Journal of Autoimmunity

Increased p16INK4a-expressing senescent bile ductular cells are associated
with inadequate response to ursodeoxycholic acid in primary biliary
cholangitis
ABSTRACT
Background & aims: Senescent biliary epithelial cells (BECs) may be involved in the pathophysiology of primary
biliary cholangitis (PBC) by secreting senescence-associated secretory phenotypes. We examined an association
of the extent of cellular senescence in BECs with clinicopathological features including response to urso￾deoxycholic acid (UDCA) and a possibility of senolytic therapy in PBC.
Methods: The expression of senescent markers (p21WAF1/Cip1, p16INK4a) and B-cell lymphoma–extra large (Bcl￾xL), a key regulator of senescent cell anti-apoptotic pathway, was immunohistochemically examined in livers
from patients with PBC (n = 145) and 103 control livers. Senolytic effect of Bcl-xL inhibitors (A-1331852 and
Navitoclax) was examined in senescent murine BECs.
Results: Senescent BECs were increased in small bile ducts in PBC, compared with control livers (p < 0.01).
Senescent BECs were increased in ductular reactions in PBC, stage 3–4, compared with PBC, stage 1–2 and
control livers (p < 0.01). The extent of senescent BECs in bile ductules was significantly correlated with stage
and hepatitis activity (p < 0.01) and the expression of p16INK4a in bile ductules was significantly correlated to
inadequate response to UDCA in PBC (p < 0.01). Double immunofluorescence revealed an increased expression
of Bcl-xL in p16INK4a-positive senescent BECs in PBC. Bcl-xL inhibitors selectively induced apoptosis in senescent
murine BECs (p < 0.01).
Conclusion: The extent of senescent BECs in small bile ducts and bile ductules was closely related to stage and
activity of PBC and the increased expression of p16 INK4a in bile ductules was correlated with inadequate re￾sponse to UDCA.
1. Introduction
Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver
disease characterized by serum anti-mitochondrial antibodies (AMAs)
and a unique chronic non-suppurative destructive cholangitis in small
bile ducts [1–4]. The pathogenesis of PBC has not been fully clarified, so
far [1–4]. PBC presents chronic, progressive cholestasis, biliary fibrosis
and subsequently liver failure and ursodeoxycholic acid (UDCA) is the
only accepted first-line agent for the treatment of PBC [1–9]. Ap￾proximately two-thirds of patients treated with UDCA in early-stage
PBC have a good prognosis without additional therapies, however, the
remaining patients, “inadequate responders” to UDCA, are at risk of
disease progression and additional therapies are demanded [2,5,6]. The
farnesoid X receptor agonist obeticholic acid was recently approved for
the treatment of PBC [10] and addition of bezafibrate to UDCA
monotherapy are also promising therapy for “inadequate responders” to
UDCA [11,12].
Biliary epithelial senescence in small bile duct and bile ductules is a
characteristic of PBC [7,13–19]. We reported that cellular senescence is
observed in the damaged small bile duct involved in chronic non-sup￾purative destructive cholangitis and also bile ductular cells in ductular
reactions [13,14]. Senescent BECs may play various roles in aggravated
inflammation, bile duct loss and progression of fibrosis by secreting
senescence-associated secretory phenotypes (SASPs) [16,20]. Further￾more, accumulating evidences suggest that cellular senescence is in￾volved in the pathogenesis and progression of other hepatobiliary dis￾eases such as primary sclerosing cholangitis (PSC) and biliary atresia
[18,21–23].

We hypothesized that the extent of cellular senescence may be re￾lated to clinicopathological features of PBC including inadequate re￾sponse to UDCA and that senescent BECs may be a therapeutic target in
PBC, especially for UDCA inadequate responders. We examined the
expression of senescent markers p21WAF1/Cip1 and p16INK4a in a large
series of PBC patients (145 patients) and analyzed an association of
cellular senescence in BECs with various clinicopathological features.
We also examined the expression of Bcl-xL, a key regulator of senescent
cells and anti-apoptotic pathways [26,28,29] in PBC and control livers.
Taking advantage of cultured BECs, we examined the effect of several
senolytic agents such as Bcl-xL inhibitors on senescent cell.
2. Materials and methods
2.1. Human study
Classification of intrahepatic biliary tree. The intrahepatic
biliary tree is classified into intrahepatic large and small bile ducts
(septal and interlobular bile ducts) by their size and distributions in the
portal tracts [31]. Bile ductules, which are characterized by tubular or
glandular structures with a poorly defined lumen and located at the
periphery of the portal tracts [31,32], are not included in the small bile
ducts and evaluated separately. BECs are the cells lining the biliary tree
including both large and small intrahepatic bile ducts and bile ductules.
2.2. Liver tissue preparation
A total of 255 liver tissue specimens (all were biopsied or surgically
resected) were collected from the liver disease file of our laboratory and
affiliated hospitals. The Ethics Committee of Kanazawa University ap￾proved this study. The liver specimens in this study were 145 PBC (23
male and 122 female, age range: 25–87 yrs, mean ± SD,
59 ± 11.0yrs), 39 chronic viral hepatitis (10 male and 29 female, age
range: 16–80 yrs, mean ± SD, 64 ± 8.3yrs), 21 PSC (10 male and 11
female, age range: 16–73 yrs, mean ± SD, 32 ± 18.4yrs), 18 extra￾hepatic biliary obstruction (8 male and 10 female, age range: 50–74 yrs,
mean ± SD, 66 ± 8.7yrs) and 32 “histologically normal” livers (10
male and 22 female, age range: 29–74yrs, mean ± SD, 56 ± 15.0yrs).
All PBC were from patients fulfilling the clinical, serological and his￾tological characteristics consistent with the diagnosis of PBC [3,4].
Table 1 summarized the clinicopathological features of PBC patients
examined. PBC livers were staged histologically according to Naka￾numa classification [3,33], and 97 and 48 of PBC were stages 1, 2 (early
PBC) and stages 3, 4 (advanced PBC), respectively. One hundred forty￾five PBC livers were divided into 79 CA3 (presence of chronic non￾suppurative destructive cholangitis) and 66 CA0-2 (absence of chronic
non-suppurative destructive cholangitis), respectively, according to
Nakanuma classification [33]. Thirty-five PBC livers were after UDCA
therapy and 24 were UDCA inadequate responders. “UDCA inadequate
responders” were selected being based on descriptions in the medical
records. Globe score or Paris –II criteria [4,5] were applied for recent
patients, although definition of UDCA inadequate response varied with
the period. Twenty-one and 17 chronic viral hepatitis were regarded as
F0-2 and as F3, 4, respectively [34]. Eight and 31 of chronic viral he￾patitis cases were serologically positive for hepatitis B surface antigen
and anti-hepatitis C viral antibody, respectively. All HCV patients were
before antiviral therapy and viremic. Seven and 14 PSC were stages 1, 2
(early PSC) and stages 3, 4 (advanced PSC), respectively. Causes of
extrahepatic biliary obstruction were obstruction of the bile duct at the
extrahepatic bile ducts or the hepatic hilum due to stone or carcinoma,
and the duration of jaundice was less than 1 month. “Histologically
normal” livers were obtained from surgically resected livers for meta￾static liver tumor or traumatic hepatic rupture. Normal liver tissues
were obtained from an area apart from the tumor and carcinoma tissues
were not evaluated. Chemotherapy was not performed before liver re￾section in all patients with histologically normal liver.
Liver tissue samples were fixed in 10% neutral-buffered formalin,
and embedded in paraffin. More than twenty serial sections, 4 μm-thick,
were cut from each block. Several sections were processed routinely for
histologic study, and the remainder was processed for the subsequent
immunohistochemistry.
Immunohistochemistry. We examined immunohistochemically
senescence-related markers p16INK4a, p21WAF1/Cip1 and Bcl-xL, as de￾scribed previously [19]. The primary antibodies used were anti￾p16INK4a (mouse, clone JC8, Neomarkers, Freemont, CA), anti-p21WAF1/
Cip1 (mouse, clone 70, BD Transduction, San Jose, CA) and anti-Bcl-xL
(rabbit, clone 54H6, Cell signaling, Danvers, MA). A similar dilution of
the control mouse or rabbit Immunoglobulin G (Dako) was applied
instead of the primary antibody as a negative control. Positive and
negative controls were routinely included. Histological analysis was
performed in a blinded manner. BECs in small bile ducts and bile
ductules were separately evaluated.
Double immunofluorescence. We also performed double im￾munofluorescence for Bcl-xL with senescent markers p16INK4a and
p21WAF1/Cip1. In brief, either of p16INK4a or p21WAF1/Cip1 was detected
using Vector Red Alkaline Phosphatase Substrate Kit (Vector Lab,
Burlingame, CA), followed by second staining for Bcl-xL using Alexa-
488-labeled anti-rabbit IgG. The sections were counterstained with
DAPI and evaluated under a conventional fluorescence microscope.
2.3. Culture study
Cell culture and treatments. Mouse BECs from intrahepatic small
bile duct were isolated from 8-week-old female BALB/c mice and were
purified and cultured as described previously [35]. The cell density of
the cells was less than 80% during experiments. Cellular senescence
was induced in cultured BECs by treatment with Etoposid (50 μM)
(Sigma-Aldrich, St Louis, MO), serum depletion or glycochenodeoxy￾cholic acid (GCDC, 200 μM)) for 4 days, as described previously. Then,
the effect of senolytic reagents (A-1331852 [0.1–1 μM], Navitoclax/
ABT-263 [1–10 μM], Dasatinib [0.2–1 μM], Quercetin [20–1000 μM],
Dasatinib [0.2–1 μM]+Quercetin [20–1000 μM] and Fisetin
[0.5–5 μM]) on induction of apoptosis and clearance of senescent cells

were examined.

Assay for cell number. BECs were seeded into 96-well microplates
(1 × 104 cells/well), and incubated in a final volume of 100 μl medium.
The cell number was assessed after the induction of cellular senescence and
a treatment with senolytic reagents using a Cell Proliferation Reagent WST-
1 (Roche, Basel, Switzerland) according to manufacture’s recommendation.
Assay for cellular senescence. The activity of senescence-asso￾ciated β-galactosidase (SA-β-gal) was detected after the induction of
cellular senescence and a treatment with senolytic reagents by using the
senescence detection kit (Bio Vision, Mountain View, CA) according to
manufacturer’s protocol [36]. The proportion of senescent cells was
assessed by counting SA-β-gal-positive cells in at least 1 × 103 total
cells using light microscopy.
Assay for apoptosis. The apoptotic cells in each condition were
assessed after the induction of cellular senescence and a treatment with
senolytic reagents by using CellEvent™ Caspase-3/7 Green Detection
Reagent (Life Technologies, Carlsbad, CA) according to manufacturer’s
protocol. The nuclei were simultaneously stained with DAPI. At least
1 × 103 total cells were checked and counted to assess the percentage
of apoptotic cells showing Caspase-3/7 activity with a conventional
fluorescence microscope (Olympus).
2.4. Statistical analysis
Statistical analysis of differences was performed using the Kruskal￾Wallis test with Dunn’s posttest. When the number of groups is 2, sta￾tistical analysis of difference was performed using the Mann-Whitney
test. The Chi-square test or Fisher’s exact test was used to analyze ca￾tegorical data. The correlation coefficient of 2 factors was evaluated
using Spearman’s rank correlation test. When the P value was less than
0.05, the difference was regarded as significant. All analyses were
performed using the GraphPad Prism software (GraphPad Software, San
Diego, CA, USA).
3. Results
3.1. Human study
Increased expression of senescent markers p16INK4a and
p21WAF1/Cip1 in damaged small bile ducts in PBC.
As we reported previously [13,14], p21 WAF1/Cip1 was expressed in
the nucleus and p16INK4a in the nucleus and cytoplasm in BECs, when
Fig. 1. Increased expression of p21WAF1/Cip1 and p16INK4a in damaged small bile ducts and bile ductular cells in PBC A) p21WAF1/Cip1 is not expressed in small
bile duct (arrow) in normal liver. B) p16INK4a is not expressed in small bile duct (arrow) in normal liver. C) p21WAF1/Cip1 is expressed in the nucleus of biliary
epithelial cells involved in chronic nonsuppurative destructive cholangitis (arrow) in PBC. D) p16INK4a is expressed in the nucleus and the cytoplasm of biliary
epithelial cells involved in chronic nonsuppurative destructive cholangitis (arrow) in PBC. E) p21WAF1/Cip1 is expressed in the nucleus of bile ductular cells in ductular
reactions in PBC. F) p16INK4a is expressed in the nucleus and the cytoplasm of bile ductular cells in ductular reactions in PBC. Immunostaining for
PBC, primary biliary cholangitis; CVH, chronic viral hepatitis; PSC, primary sclerosing cholangitis; EBO, extrahepatic biliary obstruction; a, p < 0.01 versus CVH; b,
p < 0.01 versus NSR; c, p < 0.05 versus EBO; d, p < 0.01 versus PBC, stage 1/2; n, number; [ ], number of cases showing 1+ (focal, positive cells are detected in
one third or fewer portal tracts), and 2+ (extensive, positive cells are detected in small bile ducts in more than one third of portal tracts).
M. Sasaki, et al. Journal of Autoimmunity xxx (xxxx) xxxx
4
of senescent markers p16INK4a and p21WAF1/Cip1 was significantly more
extent in small bile ducts in PBC, compared with control livers in￾cluding PSC (p < 0.01). The expression of senescent markers was
significantly more increased in PBC, CA3, compared with small bile
ducts in PBC, CA0-2 in PBC, stages 3 and 4 (p < 0.01).
Increased expression of senescent markers p16INK4a and
p21WAF1/Cip1 in bile ductular cells in ductular reaction in PBC.
The expression of senescent markers p16INK4a and p21WAF1/Cip1 was
frequently observed in BECs in bile ductular cells in ductular reactions
in PBC (Fig. 1E and F). Table 3 summarized the extent of senescent
markers p16INK4a and p21WAF1/Cip1 expression in bile ductular cells in
ductular reaction in PBC and control livers. The expression of senescent
markers was significantly more extensive in bile ductular cells in
ductular reactions in PBC, stages 3 and 4, compared with PBC, stages 1
and 2 and control normal livers (p < 0.01). The expression of p16INK4a
in bile ductules was significantly more extensive in chronic viral he￾patitis and PSC, especially stages 3, 4, compared with normal livers
White cell, significant correlation (p<0.05); light grey cell, significant inverse correlation (p<0.05); dark grey cell, no significant correlation
(p>0.05); UDCA, ursodeoxicholic acid; r, Spearman’s r; p, P value
Fig. 2. Increased expression of B-cell lymphoma-extra large (Bcl-xL) in p16INK4a-positive senescent biliary epithelial cells in damaged small bile ducts and
bile ductular cells in PBC. A) Increased expression of Bcl-xL is seen in a damaged small bile duct (arrow) showing expression of p16INK4a in PBC. B) Increased
expression of Bcl-xL is seen in p16INK4a –expressing bile ductular cells (arrows) in ductular reaction in PBC. Double Immunofluorescent staining for Bcl-xL (green) and
a senescent marker p16INK4a (red). Original magnification, ×400.
M. Sasaki, et al. Journal of Autoimmunity xxx (xxxx) xxxx
5
Association of cellular senescence in small bile ducts and bile
ductular cells with clinicopathological features.
Table 4 summarizes the association of the extent of cellular senes￾cence and clinicopathological features in PBC. The expression of se￾nescent markers p16INK4a and p21WAF1/Cip1 in small bile ducts was
significantly correlated with cholangitis activity in PBC (p < 0.01). In
contrast, the expression of senescent markers p16INK4a and p21WAF1/Cip1
in bile ductules was inversely correlated with cholangitis activity in
PBC (p < 0.01). The expression of p21WAF1/Cip1 in small bile ducts was
significantly correlated with gender (male) and inversely correlated
with inadequate response to UDCA (p < 0.05). There were no sig￾nificant correlations between patients’ age and the expression of se￾nescent markers in small bile ducts.
The expression of senescent markers in bile ductules was sig￾nificantly correlated with Scheuer stage, Nakanuma stage and hepatitis
activity in PBC (p < 0.01). The expression of p16INK4a in bile ductules
was significantly correlated with inadequate response to UDCA in PBC
(p < 0.01). The expression of senescent marker p21WAF1/Cip1 in small
bile ducts and bile ductules was correlated to gender (male-pre￾dominance) and presence of family history. There were no significant
correlations between patients’ age and the expression of senescent
markers in bile ductules.
Increased expression of Bcl-xL expression in senescent BECs in
damaged small bile ducts and bile ductules in PBC.
Double immunostaining revealed that the expression of Bcl-xL was
frequently increased in BECs in the damaged bile ducts showing ex￾pression of senescent marker p16INK4a in PBC (Fig. 2A). The expression
of Bcl-xL was frequently increased in bile ductular cells in ductular
reaction showing expression of senescent marker p16INK4a in PBC
3.2. Culture study
Senescent BECs are effectively cleared by the treatment with
Bcl-xL inhibitor A-1331852 and Navitoclax.
WST-1 assay. Cell number was assessed using WST-1 assay after the
induction of cellular senescence for 4 days and following 1 day treat￾ment with senolytic reagents. Cell number was significantly decreased
by the treatment with A-1331852, Navitoclax Dasatinib, or
Dasatinib + Quercetin in all conditions inducing cellular senescence
(p < 0.01) (Fig. 3A). Cell number was significantly decreased by the
treatment with Fisetin in senescent BECs induced by Etoposid and
serum depletion (p < 0.01) (Fig. 3A).
SA-beta-Gal assay. Cellular senescence was assessed by the activity
of SA-β-gal after treatment with Etoposid (100 μM), serum deprivation
or GCDC (500 nM) for 4 days and following 1 day treatment with sol￾vent control, A-1331852 (50 nM), Navitoclax (100 μM), Dasatinib
(200 nM), Quercetin (200 μM), Dasatinib + Quercetin and Fisetin
(5 μM). Cellular senescence was significantly induced by treatment with
Etoposid, serum deprivation or GCDC (p < 0.01). The senescent BECs
were significantly decreased by treatment with A-1331852, Navitoclax,
Dasatinib or Dasatinib + Quercetin (p < 0.01 or p < 0.05) (Fig. 3B).
Apoptosis assay. Apoptosis was assessed by detecting caspase-3/7
activity after the induction of cellular senescence for 4 days and fol￾lowing 1 h treatment with senolytic reagents. Apoptotic cells showed
caspase-3/7 activity with green fluorescence (Fig. 4A). The percentage
of apoptotic cells was significantly increased in senescent BECs by a
treatment with A-1331852, Navitoclax, Dasatinib, Dasatinib + Quer￾cetin or Fisetin (p < 0.01 or p < 0.05). (Fig. 4B).
4. Discussion
The findings obtained in this study are summarized as follows; 1)
The expression of senescent markers p16INK4a and p21WAF1/Cip1 was
significantly more extent in small bile ducts in PBC, compared with
control livers (p < 0.01) and was significantly correlated with cho￾langitis activity in PBC (p < 0.01); 2) The expression of senescent
markers p16INK4a and p21WAF1/Cip1 was significantly more extent in bile
ductular cells in ductular reactions in PBC, stage 3–4, compared with
PBC, stage 1–2 and control livers (p < 0.01); 3) The expression of
senescent markers in bile ductules was significantly correlated with
stage and hepatitis activity in PBC (p < 0.01); 4) The expression of
p16INK4a in bile ductules was significantly correlated to inadequate
response to UDCA in PBC (p < 0.01); 5) The expression of senescent
marker p21WAF1/Cip1 in small bile ducts and bile ductules was correlated
to sex (male-predominance) and presence of family history; 6) Double
immunofluorescence revealed an increased expression of Bcl-xL in
p16INK4a-positive senescent BECs in the bile duct lesion and ductular
reactions in PBC; 7) The treatment with Bcl-xL inhibitors (A-1331852,
Navitoclax), Dasatinib and Dasatinib + Quercetin induced apoptosis in
senescent BECs and eliminated them (p < 0.01).
In this study, we examined cellular senescence in small bile ducts
Fig. 3. Senescent BECs are effectively cleared by the treatment with Bcl-xL inhibitor A-1331852 and Navitoclax. A) Cell number was assessed using WST-1
assay after the induction of cellular senescence with Etoposid (Etop, 100 μM), serum deprivation (Dep) or GCDC (500 nM) for 4 days and following 1 day treatment
with solvent control, A-1331852 (50 nM), Navitoclax (100 μM), Dasatinib (D) (200 nM), Quercetin (Q) (200 μM) and Fisetin (5 μM). n = 5 for each group.
*p < 0.01 and #p < 0.05 compared to control (solvent) in each group. B) Cellular senescence was assessed by senescence-associated β-galactosidase activity (SA-β-
gal) after treatment with Etoposid (Etop, 100 μM), serum deprivation (Dep) or GCDC (500 nM) for 4 days and following 1 day treatment with solvent control, A-
1331852 (50 nM), Navitoclax (100 μM), Dasatinib (D) (200 nM), Quercetin (Q) (200 μM) and Fisetin (5 μM). n = 10 for each group. **p < 0.01 compared tocontrol-control solvent. *p < 0.01 and #p < 0.05 compared to control (solvent) in each group.

and bile ductular cells in a large number of PBC livers and control li￾vers. This study confirmed our previous studies reporting that cellular
senescence was frequently observed in damaged small bile ducts and
bile ductules in ductular reactions in PBC [13,14,17]. In this study, we
analyzed the correlation of extent of cellular senescence in small bile
duct and bile ductules with clinicopathological features in PBC. As
expected, cellular senescence in small bile ducts correlated cholangitis
activity, whereas cellular senescence in bile ductular cells correlated to
Nakanuma stages. Expression of senescent markers was higher in
ductular reaction in the advanced stage of PBC compared with the early
stage PBC, which suggests that senescent BECs in ductular reaction may
contribute to further progression of fibrosis in the advanced stage of
PBC. This study also disclosed for the first time that hepatitis activity is
significantly correlated to cellular senescence in bile ductular cells. In
previous study, piecemeal necrosis (interface hepatitis), which re￾presents hepatitic activity, is reportedly a risk factor for disease pro￾gression in PBC [2,5,6]. Taken together, cellular senescence in bile
ductular cells may play a role in the progression of PBC. These finding
confirmed that cellular senescence may participate in pathobiology of
PBC from 2 aspects; a unique cholangitis in small bile ducts in the early
stage and ductular reactions in bile ductular cells in the advanced stage.
The former is a unique finding in PBC, which may be closely related to
unique pathogenesis of PBC [13,14]. The latter is observed in PSC and
biliary atresia and other chronic cholestatic diseases in addition to PBC
[13,14,17,21–23], which may be caused by cholestasis and participate
in the progression of fibrosis.
Interestingly, we revealed for the first time that the expression of
p16 INK4a in bile ductules is correlated to inadequate response to UDCA
therapy (inadequate responders). This finding suggests that senescent
BECs in bile ductules are accumulating in the liver of UDCA inadequate
responders. The reason why the expression of p16 INK4a in bile ductules
is correlated to inadequate response to UDCA therapy remains unclear.
It is known that UDCA inadequate responders are generally at higher
stage and UDCA may not be useful for patients with advanced PBC,
because the treatment is unable to prevent the development of the
consequences of portal hypertension in patients with cirrhosis [6,37].
Accumulated senescent BECs may worsen microenvironment by se￾creting SASPs and may disturb the effect of UDCA. Furthermore, the
expression of AE2, which play a role in protecting BECs by producing
bicarbonate umbrella, is decreased in senescent BECs in PBC [38–40].
Therefore, senescent BECs may be much more exposed to hydrophobic
bile acids and UDCA could not compensate the pathological condition
for senescent BECs.
It is now well known that senescent BECs express various chemo￾kines cytokines and factors as SASPs in PBC and PSC, which modulate
inflammatory cell infiltration and fibrosis in cholangiopathies
[8,16,20,23]. Irrespective of causal relationship between p16INK4a and
inadequate response to UDCA, the senescent cell could be a target of
senolytic therapy for UDCA inadequate responders. Furthermore, this
study disclosed for the first time that the expression of Bcl-xL, a re￾presentative senescent cells and anti-apoptotic pathways [26,28,29],
was significantly increased in p16INK4a -positive senescent BECs in PBC.
This finding also may support the hypothesis that senescent BECs could
Fig. 4. Apoptosis is effectively induced in senescent BECs by the treatment
with Bcl-xL inhibitor A-1331852 and Navitoclax. Apoptosis was assessed by
detecting caspase-3/7 activity after the induction of cellular senescence with
Etoposid (Etop, 100 μM), serum deprivation (Dep) or GCDC (500 nM) for 4 days
and following 1 h treatment with solvent control, A-1331852 (50 nM),
Navitoclax (100 μM), Dasatinib (D) (200 nM), Quercetin (Q) (200 μM) and
Fisetin (5 μM). A) Apoptotic cells showed caspase-3/7 activity with green
fluorescence. B) n = 5 for each group. *p < 0.01 and #p < 0.05 compared to
control (solvent) in each group.
Fig. 3. (continued)
M. Sasaki, et al. Journal of Autoimmunity
be a target of senolytic therapy for UDCA inadequate responders.
In cultured BECs, we found that several representative senolytic
reagents; A-1331852, Navitoclax, Dasatinib and Dasanitib + Quercetin
induced apoptosis and consequently eliminated senescent BECs.
Although senolytic therapy is getting attention recently and several
senolytic agents are reportedly effective under clinical trial for idio￾pathic pulmonary fibrosis and osteoarthrosis [26,27], there were few
studies in terms of BECs and other hepatic resident cells in liver diseases
[30]. Moncsek et al. reported effects of Bcl-xL inhibitors A-1331852 and
Navitoclax in BECs and fibroblasts. In addition, they showed the ef￾fective elimination of senescent cells and reduction of fibrosis in PSC
model mice [30]. Our present study agrees with their study [30] in
terms of effective elimination of senescent BECs by A-1331852 and
Navitoclax. Furthermore, our study revealed for the first time that
Dasatinib, Dasanitib + Quercetin and Fisetin, other known senolytic
reagents [26,27] are effective for the elimination of senescent BECs.
Taken together, senolytic reagents may be candidates for therapeutic
drugs for PBC, especially for UDCA inadequate responders. Further
studies are needed to confirm the effects of senolytic reagents on se￾nescent BECs using cultured human BECs.
In conclusion, the extent of senescent BECs in small bile ducts and
bile ductules was closely related to stage and activity of PBC and the
increased expression of p16 INK4a in bile ductules was correlated with
inadequate response to UDCA. Assessment of biliary epithelial senes￾cence may be useful for the prediction of response to UDCA-therapy.
Elimination of senescent BECs by senolytic agents such as Bcl-xL in￾hibitors A-1331852 may be an effective treatment for inadequate re￾sponders to UDCA.
A statement of financial support
This study was supported in part by a Grant-in-Aid for Scientific
Research (C) from the Ministry of Education, Culture, Sports and
Science and Technology of Japan (18K06985).
CRediT authorship contribution statement
Motoko Sasaki: Conceptualization, Methodology, Software, Formal
analysis, Investigation, Writing – original draft, Writing – review &
editing, Funding acquisition. Yasunori Sato: Validation, Investigation,
Resources, Data curation, Visualization. Yasuni Nakanuma:
Resources, Data curation, Writing – review & editing, Supervision.
Declaration of competing interest
The authors declare that they have no conflict of interest.
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