Smad signaling pathway

Journal Pre-proof Deltamethrin induces liver fibrosis in quails via activation of the TGF-β1/Smad signaling pathway Bing Han, Zhanjun Lv, Xiaoya Zhang, Yueying Lv, Siyu Li, Pengfei Wu, Qingyue Yang, Jiayi Li, Bing Qu, Zhigang Zhang PII:

S0269-7491(19)35178-4

DOI:

https://doi.org/10.1016/j.envpol.2019.113870

Reference:

ENPO 113870

To appear in:

Environmental Pollution

Received Date: 12 September 2019 Revised Date:

20 December 2019

Accepted Date: 20 December 2019

Please cite this article as: Han, B., Lv, Z., Zhang, X., Lv, Y., Li, S., Wu, P., Yang, Q., Li, J., Qu, B., Zhang, Z., Deltamethrin induces liver fibrosis in quails via activation of the TGF-β1/Smad signaling pathway, Environmental Pollution (2020), doi: https://doi.org/10.1016/j.envpol.2019.113870. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Deltamethrin induces liver fibrosis in quails via activation of

2

the TGF-β1/Smad signaling pathway

3

Bing Han a, Zhanjun Lv a,b, Xiaoya Zhang a, Yueying Lv a, Siyu Li a, Pengfei Wu a,

4 5

Qingyue Yang a, Jiayi Li a, Bing Qu a, Zhigang Zhang a,b,* a

College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030,

6 7

China b

Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine,

8 9 10

Harbin, 150030, China * Corresponding author. E-mail address: [email protected] (Z.G. Zhang).

11 12

ABSTRACT

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Deltamethrin (DLM) is an important member of the pyrethroid pesticide family, and

14

its widespread use has led to serious environmental and health problems. Exposure to

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DLM causes pathological changes in the liver of animals and humans and can lead to

16

liver fibrosis. However, the mechanism of DLM-induced liver fibrosis remains

17

unclear. Therefore, to address its potential molecular mechanisms, we used both in

18

vivo and in vitro methods. Quails were treated in vivo by intragastric administration of

19

different concentrations of DLM (0, 15, 30, or 45 mg kg-1), and the chicken liver

20

cancer cell line LMH was treated in vitro with various doses of DLM (0, 50, 200, or

21

800 µg mL-1). We found that DLM treatment in vivo induced liver fibrosis in a

22

dose-dependent manner through the promotion of oxidative stress, activation of 1

23

transforming growth factor-β1 (TGF-β1) and phosphorylation of Smad2/3. Treatment

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of LMH cells with different concentrations of DLM similarly induced oxidative stress

25

and also decreased cell viability. Collectively, our study demonstrates that

26

DLM-induced liver fibrosis in quails occurs via activation of the TGF-β1/Smad

27

signaling pathway.

28 29

Keywords: Deltamethrin; Liver; Oxidative stress; Fibrosis; TGF-β1/Smad

30 31

Deltamethrin induces liver fibrosis in quails via activation of the TGF-β1/Smad

32

signaling pathway

33 34

1. Introduction

35

Insecticides have been used in agriculture for centuries and have had a major

36

impact on improving agricultural productivity (Kurek et al., 2017). The use of

37

pyrethroid insecticides has increased significantly since the implementation of

38

restrictions on organophosphates, and over the past two decades, pyrethroids have

39

become the preferred pesticide in many agricultural countries (Kumar et al., 2016). As

40

an important member of the pyrethroid family, deltamethrin (DLM) is a

41

broad-spectrum insecticide that can be used for pest prevention in agriculture (Milam

42

et al., 2000), as well as insecticidal and parasitic control of poultry (Zeman and

43

Železny, 1985; Soderlund et al., 2002; Khater et al., 2013). Compared with similar

44

pesticides in its class, DLM is the most frequently used. Consequently, its residues on 2

45

crops and its accumulation in water have led to serious environmental pollution, as

46

well as health problems for humans and many animals, including aquatic organisms

47

(Abdel-Daim et al., 2014; Abdelkhalek Nevien et al., 2015). Direct contact with DLM

48

vapors or consumption of DLM-contaminated food and water is the most common

49

route of poisoning (Barlow et al., 2001). DLM induces a variety of pathological

50

changes, including the inhibition of mitosis and chromosomal aberrations (Agarwal et

51

al., 1994), as well as histological changes in some vital organs, such as liver (Shona et

52

al., 2010; Arora et al., 2016).

53

Liver is the main site for the metabolism and detoxification of exogenous chemicals

54

(e.g., pesticides, drugs, and metals), and exposure to DLM has been reported to lead

55

to pathological changes in liver (Toś-Luty et al., 2001; Dubey et al., 2013; Xu et al.,

56

2015). DLM-induced hepatotoxicity can occur by a variety of mechanisms, such as

57

free radical production, lipid peroxidation, inflammation, and apoptosis (Khater et al.,

58

2013; Anoop et al., 2015). Liver fibrosis is a response to wound healing during

59

chronic liver injury and results in scarring of the tissue. Repetitive or long-term injury

60

causes excessive accumulation of scar tissue and eventually, leads to cirrhosis and

61

liver cancer. Liver fibrosis is a widespread health problem, with global deaths caused

62

by cirrhosis and primary liver cancer at approximately 1.4 million per year (Liteplo et

63

al., 2002). However, no studies to date have addressed the mechanism by which DLM

64

induces liver fibrosis.

65

In several liver diseases, oxidative stress is the major cause of liver damage.

66

Studies have shown that DLM poisoning leads to an imbalance in homeostasis and 3

67

causes oxidative stress (Dinu et al., 2010), which promotes inflammation and

68

apoptosis (Liu et al., 2018; Tan et al., 2018; Li et al., 2019b). Transforming growth

69

factor-β1 (TGF-β1) is one of the most potent factors in promoting liver fibrosis and

70

plays a key role in the occurrence and maintenance of fibrosis (Cui et al., 2003;

71

Schuppan et al., 2003; Wells et al., 2004; Liu et al., 2010). Oxidative stress can also

72

enhance the levels of TGF-β1 (Meng et al., 2019; Rashid et al., 2019). The profibrotic

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effect of TGF-β1 is extensive and complex, and the most important pathway in the

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development of fibrosis is the TGF-β1/Smad signaling pathway (Yao et al., 2018; Liu

75

et al., 2019a; Liu et al., 2019b).

76

Quail (Coturnix coturnix) is a popular avian model species commonly used for

77

toxicity testing and assessment of pesticide safety. Quail is also a source of food for

78

humans. DLM enters and accumulates in the body of the quail, posing a potential

79

threat to human health. Therefore, our study investigated DLM-induced liver fibrosis

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and its potential molecular mechanism.

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2. Materials and methods

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2.1. Reagents and antibodies

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DLM (25 mg mL-1) was purchased from Nanjing Red Sun Co., Ltd. (Nanjing,

84

China). Assay kits for superoxide dismutase (SOD), malondialdehyde (MDA),

85

glutathione (GSH), and hydroxyproline (HYP) were obtained from Jiancheng

86

Bioengineering Institute (Nanjing, China). Kits for CCK-8 and ROS detection, protein

87

extraction, bicinchoninic acid protein assay, as well as phenylmethylsulfonyl fluoride

88

and radio immunoprecipitation assay lysis buffer were obtained from Beyotime 4

89

Biotechnology (Shanghai, China). TRIzol was purchased from Ambion (Foster City,

90

CA, USA), a cDNA synthesis kit was purchased from Vazyme Biotech Co., Ltd

91

(Nanjing, China), and 2X PCR Taq Plus Master Mix with dye was purchased from

92

Applied Biological Materials (Vancouver, Canada). DNA markers were purchased

93

from Tiangen Biotech Co., Ltd. (Beijing, China). Antibodies to collagen-Ι (Col-Ι),

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alpha-smooth muscle actin (α-SMA), phospho-Smad2, Smad2, phospho-Smad3, and

95

Smad3 were obtained from Bioss Biotechnology (Beijing, China). All secondary

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antibodies were purchased from ZSGB-BIO (Beijing, China). An antibody to

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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Hangzhou

98

Goodhere Biotechnology (Hangzhou, China).

99

2.2. Animals and treatments

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Healthy male quails (21 days old, weighing 80 ± 15 g) were purchased from the

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Wanjia poultry farm (Heilongjiang, China) and were acclimated for one week before

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the start of the experiment. The quails were maintained under specific pathogen-free

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conditions on a 12 h light/12 h dark cycle, at a room temperature of 22 ± 2 °C and

104

relative humidity of 55 ± 5%. Food and water were provided ad libitum. The animal

105

study was conducted according to the Ethical Committee for Animal Experiments of

106

Northeast Agricultural University. Quails were randomly distributed into four groups

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(n = 10 per group) and given 0.9% (w/v) saline as a control or different doses of DLM

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(15, 30, 45 mg kg-1) by intragastric administration. All treatments were given daily for

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a total of 12 weeks, and 24 hours after the last administration, the quails were

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sacrificed. Livers were immediately removed, frozen in liquid nitrogen, and stored at 5

111

–80 °C until the time of analysis.

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2.3. Biochemical analysis

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Quail blood was collected and centrifuged at 3000 rpm for 10 min. The activities of

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alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the

115

concentrations of triglyceride (TG) and total cholesterol (TC) in serum were measured

116

using a Beckman DXC 800 biochemical analyzer (Kong et al., 2019; Lv et al., 2020a;

117

Yang et al., 2019).

118

Liver tissue was homogenized in phosphate-buffered saline (pH 7.4) for 10 min

119

using an Ultra-Turrax T25 Homogenizer and then centrifuged at 2500 rpm for 10 min

120

at 4 °C. SOD activity and the concentration of MDA and GSH in the supernatant were

121

assessed using commercially available kits.

122

Liver HYP content was measured by an alkaline hydrolysis method, following the

123

manufacturer’s instructions. Liver tissue was hydrolyzed, and the pH was adjusted to

124

6.0-6.8. The samples were then centrifuged at 3500 rpm for 10 min at 4 °C, and the

125

absorbance at 550 nm was measured in a microplate reader (BioTek Epoch, Vermont,

126

USA).

127

2.4. Quantitative reverse-transcription PCR

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Total RNA was extracted from liver tissue using TRIzol reagent, and cDNA was

129

synthesized using a High Capacity cDNA Reverse Transcription kit. The

130

concentration of purified RNA was determined by UV spectrum at 260 nm (Zhu et al.,

131

2019). Quantitative reverse-transcription PCR (RT-qPCR) was performed using a

132

SYBR Green RT-qPCR SuperMix kit with gene specific primers (shown in Table 1) 6

133

synthesized by Sangon Biotech (Shanghai, China). Relative mRNA levels were

134

calculated by standard methods (2−∆∆Ct). (Dong et al., 2019a; Dong et al., 2019b; Han

135

et al., 2019).

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Table 1 Primers sequences for qPCR Genebank Gene

Primer sequence (5’ → 3’) Number

β-actin

AB199913

Forward: CAGGATGCAGAAGGAGATCACAGC Reverse: GGATAGAGCCTCCGATCCAGACAG

TGF-β1

XM_015850545.1 Forward: CCGACTACTGCTTCGGGACT Reverse: TACTGTGTGTCTGCGCTCCA

ColΙ-α1

XM_015885868

Forward: CGTCGCCATCCAACTGACCTTC Reverse: TGCCAGTCTCCTCGTCCATGTAG

FN1

XM_015867978

Forward: GTGGCGAAGAAGACACTGCTGAG Reverse: AGTTGACGGTAAGGCTGGTAGGAG

α-SMA

XM_015866808

Forward: GGGATGGAATCTGCTGGCAT Reverse: GCCCGGGTACATTGTAGTGC

137

2.5. Histology

138

Liver tissue was fixed in 10% formalin, embedded in paraffin, cut into 5–6 µm

139

thick sections (Sonne et al., 2018; Li et al., 2019a), and then stained with hematoxylin

140

and eosin (HE) or Masson’s trichrome. The sections were scanned using the

141

Pannoramic MIDI slide scanner (Budapest, Hungary).

142

For transmission electron microscopy (TEM), liver tissue (about 1 mm3) was 7

143

rapidly fixed in 2.5% glutaric dialdehyde at 4 °C. After washing in 0.1 M

144

phosphate-buffered saline (PBS), the samples were fixed with 1% osmic acid,

145

dehydrated with ethanol and acetone, and then impregnated with acetone and

146

embedding solution. After embedding, the samples were cut into 50-60 nm thick

147

sections and stained with uranyl acetate and lead citrate. The sections were examined

148

by transmission electron microscopy (TEM, H-7650, Hitachi, Japan).

149

To measure lipids in the liver, tissues were fixed and dehydrated. Samples were

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then pre-cooled in a cryostat, cut into 10 µm thick sections, and then stained with Oil

151

Red O. The sections were examined by light microscopy (Olympus BX-FM, Tokyo,

152

Japan).

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2.6. Western blotting

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Liver tissue was homogenized in radio immunoprecipitation assay buffer

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supplemented with 1 mM phenylmethanesulfonyl fluoride (Li et al., 2020), and total

156

protein lysates were prepared using a protein extraction kit, following the

157

manufacturer’s instructions. Protein concentrations were determined by the

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bicinchoninic acid method, and 5 µL samples containing 5 µg of total protein were

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resolved by SDS-PAGE and transferred to a polyvinylidene fluoride membrane for

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western blotting. Nonspecific binding to the membrane was blocked by incubation in

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5% nonfat milk in Tris-buffered saline plus Tween 20 for 2 h at room temperature.

162

Membranes were then incubated overnight at 4 °C with the appropriate concentration

163

of protein-specific antibodies. The blots were washed in Tris-buffered saline plus

164

Tween-20, incubated with horseradish peroxidase-conjugated secondary antibodies at 8

165

37 °C for 30 min, and then washed six times with Tris-buffered saline plus Tween-20.

166

Finally, densitometry was performed using Image Pro-Plus 6.0 software (General

167

Electric Company, Fairfield, CT, USA). The experiments were repeated three times,

168

and representative results are shown. GAPDH was used as a protein loading control

169

(Lv et al., 2020b).

170

2.7. Cell culture and treatment

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The chicken liver cancer cell line LMH was purchased from American Type

172

Culture Collection (Manassas, VA, USA) and cultured in 1640 medium containing

173

10% heat-inactivated fetal bovine serum, 100 U mL-1 penicillin, and 100 µg mL-1

174

streptomycin. The cells were maintained in a humidified 95% air/5% CO2 incubator at

175

37 °C. For experiments, after dilution of DLM with sterile PBS, the cells were treated

176

with DLM (50 µg mL-1, 200 µg mL-1, 800 µg mL-1) or sterile PBS as a control, and

177

then incubated for 24 h.

178

2.8. Cell viability and intracellular ROS assays

179

LMH cells were seeded in 96-well plates at 5×105 cells/well and treated as

180

described above for 24 h. Cell viability was determined using a CCK-8 kit, according

181

to the manufacturer’s protocol.

182

Intracellular ROS levels were measured using a 2′,7′-dichlorfluoresceindiacetate

183

(DCFH-DA) fluorescent dye assay kit according to the manufacturer’s instructions

184

(Gasparotto et al., 2013). Briefly, LMH cells were seeded in 12-well plates at 5×104

185

cells/well and cultured overnight. The cells were then treated with DLM as described

186

above. After 24 h, DCFH-DA was added to the cells in serum-free medium at a final 9

187

concentration of 10 µM, and the plates were incubated at 37 °C for an additional 20

188

min. The cells were then washed with serum-free medium. Fluorescence was

189

measured at 488 nm (excitation) and 525 nm (emission) (Cao et al., 2017; Cao et al.,

190

2019; Zhang et al., 2019) using a SpectraMax® iD3 plate reader equipped with

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SoftMax® Pro 7 software (Molecular Devices, San Jose, CA, USA).

192

2.9. Protein–protein interaction analysis

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Analysis of the Protein–Protein Interaction (PPI) networks of differentially

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expressed proteins identified in this study was performed using the STRING database

195

(http://string-db.org/). We constructed networks for the species available in the

196

database by extracting target gene sequences. The networks were then built according

197

to known protein interactions in the selected reference species (rat).

198

2.10. Statistical analysis

199

All analyses were performed using SPSS 19.0 software (IBM, Armonk, NY, USA).

200

Data are expressed as the mean ± standard error (SEM). Group means were compared

201

using one-way analysis of variance (Chen et al., 2019) followed by a Tukey’s

202

post-hoc test (Sonne et al., 2017). P values of < 0.05 was considered significant.

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3. Results

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3.1. DLM induced pathological changes in the liver

205

To examine the effect of DLM treatment on quail liver, we first assessed tissue

206

morphology by histological staining. HE staining of the control showed normal liver

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tissue structures. However, the DLM-treated animals showed dose-dependent changes

208

in liver morphology. In the low-dose DLM group (15 mg kg-1), the structure of the 10

209

hepatocytes was altered, the hepatic cord and hepatic sinus were disordered, and

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inflammatory cells had infiltrated and aggregated in the liver. The changes caused by

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DLM treatment in the middle-dose DLM group (30 mg kg-1) were similar to those in

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the low-dose DLM group, but slightly more severe. The morphology of the liver in

213

the high-dose DLM group (45 mg kg-1) was altered dramatically. The hepatic cord

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and hepatic sinus were severely disordered, and the hepatocytes were swollen and

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some had ruptured. The marginal hepatocytes appeared flaky and necrotic, and a large

216

number of fat vacuoles were present and scattered throughout the tissue. There was

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also a large number of inflammatory cells present in the liver (Fig. 1).

218

Transmission electron microscopy of liver tissue showed that compared with the

219

control group, the morphology of hepatocytes in the low-dose DLM group (15 mg

220

kg-1) was altered. The nuclei were not obvious, and some mitochondrial cristae were

221

broken. The morphology of hepatocytes in the middle-dose DLM group (30 mg kg-1)

222

were similar, but more severe than those in the low-dose DLM group. The

223

morphology of the hepatocytes in the high-dose DLM group (45 mg kg-1) showed

224

significant changes in structure, including a large number of lipid droplets around the

225

nucleus and mitochondrial degeneration, with swelling and loss of mitochondrial

226

structure. Mitochondrial cristae were severely broken or missing, and some

227

mitochondrial vacuolization was observed (Fig. 1).

228

3.2. DLM induced an increase in the activity of liver biomarkers

229

ALT and AST are commonly used biomarkers for liver damage. Compared with

230

the control group, DLM administration significantly increased ALT and AST 11

231

activities in a dose-dependent manner (Fig. 2A and 2B).

232

3.3. DLM induced oxidative stress in the liver

233

To examine whether DLM treatment induced oxidative stress in the liver, we

234

measured several markers, including MDA, GSH, and SOD. As shown in Figure 2,

235

DLM administration significantly increased MDA concentration (Fig. 2C), reduced

236

GSH levels (Fig. 2D), and decreased SOD activity (Fig. 2E) in the liver in a

237

dose-dependent manner.

238

3.4. DLM decreased cell viability and increases ROS levels in LMH cells

239

To test the effect of DLM in vitro, we treated LMH cells with different

240

concentrations of DLM. We found that the growth of LMH cells was significantly

241

inhibited by DLM treatment, as measured by the CCK-8 cell proliferation assay (Fig.

242

3A).

243

To assess oxidative stress in the DLM-treated LMH cells, we measured the

244

concentration of intracellular ROS. The results showed that treatment with DLM

245

significantly increased ROS levels (Fig. 3B).

246

3.5. DLM induced steatosis in the liver

247

Steatosis is another indicator of liver damage. To determine whether DLM causes

248

steatosis, we measured lipid levels in the DLM-treated quails. As shown in Figure 4,

249

DLM administration significantly increased TG and TC concentrations, and this effect

250

was dose-dependent. The results of Oil Red O staining showed that there were

251

multiple areas of lipid accumulation in the DLM-treated groups, and these were

252

significantly increased compared with the control group. 12

253

3.6. DLM induced fibrosis in the liver

254

Damage to the liver leads to fibrosis, which is the formation and accumulation of

255

scar tissue and primarily composed of the protein collagen. HYP is the main

256

component of collagen and a good indicator of collagen levels. As shown in Figure

257

5A, we measured HYP levels and found that they were significantly higher in the

258

liver of DLM-treated quails compared with the control group, and this effect was

259

dose-dependent. Histological staining with Masson’s trichrome, which also measures

260

collagen levels, showed that collagen fibers increased significantly in the hepatic

261

sinusoids in a dose-dependent manner in the DLM groups (Fig. 5B).

262

3.7. DLM induced liver fibrosis via the TGF-β1/Smad signaling pathway

263

The livers of DLM-treated quails contained significantly higher levels of the Col-Ι,

264

α-SMA, p-Smad2, and p-Smad3 proteins, and this occurred in a dose-dependent

265

manner compared with the control group (Fig. 6A and 6B).

266

RT-qPCR analysis showed that mRNA levels of α-SMA, ColΙ-α1, FN1, and

267

TGF-β1 in the DLM groups were significantly increased in a dose-dependent manner

268

compared with the control group (Fig. 6E).

269

3.8. PPI analysis

270

To confirm our findings, we constructed a PPI network of fibrosis related genes

271

using the STRING 10 database (Fig. 7). The dynamic clusters for fibrosis included

272

TGF-β1, ColΙ-α1, FN1, Smad2, and Smad3. These functional interaction networks

273

revealed a link between fibrosis and the TGF-β1/Smad signaling pathway.

274

4. Discussion 13

275

Because of the widespread use of DLM as an insecticide, its impact on the

276

environment and human health has become an ever-increasing issue (Sibiya et al.,

277

2019). Exposure to DLM has been shown to cause histological changes in several

278

vital organs (Shona et al., 2010). Many studies have shown that in young and adult

279

rats, DLM is found in plasma and accumulates in multiple tissues, including brain,

280

liver, fat, and muscle (Berkowitz et al., 2003; Morgan et al., 2007; Kim et al., 2010;

281

Naeher et al., 2010). Moreover, DLM can lead to excessive ROS production in rat

282

liver, kidney, and brain, thereby inducing oxidative stress (Abdou and Abdel-Daim,

283

2014; Abdel-Daim et al., 2014; Abdel-Daim et al., 2016). In addition, DLM has been

284

shown to induce oxidative stress in freshwater Nile tilapia, causing damage to the

285

body (Abdel-Daim et al., 2015; Abdelkhalek Nevien et al., 2015). These results are

286

consistent with our investigation of DLM induced hepatic fibrosis in quails.

287

The liver enzymes, AST and ALT, are important biochemical markers for liver

288

dysfunction and liver damage (Sonne et al., 2008; Abdel-Daim et al., 2016). In our

289

study, the increase in the activities of AST and ALT in serum reveals a loss of

290

functional integrity of the hepatocyte membrane and liver dysfunction.

291

Pesticide poisoning causes oxidative stress by producing excess free radicals or

292

ROS (Singh and Prasad, 2018) and inducing lipid peroxidation in tissues in mammals

293

and other organisms (Mansour and Mossa, 2009; Liu et al., 2017a; Baiyun et al.,

294

2018). ROS are involved in the toxic effect of pyrethroid insecticides (Mossa et al.,

295

2013), and oxidative stress is the main cause of liver damage (Yang et al., 2017;

296

Zhang et al., 2017). MDA is a product of lipid peroxidation and has been widely used 14

297

as a marker for oxidative stress (Lu et al., 2018; Wei et al., 2018; Li et al., 2019c).

298

GSH is an antioxidant that prevents cell damage caused by ROS. All cells in the

299

human body are capable of synthesizing GSH, which has been shown to be essential

300

for protection against oxidative stress (Pompella et al., 2003; Yang et al., 2016a). A

301

decrease in SOD activity can lead to the accumulation of peroxides, which also leads

302

to oxidative stress (Dinu et al., 2010; Su et al., 2019). Our results indicate that

303

oxidative stress is a key factor in the liver damage caused by DLM.

304

TG and TC are major lipids and good indicators of lipid accumulation (Liu et al.,

305

2017b; Cui et al., 2019). As the central site for lipid synthesis and utilization, the liver

306

is the main organ regulating lipid metabolism (Mio and Bloomston, 2016). Steatosis

307

can exacerbate liver damage caused by oxidative stress (Rouvinen-Watt et al., 2014;

308

Amuno et al., 2018), and once hepatic steatosis is established, inflammation,

309

mitochondrial dysfunction, and ROS-induced oxidative stress are enhanced. Lipid

310

peroxidation occurs and adipokines are produced, which can lead to further

311

hepatocyte damage and fibrosis (Day and James, 1998). Hepatic fibrosis is

312

characterized by excessive deposition of extracellular matrix (ECM) proteins, within

313

parenchymal and non-parenchymal hepatocytes, and infiltrating immune cells (Toosi,

314

2015; van Dijk et al., 2015; Baiocchini et al., 2016). The main components of ECM

315

are Col-I and FN1, and HYP is an amino acid unique to collagen. Oxidative stress has

316

also been reported to play a major role in liver fibrosis (Novo et al., 2014). In this

317

study, the oxidative stress associated with liver fibrosis is caused by increased ROS

318

and decreased antioxidant capacity. This appears to induce hepatic stellate cell (HSC) 15

319

proliferation and collagen synthesis, thereby promoting liver fibrosis in quails.

320

TGF-β1 is the most potent factor in promoting liver fibrosis and plays a key role in

321

its occurrence and maintenance (Cui et al., 2003; Schuppan et al., 2003; Wells et al.,

322

2004; Liu et al., 2010). TGF-β1 strongly stimulates HSC to produce Col-Ι and Col-III,

323

thereby producing a large amount of ECM and inhibiting ECM degradation (Yang et

324

al., 2016b). TGF-β1 also binds to receptors on the cell membrane, phosphorylating

325

the receptor-associated Smad2/3 proteins. The conformational changes of activated

326

Smad2/3 results in its release from the receptor complex, which, in turn, leads to

327

interaction with Smad4 to form the Smad2/3-Smad4 complex (Ma et al., 2017). The

328

transport of this complex into the nucleus enhances the expression of α-SMA and

329

Col-Ι and promotes fibrosis. Our data are completely consistent with these events and

330

suggest that long-term exposure to DLM induces oxidative stress in the liver, thereby

331

activating the TGF-β1/Smad pathway and resulting in liver fibrosis in quail.

332

5. Conclusion

333

We discovered in quails that DLM induced liver fibrosis occurs via activation of

334

the TGF-β1/Smad pathway (Figure 8). Inhibition of TGF-β1 production may therefore

335

be a potential therapeutic target for the treatment of DLM-induced liver fibrosis.

336

Declaration of Competing Interest

337 338

The authors declare no conflict of interest.

Acknowledgement

339

This work was funded by the National Natural Science Foundation of China

340

(31972754), Scientific Research Foundation for the Returned Overseas Chinese 16

341 342

Scholars of Heilongjiang Province (LC2017007). We

thank

Kathy

Tamai,

from

Liwen

Bianji,

Edanz

Group

China

343

(www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

344

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638 639 640 641 642 643 644 645 646 647 648 30

649

Figure legends

650

Fig. 1. Morphological characteristics and ultrastructure of liver tissues in quails. (A)

651

Control group. (B) 15 mg kg-1 DLM group. (C) 30 mg kg-1 DLM group. (D) 45 mg

652

kg-1 DLM group. Red arrow: inflammatory cell infiltration. Blue arrow: hepatic cord

653

and

654

200×magnification; Ultrastructure, 10000×magnification).

655

Fig. 2. DLM dose-dependently affected the ALT and AST activity and livers

656

oxidative stress in quails. (A) The activity of ALT. (B) The activity of AST. (C) The

657

concentration of MDA. (D) The concentration of GSH. (E) The activity of SOD. Data

658

are presented as mean ± SEM (n = 10). * Statistically different (p < 0.05) VS. control

659

group.

660

Fig. 3. DLM-induced LMH cell injury and oxidative stress. (A) Cell viability (n = 6).

661

(B) The concentration of ROS. (n = 6). Data are presented as mean ± SEM. *

662

Statistically different (p < 0.05) VS. control group.

663

Fig. 4. DLM induced steatosis in the livers. (A) The concentration of TG. (B) The

664

concentration of TC. (C) Oil red O staining (200×magnification). Data are presented

665

as mean ± SEM (n = 10). * Statistically different (p < 0.05) VS. control group.

666

Fig. 5. DLM induced liver fibrosis. (A) The concentration of HYP. (B) Masson's

667

trichrome staining (200×magnification). Data are presented as mean ± SEM (n = 10).

668

* Statistically different (p < 0.05) VS. control group.

669

Fig. 6. The effect of DLM on the liver fibrosis pathway. (A) The relative protein

670

levels of Col-Ι and α-SMA. (B) The relative protein levels of p-Smad2 and p-Smad3.

hepatic

sinus

disorder.

Yellow

31

arrow:

fat

vacuole.

(HE

staining,

671

(C, D) Values of quantitative analysis (n = 4). (E) TGF-β1, FN1, α-SMA, and ColΙ-α1

672

were detected by qPCR (n = 5). Data are presented as mean ± SEM. * Statistically

673

different (p < 0.05) VS. control group.

674

Fig. 7. Protein network. Protein network of proteins regulated to fibrosis-related genes

675

expressed in rats.

676

Fig. 8. Schematic diagram of the mechanism of DLM-induced liver fibrosis in quails.

677

DLM induces liver fibrosis via activation of the TGF-β1/Smad signaling pathway.

678

32

Highlights 1. Chronic exposure of deltamethrin (DLM) induces liver fibrosis in quail. 2. DLM induces fatty degeneration of liver in quail. 3. DLM induces oxidative stress in LMH cells. 4. Oxidative stress is a key of DLM-induced liver fibrosis.

Bing Han: Conceptualization, Methodology, Validation, Data Curation, Writing-Original Draft Zhanjun Lv: Conceptualization, Writing-Original Draft, Project administration Xiaoya Zhang: Conceptualization, Methodology, Validation Yueying Lv: Validation, Formal analysis Siyu Li: Software, Formal analysis Pengfei Wu: Validation Qingyue Yang: Validation Jiayi Li: Software, Formal analysis Bing Qu: Writing-Review & Editing Zhigang Zhang: Conceptualization, Methodology, Writing-Review & Editing, Project administration

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.