Dihydromyricetin Inhibits Lead-Induced Cognitive Impairments and

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Bioactive Constituents, Metabolites, and Functions

Dihydromyricetin inhibits lead-induced cognitive impairments and inflammation by AMPK pathway in mice Chan-Min Liu, Wei Yang, Jieqiong MA, Hui-Xin Yang, ZhaoJun Feng, Jian-Mei Sun, Chao Cheng, and Hong Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02433 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Journal of Agricultural and Food Chemistry

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Dihydromyricetin inhibits lead-induced cognitive impairments and

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inflammation by AMPK pathway in mice

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Chan-Min Liu1,*, Wei Yang1, Jie-Qiong Ma2, Hui-Xin Yang1, Zhao-Jun Feng1,

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Jian-Mei Sun1, Chao Cheng1, Hong Jiang1

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1

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Tangshan New Area, 221116, Xuzhou City, Jiangsu Province, PR China

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2

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and Engineering; Xuyuan road, 643000, Zigong City, Sichuan Province, PR China

School of Life Science, Jiangsu Normal University, No.101, Shanghai Road,

School of Chemistry and Pharmaceutica Engineering, Sichuan University of Science

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*Correspondence to: Chan-Min Liu; Phone number: +86 516 83403170; Fax number:

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+86 516 83500171. E-mail addresses: [email protected]

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ACS Paragon Plus Environment


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Abbreviations:

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Aβ, amyloid-beta; AMPK, 5’ denosine monophosphate-activated protein kinase; Bax,

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Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; DHM, Dihydromyricetin;

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CAT, catalase; GSK-3, Glycogen synthase kinase-3; IL-1β: interleukin-1beta; LPO,

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lipid peroxidation; JNK: the c-Jun N-terminal kinases; NF-κB, nuclear factor-κB;

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MyD88, myeloid differentiation factor 88; Pb, lead; PCO, protein carbonyl; ROS,

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reactive oxygen species; TLR4, Toll-like receptor 4; TNF-α: tumor necrosis

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factor-alpha.

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ABSTRACT

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Dihydromyricetin (DMH), a natural flavonoid derived from the medicinal and edible

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plant Ampelopsis grossedentata, exhibits anti-oxidant, anti-apoptosis, anti-tumor, and

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anti-inflammatory bioactivities. This study evaluated the effects of DHM on

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Pb-induced neurotoxicity and explored the underlying mechanisms. DHM

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significantly ameliorated behavioral impairments of Pb-induced mice. It decreased the

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levels of lipid peroxidation and protein carbonyl, and increased the activities of

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superoxide dismutase and catalase in the brains. DHM suppressed Pb-induced

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apoptosis, as indicated by the decreased levels of Bax and cleaved caspase-3. DHM

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also decreased inflammatory cytokines in the brains of Pb-treated mice. DHM

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decreased amyloid-beta (Aβ) level and nuclear factor-κB nuclear translocation.

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Moreover, DHM induced the adenosine 5’-monophosphate-activated protein kinase

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phosphorylation and inhibited the activation of p38, Toll-like receptor 4, myeloid

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differentiation factor 88 and glycogen synthase kinase-3. Collectively, this is the first

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report indicating that DHM could improve Pb-induced cognitive functional

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impairment by preventing oxidative stress, apoptosis and inflammation and that the

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protective effect was mediated partly through the AMPK pathway.

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Keywords: Dihydromyricetin; Lead; Oxidative stress; Inflammation; Apoptosis;

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AMPK.

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INTRODUCTION

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Dihydromyricetin (DHM) is a bioactive flavanonol compound found in the

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medicinal and edible plant Ampelopsis grossedentata, which grows widely in the

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south of China.1 DHM is found as the most abundant (with a content of more than

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30%) component in A. grossedentata, though it shows poor bioavailability due to the

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highly hydrophilic character.2,3 A. grossedentata is used for tea by the Yao people in

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China to treat sore throat, cough, fever, pharyngitis, and allergenic skin disease. It is

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also used for the prevention and treatment of nephritis, hepatitis, halitosis, and

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polyorexia.1,2 DHM was reported to exert multiple pharmacological activities,

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including antioxidant, anti-inflammatory, anti-microbial, anti-cancer, neuroprotective,

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cardio-protective, and hepatoprotective.1-4 Studies have demonstrated that DHM (20

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mg/kg/d) improves 3NP-induced behavioral disorders and striatal damage in rats.5

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DHM (125 and 250 mg/kg/d) could ameliorate cognitive dysfunction and improve

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disorder of glucose and lipid metabolism in type II diabetes mellitus mic by inhibiting

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oxidative stress.6 DHM (2, 5 and 10 mg/kg/d) also has a neuroprotective effect in

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models of Parkinson's disease (PD) by inhibiting glycogen synthase kinase-3 beta

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(GSK-3β) activity and dysfunction of GABAergic neurotransmission.7,8 DHM (20

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Μm/L) inhibited the methylglyoxal-induce oxidative stress response via the AMPK

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pathway in PC12 cells.9

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Adenosine 5’-monophosphate-activated protein kinase (AMPK) is a highly

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conserved serine/threonine protein kinase that plays a pivotal role in regulating

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cellular energy metabolism, oxidative stress, inflammatory response and apoptosis.8

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Overwhelming evidence has demonstrated that AMPK activation has protective

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effects against neuroinflammation of the central nervous system (CNS).9-11 Research

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has indicated that AMPK activation inhibited IFN-γ-induced generation of

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pro-inflammatory cytokines in primary astrocytes.11 AMPK activation exerts

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inhibitory activity against LPS-induced inflammation by decreasing nuclear

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translocation of NF-κB and consequently reducing pro-inflammatory mediators,

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including IL-6, TNF-α, iNOS, and COX-2.12,13

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Lead (Pb) is a nonessential heavy metal in the environment, which can be

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absorbed into the body through food, water, and air. The main target of Pb is the

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nervous system.14 Pb can interfere with neuronal functions by inducing apoptosis,

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inflammation, oxidative stress, nerve conduction dysfunction, and energy metabolism

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disorders.14,15 Compelling evidence indicates that Pb can induce an inflammatory

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response and affect the inflammatory processes in the nervous system by increasing

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the production and activation of pro-inflammatory cytokines.14,16,17 A recent study

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indicates that Pb can induce cognitive deficit and oxidative stress and amyloidogenic

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process via the AMPK pathway.18

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Thus, this study investigated the protective effects of DHM on Pb-induced

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neurotoxicity in mice and its possible mechanism. We hypothesized that DHM

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protects against Pb-induced cognitive impairments and inflammation via the AMPK

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pathway.

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MATERIALS AND METHODS

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Chemicals and reagents Dihydromyricetin and lead acetate (Pb(CH3COO)2)

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were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The IL-1β, TNF-α,

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phospho-p38, p38, phospho-AMPK, AMPK, phospho-GSK-3β (Ty216), NF-κB p65,

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TLR4 and MyD88 antibodies were provided by Abcam (Cambridge, MA, USA) and

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Santa Cruz Biotechnology (Santa Cruz, CA).

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Animals and Treatment

Male ICR mice (9-11 g) were provided by the

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Beijing HFK Bioscience CO., LTD (Beijing, China). Forty mice were housed under

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the standard laboratory conditions for 1 week.17 Then, the mice were randomly

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divided into four groups: Control group (saline), Pb group (250 mg Pb/L),

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Pb+DHM(125 mg/kg) and Pb+DHM(250 mg/kg). For the groups with Pb treatment,

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mice received an aqueous solution of lead acetate (Pb(CH3COO)2) at a concentration

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of 250 mg Pb/L of drinking water. For the group with Pb and DHM treatment, mice

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were subjected to daily oral gavage administration of DMH at a dose of 125 and 250

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mg/kg/body weight (during Pb administration). The choice of Pb and DMH dose was

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based on previous studies.6,19

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The experiment lasted for 3 months. After behavioral tests, the mice were

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sacrificed by decapitation. Brain tissues were rapidly collected and preserved at -80°C

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for further analysis.

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All experimental procedures were approved by the local ethics committee of the

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university (No, IACUC-1.0,1) and conducted in accordance with Chinese laws and

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the National Institutes of Health Guidelines for the Care and Use of Animals (NIH

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Publications No. 80-23).

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Behavioral assessments The behaviors were analyzed by the methods described

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previously.20 In brief, habituation to an open field was carried out in a 40 cm×60 cm

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open field surrounded by 50 cm high walls made of brown plywood with a frontal

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glass wall. The floor of the open field was divided into 9 equal rectangles by black

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lines. The animals were gently placed on the left rear quadrant and left to explore the

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area for 5 min (training session). Then, the mice were immediately taken back to their

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home cage and submitted again to a similar open field session 24 h later (test session).

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The number of crossed lines and rearing in both sessions were manually recorded.

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The decrease in the number of crossings and rearing between the two sessions was

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taken as a measure of the retention of habituation.

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Oxidative stress assays The levels of lipid peroxidation (LPO), protein carbonyl

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(PCO) and the activities of superoxide dismutase (SOD) and catalase (CAT) in brain

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tissues were determined using commercial assay kits (Jiancheng, Nanjing, China),

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according to the manufacturers’s protocol.

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Western blot analyses The levels of protein expressions were confirmed by

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Western blot analysis as described in our previous studies.19,20 The brain was

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homogenized with ice-cold RIPA buffer.

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Statistical analysis Differences between groups and the individual comparisons

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were obtained by one-way analysis of variance (ANOVA) followed by Tukey’s HSD

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post hoc test.

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RESULTS

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DMH rescues Pb-induced behavioral deficits The effects of Pb on motor

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activity in mice were assessed using the open-field test. Compared with that in the

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control group, the decreased number of crossing and rearing behaviors increased in Pb

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group (Table 1). However, treatment with a low (125mg/kg) or high (125mg/kg) dose

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of DMH dramatically blocked Pb-induced changes in these behaviors (P < 0.01).

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DMH attenuated Pb-induced oxidative stress in brains

To further examine

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the antioxidant effect of DMH, we assayed the levels of LPO and PCO, and activities

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of SOD and CAT in the brains. As shown in Table 2, the levels of LPO and PCO

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were dramatically elevated in the Pb-treated group, when compared to the control

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group. However, DMH supplementation remarkably reduced the levels of LPO and

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PCO. Similarly, the activities of SOD and CAT in the brains remarkably decreased in

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the Pb group, but treatment with DMH effectively restored these antioxidant

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parameters (P < 0.01).

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DMH inhibited Pb-induced apoptosis in the brains of mice It is reported that

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Pb could induce apoptosis in nervous tissues.21,22 To reveal whether DMH could

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inhibit Pb-induced apoptosis in the brains of mice, we measured the protein

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expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and

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caspase-3. As shown in Figure 1, Pb exposure significantly increased the expression

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of Bax and cleaved caspase-3, while decreasing expression of the anti-apoptotic

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protein Bcl-2 in comparison to control group. However, treatment with DMH

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effectively blocked Pb-induced changes in these apoptotic proteins.

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DMH inhibited Pb-induced inflammation in the brains of mice NF-κB

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nuclear translocation plays a key role in the occurrence and development of

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inflammation.10,14,20 We further performed western blotting assay to assess changes in

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the levels of pro-inflammatory cytokines. As seen in Figure 2, the increases in TNF-α,

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IL-1β and nuclear translocation of NF-κB p65 were caused in the brains by Pb

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exposure, and were significantly inhibited by treatment with a low (125mg/kg) or high

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(125mg/kg) dose of DMH (P < 0.01).

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AMPK was involved in the protective effects of DMH in the brains of mice

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AMPK was reported to be involved in oxidative stress and chronic inflammatory

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disorders.10 We next examined whether DMH could inhibit Pb-induced brain damage

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via AMPK activation. Pb exposure significantly suppressed AMPK phosphorylation

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in the mouse brains (P < 0.01). However, treatment with DMH markedly rescued the

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decreased phosphorylation levels of AMPK (Figure 3A).

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DMH decreased Aβ production in the brains of mice exposed to Pb

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Amyloid-beta (Aβ) peptides can induce oxidative stress and inflammation,23 so we

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measured the Aβ expression in the mouse brains (Figure 3B). Pb exposure

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significantly increased Aβ production. Interestingly, treatment with DMH markedly

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prevented the increased Aβ protein expressions.

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TLR4 signaling was involved in the anti-inflammatory roles of DMH

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Activation of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88

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(MyD88) pathway could induce excessive expressions of inflammatory cytokines.24

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In this study, the protein expression of TLR4 and MyD88 was remarkably

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up-regulated in the brains of mice exposed to Pb (Figure 4). However, treatment with

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DMH significantly decreased these protein levels in the Pb group (P < 0.01).

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Activation of GSK-3β and p38 pathway mediated the protective effects of

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DMH Glycogen synthase kinase-3 (GSK-3) and p38 MAPK play important roles in

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inflammation and apoptosis.10,22,25 Thus, we measured the phosphorylation levels of

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GSK-3β and p38 (Figure 5). We observed that the protein phosphorylation of

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GSK-3β (Ty216) and p38 was reduced due to Pb exposure, whereas oral

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administration of DMH markedly inhibited the phosphorylation levels of GSK-3β

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(Ty216) and p38 (P < 0.01).

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DISCUSSION

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DHM is the most abundant flavonoid extracted from A. grossedentata, which was

207

shown

to

multiple

pharmacological

functions,

including

antioxidant,

208

anti-inflammatory, anti-microbial, anti-cancer, cardio-protective, neuroprotective and

209

hepatoprotective properties.1,6,26 In our study, DHM ameliorated cognitive

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impairments by regulating the expression of AMPK, Aβ, TLR4, MyD88, p38 and

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GSK-3β in mice.

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Pb is a widespread environmental toxin, which can cause acute or chronic

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poisoning, including neuronal injury of the central nervous system. DHM has been

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reported to ameliorate behavioral deficits in 3NP-induced animal model of Huntington

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disease (HD) and transgenic mouse models of Alzheimer’s disease (AD).5,7

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Consistently, in the present study, we observed DHM administration markedly

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attenuated the behavioral impairments in mouse model of Pb exposure, illustrating the

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neuroprotective effects of DHM (Table 1). Several lines of evidences show that

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oxidative stress played a crucial role in Pb-induced neurotoxicity.18,20 Pb can induce

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excess reactive oxygen species (ROS), which can damage proteins, lipids,

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carbohydrates and DNA.15,18 In this research, we observed that Pb exposure

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remarkably increased the levels of PCO and LPO, while the activities of SOD and

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CAT decreased markedly in comparison with the control group, thereby increasing

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oxidative stress. Interestingly, DHM obviously decreased the levels of LPO and PCO,

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and increased the activities of SOD and CAT in the brains of Pb-treated mice (Table

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2), which could be due to the notable antioxidant capacity of DHM.1,6,9 These results

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indicated that the neuroprotective effects of DHM may be attributed to the

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suppression of oxidative stress in the brains of mice exposed to Pb.

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Overwhelming evidence indicated that Pb exposure could cause neuronal

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apoptosis in animals and cultured cells.19,21,22 Bax is a pro-apoptotic regulator. Bcl-2 is

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an important anti-apoptotic protein.21, 22 In this study, Pb exposure decreased Bcl-2

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expression, concomitant with an increase in Bax protein expression. However, DHM

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treatment markedly inhibited these Pb-induced changes, suggesting that DHM could

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inhibit the activation of the apoptotic pathway (Figure 1). Moreover, DHM decreased

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Pb-induced cleavage of caspase-3 (one of the key executioners of apoptosis), further

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confirming that the neuroprotective effects of DHM are mediated by suppression of

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apoptosis.

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Multiple researches indicated that Pb could affect the immune system and induce

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inflammatory response.14,20 Pb exposure could activate the transcription factor NF-κB,

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which further regulate the inflammatory responses and the immune response.14,17,20

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Activation of NF-κB could result in increased synthesis of pro-inflammatory

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cytokines, including IL-1β and IF-6.14,27 Oxidative stress induced by Pb exposure

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could also boost ROS generation and stimulate inflammatory responses in the nervous

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system.14,17 Results of the present study showed that Pb exposure obviously activated

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NF-κB, while subsequently increasing the release of proinflammatory cytokines,

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including IL-1β and TNF-α, in the mouse brains. However, the supplementation of

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DHM significantly attenuated inflammation by inhibiting NF-κB nuclear translocation

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(Figure 2). Hence, the results of this study suggested that DHM could protect the

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brain against Pb-induced injury by suppressing inflammatory response.

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AMPK is a multifaceted enzyme that participates in the cellular response to

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metabolic stress. AMPK plays a permissive role in oxidative stress, inflammatory

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response and apoptosis.9-12 AMPK activation could inhibit oxidative stress,

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inflammation and cognitive impairment.28 Aβ is a fragment amyloid precursor protein

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(APP), which is reported to cause neurodegeneration in the hippocampus of brains by

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promoting inflammatory response and oxidative stress.23 Activated AMPK could

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decrease the generation and accumulation of Aβ production.29,30 Recent research

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showed that Aβ caused neuronal impairment and neurodegenerative disorder

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associated with suppression of hippocampal AMPK activity.31 Aβ could induce the

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production and release of proinflammatory cytokines by modulating NF-κB

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activation.14,20 Activated AMPK could inhibit expression of pro-inflammatory

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cytokines by regulating NF-κB in brains.32 It is reported that Pb could decrease

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AMPK activity and increase Aβ accumulation in the hippocampus, resulting in

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cognitive

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methylglyoxal-induced apoptosis, oxidative stress and abnormal glucose metabolism

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in PC12 cell by increasing AMPK activity.9 DHM can inhibit neuroinflammation and

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neurodegeneration by activating the AMPK pathway.10 DHM has been seen to

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decrease Aβ generation and reverse progressive neuropathology in mouse models of

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AD.7 This study revealed that Pb inhibited AMPK activation and increased Aβ

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accumulation in the brains of mice. However, DHM noticeably decreased the Aβ level

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and increased phosphorylation of AMPK (Figure 3). These results indicated that

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AMPK and Aβ were involved in the neuroprotective effects of DHM against Pb.

impairment.18

Research

has

revealed

that

DHM

can

inhibit

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TLR4 is a membrane-bound receptor that plays a critical role in the innate

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immune response. TLR4 could regulate inflammatory response through MyD88

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pathway.16,17,24 Previous research showed that Pb exposure could disturb hippocampal

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neurogenesis and functional plasticity by the TLR4/MyD88/NF-κB signaling

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pathway.10,16,17 It is reported that p38 may be involved in TLR4 signaling pathway in

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the hippocampus of young mice exposed to Pb.14,17 AMPK could also regulate

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neuroinflammation and neurodegeneration by TLR4-related signal pathways.10 DHM

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was reported to protect against LPS-induced cardiomyocyte injury by inhibiting TLR4

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activation and subsequent regulating the associated downstream signaling pathway of

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NF-κB.33 Our data reveal that Pb exposure notably inhibited the phosphorylation of

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AMPK and increased the expression levels of TLR4 and MyD88, which might

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activate NF-κB and regulate inflammation.16,17 However, DHM treatment decreased

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the expression levels of TLR4, MyD88 and inflammatory cytokines (Figure 4). These

285

data demonstrated that DHM might ameliorate Pb-induced neuroinflammation via the

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AMPK/TLR4/MyD88 pathway.

287

P38 MAPK is a class of MAPKs, which processes and regulates cellular

288

properties in response to stress stimuli such as ROS and inflammatory cytokines.34

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Overwhelming evidence has identified p38 as a central mediator with an important

290

role in Pb-induced neurotoxicity.25,35,36 Pb can affect the system of MAP kinases by

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activating

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AMPK

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proinflammatory cytokines.32,38 It has been reported that p38 is involved in the

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development process of AD, such as tau phosphorylation, Aβ deposition,

295

neurotoxicity, neuroinflammation and cognitive impairments.34 Research has also

296

shown that DHM can inhibit inflammation by suppressing p38 and NF-κB.2 In this

297

study, DHM significantly inhibited the p38 signaling pathway and its downstream

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inflammatory mediators’ release to alleviate Pb-induced damage (Figure 5),

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suggesting that DHM supplementation may have an important role in preventing the

300

progression of neuroinflammation diseases.

protein activation

kinase C (PKC) in rat brain and human astrocytoma cells.14,37 could

reduce

p38

phosphorylation,

further

inhibiting

301

The GSK-3 signaling pathway plays a key role in the pathogenesis of nervous

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disorders, and is also the target for a wide variety of immune and metabolic

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diseases.39,40 Several studies have shown that AMPK and GSK-3 interact with each

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other.41 Inhibition of GSK-3 by the AMPK signaling pathway is involved in the

305

regulation of the NF-κB activation and inflammatory pathway. GSK-3 inhibitors

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could suppress inflammatory response by regulating TLRs, MAPK, β-catenin, STATs,

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and the NF-κB pathway.39,40 Previous research showed that Pb exposure significantly

308

increased GSK-3β expression and activation in brains.42-44 DHM was reported to

309

confer the neuroprotective effects on DA neurons by suppressing the GSK-3β

310

activity.8 DHM also elicits potent neuroprotective effects in a model of major

311

depressive disorder (MDD) by inhibiting GSK-3β activation.26 In this study, Pb

312

induced GSK-3β phosphorylation at Tyr216, which might promote an inflammatory

313

response (Figure 5). However, DHM treatment inhibited Pb-induced inflammation

314

and suppressing the GSK-3β activity in the brains of mice. These results provide the

315

first evidence that DHM exerts protective effects on Pb-induced nerve damage via the

316

AMPK/GSK-3β pathway.

317

In summary, our present study for the first time demonstrated that DHM reversed

318

Pb-induced behavioral impairments by suppressing oxidative stress, inflammation and

319

apoptosis (Figure 6). Specifically, DHM ameliorated Pb-induced oxidative damage in

320

the brain. DHM inhibited apoptosis by decreasing the levels of Bax and cleaved

321

caspase-3 in the brains of mice exposed to Pb. Finally, DHM inhibited Pb-stimulated

322

inflammation by regulating the AMPK, Aβ, TLR4, MyD88, p38 and GSK-3β

323

pathways. The neuroprotection of the bioactive metabolites of DMH warrants further

324

investigation in our future research.

325

Conflict of interest statement

326 327

None. Acknowledgments

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328

This study was supported by the Project Funded by the Priority Academic Program

329

Development of Jiangsu Higher Education Institutions (PAPD) and Graduate Student

330

Scientific Research Innovation Projects of Jiangsu Normal University (2017YXJ127).

331

This work is sponsored by grants from Scientific Research Fund of SiChuan

332

Provincial Education Department (15ZA0236).

333

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500

Figure captions:

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Figure 1. Dihydromyricetin (DHM) inhibited Pb-induced apoptosis in the brains of

502

mice. (A) Western blot analysis of the Bax, Bcl-2 and caspase-3 proteins in the brains;

503

(B) Relative density analysis of the Bax protein bands; (C) Relative density analysis

504

of the Bcl-2 protein bands; (D) Relative density analysis of the cleaved caspase-3

505

protein bands; β-Actin was probed as an internal control in relative density analysis.

506

The vehicle control is set as 1.0. Values are averages from three independent

507

experiments. Each value is expressed as mean±S.E.M. ## P < 0.01, compared with the

508

control group; ** P < 0.01, vs. the Pb-treated group.

509 510

Figure 2. Dihydromyricetin (DHM) decreased the levels of inflammatory cytokines

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512

and IL-1β proteins in the brains; (B) Relative density analysis of the NF-κB p65

513

protein bands in cytosol and nucleus; (C) Relative density analysis of the TNF-α

514

protein bands; (D) Relative density analysis of the IL-1β protein bands; β-Actin was

515

probed as an internal control in relative density analysis. Lamin A was used as a

516

reference for the measurements of the NF-κB p65 protein bands in nucleus. The

517

vehicle control is set as 1.0. Values are averages from three independent experiments.

518

Each value is expressed as mean±S.E.M. ** P

Dihydromyricetin Inhibits Lead-Induced Cognitive Impairments and

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