Effects of Baicalein on Cortical Proinflammatory Cytokines and the

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The effects of baicalein on cortical pro-inflammatory cytokines and the intestinal microbiome in senescence accelerated mouse prone 8 Li Gao, Jia-Qi Li, Yu-Zhi Zhou, Xudong Huang, Xue-mei Qin, and Guan-Hua Du ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00074 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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ACS Chemical Neuroscience

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The effects of baicalein on cortical pro-inflammatory cytokines and

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the intestinal microbiome in senescence accelerated mouse prone 8

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Li Gao†1*, Jiaqi Li†1,2, Yuzhi Zhou1, Xudong Huang3, Xuemei Qin1*, Guanhua Du1,4 1

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Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, PR China;

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6

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College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, PR China;

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Hospital and Harvard Medical School, Boston, MA, USA.

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Neurochemistry Laboratory, Department of Psychiatry, Massachusetts General

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Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100050, PR China.

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* Corresponding authors

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†These authors contributed equally to this work

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Li Gao* (E-mail: [email protected]); Tel & Fax: 86-351-7018379; Address: No.92

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Wu Cheng Road, Taiyuan 030006, China

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Xuemei Qin* (E-mail: [email protected]); Tel & Fax: 86-351-7011501; Address:

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No.92 Wu Cheng Road, Taiyuan 030006 China 1

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Baicalein, a flavonoid derived from the roots of Scutellariae baicalensis Georgi, has

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shown health benefits for an array of human diseases including dementia. The

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senescence-accelerated mouse prone 8 (SAMP8) strain is extensively used as a senile

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dementia model. To further investigate the effects of baicalein in SAMP8 mice,

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behavioral testing, biochemical detection and gut microbiota analysis were performed.

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The results demonstrated that treatment with baicalein ameliorated the senescence

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status of the SAMP8 mice, as manifested by reducing the grading score of senescence.

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Additionally, baicalein improved the cognitive functions of the SAMP8 mice,

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including spatial learning and memory abilities, object recognition memory and

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olfactory memory. Furthermore, baicalein significantly inhibited the release of

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pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1 beta (IL-1β),

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and tumor necrosis factor-α (TNF-α) in the brain cortex of SAMP8 mice. Gut

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microbiota analysis revealed that treatment with baicalein markedly altered the

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abundance of 6 genera in SAMP8 mice. Correlation analysis indicated that the

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abundances of Mucispirillum, Bacteroides and Sutterella were negatively correlated

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with cognitive abilities and that Christensenellaceae was positively correlated with

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cognition. Furthermore, the abundance of Christensenellaceae was negatively

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correlated with the levels of IL-6 and TNF-α, while [Prevotella] was positively

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correlated with the levels of IL-1β and IL-6. In addition, Mucispirillum and

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Bacteroides were positively correlated with the levels of IL-6 in the brain cortex.

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These data indicated that baicalein ameliorates senescence status and improves 2


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cognitive function in SAMP8 mice and that this effect might be attributable to

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suppression of cortical pro-inflammatory cytokines and modulation of the intestinal

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microbiome.

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Keywords: baicalein, aging, learning and memory, SAMP8 mice, cytokines,

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intestinal microbiome

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Introduction

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Aging is a polygenic, multifactorial, and complex pathological process accompanied

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by the deterioration of multiple organ systems. Central nervous system (CNS)

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dysfunction due to aging has become one of the greatest threats to the health of the

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elderly, and cognitive functional decline is extremely common at an advanced age (1).

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Generally, brain aging can lead to systemic damage by triggering hormonal

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dyshomeostasis and then establish a vicious pathophysiological cycle, indicating that

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the CNS plays a central regulatory role in aging (2, 3).

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Immunosenescence is a suspected factor in aging. Immunosenescence, an

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imbalance of pro-inflammatory and anti-inflammatory function, triggers a low grade

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chronic inflammatory status (4). Recent studies have advanced our understanding that

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inflammation is strongly linked to the changes in intestinal flora during the aging

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process. With the development of metagenomics, the role of intestinal flora has been

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gradually investigated over recent years. Accumulated evidence has shown that a

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healthy intestinal flora plays an important role in maintaining a healthy body,

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including participation in nutrition metabolism, regulation of immunity, resistance to

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pathogenic strains, and regulation of the gut-brain axis. 3



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Notably, the structure and function of intestinal flora are gradually transformed

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during the aging process and even produce side effects in the host, such as increasing

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blood glucose levels (5) and affecting the absorption of certain amino acids (6).

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Furthermore, senescence-related changes in intestinal bacteria can increase intestinal

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permeability and release pro-inflammatory products into the bloodstream, eventually

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increasing the level of inflammation (7-9).

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Baicalein is a flavonoid derived from the roots of Scutellariae baicalensis Georgi.

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Baicalein is known for its various pharmacological effects, such as antioxidant,

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anti-inflammatory, and antitumor effects. In previous research, our group found that

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baicalein extended longevity by attenuating oxidative stress in Drosophila

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melanogaster (10) and ameliorated memory deficits by reducing inflammation and

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metabolic dysfunction in D‐galactose-induced aging rats (11). In addition, the results

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from other studies showed that baicalein significantly improved cognition by

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facilitating the induction of hippocampal long-term potentiation (LTP) in SD rats (12)

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or intensifying synaptic plasticity in APP/PS1 mice (13). However, the mechanisms

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underlying the neuroprotective effect of baicalein have not been fully explored.

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The senescence-accelerated mouse (SAM) is an accelerated aging model that

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was established from AKR/J inbred strains of mice. The SAM was divided into

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senescence-accelerated-prone mice (SAM-P) and senescence-accelerated-resistant

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mice (SAM-R) subtypes based on the degree of senescence, the lifespan, and the

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age-associated pathologic phenotypes (14). Senescence accelerated mouse prone 8

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(SAMP8) is currently the ideal model for neurodegeneration and dementia research 4


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with comprehensive brain pathological changes, including cortical atrophy, neuronal

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cell loss, gliosis, and vascular impairment, starting at a young age (15). Moreover,

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these changes are spontaneous and nontransgenic, which is a great advantage when

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studying aging cognitive impairment. Senescence accelerated mouse resistant 1

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(SAMR1) is commonly used as a normal control.

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In the present work, the effects of baicalein on senescence status and hypomnesis

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were evaluated in SAMP8 mice by examining the levels of cortical pro-inflammatory

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cytokines and intestinal microbial flora in mice. Herein, we are the first to report the

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ameliorative and modulating effects of baicalein on cortical pro-inflammatory

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cytokines and the intestinal microbiome in SAMP8.

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Results

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Baicalein ameliorated the senescence status of SAMP8 mice

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As shown in Figure 1, the grading score of senescence in SAMP8 mice was

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significantly higher than that in SAMR1 mice after the age of 8 months. However,

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after 4 weeks of treatment with baicalein, the grading score in SAMP8 mice was

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significantly reduced with reduction percentages of 29.1%, 30.3% and 36.8% after 4,

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6 and 8 week treatments, respectively. These results implied that baicalein could delay

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the senescence status of SAMP8 mice.

5



Figure 1. The effects of baicalein on the grading scores of senescence were assessed at 2, 4, 6 and 8 weeks, and data corresponding to 0 weeks were obtained before baicalein administration. n = 10 to 15 mice per group. Data are expressed as the mean ± SEM;

###

p < 0.001 versus SAMR1 mice; * p < 0.05, **p < 0.01 versus SAMP8

mice. 106

Baicalein improved cognitive functions in the Morris water maze test

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The results showed that the spatial learning and memory abilities of 8-month-old

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SAMP8 mice were significantly lower than those of SAMR1 mice (Supplementary

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Figure 1). Six weeks later, SAMP8 mice showed a longer escape latency from the first

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day to the fifth day than SAMR1 mice, while baicalein-treated SAMP8 mice showed

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a significant decrease in escape latency in comparison with SAMP8 mice on days 2, 4

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and 5 in the place navigation test (Figure 2a). In the spatial probe test, the escape

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latency was noticeably longer in the SAMP8 mice (36.15 ± 5.50 s) than the SAMR1

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mice (10.64 ± 1.76 s). However, baicalein treatment significantly decreased the

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escape latency (17.24 ± 3.45 s) (Figure 2b). As shown in Figure 2c, the time in the

116

target quadrant was significantly decreased in SAMP8 mice (12.60 ± 1.44 s) 6


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compared with SAMR1 mice (17.95 ± 1.68 s), while the administration of baicalein

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significantly increased with time (16.81 ± 1.42 s). The number of platform crossings

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was significantly decreased in SAMP8 mice (2.58 ± 0.65) compared with SAMR1

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mice (8.60 ± 0.79), and baicalein had a tendency to increase the platform crossing

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numbers (4.00 ± 0.47) (Figure 2d). These results demonstrated that baicalein could

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improve spatial learning and memory in SAMP8 mice.

Figure 2. The effects of baicalein on spatial learning and memory were assessed with the Morris water maze test. (a) Escape latencies during the 5 day acquisition phase. (b) Latency time in the retention phase. (c) Time in the target quadrant. (d) The number of platform crossings. n = 10 to 15 mice per group. Data are expressed as the mean ± SEM; #p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1 mice; * p < 0.05, **p < 0.01, versus SAMP8 mice. 7



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Baicalein improved cognitive abilities in the novel object recognition test and

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olfactory memory test

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The results showed that the exploration time for a new object was significantly longer

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than that for a familiar object in SAMR1 and baicalein-treated SAMP8 mice; however,

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the exploration time for new and familiar objects showed no difference in SAMP8

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mice (Figure 3a). The recognition index could reflect the cognitive memory ability of

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mice. Compared with that in SAMR1 mice, the recognition index in SAMP8 mice

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was significantly decreased (28.9%); nevertheless, it was dramatically increased in

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baicalein-treated SAMP8 mice (27.6%) in comparison with SAMP8 (Figure 3b). The

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present results revealed that baicalein exerted a protective influence on recognition

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memory.

Figure 3. Effects of baicalein on novel object recognition behavior. (a) Time spent on the familiar object (object A) and novel object (object C). (b) Recognition index. n = 10 to 15 mice per group.

&&

p < 0.01,

expressed as the mean ± SEM;

##

&&&

p < 0.001 versus object A. Data are

p < 0.01 versus SAMR1 mice; *p < 0.05 versus

SAMP8 mice. 134

In the olfactory memory test, with an increase in the number of exposures, the 8


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mice gradually adapt to the smell, that is, olfactory memory. The results (Figure 4)

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showed that as the number of exposures increased, SAMP8 mice spent longer on odor

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investigation than SAMR1 mice, and baicalein treatment markedly increased the time

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in the first trial, decreased the time in the third trial of amyl acetate odor investigation

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(Figure 4a) and decreased the time in the fourth trial of ethyl valerate odor

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investigation (Figure 4b). In other words, SAMP8 mice had a slower odor adaptation

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process than SAMR1 mice; conversely, baicalein treatment improved odor habituation

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behavior in SAMP8 mice. This indicated that baicalein had a protective effect on

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olfactory memory in SAMP8 mice.

Figure 4. Effects of baicalein on odor habituation behavior. (a) Amyl acetate. (b) Ethyl valerate. n = 10 to 15 mice per group. Data are expressed as the mean ± SEM, #

p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1 mice; * p < 0.05, **p < 0.01, ***p

< 0.001 versus SAMP8 mice. 144

Baicalein decreased the levels of pro-inflammatory cytokines in the brain cortex

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of SAMP8 mice

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To explore the effect of baicalein on brain inflammation, the levels of several

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pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the brain cortex were 9



148

analyzed. The results (Figure 5) showed that IL-1β and TNF-α were significantly

149

higher in the SAMP8 mice than in SAMR1 mice (22.0% and 23.4%), while IL-1β,

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IL-6, and TNF-α levels were significantly reduced after treatment with baicalein in

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SAMP8 mice (9.4%, 10.7% and 11.9%, respectively) for 8 weeks. These data

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indicated that baicalein could attenuate the secretion of pro-inflammatory cytokines in

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old SAMP8 mice to suppress senescence-related neuroinflammation.

Figure 5. Effects of baicalein on the levels of pro-inflammatory cytokines in the brain cortex. (a) IL-1β. (b) IL-6. (c) TNF-α. n = 6 or 9 mice per group. Data are expressed as the mean ± SEM; ##p < 0.01 versus SAMR1 mice; *p < 0.05 versus SAMP8 mice. 154

Baicalein modulated several gut microbiotas in SAMP8 mice

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Although our data demonstrated that baicalein could exert ameliorative effects on

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age-associated brain lesions in SAMP8 mice, we next investigated whether baicalein

157

could affect the gut microbiota that may regulate the CNS. 10


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By performing 16S rRNA amplicon sequencing on 24 samples, 1440262

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high-quality sequences were obtained and were subsequently clustered into

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operational taxonomic units (OTUs) at 97% sequence identity (Supplementary Table

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1). The comparison of alpha diversity indices revealed that SAMP8 mice increased

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alpha diversities compared to SAMR1 mice (Simpson index, p = 0.0190; Shannon

163

diversity index, p = 0.0199) and that there were no significant differences between

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SAMP8 mice and baicalein-treated SAMP8 mice (Figure 6a~c and Supplementary

165

Figure 2). Principal Coordinate Analysis (PCoA) based on unweighted UniFrac

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distances revealed that the gut microbiotas of SAMR1 mice, SAMP8 mice and

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baicalein-treated SAMP8 mice could be clearly distinguished (Figure 6d). PCoA

168

based on weighted UniFrac distances also showed similar trends (Figure 6e). Gut

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microbiota diversity analysis showed that intestinal microbiome diversity changed

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during the aging process. Therefore, we further explored whether the composition of

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intestinal flora was different between the two groups.

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Figure 6. Effects of baicalein on intestinal flora. (a) The rarefaction curves, (b) the Chao1 estimator, and (c) Shannon alpha diversity index based on randomly subsampling the reads of each sample. (d, e) Unweighted and weighted UniFrac distance PCoA by the first two principal coordinates PC1 and PC2. (f) The histogram of the composition and abundance distribution of the samples at the genus level. Only the genera with abundance > 0.1% are presented in the legend. n = 8 mice per group. 172

As shown in Figure 6f, a total of 81 genera were obtained at the genus level. We

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used Student’s t-test to identify the specific bacterial phylotypes that were different

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between baicalein-treated and untreated SAMP8 mice. The results showed that

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treatment with baicalein altered 6 strains in SAMP8 mice. Among these genera,

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Mucispirillum, Parabacteroides, [Prevotella], Bacteroides, and Sutterella were 12


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significantly reduced by 96.2%, 97.6%, 77.5%, 68.0% and 75.8% after treatment with

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baicalein, respectively. In contrast, Christensenellaceae was significantly increased by

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baicalein administration in SAMP8 mice (Figure 7 and Table 1).

Figure 7. Effects of baicalein on 6 kinds of genera in SAMP8 mice. n = 8 mice per 13



group. Data are expressed as the mean ± SEM

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###

p < 0.001, versus SAMR1 mice; *p

< 0.05, **p < 0.05 versus SAMP8 mice. Table 1. The bacterial taxa information (genus, family, order class, phylum) of 6 strains affected by baicalein. Genus

Family

Order

Class

Phylum

Unclassified

Christensenellaceae

Clostridiales

Clostridia

Firmicutes

Mucispirillum

Deferribacteraceae

Deferribacterales

Deferribacteres

Deferribacteres

Parabacteroides

Porphyromonadaceae

Bacteroidales

Bacteroidia

Bacteroidetes

[Prevotella]

[Paraprevotellaceae]

Bacteroidales

Bacteroidia

Bacteroidetes

Bacteroides

Bacteroidaceae

Bacteroidales

Bacteroidia

Bacteroidetes

Sutterella

Alcaligenaceae

Burkholderiales

Betaproteobacteria

Proteobacteria

180

The effect of baicalein on bacteria correlated with learning and memory abilities

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and levels of pro-inflammatory cytokines in SAMP8 mice

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To investigate whether the effect of baicalein on gut microbiota was associated with

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learning and memory abilities or inflammation in the brain cortex, the correlation

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coefficients between the differentially abundant OTUs and the cognitive abilities or

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the levels of pro-inflammatory cytokines in the brain cortex were calculated using

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Spearman’s correlation analyses in SAMP8 mice (n = 8 or 6) and baicalein-treated

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SAMP8 mice (n = 8 or 6).

188

The

results

(Figure

8)

showed

that

the

community

abundance

of

189

Christensenellaceae was positively correlated with the recognition index in the novel

190

object recognition test (NORT), while the community abundance of Mucispirillum 14


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191

was negatively correlated with the recognition index. The community abundances of

192

Sutterella and Bacteroides were negatively correlated with time in the target quadrant

193

in the MWM. In addition, Bacteroides and escape latency in the spatial probe test of

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the MWM were positively correlated. In short, the community abundances of

195

Mucispirillum, Bacteroides and Sutterella were negatively correlated with the

196

cognitive ability of SAMP8 mice, whereas Christensenellaceae was positively

197

correlated with the cognitive ability of SAMP8 mice. These results indicated that

198

intestinal microbiota might be linked to learning and memory dysfunction and that

199

baicalein improved cognitive ability, possibly by modulating certain specific flora in

200

the gut.

201

In addition, the community abundance of Christensenellaceae was negatively

202

correlated with the levels of IL-6 and TNF-α, while [Prevotella] was positively

203

correlated with the levels of IL-1β and IL-6. In addition, Mucispirillum and

204

Bacteroides were positively correlated with IL-6 in the brain cortex of SAMP8 mice.

205

These data indicated that intestinal microbiota might be linked to inflammation in the

206

brain cortex and that the anti-inflammatory role of baicalein was associated with the

207

modulation of specific intestinal flora to a certain degree.

15



Figure 8. Correlation between genera and learning and memory behaviors or pro-inflammatory cytokines. The red rhombus node represents the bacteria, the purple hexagon node represents the behavioral index, and the green triangle node represents the pro-inflammatory cytokines. The yellow edge represents the positive correlation (r > 0.6), the blue edge represents the negative correlation (r < -0.6), and the gray dotted edge represents no correlation (| r | < 0.6); r represents Spearman’s correlation coefficient. Data were collected from SAMP8 mice (n = 8 or 6) and baicalein-treated SAMP8 mice (n = 8 or 6). One-tailed Spearman’s analysis, confidence interval 95%. 208

Discussion

209

The grading score system was a valid and convenient method for evaluating the

210

degree of senescence in SAM mice. Consistent with previous reports (16), we

211

discovered that SAMP8 mice had significantly higher scores than SAMR1 mice at 8

212

months of age. After 4 weeks of baicalein administration, the grading score of 16


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senescence was significantly reduced in SAMP8 mice. Specifically, baicalein

214

improved the reactivity and passivity, fur glossiness and density, and attenuated skin

215

ulcers and periophthalmic lesions, which indicate that baicalein exerted an anti-aging

216

effect.

217

Cell senescence is the essence of organism aging. Cell senescence is

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accompanied by the acquisition of the senescence-associated secretory phenotype

219

(SASP),

220

pro-inflammatory cytokines and chemokines (17), and therefore, the accumulation of

221

senescent cells during aging induces a chronic low-grade inflammatory state.

222

Moreover, aging could drive the dysfunction of microglial cells, which in turn

223

increases the level of pro-inflammatory cytokines in the brain (18).

a

distinctive

phenotype

characterized

by enhanced

secretion of

224

An astonishing number of studies have demonstrated that the decreases in

225

learning and memory capabilities in SAMP8 mice are related to increased

226

inflammation in the cortex and hippocampus at a young age, manifesting as increases

227

in IL-1β, IL-6 and TNF-α, and a decrease in the protein expression of interleukin-10

228

(IL-10) (19, 20). In particular, an elevated level of IL-1β can inhibit LTP (21). The

229

potential cellular mechanisms are related to decreased mRNA expression of

230

brain-derived neurotrophic factor, induction of reactive oxygen species production,

231

and activation of c-jun N-terminal kinase and p38, which induce cell death and finally

232

lead to memory dysfunction (22).

233

In line with previous reports, we found that the levels of IL-1β and TNF-α in the

234

cortex of 10-month-old SAMP8 mice were higher than those in SAMR1 mice. 17



Page 18 of 40

235

However, the level of IL-6 did not change significantly (P = 0.0817) in the cortex of

236

10-month-old SAMP8 mice compared with SAMR1 mice. The reports on IL-6 were

237

inconsistent in different experiments (19, 23), which might be related to the age of the

238

mice and the experimental conditions. The levels of pro-inflammatory cytokines were

239

reduced after baicalein treatment for 8 weeks in SAMP8 mice. Our data suggested

240

that the ameliorative effect of baicalein on learning and memory impairment might be

241

through attenuation of cortical pro-inflammatory cytokines.

242

As early as the mid-19th century, the idea of the gut-brain axis (i.e., the

243

reciprocal impact of the gastrointestinal tract on brain function) was proposed.

244

Recently, increasing evidence has shown that the intestinal microflora can affect the

245

gut-brain axis, which in turn affects cognitive function (24). Surprisingly, the

246

formation of stable intestinal flora in humans occurs at the age of 2-3 years, the same

247

time frame for intestinal barrier maturation and hippocampal neurogenesis, which

248

together form the microbiota–gut–brain axis (25). Moreover, studies have shown that

249

some

250

norepinephrine, serotonin and acetylcholine). The neurotransmitters and cell wall

251

components of these microorganisms in the intestine can induce epithelial cells to

252

release the abovementioned molecules, thus regulating CNS function (26, 27).

intestinal

flora

can

produce

neurochemicals

(γ–aminobutyric

acid,

253

However, due to changes in intestinal microflora and permeability of the

254

intestinal barrier with aging, microbial products can enter the circulating blood by

255

penetrating the intestinal wall (9), cross the blood-brain-barrier and induce brain

256

inflammation (28). In addition, Erny et al. revealed that intestinal microbiota could 18


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257

control the maturation and function of microglia that secrete pro-inflammatory

258

cytokines by affecting system immune function (29). These studies hint that an

259

imbalance of intestinal flora is possibly related to the level of pro-inflammatory

260

cytokines in the CNS.

261

The present study showed that the alpha diversity increased and that the profile

262

of intestinal flora significantly changed in 10-month-old SAMP8 mice compared with

263

SAMR1 mice, which might be caused by the fact that some frailty-associated

264

alterations may increase the diversity of the age-related microbiome and change the

265

original flora profile (30). In addition, the profile of the intestinal flora significantly

266

changed in baicalein-treated SAMP8 mice compared with SAMP8 mice. We observed

267

that 5 strains (Mucispirillum, Parabacteroides, [Prevotella], Sutterella, and

268

Bacteroides) were apparently decreased and that 1 strain (Christensenellaceae) was

269

apparently increased after treatment with baicalein in SAMP8 mice. The results from

270

Spearman’s analysis indicated that Mucispirillum, Bacteroides and Sutterella were

271

related to cognitive deterioration and were modulated by baicalein and that

272

Mucispirillum, [Prevotella] and Bacteroides were associated with an increase in

273

inflammation. Moreover, Christensenella, a strain closely related to longevity, was

274

modulated by baicalein, helping reduce inflammation and protect cognition in

275

SAMP8 mice. Many studies have shown that changes in Mucispirillum,

276

Parabacteroides, [Prevotella], Sutterella, and Bacteroides in the intestinal flora were

277

closely related to inflammation (31-33). Magnusson et al. revealed that the proportion

278

of Bacteroidales was closely related to cognitive function (34). Interestingly, 19



279

Christensenellaceae increased in relative abundance and prevalence in the gut

280

microbiota of centenarians and is considered an important flora to maintain the health

281

of the elderly (35). These results indicate that the gut microbiome is closely related to

282

cognition and inflammation.

283

However, the underlying mechanisms of baicalein regulating intestinal

284

microbiota remain elusive. Studies have shown host's immune system plays an

285

important role in determining the composition of the gut microbiota (36). Niess et al.

286

found dendritic cells, a kind of mononuclear phagocyte of the intestinal lamina

287

propria, could form transepithelial dendrites depending on the chemokine receptor

288

CX3CR1, which enable the cells take up bacteria and drive downstream immune

289

responses in order to provide defense against pathogenic micro-organisms (37).

290

Several studies have revealed that baicalein could regulate immune-related pathways

291

including NF-κB and MAPK signaling pathways (38, 39). Meanwhile, our results

292

suggest that baicalein down-regulated the levels of cortical pro-inflammatory

293

cytokines. Therefore, we speculated that baicalein may affect intestinal flora by

294

regulating immunity, while the exact mechanism warrant further exploration.

295

The above results indicate that baicalein has a beneficial effect on SAMP8 mice.

296

Unfortunately, its low solubility causes baicalein to have a low oral bioavailability.

297

Nevertheless, some studies have shown that baicalein is well absorbed in the small

298

intestine but that during this process, it is subjected to extensive glucuronidation and

299

sulfation metabolism, which may be the reason for its low oral bioavailability (40).

300

Moreover, it has been shown that a higher loading dose of baicalein contributed to a 20


Page 20 of 40

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301

reduction in metabolism in the intestine during preliminary metabolic studies (41).

302

Recently, some studies have found that baicalein-loaded nanoemulsions or

303

nanocrystals could ameliorate the oral bioavailability of baicalein (42, 43). In addition,

304

Tsai et al. showed that baicalein is capable of penetrating the blood-brain barrier (44).

305

These results revealed that baicalein is a potential compound for improving

306

aging-related learning and memory impairment.

307

In summary, using the SAMP8 model, we have demonstrated that baicalein

308

treatment (i) lowered the grading score of senescence and improved behavior and

309

cognitive functions as evaluated by spatial learning and memory, novel object

310

recognition, and olfactory memory; (ii) ameliorated cortical pro-inflammatory

311

cytokines by reducing the cortical concentrations of pro-inflammatory cytokines such

312

as IL-1β, IL-6, TNF-α; and (iii) shifted the intestinal microbiome profile towards

313

antiaging. Nevertheless, the intricate relationship between the gut microbiome and

314

aging-related learning and memory decline needs to be further investigated.

315

Methods

316

Chemicals and kits

317

Baicalein (purity 98%) was purchased from Jingzhu Biotechnology Co., Ltd. (Nanjing,

318

China). Ethyl valerate (purity 99.5%) and amyl acetate (purity 99.5%) were

319

acquired from Guangfu Fine Chemical Research Institute (Tianjin, China). Paraffin

320

liquid was purchased from Dengfeng Chemical Co., Ltd. (Tianjin, China). The ELISA

321

kits (IL-6, IL-1β and TNF-α) were obtained from Beijing Andy Huatai Technology

322

Co., Ltd. (Beijing, China). The Fast DNA SPIN extraction kits were obtained from 21



323

MP Biomedicals (Santa Ana, CA, USA); Agencourt AMPure Beads were

324

purchased from Beckman Coulter, Inc. (Indianapolis, IN); and the PicoGreen dsDNA

325

Assay Kit was obtained from Invitrogen (Carlsbad, CA, USA).

326

Animals and drug administration

327

Eight-month-old male SAMP8 and SAMR1 mice were provided by the First Teaching

328

Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China). Mice

329

were housed in single cages and allowed unrestricted access to food and water on a

330

light/dark (1:1) cycle under controlled conditions (22 ± 2‐, 50 ± 10% humidity). The

331

mice were acclimated to the new environment for 7 days prior to the commencement

332

of the experiment.

333

Baicalein was dissolved in saline solution-0.9% NaCl to form a suspension, and

334

it was used after ultrasonic treatment. The dose of baicalein (200 mg/kg/d) was

335

determined based on the previous experiments by Duan et al. (11) and the work of

336

Jeong et al. (45). SAMP8 mice were split into two groups to be intragastrically

337

administered baicalein (200 mg/kg/d, n = 14, SAMP8 + baicalein group) or

338

intragastrically administered saline solution-0.9% NaCl (n = 13, SAMP8 group). The

339

SAMR1 mice were used as a control group and were intragastrically administered

340

saline solution-0.9% NaCl (n = 15, SAMR1 group). After administration of baicalein

341

for 6 weeks, the behavioral experiments were performed. Following the behavioral

342

experiments, their fresh stool was collected. After 8 weeks of administration, the mice

343

were sacrificed by cervical dislocation. The brain cortex was collected and stored at

344

-80‐ until processing. All experimental procedures complied with institutional animal 22


Page 22 of 40

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345

care and use committee (IACUC)-approved animal protocols that were based on the

346

Guide for the Care and Use of Laboratory Animals of the National Institutes of

347

Health.

348

The grading score of senescence

349

The grading score of senescence, developed by Prof. Takeda Toshio of Kyoto

350

University (46), has been widely used in aging-related studies to assess the

351

senescence status of the senescence-accelerated mouse (47, 48). In this evaluation

352

system, 11 aging-related indicators (reactivity, passivity, glossiness, coarseness, hair

353

loss, ulcer, periophthalmic lesions, cataract, corneal ulcer, corneal opacity and

354

lordokyphosis) were observed (49). According to the detailed criteria, the degree of

355

senescence in each category was graded from 0 to 4, where grade 0 indicated no

356

apparent changes and grade 4 indicated severe changes. For each mouse, the total

357

score was calculated.

358

Morris water maze test

359

The Morris water maze (MWM) test is a behavioral experiment applied to assess

360

spatial learning and memory abilities (50). The MWM test was conducted according

361

to previous reports by Chun et al. (51) and Wang et al. (52). The behavioral task

362

consists of two phases: acquisition (spatial learning) and retention (spatial memory).

363

In the acquisition phase (5 sequential days), each animal was allowed 4 daily trials.

364

When the mice climbed the platform, they had to remain on it for 10 s. If the mouse

365

failed to climb the platform within 60 s, it would be placed on the platform for 10 s. In

366

the retention phase, mice were allowed to swim to search for the platform for 60 s, 23



367

during which the platform was removed. The escape latencies, time in the target

368

quadrant and times of passing through the removed platform in the spatial probe test

369

were recorded and analyzed.

370

Novel object recognition test

371

The novel object recognition test (NORT) is a behavioral experiment that examines

372

memory ability based on differences between familiar and new objects, and this

373

method is comparable to the memory test used for humans (53). The NORT was

374

conducted according to previous reports by Wang et al. (52) and Chun et al. (51). The

375

procedure was divided into three phases: habituation, acquisition, and retention. In the

376

habituation phase (three days), the animal was allowed to explore freely in the empty

377

box for 10 minutes to familiarize with the test environment. In the acquisition phase

378

(the fourth day), the mice were permitted a single 10 minute exploration session,

379

during which two stably identical objects (A and B) were placed in a symmetrical

380

position in the center of the box with enough space between them and the walls. In the

381

retention phase (the fifth day), mice were subjected to a single 4 minute exploration

382

session. In the course of this session, the mice were placed in the box, and one of the

383

two identical objects was switched for a new one with a different color and shape

384

(novel object C). The exploration time for objects A and C was recorded in the

385

acquisition and retention phases. The recognition index for each mouse was expressed

386

as a ratio of the amount of time spent exploring the novel object C, (Tc-Ta)/(Tc+Ta),

387

where Ta and Tc were the time exploring objects A and C, respectively, during the

388

retention phase. 24


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389

Olfactory memory test

390

The olfactory memory deficits of mice were detected using an odor cross-habituation

391

test (54) with slight modifications. The olfactory memory test was conducted referring

392

to previous reports by Yang & Crawley (55) and Cramer et al. (56). The experiment

393

was divided into the adaptation period (1st day) and the detection period (2nd day).

394

Three 50 ml centrifuge tubes were taken, and a drill was used evenly on the edge of

395

the lid. Then, cotton balls dipped in paraffin liquid, ethyl valerate, and amyl acetate

396

were placed into the centrifuge tubes. An empty mouse cage was used as the

397

experimental site and was kept clean. During the adaptation period, a centrifuge tube

398

containing liquid paraffin was fixed in the center of the cage and mice were allowed a

399

single 10 minute exploration session to familiarize with the test environment and

400

eliminate the visual impact of the centrifuge tube. During the test period, a centrifuge

401

tube containing paraffin liquid was placed in the center of the cage first, and the mice

402

were allowed to explore freely for 30 s, and then the centrifuge tube was removed.

403

After an interval of 30 s, the centrifuge tube was put back in its original position. Each

404

odor (ethyl valerate and amyl acetate) was delivered for 4 successive trials in this

405

manner. The sniffing time was defined as when the animal was orienting towards the

406

centrifuge tube with its nose being 1 cm or closer to the tube, and time spent biting or

407

climbing the centrifuge tubes was excluded.

408

Assay of pro-inflammatory cytokines

409

The brain cortex was homogenized in a volume of 9 times ice-cold, 0.1 M phosphate

410

buffer saline (pH 7.4). The supernatant was used to detect the levels of IL-6, IL-1β 25



Page 26 of 40

411

and TNF-α using ELISA kits. The ELISA was performed according to the

412

manufacturer’s instructions.

413

Intestinal microbial community analysis

414

All fecal samples (180 ~ 200 mg) were quickly collected after free defecation in mice

415

(n = 8 per group). Total DNA was extracted using the Fast DNA SPIN extraction kits

416

according to the manufacturer’s instructions. The primers that were used for PCR

417

amplification of the bacterial 16S rRNA genes V3–V4 region were forward primer

418

5’-ACTCCTACGGGAGGCAGCA-3’

419

5’-GGACTACHVGGGTWTCTAAT-3’, which were inserted in sample-specific 7-bp

420

barcodes for multiplex sequencing. The PCR was performed in a volume of 25 µL

421

consisting of Q5 reaction buffer (5 µL), Q5 High-Fidelity GC buffer (5 µL), Q5

422

High-Fidelity DNA Polymerase (0.25 µL), dNTPs (2 µL), primers (1 µL), cDNA

423

template (2 µL), and ddH2O (8.75 µL). Thermal cycler conditions consisted of initial

424

denaturation for 2 minutes at 98 °C, followed by 25 cycles of amplification (15 s at

425

98 °C, 30 s at 55 °C, 30 s at 72 °C), with a final incubation of 5 minutes at 72 °C.

426

After purification using the Agencourt AMPure Beads and quantification using the

427

PicoGreen dsDNA Assay Kit, PCR amplicons were mixed in equal amounts, and

428

2×300-bp pair-end sequencing was conducted on the Illumina MiSeq platform with

429

MiSeq Reagent Kit v3.

and

the

reverse

primer

430

The Quantitative Insights Into Microbial Ecology (QIIME, v1.8.0) pipeline was

431

employed to eliminate failed sequences (57), and FLASH was used to assemble

432

paired-end reads (58) as previously described. The rest of the sequences were 26


Page 27 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


433

clustered into OTUs at 97% sequence identity by UCLUST following chimera

434

detection. OTU taxonomic classification was carried out by BLAST searching (59)

435

for representative sequences set against the Greengenes Database (60) using the best

436

hit.

437

Sequence data analyses were processed primarily for QIIME and R packages

438

(v3.2.0). OUT-level alpha diversity indices that were mainly used to investigate the

439

species diversity within the community such as Rarefaction curves, Chao1 richness

440

estimator, ACE metric (Abundance-based Coverage Estimator), Simpson index, and

441

Shannon diversity index were calculated in QIIME. The rarefaction curve is used to

442

determine whether the current sequencing depth is sufficient to reflect the diversity of

443

microorganisms contained in each sample. Chao1 and ACE are used to estimate the

444

number of species actually present in the community, and the larger the value is, the

445

higher the abundance of the community. Simpson and Shannon are used to estimate

446

the diversity of the community, and the higher the value is, the more diverse the

447

community. Beta diversity analysis was performed to examine the similarity of the

448

community structure between different samples using UniFrac distance metrics (61,

449

62) and visualized via PCoA (63). The taxonomy compositions and abundances were

450

visualized using MEGAN (64) and GraPhlAn (65). Intestinal microbial community

451

analysis was performed by Shanghai Personal Biotechnology Co., Ltd. (Shanghai,

452

China).

453

Statistical analysis

454

All data are expressed as the mean ± SEM. Repeated measures and multivariate 27



455

analysis of variance (ANOVA) in SPSS 16.0 (SPSS Software, Inc., Chicago, IL) were

456

used for analysis of the data from the place navigation test. Other comparisons

457

between groups were performed using an unpaired two-tailed Student’s t-test by

458

GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). P < 0.05 was

459

considered statistically significant. Correlations were performed by one-tailed

460

Spearman’s analysis with 95% confidence intervals.

461

Supporting Information

462

Morris water maze before administration with baicalein in 8-month-old mice,

463

OUT-level alpha diversity indices, and the results of OTU classification and

464

taxonomic status identification.

465

Abbreviations

466

ACE: Abundance-based Coverage Estimator; Aβ: amyloid-β; AD: Alzheimer’s;

467

CNS: central nervous system; IACUC: institutional animal care and use committee;

468

IL-6: Interleukin-6; IL-1β: interleukin-1 beta; TNF-α: tumor necrosis factor-α; LTP:

469

long-term potentiation; MWM: Morris water maze; NORT: novel object recognition

470

test; OUT: operational taxonomic unit; PCoA: principal coordinate analysis; SAMP8:

471

senescence-accelerated mouse prone 8; SAMR1: senescence accelerated mouse

472

resistant 1.

473

Author Information

474

Corresponding authors:

475

Li Gao, Modern Research Center for Traditional Chinese Medicine, Shanxi University,

476

No.92 Wu Cheng Road, Taiyuan 030006 China. E-mail: [email protected]. Tel & 28


Page 28 of 40

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477

Fax: 86-351-7018379.

478

Xuemei Qin, Modern Research Center for Traditional Chinese Medicine, Shanxi

479

University,

480

[email protected]. Tel & Fax: 86-351-7011501.

481

Author Contributions

482

LG, conception and design, interpretation of data, writing, revising and final approval

483

of the manuscript submitted. JL, performed the experiments and drafting of the

484

manuscript. XH, revising of the manuscript. YZ, technical or material support; study

485

supervision. XQ and GD, design of the study and writing the protocol.

486

Funding Sources

487

This work is supported by the National Natural Science Foundation of China

488

(81603319), Programs of Science and Technology and Higher Education of Shanxi

489

Province (2015118), Science and Technology Innovation Team of Shanxi Province

490

(201605D131045-18), and Key laboratory of Effective Substances Research and

491

Utilization in TCM of Shanxi province (201705D111008-21).

492

Conflict of Interest

493

The authors declare that thy have no competing interests.

494

Acknowledgements

495

Not applicable.

No.92

Wu

Cheng

Road,

Taiyuan

29


030006

China.

E-mail:


Page 30 of 40

496

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The effects of baicalein on cortical pro-inflammatory cytokines and

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the intestinal microbiome in senescence accelerated mouse prone 8

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Li Gao†1*, Jiaqi Li†1,2, Yuzhi Zhou1, Xudong Huang3, Xuemei Qin1*, Guanhua Du1,4

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Effects of Baicalein on Cortical Proinflammatory Cytokines and the

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