Intranasal Pretreatment with Z-Ligustilide, the Main Volatile

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Intranasal Pretreatment of Z-ligustilide, the Main Volatile Component of Rhizoma Chuanxiong, Confers Prophylaxis Against Cerebral Ischemia via Nrf2 and HSP70 Signaling Pathway Juan Li, Jie Yu, Hui Ma, Na Yang, Li Li, Dingding Zheng, Mingxia Wu, Zhilong Zhao, and Hongyi Qi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04979 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 7, 2017

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Journal of Agricultural and Food Chemistry

Intranasal Pretreatment of Z-ligustilide, the Main Volatile Component of Rhizoma Chuanxiong, Confers Prophylaxis Against Cerebral Ischemia via Nrf2 and HSP70 Signaling Pathways

Juan Li†#, Jie Yu†#, Hui Ma†, Na Yang§, Li Li†, Ding-ding Zheng†, Ming-xia Wu†, Zhi-long Zhao§, Hong-yi Qi†*

†

College of Pharmaceutical Sciences, Southwest University, 2 Tiansheng Road,

Beibei District, Chongqing 400716, China; §

Institute of Laboratory Animals, Sichuan Academy of Medical Sciences and Sichuan

Provincial People’s Hospital, Chengdu 610212, Sichuan, China #

equal contributor

*Corresponding author at: College of Pharmaceutical Sciences, Southwest University, 2 Tiansheng Road, Beibei District, Chongqing 400716, China. Tel./Fax: +86 23 68251225; E-mail: [email protected]

Running title: Z-ligustilide for Prophylaxis against Cerebral Ischemia

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ACS Paragon Plus Environment


1

ABSTRACT

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Z-ligustilide is a major component in Rhizoma Chuanxiong, which has been

3

traditionally used as a health food supplement for the prevention of cerebrovascular

4

disease in China. This study investigates the ability of intranasal Z-ligustilide

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pretreatment to enhance protection against neuronal damage in rats with middle

6

cerebral artery occlusion (MCAO) and the role of cellular stress response mechanisms

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Nrf2 and HSP70. Z-ligustilide significantly mitigated infarct volume, neurological

8

dysfunction, blood-brain barrier disruption, and brain edema (p < 0.01). Moreover,

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Z-ligustilide prevented the loss of collagen IV, occludin, and ZO-1 (p < 0.05) and

10

decreased MMP-2 and -9 levels (p < 0.01). Meanwhile, Z-ligustilide upregulated

11

NQO1 and HSP70. Notably, blockage of Nrf2-driven transcription or downregulation

12

of HSP70 remarkably attenuated the preventive effect of Z-ligustilide (p < 0.05).

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Together, intranasal delivery of Z-ligustilide enhanced protection against ischemic

14

injury via Nrf2 and HSP70 signaling pathways and has prophylactic potential in the

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population at high risk of stroke.

16 17

KEYWORDS: Z-ligustilide, Nrf2, HSP70, ischemic stroke, intranasal delivery

18 19 20 21 22 2


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INTRODUCTION Stroke is the second largest cause of death and a main contributor of serious 1, 2

25

neurological disability worldwide

. In China, stroke has become the leading cause

26

of adult mortality and disability, with an estimated 2.5 million incident strokes and 1.6

27

million deaths annually 1. Patients with stroke often require long-term rehabilitation,

28

which considerably impairs their quality of life and leads to a major economic burden

29

on society. It is predicted that there will be an unprecedented 50% increase in stroke

30

incidence in China during the next 20 years 1. Nowadays, stroke intervention is no

31

longer limited to first aid treatment following disease onset. Prophylactic treatment,

32

especially targeting recurrent stroke in a variety of pathological circumstances, has

33

become a focus in recent years 3.

34

Ischemic stroke accounts for 85% of all strokes and is characterized by rapid

35

initiation of the ischemic cascade, which comprises a series of subsequent deleterious

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mechanisms that eventually lead to cellular injury and infarct progression 4. At the

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onset of MCAO-reperfusion, ischemic injury is mainly caused by excessive

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generation of reactive oxygen species (ROS), which leads to direct cellular injury.

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ROS-induced activation of immune cells, which in turn releases multiple

40

inflammation factors, including cytokines, chemokines, enzymes, and oxygen derived

41

free radicals, further aggravates ischemic injury

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MCAO-reperfusion, inflammation has been described as the main factor responsible

43

for ischemic injury 6. Meanwhile, it has also been demonstrated that severe protein

44

aggregation was observed in hippocampal CA1 neurons of animal models of stroke,

5

. At the late stage of

3



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suggesting that protein aggregation is part of the etiology of ischemic stroke 7. Thus,

46

control of oxidative stress, neuroinflammation, and proteotoxic stress may provide a

47

promising approach for prevention and treatment of ischemic stroke.

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Cells constantly subjected to various exogenous stresses have adaptively evolved

49

diverse survival mechanisms called cellular stress responses, which may confer

50

resistance or tolerance to a more overwhelming stress that might otherwise be lethal

51

or lead to disease 8, 9. The transcription factor nuclear factor erythroid 2-related factor

52

2 (Nrf2) is widely accepted as a critical modulator of an endogenous inducible

53

defense system against oxidative stress in the body. Under physiological conditions,

54

Nrf2 is kept in the cytoplasm. Upon activation, it enters into the nucleus and binds

55

specifically to a gene promoter called the antioxidant responsive element (ARE)

56

initiating transcription of cellular antioxidant and detoxifying enzymes

57

Furthermore, emerging evidence has suggested that a functional Nrf2 is indispensable

58

to regulate neuroinflammation in response to oxidative stress in the brain

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example, inflammation and microglial activation in the brain are much more

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pronounced in response to LPS in Nrf2-deficient mice than in wild-type mice 12. Heat

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shock protein 70 (HSP70) is a main stress-inducible heat shock protein that plays a

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pivotal role in assisting protein folding 13. Overexpression of HSP70 in hippocampal

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CA1 neurons reduced evidence of protein aggregation and enhanced neuronal survival

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in an animal ischemic stroke model 14. Moreover, it is also increasingly evident that

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pharmacological induction of HSP70 remarkably mitigates neuroinflammation and

66

leads to neuroprotection from stroke or traumatic brain injury 15. Thus, targeting both 4


10

.

11

. For

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Nrf2 and HSP70 may provide an effective strategy for simultaneously controlling

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oxidative stress, neuroinflammation, and proteotoxic stress implicated in ischemic

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cascade.

70

Ligusticum chuanxiong Hort. is a valuable medicinal plant of the family

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Umbelliferae. Rhizoma Chuanxiong, the dried rhizome of L. chuanxiong Hort., is one

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of the most popular herbal medicines used in East Asia. Traditionally, it is believed to

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have beneficial effect on ischemic disorders, headache, and menstrual symptoms.

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Meanwhile, L. chuanxiong Hort. is also widely used as a health food product in China.

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For example, its tender leaves and stems are frequently used as tossed salad or fried

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vegetables. It is also stewed with chicken or fish. It is believed that regular

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consumption of food derived from L. chuanxiong Hort. can prevent cerebrovascular

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disease. Z-ligustilide (Z-LIG) is the major component of the volatile oil of L.

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chuanxiong Hort. (ranging from 7.57 to 20.74 mg/g in dried Rhizoma Chuanxiong

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samples 16) and reported to have potent insecticidal activity

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treatment with Z-LIG has been shown to reduce ischemic injury in rodents and could

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be developed as a therapeutic agent for ischemic stroke treatment

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bioavailability of orally administered Z-LIG is reported to be low; this effect is partly

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due to extensive first-pass metabolism 24, 25. Moreover, Z-LIG could not be detected in

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brain tissue after oral administration 24, 26, which may limit its clinical translation. It is

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worth noting that Z-LIG is detectable in the brain only 5 min after intranasal delivery

87

26

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alternative to the conventional oral route for ischemic stroke treatment needs to be

17-20

. In recent years, oral

21-23

. However, the

. Thus, whether intranasal administration of Z-LIG could act as a promising

5



89

further investigated. Our recent investigation indicated that Z-LIG pretreatment

90

remarkably enhanced the tolerance of neuron-like PC12 cells to oxygen-glucose

91

deprivation (OGD) injury, and the cellular stress response pathways Nrf2 and HSP70

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are found to be the key underlying mechanisms responsible for this preventive effect

93

of Z-LIG

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pathway may be, at least in part, responsible for the neuroprotection of Z-LIG via

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intravenous delivery against damage caused by cerebral ischemic injury 25. However,

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whether intranasal delivery of Z-LIG could target both Nrf2 and HSP70 pathways and

97

provide direct prevention against damage caused by cerebral ischemic injury is still

98

unclear.

27, 28

. Meanwhile, another study from Peng et al. also revealed that the Nrf2

99

In this study, the prophylactic effect of Z-LIG administered by intranasal

100

pretreatment was evaluated in an ischemic stroke model with middle cerebral artery

101

occlusion (MCAO). The influence of Z-LIG on the tolerance of rats to

102

MCAO-induced infarct volume, neurological dysfunction, and blood-brain barrier

103

(BBB) permeability derangements was determined. Then, the potential role of the

104

cellular stress response pathways Nrf2 and HSP70 in preventive treatment with Z-LIG

105

against cerebral ischemic injury was further investigated.

106 107

MATERIALS AND METHODS

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Chemicals and Antibodies. Z-LIG with purity more than 98 % was extracted and

109

purified from Rhizoma Chuanxiong by a well-established procedure in our lab

110

described in our previous study

27

and stored in -80 °C before use. The antibodies 6


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against ZO-1, Nrf2, occludin, collagen IV, MMP-2, MMP-9, cleaved caspase 3 and

112

NQO1 were obtained from Santa Cruz Biotechnology (CA, USA). Antibody against

113

HSP70 was obtained from Beyotime (Jiangsu, China). The antibodies against β-actin

114

and rabbit IgG were obtained from Sigma-Aldrich (St. Louis, MO, USA).

115

Animals. Male Sprague-Dawley (230-260 g) rats were provided by the Institute of

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Laboratory Animals, Sichuan Academy of Medical Sciences and Sichuan Provincial

117

People’s Hospital. All rats were housed in controlled temperature (23 to 25 ºC) and

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lighting (12 h light and 12 h dark) and with free access to standard food and drinking

119

water. This study was performed strictly complying with guidelines of the Animal

120

Care and Use Committee of Sichuan Academy of Medical Sciences & Sichuan

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Provincial People’s Hospital and the Sichuan Province Animal Care Ethics

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Committee. MCAO Model. MCAO surgery was conducted according to previous reports

123

27, 29,

124

30

125

injection. To generate a focal ischemia model, the right middle cerebral artery of rat

126

was occluded by a silicone-coated 4-0 nylon suture. Reperfusion was allowed 1 h

127

after MCAO by withdrawing the monofilament. Animals after 24 h from the

128

beginning of MCAO were first performed with neurological behavior examination,

129

followed by various evaluations. Sham-operated rats received the same surgical

130

procedures except for nylon filament insertion. Animals were kept at 37 °C

131

throughout the surgery.

132

Z-LIG, Decoy Oligonucleotide and Lentivirus Administrations. For intranasal

. The rats were anesthetized with chloral hydrate (350 mg/kg) via intraperitoneal

7



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administration, Z-LIG was dissolved in peanut oil and directly applied into each

134

nostril of rat under anesthesia using micropipette. To ensure that the drops were

135

completely inhaled into the nasal cavity, the opposite nostril and mouth of the rat were

136

kept closed during the administration. To evaluate the potential protective effect

137

against cerebral ischemic damage, rats were pretreated with one dose of Z-LIG (15

138

mg/kg) via intranasal route for 3 days. Lentivirus infusion was performed by the

139

injection of lentiviral vectors encoding HSP70, empty lentiviral vectors or vehicle into

140

the lateral ventricle of the ischemic side by intracerebroventricular injection at 5 days

141

before MCAO surgery: rats under anesthesia were mounted in a stereotactic frame

142

(Shenzhen RWD Life Science technology Co., LTD) and injected at the rate of 1

143

ߤL⋅min−1 with the volume of 10 ߤL containing 5 ߤg HSP70-shRNA. The stereotaxic

144

coordinates used in this study was 0.8 mm posterior to the bregma, 1.8 mm left/right

145

to the midline, and 3.6mm ventral to the bregma. For decoy oligonucleotide infusion,

146

ARE decoy oligonucleotide (ODN), mut ODN or vehicle control were injected in the

147

same way and site as lentivirus infusion.

148

Measurement of Neurological behavior and Infarct Volume. The neurologic 31

149

behavior was blindly evaluated using the 18-point scale as previously described

.

150

This scoring system includes six tests as followings-spontaneous activities, symmetry

151

of movements, symmetry of forelimbs, climbing, reaction to touch on either side of

152

trunk, and response to vibrissae touch.

153

After 24 h from the beginning of MCAO, rats were enthanized by decapitation and

154

brains were cut into 2 mm thick of 5 coronal slices. The slices were stained with 0.5 % 8


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2,3,5-triphenyltetrazolium chloride (TTC) for 0.5 h at 37 °C. Then, each brain slice

156

was dried with a tissue paper and taken a photo with a camera. ImageJ software (NIH,

157

MD, USA) was applied to draw and quantify the infarct volume of each slide. The

158

percentage of infarct volume was calculated against the volume of the contralateral

159

structure with the aim to compensating the brain swelling.

160

Brain Water Content Analysis. The brain water content was analyzed with a

161

standard wet-dry way according to the previous report32. Rats were enthanized by

162

decapitation 24 h after MCAO. The brains were divided into the right and left

163

hemispheres without cerebellums. Each hemisphere was measured to get the wet

164

weight. Then, the hemispheres were warmed at 100 °C overnight with an oven. Each

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hemisphere was measured again to get the dry weight. %H2O was obtained as (wet

166

weight−dry weight) ×100/wet weight.

167

Determination of Blood-brain Barrier Permeability. The permeability of BBB

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was evaluated by determining the extravasation Evans Blue (EB) dye according to the

169

previous report

170

Two hours later, rats were perfused with saline and then with 4 % paraformaldehyde.

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After perfusion, brains were removed and each hemisphere was measured to get the

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wet weight, followed by incubated in formamide (1 ml/100 g) at 60 °C for 24 h. Then,

173

each hemisphere was homogenized and centrifuged at 1500 rpm for 10 min. The

174

absorbance was detected at 620 nm to calculate the relative content of EB dye.

175 176

33

. Briefly, rats were first injected with 2 % of EB (0.4 ml/ 100 g).

Western Blotting Analysis. The total protein of animal tissue was extracted according to the previous report

34

. Briefly, tissue was homogenized on ice with 9



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ice-cold RIPA buffer (Cell Signaling Technologies, USA) containing 1 % (v/v) protein

178

inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride. Thirty micrograms of the

179

cellular proteins were separated in 10 % SDS-polyacrylamide gel at 110 V for 60 min,

180

and then transferred at 100 V for 90 min to a polyvinylidene difluoride (PVDF)

181

membrane. Then, the membrane was blocked in 5% BSA-TBS (10 mM Tris (pH 8.0)

182

and 150 mM NaCl) containing 0.1% Tween-20 (TBST) for 1 h, followed by washed

183

in TBST buffer for 3 times and 5 min for each time. The specific primary antibodies

184

were incubated overnight with the corresponding blots to probe the target proteins at

185

4 °C. After washed with TBST buffer again, the blots were incubated with the

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HRP-conjugated secondary antibodies. Finally, the blots were detected with an

187

enhanced chemiluminescence (ECL) reagent (GE Healthcare, Sweden). The gel

188

imaging system (Tanon, China) was applied to acquire the bands and make a

189

quantification. For each protein, the signal intensity was normalized to that of internal

190

control. The quantitative value was given as each normalized data relative to control.

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Immunohistochemical Staining. After perfusion with saline solution and fixation

192

with 4% formalin, the brains were instantly removed and cut into coronal sections for

193

immunohistochemistry. The immunostaining procedure was performed as previously

194

described

195

collagen-IV, ZO-1, occludin at 4°C overnight, followed by incubated with the

196

corresponding

197

6-diamidino-2-phenylindole (DAPI) (Invitrogen) was used to co-stain the nuclei.

198

Fluorescence images were taken by a microscope system (Leica DM4000B, Wetzlar,

35

. The sections were probed with the primary antibodies against

fluorescent-labeled

secondary

antibodies.

10


Meanwhile,

4′,

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199 200

Germany). Decoy Oligonucleotide Design. ARE decoy ODN was applied to block Nrf236

201

mediated gene expression described as our previous report

202

reverse-complement phosphorothioated ODNs were designed and prepared by Sangon

203

Biotech Inc. (Shanghai, China). Double-stranded decoy ODNs were obtained through

204

an annealing procedure in sterile saline with complimentary single strands. The

205

sequences

206

5′-CTAATGGTGACAAAGCAACTTT-3′

207

3′-GATTACCACTGTTTCGTTGAAA-5′. The underlining part represents the ARE

208

core binding sequence. Additionally, a decoy ODN with scrambled sequence (mut

209

ODN) was applied as control to evaluate the specificity and the sequences are listed as

210

5′-CGACTGCCTTCAAAATAACTTT-3′

211

3′-GCTGACGGAAGTTTTATTGAAA-5′.

212

designed

Construction

and

for

ARE

Production

decoy

ODN

. Upper-strand and

are

listed

as and

and

of

HSP70-shRNA

Lentivirus

Vector.

213

HSP70-shRNA lentivirus vectors were constructed and produced according to our

214

previous report 27. Briefly, the rat HSP70 (Gene ID: 24472) cDNA was PCR amplified

215

and subcloned into the lentivirus-based RNAi vector pGPU6/GFP/Neo (GeneChem

216

Co.

217

5′-GATCCGAGGTGCAGGTGAACTACAAGGTTCAAGAGACCTTGTAGTTCAC

218

C-TGCACCTCTTTTTTG-3′,

219

5′-AATTCAAAAAAGAGGTGCAGGTG-AACTACAAGGTCTCTTGAACCTTGT-

220

AGTTCACCTGCACCTCG-3′. A negative control (NC) was generated by inserting a

Ltd.).

Primer

designed

for

HSP70

cDNA is

and

11


shown

as

HSP70-F,

HSP70-R


221

non-targeting sequence into pGPU6/GFP/Neo vector. Lentiviral vectors were

222

packaged through co-transfecting 293T cells with the ViraPower™ Lentiviral

223

Expression Systems (invitrogen, USA) following the instruction provided by the

224

manufacturer. After 48 h, the transfection efficiency was monitored by GFP

225

expression. Lentiviral particles were collected within 48-72 h after transfection.

226

Terminal Dexynucleotidyl Transferase-mediated dUTP Nick End Labeling

227

(TUNEL) Assay. Apoptosis was determined with a standard in situ TUNEL method.

228

The rats were fixed by perfusion of 4% paraformaldehyde for 24 h under the depth of

229

anesthesia. Brains were cunt into coronal sections (5-mm thick). Then, the staining

230

procedure was followed the TUNEL kit’s instruction provided by manufacture. The

231

Photographs were acquired by a digital CCD Camera and images were analyzed by a

232

person who was blinded to the animal group information.

233

Statistical Analysis. The experimental results were presented as means ± SD.

234

Two-tail Student’s t-test or ANOVA test was used to calculate the significant

235

difference in the study. A p-value of < 0.05 was considered to be statistically

236

significant.

237

RESULTS

238

Intranasal Z-LIG Pretreatment Reduced Infarct Volume and Neurological

239

Dysfunction. To examine the efficacy of Z-LIG via intranasal administration on

240

cerebral ischemic injury, rats were intranasally pretreated with Z-LIG for 3 days and

241

then subjected to MCAO/reperfusion, which mimics the most common type of stroke

242

in humans. As shown in Figure 1A and B, MCAO injury led to a 28% infarct volume 12


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in rats of the vehicle group compared with that of the sham group, whereas intranasal

244

pretreatment of Z-LIG (15 mg/kg) obviously reduced the infarct volume caused by

245

MCAO (p < 0.01). Further neurological deficit score evaluation demonstrated that

246

intranasal pretreatment of Z-LIG remarkably mitigated neurological dysfunction,

247

determined at 24 h after MCAO injury (p < 0.01) (Figure 1C). These findings suggest

248

that intranasal delivery of Z-LIG may provide efficient neuroprotection after

249

ischemia.

250

Intranasal Z-LIG Pretreatment Mitigated BBB Permeability and Brain

251

Edema. To evaluate the BBB permeability caused by cerebral ischemia, the

252

extravasation of EB dye into the brain was measured. Figure 2A shows that ischemic

253

injury produced a significant increase in EB leakage (p < 0.01), indicating damage to

254

the BBB and an increase in permeability. Intranasal pretreatment of Z-LIG

255

remarkably reduced the extravasation of EB dye (p < 0.01) in injured animals

256

compared with that detected in the model group, demonstrating that Z-LIG prevented

257

BBB disruption after ischemic injury. Then, we further determined brain water

258

content, which reflects the brain edema level. As shown in Figure 2B, the water level

259

in the brains of the vehicle group was remarkably greater than that of the sham group

260

(p < 0.01), indicating that brain edema was evident after ischemic injury. However,

261

the water content in the brains of injured rats pre-treated with intranasal

262

administration of Z-LIG was remarkably less than that of the vehicle group (p < 0.01),

263

suggesting that Z-LIG reduced the brain edema caused by ischemic injury.

264

Intranasal Z-LIG Pretreatment Attenuated the Loss of Vascular Basement 13



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Membrane Protein and Tight Junction Proteins. To assess the effect of Z-LIG on

266

the vasculature after cerebral ischemic injury, we first examined the vascular

267

basement

268

immunohistochemical staining. Figure 3A shows that ischemic injury caused a

269

significant degradation of collagen IV. Similarly, ZO-1 and occludin were also

270

degraded after brain injury. To determine whether Z-LIG could prevent the decrease

271

of those proteins, rats were pre-treated with intranasal administration of Z-LIG before

272

MCAO injury. Further immunohistochemical staining revealed that the loss of

273

collagen IV, ZO-1, and occludin due to ischemic injury was remarkably reduced in

274

animal pre-treated with Z-LIG. Then, we further determined the levels of vascular

275

basement membrane protein and tight junction proteins. As shown in Figure 3B, the

276

immunoreactivities of collagen IV, ZO-1, and occludin were all obviously mitigated in

277

the vehicle group compared to those in the sham group. It is worth noting that the loss

278

of those proteins was significantly prevented by the intranasal pretreatment of Z-LIG.

membrane

protein

and

tight

junction

proteins

by

using

279

Intranasal Z-LIG Pretreatment Reduced level of Matrix Metalloproteinases 2

280

and -9. We further determined the influence of Z-LIG on matrix metalloproteinases 2

281

and -9 (MMP-2 and MMP-9), which are usually induced after ischemic injury and

282

lead to the degradation of extracellular matrix (ECM) and BBB disruption 37. Figure 4

283

shows that the level of both MMP-2 and MMP-9 was remarkably elevated in the

284

vehicle group as compared to those in the sham group. Importantly, intranasal

285

pretreatment of Z-LIG significantly reduced the level of both MMP-2 and MMP-9 in

286

rats subjected to MCAO-reperfusion. 14


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Inhibition of Nrf2 and HSP70 Diminished the Protective Effect of Z-LIG

288

against Ischemic Injury. Our previous studies demonstrated that both Nrf2-mediated

289

antioxidant phase II enzymes and HSP70 contribute to the protection of Z-LIG against

290

OGD-Rep injury

291

antioxidant phase II enzymes and HSP70 in Z-LIG’s protection against MCAO injury

292

in rats. Nrf2-driven transcription of phase II enzymes and HSP70 were inhibited by

293

ARE decoy ODNs and small hairpin RNA targeting HSP70 (shHSP70), respectively.

294

First, we analyzed the protein expression of NQO1, a key enzyme downstream of

295

Nrf2, and HSP70 in the cortical tissue surrounding the contusion core. Figure 5A

296

shows that ischemic injury caused a remarkable increase in NQO1 protein expression

297

(p < 0.05). Intranasal pretreatment of Z-LIG further promoted NQO1 protein

298

expression in rats with ischemic injury (p < 0.05). However, ARE decoy ODNs

299

significantly reduced Z-LIG-induced NQO1 protein expression in rats with ischemic

300

injury (p < 0.01), whereas ARE mut ODNs did not exhibit a similar effect. Figure 5A

301

also presents a similar variation trend of HSP70 as NQO1. The protein expression of

302

HSP70 was increased in cortical tissues of rats with MCAO injury and further

303

augmented after intranasal pretreatment of Z-LIG. The silencing of the HSP70 gene

304

by shHSP70 notably attenuated the upregulation of HSP70 expression by Z-LIG in

305

MCAO-injured rats compared with that exerted by NC-shRNA (p < 0.05). Then, we

306

determined the influence of ARE decoy ODNs and shHSP70 on the improvement of

307

infarct volume and neurological dysfunction by Z-LIG. As shown in Figure 5B,

308

intranasal pretreatment with Z-LIG predictably ameliorated the infarct volume caused

27, 28

. In this study, we further determined the role Nrf2-mediated

15



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309

by ischemic injury (p < 0.01). Importantly, ARE decoy ODNs greatly reversed the

310

effect of Z-LIG as it increased the infarct volume compared with ARE mut ODNs (p

Intranasal Pretreatment with Z-Ligustilide, the Main Volatile

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