Canadian Journal of Physiology and Pharmacology

Protective Effect of Trillium Tschonoskii Maxim Saponin on CCl4-induced Acute Liver Injury of Rats through Apoptosis Inhibition

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2016-0228.R1

Manuscript Type: Article

Date Submitted by the Author: 27-May-2016

Complete List of Authors: Wu, Hao; University for Nationalities, College of Science and TechnologyDraft Qiu, Yong; for Nationalities, College of Medicine Shu, Ziyang; Sports University, College of Health Science Zhang, Xu; , College of Health Science Li, Renpeng; Hubei University for Nationalities, College of Science and Technology Liu, Su; Affiliated Hospital of Hubei University for Nationalities Chen, Longquan; Hubei University for Nationalities, College of Medicine Liu, Hong; Hubei University for Nationalities, College of Medicine Chen, Ning; Wuhan Sports University, College of Health Science

Trillium tschonoskii Maxim (TTM), liver injury, hepatomegaly, inflammatory Keyword: factor, apoptosis

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Manuscript type: Original paper

Protective Effect of Trillium Tschonoskii Maxim Saponin on CCl 4-induced

Acute Liver Injury of Rats through Apoptosis Inhibition

Hao Wu 1, 2, #, Yong Qiu 2, #, Ziyang Shu 3, Xu Zhang 3, Renpeng Li 1, Su Liu 4, Longquan Chen 2, Hong Liu 2, *, Ning Chen 3, *

1College of Science and Technology, Hubei University for Nationalities, Enshi 445000, 2College of Medicine, Hubei University for Nationalities, Enshi 445000, China 3Hubei Key Laboratory of Sport Training and Monitoring, College of Health Science, Wuhan Sports University, Wuhan 430079, China 4Affiliated Hospital of Hubei UniversityDraft for Nationalities, Enshi 445000, China

Running title: TTM against liver injury #These authors contributed equally to this project.

*Corresponding author: Ning Chen, PhD, Professor of Biochemistry College of Health Science, Wuhan Sports University, Wuhan 430079, China Tel: 862767846140 Fax: 862767846140 Email: [email protected]

Hong Liu, Professor of Pharmacology School of Medicine, Hubei University for Nationalties, Enshi 445000, China Tel: 867188437479 Fax: 867188437479 Email: [email protected]

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Abstract

In order to explore hepatoprotective role and underlying mechanisms of TTM, 36 rats were randomly divided into control, CCl 4induced liver injury model, and DDB and low, moderate and highdose TTM treatment groups. After CCl 4induced model establishment, the rats from

DDB and TTM groups were administrated with DDB at 0.2 g/kg ⋅d and TTM at 0.1, 0.5 and

1.0 g/kg ⋅d, while the rats from control and model groups were administrated with saline.

Upon 5 days treatments, all rats were sacrificed for determining serum ALT and AST levels and liver index, examining histopathological changes in liver through HE and TUNEL staining, and evaluating TNFα and IL6 mRNA expression by RTPCR, and Caspase3,

Bcl2 and Bax expression by WesternDraft blot. Results indicated that CCl 4 could induce acute liver injury and abnormal liver function in rats with obvious hepatomegaly, increased liver index, high ALT and AST levels, upregulated TNFα and IL6, and overexpressed Bax and

Caspase3. However, DDB and TTM could execute protective role in CCl 4induced liver injury in rats through reducing ALT and AST levels, rescuing hepatomegaly, downregulating inflammatory factors and inhibiting hepatocyte apoptosis in a dosedependent manner. Therefore, TTM has obvious protective role in CCl 4induced liver injury of rats through inhibiting hepatocyte apoptosis.

Key words: Trillium tschonoskii Maxim (TTM), liver injury, hepatomegaly, inflammatory factor, apoptosis.

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Introduction

Liver plays an important role in metabolism and detoxification in human body. It is also

one of the organs sensitive to a series of stimuli such as trauma, viral infection, autoimmune

disease and other exogenous materials including drugs, alcohol and toxins, thus resulting in

its acute or chronic damage (Shi et al. 2014; Wang et al. 2007). Severe acute liver injury

could result in lifethreatening clinical problems such as jaundice, serious blood coagulation

disorders and high death rate (Bernal et al. 2010). At the same time, the longterm

accumulated liver injury also can lead to liver fibrosis, cirrhosis and hepatocellular carcinoma

(HCC), so it is very important for the prevention of liver injury (Dong et al. 2016). Although there is an increasing drug demand Draftfor the protection of the liver, modern medicine is still lack of reliable liverprotective drugs (Lu et al. 2016), and severe acute liver injury is still a

great challenge in the field of clinical medicine (Auzinger and Wendon 2008).

During long application history in China, Chinese herbs or natural products have

obvious treatment efficacy for a series of diseases with little side effects and low cost (Kou

and Chen 2012; Kou et al. 2013; Kou et al. 2015; Zhang et al. 2014). Therefore, more and

more people are exploring a novel strategy for the prevention and treatment of liver diseases

through Chinese herbs (Akhter et al. 2015; Yan et al. 2015).

Trillium tschonoskii Maxim (TTM), a pharmacologically important medicinal plant

containing steroidal saponins and alkaloids from Liliaceae, is widely distributed in central

and western China (Li et al. 2005; Zhang et al. 2013). Its dried rhizome and root have

medicinal roles in promoting blood circulation and stanching bleeding, detoxification,

tranquilizing and allaying excitement, so that it is usually used for the treatment of

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hypertension, neurasthenia, dizziness, headache, traumatic injury and bleeding as well as a series of liver disorders (Fu 1992). TTM is honored as one of four magical herbs from Tujia nationality due to its obvious curative effects for a series of diseases. However, as it’s a kind of medicinal plant from the minority in China, its clinical application is limited in the regions of Tujia and Miao nationalities without extension all over the China.

According to previous reports, saponins from several herbs are known to induce apoptosis in cancer cells and are proposed to be promising modulators of drug resistance.

Paris saponin VII extracted from T. tschonoskii can reverse multidrug resistance of adriamycinresistant MCF7/ADR cells via Pglycoprotein inhibition and apoptosis augmentation (Li et al. 2014), inhibitDraft metastasis by modulating matrix metalloproteinases in colorectal cancer cells (Fan et al. 2015 b) and reduce migration and invasion in human A549 lung cancer cells (Fan et al. 2015 a). The saponin TTB2 isolated from T. tschonoskii Maxim can exert anticancer effects and may be a potential candidate for the development of anticancer drugs (Huang and Zou 2015). The true incidence of phytotherapyrelated hepatotoxicity and its pathogenic mechanisms are largely unknown (Tarantino et al. 2009). It is important to increase the awareness of both clinicians and patients about the potential dangers of herbal remedies. Meanwhile, the role of TTM in liver disorders and underlying mechanisms are also not fully understood. In order to further confirm its treatment efficacy and extend its clinical application, we established CCl 4induced acute liver injury model in rats to explore its protective roles and underlying mechanisms for diseases associated with acute liver injury.

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Materials and methods

The preparation of TTM saponin

TTM provided by Specimen Center of Hubei University for Nationalities was smashed

after dried in 60 °C constant temperature oven, then sequentially immersed in 75% alcohol

for 2 hours and completed ethanol reflux extraction for three times. The alcohol extract after

filtration was concentrated to 1 g/mL for future use. The saponin in TTM extract was

analyzed by highperformance liquid chromatography to reveal total saponin of 18.54%.

Animal grouping and drug administration

The healthy male rats with body weights of 180220 g were ordered from Hubei Research Center of Laboratory AnimalsDraft (License number: 20150018). All rats were

randomly divided into 6 groups including control group, CCl 4induced acute liver injury

model group, biphenyl dimethyl dicarboxylate (DDB) group at the dose of 0.2 g/kg ⋅d, and

TTM groups at low, moderate and high dosages (0.1, 0.5 and 1.0 g/kg ⋅d), respectively. Six

rats were in each group. All rats were subjected to adaptive feeding with regular feeds for 1

week. The rats were subjected to diet restriction for 12 h prior to model establishment. The

rats from the control group were subjected to subcutaneous injection of peanut oil at the dose

of 5 mL/kg, while the rats from other groups were subjected to the administration of peanut

oil mixed with 25% CCl 4 at the dose of 5 mL/kg (equal to 2 g/kg for CCl 4) for the

establishment of acute liver injury model. After model establishment, the rats from treatment

groups were administrated with DDB and TTM at the designed dosages through gavage twice

a day with an interval of 12 h for 5 successive days. The diets of the rats were restricted

except water after the last drug administration. Twentyfour hours later, the rats were

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anesthetized with 10% chloraldurate, and blood and liver tissue samples were harvested for analysis.

Liver index determination

The body weight of each rat was measured prior to being sacrificed. Similarly, the wet weight of rat liver was measured after sacrificed. The liver index = liver weight/rat body weight × 100%.

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level measurement

Blood samples of the rats after last drug administration for 12 h were harvested in a common anticoagulant tube, and thenDraft subjected to the centrifugation at the speed of 1000 ×g for 5 min. The supernatant was collected, and then serum ALT and AST levels were determined by immunity transmission turbidity method using commercially available assay kits (Mindray Biomedical Electronics Co., Ltd, Shenzhen, China) in an automatic biochemical analyzer (BS800M, Mindray Biomedical Electronics Co., Ltd, Shenzhen,

China).

Real-time polymerase chain reaction (RT-PCR) detection

Approximately 0.1 g liver tissue was subjected to complete homogenate. Total RNA was extracted using Trizon reagent (Invitrogen, USA) following manufacturer’s instructions and then reversely transcribed into cDNA. The mRNA expression levels of IL6 and TNFα were determined through fluorescence quantitative RTPCR. The forward and reversed primers for

IL6 were 5’GCTCTCCGCAAGAGACTTCCA3’ and

5’GGTCTGTTGTGGGTGGTATCC3’, respectively. The forward and reversed primers for

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TNFα were 5’GGTGATTGGTCCCAACA3’ and 5’GTCTTTGAGATCCATGCC3’,

respectively. The forward and reversed primers for βactin as the internal control were

5’CACGATGGAGGGGCCGGACTCATC3’ and

5’TAAAGACCTCTATGCCAACACAGT3’, respectively. The relative expression level of

IL6 or TNFα was calculated by the ratio of IL6 or TNFα to βactin.

Histology and immunohistochemistry

The small slice of liver tissue from each rat was subjected to the fixing by 4%

paraformaldehyde, regular embedding by paraffin, sectioning, hematoxylineosin (HE)

staining and Ki67 staining (Abcam, Cambridge, USA), and the slides were observed for conventional morphological evaluationDraft under a light microscope (Nikon Eclipse TE2000U, NIKON, Japan) and photographed at 100 × magnification.

Terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) staining

The sections of liver tissues were analyzed by terminal deoxynucleotidyl transferase

dUTP nick end labeling (TUNEL) assay using the in situ cell death detection kit (Roche

Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions.

TUNELpositive cells were captured under a light microscope (Nikon Eclipse TE2000U,

NIKON, Japan) and analyzed through Image proplus 6.0 software (Media Cybernetics Inc.,

MD, USA).

Apoptosis-related protein expression evaluated by Western blot

Approximately 0.1 g of liver tissue after homogenizing was lysed with RIPA buffer

(Santa Cruz Biotechnology, Santa Cruz, CA) in the presence of PMSF (Beyotime Institute of

Biotechnology, Jiangsu, China) at the speed of 4000 ×g at 4 °C for 20 min. The collected

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supernatant was added with loading buffer and boiled at 95 °C water batch for 5 min. Then,

20 g total protein was separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE), and transferred to PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 3 h. The targeted protein on the membrane was probed with primary antibody such as Caspase3, Bax or Bcl2 (Cell

Signaling Technology, USA, 1:1000) at 4 °C overnight. The membrane was then washed with TBST buffer for three times with 15 min in each time. Sequentially, the membrane was incubated with secondary antibody, HRPlabeled antirabbit IgG (Cell Signaling Technology,

USA, 1:30000) for 2 h, and washed with TBST buffer for three times. Then, the chemiluminescence agent was incubatedDraft with the membrane in dark room for 2 min and the protein probed by primary antibody in the membrane was imaged. The βactin was used as an internal reference. The data were analyzed by the software Quantity one 4.6.1 (BioRad

Software Inc., California, USA).

Statistical analysis

All data were expressed as mean ± standard deviation (M ± SD). All statistical analyses were conducted using SPSS19.0 software (SPSS, Inc., Chicago, USA) and GraphPad Prism

6.0 (GraphPad Software, Inc., San Diego, USA) through ANOVA with posthoc tests. The significant difference and very significant difference were considered at P < 0.05 and P <

0.01, respectively.

Results

TTM rescues CCl 4-induced acute liver injury

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Serum ALT and AST levels, as the golden biomarkers for liver damage, are

wellaccepted indicators to evaluate the severity of liver injury (Shi et al. 2014). In order to

validate the treatment efficacy of TTM on acute liver injury, we established the rat model

with CCl 4induced acute liver injury to explore the treatment efficacy of TTM. Compared

with the control group, the levels of serum ALT and AST in the model group revealed an

obvious increase ( P < 0.01 and P < 0.05, respectively), indicating that the establishment of

CCl 4induced acute liver injury model was successful. In contrast, after treatment with DDB

and TTM, the serum ALT levels of the rats treated with DDB and TTM at low, moderate and

high dosages revealed a very significant reduction ( P < 0.01); the serum AST levels of the rats treated with DDB and highdoseDraft TTM exhibited a significant decrease ( P < 0.05), indicating that TTM treatment can decrease serum ALT and AST levels in a dosedependent

manner (Figures 1A and 1B).

TTM inhibits CCl 4-induced hepatomegaly and liver index

In order to explore the effect of TTM on CCl 4induced histopathological change of liver

tissues in rats, we compared fresh liver tissues of the rats from various treatments. As shown

in Figure 2A, the rats from CCl 4induced model group revealed obvious hepatomegaly when

compared with the rats from control group and treatment groups. Subjected to the treatments

with DDB and TTM, the hepatomegaly of the rats exhibited an obvious inhibition when

compared with that of the CCl 4induced model rats, suggesting that both DDB and TTM have

obvious protective roles in CCl 4induced hepatomegaly.

Liver index reflects liver pathological changes and nutritional status in the body (Chen

1993). Based on the results of liver index, its significant difference between model group and

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control group was observed ( P < 0.01), suggesting that CCl 4 could result in hepatomegaly, while the treatment with BBD and TTM at various dosages could inhibit CCl 4induced hepatomegaly in rats (Figure 2B).

In addition, the liver tissues were examined with HE staining. As shown in Figure 2C, normal liver lobule structure and liver cells in rats from normal control group were observed.

In contrast, in the CCl 4induced model group, diffused edema, necrosis and inflammatory cell infiltration in liver tissues were observed. However, the treatments with DDB and TTM at various dosages could rescue the liver damage with the significant reduction of diffused edema, necrosis and inflammatory cell infiltration in liver tissues when compared with the model group. Moreover, the protectionDraft of TTM on liver tissues revealed an obvious dosedependent manner.

TTM reduces inflammatory factors for CCl 4-induced acute liver injury

TNFα and IL6 are two important proinflammatory factors causing cell inflammation and death (Diehl 2000; Schmich et al. 2011; Tilg 2001). Then, we examined the mRNA expression of both factors through RTPCR. Results indicated that acute liver injury induced by CCl 4 could result in an obvious increase of TNFα and IL6 ( P < 0.05, and P < 0.01, respectively) (Figures 3A and 3B). On the other hand, after treatment with DDB and TTM at various dosages, the expression of TNFα and IL6 in DDB and TTM groups revealed an obvious reduction. Therefore, TTM can alleviate inflammatory response of liver cells to execute the protective function for liver.

TTM inhibits apoptosis of liver cells induced by CCl 4

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In order to further understand the protective mechanism of TTM for acute liver injury,

the cell apoptosis in liver tissues from the rats subjected to CCl 4 induction and DDB and

TTM treatments at various dosages were examined through TUNEL staining. Compared with

normal control group, a large number of cell apoptosis in liver tissues due to the CCl 4

induction in the model group was observed (Figure 4A); in contrast, subjected to the

treatments with DDB and TMM at moderate and high dosages, the apoptotic cell number

revealed a significantly decrease and the reduced apoptosis of liver cells exhibited as a

dosedependent manner for TTM (Figure 4B). Similarly, compared with the normal control

group, CCl 4induced liver tissues revealed an obviously increased Caspase3 and Bax and reduced Bcl2; on the other hand, theDraft treatments with DDB and TTM at moderate and high dosages could result in the downregulation of Caspase3 and the upregulation of Bcl2

when compared with those in CCl 4induced liver injury model group (Figure 4C). Therefore,

the protective role of TTM in acute liver injury should be correlated with the inhibition of

liver cell apoptosis.

Discussion

The mechanisms of the diseases with acute liver injury are very complex. Current reports

are mainly focused on oxidative stress, inflammation and apoptosis (Abouzied et al. 2016;

Chan et al. 2014; Schmich et al. 2011). The CCl 4induced acute liver injury rat model is well

accepted, which can result in substantial liver cell damage and liver dysfunction (Masuda

2006; Weber et al. 2003). Once CCl 4 is absorbed by the body, it can activate liver

cytochrome (P450) to generate trichloromethyl free radicals (CC 3•) and peroxide

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trichloromethyl free radicals (CCl 3O2•), thus attacking phospholipid molecules on cell membrane of liver, increasing cell membrane permeability, and finally leading to liver cell swelling, necrosis, and symptoms of acute liver injury (LeSage et al. 1999; Weber et al.

2003).

In the present study, we utilize CCl 4induced rat model with acute liver injury to clarify the treatment efficacy of TTM and underlying mechanisms. The administration of TTM at various dosages for model rats with acute liver injury by lavage for five days can reduce the levels of serum ALT and AST and liver index, and downregulate the expression of TNFα and IL6. HE staining revealed that TTM treatment could alleviate inflammatory necrosis of liver tissues, and present an obvious protectiveDraft effect on liver damage. It is well known that the liver has strong capacity of regeneration and recovery (Kim and

Kim 2013). Therefore, we suspect TTM protection against acute liver injury may be due to the promotion from liver cell regeneration. In our study, compared with CCl 4 model group, the expression of Ki67 revealed an obvious increase after the treatment with DDB; in contrast, no obvious expression of Ki67 was observed in liver tissues from the rats treated with TTM at various dosages (Figure 5), suggesting that the protective effect of TTM on acute liver injury may be not correlated with the regeneration of liver cells.

Mitochondrial apoptotic pathway is one of the major pathways of apoptosis. Bcl2 protein family plays a vital role in mitochondrial apoptotic pathway (Tischner et al. 2010).

Bax, Bcl2, and Bcl2 protein family can promote or inhibit the release of cytokines and other apoptotic factors through changing the mitochondrial outer membrane permeability (Rosse et al. 1998). Cytochrome C could mediate the activity of Caspase3, and the activated Caspase3

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can induce cell apoptosis (Cusack et al. 2013). In the present study, CCl 4induced liver cell

apoptosis could be obviously alleviated in the presence of TTM treatment under the

examination by TUNEL staining; similarly, based on the analysis of Western blot, TTM

could reduce the expression of cleaved Caspase3 and Bax, and improve the expression of

Bcl2, thus further verifying the inhibitory role of TTM in apoptosis of liver cells for the

protection of acute liver injury. However, apoptotic cell death can be also associated with

mitochondrial membrane depolarization and cytochrome C release. The exposure of

hepatocytes to hepatototoxic substances may result in increased ROS production and

mitochondrial damage, and is balanced by the presence of antioxidant substances. Therefore, circulating levels of cytochrome Draft C, gammaglutamyl transferase, triglycerides and unconjugated bilirubin, as well as Bcl2 in overweight/obese patients is highly associated

with nonalcoholic fatty liver diseases (Tarantino et al. 2011 a; Tarantino et al. 2011 b).

Whether TTM can inhibit the release of cytochrome C, alleviate the production of ROS,

reduce the damage of mitochondria, and be benefit for nonalcoholic fatty liver diseases

needs to be further explored. As the crosstalk between apoptosis and autophagy for

maintaining cellular hemostasis in a series of diseases (Chen and Karantza 2011; Chen and

KarantzaWadsworth 2009; Fan et al. 2016), whether TTM can execute the prevention and

treatment of CCl 4induced acute liver injury through the functional status of autophagy also

needs to be further explored.

In conclusion, TTM has a protective effect against acute liver injury through reducing

liver cell apoptosis rather than promoting liver cell proliferation. This study provides the

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experimental basis for TTM application in acute liver injury to extend its application fields as a novel drug against acute liver injury.

Draft

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Acknowledgements

This study was finically supported by grants from Hubei Province Scientific Research

Foundation (D200729005), Chutian Scholar Program from Education Department of Hubei

Province, Hubei Superior Discipline Groups of Physical Education and Health Promotion and

Innovative Startup Foundation from Wuhan Sports University to NC. The authors would like

to express their gratitude to the School of Basic Medical Sciences from for

providing a partial experimental platform.

Conflict of interest All authors have declared no conflictDraft of interest associated with this project.

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Figure legends

Figure 1. TTM rescued CCl 4induced acute liver damage based on examination of ALT (A) and AST (B) levels. All data were expressed as Mean ± SD ( n = 6). #P < 0.05 and ## P < 0.01 compared with the control group; * P < 0.05 and ** P < 0.01 compared with CCl 4induced acute liver injury model group.

Figure 2. TTM inhibited CCl 4induced hepatomegaly (A) and reduced liver index (B) as well as alleviated CCl 4induced cell necrosis or cell death (C) through the evaluation by HE staining. Draft

Figure 3. TTM reduced inflammatory responses from CCl 4induced acute liver injury in rats through the determination of mRNA expression of TNFα (A) and IL6 (B) by RTPCR. All data were expressed as Mean ± SD ( n = 3) from three independent experiments. #P < 0.05 and

## P < 0.01 compared with the control group; * P < 0.05 and ** P < 0.01 compared with the

CCl 4model group.

Figure 4 . TTM executed hepatoprotection through inhibited apoptosis. TTM could obviously reduce liver cell apoptosis of rats induced by CCl 4 based on the TUNEL staining (A) and its statistical analysis (B). Similarly, TTM could result in downregulation of Bax and Caspase3 and upregulation of Bcl2 based on western blot analysis (C). ## P < 0.01 compared with the

* ** control group; P < 0.05 and P < 0.01 compared with the CCl 4model group.

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Figure 5. Effect of TTM on liver cell regeneration in the presence of CCl 4 induction.

Compared with the CCl4induced model group, DDB could promote liver cell regeneration

but TTM at various dosages did not exhibit the promotion role in liver cell regeneration,

which was evaluated by Ki67 staining (A) and corresponding statistical analysis (B). ## P <

0.01 compared with the control group; ** P < 0.01 compared with the CCl 4induced model

group.

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