Curcumin ameliorates oxidative stress-induced intestinal barrier injury and mitochondrial damage by promoting Parkin dependent mitophagy through AMPK-TFEB signal pathway

Journal Pre-proof Curcumin ameliorates oxidative stress-induced intestinal barrier injury and mitochondrial damage by promoting Parkin dependent mitophagy through AMPKTFEB signal pathway Shuting Cao, Chunchun Wang, Jintao Yan, Xin Li, Jiashu Wen, Caihong Hu PII:

S0891-5849(19)31488-1

DOI:

https://doi.org/10.1016/j.freeradbiomed.2019.12.004

Reference:

FRB 14513

To appear in:

Free Radical Biology and Medicine

Received Date: 12 September 2019 Revised Date:

23 November 2019

Accepted Date: 3 December 2019

Please cite this article as: S. Cao, C. Wang, J. Yan, X. Li, J. Wen, C. Hu, Curcumin ameliorates oxidative stress-induced intestinal barrier injury and mitochondrial damage by promoting Parkin dependent mitophagy through AMPK-TFEB signal pathway, Free Radical Biology and Medicine (2020), doi: https://doi.org/10.1016/j.freeradbiomed.2019.12.004. 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 Inc.

Graphical abstract

1

Curcumin ameliorates oxidative stress-induced intestinal barrier injury and

2

mitochondrial damage by promoting Parkin dependent mitophagy through

3

AMPK-TFEB signal pathway a

4

Shuting Cao1, Chunchun Wang1, Jintao Yan2, Xin Li1, Jiashu Wen1, Caihong Hu 1*

5 6

7 8

9

1

Animal Science College, Zhejiang University; Key Laboratory of Molecular

Animal Nutrition, Ministry of Education; Hangzhou, 310058, China; 2

UoG-UESTC Joint School, University of Electronic Science and technology of

China, Chengdu 611731, China. a

This research was supported by National Natural Science Foundation of China

10

(31872387), Zhejiang Provincial Natural Science Foundation (LZ20C170003), and

11

National Key R & D Program (2016YFD0501210).

12 13

14 15 16 17 18 19 20 21 22 23

Key words: curcumin, oxidative stress, intestinal injury, mitochondrial function, mitophagy, Parkin, AMPK-TFEB; *Corresponding author: Dr. C. H. Hu, Email: [email protected];

24

Abstract

25

The gut epithelial is known as the most critical barrier for protection against harmful

26

antigens and pathogens. Oxidative stress has been implicated in the dysfunction of the

27

intestine barrier. Hence, effective and safe therapeutic approaches for maintaining

28

intestinal redox balance are urgently needed. Curcumin has gained attention for its

29

vast beneficial biological function via antioxidative stress. However, whether the

30

curcumin can relief intestine damage and mitochondrial injury induced by oxidative

31

stress is still unclear. In this study, we found that curcumin can effectively ameliorate

32

hydrogen peroxide (H2O2)-induced oxidative stress, intestinal epithelial barrier injury

33

and mitochondrial damage in porcine intestinal epithelial cells (IPEC-J2 cells) in a

34

PTEN-induced

35

Mechanistically, depletion of Parkin (a mitophagy related protein) abolished

36

curcumin’s protective action on anti-oxidative stress, improving intestinal barrier and

37

mitochondrial function in porcine intestinal epithelial cells (IPEC-J2) induced by

38

H2O2. Consistently, the protective effect of curcumin was not found in cells

39

transfected with GFP-Parkin∆UBL, which encodes a mutant Parkin protein without

40

the ubiquitin E3 ligase activity, indicating that the ubiquitin E3 ligase of Parkin is

41

required for curcumin’s protective effects. On the other hand, we also found that the

42

protective function of curcumin was diminished when PRKAA1 was depleted in

43

IPEC-J2 cells treated with H2O2. Immunofluorescence and luciferase assay showed

44

that curcumin dramatically enhanced nuclear translocation and transcriptional activity

45

of transcription factor EB (TFEB) in IPEC-J2 cells treated with H2O2, and it was

46

ameliorated by co-treated with compound C, an Adenosine 5‘-monophosphate

47

(AMP)-activated protein kinase (AMPK) inhibitor, which means curcumin promotes

48

TFEB transcript via AMPK signal pathway. Consistent with in vitro data, dietary

49

curcumin protected intestinal barrier function, improved redox status, alleviated

50

mitochondrial damage, triggered mitophagy and influenced AMPK-TFEB signal

51

pathway in a well-established pig oxidative stress model by challenging with diquat.

52

Taken together, these results unveil that curcumin ameliorates oxidative stress,

53

enhances intestinal barrier function and mitochondrial function via the induction of

54

Parkin dependent mitophagy through AMPK activation and subsequent TFEB nuclear

55

translocation.

putative

kinase

(PINK1)-Parkin

mitophagy

dependent

way.

56

Key words: curcumin; oxidative stress; intestinal injury; mitochondrial function;

57

mitophagy; Parkin; AMPK-TFEB;

58

1. INTRODUCTION

59

The gut epithelial is known as the most critical barrier for protection against

60

endogenous and exogenous harmful antigens and pathogens [1]. Oxidative stress has

61

been implicated in the dysfunction of the intestine barrier [2]. Oxidative stress reflects

62

an imbalance between the reactive oxygen species (ROS) and the antioxidative

63

system. Superfluous of ROS may destroy the redox balance, and then damage the

64

proteins, lipids, and DNA, eventually leading to intestinal injury and gut dysfunction.

65

Hence, effective and safe therapeutic approaches are urgently needed to maintain

66

intestinal redox balance.

67

Curcumin is the primary active component found in powdered dry rhizomes of

68

Curcuma longa Linn [3]. Curcumin has been shown as having a broad range of

69

beneficial biological properties, such as antioxidant, anti-inflammatory, antiviral,

70

anticancer and antimicrobial [4], etc. Several papers addressing the effect of curcumin

71

on mitochondrial function and mitochondrial biogenesis and mitophagy [5-8].

72

Generally, under oxidative stress, ROS is overproduced particularly in oxidative stress

73

damaged mitochondria. Therefore, the powerful antioxidant effect of curcumin may

74

be due to the targeting on mitochondria, the primary source of ROS.

75

The intestine has the high energy requirements that rely on mitochondrial

76

oxidative phosphorylation (OXPHOS) to provide energy to support its energy

77

requirements. Mitochondria are the major power factory in various cells, while the

78

process of energy generation companies with the formation of ROS by the

79

mitochondrial oxidative respiratory chain. However, the oxidative respiratory chain is

80

the primary source of ROS, and also more prone to be attacked by ROS, in turns may

81

generate the superfluous ROS. ROS overproduction induces mitochondria injured and

82

oxidation respiratory chain disrupted, finally lead to cell death [9]. Recent research

83

indicated that damaged mitochondria may experience a selective elimination process

84

via lysosome degradation to maintain mitochondria homeostasis and prevent cell

85

death, called mitophagy. A recent study reported that curcumin may serve as an

86

inducer of mitochondrial biogenesis and mitophagy [10]. However, so far, no report is

87

available concerning the influence of curcumin on mitochondrial function and

88

mitophagy in oxidative stress-induced intestinal injury of piglets. It would be of great

89

interest to focus on whether curcumin could prevent intestinal injury induced by

90

oxidative stress via advancing the mitochondria function, mitophagy level of piglets

91

and its underlying molecular mechanisms.

92

Adenosine monophosphate-activated protein kinase (AMPK) has essential roles

93

in the maintenance of cellular energy homeostasis and regulation on mitochondrial

94

function and mitophagy [11]. Transcription factor EB (TFEB) serves as the main

95

regulator of lysosomal biogenesis and autophagy [12]. A previous study has shown

96

that the AMPK- TFEB pathway serves as a dominant regulator of cell fate

97

determination under stress [12]. To be specific, phosphorylated of AMPK under stress

98

promotes the dephosphorylated TFEB and drives its import into the nucleus [13]. The

99

activated TFEB is capable to induce transcription of genes related to autophagosome

100

and lysosome biogenesis. Recently, it was shown that AMPK is activated under

101

oxidative

102

ischemia/reperfusion-induced intestine disorder [14]. Therefore, we speculated that

103

AMPK-TFEB signal pathway may engage in the modulation of curcumin on

104

mitophagy and mitochondria function.

105

stress

situations

and

regulated

mitophagy

to

relieve

intestinal

In this study, we found that curcumin alleviated H2O2 induced oxidative stress,

106

intestinal

epithelial

barrier

injury

and

mitochondrial

damage

in

a

107

mitophagy-dependent way through the PINK1-Pakin pathway in IPEC-J2 cells.

108

Ubiquitin E3 ligase of Parkin was required for curcumin’s protective effects.

109

Furthermore, curcumin promoted Parkin dependent mitophagy via AMPK activation

110

and subsequent TFEB nuclear translocation. These results illustrate the underlying

111

molecular mechanisms of curcumin against oxidative stress-induced intestinal injury

112

and mitochondrial dysfunction. Moreover, the interplay among the intestinal barrier,

113

mitochondrial and mitophagy provides insight into the development of therapeutic

114

strategies in the prevention and treatment of intestinal oxidative stress.

115 116

2. MATERIALS AND METHODS

117

2.1 Reagents and antibodies

118

H2O2, diquat, and Curcumin were purchased from Sinopharm Chemical Reagent Co.,

119

Ltd. (Shanghai, China), Sigma-Aldrich (St. Louis, MO, USA) and Sangon Biotech

120

Co., Ltd. (Shanghai, China), respectively. Dulbecco’s modified eagle medium

121

(DMEM)/Ham’s F-12 (DMEM-F12) was obtained from Sigma Chemical (St. Louis,

122

MO,

123

bromide (MTT) were obtained from Beyotime Institute of Biotechnology (Shanghai,

124

China). Penicillin-streptomycin and sterile phosphate buffered saline (PBS) were

125

purchased from Solarbio life science Co., Ltd. (Beijing, China). Fetal bovine serum

126

(FBS) was obtained from Hangzhou Sijiqing Bio-Engineering Material Co., Ltd.

127

(Hangzhou, China). Fluorescein isothiocyanate dextran 4 kDa (FD4) was obtained

128

from Sigma Aldrich (St. Louis, Missouri, USA). Lipofectamine RNAiMAX and

129

Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA, USA). Mdivi-1,

130

compound C and MG-132 were obtained from Selleck (Westlake Village, CA, USA).

131

Occludin, ZO-1, Claudin-1, SQSTM1, Beclin-1, PRKAA1, GAPDH, Histone H3,

132

HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG antibodies were

133

purchased from HUABIO (Hangzhou, China). Ubiquitin, PINK1, Parkin, LC3B,

134

AMPK, p-AMPK, TFEB antibodies were purchased from Abcam (MA, USA).

135

2.2 Cell culture

136

IPEC-J2 Cells were cultured in DMEM-F12 supplemented with 10% FBS, 1% of

137

penicillin (100 U/mL) and streptomycin (0.1 mg/mL) in a humid incubator with 5%

138

CO2 and 95% air at 37°C. Cells were tested negative for mycoplasma contamination

139

before use. Cells were treated with Mdivi-1 (1 µM) for 1 h, and then treated with

USA).

Trypsin

and

3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium

140

curcumin (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. And then, cells were

141

collected for ELISA, qPCR, western blot and flow cytometer after washed twice by

142

cold PBS.

143

2.3 Animal care and sample collection

144

The experiment processes in this trial were conducted according to the guidelines of

145

the Zhejiang University Animal Care and Use Committee (No. 11844). A total of 35d

146

24 piglets (Duroc×Landrace×Yorkshire, weaned at 21d, n = 6/ treatment; 9.2±0.17kg)

147

were randomly allotted into four groups. Feed and water were freely available. The

148

four treatment groups including: (1) non-challenged control group: pigs receiving a

149

control diet and injected 0.9% NaCl solution; (2) control+curcumin group: piglets fed

150

the diet inclusion of 200 mg/kg curcumin injected 0.9% NaCl solution; (3)

151

diquat-challenged group: pigs receiving the control diet and injected diquat; (4)

152

H2O2+curcumin group: piglets fed the diet inclusion of 200 mg/kg curcumin and

153

administered diquat. Diets were formulated in accordance with NRC 2012. At the

154

starting of the trial, the pigs were administered by injecting abdominal with diquat or

155

saline (10 mg/kg of BW) in accordance with a previous report [15]. On day 14, blood

156

samples were obtained through anterior vena cava in 10 ml vacuum tubes and

157

centrifuged (4000×g, 5 min) to separate serum. Pigs were euthanasia with sodium

158

pentobarbital (200 mg/kg BW) as previous research [16]. Separating of mitochondrial

159

was using the proximal jejunum. Mucosa was obtained from the adjacent jejunum,

160

then immediately frozen in liquid N2 and stored at -80℃.

161

2.4 Cell viability assay

162

IPEC-J2 cells were seeded in a 96-well plate with a density of 1 × 104 cells per well.

163

After 24 h, cells were treated with agents for the indicated time. Then IPEC-J2 cells

164

were incubated with 20 µl of 0.5% MTT for 4 h (Sangon Biotech, Shanghai, China).

165

Absorbance at 570 nm was measured using a fluorescence microplate reader (FLx800,

166

Bio-Tek Instruments Inc., Winooski, USA).

167

2.5 Measurement of redox status

168

In accordance with the manufacturer’s guidelines, the activity of superoxide

169

dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) level were detected

170

by the ELISA kits from Beyotime Institute of Biotechnology (Shanghai, China) [17].

171

2.6 Transepithelial electrical resistance (TER) and FD4 flux of IPEC-J2 cells in

172

the transwell system

173

TER of IPEC-J2 cell monolayer cultured in the transwell (12mm diameter inserts, 0.4

174

µm pore size were from Costar (Corning Incorporated)) was measured using the

175

Millicell-ERS resistance system (Millipore; Bedford, MA). TER was measured at 3

176

different points in each transwell and the background obtained from the blank control

177

was subtracted. As the previous report, the net resistance was multiplied by the

178

membrane area to give the resistance in Ω cm2 [18]. FD4 was dissolved in complete

179

DMEM medium containing 10% FBS at 500 µg/mL concentration and then applied to

180

the apical side of the cell monolayers on the Transwells. FD4 flux detected through

181

collecting the basolateral medium for every 15 min for 1.5 h and replacing the

182

sampled volume with fresh medium without FD4 after sampling. The FD4 flux was

183

quantified using a fluorescence microplate reader (FLx800, Bio-Tek Instruments

184

Inc.,) [19].

185

2.7 Ultrastructure of intestinal mitochondria

186

The IPEC-J2 cells or fresh jejunal segments were fixed with 2.5% glutaraldehyde in

187

phosphate buffer (0.1M，pH7.0) for 24h and 1% OsO4 in phosphate buffer (0.1M，

188

pH7.0) for 1.5h. The IPEC-J2 cells and jejunal segments were dehydrated by ethanol

189

and acetone and embedded in resin. The specimen was sectioned in LEICA EM UC7

190

ultratome and stained by uranyl acetate and alkaline lead citrate for 5 to 10min

191

respectively and observed in Hitachi Model H-7650 TEM (Tokyo, Japan).

192

2.8 Mitochondrial swelling assay

193

The procedure of mitochondrial swelling measurement was according to the method

194

of Du et al. (2010) [20]. The mitochondria were suspended in 1 mL swelling assay

195

buffer [150 mM KCl, 5 mM Hepes, 2 mM K2HPO4, 5 mM glutamate (pH 7.2)].

196

Mitochondrial swelling induced by the addition of calcium (500 nmol/mg of protein).

197

The swelling degree was observed by recording changes in OD540

198

fluorescence microplate reader (FLx800, Bio-Tek Instruments Inc.,) at 37°C for 600s.

199

2.9 Measurement of mitochondria membrane potential (△ △Ψm)

200

The △Ψm was detected by △Ψm assay kit (Beyotime Institute of Biotechnology,

201

Shanghai, China) and the procedures were in accordance with the guidance. When at

202

a high ∆Ψm, JC-1 monomers are able to form aggregates in the mitochondrial matrix,

203

which fluoresce red (OD590

204

[fluoresces green (OD529

205

mitochondria was calculated as the fluorescence ratio of aggregates (red) to

206

monomers (green) using fluorescence microplate reader (FLx800, Bio-Tek

207

Instruments Inc.,) [21].

208

2.10 ATP production capacity

209

Measurement of ATP production used an ATP detection kit (Beyotime Institute of

210

Biotechnology) following the manufacturer’s instructions. This method based on the

211

firefly luciferase catalyzes the production of fluorescence by fluorescein, which

212

requires ATP to provide energy. Fluorescence is proportional to the concentration of

213

ATP in a certain concentration range and the ATP production was calculated using an

214

ATP standard curve.

215

2.11 Activity of electron transfer chain complexes I–III in mitochondria

216

The activity of mitochondria electron transfer chain complexes I-III was measured by

217

the quantitative determination kits in accordance with the guidance of Genmed

218

Scientifics (Shanghai, China), as a previous study [22]. Complex I (NADH

219

dehydrogenase) activity was detected using the changing of NADH oxidation

220

absorption at 340 nm. Complex II (succinate dehydrogenase) activity was determined

221

by recording the alteration of the absorbance of 2, 6-dichlorophenolindo-phenol

222

(DCPIP) at 600 nm. Complex III (cytochrome c reductase) activity detected by

223

calculating the alteration of the absorbance of cytochrome c at 550 nm.

nm).

nm)]

nm

using a

When the ∆Ψm is declined, the JC-1 monomers

unable to assembled. Since, the △Ψm of intestinal

224

2.12 Immunofluorescence analysis

225

IPEC-J2 cells were seeded on the glass bottom dishes and treated with or without

226

indicated agents. The cells washed with PBS and fixed with 4% paraformaldehyde for

227

10 min, washed 3 times and permeabilized with Triton X-100 for 10 min (Beyotime

228

Biotechnology, Shanghai, China). The cells were incubated with primary Abs (TFEB,

229

ZO-1) overnight at 4°C, and incubated with FITC-conjugated secondary Abs (1:200)

230

for 1 h. Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) or Hoechst

231

33258 staining. Images were obtained with an LSM 510 META confocal laser

232

microscope (Carl Zeiss Ltd, Oberkochen, Germany) and analyzed using Zeiss

233

LSM800.

234

2.13 siRNA and transfection

235

IPEC-J2 cells were seeded into 6-well plates to grow about 80% confluent. The next

236

day, individual targeted siRNA and plasmid were mixed with Lipofectamine

237

RNAiMAX or lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), respectively.

238

The RNAiMAX/siRNA mixture was added to IPEC-J2 cells in antibiotic-free medium

239

and cultured for 8 h. Medium containing siRNA was refreshed with the general

240

medium for another 12 h before other treatments. Small interfering RNA (siRNA)

241

targeting pig Parkin (5’- GCA TCA CCT GTA CGG ACA TTT -3’), pig PRKAA1

242

(5’- GCT GCA CCA GAA GTA ATT TTT-3’) and scrambled control siRNA

243

(5’-UUC UCC GAA CGU GUC ACG UTT-3’) were synthesized by GenePharm. To

244

directly visualize autophagosomes-containing mitochondria in IPEC-J2 cells, the

245

Ad-GFP-LC3 (Hanbio, Shanghai, China) used to detect autophagosomes and

246

Ad-HBAD-Mito-dsred (Hanbio, Shanghai, China) used for fluorescent labeling of

247

mitochondria which were incubated with IPEC-J2 cells for 24 h.

248

2.14 Luciferase reporter assay

249

IPEC-J2 cells were seeded in a 24-well plate for 18 h to reach 70% confluence. The

250

cells were then co-transfected with luciferase reporter plasmid with TFEB promoter

251

or empty inserted into pmirGLO Dual-Luciferase vector (Promega, Madison, WI,

252

USA) using LipofectAMINE™ 2000 reagent (Invitrogen). After 48 h post

253

transfection, IPEC-J2 cells were co-treated with curcumin (10 µM) and compound C

254

(10 µM) in Luciferase assay for 12 h, followed by H2O2 (600 µM) for 8 h. The

255

luminescence intensities were measured using a Dual-Luciferase Reporter Assay

256

system (Promega, WI, USA). Renilla luciferase signals were normalized to the

257

internal firefly luciferase transfection control. Transfections were performed in at

258

least triplicate for each independent experiment.

259

2.15 Intestinal mitochondrial isolation

260

Extracting of intestinal mitochondria was using proximal jejunum. All the processes

261

were in accordance with the guidelines of mitochondrial isolating kits (Beyotime

262

Institute of Biotechnology, Shanghai, China).

263

2.16 Intestinal barrier function

264

The intestinal barrier function was measured by Ussing chamber system (model VCC

265

MC6; Physiologic Instruments, San Diego, CA, USA) and the procedures were in

266

accordance with previously reported [23].

267

2.17 Histopathology

268

Fixed in 10% formalin, massive specimens of intestine were embedded in paraffin

269

and cut into 5 µm thick slides. Staining with hematoxylin and eosin (H & E), slides

270

were scanned with a scanner (Leica Aperio CS2, Germany), with histopathological

271

analysis using Aperio XT system (Nikon Instruments Europe, Aperio Technologies,

272

Vista, CA, USA).

273

2.18 Quantitative real-time PCR (qPCR) analysis.

274

Total RNA from cells was extracted using the TRIzol reagent (Beyotime Institute of

275

Biotechnology, Shanghai, China) and reverse transcribed into cDNA using M-MLV

276

reverse transcriptase (Beyotime Institute of Biotechnology, Shanghai, China). qPCR

277

analysis was performed using the SYBR Green PCR Master Mix (Beyotime) with the

278

CFX96 Real-Time PCR System. The data were analyzed following the 2-∆∆Ct method

279

and calculated using β-Actin as the normalization control. The sequences of primers

280

used are presented in Table S1.

281

2.19 Western blot analysis

282

The procedures of western blot assay were according to the description of Xiao et al.

283

(2016)[23].

284

radioimmunoprecipitation lysis buffer (Solarbio life science. Beijing, China), and then

285

total cellular proteins were gathered. Nuclear proteins were collected by nuclear and

286

cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology). After

287

electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes

288

(Millipore, Bedford, MA, USA). The membranes were incubated with first antibodies

289

(claudin-1, occludin, ZO-1, P62, Beclin-1, LC3 I, II, GAPDH, PINK1, Parkin,

290

Ubiquitin, AMPK, p-AMPK, TFEB, Histone H3) for 12-16 h at 4℃ and then

291

incubated with the secondary antibodies (HRP conjugated anti-rabbit Ab) for 2 h at

292

21-25℃. Chemiluminescence signals were detected by ECL western blotting detection

293

reagent (Amersham), and visualized using ChemiScope 3400 (Clinx Science

294

Instruments, China). The arbitrary densitometric units for each protein of interest

295

were normalized using those of GAPDH.

296

2.20 Statistical analyses

297

Statistics analyses were conducted using GraphPad Prism 6. Differences among

298

means were tested using Turkey multiple-comparisons test. The difference was

299

considered to be significant if P < 0.05.

IPEC-J2

cells

and

intestinal

samples

were

lysed

by

300 301

3. RESULTS

302

3.1

303

mitophagy-dependent way in IPEC-J2 cells

304

To explore the mechanism that curcumin ameliorates oxidative stress-induced

305

intestinal barrier injury, we established oxidative stress models in IPEC-J2 cells by

306

H2O2. As presented in Fig. 1A, H2O2 treatment decreased the cell viability after

Curcumin

ameliorates

H2 O2

induced

oxidative

stress

in

a

307

treatment with 600 µM, 800 µM and 1000 µM H2O2 for 8 h. So, 600 µM H2O2 for 8h

308

was used to establish the model of oxidative stress in the current experiment.

309

Furthermore, Fig. 1B shows the viability of IPEC-J2 cells after treated with different

310

concentrations of curcumin. Compared with the control cells, exposure to 5 µM and

311

10 µM curcumin for 12 h increased the cell viability, while exposure to 60 and 80 µM

312

curcumin for 12h decreased the cell viability. Furthermore, the protective function of

313

curcumin on H2O2 induced cell oxidative stress was observed for IPEC-J2 cells at 10

314

µM and 20 µM (Fig. 1C). Based on the above results, 10 µM dosage of curcumin was

315

utilized in the subsequent experiments.

316

Next, we determined the activity of SOD, CAT, and level of MDA. As shown in

317

Fig. 1D, E, F, the curcumin treatment can effectively prevent the decline of activity of

318

SOD, CAT, and enhancement of MDA level induced by H2O2. Moreover, H2O2

319

treatment decrease the expression of Cu/Zn-SOD, Mn-SOD, GPX-1 and GPX-4,

320

which was increased by curcumin (Fig. G). Furthermore, we detected the ROS

321

generation by flow cytometer and fluorescence staining. As shown in Fig. H, I,

322

curcumin addition significantly inhibited the increased ROS production induced by

323

H2O2 treatment in IPEC-J2 cells.

324

Our previous studies found that oxidative stress-induced mitochondrial

325

dysfunction and triggered protective mitophagy in a pig oxidative stress model,

326

suggesting that induction of mitophagy in the intestine may play an important role in

327

the host response to intestinal barrier dysfunction [24]. Additionally, Wang et al.

328

(2012) detected curcumin induced the initiation of mitophagy through TEM after

329

ultrasound treatment in CNE2 cells [25]. In order to elucidate whether curcumin plays

330

a protective role through the regulation of mitophagy, we treated the IPEC-J2 cells

331

with a mitophagy inhibitor, Mdivi-1. Interestingly, we found that the protective effect

332

of curcumin on anti-oxidative stress was almost blocked by Midivi-1 in IPEC-J2 cells

333

treated with H2O2. These results suggest that curcumin can effectively ameliorate

334

H2O2 induced oxidative stress in IPEC-J2 cells, and mitophagy may account for the

335

anti-oxidative stress effect of curcumin.

336

3.2

337

mitochondria dysfunction in a mitophagy-dependent way in IPEC-J2 cells

338

In Fig. 2A, B, we found that curcumin showed a protective effect on intestinal barrier

339

function, as indicated by prevent the decreased TER (Fig. 2A) and increased FD4 flux

340

(Fig. 2B) induced in H2O2 treated cells, which was counteracted by Mdivi-1. The

341

integral membrane components of tight junction proteins to regulate the selective

342

permeability between epithelial cells. Accordingly, we found that curcumin increased

343

the expression of tight junction proteins (occludin, ZO-1, and claudin-1) in IPEC-J2

344

cells challenged with H2O2, and these effects of curcumin were suppressed by

345

Mdivi-1 (Fig. 2 C), which was consistent with the immunofluorescence of Claudin-1.

346

Curcumin inhibits IPEC-J2 cells barrier function disrupted by the H2O2 through

347

enhanced the distribution of claudin-1 along the cell membrane, while this protective

348

function was blocked by Mdivi-1 (Fig. 2 D). Additionally, we indicated the

349

mitochondria function by ATP production, mitochondria complexes activity, and

350

mitochondria membrane potential. We found that the curcumin treatment effectively

351

prevented the decline of ATP production (Fig. 2E), mitochondria complexes activity

352

(Fig. 2F) and mitochondria membrane potential (Fig. 2G) induced by H2O2 in

353

IPEC-J2 cells and Mdivi-1 counteract these protective functions. To determine

354

whether the mitochondria ultrastructure affected by curcumin and H2O2 treatment, we

355

examined mitochondria ultrastructure by transmission electron microscope (TEM)

356

(Fig. 2H). the data showed that curcumin significantly ameliorated mitochondrial

357

swelling and disruption of mitochondrial cristal membrane induced by H2O2, while

358

this effect was diminished by Mdivi-1. These results suggest that curcumin can

359

effectively ameliorate H2O2 induced disrupted epithelial barrier and mitochondria

360

dysfunction in IPEC-J2 cells, and mitophagy may account for this protective effect of

361

curcumin.

362

3.3 Curcumin activates mitophagy through PINK1-Parkin pathway in H2O2

Curcumin

ameliorates

H2 O2

induced disrupted

epithelial barrier,

363

treated IPEC-J2 cells

364

Mitophagy is a special kind of autophagy that means a lot to keep cell homeostasis

365

through clearing disrupted mitochondrial with declined membrane potential. To

366

further determine the underlying mechanism through which curcumin regulated

367

mitophagy to prevented IPEC-J2 cells form H2O2 stimulation, we examined the

368

related protein expression, gene expression, and mitochondrial autophagosome

369

formation. We found that curcumin significantly alters the expression of autophagy

370

markers in IPEC-J2 cells treated with H2O2 (Fig. 3A), while the Mdivi-1 suppressed

371

them. Interestingly, the curcumin addition increased the gene and protein expression

372

of PINK-1 and Parkin compared with the H2O2 treated cells and these effects of

373

curcumin were suppressed by Mdivi-1, while it did not alter the expression of

374

BNIP3L, BNIP3 and FUNDC-1 (Fig.3 B, C). To directly confirm the activation of

375

mitophagy, we transinfected Ad-GFP-LC3 and Ad-HBAD-Mito-dsred to monitor

376

mitochondrial autophagosome formation in IPEC-J2 cells. Confocal microscopy

377

analysis showed curcumin significantly increased the co-location of Ad-GFP-LC3 and

378

Ad-HBAD-Mito-dsred in cells treated with H2O2, while the Mdivi-1 declined the

379

co-location (Fig.3D). We utilized TEM to directly monitor mitochondrial

380

autophagosome formation and found that a lot of autophagosomes to parcel the

381

mitochondria in curcumin + H2O2 group, while we cannot find in Mdivi-1 group

382

(Fig.3E). These data suggest that the curcumin significantly activated mitophagy

383

through the PINK1-Parkin pathway in H2O2 treated IPEC-J2 cells. Moreover, we

384

indicated the colocalization of Parkin/PINK and LC3 puncta using confocal

385

microscopy and showed curcumin significantly increased the co-location of

386

Ad-GFP-LC3 and Parkin/PINK in cells treated with H2O2, while the Mdivi-1 declined

387

the co-location (Fig.S1A, B).

388

3.4 Parkin dependent mitophagy is necessary for curcumin’s protection

389

functions in H2O2 treated IPEC-J2 cells

390

Parkin has been demonstrated to play a crucial role in mitophagy induction in

391

mammalian cells. We previously provided evidence indicating that Parkin may be

392

involved in the mitophagy process of IPEC-J2 cells treated with H2O2. To determine

393

if Parkin is required for curcumin-activated mitophagy, we depleted Parkin and

394

examined mitophagy related genes. We used siRNA to knockdown the Parkin gene

395

and found that knockdown of Parkin decreased cell viability upon H2O2/curcumin

396

treatment (Fig. 4A). The western blot showed that the Parkin protein expression was

397

dramatically reduced (Fig. 4 B). Correspondently, Parkin depletion decreased the

398

mitophagy related proteins, PINK-1, LC3-II, and Beclin-1, indicating the inhibition of

399

mitophagy. Most importantly, Parkin depletion significantly reduced H2O2-induced

400

decreased the activity of SOD, CAT and increased MDA level in IPEC-J2 cells (Fig.4

401

C, D, E). And also, we examined the expression of antioxidative stress gene

402

(Cu/Zn-SOD, Mn-SOD, GPX-1, GPX-4) and found that Parkin knockdown

403

dramatically attenuated the curcumin induced enhancement of Cu/Zn-SOD, Mn-SOD,

404

GPX-1 and GPX-4 in IPEC-J2 cells (Fig. 4F).

405

Furthermore, we noticed that Parkin knockdown exacerbated the impaired intestinal

406

barrier function induced by H2O2, indicated by decreased TER and increased FD4

407

flux in IPEC-J2 cells (Fig.4 G, H). The intestinal protection of curcumin can be

408

significantly counteracted by Parkin knockdown in IPEC-J2 cells treated with H2O2

409

(Fig.4 G, H). We also measured the mitochondrial function after the depletion of

410

Parkin. We found that Parkin depletion exacerbated the disrupted mitochondrial

411

function induced by H2O2, indicated by decreased ATP production and mitochondrial

412

membrane potential (Fig.4 I, J). Meanwhile, the curcumin’s protective effect on

413

mitochondria was blocked by Parkin knockdown (Fig.4 I, J). Further, the protection

414

was not found in cells transfected with GFP-Parkin∆UBL, which encodes a mutant

415

Parkin protein without the ubiquitin E3 ligase activity, as indicated by decreased

416

ubiquitin level (Fig.4 K). Consistently, Parkin transfection but not Parkin∆UBL

417

transfection, enhanced mitophagy, as reflected by a decreased TOMM20 level,

418

suggesting that the E3 ligase function of Parkin was required for curcumin activated

419

mitophagy and cell viability against oxidative stress (Fig. 4 L, M). Furthermore,

420

Parkin∆UBL transfection increased ROS levels and decreased ATP production and

421

mitochondrial membrane potential (Fig. S2A, B, C). Thus, these data indicated that

422

ubiquitin E3 ligase of Parkin mediated mitophagy is necessary for curcumin’s

423

protective effects.

424

3.5 Curcumin promotes mitophagy through AMPK-TFEB signal pathway in

425

H2O2 treated IPEC-J2 cells

426

Since the AMPK plays an essential role in the transcriptional regulation of autophagy

427

and TFEB is a key positive regulator of autophagy and lysosome biogenesis. To

428

further explore the mechanisms by which curcumin regulates Parkin dependent

429

mitophagy, the AMPK-TFEB signal pathway was investigated. We found that

430

curcumin increased the phosphorylation of AMPK and TFEB nuclear translocation in

431

IPEC-J2 cells treated with H2O2 (Fig. 5A). To verify that AMPK is required for

432

curcumin-induced mitophagy, we utilized the siRNA of PRKAA1 (protein kinase

433

AMP-activated catalytic subunit a1, the dominating AMPK catalytic subunit) to

434

knockdown PRKAA1. Depletion PRKAA1 decreased the PINK1 and Parkin

435

expression (Fig. 5B), which indicated that curcumin was not able to induce mitophagy

436

after knockdown PRKAA1. Then we examined whether the AMPK is involved in the

437

effect of curcumin on antioxidative stress, mitochondrial protection, and intestinal

438

barrier protection. The PRKAA1 depletion abolished the curcumin’s enhanced effect

439

on SOD, CAT and decreased the effect on MDA in IPEC-J2 cells (Fig. 5C, D, E). We

440

also found that PRKAA1 depletion aggravated H2O2 induced low expression of

441

Cu/Zn-SOD, Mn-SOD, GPX-1 and GPX-4 (Fig. 5F). We noticed that Curcumin

442

prevented the decline of TER and enhancement of FD4 flux induced by H2O2 in

443

wild-type IPEC-J2 cells but not in PRKAA1 depletion cells (Fig.5G, H). We also

444

detected that PRKAA1 depletion exacerbated the impaired mitochondrial function

445

induced by H2O2, indicated by decreased ATP production and mitochondrial

446

membrane potential in IPEC-J2 cells (Fig.5I, J). The mitochondrial protection of

447

curcumin can be markable counteracted by PRKAA1 depletion in IPEC-J2 cells

448

treated with H2O2 (Fig.5I, J). Furthermore, we utilized the fluorescence microscopy to

449

detect nuclear translocation of TFEB in IPEC-J2 cells, TFEB nuclear translocation

450

was observed in curcumin+H2O2 group compared with controls and H2O2 group (Fig.

451

5K). To determine whether curcumin regulates TFEB transcription, TFEB promoter

452

activity in IPEC-J2 cells was detected by luciferase assay. Curcumin dramatically

453

enhanced TFEB transcriptional activity in IPEC-J2 cells treated with H2O2 compared

454

with the H2O2 group, and it was ameliorated by co-treated with compound C, an

455

AMPK inhibitor, which means curcumin promotes TFEB transcript via AMPK signal

456

pathway (Fig. 5L). Moreover, we found that compound C can counteract curcumin’s

457

protective functions as Mdivi-1 did, indicating by decreased antioxidant activity,

458

disrupted intestinal barrier and mitochondrial function (Fig. S3 A-I). These results

459

suggest that the AMPK-TFEB signal pathway may be responsible for curcumin’s

460

mitophagy induction, antioxidative stress, mitochondrial and intestinal protection

461

functions.

462

3.6 Curcumin alleviates intestinal injury, improves mitochondrial function,

463

induces mitophagy and influences AMPK-TFEB signal pathway in a piglet’s

464

model

465

To determine whether curcumin can exert protective functions in vivo, we utilized pig

466

as a model, which is an excellent model for human disease and clinical medicine

467

applications, due to the high similarity in intestinal physiological properties [26]. We

468

made use of a well-established pig intestinal oxidative stress model by challenging

469

with diquat, which could induce dysfunction of intestinal barrier function and

470

nutrition metabolism [24, 27]. In the present study, challenging with diquat

471

successfully led to oxidative stress in the jejunum of pigs, exhibited by the pigs

472

challenged with diquat had a lower activity of SOD, CAT and a higher MDA level in

473

the intestine than pigs in the control group (Fig. 6A, B, C). Supplementation with

474

curcumin in pigs injected with diquat increased the SOD, CAT activity and reduced

475

the MDA level in the intestinal mucosa, in comparison with pigs challenged with

476

diquat (Fig. 6A, B, C). Compared with pigs injected with saline, pigs challenged with

477

diquat had a lower TER and higher FD4 activity in the jejunum (Fig. 6D, E). Addition

478

with curcumin in pigs injected with diquat enhanced TER and declined (P < 0.05)

479

FD4 in comparison with the diquat group (Fig. 6D, E). Furthermore, in comparison

480

with pigs injected with saline, western blotting results showed that pigs treated with

481

diquat had a lower level of occludin, claudin-1 and ZO-1 (Fig. 6F). Supplied with

482

curcumin in pigs treated with diquat reversed the decline of the occludin, claudin-1

483

and ZO-1 expression induced by diquat (Fig. 6F). As compared to the control group,

484

Diquat challenge has resulted in atrophy of the intestinal mucosa, while the intestinal

485

morphology was improved by curcumin supplementation (Fig. G).

486

Through TEM observation, the mitochondria of the pigs treated with saline and

487

supplied curcumin in control pigs showed intact membranes of mitochondria (Fig.

488

6H). On the contrary, some swelling mitochondria with disrupted respiratory cristae

489

were showed in pigs treated with diquat (Fig. 6H), which were prevented by curcumin

490

supplementation (Fig. 6H). Meanwhile, compared with control group, the diquat

491

challenge dramatically promoted the mitochondria swelling, while curcumin in pigs

492

treated with diquat decreased the mitochondria swelling degree in the intestine (Fig.

493

6I). In comparison to the control group, dietary curcumin in control pigs had no effect

494

on △Ψm and mitochondrial complexes activity. After the diquat challenge, the △Ψm

495

and mitochondrial complexes I, II, III activity were declined in the jejunum

496

mitochondria in comparison with those treated with saline (Fig. 6J, K). However,

497

dietary curcumin in pigs treated with diquat significantly increased △Ψm and

498

mitochondrial complexes I, II, III activity in the jejunum, relative to those injected

499

with diquat (Fig. 6 J, K).

500

Addition with curcumin in control group and injecting with diquat had no effect

501

on SQSTM1, Beclin-1, LC3 I, II, PINK1 and Parkin expression, in comparison with

502

pigs treated with saline (Fig. 6L). Dietary curcumin in those pigs treated with diquat

503

had a lower SQSTM1 level, and higher Beclin-1, LC3 II expressions compared with

504

diquat treated pigs (Fig. 6L). Additionally, compared with pigs injected with diquat,

505

dietary curcumin in pigs treated with diquat enhanced PINK1 and Parkin level in the

506

intestine mitochondrion (Fig. 6L). There was no difference in the expression of

507

pAMPK and TFEB between the control group and addition curcumin in the control

508

group (Fig. 6M). Compared to pigs treated with saline, supplementation with

509

curcumin in diquat challenged group dramatically enhanced the phosphorylation level

510

of AMPK and TFEB nuclear translocation in comparison with diquat piglets (Fig.

511

6M). Taken together, these data suggested that dietary curcumin protected intestinal

512

barrier function, improved redox status, alleviated mitochondrial damage, triggered

513

mitophagy and influenced AMPK-TFEB signal pathway in the pig model, which was

514

consistent with in vitro results. Furthermore, we indicated the Parkin, phospho-AMPK

515

and TFEB nuclear translocation by immunofluorescence using a confocal microscope.

516

We found that curcumin treatment significantly increased Parkin, phospho-AMPK

517

expression levels and TFEB nuclear translocation in the intestine in the diquat

518

challenged piglets, which strongly support our mechanism in vivo (Fig. S4 A-C).

519

4. DISCUSSION

520

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a

521

primary active constituent of turmeric (Curcuma Longa), which has been shown its

522

antioxidative stress effect in vitro [28] and in vivo [29]. Additionally, an increasing

523

number of reports found that curcumin plays a vital role in preserving intestinal

524

barrier

525

ischemia/reperfusion-induced intestinal injury in rats, due to its antioxidant capacity

526

[30, 31]. Till now, there is no data concerning the influence of curcumin on intestine

527

damage induced by oxidative stress in IPEC-J2 cells. Hence, we made use of H2O2, a

528

strong oxidant capable to oxidize various moieties, to induce oxidative stress in

529

IPEC-J2 cells as previous reports [32, 33]. In the present study, we demonstrated that

function

in

H2O2-induced

Caco-2

enterocytic

monolayers

or

530

curcumin at 10 µM protected IPEC-J2 cells from H2O2 induced oxidative stress. Even

531

if there were reports outlining the protective effects of curcumin against oxidative

532

stress [34, 35]. As far as I know, this is the first study to reveal the underlying

533

molecular mechanism by which the curcumin protects IPEC-J2 cells from oxidative

534

stress. We indicated that curcumin counteracted the H2O2 induced oxidative stress by

535

decreasing MDA level, increasing SOD, CAT activity and upregulating Cu/Zn-SOD,

536

Mn-SOD, GPX-1 and GPX-4. Moreover, curcumin decreased the H2O2 induced

537

generation of ROS in IPEC-J2 cells. Our result was supported by the consequences of

538

Dai et al (2015) discovered that curcumin pre-treated induced higher SOD, GSH

539

activity and lower ROS production, in hepatocyte L02 cells [36]. To further

540

investigate whether the antioxidative stress function of curcumin is exerting via

541

regulation on mitophagy in IPEC-J2, we utilized the mitochondrial division inhibitor

542

1, Mdivi-1, to inhibit mitophagy in this experiment as previous studies [37, 38].

543

Intriguingly, the curcumin’s protective effect on antioxidative stress was almost

544

inhibited by Mdivi-1. These data revealed that curcumin can effectively ameliorate

545

H2O2 induced oxidative stress in a mitophagy dependent way in IPEC-J2 cells.

546

An intact intestinal barrier plays a vital role in protecting gut health [1]. In the

547

current experiment, the data showed that treated with H2O2 damaged intestinal

548

epithelial barrier function in IPEC-J2, which was similar to a previous report [39].

549

However, addition curcumin significantly increased TER and decrease FD4 flux

550

compared with the H2O2 group. Similarly, Wang et al. (2012) had reported that dietary

551

curcumin exerted a beneficial effect on intestinal barrier function as indicated by

552

decreasing sodium fluorescein permeability of Caco-2 challenged with H2O2 [30]. The

553

integral membrane components of tight junction regulate the selective permeability

554

between intestinal epithelial cells [40]. In this trial, we found that curcumin prevented

555

the declined expression of tight junction proteins ZO-1, Occludin, and Claudin-1

556

induced by H2O2. Similarly, Trujillo et al (2016) had reported that curcumin inhibits

557

cisplatin-induced decrease of occludin, β-catenin and E-cadherin in rats [41]. Apart

558

from appropriate expression, suitable distribution of the tight junction proteins is also

559

significant for maintaining an integrated intestinal epithelial. So, we detected the

560

Claudin-1 organization in IPEC-J2 cells using immunofluorescence and found that

561

curcumin prevents H2O2-induced disorganization of claudin-1 in accordance with

562

published data [42]. At present, by using TEM and mitochondria swelling degree

563

assay, we found that H2O2-induced oxidative stress induced the mitochondrion

564

became cristae disrupted, swelled and vacuolated. And then, we firstly indicated that

565

curcumin improved the H2O2-induced disruption of mitochondria morphology in

566

IPEC-J2 cells, reflected by unbroken membrane and cristae. Similarly, Zhang et al

567

(2014) had shown that curcumin effectively alleviated irregular-shaped and swelling

568

of hepatic mitochondria in mice challenged with D-galactosamine/Lipopolysaccharide

569

[43]. Furthermore, the damage mitochondria structure will accompany with loss of

570

mitochondrial membrane potential [44]. Disruption of mitochondria structure might

571

result in the opening of mitochondrial permeability transition pore, which can bring

572

about the depolarization of mitochondrial and decline of ATP production. In this trial,

573

we found that curcumin can inhibit the decline of mitochondrial membrane potential,

574

ATP production and mitochondrial complexes activity induced by H2O2. Priyanka et

575

al. (2017) curcumin exerts a mitochondria protective effect in 3T3-L1 adipocytes

576

challenged with hypoxia by protecting mitochondria, reflect an increased

577

mitochondria membrane potential and integrity of mitochondria permeability

578

transition pore [45]. Garcin-Nino et al. (2013) had reported that treatment with

579

curcumin prevented potassium dichromate-induced decline of respiratory complex I

580

activity in liver of rats [46]. Thus, we speculated that the beneficial influence of

581

curcumin on intestinal injury induced by oxidative stress maybe partially through

582

prevent mitochondria swelling and depolarization of intestinal mitochondria.

583

Furthermore, we found that the curcumin’s protective effect on intestinal barrier

584

function and mitochondrial were almost counteracted by Mdivi-1, means the

585

mitophagy may engage in this process.

586

Mitophagy has been regarded as one of the important pathways to remove

587

dysfunctional mitochondria before it activates cell apoptosis [9]. It is a complex

588

process that the damaged mitochondria have been swallowed into vesicles coated with

589

the autophagosome marker LC3. However, there is no report concerning the effect

590

and mechanism of curcumin on mitophagy in the H2O2-oxidative stress of IPEC-J2

591

cells. Here, we first determined the influence of curcumin on mitophagy level in

592

H2O2-oxidative stress of IPEC-J2 cells. We found that curcumin enhanced the level of

593

Beclin-1 and LC3-II, declined the P62 level, which means a higher autophagy level in

594

cells treated with curcumin and H2O2. Currently, a number of studies had reported

595

that PINK1, Parkin, BNIP3L, BNIP3, and FUNDC-1 implicated in modulating

596

mitophagy [47, 48]. In the present experiment, we demonstrated that curcumin

597

dramatically increased the expression of PINK1 and Parkin, but it has no influence on

598

BNIP3L, BNIP3 and FUNDC-1 in cells treated with H2O2. Correspondingly, we

599

verify that curcumin promoted mitophagy indicated by the enhanced autophagosomes

600

under TEM and colocalization of Ad-GFP-LC3, Ad-HBAD-Mito-dsred in IPEC-J2

601

cells treated with H2O2. de Oliveira et al. (2016) had stated that curcumin may

602

regulate mitophagy and the clearance of dysfunctional mitochondria through

603

modulating autophagy related signaling pathways [49]. Wu et al. (2012) had found

604

that curcumin is able to trigger mitophagy in nasopharyngeal carcinoma CNE2 cells

605

after challenged with ultrasound [50]. So, it is reasonable to assume that curcumin can

606

regulate PINK1, Parkin and trigger mitophagy to obliterate the damaged mitochondria

607

caused by oxidative stress, which can prevent the intestine from metabolism

608

disorders.

609

Currently, several reports indicated that Parkin is tightly linked with mitophagy

610

[48, 51]. However, whether Parkin dependent mitophagy link with H2O2 induced

611

oxidative stress in IPEC-J2 cells is still a mystery. In this trial, we knockdown the

612

Parkin to detected the antioxidant ability, intestinal epithelial barrier function, and

613

mitochondrial function. We found that Parkin depletion almost blocked the

614

curcumin’s protective effect on antioxidant, intestinal epithelial barrier function and

615

mitochondrial function. So, we clarified the contributions of Parkin dependent

616

mitophagy in curcumin’s protection effect. However, it is still largely unknown which

617

biochemical structure part of Parkin is involved in curcumin’s protection function. To

618

our knowledge, Parkin is a cytosolic E3 ubiquitin ligase that is recruited to

619

mitochondria depolarization or ROS and ubiquitinates mitochondrial outer membrane

620

proteins [52]. Therefore, we transfected the GFP-Parkin∆UBL in IPEC-J2 cells,

621

which encodes a mutant Parkin protein without the ubiquitin E3 ligase activity. We

622

demonstrated that control cells treated with curcumin, but not the Parkin∆UBL cells,

623

induced mitophagy and increased cell viability in IPEC-J2 cells challenged with H2O2.

624

Hence, the E3 ligase of Parkin seems to be indispensable for curcumin’s protection

625

against oxidative stress, as a mutant Parkin could ameliorate this effect.

626

The molecular mechanisms by which oxidative stress leads to the disruption in

627

mitochondria and induction of mitophagy are still unclear. Several studied had

628

reported that AMPK played a central protective role in attenuating oxidative injury

629

and regulating mitochondrial function [53, 54]. AMPK has been implicated in

630

involving in the regulation of oxidative stress, through phosphorylating some

631

transcription factors, including the master transcriptional regulator of lysosomal genes,

632

TFEB. TFEB is tightly connected with stress, with non-stressed conditions reducing

633

hyperphosphorylation and cytoplasmic reservation and under stress facilitating

634

hypophosphorylation and nuclear translocation. We demonstrated that addition with

635

curcumin in IPEC-J2 cells challenged with H2O2 unregulated the AMPK

636

phosphorylation and TFEB level in the nucleus. Furthermore, we utilized the siRNA

637

of PRKAA1 to knockdown AMPKα1, which is known as the dominating AMPK

638

catalytic subunit. Importantly, we showed that siRNA PRKAA1 treatment reversed

639

the antioxidative stress, enhancing intestinal barrier function and protecting

640

mitochondrial function effects of curcumin in IPEC-J2 cells. Similarly, Xiao et al

641

(2013) demonstrated that curcumin significantly enhances the phosphorylation of

642

AMPK in lung adenocarcinoma cells [55]. Interestingly, we found that depletion

643

PRKAA1 decreased the PINK1 and Parkin expression, which indicated that curcumin

644

was not able to induce mitophagy after knockdown PRKAA1. Furthermore, we

645

utilized immunofluorescence to detect nuclear translocation of TFEB and found that

646

curcumin dramatically increases the nuclear translocation of TFEB in IPEC-J2 cells

647

treated with H2O2, which was consistent with previous data in HCT116 cells [56]. To

648

determine whether the curcumin affects the transcriptional activity of TFEB through

649

the AMPK signal pathway, we make use of the dual-luciferase assay to determine the

650

activity of a TFEB promoter. We showed that curcumin dramatically enhanced TFEB

651

transcriptional activity in IPEC-J2 cells treated with H2O2, and it was ameliorated by

652

co-treated with compound C, an AMPK inhibitor. Similarly, Zhang et al (2016) had

653

shown that curcumin can directly bind with TFEB and increase its transcriptional

654

activity in HCT116 cells [56]. Accordingly, our results propose that treatment with

655

curcumin may promote Parkin dependent mitophagy via AMPK activation and

656

subsequent TFEB nuclear translocation and then exert a protective effect on intestinal

657

epithelium and mitochondria.

658

To confirm the effect of curcumin on intestinal oxidative stress status, epithelial

659

barrier and mitochondrial function through regulating mitophagy in vivo, we utilized a

660

well-established pig model for leading to oxidative stress by intraperitoneal injection

661

with diquat, which is an excellent model system for studying the intestinal oxidative

662

stress. Consistent with the in vitro results, the in vivo data also showed that dietary

663

curcumin protected intestinal barrier function and morphology, improved redox status,

664

alleviated mitochondrial damage, triggered mitophagy and influenced AMPK-TFEB

665

signal pathway of piglets after challenged with diquat. Our result was supported by

666

the consequences of Badria et al. (2015) indicated curcumin attenuated oxidative

667

stress, indicated by curcumin-treated rats had higher SOD activity and lower MDA,

668

NO contents in liver and spleen compared with iron overload rats [5]. Similarly,

669

González-Salazar et al. (2011) had found that dietary with curcumin protects from

670

cardiac ischemia and reperfusion injury by alleviating of oxidant stress and

671

mitochondrial dysfunction in rats’ heart [57].

672

In conclusion, our work proposes that curcumin ameliorated oxidative stress,

673

enhanced intestinal barrier function and mitochondrial function through induction of

674

Parkin dependent mitophagy via AMPK activation and subsequent TFEB nuclear

675

translocation.

676 677

Conflicts of Interest statement

678

The authors declare that there are no conflicts of interest regarding the

679

publication of this article.

680

Acknowledgments

681

This research was supported by National Natural Science Foundation of China

682

(31872387), Zhejiang Provincial Natural Science Foundation (LZ20C170003), and

683

National Key R & D Program (2016YFD0501210). We are grateful to the

684

Bio-ultrastructure analysis Lab. of Analysis center of Agrobiology and environmental

685

sciences, Zhejiang University.

686

Reference

687 688

[1] Zhu HL, Wang HB, Wang SH, Tu ZX, Zhang L, Wang XY, et al. Flaxseed

689

Oil Attenuates Intestinal Damage and Inflammation by Regulating Necroptosis and

690

TLR4/NOD Signaling Pathways Following Lipopolysaccharide Challenge in a Piglet

691

Model. Mol Nutr Food Res. 2018;62(9).

692

[2] Bhattacharyya A, Chattopadhyay R, Mitra S, Crowe SE. Oxidative Stress: An

693

Essential Factor in the Pathogenesis of Gastrointestinal Mucosal Diseases. Physiol

694

Rev. 2014;94(2):329-354.

695

[3] Tian SY, Guo RX, Wei SC, Kong Y, Wei XL, Wang WW, et al. Curcumin

696

protects against the intestinal ischemia-reperfusion injury: involvement of the tight

697

junction protein ZO-1 and TNF-alpha related mechanism. Korean J Physiol Pha.

698 699 700

2016;20(2):147-152. [4] Cikrikci S, Mozioglu E, Yilmaz H. Biological Activity of Curcuminoids Isolated from Curcuma longa. Rec Nat Prod. 2008;2(1):19-24.

701

[5] Badria FA, Ibrahim AS, Badria AF, Elmarakby AA. Curcumin Attenuates

702

Iron Accumulation and Oxidative Stress in the Liver and Spleen of Chronic

703

Iron-Overloaded Rats. PLoS One. 2015;10(7):e0134156.

704

[6] Ortega-Dominguez

B,

Aparicio-Trejo

OE,

Garcia-Arroyo

FE,

705

Leon-Contreras JC, Tapia E, Molina-Jijon E, et al. Curcumin prevents

706

cisplatin-induced renal alterations in mitochondrial bioenergetics and dynamic. Food

707

Chem Toxicol. 2017;107(Pt A):373-385.

708

[7] Aparicio-Trejo OE, Tapia E, Molina-Jijon E, Medina-Campos ON,

709

Macias-Ruvalcaba NA, Leon-Contreras JC, et al. Curcumin prevents mitochondrial

710

dynamics disturbances in early 5/6 nephrectomy: Relation to oxidative stress and

711

mitochondrial bioenergetics. Biofactors. 2017;43(2):293-310.

712

[8] Molina-Jijon E, Aparicio-Trejo OE, Rodriguez-Munoz R, Leon-Contreras JC,

713

Del Carmen Cardenas-Aguayo M, Medina-Campos ON, et al. The nephroprotection

714

exerted by curcumin in maleate-induced renal damage is associated with decreased

715

mitochondrial fission and autophagy. Biofactors. 2016;42(6):686-702.

716 717

[9] Palikaras K, Lionaki E, Tavernarakis N. Mitophagy: In sickness and in health. Mol Cell Oncol. 2016;3(1).

718

[10] de Oliveira MR, Jardim FR, Setzer WN, Nabavi SM, Nabavi SF. Curcumin,

719

mitochondrial biogenesis, and mitophagy: Exploring recent data and indicating future

720

needs. Biotechnol Adv. 2016;34(5):813-826.

721 722

[11] Herzig S, Shaw RJ. AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol. 2018;19(2):121-135.

723

[12] Young NP, Kamireddy A, Van Nostrand JL, Eichner LJ, Shokhirev MN,

724

Dayn Y, et al. AMPK governs lineage specification through Tfeb-dependent

725

regulation of lysosomes. Gene Dev. 2016;30(5):535-552.

726

[13] Wu HQ, Ding J, Li SH, Lin JT, Jiang RH, Lin C, et al. Metformin Promotes

727

the Survival of Random-Pattern Skin Flaps by Inducing Autophagy via the

728

AMPK-mTOR-TFEB signaling pathway. Int J Biol Sci. 2019;15(2):325-340.

729

[14] Wen J, Xu B, Sun Y, Lian M, Li Y, Lin Y, et al. Paeoniflorin protects against

730

intestinal ischemia/reperfusion by activating LKB1/AMPK and promoting autophagy.

731

Pharmacol Res. 2019;146:104308.

732

[15] Yin J, Liu MF, Ren WK, Duan JL, Yang G, Zhao YR, et al. Effects of

733

Dietary Supplementation with Glutamate and Aspartate on Diquat-Induced Oxidative

734

Stress in Piglets. PloS one. 2015;10(4).

735

[16] Cao ST, Wang CC, Wu H, Zhang QH, Jiao LF, Hu CH. Weaning disrupts

736

intestinal antioxidant status, impairs intestinal barrier and mitochondrial function, and

737

triggers mitophagy in piglets. J Anim Sci. 2018;96(3):1073-1083.

738

[17] Song Z, Tong G, Xiao K, Jiao le F, Ke Y, Hu C. L-cysteine protects

739

intestinal integrity, attenuates intestinal inflammation and oxidant stress, and

740

modulates NF-kappaB and Nrf2 pathways in weaned piglets after LPS challenge.

741

Innate Immun. 2016;22(3):152-161.

742

[18] Chalubinski M, Wojdan K, Gorzelak P, Borowiec M, Broncel M. The effect

743

of oxidized cholesterol on barrier functions and IL-10 mRNA expression in human

744

intestinal epithelium co-cultured with dendritic cells in the transwell system. Food

745

Chem Toxicol. 2014;69:289-293.

746

[19] Tuncer S, Colakoglu M, Ulusan S, Ertas G, Karasu C, Banerjee S.

747

Evaluation of colloidal platinum on cytotoxicity, oxidative stress and barrier

748

permeability across the gut epithelium. Heliyon. 2019;5(3).

749

[20] Du H, Guo L, Yan SQ, Sosunov AA, McKhann GM, Yan SS. Early deficits

750

in synaptic mitochondria in an Alzheimer's disease mouse model. P Natl Acad Sci

751

USA. 2010;107(43):18670-18675.

752

[21] Li WJ, Chen Y, Nie SP, Xie MY, He M, Zhang SS, et al. Ganoderma atrum

753

polysaccharide induces anti-tumor activity via the mitochondrial apoptotic pathway

754 755

related to activation of host immune response. J Cell Biochem. 2011;112(3):860-871. [22] Wu J, Zhang M, Hao S, Jia M, Ji M, Qiu L, et al. Mitochondria-Targeted

756

Peptide

Reverses

Mitochondrial

Dysfunction

and

Cognitive

Deficits

757

Sepsis-Associated Encephalopathy. Mol Neurobiol. 2015;52(1):783-791.

in

758

[23] Xiao K, Jiao L, Cao S, Song Z, Hu C, Han X. Whey protein concentrate

759

enhances intestinal integrity and influences transforming growth factor-beta1 and

760

mitogen-activated

761

lipopolysaccharide challenge. Br J Nutr. 2016;115(6):984-993.

762

protein

kinase

signalling

pathways

in

piglets

after

[24] Cao ST, Wu H, Wang CC, Zhang QH, Jiao LF, Lin FH, et al.

763

Diquat-induced

oxidative

stress

increases

intestinal

permeability,

impairs

764

mitochondrial function, and triggers mitophagy in piglets. J Anim Sci.

765

2018;96(5):1795-1805.

766

[25] Wang X, Leung AW, Luo J, Xu C. TEM observation of ultrasound-induced

767

mitophagy in nasopharyngeal carcinoma cells in the presence of curcumin. Exp Ther

768

Med. 2012;3(1):146-148.

769

[26] Guilloteau P, Zabielski R, Hammon HM, Metges CC. Nutritional

770

programming of gastrointestinal tract development. Is the pig a good model for man?

771

Nutr Res Rev. 2010;23(1):4-22.

772

[27] Lv M, Yu B, Mao XB, Zheng P, He J, Chen DW. Responses of growth

773

performance and tryptophan metabolism to oxidative stress induced by diquat in

774

weaned pigs. Animal. 2012;6(6):928-934.

775

[28] Zhong W, Qian K, Xiong J, Ma K, Wang A, Zou Y. Curcumin alleviates

776

lipopolysaccharide induced sepsis and liver failure by suppression of oxidative

777

stress-related inflammation via PI3K/AKT and NF-kappaB related signaling. Biomed

778

Pharmacother. 2016;83:302-313.

779

[29] Xie YL, Chu JG, Jian XM, Dong JZ, Wang LP, Li GX, et al. Curcumin

780

attenuates

lipopolysaccharide/D-galactosamine-induced

acute

liver

injury

by

781

activating Nrf2 nuclear translocation and inhibiting NF-kB activation. Biomed

782

Pharmacother. 2017;91:70-77.

783

[30] Wang N, Wang G, Hao J, Ma J, Wang Y, Jiang X, et al. Curcumin

784

ameliorates hydrogen peroxide-induced epithelial barrier disruption by upregulating

785

heme oxygenase-1 expression in human intestinal epithelial cells. Dig Dis Sci.

786

2012;57(7):1792-1801.

787

[31] Yucel AF, Kanter M, Pergel A, Erboga M, Guzel A. The role of curcumin on

788

intestinal oxidative stress, cell proliferation and apoptosis after ischemia/reperfusion

789

injury in rats. J Mol Histol. 2011;42(6):579-587.

790

[32] Zou Y, Wang J, Peng J, Wei H. Oregano Essential Oil Induces SOD1 and

791

GSH Expression through Nrf2 Activation and Alleviates Hydrogen Peroxide-Induced

792

Oxidative Damage in IPEC-J2 Cells. Oxid Med Cell Longev. 2016;2016:5987183.

793

[33] Yin J, Wu M, Li Y, Ren W, Xiao H, Chen S, et al. Toxicity assessment of

794

hydrogen peroxide on Toll-like receptor system, apoptosis, and mitochondrial

795

respiration in piglets and IPEC-J2 cells. Oncotarget. 2017;8(2):3124-3131.

796

[34] Jaroonwitchawan T, Chaicharoenaudomrung N, Natnkaew J, Noisa P.

797

Curcumin attenuates paraquat-induced cell death in human neuroblastoma cells

798

through modulating oxidative stress and autophagy. Neurosci Lett. 2017;636:40-47.

799

[35] Dai C, Ciccotosto GD, Cappai R, Tang S, Li D, Xie S, et al. Curcumin

800

Attenuates Colistin-Induced Neurotoxicity in N2a Cells via Anti-inflammatory

801

Activity, Suppression of Oxidative Stress, and Apoptosis. Mol Neurobiol.

802

2018;55(1):421-434.

803

[36] Dai C, Tang S, Li D, Zhao K, Xiao X. Curcumin attenuates

804

quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells.

805

Toxicol Mech Methods. 2015;25(4):340-346.

806

[37] Lin C, Chao H, Li Z, Xu X, Liu Y, Hou L, et al. Melatonin attenuates

807

traumatic brain injury-induced inflammation: a possible role for mitophagy. J Pineal

808

Res. 2016;61(2):177-186.

809

[38] Zhou M, Xia ZY, Lei SQ, Leng Y, Xue R. Role of mitophagy regulated by

810

Parkin/DJ-1 in remote ischemic postconditioning-induced mitigation of focal cerebral

811

ischemia-reperfusion. Eur Rev Med Pharmacol Sci. 2015;19(24):4866-4871.

812

[39] Song D, Cheng Y, Li X, Wang F, Lu Z, Xiao X, et al. Biogenic

813

Nanoselenium Particles Effectively Attenuate Oxidative Stress-Induced Intestinal

814

Epithelial Barrier Injury by Activating the Nrf2 Antioxidant Pathway. ACS Appl

815

Mater Interfaces. 2017;9(17):14724-14740.

816

[40] Liu YL, Chen F, Odle J, Lin X, Jacobi SK, Zhu HL, et al. Fish Oil Enhances

817

Intestinal Integrity and Inhibits TLR4 and NOD2 Signaling Pathways in Weaned Pigs

818

after LPS Challenge. J Nutr. 2012;142(11):2017-2024.

819

[41] Trujillo J, Molina-Jijon E, Medina-Campos ON, Rodriguez-Munoz R,

820

Reyes JL, Loredo ML, et al. Curcumin prevents cisplatin-induced decrease in the tight

821

and adherens junctions: relation to oxidative stress. Food Funct. 2016;7(1):279-293.

822

[42] Wang J, Ghosh SS, Ghosh S. Curcumin improves intestinal barrier function:

823

modulation of intracellular signaling, and organization of tight junctions. Am J

824

Physiol Cell Physiol. 2017;312(4):C438-C445.

825

[43] Zhang JF, Xu L, Zhang LL, Ying ZX, Su WP, Wang T. Curcumin Attenuates

826

D-Galactosamine/Lipopolysaccharide-Induced

Liver

827

Dysfunction in Mice. J Nutr. 2014;144(8):1211-1218.

Injury

and

Mitochondrial

828

[44] Indo HP, Davidson M, Yen HC, Suenaga S, Tomita K, Nishii T, et al.

829

Evidence of ROS generation by mitochondria in cells with impaired electron transport

830

chain and mitochondrial DNA damage. Mitochondrion. 2007;7(1-2):106-118.

831

[45] Priyanka A, Shyni GL, Anupama N, Raj PS, Anusree SS, Raghu KG.

832

Development of insulin resistance through sprouting of inflammatory markers during

833

hypoxia in 3T3-L1 adipocytes and amelioration with curcumin. Eur J Pharmacol.

834

2017;812:73-81.

835

[46]

Garcia-Nino

WR,

Tapia

E,

Zazueta

C,

Zatarain-Barron

ZL,

836

Hernandez-Pando R, Vega-Garcia CC, et al. Curcumin Pretreatment Prevents

837

Potassium

Dichromate-Induced

Hepatotoxicity,

Oxidative

Stress,

Decreased

838

Respiratory Complex I Activity, and Membrane Permeability Transition Pore Opening.

839

Evid-Based Compl Alt. 2013.

840

[47] Lampert MA, Orogo AM, Najor RH, Hammerling BC, Leon LJ, Wang BYJ,

841

et al. BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial

842

network remodeling during cardiac progenitor cell differentiation. Autophagy.

843

2019;15(7):1182-1198.

844 845

[48] Saita S, Shirane M, Nakayama KI. Selective escape of proteins from the mitochondria during mitophagy. Nat Commun. 2013;4.

846

[49] de Oliveira MR, Jardim FR, Setzer WN, Nabavi SM, Nabavi SF. Curcumin,

847

mitochondrial biogenesis, and mitophagy: Exploring recent data and indicating future

848

needs. Biotechnol Adv. 2016;34(5):813-826.

849

[50] Wu Q, Hou YL, Sun B, Zhuang K, Zhang HC, Jin DJ. Curcumin induce

850

apoptosis of CNE-2z cells via caspase-dependent mitochondrial intrinsic pathway. Afr

851

J Pharm Pharmaco. 2011;5(15):1748-1756.

852

[51] Yuan Y, Zheng YR, Zhang XN, Chen Y, Wu XL, Wu JY, et al.

853

BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independent

854

of PARK2. Autophagy. 2017;13(10):1754-1766.

855 856

[52] Sauve V, Lilov A, Seirafi M, Vranas M, Rasool S, Kozlov G, et al. A Ubl/ubiquitin switch in the activation of Parkin. Embo J. 2015;34(20):2492-2505.

857

[53] Wu SB, Wu YT, Wu TP, Wei YH. Role of AMPK-mediated adaptive

858

responses in human cells with mitochondrial dysfunction to oxidative stress. Bba-Gen

859

Subjects. 2014;1840(4):1331-1344.

860 861

[54] Jeon SM, Chandel NS, Hay N. AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress. Nature. 2012;485(7400):661-+.

862

[55] Xiao K, Jiang JH, Guan CX, Dong CL, Wang GF, Bai L, et al. Curcumin

863

Induces Autophagy via Activating the AMPK Signaling Pathway in Lung

864

Adenocarcinoma Cells. J Pharmacol Sci. 2013;123(2):102-109.

865

[56] Zhang JB, Wang JG, Xu J, Lu YQ, Jiang JK, Wang LM, et al. Curcumin

866

targets the TFEB-lysosome pathway for induction of autophagy. Oncotarget.

867

2016;7(46):75659-75671.

868

[57] Gonzalez-Salazar A, Molina-Jijon E, Correa F, Zarco-Marquez G,

869

Calderon-Oliver M, Tapia E, et al. Curcumin Protects from Cardiac Reperfusion

870

Damage by Attenuation of Oxidant Stress and Mitochondrial Dysfunction. Cardiovasc

871

Toxicol. 2011;11(4):357-364.

872 873 874

Figure Legend Fig 1. Curcumin ameliorates hydrogen peroxide (H2O2) induced oxidative stress in a mitophagy-dependent way in porcine intestinal epithelial cells (IPEC-J2) (A, B, C) The cell viability after IPEC-J2 cells treated with H2O2 and curcumin was determined by Thiazolyl blue (MTT) assay. (D, E, F) The activity of Superoxide dismutase (SOD) (D), Catalase (CAT) (E) and Malondialdehyde (MDA) level (F) were determined by ELISA in IPEC-J2 cells. (G) Relative mRNA levels of Cu/Zn-SOD, Mn-SOD, GPX-1 and GPX-4 in IPEC-J2 cells. (H) Flow cytometry was used to determine cellular reactive oxygen species (ROS) level and quantification. (I) ROS staining of IPEC-J2 cells and quantification. Scale bar, 100 µm. Cells were treated with mitochondrial division inhibitor 1 (mdivi-1) (1 µM) for 1 h, and then treated with curcumin (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01 and *** P < 0.001vs. Control group, #P < 0.05, ##P< 0.01 and ### P < 0.001 vs. H2O2 group. ɸ P < 0.05, ɸ ɸ P< 0.01 and ɸ ɸ ɸ P < 0.001 vs. Curcumin+H2O2 group.

Fig 2. Curcumin ameliorates hydrogen peroxide (H2O2) induced disrupted epithelial barrier, mitochondria dysfunction in a mitophagy-dependent way in porcine intestinal epithelial cells (IPEC-J2) Cells were treated with mitochondrial division inhibitor 1 (mdivi-1) (1 µM) for 1 h, and then treated with curcumin (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. (A, B) Detection of transepithelial electrical resistance (A) and flux of fluorescein isothiocyanate dextran 4 kDa (FD4) (B) in IPEC-J2 cells. (C) Protein expression and quantitation of ZO-1, Occludin and Claudin-1 determined by western blotting in IPEC-J2

cells

(scale

bars

represent

10,000

nm).

(D)

Representative

immunofluorescence images of Claudin-1 and quantification. (E, F, G) Detection of ATP production (E), mitochondria membrane potential (F) and activity of electron transfer chain complexes I–III (G) in IPEC-J2 cells. (H) Detection of ultrastructure of mitochondria using transmission electron microscope (TEM)in IPEC-J2 cells (scale

bars represent 0.5 µm). Red arrows indicate swelling mitochondria with disrupted respiratory cristae. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01 and *** P < 0.001vs. Control group, #P < 0.05, ##P< 0.01 and ### P < 0.001 vs. H2O2 group. ɸ P < 0.05,

ɸɸ

P< 0.01 and

ɸɸɸ

P < 0.001 vs. Curcumin+H2O2

group.

Fig 3. Curcumin activated mitophagy through PINK1-Parkin pathway in hydrogen peroxide (H2O2) treated porcine intestinal epithelial cells (IPEC-J2) Cells were treated with mitochondrial division inhibitor 1 (mdivi-1) (1 µM) for 1 h, and then treated with curcumin (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. (A) Protein expression and quantitation of LC3-I/II, SQSTM-1, Beclin-1 determined by western blotting in IPEC-J2 cells. (B) Gene expression of BNIP3L, BNIP3, FUNDC-1, PINK-1 and Parkin in IPEC-J2 cells. (C) Protein expression and quantitation of PINK-1 and Parkin determined by western blotting in IPEC-J2 cells. (D) Mitiphagy of IPEC-J2 cells detected under confocal microscopy after co-transfection of Ad-GFP-LC3 and Ad-HBAD-Mito-dsred and quantification colocalization. The data were obtained from 3 independent experiments (scale bars represent 10,000 nm). Green: Ad-GFP-LC3, Red: Ad-HBAD-Mito-dsred, Blue: Hochest33258, Yellow: co-localization of Ad-GFP-LC3 and Ad-HBAD-Mito-dsred. (E) Mitochondrial autophagosomes assessed by transmission electron microscope (TEM)and quantized in IPEC-J2 cells (scale bars represent 1 µm). Red arrows indicate mitochondrial autophagosomes. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01 and *** P < 0.001vs. Control group, #P < 0.05, ##

P< 0.01 and ### P < 0.001 vs. H2O2 group. ɸ P < 0.05, ɸ ɸ P< 0.01 and ɸ ɸ ɸ P < 0.001

vs. Curcumin+H2O2 group.

Fig 4. Parkin dependent mitophagy is necessary for curcumin’s protection functions in hydrogen peroxide (H2O2) treated porcine intestinal epithelial cells (IPEC-J2) Cells treated with curcumin (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. (A) The cell viability after IPEC-J2 cells treated with si Parkin was determined by Thiazolyl blue (MTT) assay. (B) Protein expression and quantitation of PINK-1,

Parkin, LC3-I/II, SQSTM-1 and Beclin-1 determined by western blotting in IPEC-J2 cells transfected with control siRNA or siRNA targeting Parkin. (C, D, E) The activity of Superoxide dismutase (SOD) (C), Catalase (CAT) (D) and Malondialdehyde (MDA) level (E) were determined by ELISA in IPEC-J2 cells transfected with control siRNA or siRNA Parkin. (F) Relative mRNA levels of Cu/Zn-SOD, Mn-SOD, GPX-1 and GPX-4 in IPEC-J2 cells transfected with control siRNA or siRNA targeting Parkin. (G, H) Detection of transepithelial electrical resistance (G) and FD4 flux (H) in IPEC-J2 cells transfected with control siRNA or siRNA Parkin. (I, J) Detection of ATP production (I) and activity of electron transfer chain complexes I–III (J) in IPEC-J2 cells transfected with control siRNA or siRNA targeting Parkin. (K) The amino acid sequence of Parkin and Parkin ∆UBL. IPEC-J2 cells were either transfected with plasmids encoding pEGFP-Parkin or pEGFP-Parkin ∆UBL. The Parkin ∆UBL encodes a mutant Parkin protein without the ubiquitin E3 ligase activity. To detect the ubiquitin level, the proteasome inhibitor MG132 (5µM) was used to treat cells for 8h before collecting samples. (L) Protein expression and quantitation of TOMM20 determined by western blotting in IPEC-J2 cells transfected with plasmids encoding GFP-Parkin or GFP-Parkin ∆UBL. (M) The cell viability IPEC-J2 cells transfected with plasmids encoding GFP-Parkin or GFP-Parkin ∆UBL determined by the Thiazolyl blue (MTT) assay. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01 and *** P < 0.001, NS, not significant.

Fig 5. Curcumin promote mitophagy through AMPK-TFEB signal pathway hydrogen peroxide (H2O2) treated porcine intestinal epithelial cells (IPEC-J2) Cells treated with curcumin (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. (A) Protein expression and quantitation of pAMPK, AMPK and TFEB determined by western blotting in IPEC-J2 cells transfected with control siRNA or siRNA PRKAA1. (B) Protein expression and quantitation of PRKAA1, PINK-1 and Parkin determined by western blotting in IPEC-J2 cells transfected with control siRNA or siRNA targeting PRKAA1. (C, D, E) The activity of Superoxide dismutase (SOD) (C), Catalase (CAT) (D) and Malondialdehyde (MDA) level (E) were determined by ELISA in IPEC-J2 cells transfected with control siRNA or siRNA PRKAA1. (F) Relative mRNA levels of Cu/Zn-SOD, Mn-SOD, GPX-1 and GPX-4 in IPEC-J2 cells. (G, H) Detection of transepithelial electrical resistance (G) and FD4 flux (H) in

IPEC-J2 cells transfected with control siRNA or siRNA targeting PRKAA1. (I, J) Detection of ATP production (I) and activity of electron transfer chain complexes I– III (J) in IPEC-J2 cells transfected with control siRNA or siRNA PRKAA1. (K) Fluorescence microscopy images of TFEB in IPEC-J2 cells (scale bars represent 10,000 nm). (L) Luciferase assay of IPEC-J2 cells transfected with TFEB-luciferase reporter or TFEB-empty. In Luciferase assay, IPEC-J2 cells were co-treated with curcumin (10 µM) and compound C (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01 and *** P < 0.001, NS, not significant.

Fig 6. Curcumin alleviated intestinal injury, improved mitochondrial function, induced mitophagy and influenced AMPK-TFEB signal pathway in a piglet’s model Control group: pigs receiving a control diet and injected 0.9% NaCl solution; Control+curcumin group: piglets fed the diet inclusion of 200 mg/kg curcumin injected 0.9% NaCl solution; Diquat-challenged group: pigs receiving the control diet and injected diquat; Curcumin + Diquat: piglets fed the diet inclusion of 200 mg/kg curcumin and administered diquat. (A, B, C) The activity of Superoxide dismutase (SOD) (A), Catalase (CAT) (B) and Malondialdehyde (MDA) level (C) were determined by ELISA of piglets’ intestinal mucosa. (D, E) The transepithelial electrical resistance (D) and FD4 flux (E) of piglets’ jejunum were determined by Ussing chambers. (F) Protein expression and quantitation of ZO-1, Occludin and Claudin-1 determined by western blotting in piglets’ intestinal mucosa. (G) Images of the piglets’ jejunum villus morphology and quantification (scale bars represent 100 µm). (H) Detection of ultrastructure of piglets’ jejunum mitochondria using transmission electron microscope (TEM) and quantification (scale bars represent 1 µm). Red arrows indicate swelling mitochondria with disrupted respiratory cristae. (I, J, K) Detection of ATP production (I), mitochondria membrane potential (J) and activity of electron transfer chain complexes I–III (K) of piglets’ jejunum mitochondria. (L, M) Protein expression and quantitation of PINK-1, Parkin, LC3-I/II, SQSTM-1, Beclin-1, pAMPK, AMPK and TFEB determined by western blotting of

piglets’ intestinal mucosa. Values were means and SD represented by vertical bars (n=6). *P < 0.05, **P < 0.01 and *** P < 0.001 vs. control; #P < 0.05, ##P< 0.01 and ###

P < 0.001 vs. diquat group. NS, not significant.

Fig S1. Colocalization of Parkin/PINK-1 and LC3 puncta in hydrogen peroxide (H2O2) treated porcine intestinal epithelial cells (IPEC-J2) Cells were treated with mitochondrial division inhibitor 1 (mdivi-1) (1 µM) for 1 h, and then treated with curcumin (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. (A) Colocalization of PINK-1 and LC3 puncta in IPEC-J2 cells detected under confocal microscopy after co-transfection of Ad-GFP-LC3 and quantification colocalization. The data were obtained from 3 independent experiments (scale bars represent 10,000 nm). Green: Ad-GFP-LC3, Red: PINK-1, Blue: DAPI. (B) Colocalization of Parkin and LC3 puncta in IPEC-J2 cells detected under confocal microscopy after co-transfection of Ad-GFP-LC3 and quantification colocalization. The data were obtained from 3 independent experiments. Green: Ad-GFP-LC3, Red: Parkin, Blue: DAPI. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01 and *** P < 0.001vs. Control group, #P < 0.05, ##P< 0.01 and ### P < 0.001 vs. H2O2 group. ɸ P < 0.05, ɸ ɸ P< 0.01 and ɸ ɸ ɸ P < 0.001 vs. Curcumin+H2O2 group.

Fig S2. The effect of Parkin E3 ligase-defective mutant on ROS levels, ATP production and mitochondrial membrane potential upon hydrogen peroxide (H2O2) /curcumin treatment in porcine intestinal epithelial cells (IPEC-J2) Cells treated with curcumin (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. IPEC-J2 cells were either transfected with plasmids encoding pEGFP-Parkin or pEGFP-Parkin ∆UBL. (A, B, C) Detection of reactive oxygen species (ROS) levels (A), ATP production (B) and mitochondria membrane potential (C) in IPEC-J2 cells. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01 and *** P < 0.001, NS, not significant.

Fig S3. p-AMPK is necessary for curcumin’s protection functions in hydrogen peroxide (H2O2) treated porcine intestinal epithelial cells (IPEC-J2)

IPEC-J2 cells were co-treated with curcumin (10 µM) and compound C (10 µM) for 12 h, followed by H2O2 (600 µM) for 8 h. (A, B, C) The activity of Superoxide dismutase (SOD) (A), Catalase (CAT) (B) and Malondialdehyde (MDA) level (C) were determined by ELISA in IPEC-J2 cells. (D) Relative mRNA levels of Cu/Zn-SOD, Mn-SOD, GPX-1 and GPX-4 in IPEC-J2 cells. (E, F, G) Detection of ATP production (E), mitochondria membrane potential (F) and activity of electron transfer chain complexes I–III (G) in IPEC-J2 cells. (H, I) Detection of transepithelial electrical resistance (H) and flux of fluorescein isothiocyanate dextran 4 kDa (FD4) (I) in IPEC-J2 cells. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01 and *** P < 0.001, NS, not significant.

Fig S4. The Parkin, phospho-AMPK expression levels and TFEB nuclear translocation in intestine in the diquat challenged piglets Control group: pigs receiving a control diet and injected 0.9% NaCl solution; Control+curcumin group: piglets fed the diet inclusion of 200 mg/kg curcumin injected 0.9% NaCl solution; Diquat-challenged group: pigs receiving the control diet and injected diquat; Curcumin + Diquat: piglets fed the diet inclusion of 200 mg/kg curcumin and administered diquat. (A, B, C) Localization and quantification of Parkin, p-AMPK, TFEB and DAPI (DNA) within the jejunum of weaned pigs was assessed by immunofluorescence (scale bars represent 5000 nm). Parkin, p-AMPK, TFEB (red), DAPI stain (blue), as well as merged claudin-1 protein and DAPI are presented. Values were means and SD represented by vertical bars. *P < 0.05, **P < 0.01 and *** P < 0.001 vs. control; #P < 0.05, ##P< 0.01 and ### P < 0.001 vs. diquat group. NS, not significant.

Fig 1

Fig 2

Fig 3

Fig 4

Fig 5

Fig 6

Highlights 1. Curcumin alleviates oxidative stress, intestinal barrier and mitochondrial injury. 2. Curcumin exerts protective functions in a mitophagy-dependent way via PINK1-Parkin. 3. Ubiquitin E3 ligase of Parkin is required for curcumin’s protection effects. 4. Curcumin promotes Parkin dependent mitophagy through AMPK-TFEB signal pathway. 5. Curcumin also exerts protective functions in the pig intestinal oxidative stress model.