased the elevated serum ALT/AST activity and serum TBil and TBA amounts induced by MCT (90 mg/kg) in rats. CAT (40 mg/kg) alone had no obvious effect on serum ALT/AST activity and TBil and TBA amounts. The results of liver histological evaluation showed that MCT-treated rats showed severe liver damage, indicating by intrahepatic hemorrhage, lymphocytes infiltration, the dilatation and detachment of hepatic sinusoidal endothelial cells, and the destruction of liver structure (Fig.1D-b). After CAT treatment, all these phenomena were ameliorated (Fig.1D-c), which further confirmed the protection of CAT against MCT-induced liver injury. In addition, normal control rats and rats treated with CAT alone did not show any obvious liver damage. The results of hepatic MMP-9 expression showed that CAT (40 mg/kg) reduced the elevated expression of MMP-9 induced by MCT (90 mg/kg) in rats (Figs.2A-B). Next, scanning electron microscopy was used to evaluate the potential damage of hepatic sinusoidal endothelial cells (SECs). The results showed that MCT induced the dilatation of sinusoids, enlargement of fenestrae, exposure of the Disse space and the damage of endothelium (Fig.2C). However, all those phenomena were ameliorated in rats treated with CAT (40 mg/kg) (Fig.2C). CAT was found to reduce the increased number of rats with liver ascites induced by MCT in rats (Tab.2). Data in Tab.3 showed that CAT reversed the decreased number of platelets, reticulocytes, white blood cells and red cells induced by MCT. Moreover, MCT decreased the amount of hemoglobin in rat plasma, but CAT reversed such decrease (Tab.3). To assess the effects of CAT and MCT on rat plasma coagulation, PT, APTT and TT were detected. Data in Fig.2D showed that MCT (90 mg/kg) increased PT and APTT of rat plasma, but CAT (40 mg/kg) reversed such increase. MCT and CAT had no effect on TT of rat plasma. Additionally, MCT decreased the amount of FIB in rat plasma. The amount of FIB in plasma was restored to the normal level after rats were treated with CAT (40 mg/kg) (Fig.2E). CAT attenuated MCT-induced liver oxidative injury in rats As shown in Figs.3A-B, CAT (40 mg/kg) reduced the elevated liver MDA amount and ROS level induced by MCT (90 mg/kg) in rats. MCT decreased hepatic GSH/GSSG in rats, but CAT (40 mg/kg) reversed this decrease (Fig.3C).Data in Fig.3D showed that CAT reduced the increased amount of liver protein carbonylation induced by MCT in rats. MCT decreased liver GST activity, but CAT (40 mg/kg) reversed this decrease (Fig.3E). CAT induced the activation of Nrf2 signaling pathway in vivo To further search the critical signals involved in CAT-provided the protection against MCT-induced HSOS in rats, we observed the effects of CAT on the activation of Nrf2, which is the main and critical transcription factor regulating antioxidative responses [29-31]. As shown in Fig.4A, CAT (40 mg/kg) reversed the decreased hepatic mRNA expression of Nrf2 induced by MCT (90 mg/kg) in rats. The results of western-blot analysis showed that MCT decreased hepatic Nrf2 expression in both cytosol and nucleus, but CAT (40 mg/kg) reversed the reduced expression of Nrf2 in both cytosol and nucleus (Figs.4B-C). GCLC, GCLM, NQO1 and HO-1 are important antioxidative enzymes, and they are all Nrf2 target genes [29,30]. Further results showed that MCT reduced hepatic mRNA expression of GCLC, GCLM and NQO1, and increased HO-1 mRNA expression (Figs.4D-E). However, CAT (40 mg/kg) reversed MCT-induced the decrease in GCLC, GCLM and NQO1 mRNA expression and the increase in HO-1 mRNA expression (Figs.4D-E). The results of western-blot further evidenced that CAT (40 mg/kg) reversed the decreased expression of GCLM and NQO1, and the elevated HO-1 expression in liver tissues from MCT-treated rats (Figs.4F-G). MCT had no effect on hepatic GCLC expression, but CAT (40 mg/kg) increased GCLC expression in MCT-treated rats (Figs.4F-G). CAT attenuated MCT-induced cytotoxicity and induced Nrf2 activation in HHSECs MCT (10 mM) reduced the number of survival cells in HHSECs after incubated with cells for 72 h, but CAT (1-50 μM) reversed such decreased cell viability induced by MCT in a concentration-dependent manner (Fig.5A). CAT (50 μM) reduced the elevated cellular ROS level induced by MCT in HHSECs (Fig.5B). Further results showed that CAT (50 μM) reversed the decreased Nrf2 mRNA expression induced by MCT in HHSECs (Fig.5C). In addition, CAT (10, 25, 50 μM) also enhanced the nuclear translocation of Nrf2 in MCT-treated HHSECs, but had no obvious effect on the expression of cytosolic Nrf2 (Figs.5D-E). The involvement of GCL, HO-1 and NQO1 in CAT-provided the protection against MCT-induced cytotoxicity in HHSECs As shown in Figs.6A-B, MCT reduced cellular protein expression of GCLM and HO-1, but CAT reversed such decrease in a concentration-dependent manner. MCT enhanced cellular NQO1 expression but had no significant effect on GCLC expression, and CAT further enhanced cellular expression of GCLC and NQO1 in MCT-treated HHSECs (Figs.6A-B). Data in Fig.6C showed that MCT decreased mRNA expression of GCLC, GCLM, HO-1 and NQO1 in HHSECs, but CAT (50 μM) reversed such decrease. Next, GCL (composed of GCLC and GCLM subunits) inhibitor BSO, HO-1 inhibitor ZnPP and NQO1 inhibitor Dim were used, respectively. The results showed that BSO, ZnPP and Dim all abrogated CAT- provided the protection against MCT-induced cytotoxicity in HHSECs (Fig.6D). Results of Keap1 and p62 expression and molecular docking analysis Next, we observed the effects of CAT on the expression of Keap1 and p62 in vitro. As shown in Figs.7A-B, MCT alone and MCT plus CAT (10, 25, 50 μM) all had no obvious effect on cellular expression of Keap1 and p62 in HHSECs. Further, the molecular docking analysis was conducted to detect the potential interaction of CAT with Keap1 kelch domain. The chemical structure of CAT was shown in Fig.7C. The docking mode of CAT in the binding site of human Keap1 was illustrated in Fig.7D (Front view) and Fig.7E (Top View). The three-dimension (Fig.7F) and two-dimension (Fig.7G) interaction-map showed that CAT had an H-Bond interaction between the Ser508 bridging by one water molecule, and other two H-Bond interactions with Asn382 and Ser602. All those interactions help CAT anchored in the binding site of Keap1. Discussion HSOS induced by MCT in rats has been generally used for experimental HSOS study [13,32]. In this study, the results of serum ALT/AST activities, TBil and TBA amounts, and liver histological observation all evidenced the protection of CAT against MCT-induced liver injury in rats. HSOS is characterized by the damage on SECs and the degradation of basement membrane collagen mediated by matrix metalloproteinases such as MMP-9, and thus leads to the loss of endothelium [33]. Moreover, liver ascites is a classic symptom for HSOS in clinic. Next, the results of liver ascites detection and scanning electron microscopy observation, the change of blood cells and hepatic MMP-9 expression all demonstrated that CAT attenuated MCT-induced HSOSin rats. The balance of coagulation-fibrinolysis has already been shown to be critically involved in the development of HSOS, and promoting fibrinolysis with concomitant anticoagulation therapy has been a strategy for HSOS treatment [34]. However, it seems that only aiming at restoring coagulation-fibrinolysis balance was not sufficient enough for HSOS treatment in clinic. Recently, defibrotide was suggested for serve HSOS treatment due to its extensive biological functions including maintaining coagulation-fibrinolysis homeostasis and protecting endothelial cells [35]. APTT, PT, TT and FIB are four commonly used tests to evaluate blood coagulation. A previous study reported the occurrence of the prolonged APTT in clinical cases of HSOS [36]. Further, the results of PT, APTT and FIB amount in rat plasma all demonstrated that MCT led to the disorder of coagulation-fibrinolysis balance, but CAT reversed such disorder induced by MCT. These results further evidenced the protection of CAT against MCT-induced HSOS in rats. The occurrence of HSOS is reported to be associated with the free radical damage to SECs, and supporting cellular GSH amount that is the main antioxidant prevents MCT-induced HSOS in rats [37]. Moreover, a variety of reports showed that butylated hydroxyanisole, chlorogenic acid, quercetin, baicalein and sesame oil attenuated HSOS by inhibiting liver oxidative stress injury [38-41]. In this study, the results of ROS, MDA, GSH/GSSG, protein carbonylation and GST all evidenced that CAT attenuated MCT-induced liver oxidative stress injury in rats, and CAT also reduced MCT-induced the formation of cellular ROS in HHSECs. All these results imply that CAT can prevent MCT-induced HSOS by attenuating liver oxidative stress injury. Transcription factor Nrf2 regulates the expression of a variety of antioxidant enzymes by binding with the antioxidant response element (ARE) [29]. Recently, the important protective role of Nrf2 in various liver injuries was identified [31]. Next, the effects of CAT on Nrf2 activation in vivo and in vitro were observed. The results showed that CAT reversed the reduced mRNA expression of Nrf2 induced by MCT in rats and in HHSECs.CAT also increased the nuclear translocation of Nrf2 at the present of MCT in vivo and in vitro. A previous study showed that CAT had a protective effect on gastrointestinal ulcers by inhibiting oxidative injury via inducing Nrf2 activation [42]. This study evidenced the potential contribution of Nrf2 activation to CAT-provided the protection against MCT-induced HSOS. GCL, constituted by GCLC and GCLM subunits, is a rate-limiting enzyme for cellular GSH synthesis and is critical for maintaining cellular GSH homeostasis [43]. The expression of GCLC, GCLM and NQO1 was found to be regulated by Nrf2 [29]. CAT enhanced GCLC, GCLM and NQO1 expression both in livers from MCT- treated rats and in MCT-treated HHSECs. Moreover, GCL inhibitor BSO and NQO1 inhibitor Dim both reduced CAT-provided the protection against MCT-induced cytotoxicity in HHSECs. All these results imply that GCL and NQO1 may play important roles in regulating the protection of CAT against MCT-induced HSOS. HO-1 is another downstream antioxidant enzyme of Nrf2/ARE [29]. We found that CAT reversed the decreased expression of HO-1 induced by MCT, and HO-1 inhibitor ZnPP reduced CAT-provided the protection against MCT-induced cytotoxicity in HHSECs. These results indicate the important role of HO-1 in regulating the protection of CAT against MCT-induced damage on HHSECs. However, the results in rats showed that MCT enhanced hepatic HO-1 expression, which is totally different from the results obtained from HHSECs. The main reason leading to the difference may be due to the complication of in vivo experiment, and the responses of whole body is not only limited to SECs. Previous studies showed that HO-1 expression was not only dependent on Nrf2, but also regulated by other transcription factors including activating transcription factor 4 (ATF4), cAMP- response element binding protein (CREB) and forkhead box P3 (Foxp3) [44-46], that may be the reason why MCT reduced Nrf2 transcriptional activation but not decreased HO-1 expression in rats. Also, HO-1 induction is generally seen in liver injuries, which is helpful for the improvement of liver damage and down-regulation of pro-inflammatory cytokines [47]. In this study, MCT enhanced HO-1 expression in rats, which may be due to the body self-defensive capacity. Meanwhile, CAT reduced the increased HO-1 expression induced by MCT, which may attribute to the attenuation of HSOS. Keap1 is an inhibitor protein for Nrf2 activation through binding with Nrf2 and leading to its subsequent degradation, whereas p62 can bind with Keap1 and thus lead to the dissociation of Keap1 and Nrf2 [30,48]. Next results showed that CAT had no effect on the expression of Keap1 and p62 in HHSECs. Further results of molecular docking analysis indicate that CAT may interact with the Nrf2 binding site in Keap1 protein, which will lead to the dissociation Nrf2/Keap1 complex and thus cause Nrf2 transcriptional activation. In conclusion, this study showed that CAT attenuated MCT-induced HSOS by attenuating liver oxidative stress injury via inducing Nrf2-mediated transcriptional activation of downstream antioxidant enzymes including GCL and NQO1. Our results evidenced the huge potential for the development of (+)-Catechin hydrate as a candidate drug for HSOS treatment. This study also contributed to the promotion of the medicinal value of catechins.