Therapeutic effect of targeting Substance P on the progression of osteoarthritis - Oxford Academic Journals
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Modern Rheumatology, 00, 2021, 1–11
DOI: https://doi.org/10.1093/mr/roab089
Advance access publication date: 6 October 2021
Original Article
Therapeutic effect of targeting Substance P on the
progression of osteoarthritis
Yoshiko Shirakawaa , Tomoyuki Nakasaa,b,* , Munekazu Kanemitsua , Akinori Nekomotoa ,
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Masakazu Ishikawaa , Dilimulati Yimitia , Shigeru Miyakia,b and Nobuo Adachia
a
Department of Orthopaedic Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
b
Medical Center for Translational and Clinical Research, Hiroshima University Hospital, Hiroshima, Japan
*Correspondence: Tomoyuki Nakasa; tnakasa0@gmail.com; Department of Orthopaedic Surgery, Graduate School of Biomedical and Health Sciences,
Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
ABSTRACT
Objectives: Substance P (SP) modulates NK1 and has various functions such as regulation of pain response, bone metabolism, and angiogenesis,
which are recognized as important factors in osteoarthritis (OA). We aimed to evaluate the therapeutic effect of targeting SP on OA progression.
Methods: SP expression patterns were analysed histologically in articular cartilage and subchondral bone of human knees from OA patients
and autopsy donors as non-OA samples and in mouse articular cartilage. Moreover, to examine the effect of SP on the progression of OA, we
administered drugs to mice following the surgical destabilization of the medial meniscus: Phosphate-buffered saline (PBS), septide (NK1 receptor
agonist), or aprepitant (NK1 receptor antagonist). Histological analysis and bone morphologic analysis using micro-computed tomography were
performed.
Results: In human analysis, the expression of SP in mild OA samples was significantly higher than that in severe OA, and that in healthy
cartilage was significantly higher than that in OA. In mouse analysis, Osteoarthritis Research Society International scores in the septide group
were significantly lower than those in the control group. Computed tomography analysis showed that the subchondral bone’s epiphysis in the
control group had sclerotic change, not observed in the septide group.
Conclusions: The administration of septide ameliorates OA progression through preventing subchondral bone sclerosis.
KEYWORDS: NK1 receptor agonist; osteoarthritis; septide; Substance P
Introduction OA using neuropeptide receptor agonists or antagonists may
Osteoarthritis (OA) is a multifactorial degenerative disorder be promising.
of the synovial joints that causes the irreversible destruction SP, a neuropeptide composed of 11 amino acids, is a highly
of the articular cartilage [1, 2]. Although OA causes signifi- conserved member of the tachykinin peptide family that is
cant health and social problems, the precise mechanism of OA widely distributed in both the central and the peripheral ner-
initiation and progression has not been completely elucidated vous systems. SP preferentially activates the neurokinin-1
[3]. Therefore, there are currently no effective preventive receptor (NK1R) and transmits nociceptive signals via pri-
measures or treatments for OA. Various pathomechanisms mary afferent fibres to spinal and brainstem second-order
of OA have been advocated, and among them is the the- neurons [9]. SP is localized and distributed throughout the
ory that sensory and sympathetic joint innervation plays an subchondral bone where it may contribute to OA pain
important role in the perturbation of joint homeostasis in OA [10–12]. SP has various biological functions other than reg-
[4]. It has been reported that the peripheral nervous system ulating pain responses. NK1R is expressed by osteoblasts and
is critically involved in bone metabolism [5, 6]. Neuropep- osteoclast precursors and stimulates osteoblast and osteoclast
tides such as Substance P (SP), calcitonin-gene-related peptide differentiation and function [6, 13]. Several animal studies
(CGRP), and vasoactive intestinal peptide, which are recog- have reported cartilage degeneration accompanying changes
nized as pain-related neurotransmitters, play roles in bone in subchondral bone conditions such as in the cases of bone
metabolism, angiogenesis, and inflammation. The expression sclerosis or absorption during the progression of OA [14–16].
of these neuropeptides is increased in the subchondral bone SP regulates bone remodelling, which affects osteoblastic dif-
in OA, suggesting that sensory-neuron-released neuropeptides ferentiation and osteoclastogenesis in the subchondral bone.
are involved in joint homeostasis, and possibly the subsequent Moreover, SP play roles in anti-inflammatory responses and
bone deformity, cartilage degeneration, and inflammation tissue repair through the recruitment of mesenchymal stem
during OA progression [7, 8]. Considering these neuropep- cells (MSCs) [17–19]. Given these functions of SP, the reg-
tides play a crucial role in OA pathogenesis, drug therapy for ulation of the SP expression can be beneficial or harmful in
Received 29 March 2021; Accepted 29 September 2021
© Japan College of Rheumatology 2021. Published by Oxford University Press. All rights reserved.
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2 Shirakawa et al.
OA pathogenesis. As SP expression is increased in OA, it may meniscotibial ligament of the right knee joint was resected
play an important role in the pathogenesis of OA. However, (DMM) according to the previous reports [20]. Other ani-
the precise expression pattern and role of SP as anabolic or mals in the sham group underwent sham surgery, which
catabolic factors in OA remain unclear. The study of SP in consisted of a skin incision in the right knee joint [21]. To
OA is necessary as it could serve as a potential therapeutic examine the effect of SP on the progression of OA, we admin-
target to ameliorate OA symptoms. We hypothesized that the istered the following drugs to the animals of each group
regulation of SP could inhibit OA progression through mod- including DMM and sham mice through an intraperitoneal
ulating bone remodelling of the subchondral bone its anti- injection immediately after surgery: control group: PBS at a
inflammatory effects and by inducing tissue repair through the dose of 100 µl/animal, aprepitant group: NK1R antagonist
recruitment of MSCs. (Aprepitant; MK-0869, Adooq Bioscience, Irvine, CA, USA)
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The purpose of this study is to analyse the changes in dissolved in 100 µl distilled water containing 2.5% dimethyl
the subchondral bone and cartilage during OA progression, sulfoxide at a dose of 3 mg/kg [22], or septide group: NK1R
focusing on SP expression. Further, this study aimed to exam- agonist (Septide; Bachem, Bubendorf, Switzerland) dissolved
ine the therapeutic effect of SP on OA progression using an in 100 µl of PBS at a dose of 10−8 mol/kg [23].
SP receptor agonist and antagonist in mice that underwent Animals were sacrificed at 2 days, 1 week, 4 weeks, and
surgical destabilization of the medial meniscus (DMM). 8 weeks after surgery or sham surgery by anaesthesia (n = 8
each time point). After fixation of the knee joints in 4% PFA
for 24 h, the samples were analysed using micro-computed
Methods tomography (m-CT). Then, they were decalcified in 20%
Human samples EDTA for 10 days and embedded in paraffin. The knee joints
This study was reviewed and approved by the ethics commit- were sectioned (4.0 µm) in the sagittal plane through the cen-
tee of our institution, and informed consent was obtained tral weight-bearing region of the medial femorotibial joint.
from all patients. For the analysis of SP expression pat- The sections were stained with Safranin O/fast green, and at
terns in OA patients, articular cartilage and bone samples least two different sections per sample were analysed micro-
were obtained from six patients who underwent total knee scopically. All sections were graded by two independent
arthroplasty. This group included one male and five females, observers, and median scores were determined for statistical
with a mean age of 73 years (range: 66–86 years). Knee analysis.
OA was diagnosed based on standing radiographs using the
American Rheumatism Association’s criteria for OA, with Micro-computed tomography
Kellgren–Lawrence Grades III (four cases) and IV (two cases)
Samples were analysed under high-resolution m-CT (Sky-
included in this group. Patients with a history of knee trauma
Scan1176, Toyo Corporation, Tokyo, Japan) using the fol-
and systemic diseases, such as rheumatoid arthritis, were
lowing parameters: source voltage, 40 kV; source current,
excluded. The entire proximal tibia was obtained using stan-
580 µA; pixel size, 12.47 µm; and spatial resolution, 9 µm.
dard surgical techniques for total knee arthroplasty and was
Images were reconstructed (NRecon, Toyo Corporation,
divided into six regions, as the degeneration of cartilage and
Tokyo, Japan) for analysis (CT-analyzer, Toyo Corporation,
subchondral bone in OA varies across the different regions of
Tokyo, Japan). In mice, the ratio of bone volume (BV) and
the proximal tibia. Specifically, the medial and lateral tibial
total volume (TV) in the tibial plateau subchondral bone’s
plateau was subdivided into three regions each, with 30 sam-
epiphysis (BV/TV, %) was measured, as previously described
ples obtained for analysis from each proximal tibia specimen.
[24]. In setting of the region of the interest for BV/TV, the sub-
Six regions were not included because they did not contain the
chondral bone plate, subchondral bone plate of growth plate,
osteochondral unit due to severe deformity.
and cortical bone were not included for the measurement
For the analysis of non-OA samples, human articular car-
[25]. In addition, the ratio of the medial and lateral height
tilages and subchondral bone from knee joints were obtained
of the epiphysis at 8 weeks was calculated in the sham, con-
from six autopsy donors (two males and four females) with
trol, aprepitant, and septide groups according to the previous
a mean age of 38 years (range: 23–48 years old) as healthy
report [26].
young samples and from seven donors (four males and three
females) with a mean age of 69 years (range: 62–76 years old)
as healthy aged samples. Tissue collection was approved by Histological analysis
the Scripps Human Subjects Committee. All samples were Samples were stained using Safranin O/fast green and
fixed in 4% paraformaldehyde (PFA), decalcified in 20% haematoxylin–eosin. The pathological changes in the tibial
ethylenediaminetetraacetic acid (EDTA) for 2 weeks, and sub- plateau for both human and mouse samples were scored using
sequently embedded in paraffin. Coronal sections of 4 µm in the Osteoarthritis Research Society International’s (OARSI)
thickness were prepared for histological analysis. scoring system for OA [27, 28]. The average OARSI grade
(0–6.5) was calculated. Human samples were classified into
Animal models three groups, as follows: mild OA (OARSI grade: 1.0–2.5),
The study protocols involving animals were approved by the moderate OA (OARSI grade: 3.0–4.5), and severe OA (OARSI
Ethics Committee for Experimental Animals of Hiroshima grade: 5.0–6.5) groups according to the previous report [21].
University and were performed in strict accordance with the Synovitis after injection for mouse models was evaluated at
committee guidelines. Ten-week-old male C57BL/6 mice were 1, 4, and 8 weeks using the established synovitis score for
used in this study. The animals were provided free access changes in the synovial lining thickness and cellular density
to food and water and allowed unrestricted weight bearing in the synovial stroma (0–3 points, maximum score: 6 points)
prior to surgical intervention. For the OA model, the medial [29]. Other sections were used for immunohistochemistry
Therapeutic effect of Substance P on progression of osteoarthritis 3
tests and tartrate-resistant acid phosphatase (TRAP) staining. metalloproteinase with thrombospondin motifs 5 (ADAMTS-
Subchondral bone plate was identified as the region between 5; 1:100 dilution, Gene Tex, Irvine, CA), or anti-type II col-
the osteochondral junction and the bone marrow cavity, and lagen (1:10 dilution, The Developmental Studies Hybridoma
the thickness of the subchondral bone plate and BV/TV was Bank, IA), using a 3,3′ -diaminobenzidine substrate according
measured by Image J (National Institution of Health) accord- to the method of a previous report [30–32].
ing to the previous report [14, 25]. In addition, angiogene- SP-positive cells in the articular cartilage and the subchon-
sis, which was identified as two or more red blood cells in dral bone were counted and the area was quantified using
the luminal structure lined with endothelial cells in the sub- Image J. Three areas were randomly set in the articular car-
chondral bone, was evaluated [25]. Then, subchondral bone tilage and subchondral bone of the tibia, just below the
changes were assessed using the histopathological scoring sys- subchondral bone plate, and the cell number was counted,
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tem by Nagira et al. [25]. This subchondral bone scoring and the calculated mean values per unit area of 1 mm2 for
system has three parameters: subchondral bone plate consist- human and mouse samples were recorded. In addition, the
ing of the combination of subchondral bone plate thickness percentage of SP-positive cells to total chondrocytes in human
and angiogenesis, BV/TV, and osteophytes in the horizon- articular cartilage was calculated. In human samples with a
tal plane. In our study, the score for osteophyte formation loss of the articular cartilage layer, only subchondral bone
was omitted because it was difficult to evaluate the osteo- was evaluated.
phyte formation in the sagittal section. After quantifying each For mouse samples, TRAP staining was performed using a
parameter except for osteophytes, they were graded on a scale commercially available kit (Wako Pure Chemical Industries,
of subchondral bone plate 0–6; BV/TV 0–3. The minimum Ltd., Osaka, Japan), according to the manufacturer’s proto-
score is 0 points and the maximal score is 9. col. TRAP-positive multinucleated cells containing more than
three nuclei were recognized as osteoclasts and counted using
Image J in the subchondral bone with the cell count per unit
Immunohistochemical analysis area reported. To analyse bone formation, we counted the
Each section was immunostained with an anti-SP antibody total number of osteocalcin-positive cells and osteoclasts in
(1:100 dilution, Santa Cruz Biotechnology: sc-58591), anti- the subchondral bone per unit area.
osteocalcin (1:100 dilution, Santa Cruz Biotechnology, Dal- For MMP-13 and ADAMTS-5 expression, three fields of
las, TX), anti-matrix metallopeptidase 13 (MMP 13) (1:20 the cartilage were randomly selected and positive cells in each
dilution, Neomarkers, Fremont, CA), anti-A disintegrin and area were counted at 200× magnification. The percentage of
Figure 1. Expression pattern of SP in cartilage and SBP of OA patients. (a–d) Safranin O staining and immunohistochemistry of SP in the tibial plateau of
moderate OA patients. The OARSI score is 3. SP is expressed in the articular cartilage and SCB. (e–h) Safranin O staining and immunohistochemistry of
SP in the tibial plateau of severe OA patients. The OARSI grade is 5. Arrow indicates SP-positive cells. Scale bars represent 100 µm. (i) The number of
SP-positive cells (/mm2 ) in the articular cartilage. (j) The number of SP-positive cells (/mm2 ) in the SCB. The number of SP-positive cells in the cartilage
and SCB of mild OA samples were significantly higher than those in severe OA samples (i, j). SCB: subchondral bone, **p < .01, *p < .05.
4 Shirakawa et al.
MMP-13- and ADAMTS-5-positive cells in each field was (Figure 2). Additionally, in mild OA samples, SP expression
calculated. in the superficial cartilage layer decreased and was mainly
expressed in the middle layer. Thus, SP is expressed at high
levels in normal cartilage and decreases with the progression
Statistical analysis of OA. The percentage of SP-positive cells to total chondro-
All results in this study were expressed as the mean ± standard cytes in the healthy individuals was significantly higher than
deviation. A comparison among three or four groups was that in the OA group (Figure 2).
done using the Tukey–Kramer post hoc test, and the Mann–
Whitney U-test was used for the detection of the differences
between the two groups using SPSS for Windows, version The effect of aprepitant and septide administration
22.0 (IBM Corporation, Armonk, NY, USA). P values of less on OA progression
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than.05 were considered to be statistically significant. In mice from the control group that were injected with PBS
following surgical DMM, the degeneration of the articu-
lar cartilage progressed gradually over time. Even at 2 days
post-surgery, the loss of proteoglycan was observed. At
Results 1 week, erosion, clefts, and progressive proteoglycan loss
Expression pattern of SP in humans were observed. In the subchondral bone of the tibia’s epiph-
In the human tibia, SP is expressed in the articular cartilage ysis, the bone marrow cavity decreased and the BV increased,
and subchondral bone. In patients with mild OA whose bone which suggested sclerotic changes. The expression of SP
marrow cavity was maintained, SP was expressed along the was maximal on 2 days post-surgery and gradually decreased
bone marrow cavities of the tibia. In severe cases of OA where with the progression of OA (Figure 3), which was consis-
the bone marrow cavity was decreased and BV was increased, tent with the expression patterns seen in humans. SP was
SP expression decreased. The expression of SP in the mild expressed intensely in the cartilage on day 2 and decreased
OA group was significantly higher than in the severe OA substantially particularly in the superficial cartilage layer. The
group in not only the subchondral bone but also the cartilage. expression pattern of SP in sham mice did not change over
(Figure 1). Furthermore, the expression of SP in the articu- time.
lar cartilage was significantly higher in samples from healthy To examine the effect of SP regulation on OA progression,
individuals compared to those with moderate or severe OA an NK1R antagonist or agonist was administered to the mice
Figure 2. Expression pattern of SP in healthy and OA samples. (a–i) Safranin O staining and immunohistochemistry of SP of the tibial plateau from
autopsy donors and OA patients. Young healthy (a–c) and aged healthy (d–f) and OA patient samples with 6.5 of the OARSI grade (g–i). Scale bars
represent 100 µm. (j) The number of SP-positive cells in the articular cartilage. The number of SP-positive cells in the articular cartilage of young healthy
samples was significantly higher than that of OA patients. (k) The percentage of the SP-positive cells to total chondrocytes. The percentage of
SP-positive cells in the articular cartilage of healthy individuals was significantly higher than that of OA patients. **p < .01, *p < .05.
Therapeutic effect of Substance P on progression of osteoarthritis 5
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Figure 3. Expression pattern of SP in the DMM mice. (a) Immunohistochemistry of SP in the knee joint of control DMM and sham mice. (b) The number
of SP-positive cells in the articular cartilage of tibia of control DMM and sham mice. The expression of SP was maximal on 2 days post-surgery and
gradually decreased with the progression of OA. Scale bars represent 100 µm. **p < .01, *p < .05.
following surgery (Figure 4(a)). Like the control group, the At 1 week, the subchondral bone score in the apprepitant
mice in the aprepitant group exhibited progressive degenera- group was significantly lower than those in the control and
tive OA over time, while the mice in the septide group showed septide groups (p < .05) (Figure 4(d)). The thickness of the
reduced cartilage degeneration. Although there was no sig- subchondral bone plate in the control and aprepitant groups
nificant difference in each at 2 days post-surgery, the OARSI became thicker as OA progressed, while the subchondral bone
score in the septide group was significantly lower than those thickness in the septide group did not increase at 8 weeks
in the control and aprepitant groups at 1 week, 4 weeks, and (Figure 4(e)).
8 weeks post-surgery. There was no significant difference in On m-CT analysis, the subchondral bone’s epiphysis in the
the OARSI score between the control group and the aprepi- control group exhibited sclerotic changes. The BV/TV of the
tant group at 4 weeks, but that in the aprepitant group was subchondral bone’s epiphysis of the septide group was sig-
lower than that in the control group at 8 weeks (Figure 4(b)). nificantly lower than that in the control group at 1 week,
There were no adverse events such as death following admin- 4 weeks, and 8 weeks post-surgery (Figure 5). The BV/TV
istration. To examine the effect of the aprepitant and septide in the control, aprepitant, and septide group was compared
on the normal architecture of the articular cartilage, subchon- between day 2 and 1, 4, and 8 weeks. In the control group,
dral bone, and synovium, sham mice with administration there were significant differences between day 2 and 4 and
of these drugs were evaluated. Any changes including carti- 8 weeks (p < .05 respectively), while the apprepitant and sep-
lage degeneration and subchondral bone thickness or bone tide groups did not exhibit any significant difference between
atrophy were observed. The synovitis score of the aprepitant each time point. The ratio of the medial/lateral height of the
group was the highest and that of the septide group was sig- epiphysis in the control group was significantly lower than
nificantly lower than that of the control group at 1 week after that in the sham and septide groups (sham: 79.3 ± 4.5%,
surgery (p < .01). There was no significant difference of the control: 65.6 ± 9.4%, aprepitant: 70.2 ± 10.9%, and septide:
synovitis score in all groups at 4 and 8 weeks (Figure 4(c)). 79.7 ± 2.7%) at 8 weeks (Figure 5).
The subchondral bone score in the septide group at 8 weeks In the control group, osteocalcin was intensely expressed in
was significantly lowest among the three groups (p < .01). the subchondral bone, while it was expressed at lower levels
6 Shirakawa et al.
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Figure 4. The effect of aprepitant and septide administration on the OA progression in the DMM mice. (a) Safranin O staining of the knee joint of DMM
and sham mice. Scale bars represent 100 µm. (b) OARSI score of DMM and sham mice. There was no significant difference between the three groups
at 2 days post-surgery. The OARSI scores of the septide group were significantly lower than those of the control and aprepitant group at 1 week,
4 weeks, and 8 weeks post-surgery. (c) Synovitis score of DMM and sham mice at 1, 4, and 8 weeks. (d) Subchondral bone score at 1 week, 4 weeks,
and 8 weeks post-surgery. (e) The thickness of the subchondral bone plate at 2 days and 1, 4, and 8 weeks. **p < .01, *p < .05.
in the septide group (Figure 6). Instead, the septide group in the control group was significantly higher than that in
showed abundant TRAP-positive cells in the subchondral the septide group at 4 weeks and 8 weeks post-surgery, and
bone (Figure 6). The number of osteocalcin-positive cells the number of TRAP-positive cells in the control group was
Therapeutic effect of Substance P on progression of osteoarthritis 7
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Figure 5. m-CT analysis. (a) m-CT images of the knee joint from DMM mice. The broken line indicates measured area of BV/TV. (b) The ratio of the BV
and TV in the subchondral bone’s epiphysis. The BV/TV of the septide group was significantly lower than that of the control group at 1 week, 4 weeks,
and 8 weeks post-surgery. **p < .01, *p < .05. (c) m-CT images of the knee joint in coronal images. Arrows indicate the height of lateral (yellow) and
medial (red) epiphysis. (d) The ratio of medial/lateral height of the epiphysis in the tibia plateau.
significantly lower than that in the septide group at 4 weeks subchondral bone sclerosis, which suggests that SP regulates
post-surgery. Thus, SP seems to inhibit subchondral bone the balance of osteoblast and osteoclast differentiation in the
sclerosis through preventing osteoblast differentiation and subchondral bone.
increasing osteoclastogenesis. We investigated the expression pattern of SP in the artic-
Immunohistochemistry revealed that septide administra- ular cartilage of the human tibia. Healthy cartilage does
tion could suppress MMP-13 and ADAMTS-5 expression in not contain blood vessels and is not innervated by nerve
the articular cartilage, while the control OA mice exhibited fibres. However, despite the lack of innervation, cartilage
MMP-13 and ADAMTS-5 expression intensely as cartilage metabolism is modulated and influenced by neurotransmit-
degeneration progressed. The percentage of MMP-13-positive ters. Thus, evaluation of SP expression patterns in the articu-
cells in the control group was significantly higher than that in lar cartilage might be useful to understand OA pathogenesis
the septide group at 4 weeks and 8 weeks post-surgery. Addi- [8]. Nerves grow into joint structures through vascular chan-
tionally, the percentage of ADAMTS-5-positive cells in the nels from the subchondral bone and distribute toward the
control group was significantly higher than that in the septide articular cartilage [4, 10]. Previous reports showed that vas-
group at 4 weeks after surgery (Figure 7). cular channels invade the articular cartilage with nerve fibres
containing neuropeptides in the OA patients [21]. SP was
reported to express in the chondrocytes of the middle and deep
Discussion zones of normal human articular cartilage. However, it was
This study showed that SP is expressed in the articular carti- not expressed in the superficial zone [33]. In our study, SP
lage and subchondral bone, and its expression decreased in the was expressed in the superficial zone of healthy young car-
cartilage with the progression of OA in both human OA and tilage samples, but its expression decreased in the healthy
surgical mouse OA. The administration of an NK1R agonist aged sample. SP expression also gradually decreased from
could ameliorate OA progression through the inhibition of the superficial zone as OA progressed. The percentage of8 Shirakawa et al.
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Figure 6. The expression pattern of the osteoblast and osteoclasts. (a) Immunohistochemistry of OC in DMM and sham mice. (b) Rectangles indicate
the measured area for the cell number. (c) The number of OC-positive cells in the subchondral bone’s epiphysis of tibia. The number of OC-positive cells
in the control group was significantly higher than in the septide group at 4 weeks and 8 weeks post-surgery. Scale bars represent 100 µm. OC:
osteocalcin. (d) TRAP staining of DMM and sham mice. (e) Rectangles indicate the measured area for the cell number. (f) The number of TRAP-positive
cells in the subchondral bone’s epiphysis of tibia. The number of TRAP-positive cells in the septide group was significantly higher than that in the control
and aprepitant groups at 4 weeks post-surgery. There were no significant differences at 8 weeks. Scale bars represent 100 µm. **p < .01, *p < .05.
the SP-positive cells to total chondrocytes was decreased in compared to humans. In a previous report, immunocytochem-
the OA, which suggested that SP-positive cells are decreas- istry showed that the percentage of neurons expressing SP
ing and not total chondrocyte. In mouse samples, SP was and CGRP increased with age [33]. These findings are con-
expressed intensely during the initiation of cartilage degener- sistent with the hypothesis that an age-related change in joint
ation, and then its expression gradually decreased. The rapid innervation may contribute to the development of OA [7].
progression of OA in DMM mice might have resulted in dif- Thus, decreased expression of SP might affect the progression
ferences in SP expression patterns in the early phase of OA, of OA.Therapeutic effect of Substance P on progression of osteoarthritis 9
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Figure 7. Expression analysis of catabolic factors in the articular cartilage. (a) Immunohistochemistry of SP of the knee joint from DMM mice. SP
expression in the septide group was significantly higher than that in the control and aprepitant groups at 4 weeks and 8 weeks post-surgery. (b) (c)
Immunohistochemistry of MMP13 and ADAMTS-5 in DMM mice, respectively. MMP13 expression in the septide group was significantly lower than that
in the control group at 4 weeks and 8 weeks post-surgery. ADAMTS-5 expression was significantly lower in the septide group than in the control group
at 4 weeks post-surgery, but there were no significant differences at 8 weeks. Scale bars represent 100 µm. **p < .01, *p < .05.
We also examined the effect of SP on cartilage as an mice exhibited a significantly higher OARSI score than sham
anabolic factor. We proved that septide treatment could sup- mice at 12 weeks [40]. They concluded that SP is required
press MMP-13 and ADAMTS-5 expression compared to con- for bone and cartilage homeostasis, which suggests that
trol OA mice, which exhibited high MMP-13 and ADAMTS-5 aprepitant treatment induced severe OA changes. These
expression as cartilage degeneration progressed. Opolka et al. reports support our results that SP expression is impor-
demonstrated that chondrocyte proliferation increases in both tant to maintain the homeostasis of the osteochondral unit
monolayer and micromass pellet cultures in a dose-dependent in OA.
manner by the stimulation of exogeneous SP [34]. More- Previous reports showed that the administration of a
over, there were several reports on the positive effect of CGRP receptor antagonist could ameliorate OA progression
SP on cartilage degeneration in vivo. Hong et al. showed in DMM mice through the inhibition of subchondral bone
that SP has a novel ability to mobilize MSCs and modu- sclerosis, which indicates the importance of the subchondral
late injury-mediated inflammation, while also ameliorating bone in OA progression [26]. Specifically, this report suggests
collagen-II-induced arthritis in mice via the suppression of that neuropeptides could be a therapeutic target for subchon-
the inflammatory response [35]. In an OA animal study, Kim dral bone sclerosis prevention in OA. In this study, we focused
et al. reported that intra-articular injection of SP coupled on SP because SP has various functions for tissue healing such
with self-assembled peptide hydrogels markedly improved as restoration of stem cells, anti-inflammation, and immune
cartilage regeneration through the recruitment of MSCs [17]. modulation in addition to the bone metabolism [34, 41–43].
SP also has the ability to mobilize endogenous stem cells SP regulates bone remodelling where it stimulates osteoblast
from the bone marrow to injured sites and accelerate tis- and osteoclast differentiation and function in vitro. SP neu-
sue repair [36–39]. Recent studies have noted that SP plays rotransmitter release from sensory neurons could potentially
an important role in the wound-healing process by recruit- regulate local bone turnover [6, 13, 44]. In our study, DMM
ing bone marrow stem cells to the injured tissue. Jiang mice administered SP exhibited the best subchondral bone
et al. showed that SP treatment can enhance recovery from score, which suggests that SP may regulate bone remodelling
spinal cord injury in rats through its function of stem cell through the inhibition of increasing bone turnover. Besides,
mobilization and/or through the modulation of inflamma- a single administration of septide could inhibit OA change
tion [18]. These functions of SP may act together to pre- compared to the control and aprepitant groups. Septide might
vent cartilage degeneration in OA. Besides, Muschter et al. be able to ameliorate the damage to the subchondral bone
demonstrated that DMM mice of SP (tachykinin 1)-deficient by DMM at an initial phase, which could maintain the10 Shirakawa et al.
homeostasis of the osteochondral unit until 8 weeks. In the Acknowledgements
DMM model, the loading stress at the medial compartment We thank T. Miyata for her technical support.
increases due to the destabilization of the medial meniscus as
OA progress. Therefore, the height of the medial epiphysis
decreases with subchondral bone sclerosis [26]. In our study, Conflict of interest
the ratio of the medial and lateral height of the epiphysis in
None declared.
the septide group at 8 weeks was almost the same as that of
the sham mice, while the control group exhibited significantly
lower medial and lateral height of the epiphysis, which means Funding
that the administration of the septide could maintain the mor-
The author(s) disclosed receipt of the following financial
Downloaded from https://academic.oup.com/mr/advance-article/doi/10.1093/mr/roab089/6382327 by guest on 13 November 2021
phology of the epiphysis. Besides, the synovitis score in the
septide group was significantly lower than those in the control support for the research, authorship, and/or publication of
and aprepitant group at 1 week, although synovitis caused by this article: This research was supported by MEXT KAK-
DMM is usually stronger in an earlier phase. This is thought ENHI Grant-in-Aid for Scientific Research (C); Grant No.
to be due to the anti-inflammatory effect and immune modu- 18K09066 (T.N.). Research grant from the Nakatomi Foun-
lation of SP, which supported that blocking SP by aprepitant dation (T.N.).
induced the worst synovitis score at 1 week [41, 42]. From
these pieces of evidence, SP may function as the anabolic
factor to OA pathogenesis. The reason why the expression Author contributions
of SP was the highest after 2 days of DMM is thought to Study concepts and design: Y.S. and T.N.
be that the stimulation of DMM increased the number of Data acquisition, analysis, and interpretation: Y.S., M.K.,
cells expressing SP in an attempt to enhance the anabolic A.N., M.I., D.Y., S.M., and T.N.
effect. Various functions of SP may ameliorate the progression Drafting and proofreading of the manuscript: Y.S., T.N.,
of OA. and N.A.
There are several limitations in this study. First, only a
single dose of the NK1R agonist and antagonist was system-
ically administered to DMM mice. This OA model exhibited References
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