Flavonoids from Scutellaria baicalensis inhibit senescence-associated secretory phenotype production by interrupting IκBζ/C/EBPβ pathway: Inhibition of age-related inflammation

Article abstract of DOI:10.1016/j.phymed.2020.153255

Background: Prolonged exposure to the senescence-associated secretory phenotype (SASP) with age leads to chronic low-grade inflammation in neighboring cells and tissues, causing many chronic degenerative diseases. Purpose: The effects on SASP production of the ethanol extract from Scutellaria radix and 17 isolated flavonoid constituents were examined in vitro and in vivo. Methods: Cellular senescence was induced by bleomycin. Expression of the SASP and cell signaling molecules was detected using ELISA, RT-qPCR, Western blotting, and immunofluorescence staining. To investigate the in vivo effects, 21-month-old aged rats were used. Results: The ethanol extract and 5 compounds including 1 (Oroxylin A; 5,7-dihydroxy-6-methoxyflavone), 5 (2′,6′,5,7-tetrahydroxy-8-methoxyflavone), 8 (2′,5,7-trihydroxyflavone), 10 (2′,5,7-trihydroxy-8-methoxyflavone) and 11 (2′,5,7-trihydroxy-6-methoxyflavone) potently reduced IL-6 and IL-8 production and gene expression of the SASP, including IL-1α, IL-1β, IL-6, IL-8, GM-CSF, CXCL1, MCP-2, and MMP-3. This finding indicates the important role of the B-ring 2′?hydroxyl group in flavonoid molecules. Furthermore, compounds 8 and 11, the strongest SASP inhibitors, decreased the expression of IκBζ and C/EBPβ protein without affecting either BrdU uptake or the expression of senescence markers, such as pRb and p21. Finally, the oral administration of compound 8 to aged rats at 2 and 4 mg/kg/day for 10 days significantly inhibited the gene expression of SASP and IκBζ in kidneys. This is the first report of the strong SASP inhibitory action of flavonoids from Scutellaria radix on in vitro and in vivo senescence models. The inhibitory action was shown to be mediated mainly by interfering with the IκBζ/C/EBPβ signaling pathway. Conclusion: Targeting production of the SASP using flavonoids from Scutellaria radix or its extract might help reduce low-grade sterile inflammation and control age-related diseases.

Full text of DOI:10.1016/j.phymed.2020.153255

Journal Pre-proof
Flavonoids from Scutellaria baicalensis inhibit
senescence-associated secretory phenotype production by
interrupting IκBζ/C/EBPβ pathway: Inhibition of age-related
inflammation
Hyun Lim , Yong Soo Kwon , Donghoon Kim , Jongkook Lee ,
Hyun Pyo Kim
PII:
S0944-7113(20)30086-6
DOI:
Reference:
https://doi.org/10.1016/j.phymed.2020.153255
PHYMED 153255
To appear in:
Phytomedicine
Received date:
Revised date:
Accepted date:
23 December 2019
6 May 2020
23 May 2020
Please cite this article as: Hyun Lim , Yong Soo Kwon , Donghoon Kim , Jongkook Lee ,
Hyun Pyo Kim , Flavonoids from Scutellaria baicalensis inhibit senescence-associated secretory phe-
notype production by interrupting IκBζ/C/EBPβ pathway: Inhibition of age-related inflammation, Phy-
tomedicine (2020), doi: https://doi.org/10.1016/j.phymed.2020.153255
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier GmbH.

1
2
Flavonoids from Scutellaria baicalensis inhibit senescence-associated secretory
phenotype production by interrupting IκBζ/C/EBPβ pathway: Inhibition of age-
related inflammation
3
4
5
6
Hyun Lim, Yong Soo Kwon, Donghoon Kim, Jongkook Lee, and Hyun Pyo Kim*
7
College of Pharmacy, Kangwon National University, Chuncheon 24341, Republic of Korea
8
9
10
11
12
13
14
*Corresponding author
Hyun Pyo Kim, College of Pharmacy, Kangwon National University, 1, Gangwondaehak-gil,
Chuncheon 24341, Republic of Korea,
Tel.: +82 33 250 6915, Fax.: +82 33 259 5631
E-mail address: hpkim@kangwon.ac.kr (H.P. Kim).
15
16
17
Word count: 7,072
1

18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
ABSTRACT
Background: Prolonged exposure to the senescence-associated secretory phenotype (SASP)
with age leads to chronic low-grade inflammation in neighboring cells and tissues, causing
many chronic degenerative diseases.
Purpose: The effects on SASP production of the ethanol extract from Scutellaria radix and 17
isolated flavonoid constituents were examined in vitro and in vivo.
Methods: Cellular senescence was induced by bleomycin. Expression of the SASP and cell
signaling molecules was detected using ELISA, RT-qPCR, Western blotting, and
immunofluorescence staining. To investigate the in vivo effects, 21-month-old aged rats were
used.
Results: The ethanol extract and 5 compounds including 1 (Oroxylin A; 5,7-dihydroxy-6-
methoxyflavone), 5 (2‘,6’,5,7-tetrahydroxy-8-methoxyflavone), 8 (2‘,5,7-trihydroxyflavone),
10 (2’,5,7-trihydroxy-8-methoxyflavone) and 11 (2‘,5,7-trihydroxy-6-methoxyflavone)
potently reduced IL-6 and IL-8 production and gene expression of the SASP, including IL-1α,
IL-1β, IL-6, IL-8, GM-CSF, CXCL1, MCP-2, and MMP-3. This finding indicates the
important role of the B-ring 2'-hydroxyl group in flavonoid molecules. Furthermore,
compounds 8 and 11, the strongest SASP inhibitors, decreased the expression of IκBζ and
C/EBPβ protein without affecting either BrdU uptake or the expression of senescence
markers, such as pRb and p21. Finally, the oral administration of compound 8 to aged rats at
2 and 4 mg/kg/day for 10 days significantly inhibited the gene expression of SASP and IκBζ
in kidneys. This is the first report of the strong SASP inhibitory action of flavonoids from
Scutellaria radix on in vitro and in vivo senescence models. The inhibitory action was shown
2

41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
to be mediated mainly by interfering with the IκBζ/C/EBPβ signaling pathway.
Conclusion: Targeting production of the SASP using flavonoids from Scutellaria radix or its
extract might help reduce low-grade sterile inflammation and control age-related diseases.
Keywords: Scutellaria baicalensis; Senescence; SASP; IκBζ; C/EBPβ; Chronic low-grade
inflammation
Abbreviations:
BrdU, 5-bromo-2’-deoxy-uridine; C/EBPβ, CCAAT/enhancer-binding protein beta; CXCL1,
chemokine (C-X-C motif) ligand 1; ELISA, enzyme-linked immunosorbent assay; GM-CSF,
granulocyte-macrophage colony-stimulating factor; IL, interleukin; IRAK1, interleukin-1
receptor-associated kinase; MCP-2, monocyte chemoattractant protein-2; MMP-3, matrix
metalloproteinase-3; RT-qPCR, reverse transcription-quantitative polymerase chain reaction
3

56
57
1. Introduction
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
The identification of anti-aging agents that extend the lifespan of organisms is not a
simple task. The compounds such as resveratrol, curcumin, and rapamycin have been shown
to prolong lifespan of fruit flies Drosophila melanogaster and Caenorhabditis elegans (Bass
et al., 2007; Bjedov et al., 2010; Lee et al., 2010). Recently, potential targets such as senolytic
activity of fisetin, microbiome modulation of rapamycin, and proteostatic maintenance of
rapamycin with or without metformin have been demonstrated to be associated with lifespan
extension in mice (Bitto et al., 2016; Reid et al., 2020; Yousefzadeh et al., 2018). However,
evidence demonstrating the extension of the human lifespan is lacking. Agents that inhibit
cellular senescence may have some deleterious effects, possibly resulting in cancer cell
formation, because cellular senescence is one way to eliminate potential cancer-transforming
cells. Instead, eliminating the harmful effects of cellular senescence is an option for healthy
aging. As organisms age, cells can become damaged or telomerase shorten that triggers
activation of a signaling cascade that results in cell senescence, a permanent cell cycle arrest.
The senescent cells express molecules known as senescence-associated secretory phenotype
(SASP) during this process (Coppé et al., 2008; 2010). Although the SASP, consisting of the
proinflammatory cytokines, proteases, and growth factors, has some beneficial effects, such
as normal development and tissue remodeling (Coppé et al., 2010; Storer et al., 2013),
prolonged exposure to SASP factors can cause chronic low-grade sterile inflammation during
aging, also known as inflammaging, which facilitates aging and transformation of the
surrounding cells, leading to age-related chronic inflammatory diseases, such as type 2
diabetes, cardiovascular diseases, neurodegenerative diseases, and cancer (Campisi et al.,
4

79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
2011; Kumar et al., 2014; Onat and Can 2014; Singh and Newman 2011). In this regard,
finding SASP-inhibiting agents without affecting cellular senescence is a current issue in the
field of healthy aging.
Recently, several natural and synthetic flavonoids were shown for the first time to inhibit
SASP production without affecting the senescence process itself. The optimum chemical
structures are found to be basic flavone structures having hydroxyl and/or methoxyl
substitutions on C-5,7,2’,3’ and/or 4’ (Lim et al., 2015). The inhibitory activity was shown to
be associated with the activation of transcription factors, such as nuclear factor-kappa B (NF-
κB) and IkappaB zeta (IκBζ) (Lim et al., 2015). Moreover, one of the strong inhibitors,
apigenin, showed a similar response in aged-rats by the oral administration of doses as low as
2 – 4 mg/kg/day (Lim et al., 2015). The similar suppressive effect of apigenin on expression
of the SASP was also confirmed in fibroblasts and breast cancer cells (Perrott et al., 2017).
During our preliminary experiment to screen various herbal products, the alcohol extract of
the radix of Scutellaria baicalensis Georgi strongly inhibited SASP production. It is well-
known that the chemical structures of the main flavonoids in the radix of S. baicalensis are
flavones with hydroxy/methoxyl substitution(s) at C-5,6,7 or 5,7,8 in the A-ring and C-2’
and/or 6’ in the B-ring (Liu et al., 2009). However, the effects of these flavonoids on SASP
production are not known. Based on the potential importance of this SASP inhibitor, we
isolated 17 flavonoids from S. baicalensis and their inhibitory action against SASP
production, as well as the mechanisms of action, were evaluated in the present investigation.
2. Materials and methods
5

102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
2.1. Chemicals and animals
Bleomycin and apigenin were purchased from Enzo Life Sciences (Farmingdale, NY,
USA). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM) were
bought form MP biomedicals (Santa Ana, CA, USA) and Hyclone Laboratories (South Logan,
UT, USA). Phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, sodium fluoride,
protease inhibitor cocktail and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Human IL-6 and IL-8
ELISA kits were bought from BD Biosciences (San Jose, CA, USA). The origins and grades
of other chemicals, primers and antibodies were described in detail in supplementary file.
Male Sprague Dawley (SD) rats (5-month-old young and 21-month-old) were purchased
from Samtako (Osan, Korea). The animals were maintained in the animal facility at 20 - 22oC
under 40 - 60% relative humidity and a 12 h light/dark cycle for at least 7 days prior to the
experiment. The experimental design using the animals was approved by the local committee
for animal experimentation, KNU (KW-190927-3). In addition, the ethical guidelines
described in the Korean Food and Drug Administration guide for the care and use of
laboratory animals was followed throughout the experiments.
2.2. Preparation of 80% ethanol extract from Scutellaria radix and isolation of flavonoid
constituents
The dried radix of Scutellaria baicalensis was purchased in local herbal market and
taxonomically identified by one of the authors (Dr. Y. S. Kwon). A voucher specimen
6

125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
(KNUH-R-15-1) was deposited at the Herbarium of the College of Pharmacy (KNU, Korea).
The dried radix of S. baicalensis (1 kg) was refluxed in 80% ethanol three times and filtered.
The evaporation gave the dried ethanol extract (2.8 kg) and the extract was used for the assay.
Part of the ethanol extract (2.7 kg) was suspended in water and fractioned with n-hexane,
ethylacetate and n-butanol, successfully. The evaporation of each fraction under reduced
pressure provided n-hexane (38.7 g), ethylacetate (84.4 g) and n-butanol (106.0 g) fractions.
The flavonoid constituents were isolated with repetitive column chromatography. Briefly,
ethylacetate fraction (80.0 g) was separated in silica gel chromatography using
chloroform:methanol mixture as mobile phase, yielding fractions (S-1 to S-6). Fraction S-1
(12.9 g) was separated with Buchi MPLC, Rdisep silica gel, ODS and Sephadex LH20
column chromatography, yielding compound 1 (70 mg), 2 (110 mg) and 3 (160 mg). Using
similar column chromatography, fraction S-2 gave compounds 4 (65 mg), 5 (5 mg), 6 (20
mg), 7 (7 mg), 8 (6 mg), 9 (4.2 mg), 10 (4 mg) and 11 (2.4 mg). From S-3 (19 g), consecutive
silica gel and ODS column chromatography produced compounds 12 (350 mg) and 13 (1.3 g).
Fraction S-4 (11 g) was separated successfully with silica gel flash column and ODS column
chromatography, yielding compounds 14 (365 mg) and 15 (2.3 g). From n-butanol fraction
(30 g), repetitive silica gel column chromatography yielded compound 16 (40 mg) and
compound 17 (1.98 g). All the chemical structures of the isolated compounds are shown in
Fig. 1A and confirmed by the comparison of the previously reported data. The detailed
separation procedures and instrumental data are represented in supplementary file. The
isolated compounds showed one spot in two-dimensional thin-layer chromatography and the
purities (> 95%) were determined by Thermo Dynamic HPLC system using a intersil column
(ODS-2, 150 x 4.5 mm) with acetonitrile:water (4:6) as a mobile phase.
7

148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
For in vivo study, compound 8 was chemically synthesized. 2’,5,7-Trihydroxyflavone (8)
was synthesized from commercially available 1[2,4-bis(benzyloxy)-6-
hydroxyphenyl]ethanone via a aldol condensation-oxidative cyclization-debenzylation
sequence as shown in Fig. 1B.
2.3. Bleomycin-induced cellular senescence and SASP production
BJ cells (human foreskin fibroblast) purchased from the American Type Culture
Collection (ATCC, Manassas, VA) were maintained in DMEM containing 10% FBS,
glutamine, and penicillin/streptomycin. Damage-induced cellular senescence was produced
by treating BJ cells with bleomycin according to a protocol in previous reports (Lim et al.,
2015; Robles and Adami, 1998). In brief, the cells were treated with bleomycin (50 μg/ml)
for 24 h and washed twice with phosphate-buffered saline (PBS), followed by further
incubation for 6 days. Completion of cellular senescence was determined by the ratio of
incorporated BrdU into cellular DNA using the BrdU labeling and detection kit using anti-
BrdU antibody. Percentages of BrdU-positive cells of 10% or less indicate that the cellular
senescence is complete (Laberge et al., 2012).
To identify the effect of the test compounds after the completion of senescence, the cells
were treated with test compounds for the last 24 h after the completion of senescence by
bleomycin. To examine simultaneous effects, the cells were treated with the test compounds
with bleomycin (50 μg/ml) for 24 h at the initial time of senescence induction and incubated
for 6 days with the test compounds. On the 6th day, the IL-6 and/or IL-8 (representative
markers of SASP molecules) production in the media was quantified with an ELISA kit. The
8

171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
gene expression of the SASP (IL-6, IL-8, IL-1α, IL-1β, GM-CSF, CXCL1, MCP-2, and
MMP-3) was evaluated using RT-qPCR with specific primers on an C1000 Touch Thermal
Cycler (Biorad Lab., Hercules, CA, USA) according to protocols described in a previous
report (Lim et al., 2015). The primer sequences for the human genes are shown in Table 1 of
the supplementary file. Senescence-associated-β-galactosidase (SA-β-gal) activity was
determined by counting the number of blue-stained cells versus the number of total cells
using senescence cells histochemical staining kit (Sigma Aldrich, St. Louis, MO, USA). Cells
are considered to be senescent if >80% of cells are stained in blue (Itahana et al., 2013).
Cellular viability was examined using the MTT assay (Mosmann, 1983) and all tested
compounds were used at non-cytotoxic concentrations.
2.4. Cellular mechanisms associated with SASP production
To investigate the cellular mechanisms related to the inhibitory effects on SASP
production, compound 8 and 11 were selected due to their strong SASP inhibitory activity
among the 17 compounds and the mechanism study was carried out by simultaneous
treatment with bleomycin. On the 6th day, the total proteins in the cell lysates were extracted
with Proprep solution (iNtRON Biotechnology, Korea). All Western blot procedures,
including the preparation of nuclear protein, were essentially the same as previously
described (Lim et al., 2015). Each protein band on the blots was visualized using an enhanced
chemiluminescence (ECL) system (GE Healthcare, UK) with ImageQuant LAS4000 mini
and analyzed using Image J (NIH, USA). ). Images of the cells immunostained with anti-IκBζ
or CCAAT/enhancer-binding protein beta (C/EBPβ) antibody on cells were obtained using a
9

194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
super-sensitive high resolution confocal laser scanning microscope (SR-CLSM) (Carl Zeiss,
Oberkochen, Germany). The detailed procedures are found in the supplementary file.
2.5. siRNA transfection against C/EBPβ
siRNA against C/EBPβ and non-targeting siRNA were prepared in 1 × siRNA buffer (60
mM KCl, 6 mM HEPES and 0.2 mM MgCl2) as suggested in the manufacturer’s protocol
(Dharmacon Inc, Lafayette, CO, USA). siRNA against C/EBPβ or non-targeting siRNA and
DharmaFECT reagent were mixed and incubated for 20 min. To increase the transfection
efficiency, each siRNA (25 nM) was pre-transfected for 24 h, washed out with PBS twice,
and re-transfected with bleomycin (50 μg/ml) for 24 h. The gene expressions of C/EBPβ,
IκBζ, p21, and the SASP were detected by RT-qPCR.
2.6. SASP production in aged rats
Compound 8 was dissolved in 0.3% carboxymethyl cellulose (CMC) and orally
administered to 21-month-old aged rats at 2 and 4 mg/kg/day once daily for 10 consecutive
days. For the control group, only 0.3% CMC was administered to 5- and 21-month-old rats
for 10 days. Five-month-old rats were used as young controls for comparison with 21-month-
old aged rats. After 10 days, the rats were euthanized under CO2. Organs, including the
kidneys, liver, and lung were excised and blood was collected. To measure the expression of
the SASP genes in the kidneys, kidney tissue (50 mg) was homogenized in 1 ml of Trizol
reagent (Invitrogen, Carlsbad, CA, USA) at 50 Hz for 2 min using TissueLyser LT (Qiagen,
10

217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
Hilden, Germany). Total RNA was extracted with chloroform and purified with a NucleoSpin
Clean-up kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). RT-qPCR was
performed by the same method used in the BJ cells above. The primer sequences and
manufacturer's information are shown in Table 1 of the supplementary file.
2.7. Statistics
Experimental values were represented as arithmetic mean  SD. One way analysis of
variance (ANOVA) followed by Dunnett’s post hoc test was used to determine the statistical
significance (IBM SPSS Statistics, version 24).
3. Results
3.1. Effects of the ethanol extract of Scutellaria radix on cellular senescence and SASP
production
Bleomycin treatment provoked cellular senescence with SASP production. Treatment
with the extract after senescence inhibited SASP production (IL-6 and IL-8, 59.5% and
41.5%, respectively, at 100 μg/ml), whereas the level of BrdU uptake was not changed (Fig.
2A). When the expression levels of the SASP genes (IL-6 and IL-8), IκBζ and p21
(senescence marker) were examined using RT-PCR, the extract significantly inhibited the
expression of IL-6 and IL-8 (55.5% and 24.3% at 100 μg/ml), and IκBζ expression was
slightly decreased by 18.3% at 100 μg/ml, although not significant. However, the mRNA
11

240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
level of p21 was not changed (Fig. 2B).
When the extract (5, 10, and 20 μg/ml) was simultaneously treated with bleomycin from
the initial point of senescence induction, the expression of IL-6 and IL-8 in the media was
inhibited by 80.1% and 80.3%, respectively, at 20 μg/ml (Fig. 2C). The gene expression of
IL-6, IL-8 and IκBζ was strongly inhibited by 86.3%, 85.1%, and 58.8%, respectively, at 20
μg/ml (Fig. 2D). The ratio of BrdU uptake and the p21 gene level were not changed by
treatment with the extract (Fig. 2C and 2D). These results showed that the ethanol extract of
Scutellaria radix had strong SASP inhibitory activity without affecting cellular senescence.
3.2. Effects of flavonoid constituents isolated from the extract of Scutellaria radix on SASP
production
To identify the specific constituents responsible for SASP inhibitory activity in the extract
of Scutellaria radix, 17 flavonoid derivatives were isolated from the extract (Fig. 1A). The
effects of all flavonoids isolated on IL-6 production were examined at 10 μM, a
pharmacologically-relevant concentration (Rodriguez-Mateos et al., 2014). When treated
after senescence completion, only compound 5 (5,7,2’,6’-tetrahydroxy-8-methoxyflavone)
significantly inhibited IL-6 production (Fig. 3A). However, when treated concomitantly with
bleomycin from the initial point of senescence induction, eight compounds (1, 2, 5, 8, 9, 10,
11 and 16) significantly inhibited IL-6 production (Fig. 3B). Compound 1 (oroxylin A), 2
(wogonin), 5 (5,7,2’,6’-tetrahydroxy-8-methoxyflavone), 8 (2’,5,7-trihydroxyflavone), 9
(norwogonin),
10
(2’,5,7-trihydroxy-8-methoxyflavone),
11
(2’,5,7-trihydroxy-6-
methoxyflavone), and 16 (baicalin) inhibited IL-6 production by 107.5%, 69.9%, 73.0%,
12

263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
153.9%, 42.6%, 96.9%, 136.3%, and 41.6%, respectively, at 10 μM. Among them, five
compounds (compounds 1, 5, 8, 10 and 11) showing strong inhibitory activity were selected
to further examine the effects on IL-8 production and the ratio of incorporated BrdU into cells.
When treated once for the last 24 h after reaching senescence, the compounds slightly
reduced IL-8 production, although not significantly (Fig. 3C). On the other hand, in the case
of simultaneous treatment, compounds 1, 5, 8, 10, and 11 inhibited IL-8 production by 55.3%,
69.1%, 76.7%, 44.8% and 71.8%, respectively, at 10 μM (Fig. 3D). The ratio of BrdU uptake
into cells was not changed in either experiments (Fig. 3C and 3D). Thus, these results
indicate that certain flavonoid constituents were responsible for the strong SASP inhibitory
activity exerted by Scutellaria radix without affecting cellular senescence itself. Like the
extract, these flavonoids also showed more potent activity when simultaneously treated with
bleomycin.
3.3. Effects on gene expression of SASP molecules
Each compound (1, 5, 8, 10, and 11) was simultaneously treated with 50 μg/ml of
bleomycin. At 10 μM, all five compounds strongly suppressed the gene expression of SASP
molecules including IL-6, IL-8, IL-1α, IL-1β, GM-CSF, CXCL1, MCP-2, and MMP-3 (Fig.
4A). Among them, compounds 8 and 11 most strongly suppressed the gene expression of IL-6,
GM-CSF, IL-1α, IL-1β, and MCP-2 by more than 90% at 10 μM. Apigenin, a potent inhibitor
(Lim et al., 2015), also strongly inhibited the gene expression of all tested SASP molecules at
10 μM. Expression of the IκBζ gene showed a close relationship with the cellular signaling
pathway for SASP production and was also significantly decreased by these five compounds
13

286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
and apigenin (Fig. 4B). In contrast, none of the compounds changed the gene expression level
of p21, a senescence marker. Furthermore, compound 8 and 11 having potent activity on
SASP inhibition, did not affect SA-β-gal activity compared to bleomycin-treated positive
control (Fig. 4C). These results clearly showed that certain flavonoids from Scutellaria radix
inhibited the expression of SASP molecules and these inhibitory effects coincided with the
reduced expression of the IκBζ gene without changing the p21 gene expression and SA-β-gal
activity.
3.4. Cellular mechanisms related to SASP inhibition by compound 8 and 11
Compounds 8 and 11 were selected for further investigation of the cellular signaling
mechanism of SASP inhibition. Bleomycin treatment reduced the expression of IRAK1 and
IκBα protein and phosphorylation of signal transducer and activator of transcription 3
(STAT3) (Fig. 5A). Among them, the degradation of IRAK1 protein was blocked by 56.3%
and 32.0% by treatment with 10 μM 8 and 11 compounds, respectively. Also, the
phosphorylation level of STAT3 was slightly increased by 24.5 % following treatment with
compound 11 at 10 μM. The decreased expression of IκBα protein provoked by bleomycin
was not recovered by these compounds. Compounds 8 and 11 did not affect either the
degradation of pRb protein or the expression of p21 protein, senescence markers. Among the
transcription factors involved in SASP production, the elevated expression of IκBζ in the
nuclear fraction by bleomycin was decreased by 65.1% and 79.2% by treatment with
compounds 8 and 11, respectively, at 10 μM (Fig. 5B). Whereas the expression of C/EBPβ
protein was increased 3.9-fold in the nuclear fraction by bleomycin, compounds 8 and 11 at
14

309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
concentrations of 5 - 10 μM significantly inhibited the expression by more than 50%. As a
reference, apigenin also suppressed the expression of IκBζ and C/EBPβ. Reduction of the
expression of IκBζ and C/EBPβ protein by compounds 8 and 11 was also confirmed by
immunofluorescence staining using anti-IκBζ and C/EBPβ antibodies (Fig. 5C). These results
demonstrate that the inhibitory effect of compounds 8 and 11 on SASP production was
closely related to the inhibition of signaling pathways involved in IκBζ and C/EBPβ.
To further characterize the essential role of C/EBPβ, an siRNA transfection study was
designed, since the correlation between IκBζ expression and SASP production has been
shown in previous reports (Alexander et al., 2013; Lim et al., 2015). When siRNA against
C/EBPβ (25 nM) was treated with bleomycin (50 μg/ml) for 24 h, the expression of C/EBPβ
gene was successfully blocked compared to that of non-targeting siRNA-transfected cells, as
expected (Fig. 5D). The increased expression of several SASP genes by bleomycin was
significantly suppressed by transfection with C/EBPβ-targeting siRNA. The levels of IL-1α,
IL-1β, IL-6, IL-8, GM-CSF, CXCL1, MCP-2, and MMP-3 genes were inhibited by 41.8%,
50.5%, 49.1%, 47.5%, 38.9%, 56.8%, 69.5%, and 61.7%, respectively. Moreover, along with
the inhibition of SASP expression resulting from blocking the C/EBPβ gene, the IκBζ gene
level was inhibited by 32.4%, as well. However, the expression level of the p21 gene
(senescence marker) was not changed. All these results indicate that C/EBPβ expression was
essential to the signaling pathway for SASP production and IκBζ expression without affecting
senescence itself in the bleomycin-induced senescence model.
3.5. Effects of compound 8 on SASP production in aged rats
15

332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
To demonstrate the in vivo effect, compound 8 was orally administered to 21-month-old
aged rats for 10 days (2 and 4 mg/kg/day). After euthanasia, the expression of SASP genes in
the kidney was evaluated using RT-qPCR. The expression of IL-6, IL-1α, IL-1β, GM-CSF,
and CXCL1 genes in 21-month-old aged rats was significantly increased by 17.1, 20.7, 5.2,
6.7, and 8.2-fold, respectively, over those in 5-month-old rats used as young controls (Fig.
6A). The elevated expression of these SASP genes was reduced by oral treatment with
compound 8 for 10 days. In particular, IL-6 was strongly decreased by 47.1% and 49.6% at
the doses of 2 and 4 mg/kg/day, respectively. IL-1β expression was significantly inhibited by
42.9% at 4 mg/kg/day and CXCL1 was inhibited by 40.4% at 2 mg/kg/day. In parallel with
the increased SASP expression, the expression of IκBζ found as the transcription factor
associated with senescence, was also increased by 6.1-fold in the kidney of a 21-month-old
aged rat, whereas this increase was significantly reduced by 48.0% with the administration of
compound 8 at 2 mg/kg/day (Fig. 6B). The C/EBPβ gene was also slightly increased by 1.2-
fold in 21-month-old aged rats (P < 0.5). This increase in the C/EBPβ gene was down-
regulated to similar levels as those in 5-month-old aged rats at 4 mg/kg/day, although not
statistically significant. All these results showed that compound 8 was a strong SASP
inhibitor in the aged animal model. It also demonstrated that the IκBζ and C/EBPβ key
factors were downregulated by the oral administration of compound 8 in aged rats.
4. Discussion
The present study demonstrated that the ethanol extract of Scutellaria radix and flavonoid
16

355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
constituents possessed strong inhibitory activities against SASP production without affecting
cellular senescence in bleomycin-treated senescence cells. In particular, the study found that
flavone derivatives having a B-ring 2’-hydroxyl substitution among the constituents had an
enhanced inhibitory activity on SASP production. For the cellular action mechanism, the
present study strongly suggests that transcription factors, such as IκBζ and C/EBPβ, are
closely involved in SASP production and flavonoids possibly interrupt their expression.
Based on these results, the analysis of the chemical structures of 17 flavonoid derivatives
isolated from the extract of Scutellaria radix provided some structure-activity relationships
involved in SASP production, despite some limited structural diversities. Among the isolated
flavonoids, compounds 8, 10, and 11 showed strong inhibitory action. These three
compounds have B-ring 2’-hydroxyl substitution in common and showed higher inhibition
than flavonoids having the same chemical structures devoid of 2'-hydroxyl moiety
(compound 8 vs. chrysin, 10 vs. 2, 11 vs. 1). Chrysin did not show significant inhibitory
action under the same experimental conditions (Lim et al., 2015). Of note, methoxyl
substitution at C-6 in the A-ring maybe favorable (compound 1 vs. 12, baicalein, 5,6,7-
trihydroxyflavone). In our previous report, the results suggested that apigenin, kaempferol,
2’,3’-dihydroxyflavone, 3’,4’,7-trihydroxyflavone, and 2’,3’,5,7-tetrahydroxyflavone were
the flavonoid derivatives with optimum structures for SASP inhibition (Lim et al., 2015). In
the present study, the importance of a hydroxyl substitution at C-2’ in the B-ring and/or a
methoxy group substitution at C-6 in the A-ring was revealed.
Following the initial discovery of SASP inhibitory action of some flavonoids (Lim et al.,
2015), several studies demonstrated the SASP inhibitory action of certain flavonoid and
polyphenol derivatives. Quercetin was found to reduce SASP production under various
17

378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
treatment conditions alone or in combination with dasatinib in vitro and in vivo (Hickson et
al., 2019; Kim et al., 2019; Xu et al., 2018). Epigallocatechin gallate and resveratrol were
shown to suppress SASP production in 3T3-L1 preadipocytes and stromal senescent
fibroblasts, respectively (Kumar et al., 2019; Menicacci et al., 2017). In the present study,
another cluster of flavonoids with B-ring 2'-hydroxyl group was found to be strong SASP
inhibitors.
Thus far, the regulatory mechanisms of SASP production have been linked to NF-κB,
C/EBPβ, DNA damage response, and the p38 mitogen-activated protein kinase pathway
(Freund et al. 2011; Salminen et al. 2012). Mixed lineage linkage, GATA4 and reactive
oxygen species-protein kinase Cδ-protein kinase 1 cascade were also proposed to regulate
SASP expression (Capell et al., 2016; Kang et al., 2015; Wang et al. 2014). These signaling
molecules contribute to SASP production differently depending on the cell type and
senescence inducers.
The involvement of the IκBζ transcription factor was demonstrated in the signaling
pathway for IL-6 and IL-8 expression in DNA damage- and oncogene-induced senescence
(Alexander et al., 2013). IκBζ expression in the kidneys of aged rats was maintained at an
elevated level in 17- to 19-month-old aged rats and greatly increased in 21-month-old aged
rats (data not shown). Regarding the cellular mechanisms of action of the flavonoids, in our
previous report, the inhibitory action of flavonoid derivatives, including apigenin on the
SASP production, was shown to be mediated through the IκBζ and NF-κB pathways (Lim et
al., 2015). Compound 8 was also found to affect the expression of IκBζ in the present study.
Furthermore, in the present study, C/EBPβ expression was suggested to be associated with
the cellular mechanism of the SASP production, as well as IκBζ expression. C/EBPβ was
18

401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
previously reported to regulate IκBζ target genes by interacting with other transcription
factors, such as NF-κB and IκBζ (Matsuo et al. 2007). Using microarray analysis of SASP
genes by targeting against C/EBPβ, the expression of 127 SASP genes was reported to be
C/EBPβ-dependent (Flanagan et al., 2017). In our experimental results, blocking C/EBPβ by
siRNA transfection was found to significantly decrease the gene expression of SASP factors
(Fig. 5D). Furthermore, the IκBζ mRNA level was inhibited by treatment with C/EBPβ
siRNA. These results strongly support that IκBζ and C/EBPβ are involved in the signaling
pathway for SASP production in the DNA damage-induced senescence model and present an
opportunity for discovering key mediators targeting SASP production.
To treat age-associated disorders, besides the strategy targeting SASP production, the
removal of the senescent cells from the organisms seems to be a possible option. Since high-
level activation of the p16INK4a promoter is a feature of cellular senescence and coincided
with SASP production (Liu et al., 2019), the clearance of p16INK4a-positive cells in a mouse
model was shown to slow down age-related symptoms in organs, such as kidneys and heart
(Baker et al., 2016). Clearance of senescent cells using the p16-3MR transgenic mouse also
attenuated inflammatory markers in degenerative joints of the anterior cruciate ligament
transection mouse model (Jeon et al., 2017). However, some studies have reported with
conflicting results. Somatic inactivation of p16INK4a did not lower SASP production and did
not protect against aging-related osteoarthritis (Diekman et al., 2018). P16INK4a global
knockout did not attenuate the lung inflammation induced in a chronic obstructive pulmonary
disease mouse model (Sundar et al., 2018). Senescent cells have dual and opposite functions
in complex aging processes. Beneficial actions of senescent cells include tissue remodeling
by promoting wound healing and anti-cancer mechanisms by recruiting immune cells to
19

424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
eliminate damaged cells in normal tissues (Campisi, 2005; Storer et al., 2013). Therefore, the
approach targeting only SASP factors could be more safe and effective. In this regard, it is
important to mention that the properties of flavonoids may fit into a therapeutic role by
inhibiting SASP production without interfering with proliferative activity. Thus, SASP
inhibitory activity of the extract and certain constituents from the roots of S. baicalensis may
be good candidates with less toxicity to slow down age-associated chronic inflammation,
prolonging the healthy lifespan.
5. Conclusions
In conclusion, our experimental results demonstrate that the extract and certain flavonoids
from Scutellaria radix acted as strong SASP inhibitors in bleomycin-induced senescence in
BJ fibroblasts. Among the 17 flavonoids isolated from Scutellaria radix, 5 compounds
showed strong SASP suppressive action and 2’-hydroxyl substitution in the B-ring of the
flavone structure is an important moiety for SASP inhibition. Compounds 8 (2’,5,7-
trihydroxyflavone) and 11 (2’,5,7-trihydroxy-6-methoxyflavone) showed most potent SASP
inhibitory activity. Their inhibitory action was possibly mediated by blocking the signaling
pathway associated with IκBζ/C/EBPβ expression. Importantly, the oral administration of
compound 8 significantly inhibited the gene expression of SASP, IκBζ and C/EBPβ in old-
aged rats. Therefore, our data suggest that the extract and certain flavonoids from the roots of
Scutellaria baicalensis have strong SASP inhibitory activity and that treatment with the
compounds or blocking the target molecules can be helpful to prevent or slow down the
progression of age-related chronic inflammatory disorders.
20

447
448
449
450
451
452
453
454
455
456
457
458
459
Conflict of interest statement
There are no conflicts of interest.
Acknowledgements
This research was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-
2017R1A6A3A11028151), NRF grant (NRF-2018R1A2B6001314) funded by the Korean
government (MSIT) and BK21-plus by the Ministry of Education. The bioassay facility of
New Drug Development Institute (KNU) was used.
21

460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
References
Alexander, E., Hildebrand, D.G., Kriebs, A., Obermayer, K., Manz, M., Rothfuss, O.,
Schulze-Osthoff, K., Essmann, F., 2013. IκBζ is a regulator of the senescence-associated
secretory phenotype in DNA damage- and oncogene-induced senescence. J. Cell Sci. 26,
3738-3745.
Baker, D.J., Childs, B.G., Durik, M., Wijers, M.E., Sieben, C.J., Zhong, J., Saltness, R.A.,
Jeganathan, K.B., Verzosa, G.C., Pezeshki, A., Khazaie, K., Miller, J.D., van Deursen,
J.M., 2016. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature
530, 184-189.
Bass, T.M., Weinkove, D., Houthoofd, K., Gems, D., Partridge, L., 2007. Effects of
resveratrol on lifespan in Drosophila melanogaster and Caenorhabditis elegans. Mech.
Ageing Dev. 128, 546-552.
Bitto, A., Ito, T.K., Pineda, V.V., LeTexier, N.J., Huang, H.Z., Sutlief, E., Tung, H., Vizzini,
N., Chen, B., Smith, K., Meza, D., Yajima, M., Beyer, R.P., Kerr, K.F., Davis, D.J.,
Gillespie, C.H., Snyder, J.M., Treuting, P.M., Kaeberlein, M., 2016. Transient rapamycin
treatment can increase lifespan and healthspan in middle-aged mice. Elife 23, pii: e16351.
Bjedov, I., Toivonen, J.M., Kerr, F., Slack, C., Jacobson, J., Foley, A., Partridge, L., 2010.
Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster.
Cell Metab. 11, 35-46.
Campisi, J., Andersen, J.K., Kapahi, P., Melov, S., 2011. Cellular senescence: a link between
cancer and age-related degenerative disease? Semin. Cancer Biol. 21, 354-359.
Campisi, J., 2005. Suppressing cancer: the importance of being senescent. Science 309, 886-
22

483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
887
Capell, B.C., Drake, A.M., Zhu, J., Shah, P.P., Dou, Z., Dorsey, J., Simola, D.F., Donahue, G.,
Sammons, M., Rai, T.S., Natale, C., Ridky, T.W., Adams, P.D., Berger, S.L., 2016. MLL1
is essential for the senescence-associated secretory phenotype. Genes Dev. 30, 321-336.
Coppé, J.P., Desprez, P.Y., Krtolica, A., Campisi, J., 2010. The senescence-associated
secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99-118.
Coppé, J.P., Patil, C.K., Rodier, F., Sun, Y., Muñoz, D.P., Goldstein, J., Nelson, P.S., Desprez,
P.Y., Campisi, J., 2008. Senescence-associated secretory phenotypes reveal cell-
nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 6,
2853-2868.
Diekman, B.O., Sessions, G.A., Collins, J.A., Knecht, A.K., Strum, S.L., Mitin, N.K.,
Carlson, C.S., Loeser, R.F., Sharpless, N.E., 2018. Expression of p16INK 4a is a
biomarker of chondrocyte aging but does not cause osteoarthritis. Aging Cell 17, e12771.
Flanagan, K.C., Alspach, E., Pazolli, E., Parajuli, S., Ren, Q., Arthur, L.L., Tapia, R., Stewart,
S.A., 2017. c-Myb and C/EBPβ regulate OPN and other senescence-associated secretory
phenotype factors. Oncotarget 9, 21-36.
Freund, A., Patil, C.K., Campisi, J., 2011. p38MAPK is a novel DNA damage response-
independent regulator of the senescence-associated secretory phenotype. EMBO J. 30,
1536-1548.
Hickson, L.J., Langhi Prata, L.G.P., Bobart, S.A., Evans, T.K., Giorgadze, N., Hashmi, S.K.,
Herrmann, S.M., Jensen, M.D., Jia, Q., Jordan, K.L., Kellogg, T.A., Khosla, S., Koerber,
D.M., Lagnado, A.B., Lawson, D.K., LeBrasseur, N.K., Lerman, L.O., McDonald, K.M.,
McKenzie, T.J., Passos, J.F., Pignolo, R.J., Pirtskhalava, T., Saadiq, I.M., Schaefer, K.K.,
23

506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
Textor, S.C., Victorelli, S.G., Volkman, T.L., Xue, A., Wentworth, M.A., Wissler Gerdes,
E.O., Zhu, Y., Tchkonia, T., Kirkland, J.L., 2019. Senolytics decrease senescent cells in
humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals
with diabetic kidney disease. EBioMedicine 47, 446-456.
Itahana, K., Itahana, Y., Dimri, G.P., 2013. Colorimetric detection of senescence-associated β
galactosidase. Methods Mol. Biol. 965, 143-156.
Jeon, O.H., Kim, C., Laberge, R.M., Demaria, M., Rathod, S., Vasserot, A.P., Chung, J.W.,
Kim, D.H., Poon, Y., David, N., Baker, D.J., van Deursen, J.M., Campisi, J., Elisseeff,
J.H., 2017. Local clearance of senescent cells attenuates the development of post-
traumatic osteoarthritis and creates a pro-regenerative environment. Nat. Med. 23, 775-
781.
Kang, C., Xu, Q., Martin, T.D., Li, M.Z., Demaria, M., Aron, L., Lu, T., Yankner, B.A.,
Campisi, J., Elledge, S.J., 2015. The DNA damage response induces inflammation and
senescence by inhibiting autophagy of GATA4. Science 349, aaa5612.
Kim, S.R., Jiang, K., Ogrodnik, M., Chen, X., Zhu, X.Y., Lohmeier, H., Ahmed, L., Tang, H.,
Tchkonia, T., Hickson, L.J., Kirkland, J.L., Lerman, L.O., 2019. Increased renal cellular
senescence in murine high-fat diet: effect of the senolytic drug quercetin. Transl. Res. 213,
112-123.
Kumar, M., Seeger, W., Voswinckel, R., 2014. Senescence-associated secretory phenotype
and its possible role in chronic obstructive pulmonary disease. Am. J. Respir. Cell Mol.
Biol. 51, 323-333.
Kumar, R., Sharma, A., Kumari, A., Gulati, A., Padwad, Y., Sharma, R., 2019.
Epigallocatechin gallate suppresses premature senescence of preadipocytes by inhibition
24

529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
of PI3K/Akt/mTOR pathway and induces senescent cell death by regulation of Bax/Bcl-2
pathway. Biogerontology 20, 171-189.
Laberge, R.M., Zhou, L., Sarantos, M.R., Rodier, F., Freund, A., de Keizer, P.L., Liu, S.,
Demaria, M., Cong, Y.S., Kapahi, P., Desprez, P.Y., Hughes, R.E., Campisi, J., 2012.
Glucocorticoids suppress selected components of the senescence-associated secretory
phenotype. Aging Cell 11, 569-578.
Lee, K.S., Lee, B.S., Semnani, S., Avanesian, A., Um, C.Y., Jeon, H.J., Seong, K.M., Yu, K.,
Min, K.J., Jafari, M., 2010. Curcumin extends life span, improves health span, and
modulates the expression of age-associated aging genes in Drosophila melanogaster.
Rejuvenation Res. 13, 561-570.
Lim, H., Park, H., Kim, H.P., 2015. Effects of flavonoids on senescence-associated secretory
phenotype formation from bleomycin-induced senescence in BJ fibroblasts. Biochem.
Pharmacol. 96, 337-348.
Liu, G., Ma, J., Chen, Y., Tian, Q., Shen, Y., Wang, X., Chen, B., Yao, S., 2009. Investigation
of flavonoid profile of Scutellaria bacalensis Georgi by high performance liquid
chromatography with diode array detection and electrospray ion trap mass spectrometry. J.
Chromatogr. A. 1216, 4809-4814.
Liu, J.Y., Souroullas, G.P., Diekman, B.O., Krishnamurthy, J., Hall, B.M., Sorrentino, J.A.,
Parker, J.S., Sessions, G.A., Gudkov, A.V., Sharpless, N.E., 2019. Cells exhibiting strong
p16 INK4a promoter activation in vivo display features of senescence. Proc. Natl. Acad.
Sci. U S A. 116, 2603-2611.
Matsuo, S., Yamazaki, S., Takeshige, K., Muta, T., 2007. Crucial roles of binding sites for
NF-kappaB and C/EBPs in IkappaB-zeta-mediated transcriptional activation. Biochem. J.
25

552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
405, 605-615.
Menicacci, B., Laurenzana, A., Chillà, A., Margheri, F., Peppicelli, S., Tanganelli, E., Fibbi,
G., Giovannelli, L., Del Rosso, M., Mocali, A., 2017. Chronic Resveratrol Treatment
Inhibits MRC5 Fibroblast SASP-Related Protumoral Effects on Melanoma Cells. J.
Gerontol. A Biol. Sci. Med. Sci. 72, 1187-1195.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.
Onat, A., Can, G., 2014. Enhanced proinflammatory state and autoimmune activation: a
breakthrough to understanding chronic diseases. Curr. Pharm. Des. 20, 575-584.
Perrott, K.M., Wiley, C.D., Desprez, P.Y., Campisi, J., 2017. Apigenin suppresses the
senescence-associated secretory phenotype and paracrine effects on breast cancer cells.
Geroscience 39, 161-173.
Reid, J.J., Linden, M.A., Peelor, F.F., Miller, R.A., Hamilton, K.L., Miller, B.F., 2020. Brain
Protein Synthesis Rates in the UM-HET3 Mouse Following Treatment With Rapamycin
or Rapamycin With Metformin. J. Gerontol. A Biol. Sci. Med. Sci. 75, 40-49.
Robles, S.J., Adami, G.R., 1998. Agents that cause DNA double strand breaks lead to
p16INK4a enrichment and the premature senescence of normal fibroblasts. Oncogene 16,
1113-1123.
Rodriguez-Mateos, A., Vauzour, D., Krueger, C.G., Shanmuganayagam, D., Reed, J., Calani,
L., Mena, P., Del Rio, D., Crozier, A., 2014. Bioavailability, bioactivity and impact on
health of dietary flavonoids and related compounds: an update. Arch. Toxicol. 88, 1803-
1853.
Salminen, A., Kauppinen, A., Kaarniranta, K., 2012. Emerging role of NF-κB signaling in the
26

575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
induction of senescence-associated secretory phenotype (SASP). Cell. Signal. 24, 835-
845.
Singh, T., Newman, A.B., 2011. Inflammatory markers in population studies of aging. Ageing
Res. Rev. 10, 319-329.
Storer, M., Mas, A., Robert-Moreno, A., Pecoraro, M., Ortells, M.C., Di Giacomo, V., Yosef,
R., Pilpel, N., Krizhanovsky, V., Sharpe, J., Keyes, W.M., 2013. Senescence is a
developmental mechanism that contributes to embryonic growth and patterning. Cell 155,
1119-1130.
Sundar, I.K., Rashid, K., Gerloff, J., Li, D., Rahman, I., 2018. Genetic Ablation of p16INK4a
Does Not Protect against Cellular Senescence in Mouse Models of Chronic Obstructive
Pulmonary Disease/Emphysema. Am. J. Respir. Cell Mol. Biol. 59, 189-199.
Wang, P., Han, L., Shen, H., Wang, P., Lv, C., Zhao, G., Niu, J., Xue, L., Wang, Q.J., Tong, T.,
Chen, J., 2014. Protein kinase D1 is essential for Ras-induced senescence and tumor
suppression by regulating senescence-associated inflammation. Proc. Natl. Acad. Sci. U S
A 111, 7683-7688.
Xu, M., Pirtskhalava, T., Farr, J.N., Weigand, B.M., Palmer, A.K., Weivoda, M.M., Inman,
C.L., Ogrodnik, M.B., Hachfeld, C.M., Fraser, D.G., Onken, J.L., Johnson, K.O., Verzosa,
G.C., Langhi, L.G.P., Weigl, M., Giorgadze, N., LeBrasseur, N.K., Miller, J.D., Jurk, D.,
Singh, R.J., Allison, D.B., Ejima, K., Hubbard, G.B., Ikeno, Y., Cubro, H., Garovic, V.D.,
Hou, X., Weroha, S.J., Robbins, P.D., Niedernhofer, L.J., Khosla, S., Tchkonia, T.,
Kirkland, J.L., 2018. Senolytics improve physical function and increase lifespan in old
age. Nat. Med. 24, 1246-1256.
Yousefzadeh, M.J., Zhu, Y., McGowan, S.J., Angelini, L., Fuhrmann-Stroissnigg, H., Xu, M.,
27

598
599
600
601
Ling, Y.Y., Melos, K.I., Pirtskhalava, T., Inman, C.L., McGuckian, C., Wade, E.A., Kato,
J.I., Grassi, D., Wentworth, M., Burd, C.E., Arriaga, E.A., Ladiges, W.L., Tchkonia, T.,
Kirkland, J.L., Robbins, P.D., Niedernhofer, L.J., 2018. Fisetin is a senotherapeutic that
extends health and lifespan. EBioMedicine 36, 18-28.
28

602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
Figure legends
Fig. 1. The chemical structures of 17 flavonoid derivatives isolated from S. baicalensis used
in this study (A) and synthesis of 2’,5,7-trihydroxyflavone (8) (B).
Fig. 2. Effects of the ethanol extract of Scutellaria radix on the expression of IL-6, IL-8, IκBζ,
and p21 and BrdU uptake in bleomycin-treated BJ cells.
A, Effects on IL-6 and IL-8 protein production and BrdU uptake by the ethanol extract
(100, 50, and 20 μg/ml) for the last 24 h after senescence completion. B, Effects on the gene
expression of IL-6, IL-8, IκBζ, and p21 by the ethanol extract (100 and 50 μg/ml) for the last
24 h after senescence completion. C, Effects of the ethanol extract (20, 10, and 5 μg/ml) on
IL-6 and IL-8 production and BrdU uptake by concomitant treatment with bleomycin during
cellular senescence. D, Effects of the ethanol extract (20, 10, and 5 μg/ml) on the gene
expression of IL-6 IL-8, IκBζ, and p21 by concomitant treatment with bleomycin, Bl:
bleomycin, EXT: ethanol extract. *P < 0.05, **P < 0.01, significantly different from the
bleomycin-treated control group (n = 3).
Fig. 3. Effects on IL-6/IL-8 production and BrdU uptake by flavonoid derivatives isolated
from the ethanol extract of Scutellaria radix.
A, Effects on IL-6 production by 17 compounds for the last 24 h after senescence
completion. B, Effects of 17 compounds on IL-6 production when simultaneously treated
with bleomycin during cellular senescence. C, Effects on IL-8 production and BrdU uptake
by 5 compounds (compound 1, 5, 8, 10, and 11, 10 μM), having strong activity, for the last 24
29