Abstract
Frozen shoulder (FS) is a common disorder often treated with Tuina, but
the mechanisms involved remain unclear. We employed proteomics and
phosphoproteomics to investigate the mechanisms associated with the
treatment of capsule fibrosis in FS rats. We used a method composed of
three weeks of cast immobilization to establish a model of FS. We then
administered Tuina once daily for 14 days, evaluated glenohumeral range
of motion (ROM), assessed histological changes, and identified
differentially expressed proteins (DEPs) using proteomics and
phosphoproteomics. This study demonstrated that Tuina could improve
glenohumeral ROM and reserve capsule fibrosis in FS rats. Proteomics
revealed proteins regulated by Tuina belonging to the PI3K-AKT and ECM
receptor interaction signaling pathways. Phosphoproteomics detected
differentially phosphorylated proteins regulated by Tuina to be
enriched in the MAPK signaling pathway. The combination of proteomics
and phosphoproteomics for Protein–Protein Interaction (PPI) network
analysis revealed that the phosphorylation of Myh3 and Srsf1 with a
node degree larger than the average degree were considered the central
regulatory protein modulated by Tuina to reverse capsule fibrosis.
Thbs1, Vtn, and Tenascin-W were significantly enriched in PI3K-AKT and
ECM receptor interaction signaling pathways and highly expressed in
model rats. Tuina resulted in reduced expression of these proteins. Our
findings demonstrated some of mechanisms behind the reversal of FS
capsule fibrosis following Tuina, a scientific medical therapy for FS
patients.
Subject terms: Proteomic analysis, Therapeutics
Introduction
Frozen shoulder (FS), also known as adhesive capsulitis of the
shoulder, is a multifaceted disease characterized by pain and
dyskinesia. Epidemiological research demonstrated an average onset age
of 50, ranging from 30 to 70 years old^[40]1. The prevalence of FS is
approximately 2–5%, and the primary risk factors are diabetes, thyroid
disease, stroke, and autoimmune diseases^[41]2–[42]4. Studies have
demonstrated that FS patients suffer from pain and reduced function,
seriously impacting their quality of life, with symptoms such as
insomnia and anxiety causing serious harm to their physical and mental
health^[43]5,[44]6.
FS is a debilitating disorder that can be cured in approximately
1–3 years following symptom onset, but occasionally symptoms do not
completely subside^[45]7. Treatment is necessary and helpful for FS
patients, with commonly used treatment approaches including
corticosteroid injection, physical therapy, and arthroscopic
surgery^[46]8. Complementary and alternative medicine (CAM) is commonly
employed for the treatment of FS^[47]9,[48]10. The traditional Chinese
medicine (TCM) approach, Tuina, is a CAM method widely used in the
clinical treatment of diseases that provides comfort, high patient
compliance, safety, and no side effects^[49]11. Clinical research
demonstrates that Tuina can effectively relieve pain, restore shoulder
function, and enhance the quality of life of FS patients^[50]12.
However, the associated mechanisms remain unclear. Our prior study
confirmed that Tuina has excellent effects on FS patients^[51]13.
Therefore, this study aims to reveal the some of mechanisms
underlying the effects of Tuina.
Proteomics and phosphoproteomics can uncover protein activity in cells
during disease progression, providing a theoretical basis for the
elucidation of disease occurrence and progression, including early
diagnosis and treatment^[52]14,[53]15. In TCM, these technologies can
be employed for the analysis of protein differences throughout various
syndromes of the same disease to enrich current scientific knowledge,
as well as reveal critical mechanisms for acupuncture and other TCM
treatment strategies^[54]16–[55]18. Our study represents an
unprecedented examination of Tuina reversal of FS-associated capsule
fibrosis via proteomic and phosphoproteomic analysis.
Results
The effectiveness of Tuina in improving glenohumeral range of motion and
reversing capsule fibrosis
The primary criterion for evaluating the effectiveness of Tuina in FS
is the measurement of glenohumeral range of motion (ROM) (Fig. [56]1a).
There were 4 groups in this study: the control group (C), the control
combined with the Tuina treatment group (CT), the FS model group (M)
and the FS model combined with the Tuina treatment group (MT).
The details of these groups can be seen in Grouping Method secition. We
observed that the average values of glenohumeral ROM were 147.9° ± 4.3°
in the C group, 148.5° ± 3.4° in the CT group, 113.9° ± 6.3° in the M
group, and 131.7° ± 4.7° in the MT group. As shown in the Fig. [57]1b,
The difference between the glenohumeral ROM of rats in C group and CT
group had no statistical significance (P > 0.05), and the ROM in the M
group was significantly lower than that in the C group (P < 0.0001).
Moreover, the ROM in the MT group and was significantly higher than
that in the M group (P < 0.0001). The mitigating effect of Tuina on
joint capsule fibrosis was observed through H&E staining and Masson
staining. As illustrated in Fig. [58]1, H&E staining and Masson
staining indicated the normal histological structure of the capsule in
the C and CT groups (Fig. [59]1c, d, g, h). H&E staining revealed
synoviocyte hyperplasia and synovial hyperemia in the M group, which
are typical features of FS (Fig. [60]1e). These features were reduced
to an extent after Tuina (Fig. [61]1f). Masson staining indicated the
arrangement of fiber bundles throughout each group. The capsule was
composed of a loose network of reticular fibers with fiber bundles
organized in a neat direction. In the M group, the fiber bundles
exhibited a disordered arrangement, suggesting capsule fibrosis
(Fig. [62]1i). The capsules of rats in the MT group indicated that the
fiber bundles were neatly and clearly stratified (Fig. [63]1j). These
results were consistent with the results of our previous
research^[64]19.
Figure 1.
[65]Figure 1
[66]Open in a new tab
The efficacy of Tuina in FS rats. (a) Measurement of glenohumeral ROM.
(b) Glenohumeral ROM across four groups of rats. Values are
means ± S.D., n = 10. Significant differences are indicated by one-way
ANOVA (ns P > 0.05,****p < 0.0001). (c,g) The normal capsule in C group
(H&E and Masson staining). (d,h) The normal capsule in CT group (H&E
and Masson staining). (e,i) The structure of the FS capsule in M group
as follows: synovial hyperemia and capsule fibrosis (H&E and Masson
staining). (f,j) The capsule is closely related to normal in MT group
(H&E and Masson staining). Scale bar = 100 µm. Red arrow: erythrocyte
stasis and vascular proliferation; Yellow arrow: fibrosis on
histological findings.
Tuina reverses capsule fibrosis in frozen shoulder as indicated via
protemocis
Tuina can reverse fibrosis of the FS capsule, but its mechanisms has
not been fully elucidated. In this study, some of mechanisms by which
Tuina reverses fibrosis of the capsule was uncovered via proteomics.
Before analysis, we tested the differences and repeatability of the
samples in C, CT, M and MT groups. Principal component analysis (PCA)
indicated that the degree of intragroup sample aggregation was high
with limited differences, while the degree of sample aggregation
between groups was low with pronounced differences (Fig. [67]2a). The
assessment indicated that the value of relative standard deviation
(RSD) is less than 0.2 which summarized samples in each group had
adequate quantitative repeatability (Fig. [68]2b). As illustrated in
Fig. [69]2c, we identified a total of 1,741,597 spectra based on 4D
Label-free quantitative proteomics, comprising 524,118 matched spectra.
A total of 28,465 peptides were identified, of which 25,728 represented
unique peptides. In addition, we identified 3,909 proteins, of which
3,048 were comparable proteins (Table S1). Differential expression
proteins (DEPs) analysis was conducted for k-means clustering (ANOVA
p-value < 0.05), and the thermogram was drawn according to the
clustering results (Table S2). This analysis characterized 1,820 DEPs
separated into 6 clusters (Fig. [70]2d). The proteins selection for
further analysis was based on the expression trends of DEPs: the
proteins exhibiting the same expression trend in the CT/C and MT/M
groups, but with an opposite expression trend in the M/C group were
chosen. We thus selected 799 DEPs from Clusters 4 and 5 for Mfuzz
expression clustering analysis.
Figure 2.
[71]Figure 2
[72]Open in a new tab
Proteomic findings and confirmation of key proteins. (a) PCA of
proteomics. (b) RSD of proteomics. (c) Mass spectrometry fundamental
statistics. (d) K-means clustering heatmap. (e) Mfuzz analysis of
differential protein expression across the four groups. The shades red
and blue represent high expression and low expression, respectively.
Enrichment analysis is indicated on the right side of the
illustrations.
As shown in Fig. [73]2e, following a Log2 ratio transformation, 525
DEPs with SD > 0.2 were chosen for Mfuzz expression clustering analysis
(Table S3, S4, S5 and S6). In Cluster 2, the DEPs of group M were
elevated compared to group C, and decreased following Tuina treatment.
In Clusters 3 and 4, the DEPs of the M group decreased compared to the
C group, and increased following Tuina administration. According to
prior results, a large amount of type I (COL-I) and type III (COL-III)
collagen deposition exists in the FS capsule tissue, mainly occurring
in the extracellular matrix (ECM), which is reversed by Tuina acting on
the ECM^[74]20–[75]22. Therefore, we focused on DEPs associated with
ECM regulation. In Cluster 2, the DEPs regulated by Tuina were enriched
in the extracellular matrix, with GO terms denoting
extracellular-matrix binding, extracellular-matrix structural
constituent and collagen binding (molecular function), extracellular
matrix organization, and extracellular structure organization
(biological processes). Further analysis revealed that enriched KEGG
pathways included the ECM-receptor interaction signaling pathway (Table
S7). These analyses confirmed that Tuina was linked to changes in the
ECM. Moreover, the PI3K-Akt signaling pathway showed the highest
enrichment in the KEGG analysis.
Tuina reverses capsule fibrosis in frozen shoulder as indicated via
phosphoproteomics
Protein phosphorylation is the most widespread protein modification
process, and revealing the protein phosphorylation modification process
in the reversal of FS capsule fibrosis via Tuina was accomplished
through phosphoproteomics. PCA and RSD analysis indicated that the same
results as proteomics, which means the samples in C, CT, M and MT
groups showed good duplication within groups, significant
differences between groups and adequate quantitative repeatability by
phosphoproteomics (Fig. [76]3a, b). We generated a total of 2,582
proteins with 7,901 phosphorylation modification sites were identified
through 4D Label-free quantitative phosphoproteomics (Fig. [77]3c).
These numbers reflect the overall identification and detection depth of
this project, and DEPs were screened based on comparable 7901
phosphorylation sites. In Fig. [78]3d (Table S8), of the 7,901
phosphorylation sites detected, 6,799 (86.0%) occurred on serine (pS),
984 (12.5%) occurred on Threonine (pT), and 118 (1.5%) occurred on
tyrosine (pY). Differential protein expression analysis was conducted
for k-means clustering (ANOVA p-value < 0.05), and the thermogram was
constructed according to the clustering results (Table S9). The trend
of expression within and between clusters following the enrichment of
DEPs was unclear, and the protein differences within clusters were too
large, making it unsuitable for preliminary protein screening
(Fig. [79]3e). Therefore, all 7,901 differential phosphorylation sites
were directly transformed via a Log2 logarithm, and the proteins with
SD > 0.5 were screened.
Figure 3.
[80]Figure 3
[81]Open in a new tab
Phosphoproteomic analysis. (a) PCA of phosphoproteomics. (b) RSD of
phosphoproteomics. (c) Mass spectrometry fundamental statistics. (d)
Statistics of phosphorylation on serine, threonine, and tyrosine. (e)
K-means clustering heatmap. (f) Mfuzz analysis of differential protein
expression across the four groups. The shades of red and blue represent
high expression and low expression, respectively. Enrichment analysis
is depicted on the right side of the illustrations.
Following screening, the remaining 916 phosphorylation sites were
employed for clustering analysis of expression patterns using the Mfuzz
method. Based on the trends of protein sites alterations, 7 clusters
were grouped and performed the enrichment analysis (Table S10, S11, S12
and S14). As illustrated in Fig. [82]3f, in Cluster 4, the DEPs of
group M increased relative to the C group, and decreased after Tuina
treatment. In Cluster 5, the DEPs of the M group decreased in contrast
with the C group, and increased following Tuina administration.
We focused on the bioinformatics results in these two clusters.
Representative results of KEGG enrichment analysis showed that Flna
(S2120), Map3k20 (S302), Egfr (S992), Cacna1s (T395, S393) and Mapk12
(T183) were enriched in MAPK signaling pathway. MAPK signaling pathway
also showed the highest enrichment in the analysis.
Integration of proteomics and phosphoproteomics for protein–protein
interaction networks analysis
Through Protein–Protein Interaction Networks (PPI), the proteomics and
phosphoproteomics results were combined to assess and identify the
central regulatory protein modulated by Tuina to reverse capsule
fibrosis. For DEPs from phosphoproteomics and proteomics results, the
difference threshold of significant upregulation was a differential
expression change of more than 1.5, and the change threshold of
significant downregulation was less than 1/1.5 to identify DEPs.
Predicting differentially expressed protein interactions via STRING. By
calculating the level of nodes and other parameters, we identified the
key proteins playing an important role in Tuina reversing capsule
fibrosis (Tables S15, S16, and S17). In this PPI network, the average
node degree of DEPs in the CT/C group was 3.41. The average node degree
of DEPs in the M/C group was 4.86. The average node degree of DEPs in
the MT/M group was 4.52. And the DEPs with a node degree larger than
the average degree were considered to have a substantial regulatory
effect. Our study further analyzed proteins larger than the average
node, and the findings indicated that there were a total of ten
overlapping proteins and phosphorylation sites across the CT/C, M/C,
and MT/M groups. In Table [83]1, Srsf1 (S199) and Csrp3 were
upregulated in the M/C group, downregulated in the MT/M group, and
downregulated in the CT/C group. Myh3 possessed four phosphorylation
sites (S1916, S1148, S949, and T379) that were downregulated in the M/C
group, upregulated in the MT/M group, and similarly upregulated in the
CT/C group.
Table 1.
Overlapping proteins in the CT/C, M/C, and MT/M comparison groups.
Gene encoding the protein Amino acid Site CT/C M/C MT/M
Site type Protein type Site type Protein type Site type Protein type
Neb T 49 Down Down Down
Neb S 45 Down Down Down
Srsf1 S 199 Down Up Up Down Up
Myh3 T 379 Up Down Up Up Down
Myh3 S 1916 Up Down Up Up Down
Myh3 S 1148 Up Down Up Up Down
Myh3 S 949 Up Down Up Up Down
Myh7 T 446 Up Up Down
Unknown
Csrp3 Down Up Down
[84]Open in a new tab
Validation of key proteins characterized via in proteomics research through
qPCR and WB
Although multiple DEPs were found in proteomics and phosphoproteomics,
we chosed to use DEPs based on previous literature. In prior
RNA-seq-related studies, it has been determined that FS was closely
related to the PI3K-AKT signaling pathway^[85]23. The findings of the
KEGG pathway enrichment analysis of proteomics suggested a total of 9
DEPs enriched in PI3K-AKT signaling pathway. Of these, Thbs1, Vtn, and
Tenascin-W were enriched in the ECM-receptor interaction signaling
pathway and PI3K-AKT signaling pathway at the same time. Therefore, we
selected these three proteins for further examination.
In Fig. [86]4a–c, real-time quantitative polymerase chain reaction
(qPCR) results demonstrated that the mRNA expression of Thbs1, Vtn, and
Tnn in the M group was significantly up-regulated (p < 0.01, or
p < 0.0001), and was inhibited following Tuina administration (p
< 0.05, p < 0.01 or p < 0.0001). In the normal capsule, Tuina could
also play the same regulatory role (p < 0.01, p < 0.0001). In
Fig. [87]4d–f, further examination through Western Blot analysis (WB)
demonstrated a significant upregulation of Thbs1 and Vtn proteins in
the M group (p < 0.001, p < 0.0001) and inhibition following Tuina
treatment (p < 0.05, p < 0.01).
Figure 4.
[88]Figure 4
[89]Open in a new tab
Validation of key proteins. (a–c) The expression of Thbs1, Vtn, and Tnn
genes was quantified by qPCR. (g–i) Western blot analysis for Thbs1 and
Vtn proteins. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001. N = 3
rats/group. Original image of Western blot analysis was shown in the
Supplementary Fig. 1.
Discussion
The most common clinical symptom of FS is the limited active and
passive movement of the shoulder joint in all directions, which is
difficult to overcome even with the use of pain relief drugs^[90]24.
This illustrates that this limitation is predominantly caused by
pathological changes in the shoulder joint, with pain being an
important limiting factor of the resulting movement. Physiotherapy is
commonly applied in the treatment of FS with satisfactory therapeutic
results^[91]25. In traditional Chinese medicine, physiotherapy is often
known as Tuina, which plays a crucial role in the treatment of FS.
However, some studies have indicated that physical therapy cannot
relieve persistent pain and suggested a steroid injection as primary
therapy for FS^[92]26. Here, we investigated the underlying mechanisms
of Tuina in reversing capsule fibrosis, providing a more reliable basis
for future Tuina treatment. The treatments used in this study are based
on our previous clinical and experimental researches^[93]13,[94]19. In
this study, we confirmed that Tuina can improve the shoulder joint
function and reverse capsule fibrosis in the FS rat model.
In early FS stages, inflammation surrounding the capsule is the main
contributing factor to shoulder pain, and with the continuous disease
progress, the contracture and fibrosis of the capsule eventually result
in limited activity of the shoulder joint and, ultimately, capsule
fibrosis^[95]27. The capsule is composed of a thin inner synovial
membrane and connective tissue with more fibers in the outer layer.
Fibroblasts are the primary cell components in this connective tissue,
producing ECM following tissue damage^[96]28. Pathological results
indicated that Tunia reduces FS-related capsule fibrosis in rats, and
we therefore focused on ECM regulation by Tuina in our study.
In contrast with traditional proteomics technology, 4D label-free
quantitative proteomics is able to more quickly and efficiently
identify and quantify various proteins, allowing an opportunity to
investigate the mechanisms of Tuina therapy and improve clinical
practice^[97]29. In this study, we analyzed the expression trends of
DEPs across each group to assess the proteins directly regulated by
Tuina. GO enrichment revealed that FS was closely associated with the
biological process of ECM regulation (cluster 2) and that Tuina
inhibits this process. KEGG pathway enrichment analysis showed that
PI3K-AKT and ECM-receptor interaction were significantly enriched
following Tuina in the FS rat model. We note this pathway is implicated
in cell survival, growth, and apoptosis and regulates downstream
targets, such as rapamycin (mTOR), hypoxia-inducible factor-1α
(HIF-1α), and the FOX family^[98]30. Previous research indicated that
IL-6 promotes AKT phosphorylation, activates the PI3-AKT signal
pathway, increases the expression of COL-1 and COL-3, and results in
the deposition of ECM and fibrosis in FS^[99]31. ECM-receptor
interaction is a microenvironment pathway maintaining the structure and
function of cells and tissues, which is located upstream of the
PI3K-AKT pathway to act on it through various genes to generate
different biological effects^[100]32,[101]33. Moreover, genes linked to
ECM-receptor interaction can result in collagen deposition in the ECM
and produce fibrosis^[102]34. In this experiment, we identified that
the genes Vtn, Thbs1, and Tnn were significantly enriched in the
ECM-receptor interaction and the PI3K-AKT pathways and that the
expression of these genes was increased in the FS rat model but could
be limited by Tuina. We thus propose that Vtn, Thbs1, and Tenascin-W
are target proteins in FS Tuina treatment.
Vtn, Thbs1, and Tenascin-W are important constituents of the
extracellular matrix. Vtn is a glycoprotein in the extracellular matrix
that is highly expressed in fibrotic tissue^[103]35. Vtn can mediate
the regulation of collagens and elevate the expression of TGF-β to
encourage fibrosis^[104]36. Similarly, Tenascin-W is a glycoprotein of
the ECM, with four isoforms identified in mammals: Tenascin-C, -R, -W
(-N), and -X. Tenascin-W is encoded by Tnn in rats, with a specific
similarity to Tenascin-C being closely related to fibrosis,
inflammation, and cancer^[105]37. Tenascin-W is also closely related to
ECM production and tissue fibrosis^[106]38. Finally, Thbs1 is present
in the ECM and can be secreted by various cell types, combining with
ECM ligands to encourage TGF-β1 signaling in fibroblasts to form
fibrotic tissue^[107]39. Thbs1 can be secreted by cyclic stretching
force, bind with integrin receptors, transmit mechanical signals via
YAP, and regulate the perception of the mechanical microenvironment of
the ECM^[108]40,[109]41. Vtn, Tenascins-w, and Thbs1 have not been
previously reported in association with FS. Our qPCR findings confirmed
that the mRNA levels of these genes were highly expressed in the FS
model and suppressed by Tuina treatment in both normal and FS rat
models. These observations were verified by Western Blot expression
analysis of Vtn and Thbs1. However, due to a lack of antibodies, we did
not analyze the expression of Tenascin-W. The specific functions of
these proteins require further validation.
Phosphoproteomics was employed further to examine the mechanism of
Tuina intervention on capsule fibrosis. The KEGG pathway enrichment
analysis indicated that the DEPs modulated by Tuina had the highest
level of enrichment in the MAPK pathway. The MAPK pathway is a
classical pathway that includes four branching pathways: ERK, JNK, p38,
and ERK5^[110]42. The study suggested that FS is closely linked to the
abnormal mechanical force of the shoulder joint, and that mechanical
force can increase the expression of MAPK molecules via integrin-β,
with p44/42 MAPK (ERK) and JNK MAP being significantly increased in the
capsule of FS patients^[111]43. p38 MAPK was not significantly
expressed in the capsule, but P-p38 was significantly overexpressed in
the hyperplastic synovium, suggesting that p38 participated in capsule
fibrosis through phosphorylation^[112]43. Prior studies have indicated
that Tuina can suppress inflammatory responses and alleviate pain by
inhibiting the phosphorylation of p38^[113]44. This study determined
that Tuina could promote the phosphorylation of diverse proteins and
reverse capsule fibrosis through the MAPK pathway.
PPI network analysis indicated that the phosphorylation of Srsf1 and
Myh3 played a role in the Tuina intervention of FS. This study found
that in the FS model, the phosphorylation level of Srsf1 increased, and
Tuina could reduce its phosphorylation, while the phosphorylation of
Myh3 was inhibited at four sites, which Tuina could promote to
intervene in frozen shoulders. Serine/arginine-rich splicing factor 1
(Srsf1) is a shear factor that primarily participates in mRNA splicing.
Srsf1 belongs to the SR family of histones, and the in vivo activity of
SR family proteins relies upon their phosphorylation state^[114]45. In
fibroblasts, Srsf1 promotes the production, differentiation, and
collagen deposition of EDA + Fn1 subtypes, playing a critical role in
the formation of ECM, and is an important target for intervention in
fibrosis-related diseases^[115]46. Myosin is a hexameric protein
composed of a pair of myosin heavy chains and two pairs of light
chains. It is a major contractile protein, which converts chemical
energy into mechanical energy through ATP hydrolysis. Myosin heavy
chain 3 (Myh3) is a member of the MYH family, which encodes a protein
containing an IQ domain and myosin head domain^[116]47. It is highly
expressed throughout embryonic and fetal development, and is an
important part of myofilaments in skeletal muscle and nonmuscle
cells^[117]48. Past studies have focused on the correlation
between Myh3 and muscle-related disorders, but the role of Myh3 in FS
has not been reported^[118]49. Our study found that Tuina could reverse
capsule fibrosis by promoting Myh3 phosphorylation at S1916, S1148,
S949, and T379, which indicated that Myh3 phosphorylation plays a
crucial role in the mechanism of the treatment of Tuina therapy in FS.
Therefore, the mechanism of Myh3 requires further study.
Conclusion
Collectively, our results uncovered that Tuina could reverse capsule
fibrosis. In addition, we elucidated some of mechanisms behind the
reversal of FS following Tuina therapy, an effective complementary
strategy for FS patients.
Materials and methods
Ethical policy
The study received approval from the Experimental Animal Ethics
Committee of the Affiliated Hospital of Shandong University of
Traditional Chinese Medicine (Number: AWE-2022-023). This study was
conducted in accordance with relevant guidelines and regulations. All
authors complied with the ARRIVE guidelines.
Experimental animals
Pathogen-free male Sprague Dawley rats (aged seven weeks) weighing
250 g were procured from the Beijing Vital River Laboratory Animal
Technology Co., Ltd under the animal use license number of SYXK (Lu)
20170022. All rats were raised in the Experimental Animal Center of the
Affiliated Hospital of Shandong University of Traditional Chinese
Medicine. The standard laboratory conditions consisted of alternating
12-h cycles of light and dark, a temperature of 20–24 °C, and 40–60%
relative humidity. This study was performed in strict accordance with
the recommendations in the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health.
Animal modeling
Following seven days of routine feeding, 42 rats were randomly
separated into normal and model groups. The rats in the model group
were anesthetized via intraperitoneal injection of tribromoethanol
(250 mg/kg). Thereafter, the entire right arm, including the shoulder
and chest, was immobilized using a plaster bandage^[119]50, which was
removed after the right shoulder had been fully adducted and internally
rotated for a period of 21 days. The procedure included proper
immobilization, but the rats could self-feed and locomote normally.
After modeling, the physical activity of rats was examined to assess
the FS model. The stiffness of the right shoulder joint, and
contracture signals in the right forelimb, accompanied by muscle
atrophy, and an unsteady gait, were observed in the model organism.
These characteristics confirm the successful induction of FS. In this
study, the modeling success rate was 100%, achieved through the
observation of physical activities. Each group contributed one rat for
histopathological observation to further confirm the success
of the FS model ().
Grouping method
After evaluation, the rats of the normal group were randomly separated
into the C group (n = 10) and the M group (n = 10). The model group
rats were randomly separated into the M group (n = 10) and the MT group
(n = 10). From days 22 through 35, we administered Tuina to the animals
of groups CT and MT once daily. The groups of rats, including the C
group and M group, were held and fixed, with the time matching the
duration of the Tuina operation.
Tuina treatment method
Tuina was performed according to our previous research^[120]19. Before
administering Tuina, the operator held the rat in his left hand for
2 min. This ensured the animals were calm prior to the manipulation.
Tuina administration was as follows: Firstly, the operator kneaded the
right shoulder, scapula, and humerus muscles of the rat with his right
thumb for 3 min. The operator then point-pressed the four acupoints of
Jianyu, Jiquan, Tianzong, and Quchi with the vertical point of the
thumb 30 times per acupoint. Finally, the right scapula of the rat was
fixed with the left thumb, the forelimb was held with the right hand,
and the shoulder joint was stretched for 10 s in the adduction,
abduction, anterior extension, and posterior extension positions.
Measurement of glenohumeral ROM
After the animals were sacrificed, the right scapula and proximal
two-thirds of the humerus were removed. A thin thread is attached to an
injection needle inserted into the humeral shaft and pulled at the
other end with a 5 g force to make it parallel to the humeral shaft.
The angle between the lower edge of the scapula and the humeral shaft
is measured as glenohumeral ROM.
Sample extraction and preservation
After measurement, a total of three samples from each group were fixed
in 4% paraformaldehyde for three days, followed by decalcification in
an EDTA solution (pH 7.2) for two months. The capsules of the remaining
samples were dissected from the joint and stored at − 80 °C.
Histology evaluation
Specimens were embedded in paraffin and cut into 5 μM thick slices.
Standardized 5-μm thickness sections were stained using hematoxylin and
eosin (H&E) and Masson solutions. The H&E staining methods included
dewaxing and hydration, hematoxylin staining, eosin re-staining,
dehydration, and clearing, followed by sealing. Masson staining
includes dewaxing and hydration; staining with Bouin, azure blue, Mayer
hematoxylin Fuchsin, Phosphomolybdate Acid, and Aniline Blue;
dehydration and clearing, followed by sealing. Images were collected
using a fluorescence inverted microscope (Leica, Leica DMIL LED).
4D label-free proteomic analysis
A total of 16 capsule samples (4 per group) were ground with liquid
nitrogen into cell powder and transferred to a 5-mL centrifuge tube.
After this, four volumes of lysis buffer (1% SDS, 1% protease inhibitor
cocktail, and 1% phosphorylase inhibitor) were added to the cell
powder, followed by sonication (220W, with a pulse of 3 s on and 5 s
off, 3 min) on ice using a high-intensity ultrasonic processor
(Scientz). The remaining debris was eliminated through centrifugation
at 12,000 × g at 4 °C for 10 min. Finally, the supernatant was
collected, and the protein concentration was determined using the BCA
kit (Beyotime) following the manufacturer’s instructions. An aliquot of
20 μL of sample per lane was separated through SDS-PAGE and stained
with Coomassie Blue.
An equal volume of each protein sample was adjusted to be consistent
with the lysate. One volume of 4 °C precooled acetone was included with
the protein solution, as well as 4 volumes of precooled acetone
following vortex mixing. The mixture was precipitated at − 20 °C for
2 h. The precipitate was eliminated by centrifugation at 4500 × g for
5 min and washed twice with precooled acetone. After drying, 200 mM
Tetraethylammonium bromide (TEAB) was added to the precipitate and
dispersed using a high-intensity ultrasonic processor. A 1:50
trypsin-to-protein mass ratio was then used for digestion overnight.
After digestion, the protein solution was reduced using 5 mM
dithiothreitol for 30 min at 56 °C and alkylated using 11 mM
iodoacetamide for 15 min at room temperature and maintained in the
dark.
After being dissolved in solvent A (0.1% formic acid, 2% acetonitrile
in water), the tryptic peptides were separated using the EASY-nLC 1200
Ultra Performance Liquid Chromatography system (Thermo Fisher
Scientific). The flow rate was maintained at 500 nL/minute, where the
mobile phase B consisted of a solution containing 0.1% formic acid and
90% acetonitrile in water. The gradient settings were: 0–68 min, 6% to
23% B; 68.0–82.0 min, 23% to 32% B; 82.0–86.0 min, 32% to 80% B;
86.0–90.0 min 80% B. The peptides were separated by an ultra-high
performance liquid phase system and injected into an NSI ion source for
ionization followed by mass spectrometry using an Orbitrap Exploris™
480 (Thermo Fisher Scientific). The ion source voltage was set to
2.3 kV, and the peptide parent ions and their secondary fragments were
detected and analyzed using a high-resolution Orbitrap. The primary
mass spectrometry scan range was set to 400 to 1200 m/z, the scan
resolution was set at 60,000, while the scanning range of secondary
mass spectrometry was fixed at 110 m/z, the resolution of secondary
scanning was set to 15,000, and TurboTMT was turned Off. The data
acquisition mode adopted a data-dependent scanning (DDA) program (the
top 25 peptide parent ions sequentially entered into the HCD collision
pool for fragmentation using 27% of the fragmentation energy). The
secondary mass spectrum data was retrieved by the Proteome Discoverer
(v2.4.1.15).
The raw LC–MS datasets were first searched against database and
converted into matrices containing LFQ intensity (the raw intensity
after correcting the sample/batch effect) of proteins. The LFQ
intensity (I) was transformed to the relative quantitative value (R)
after centralization. The formula was listed as follow where i
represented sample and j represented protein:
[MATH: Rij=Iij/MeanIj :MATH]
1
We employed one-way analysis of variance (ANOVA) to screen all
differentially expressed proteins (DEPs) with p < 0.05 for K-means
clustering. The DEP expression trends were used to divide the
differential proteins into multiple clusters and selection for further
analysis. Following Log2 ratio transformation, DEPs with SD > 0.2 were
selected for Mfuzz clustering analysis. The clustering results were
used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
(KEGG) enrichment analyses. Significant enrichment was established if
p < 0.05 using a Fisher's test. The process of GO annotation involves
using the eggnog-mapper software (v2.1.6) to extract GO IDs from the
identified proteins based on the EggNOG database, and then performing
functional classification annotation analysis on the proteins according
to cellular components, molecular functions, and biological
processes^[121]51..Protein pathways based on the KEGG pathway database,
and identify proteins through BLAST comparison (blastp, evalue ≤ 1e−4),
for each sequence, the annotation is based on the top-scoring
comparison result^[122]52,[123]53.
4D label-free phosphoproteomics analysis
The protein extraction and trypsin digestion process were the same as
those in proteomics. Peptide mixtures were incubated with an IMAC
material with vibration in a loading buffer (50% acetonitrile and 0.5%
acetic acid). To remove non-specifically adsorbed peptides, the IMAC
materia were rinsed with 50% acetonitrile/0.5% acetic acid and 30%
acetonitrile/0.1% trifluoroacetic acid solutions, sequentially. An
elution buffer containing 10% NH[4]OH was added to elute the enriched
phosphopeptides, and the enriched phosphopeptides were eluted through
vibration. The supernatant containing phosphopeptides was obtained and
lyophilized for LC–MS/MS analysis. The same process and parameter
settings as in proteomics were employed in LC–MS/MS analysis, except
the following gradient settings were used: 0–70 min, 3% to 18% B,
70.0–82.0 min, 18% to 28% B; 82.0–86.0 min, 28% to 80% B;
86.0–90.0 min, 80% B. The peptide segments are separated by an
ultra-high performance liquid phase system and injected into the NSI
ion source for ionization, before entering Orbitrap Exploris™ 480
(Thermo Fisher Scientific) was used for analysis. The ion source
voltage is set to 2.2 kV, the FAIMS compensation voltage (CV) is set to
−65 V, −45 V, and the peptide parent ions and their secondary fragments
are detected and analyzed using high-resolution Orbitrap. The
resolution of the secondary scanning was 30,000.
The raw LC–MS datasets were first searched against database and
converted into matrices containing intensity of peptides across
samples. The relative quantitative value of each modified peptide was
then calculated based on these intensity information by the following
steps: Firstly, the intensities of modified peptides (I) were
centralized and transformed into relative quantitative values (R).
Formula wa listed as follow where i denoted the sample and j denoted
the modified peptid:
[MATH: Rij=Iij/Mean(Ij) :MATH]
2
If both Proteomics and Post-translational modification profiling were
conducted on the same cohort, the relative quantitative value of the
modified peptide was usually divided by the relative quantitative value
of corresponding protein to remove the influence from protein
expression of modifications.Then identical bioinformatic analysis was
employed as in proteomics for phosphoproteomics analysis.
Integration of proteomics and phosphoproteomics for PPI analysis
The quantitative analysis results of proteomics and phosphoproteomics
was analyed by PPI analysis. Three or more repetitions: Firstly, the
samples to be compared were selected in pairwise, and the fold change
(FC) was calculated as the ratio of the mean intensity for each protein
or modification site in two sample groups. For example, to calculate
the fold change between sample A and sample B, the formula was listed
as following wher R denoted the relative quantitative value of the
proteins or modification sites, i denoted the sample and k denoted the
proteins or modification sites:
[MATH: FCA/B,k
=Mean(Rik,i∈A)/Mean(Rik,i∈B) :MATH]
3
To calculate the significance of the difference between groups,
student's T test was performed on the relative quantitative value of
each protein or modification site in the two sample groups. P
value < 0.05 was considered as significant. The relative quantitative
value of proteins or modification sites was log2 transformed. The
formula is listed as following:
[MATH: Pik=T.test(Log2(Rik,i∈A),Log2(Rik,i∈B)). :MATH]
4
The proteins or modification sites with P value < 0.05, the fold
change > 1.5 was regarded as significantly up-regulated protein, while
P value < 0.05, the fold change < 1/1.5 was regarded as significantly
down-regulated protein. After comparisons to the STRING protein network
interaction database, proteins or modification sites with a fold
change (FC) > 1.5 (or < 0.6667) and p < 0.05 for interaction
relationships with confidence score > 0.7 (high confidence) were
extracted.
RNA extraction and real-time quantitative polymerase chain reaction
Total RNA was extracted from rat shoulder capsules using SparkZol. The
reverse transcription reactions were performed using a SPARKscript II
RT Plus Kit. Real-time PCR was conducted using a 2 × SYBR Green qPCR
Mix. The database was acquired using a LightCycler 480 real-time PCR
instrument (Roche). The primer sequences are denoted in Table [124]2.
Table 2.
The primer sequences used for qPCR.
Gene name Primer name Bidirectional primer sequence
5′–3′ Product length /bp
GAPDH GAPDH-F CATGACCACAGTCCATGCCA 104
GAPDH-R CAGGGATGATGTTCTGGGCT
Tnn Tnn-F AAGCGTTGGCGGAGTTATGTAGAAG 148
Tnn-R CGTAGGCGGATTCATTGGCAGTC
Thbs1 Thbs1-F CCGCCGATTCCAGATGATTCCTC 120
Thbs1-R GCAAGTCCAGGGTCACAGTTTACAG
Vtn Vtn-F CAGCAGGGATTGGCATGGTGTAC 148
Vtn-R TCCTCGGCGTGAACGGTAGC
[125]Open in a new tab
Western blot analysis
Proteins were pulverized using liquid nitrogen. After lysate
centrifugation, the supernatant was examined using a bicinchoninic acid
(BCA) assay kit (Thermo Fisher Scientific, Rockford, IL, USA). The
protein samples were separated through electrophoresis and transferred
to a PVDF membrane. The membranes were incubated with primary
antibodies targeting Thbs1 (Bioss, bs-2715R, 1:500), Vtn (proteintech,
15833-1-AP, 1:6000), or β-Actin (bioss, bs-0061R, 1:2000), and diluted
in a blocking solution overnight at 4 °C. This was followed by
incubation with Goat Anti-Rabbit IgG (H + L) HRP (Sparkjade, EF0002,
1:5000). The protein bands were assessed using a Tanon 5200 imaging
analysis system (Tanon Science & Technology Co. Ltd., Shanghai, China).
Density analysis of relative protein levels was conducted using Image J
software.
Statistical analysis
SPSS 26.0 was utilized for statistical analysis. Differences between
groups were analyzed by one-way analysis of variance (ANOVA). A value
of p < 0.05 was considered to be statistically significant.
Supplementary Information
[126]Supplementary Tables.^ (19MB, zip)
[127]Supplementary Figures.^ (220.3KB, pdf)
Acknowledgements