Ginsenoside compound K alleviates sodium valproate-induced hepatotoxicity in rats via antioxidant effect, regulation of peroxisome pathway and iron homeostasis

Journal Pre-proof Ginsenoside compound K alleviates sodium valproate-induced hepatotoxicity in rats via antioxidant effect, regulation of peroxisome pathway and iron homeostasis

Luping Zhou, Lulu Chen, Xiangchang Zeng, Jianwei Liao, Dongsheng Ouyang PII:

S0041-008X(19)30437-5

DOI:

https://doi.org/10.1016/j.taap.2019.114829

Reference:

YTAAP 114829

To appear in:

Toxicology and Applied Pharmacology

Received date:

19 July 2019

Revised date:

13 November 2019

Accepted date:

13 November 2019

Please cite this article as: L. Zhou, L. Chen, X. Zeng, et al., Ginsenoside compound K alleviates sodium valproate-induced hepatotoxicity in rats via antioxidant effect, regulation of peroxisome pathway and iron homeostasis, Toxicology and Applied Pharmacology (2019), https://doi.org/10.1016/j.taap.2019.114829

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© 2019 Published by Elsevier.

Journal Pre-proof Ginsenoside compound K alleviates sodium valproate-induced hepatotoxicity in rats via antio xidant effect, regulation of peroxisome pathway and iron homeostasis Luping Zhou 1,2,3,4 , Lulu Chen 1,2,3,4,5 , Xiangchang Zeng 1,2,3,4 , Jianwei Liao 1,2,3,4 , Dongsheng Ouyang 1,2,3,4,5# 1

Department of Clin ical Pharmaco logy, Xiangya Hospital, Central South University, 87 Xiangya Road,

Changsha 410008, P. R. China; 2

Institute of Clin ical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, 110

Xiangya Road, Changsha 410078, P. R. China; 3

Engineering Research Center of Applied Technology of Pharmacogenomics, M inistry of Educa tion, 110

National Clin ical Research Center for Geriatric Disorders, 87 Xiangya Road, Changsha 410008, Hunan, P.R.

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4

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Xiangya Road, Changsha 410078, P. R. China;

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China;

Hunan Key Laboratory for Bioanalysis of Complex Matrix Samples, Changsha, Hunan, 410000, P.R. China.

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Present address: Luping Zhou, Lulu Chen, Xiangchang Zeng, Jianwei Liao, and Dongsheng Ouyang,

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Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, China. The number of words: 5053 The number of figures: 8

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Running title: Effect of G-CK on the hepatotoxicity of SVP

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Name and E-mail address of the corresponding author: Dongsheng Ouyang; [email protected]; Tel:

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13307313736; Fax: (86) 0731-82354476

Journal Pre-proof Abstract Sodiu m valp roate (SVP) is a first-line treat ment for various forms of ep ilepsy; however, it can cause severe liver injury. Ginsenoside compound K (G-CK) is the main act ive ingredient of the traditional herbal med icine g inseng. According to our previous res earch, SVP-induced elevation of ALT and AST levels, as well as pathological changes of liver tissue, was believed to be significantly reversed by G-CK in LiCl-pilocarp ine induced epileptic rats. Thus, we aimed to evaluate the protective effect o f G-CK on hepatotoxicity caused by SVP. The rats treated with SVP showed liver injury with evident increases in hepatic index, transaminases activity, alkaline phosphatase level, and lipid pero xidation; a significant decreases in serum albu min level and antioxidant

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capacity; and obvious changes in histopathological and subcellular structures. All of these changes could be

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mitigated by co-administration with G-CK. Proteomic analysis indicated that hepcidin, soluble epoxide

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hydrolase (sEH, Un iProt ID P80299), and the peroxiso me pathway were involved in the hepatoprotective effect of G-CK. Changes in protein exp ression of hepcidin and sEH were verified by ELISA and Western blot analysis,

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respectively. In addition, we observed that the hepatic iron rose in SVP group and de creased in the combination

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group. In summary, our findings demonstrate the clear hepatoprotective effect of G-CK against SVP-induced hepatotoxicity through the antio xidant effect, regulation of pero xisome pathway rely ing on sEH (P80299) downregulation, as well as regulation of iron homeostasis dependent on hepcidin upregulation.

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Iron

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Keywords: Sod iu m valproate; Ginsenoside compound K; Hepatotoxicity; Hepcid in; So luble epo xide hydrolase;

Journal Pre-proof 1. Introduction Valproic acid and its salts, as one class of the most prescribed antiepileptic drugs, are nonspecific h istone deacetylase inhibitor. They have been used for more than 30 years as the first -line treat ment for various forms of epilepsy because of their broad spectrum of anticonvulsant activities and affordability (Bath and Pimentel 2017). However, chronic admin istration of sodium valproate (SVP) is accompanied by numerous side effects. Hepatotoxicity is widely regarded as the most serious side effect and is identified as the third most common cause of drug-induced liver fatalities by the World Health Organization (Vidaurre et al. 2017). Nearly 61% of patients treated with chronic ad ministration of valproic acid and its salts have been diagnosed with hepatic

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steatosis, and nearly 25% have developed non-alcoholic fatty liver d isease (Bai et al. 2017). The mechanism of liver injury induced by SVP re mains unknown. Oxidative stress (Hamzawy et al. 2018), mitochondrial

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dysfunction (Komu lainen et al. 2015), and abnormal hepatic lip id metabolis m (Bai et al. 2017) are involved in the hepatotoxicity of SVP. SVP is still widely used because of its confi rmed pharmaco logical advantages and

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effectiveness; thus, a significantly efficient treat ment for the hepatotoxicity of sodiu m valproate needs to be

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developed urgently.

Ginsenoside compound K (20-O-beta-D-g lucopyranosyl-20(S)-protopanaxadio l; also known as M1, compound K, IH901) is a main active metabolite of 20(S) -protopanaxadiol type ginsenoside (Kim 2013). Over

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the past several decades, ginsenoside compound K (G-CK) exh ibited mu ltiple pharmacological activit ies, such

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as antitumor, anti-angiogenesis, anti-in flammation, anti-allergic, and hepatoprotective effects (Yang et al. 2015).

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However, G-CK has not broadened its worldwide market. Ginsenoside Co mpound K Tablet is currently being tested as a candidate drug for rheumatoid arthritis (RA) by Hisun Pharmaceutical Co., Ltd. (Taizhou, Zhejiang, China). Results from pre -clinical and phase I clinical trials (Registration number: ChiCTR-TRC-14004824 and ChiCTR-IPR-15006107, http://www.ch ictr.org.cn/index.asp x) of G-CK have helped understand its clinical application and suggested that G-CK exh ibits favorable druggability (Chen et al. 2017a; Chen et al. 2017b; Zhou et al. 2018). G-CK has hepatoprotective activity against liver injury induced by tert-butyl hydroperoxide, CCl4, or acetaminophen (Igami et al. 2015; Lee et al. 2005; Li et al. 2011) and shows positive effects on non-alcoholic fatty liver d isease by exert ing its anti-fib rotic effect, decreasing fatty acid synthesis, and increasing fatty acid o xidation (Chen et al. 2017c; Hwang et al. 2018). Moreover, in this study about the effects of G-CK against epilepsy (Zeng et al. 2018) we also found that G-CK could significantly reverse elevated ALT and AST levels, as well as the pathological changes in liver tissue induced by SVP in LiCl -pilocarpine induced epileptic rats (Supplementary Material S1).

Journal Pre-proof On the basis of these findings, the present study aims to exp lore the protective effect and potential molecular mechanisms of G-CK on SVP-evoked hepatotoxicity. 2. Materials and methods 2.1 Materials Ginsenoside compound K with 98% purity was provided by Hisun Pharmaceutical Co., Ltd. (Taizhou, Zhejiang, Ch ina). SVP was supplied by Hunan Xiangzhong Pharmaceutical Co., Ltd. (Shaoyang, Hunan, China). L(-)-Carn itine (L-Car) as the positive contrast medicine was purchased from the Dalian Meilun Biotechnology Co., Ltd. (Dalian, Liaoning, China). SVP and L-Car were d issolved in physiological saline, and G-CK was

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prepared as a suspension in 0.5% sodium carbo xy methylcellulose (CM C-Na) for intragastric (i.g.) administration. The kits used to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST),

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alkaline phosphatase (ALP) activ ity, albu min (A LB) concentration, and triglyceride (TG) level were purchased fro m Changchun Huili Biotech Co., Ltd. (Changchun, Jilin, China). The kits used to assess hepatic

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malondialdehyde (MDA) content, superoxide dismutase (SOD) activ ity, catalase (CAT) activity, glutathione

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peroxidase (GPx) activ ity, glutathione (GSH) concentration, and iron level were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The ELISA assay kits for heme o xygenase -1 (HO-1) and hepcidin were procured fro m Wuhan Huamei Biotech Co., Ltd. (Wuhan, Hubei, China). Antibodies for sEH was

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2.2 Experimental animals

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provided by Affinity Biosciences, USA.

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Seventy SPF male Sprague Dawley rats (weight 200 g–250 g, 8–10 weeks old) fro m the Hubei Research Center of Laboratory Animals (Wuhan, China) were housed in a roo m under the following conditions: temperature, 20°C–22°C; hu mid ity, 70%–75%; 12/ 12 h light-dark cycle; and free access to normal food and water. The experimental p rotocols were approved by the Lab oratory Animal Ethics Co mmittee of Serv icebio, in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications NO. 80-23, revised 1996). 2.3 Experimental design Rats were rando mly d ivided into s even groups (10 rats per group): the 0.5% CM C-Na solvent control group (Con), h igh-dose G-CK g roup (HCK), sodium valproate group (SVP), co mbined lo w-dose G-CK and SVP group (LCK + SVP), co mbined middle -dose G-CK and SVP group (M CK + SVP), co mbined HCK and SVP group (HCK + SVP), and co mbined L-Car and SVP group (L-Car + SVP). The 0.5% CM C-Na, G-CK, and L-Car were ad ministered once daily, and physiological saline and SVP were ad ministered twice daily fo r 15

Journal Pre-proof days. All treat ments were given via the intragastric route, and the volu me was 5 mL/ kg. The time and dosage of administration are as follows:

Administration (intragastric) 8:00 a.m.

8:00 p.m.

Con

0.5%CMC-Na

Physiological saline

Physiological saline

HCK

320 mg/kg G-CK

Physiological saline

Physiological saline

SVP

0.5%CMC-Na

500 mg/kg SVP

500 mg/kg SVP

LCK + SVP

80 mg/kg G-CK

500 mg/kg SVP

500 mg/kg SVP

MCK + SVP

160 mg/kg G-CK

500 mg/kg SVP

500 mg/kg SVP

HCK + SVP

320 mg/kg G-CK

500 mg/kg SVP

500 mg/kg SVP

L-Car + SVP

500 mg/kg L-Car

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500 mg/kg SVP

500 mg/kg SVP

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2.4 Collection of blood and tissue samples

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7:30 a.m.

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Group

All rats were weighed 24 h fo llo wing the last SVP dose. Blood samples were withdrawn fro m the retro -orbital

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venous plexus of the rats before they were euthanized by inhalation of CO2 delivered at a rate of between 10%

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and 30% of the chamber volu me per minute, and then collected in tubes containing EDTA. Plas ma was separated by refrigerated centrifugation (3000 rp m, 4°C, 10 min) before they were transferred to labeled storage tubes for biochemical assay. Subsequently, the liver were removed immed iately, rinsed properly in ice-cold physiological saline, blotted dry using a sterile gauze, and weighed. Part of the hepatic tissue was then immed iately immersed in an appropriate fixative for fu rther histopathological study and t ransmission electron microscopy study of the liver. The remaining part of the liver was quick-frozen in liquid nitrogen and then stored at -80°C for biochemical assay. 2.5 Assessment of hepatic index The body weight and liver weight of each animal fro m every group were recorded. The liver weight -to-body weight ratio was calculated as the hepatic index. 2.6 Assessment of hepatic function biomarkers Liver function was assessed by estimating the levels of hepatic function biomarkers, including ALT, AST,

Journal Pre-proof ALP, and ALB by using commercially available kits (Changchun Huili Biotech Co., Ltd., China). 2.7 Histopathology and transmission electron microscopy of liver The livers were fixed with 4% paraformaldehyde for one night for h istopathological examination. The specimens were embedded in paraffin b locks, cut into 5 μm sections, and stained using a hemato xylin -eosin staining kit. The p repared slides were examined rando mly under a light microscope to assess SVP -induced histopathological changes and responses to G-CK ad min istration. The histopathologist was blinded to the experimental groups. The liver tissues (1 mm3 blocks) were fixed in 2.5% glutaraldehyde for more than 2 h at 4°C to observe the

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subcellular structures. After dehydration and embedding, the blocks were stained and cut into ultrathin sections (60 n m–80 n m) to examine the subcellular structures of the h epatic cell by transmission electron microscopy

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(TEM).

2.8 Assessment of hepatic triglyceride and oxidative/antioxidant stress biomarkers

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Liver homogenate was prepared using a t issue grinder. The homogenate was centrifuged (4000 rp m fo r 10

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min), and the supernatant was separated and immed iately used to assess the hepatic TG content, MDA content, SOD act ivity, CAT activ ity, GPx act ivity, and GSH concentration by using commercially available kits (Nanjing Jiancheng Bioengineering Institute, China) in accordan ce with the instructions provided by the manufacturer.

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HO-1 was assessed using the Rat HO-1 ELISA Assay Kit (Wuhan Huamei Biotech Co., Ltd., China) in

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accordance with the instructions provided by the manufacturer.

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2.9 Proteomic analysis and verification

Three liver t issue samples fro m the Con, SVP, and HCK + SVP groups were used for proteomic analysis to screen differentially expressed proteins (DEPs). The proteomic analysis is described in detail in Supplementary Material S2.

The proteomic analysis was verified by Western blot analysis and ELISA analysis. The liver tissues were washed with cold phosphate buffered saline, crushed with a t issue grinder, and lysed for 30 min with a RIPA lysis buffer containing protease and phosphatase inhibitor cocktails. Afte r incubation, the samples were centrifuged at 12,000 rp m for 15 min at 4°C, and supernatants were collected and stored at -80°C. The protein concentration in the supernatant was analyzed using bicinchoninic acid assay, and the pro tein expression of sEH was detected by Western blot analysis. An Enhanced Chemilu minescence Detection kit was used for band detection, and densitometric scanning was performed using the software Quantity One (Bio -Rad, USA). The Rat Hepcidin ELISA kit (Wuhan Huamei Biotech Co., Ltd. , China) was used to detect the levels of hepcidin in

Journal Pre-proof serum and liver. All operations were conducted in accordance with the instructions provided by the manufacturer. 2.10 Detection of iron level The iron levels in serum and liver were assessed using the Iron Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) in accordance with the instructions provided by the manufacturer. 2.11 Statistical analysis All results were expressed as mean ± SEM and analy zed using SPSS (v.22.0) and R studio (v.3.1.1). One-way analysis of variance followed by the least significant difference test, independent -samples t-test with the

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Benjamin i–Hochberg adjustment, and Fisher's exact test were used for statistical evaluation. Statistical significance was accepted at p <0.05.

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3. Results

3.1 Ginsenoside compound K protects rats from sodium valproate-induced liver injury

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3.1.1 Effect on hepatic index

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We exp lored whether differences in treat ment affect the hepatic index of rats in the seven groups. In Fig. 1, SVP ad ministered at 500 mg/ kg, i.g. twice daily for 15 days led to a significant increase in hepatic index relative to that of the Con g roup (fold change = 1.2). Co mpared with that of the SVP group, the hepatic index of the

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LCK + SVP, M CK + SVP, and HCK + SVP groups markedly declined by 7.6%, 8.7%, and 9.4% respectively

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(Fig. 1). The co-administration of L-Car did not restrain the SVP-induced increase in hepatic index.

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3.1.2 Effect on hepatic function biomarkers

The administration of SVP caused significant hepatic damage, as indicated by a significant increase in plas ma transaminases (ALT and AST, p <0.0001) and ALP (p <0.0001) and a decrease in plas ma A LB (p <0.05) relative to those of the Con group with the following fold changes: approximately 1.7 (62.64 ± 4.26 U/L vs. 37.02 ± 0.94 U/L), 2.8 (198.30 ± 24.77 U/L vs. 70.07 ± 1.65 U/ L), 1.7 (22.82 ± 1.04 U/ L vs. 13.40 ± 0.75 U/L), and 0.8 (34.18 ± 1.11 g/ L vs. 39.58 ± 0.30 g/L). G-CK alone exerted no effect on thes e hepatic function bio markers. Co-ad ministration of LCK, M CK, HCK, or L-Car marked ly limited the increased ALT, AST, and ALP activit ies, and the decreased ALB content caused by SVP (all p <0.05). In HCK + SVP and L-Car + SVP groups, the values of all measures could be close to those of the Con group. All aforementioned results are shown in Fig. 2. 3.1.3 Effect on histopathological changes Histopathological examination of liver slides fro m the Con and G-CK groups showed that the histological structure of hepatic tissue was normal with no signs of lesion. The SVP-treated liver showed microvesicular

Journal Pre-proof steatosis, mild necrosis, and a dilated central vein. By contrast, the hepatic pathological changes caused by SVP were significantly prevented with the co-admin istration of G-CK or L-Car (Fig. 3a), which is consistent with the results of plasma biochemical analysis. 3.1.4 Effect on hepatic triglyceride level The SVP-treated rats exhibited a significant increase in hepatic TG level relative to that of the Con group (0.27 ± 0.02 mmo L/g vs. 0.22 ± 0.01 mmo L/g, fo ld change = 1.2, p <0.05). The hepatic TG level was significantly reduced by 25%, 22%, 30%, and 38% (p <0.01, 0.05, 0.001, 0.0001) in the LCK + SVP, M CK + SVP, HCK + SVP, and L-Car + SVP groups relative to those in the SVP group (Fig. 3b).

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3.1.5 Effect on subcellular structures of liver tissues The subcellular structures of the liver tissues were examined by TEM, and the results are presented in Fig. 4.

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The hepatocellular nuclei were regular, the mitochondria an d rough endoplasmic ret icula (RER) were abundant, and the mitochondrial cristae were normal in the Con and HCK g roups. In the SVP -treated rats, less dense

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cytoplasmic and mitochondrial matrix, fewer mitochondria and RERs, shortened or absent mitochondrial cristae,

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expanded inter-ridge gap, irregular nuclei, and lip id droplet were observed. In the rats treated with co mb ined SVP and G-CK, the nu mber of mitochondria and RERs increased relat ive to those in the rats solely treated with SVP, as well as the mitochondrial and nuclear structures were nearly normal.

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3.2 Ginsenoside compound K can relieve liver injury by enhancing the antioxidant defense system

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3.2.1 Effect on hepatic lipid peroxidation

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Malondialdehyde is a product of lipid pero xidation. The hepatic M DA level in the SVP group significantly increased relative to that in the Con group (0.64 ± 0.05 mmo L/ mgprot vs. 0.40 ± 0.03 mmo L/ mgprot, p <0.0001). Meanwhile co-ad min istration of LCK, MCK, HCK or L-Car significantly suppressed the increase in M DA level induced by SVP (p <0.05, <0.05, <0.0001, <0.0001). LCK and MCK reduced the M DA content by 21% and 25%, respectively, co mpared to those of the SVP group (0.51 ± 0.02 mmo L/ mgprot and 0.49 ± 0.01 mmo L/ mgprot vs. 0.64 ± 0.05 mmo L/ mgprot). With HCK and L-Car ad min istration, the MDA level (0.31 ± 0.02 mmo L/ mgprot, 0.27 ± 0.01 mmo L/ mgprot) almost reached that of the Con group. The result of hepatic MDA level is shown in Fig. 5. 3.2.2 Effect on hepatic antioxidants As shown in Fig. 5, SVP induced significant decreases in CAT, GPx, and SOD activ ities as well as the GSH level, relative to those in the Con group by appro ximately 38% (33.66 ± 1.16 U/gprot vs. 47.47 ± 1.86 U/gprot), 34% (1813.94 ± 107.02 μmol/gprot vs. 2771.57 ± 273.81 μmo l/gprot), 36% (788.50 ± 33.88 U/ mgprot vs.

Journal Pre-proof 1231.59 ± 98.28 U/ mgprot), and 46% (11.02 ± 0.66 μmo l/gprot vs. 20.30 ± 1.89 μmo l/gprot). Co-ad ministration of LCK, M CK, HCK, or L-Car attenuated SVP induced reductions in CAT, GPx, and SOD activit ies as well as the GSH level. In the MCK + SVP and HCK + SVP groups, the CAT, GPx, and SOD activ ities as well as the GSH level were close to those of the Con group. Administration of SVP t wice daily induced decreasing trend in hepatic HO-1 content relative to that of the normal control; however, no significant difference was indicated. Meanwhile, the hepatic HO-1 contents in co mbined G-CK and SVP as well as co mbined L-Car and SVP groups had an increasing trend compared to SVP group; similarly, no significant difference was indicated. 3.3 Proteomic analysis and verification

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3.3.1 Differentially expressed proteins To explore the potential molecular mechanis ms of G-CK in SVP-induced liver in jury, the proteomic method

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was used for screening of DEPs in SVP vs. Con, and HCK + SVP vs. SVP. A total of 255 proteins significantly changed in the SVP group co mpared to the Con group (change fold >1.5). Moreover, 77 proteins significantly

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changed in the HCK + SVP group relative to the SVP group (change fold >1.5). Fifteen proteins changed in the

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SVP group relat ive to the Con group and were corrected when G-CK was co-administered with SVP. These proteins (change fold >1.5, unique peptides ≥2) might be the target of G-CK. Their UniProt accession number, protein name, gene name, and protein expression ratio are listed in Table 1.

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3.3.2 Analysis of GO and KEGG pathways

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The results of gene ontology (GO) enrich ment analysis were used to classify the cellular co mpo nents,

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mo lecular functions, and biological processes in which these DEPs were involved. The results are presented in Supplementary Material S3. No intersection of cellu lar co mponents, molecu lar functions, and biological processes was found between the downregulated proteins in SVP vs. Con and the upregulated proteins in HCK + SVP vs. SVP, and between all DEPs in SVP vs. Con and HCK + SVP vs. SVP. Ho wever, two enriched biological processes of the upregulated proteins induced by SVP, carbo xylic acid metabolic process (GO: 0019752), and oxoacid metabolic process (GO: 0043436, ch ild term of the carbo xylic acid metabolic process) were partly prevented by co-administration of G-CK. The co mmon DEPs of SVP vs. Con and HCK + SVP vs. SVP, enriched in the two aforementioned biological processes, were P80299, Q6SKG1, P18886, and F1LM K6. The KEGG pathway analysis indicated that the rno04146 pero xisome pathway was the overlapped pathway between SVP vs. Con and HCK + SVP vs. SVP (Supplementary Material S4), and the P80299 (s EH, coded by Ephx2 gene) was the overlapped DEP. On the basis of the GO enrich ment analysis and KEGG pathway analysis, P80299 was regarded as a potential target of G-CK. Data on P80299 are listed in Table 1.

Journal Pre-proof 3.3.3 Analysis of protein-protein interaction The protein-protein interaction (PPI) analysis is presented in Fig. 6. Red nodes represent DEPs in SVP vs. Con, green nodes represent DEPs in HCK + SVP vs. SVP, and yellow nodes represent DEPs at the intersection. Module analysis with the plugin M CODE were mapped using the software Cytoscape (v.3.7.1). The cluster with the highest score was selected, and these DEPs including sEH were enriched in the peroxisome pathway. 3.3.4 Effect on soluble epoxide hydrolase protein level The Western blot analysis results for sEH are presented in Fig. 7A. The figure shows that the protein expression of sEH in the SVP group was 4 t imes that of the Con group (p <0.001); meanwh ile, the protein

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expression of sEH in the LCK + SVP group was lower than that in the SVP group (p <0.05). The inhibit ions of MCK and HCK on sEH expression were weaker than that of LCK. Moreover, sEH exp ression in the HCK +

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SVP group was significantly higher than that of the Con group and was even close to that of the SVP group. Coincidentally, the proteo mic analysis results showed that Q5RKK3 exp ression in the HCK + SVP group was

Q5RKK3,

as

a

potential

isoform

of

P80299,

is

also

coded

by

the

Ephx2

gene

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The

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twice that in the Con group (p = 1.53E-05), and SVP alone did not affect the expression of Q5RKK3 (Fig. 7B).

(https://www.uniprot.org/uniprot/P80299#subcellular_ location). Only three amino acid residues in Q5RKK3

3.3.5 Effect on hepcidin and iron level

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vary from those in P80299 (Supplementary Material S5).

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Hepcidin (coded by Hamp) was a DEP at the intersection, which could be released into the blood after

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synthesis in the liver and influenced iron homeostasis. Therefore, plasma and hepatic hepcidin levels were also detected using the ELISA kit. Co mpared with the Con group, the SVP group showed lower plasma and hepatic hepcidin levels (p <0.05 fo r all). All three d ifferent doses of G-CK inhibited the decreases in plasma and hepatic hepcidin levels induced by SVP (all p <0.05), but this effect was not exhibited by L-Car (Fig. 8). Hepcidin might play a vital role in SVP-evoked liver injury and the protective effect of G-CK against the hepatotoxicity of SVP. In this study, the iron levels in plasma and liver were also assessed because of the regulating effect of hepcidin on iron ho meostasis in the body. The results revealed that SVP only caused an upward trend in p lasma iron level. However, the SVP ad min istration could evoke significant upregulation in hepatic iron level (p <0.01). Co mpared with the SVP g roup, the hepatic iron level of LCK + SVP, M CK + SVP, and HCK + SVP groups decreased significantly (p <0.01, <0.05, <0.001). The results are presented in Fig. 8. 4. Discussion

Journal Pre-proof Hepatotoxicity is the most serious side effect of valproic acid and its salts, limiting their clin ical applicatio n. STAVZOR (valproic acid delayed release capsules) received a black bo x warning for the hepatotoxicity fro m the United States Food and Drug Administration in 2014. Owing to the high potency and low cost -effectiveness ratio, SVP is still frequently prescribed as first-line anti-ep ileptic medicat ion. Moreover, its use has been expanded to various applications, such as the treatment of bipolar d isorder and migraine headaches. Consequently, high demand of SVP has been maintained. Therefore, researches into dru gs and liver support agents to reduce SVP-induced liver to xicity, in addition to the study on the mechanism of SVP -induced liver toxicity, are crucial and have been a significant concern. This study demonstrated that G-CK exerts a pro minent

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hepatoprotective effect on SVP-induced liver injury. The plasma A LT, AST, A LP, and ALB are routinely mon itored to evaluate liver function. Any increase in ALT,

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AST, and A LP or decrease in ALB level is considered as the indication of liver in jury and dysfunction. AST and ALT can be released into the circulation after cellu lar damage, and an increase in ALP indicates an obstruction

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in bile flo w cholestasis pressure (Al-A moudi 2017). Decrease in A LB level indicates an impaired ability of

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hepatic cell to synthesize proteins (Yang et al. 2018). SVP-med iated liver in jury is div ided into Type I, wh ich is characterized by a dose-dependent elevation in serum liver enzy mes and a decline in A LB, and Type II, which is man ifested as a fatal, irreversible id iosyncratic reaction (Abdel-Dayem et al. 2014). Biochemical analyses

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showed that SVP markedly increased the concentrations of ALT, AST, and A LP, and decreased the plasma A LB

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level. When G-CK was co-ad ministered with SVP, rats exh ibited reduced plasma A LT, AST, and ALP levels and

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elevated ALB level. This effect indicates the utility of G-CK in maintaining liver cell integrity, improvement of biliary outflow, and enhancement of protein synthesis. Current h istopathologic studies provide that G-CK amelio rates SVP-evoked microvesicular steatosis, decreased cytoplasmic and mitochondrial matrix density, necrosis, and dilated central vein . Valproate has been reported to cause evident accumulation of TG in the liver with no effect and even a down -regulation effect on serum TG (Bai et al. 2017; Eiris et al. 1995; Manimekalai et al. 2014; Niko laos et al. 2004) . In the current study, rats treated with SVP showed significantly higher hepatic TG level than that of the Con group. When the rats received the combination treatment of G-CK, their hepatic TG level were significantly reduced, relative to that of the SVP group. G-CK has been confirmed to modulate fatty acid-induced lipid droplets fo rmation and TG accumulat ion (Kim et al. 2013). The results of the present study corroborated the view that G-CK could significantly inhibit TG accumulation and lipid droplet formation induced by SVP.

Journal Pre-proof The hepatotoxicity of SVP has been considered as a result of interference with mitochondrial beta -oxidation, restraint of mitochondrial respiration, and triggering of mitochondrial dysfunction (Jafarian et al. 2013; Ko mulainen et al. 2015; Ponchaut and Veitch 1993; Silva et al. 2008) . Detection of subcellular structures by TEM revealed that SVP caused the mitochondrial dysfunction characterized by a decline in the number of mitochondria, shortened or absent mitochondrial cristae, and expansion of the inter-ridge gap. G-CK exhib ited an overt reverse effect on SVP-evoked mitochondrial dysfunction. The possible mechanisms by wh ich G-CK reduced SVP-evoked liver in jury were identified. The sequential relationship between oxidative stress and mitochondrial dysfunction remains unclear. Ho weve r, increased

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oxidative stress certainly causes mitochondrial dysfunction manifested as lipid pero xidation, protein o xidation, and mitochondrial DNA mutations, and mitochondria are largely involved in the occurrence of oxidative stress

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(Lenaz 1998). Imbalance between pro-o xidants and antioxidants leads to oxidative stress (Rahal et al. 2014), which is a major indicat ion of SVP-induced liver in jury and is characterized by an increase in hepatic lipid

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peroxides, a rise in total o xidant levels, and a decrease in endogenous antioxid ants (Ahmed et al. 2018; Chang

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and Abbott 2006). The statuses of endogenous antioxidants (as indicators for oxidative stress level) and MDA content (as a marker of lip id pero xidation causing damage to hepatocytes) were determined in the current study. Our results reported that SVP caused a decline in hepatic SOD, CAT, and GPx activ ities as well as GSH level, in

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addition to the increase in hepatic MDA level. Co-ad ministration with three different-doses of G-CK pro mpted

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increases in SOD, CAT, GPx, and GSH and decrease in MDA level, thus alleviat ing SVP-induced oxidative

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stress. HCK markedly replenished the hepatic antioxidant level and reduced the lip id pero xide (MDA) level to nearly normal level. These antioxidant effects of G-CK were consistent with previous studies performed in other animal models (Li et al. 2011; Shao et al. 2015; Yang et al. 2019). In addit ion, SVP (700 mg/ kg, i.p.) twice daily was reported to induce a significant decrease in hepatic HO-1 content relative to that of the normal control (Nazmy et al. 2017). The hepatic HO-1 content was also detected in the present study by ELISA, and the results suggested that SVP could only lead to a downward trend in hepatic HO-1 content. This result might be related to the variations in dosage and administration of SVP. The proteomic analysis showed that the expression levels of 15 proteins (unique peptides ≥2) and sEH (unique peptides = 1) were significantly different between the Con and SVP groups and reversed by HCK (fold change >1.5). The GO, KEGG pathway, and PPI analysis made us focus on sEH, which is a bifunctional enzy me encoded by Ephx2. sEH can metabolize endogenous aliphatic and aromatic epo xides, and plays an important role in regulating cellular redo x ho meostasis (Fretland and Omiecinski 2000). Both the expression and activity

Journal Pre-proof of sEH are influenced by a wide array of xenobiotics. Disruption or inhibit ion of sEH prevents the development of hypertension, atherosclerosis, heart failure, fatty liver, and mult iple organ fibrosis (He et al. 2016; Schuck et al. 2014; Yao et al. 2019). Moreover, s EH rapid ly hydrolyzes epoxyeicosatrienoic acids (EETs) to their corresponding diols, the less biologically active dihydro xyeicosatrienoic acid (Schuck et al. 2014). EETs are metabolites of arachidonic acid v ia cytochrome P450 (CYP) epo xygenase, which possess anti-inflammatory and antioxidant effects and inhib it the loss of mitochondrial function (Huang and Sun 2018; Lee et al. 2013; Schuck et al. 2014). Many physiological processes of sEH have recently been demonstrated to be associated with the regulation of endogenous epoxy fatty acids such as EETs (Inceoglu et al. 2007; Lee et al. 2013; Liu et al. 2011).

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In the current study, the expression of sEH in the SVP group was markedly h igher than that of the Con group , as determined by proteomic analysis and Western blot analysis. This upregulation of sEH (P80299) was evidently

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ameliorated by G-CK. Notably, the results of Western blot analysis indicated that sEH expression in the HCK group was significantly higher than that of the Con group. In addit ion, the proteomic study showed that

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Q5RKK3 expression was markedly h igher in the HCK + SVP group than in the SVP group and was not affected

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by SVP alone. On the basis of this finding, we hypothesized that G-CK could inhib it the P80299 upregulation induced by SVP, and pro mote the expression of Q5RKK3. Q5RKK3 is a potential isoform of P80299 and Western blot analysis fails to distinguish P80299 and Q5RKK3. This might be the reason that sEH exp ression

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showed an increasing trend in the MCK + SVP and HCK + SVP groups relative to the LCK + SVP group, as

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well as the HCK g roup showed a marked increase in sEH protein level relative to the Con group. Whether a

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difference in function exists between P80299 and Q5RKK3 has yet to be determined; regardless, understanding the role of sEH in SVP-induced liver injury and the hepatoprotective effect of G-CK is valuable. Another notable finding is that G-CK could correct the downregulation of plasma and hepatic hepcidin caused by SVP in our results. Hepcidin is a peptide hormone produced and released by the liver and is identified as a master regulator of iron homeostasis in the body by inhibiting the intestinal absorption of iron (Kamei et al. 2013). Decreased hepcidin in mice leads to increased liver iron content (Amin et al. 2017). Iron is a widely known pro-o xidant metal and plays a central role in pro moting o xidative stress. The increased concentration of iron is associated with the elevated ALT level and liver disease development (Basaranoglu et al. 2013; Bessone et al. 2019). Prev ious results indicated that valproate could lead to marked hepatic iron accu mulation and increased plasma iron level (Kane et al. 1992; Sarangi et al. 2014). Therefore, the plas ma and hepatic iron levels were tested in the present study, and the current results revealed that the rats treated with SVP alone exhib ited an upward trend in plas ma iron level and a significant rise in hepatic iron level. Meanwh ile, G-CK markedly

Journal Pre-proof suppressed the upregulation of iron content caused by SVP. Therefo re, iron and hepcidin were thought to be involved in SVP-induced hepatotoxicity and the hepatoprotective effect of G-CK. The association of hepcidin and sEH (P80299) with liver injury caused by SVP has not been reported. The present study suggested that hepcidin and sEH (P80299) were involved in SVP -induced liver in jury, which requires confirmat ion by further research. This study also sho wed that the CD36 significantly increased (SVP vs. Con rat io = 2.15) after t reat ment with SVP, which was consistent with the literature (Bai et al. 2017). However, in the HCK + SVP group, the expression of CD36 only exh ib ited a downward trend. The aforementioned results suggest that CD36 does not play an important role in the hepatoprotective effect of G-CK.

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In addition, the DEP with the greatest change in expression level was C-X9-C mot if-containing 4 (coded by Cmc4) with the SVP vs. Con rat io = 0.18, and the HCK + SVP vs. SVP rat io = 7.81. G-CK reversed the change

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in Cmc4 expression induced by SVP. The coding protein of Cmc4 is merely a predicted protein in Rattus norvegicus, as determined fro m the Un iProt database; thus the study of Cmc4 currently presents a challenge.

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The subcellular location of C-X9-C mot if-containing 4 was predicted to be in mitochondrial intermemb rane

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space according to the biological aspect of the ancestor (https://www.unip rot.org/uniprot/A0A0G2K2W2), suggesting that Cmc4 may play a role in the hepatotoxicity of SVP and the protective effect of G-CK. In conclusion, our findings demonstrate the clear hepatoprotective effect of G-CK on SVP-induced

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hepatotoxicity. These favorable effects were med iated by suppressing oxidative stress via the inhibition of lipid

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peroxidation and upregulation of the antioxidant defense system, regulat ion of pero xis ome pathway relying on

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sEH (P80299) downregulation, as well as regulation of iron homeostasis dependent on hepcidin upregulation.

Journal Pre-proof Acknowledgments This work was supported by the National Develop ment of Key Novel Drugs for Special Project s of China (2017ZX09304014) and Hunan Key Laboratory for Bioanalysis of Co mp lex Matrix Samples (2017TP1037). The authors wish to thank all o f the investigators and laboratory staff who participated in this study. They would also like to thank Hisun Pharmaceutical Co., Ltd. (Taizhou, Zhejiang, China) which produced and provided ginsenoside compound K, Hunan Xiangzhong Pharmaceutical Co., Ltd. (Shaoyang, Hunan, Ch ina) for the providing of SVP. Author contributions

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LZ was responsible for the study design, development of animal experiments, data collection, statistical analysis, and manuscript writing. LC assisted with the development of animal experiments and revising the

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article. XZ helped to revise the art icle. DO and JL guided the design and imp lementation o f the whole research. All authors reviewed the results and approved the final version of the manuscript.

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Conflicts of interest

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The authors confirm that there are no conflicts of interest.

Author Contribution Statement

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Luping Zhou: Conceptualization, Formal analysis, Investigation, Data Curation, Writing -

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Original Draft, Writing - Review & Editing; Lulu Chen: Investigation, Writing - Review & Editing; Xiangchang Zeng: Writing - Review & Editing; Jianwei Liao: Conceptualization,

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Writing - Review & Editing; Dongsheng Ouyang: Conceptualization, Writing - Review & Editing, Supervision

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Journal Pre-proof Figure legends Fig. 1 Effect o f SVP alone and co mbined with G-CK o r L-Car on hepatic index (liver weight-to-body weight ratio). Values are mean ± SEM of 10 rats/group for hepatic index. Con, control; SVP, sodium valp roate (500 mg/kg, twice daily); G-CK, ginsenoside compound K; LCK, lo w-dose G-CK (80 mg/kg, once daily); M CK, middle-dose G-CK (160 mg/kg, once daily); HCK, h igh-dose G-CK (320 mg/kg, once daily); L-Car, L-carnitine (500 mg/ kg, once daily). #### p <0.05, <0.0001 vs. control group; * , ** p<0.05, <0.01 vs. SVP group (one-way ANOVA followed by LSD test). Fig. 2 Effect of SVP alone and combined with G-CK or L-Car on ALT, AST, A LP and ALB levels. Values are

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mean ± SEM of 10 rats/group, except ALB detected in 6 rats/group. Con, control; SVP, sodium valproate (500 mg/kg, twice daily); G-CK, ginsenoside compound K; LCK, lo w-dose G-CK (80 mg/kg, once daily); M CK,

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middle-dose G-CK (160 mg/kg, once daily); HCK, h igh-dose G-CK (320 mg/kg, once daily); L-Car, L-carnitine (500 mg/kg, once daily). # , #### p <0.05, <0.0001 vs. control group; * , ** , *** , **** p <0.05, <0.01, <0.001, <0.0001

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vs. SVP group (one-way ANOVA followed by LSD test).

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Fig. 3 Effect of SVP alone and comb ined with G-CK or L-Car on pathological mo rphology and hepatic

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triglyceride levels. A: Effect of SVP alone and combined with G-CK or L-Car on hepatic histopathology. Liver

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sections stained by H&E (×100), SVP group showing steatosis (red arrow), necrosis (black arrow) and dilated

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central vein (asterisk); B: Effect of SVP alone and combined with G-CK or L-Car on hepatic triglyceride level, values are mean ± SEM of 6 rats/group. Con, control; SVP, sodiu m valproate (500 mg/kg, t wice daily); G-CK, ginsenoside compound K; LCK, lo w-dose G-CK (80 mg/ kg, once daily); M CK, middle -dose G-CK (160 mg/kg, once daily); HCK, high-dose G-CK (320 mg/kg, once daily); L-Car, L-carn itine (500 mg/kg, once daily).

#

p

<0.05 vs. control group; * , ** , *** , **** p<0.05, <0.01, <0.001 <0.0001 vs. SVP group (one-way A NOVA followed by LSD test). Fig. 4 Effect of SVP alone and co mbined with G-CK o r L-Car on subcellular structures. In the SVP -treated rats, less dense cytoplasmic and mitochondrial matrix (black arro w), fewer mitochondria and RERs, shortened or absent mitochondrial cristae, expanded inter-ridge gap, irregular nuclei, and lip id droplet were observed. M,

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mitochondria; RER, rough endoplasmic reticulu m; N, nucleus; L, lip id droplet; Con, control; SVP, sodium valproate (500 mg/kg, twice daily); G-CK, ginsenoside compound K; LCK, low-dose G-CK (80 mg/kg, once daily); M CK, middle -dose G-CK (160 mg/ kg, once daily); HCK, h igh-dose G-CK (320 mg/kg, once daily); L-Car, L-carnitine (500 mg/kg, once daily). Fig. 5 Effect of SVP alone and combined with G-CK or L-Car on hepatic lipid pero xidation and antio xidants.

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Values are mean ± SEM o f 6 rats/group. CAT, catalase; GPx, g lutathione peroxidase; SOD, supero xide dismutase; GSH, glutathione; MDA, malondialdehyde; HO-1, heme o xygenase-1; Con, control; SVP, sodium

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valproate (500 mg/kg, twice daily); G-CK, ginsenoside compound K; LCK, low-dose G-CK (80 mg/kg, once

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daily); M CK, middle -dose G-CK (160 mg/ kg, once daily); HCK, h igh-dose G-CK (320 mg/kg, once daily);

*** ****

p <0.05, <0.01, <0.001, <0.0001 vs. SVP group (one-way ANOVA followed by LSD test).

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,

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L-Car, L-carnit ine (500 mg/kg, once daily ). # , ## , ### , #### p <0.05, <0.01, <0.001, <0.0001 vs. control group; * , ** ,

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Fig. 6 The d ifferential exp ressed protein-protein interaction network. Red nodes represent DEPs in SVP vs. Con,

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green nodes represent DEPs in HCK + SVP vs. SVP, and yellow nodes represent DEPs at the intersection. Protein-protein interaction network and module analysis by the plugin MCODE were mapped by using the software Cytoscape. DEPs, d ifferentially exp ressed proteins; Con, control; SVP, sodium v alproate (500 mg/kg, twice daily); HCK, high-dose G-CK (320 mg/kg, once daily). Fig. 7 Effect of SVP alone and combined with G-CK or L-Car on hepatic soluble epoxide hydrolase protein level. A: Western blot analysis; B: Proteomic analysis. Values are mean ± SEM of 6 rats/group. sEH, soluble epoxide hydrolase; Con, control; SVP, sodium valp roate (500 mg/kg, twice daily); G-CK, ginsenoside compound K; LCK, low-dose G-CK (80 mg/kg, once daily); M CK, midd le-dose G-CK (160 mg/kg, once daily); HCK, h igh-dose G-CK (320 mg/kg, once daily); L-Car, L-carnit ine (500 mg/kg, once daily). # , ### , #### p <0.05,

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<0.001, <0.0001 vs. control group; * ,

***

,

****

p <0.05, <0.001, <0.0001 vs. SVP group (one-way ANOVA

followed by LSD test). Fig. 8 Effect of SVP alone and comb ined with G-CK or L-Car on hepcidin and iron levels. Values are mean ± SEM of 6 rats/group. Con, control; SVP, sodiu m valproate (500 mg/kg, twice daily); G-CK, ginsenoside compound K; LCK, low-dose G-CK (80 mg/kg, once daily); M CK, midd le-dose G-CK (160 mg/kg, once daily);

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HCK, high-dose G-CK (320 mg/kg, once daily); L-Car, L-carn itine (500 mg/kg, once daily ). # ,

###

p <0.05,

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<0.001 vs. control group; * , ** , *** p <0.05, <0.01, <0.001 vs. SVP g roup (one-way ANOVA followed by LSD

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test).

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Table 1 The proteins identified as differentially expressed of SVP vs. Con and corrected by G-CK

UniProt ID

Gene name

MW [kDa]

Coverage [%]

Peptides

Unique peptides

PSMs

SVP vs. Con Ratio

p

a

HCK + SVP vs. SVP Ratio

pa

Q6PDW6

39S ribosomal protein L17, mitochondrial

Mrpl17

20.26

9.7

2

2

2

3.257

3.93E-07

0.272

6.34E-05

Q9ESN0

Protein Niban

Fam129a

103.46

15.0

9

11

9

2.434

1.64E-05

0.632

1.56E-04

P80299

Bifunctional epoxide hydrolase 2

Ephx2

62.34

47.5

22

65

1

2.840

2.44E-02

0.226

1.99E-04

P08932

T -kininogen 2

-

47.70

46.0

17

28

7

2.144

4.41E-05

0.524

1.03E-04

F1LMK6

L-serine dehydratase/L-threonine deaminase Acyl-coenzyme A synthetase ACSM3, mitochondrial Ectonucleoside triphosphate diphosphohydrolase 8

Sds

38.28

24.0

8

13

8

2.065

4.76E-03

0.618

2.69E-06

Acsm3

65.71

41.4

23

34

23

2.063

4.49E-06

0.567

3.66E-05

Entpd8

54.36

6.7

3

5

3

1.909

2.60E-04

0.637

3.98E-04

C-X9-C motif-containing 4

Cmc4

7.61

3

4

3

0.177

8.04E-05

7.810

2.46E-03

Aldehyde oxidase 3

Aox3

146.75

32

68

2

0.343

6.08E-03

2.177

1.04E-03

18.0

12

19

2

0.531

3.18E-03

1.542

1.05E-02

Q6SKG1 F1LPV1 A0A0G2K2W2 Q5QE80

F1M1J2

Solute carrier family 2 (Facilitated glucose transporter), member 2 Nebulin

Q99MH3

Hepcidin

G3V9A4

2'-5'-oligoadenylate synthase 1A

Q63318

Ly6-C antigen

Q6AYE7

Interferon-induced protein with tetratricopeptide repeats 3

G3V645

2'-5'-oligoadenylate synthase-like protein 1

Q68FZ1

a

Protein description

26.7

Oas1a

al 41.68

25.7

8

10

8

0.598

6.55E-04

3.348

3.66E-05

LOC100911104

14.16

15.7

2

3

2

0.603

1.70E-03

3.253

1.73E-06

Ifit3

48.06

19.5

6

6

6

0.661

1.30E-03

3.072

1.98E-03

Oasl

58.97

8.6

4

5

4

0.665

1.36E-03

2.851

1.76E-05

Slc2a2 Neb

rn

Hamp

u o

J

e

r P 51.5

o r p

f o

57.07

623.38

0.7

3

4

3

0.581

7.23E-04

1.663

1.68E-03

9.29

26.2

2

3

2

0.584

1.62E-04

1.530

4.44E-04

Benjamini-Hochberg adjusted p-value. N = 3/group. SVP, sodium valproate; Con, control; G-CK, ginsenoside compound K.

Journal Pre-proof Highlights 

G-CK displayed a protective effect against SVP-induced hepatotoxicity.



The antioxidant effect mediated the action of G-CK.



Hepcidin and sEH (P80299) were the potential targets of G-CK.

Graphical abstract

f o

l a n

J

r u o

r P

e

o r p

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8