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Effects of glutaminase 1 inhibitor on rotator cuff derived cells: a preliminary report in 12 patients

BMC Musculoskeletal Disorders volume 26, Article number: 935 (2025) Cite this article

Abstract

Background

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Glutaminase 1 (GLS1) degrades glutamate into glutamine and ammonia, and is required for the survival of senescent human cells. GLS1 inhibitors contribute to the improvement of various pathological conditions associated with aging. Rotator cuff tears (RCT) increase with age, and recently the Stump classification has been proposed to evaluate the fragility of the torn rotator cuff site, with Type 3 being the most fragile and a high risk of re-tear after rotator cuff repair surgery. We hypothesized that GLS1 expression is upregulated in the degenerated rotator cuff and that GLS1 inhibitors would improve rotator cuff degeneration. In this study, we evaluated the effects of GLS1 inhibitors on human rotator cuff-derived cells.

Methods

Twelve patients who underwent surgery for RCT were included in this study. Rotator cuff tissue was harvested during arthroscopic repair for tissue and cell evaluation. Tissue evaluation involved quantitative assessment of mRNA expression of GLS1 using real-time PCR (qPCR) and immunostaining. Rotator cuff-derived cells were isolated and cultured, divided into four groups: (1) Control group (without IL-1β and GLS1 inhibitor), (2) IL-1β(-)/GLS1 inhibitor(+), (3) IL-1β(+)/GLS1 inhibitor(-), (4) IL-1β(+)/GLS1 inhibitor(+). Cell viability was evaluated by WST assay and mRNA expression was evaluated by qPCR at 48 h after treatment. The expression of p16 and Scleraxis (SCX) was also evaluated by fluorescent immunostaining.

Results

Tissue evaluation showed significantly higher expression of GLS1 in Stump classification Type 3. Cell viability was significantly decreased by IL-1β loading and increased by the GLS1 inhibitor. The mRNA expression levels of GLS1, IL-6, p16 and p21 were decreased by the GLS1 inhibitor. The mRNA expression of the tendon markers, type 1 collagen, Mohawk and SCX were increased by the GLS1 inhibitor. Immunostaining revealed that the GLS1 inhibitor decreased p16 expression and increased SCX expression.

Conclusions

This study showed that GLS1 was upregulated in the degenerated rotator cuff, and that the administration of a GLS1 inhibitor decreased inflammation and aging markers while increasing cell viability and tendon markers in rotator cuff-derived cells. These results indicate that GLS1 inhibitors exert anti-inflammatory effects in rotator cuff tears, prevent age-related degeneration of the rotator cuff, and promote tendon repair.

Peer Review reports

Background

Glutamine is the most abundant amino acid in the circulatory system and functions as a major nitrogen transporter between organs [1, 2]. Glutaminase (GLS) is an enzyme that breaks down glutamine into glutamate and ammonia [3]. Two isozymes of GLS have been identified, namely, GLS1, which is expressed in most tissues, and GLS2, mainly expressed in the liver [4,5,6]. Glutamate, metabolized through GLS1, is supplied to the tricarboxylic acid (TCA) cycle occurring in the mitochondria and is involved in the synthesis of nucleotides, amino acids, and lipids [5]. GLS1 is reported to be involved in various cancer cell proliferation and is a target for molecular targeted therapy [6,7,8,9]while it has recently been linked to aging. Johmura et al. reported that GLS1 is highly expressed in senescent cells due to lysosomal membrane damage; that is when intracellular pH is reduced due to this damage, GLS1 is expressed to promote glutamine breakdown, producing ammonia which neutralizes the low pH, improving the survival rate of senescent cells [10]. Therefore, GLS1 is thought to be required for the survival of senescent cells. GLS1 inhibitors have been reported to improve renal dysfunction, pulmonary fibrosis, serum albumin concentration, decreased grip strength associated with aging in aged mice, and glucose tolerance and insulin sensitivity in obese mice [10]. Choudhury et al., reported that tissues isolated from aged mice displayed increased GLS1 levels concomitant with decreased mitochondrial function, and that glutaminolysis inhibition in aged mice improved mitochondrial respiratory chain activity [11]. Thus, GLS1 inhibitors may ameliorate age-related pathologies. Increased expression of GLS1 was observed in the synovial cells of patients with rheumatoid arthritis, and GLS1 inhibitors improved arthritis in a mouse model of rheumatoid arthritis [12].

GLS1 is also implicated in aging and inflammation, with speculation that GLS1 inhibitors may improve pathologies associated with them. For example, the incidence of rotator cuff tears (RCT) increases with age [13]and that 1/4 and ½ of those aged > 60 and > 80 years, respectively, have RCT [14]. Advanced glycation end products (AGEs) are aging-related substances that produce reactive oxygen species (ROS) and accumulate in the rotator cuff, contributing to its fragility [15]. Increased ROS is known to produce inflammatory cytokines via activation of NF-κB [16] suggesting that GLS1 may be linked with RCT. However, only a few reports have been published regarding the relationship between GLS1 and RCT or inflammation. Recently, the Stump classification (Types 1–3) was proposed for evaluating the degree of fragility and degeneration of the rotator cuff in RCT cases with Type 3, which has the highest degree of fragility and degeneration, being identified as having a high risk of retear after rotator cuff repair [17]. The Stump classification is based on the signal intensity ratio between the rotator cuff tear site and deltoid muscle in the coronal view of T2-weighted fat-suppressed magnetic resonance imaging (MRI). Type 1 showed higher signal intensity for the deltoid. Type 2 showed the same signal intensity for the deltoid and rotator cuff tear sites. Type 3 showed higher signal intensity for the rotator cuff tear site. Shinohara et al.., reported that Stump classification Type 3 exhibited the highest accumulation of AGEs, oxidative stress and apoptosis levels, and low cellular viability in the tissue of rotator cuff tear sites [18]. In this study, we hypothesized that a GLS1 inhibitor could improve rotator cuff degeneration and inflammation, Thus, we investigated the relationship between GLS1 and Stump classification, and evaluated the anti-degenerative and anti-inflammatory effects of GLS1 inhibitors on the rotator cuff.

Methods

Prospective cohort study

The Ethics Committee of our institute approved this study. Informed consent was obtained from all patients involved. Patients with degenerative RCT were included in the study (exclusion criteria were traumatic RCTs, rotator cuff retears, and rheumatoid arthritis). Thus, 12 patients (Stump Type 1:6 cases, Stump Type 3:6 cases, four men and eight women; mean age:60.3 years; range 50–74), who underwent surgical treatment for RCT were included in this study. Based on a power analysis using G*Power 3.1. with an assumed effect size of 0.4 (equivalent to a large effect), α = 0.05, and a statistical power of 0.8, the required total sample size was estimated to be 13 participants. However, due to practical limitations, 12 participants were ultimately enrolled in the study. The patients’ backgrounds are shown in Table 1. Patients with diabetes mellitus were defined as those with a preoperative hemoglobin A1c level of 6.5% or higher, according to the WHO diagnostic criteria. Rotator cuff tear size was defined as small tears (< 1 cm), medium tears (1–3 cm), large tears (3–5 cm), and massive tears (> 5 cm). Fatty degeneration was defined regarding the Goutallier classification, with grade 0 indicating no fatty infiltration; grade 1, some fatty streaks in the supraspinatus; grade 2, less fat than muscle; grade 3, equal amounts of fat and muscle; and grade 4, more fat than muscle.

Table 1 Patient background characteristics
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Isolation of samples from the shoulder

Stump classification was based on a report by Ishitani et al. [17]. Rotator cuff tissue samples were harvested intraoperatively, and tissue specimens were excised by the same surgeon (Y. M.). For histological staining, tissue samples were fixed in formalin for 24 h and the remaining samples were minced to approximately 1 mm3 and cultured.

Tissue evaluation

Histological examination and immunohistochemistry

After formalin fixation for 24 h, tissues were dehydrated and embedded in paraffin wax. Paraffin specimens were sliced to 5 μm sections and mounted on slides, and hematoxylin and eosin (H&E) staining and immunostaining were performed [16]. For immunostaining, the sections were deparaffinized, dehydrated, and incubated with the primary antibodies anti-GLS1 (ab156876, Abcam, 1:100) overnight at 4 °C. Next, the sections were incubated with the secondary antibodies (Histofine® Simple Stain MAX Po; Nichirei Biosciences Inc., Tokyo, Japan) for 1 h at room temperature (25℃) and counterstained with H&E. Digital images of slides were obtained using a BioZero BZ-8000 microscope (Keyence, Osaka, Japan). To evaluate the immunohistochemistry, the histoscore (H-score) was calculated. This semi-quantitative analysis scored staining on a scale from 0 to 300; the H-score was calculated by multiplying the staining intensity (0, 1+, 2+, 3+) with the percentage of positive cells [19].

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of tissue

Tissues were enzymatically digested with type II collagenase for RNA isolation [20]. Total RNA was reverse transcribed into single-stranded cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). RT-PCR was performed in triplicates using an Applied Biosystems 7900HT fast real-time PCR system and SYBR Green reagent (Applied Biosystems) to analyze the mRNA expression levels of GLS1. The primer sequences were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA) (Table 2). Gene expression was normalized to that the mRNA level of GAPDH, and the stability of GAPDH expression was tested under all conditions.

Table 2 Primers for real time polymerase chain reaction (RT-PCR)
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Cell culture

The minced tissue samples were cultured in a monolayer on 100-mm-diameter culture dishes containing Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) mixed with 10% fetal bovine serum (Cansera, Rexdale, Ontario, Canada), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air and passaged every 1–2 weeks. Cells at passages 2–4 were used for the investigations conducted in this study.

Cell proliferation assay

Cell proliferation was measured using a water-soluble tetrazolium salt (WST) assay with a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) to confirm the toxicity of the GLS1 inhibitor (G-I) compound 968 (C968) and determine the optimal concentration of C968. All wells of the 96-well plates were seeded with 5,000 cells in 100 µL DMEM. Six different C968 concentrations (0, 1.25, 2.5, 5, 10, 20, and 40 µM); the cells were incubated at 37 °C in 5% CO2 atmosphere for 48 h [9]. For the WST assay, 10 µL of WST was added to each well, and the plates were incubated for an additional 4 h at 37 °C in 5% CO2 atmosphere. The conversion of WST into formazan was measured spectrophotometrically at 450 nm.

Experimental groups

Rotator cuff-derived cells were seeded onto 12-well culture plates at density of 1 × 105 cells per well and incubated in DMEM. They were divided into four groups: (1) control group (IL-1β non-load/G-I non-treated), (2) IL-1β non-load/G-I treated group, (3) IL-1β load/G-I non-treated group, (4) IL-1β load/G-I treated group. The concentration of IL-1β was 1ng/ml as previously described [21] and that of G-I (C968) was 2.5 µM based on the results of the cell proliferation assay.

Glutaminase enzyme activity

Glutaminase enzyme activity was measured using a Glutamate Assay kit (ab284547, Abcam, Cambridge, UK) according to the manufacturer’s instructions. Cultured cells were homogenized in ice-cold Glutaminase Assay Buffer and centrifuge. All reagents provided in the kit were prepared according to the kit’s protocol. The Glutaminase Assay Buffer, Developer, Enzyme Mix, and Substrate Mix were reconstituted and diluted to working concentrations as directed. 50 µL of each sample or standard was added to individual wells of a black 96-well plate. For each assay, a set of standards and a blank control were included. A reaction mix was prepared by combining 45 µL of Glutaminase Assay Buffer, 1 µL of Developer, 2 µL of Enzyme Mix, and 2 µL of Substrate Mix per reaction. 50 µL of this reaction mix was added to each well containing samples or standards. The plate was incubated at 37 °C for 30 min. Fluorescence was measured using a microplate reader at an excitation wavelength of 535 nm and an emission wavelength of 587 nm. The fluorescence readings were compared to a standard curve generated using known concentrations of glutamate. Glutaminase activity in the samples was calculated based on the standard curve and expressed as mUnits (nmol/min)/mg protein.

qRT-PCR analysis of rotator cuff derived cells

After 48 h of incubation, total RNA was extracted from each group of cells using the RNeasy Mini Kit. Reverse transcription into single-stranded cDNAs and qRT-PCR were performed as previously described in Sect. 2.3.3. The mRNA expression levels of GLS1, p16, p21, interleukin 6 (IL6), IL-1β, type 1 collagen (COL1), type 3 collagen (COL3), Mohawk (MKX), and scleraxis (SCX) were determined using the primer pairs listed in Table 1.

Immunofluorescence staining of p16

Cultured cells were rinsed three times using phosphate-buffered saline (PBS), fixed with 4% formaldehyde for 10 min. Then, the cells were treated with primary antibody against p16 (ab51243, Abcam, Cambridge, UK) for 2 h at 37 °C. The sections were washed twice with PBS and incubated with a DyLight 488-conjugated goat anti-rabbit IgG antibody (Abbkine, California, USA) for 1 h in the dark at room temperature (25℃). The cells were visualized using a BioZero BZ-8000 microscope (Keyence, Osaka, Japan). The nuclei were counterstained with DAPI (Beyotime, Shanghai, China). For quantification, the number of DAPI-positive and p16-stained cells in four fields of view (0.75 mm × 1.0 mm) on each slide was counted and the ratio of the mean values was calculated.

Immunofluorescence staining of SCX

Cultured cells were rinsed three times using phosphate-buffered saline (PBS), fixed with 4% formaldehyde for 10 min. Then, the cells were treated with primary antibody against scleraxis (ab58655, Abcam, Cambridge, UK) for 2 h at 37 °C. The sections were washed twice with PBS and incubated with a DyLight 488-conjugated goat anti-rabbit IgG antibody (Abbkine, California, USA) for 1 h in the dark at room temperature (25℃). The cells were visualized using a BioZero BZ-8000 microscope (Keyence, Osaka, Japan). The nuclei were counterstained with DAPI (Beyotime, Shanghai, China). For quantification, the number of DAPI-positive and SCX-stained cells in four fields of view (0.75 mm × 1.0 mm) on each slide was counted and the ratio of the mean values was calculated.

Statistical analyses

All data were expressed as mean ± standard deviation (SD). All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R 2.13.0 (R Foundation for Statistical Computing, Vienna, Austria) [22]. EZR is a modified version of the R commander (version 1.6-3) designed to add statistical functions that are frequently used in biostatistics. Significant differences between groups were detected using one-way analysis of variance (ANOVA) or independent t-tests. Post hoc analysis was performed using Fisher’s protected least significant difference test. Statistical significance was set at p < 0.05.

Results

Tissue evaluation

Histological examination and immunohistochemistry

H&E staining showed that the fiber structure and arrangement was near-parallel collagen fiber orientation in Type 1 and that collagen arrangement was disordered in Type 3 (Fig. 1). Immunostaining revealed that brown-colored GLS1 positive areas were mainly observed around the cell nuclei in Type 3 (Fig. 2). Type 3 showed a significantly higher H-score of 50 ± 3 than the Type 1 H-score of 21 ± 5 (p < 0.05).

Fig. 1
figure 1

Hematoxylin and eosin (H&E) staining. The fiber structure and arrangement show near-parallel collagen fiber orientation in Type 1. Collagen arrangement is disordered in Type 3

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Fig. 2
figure 2

GLS1 immunostaining. Black arrows show brown-colored GLS1 positive areas around the cell nuclei. H-score was significantly higher in Type 3 (p < 0.05). n = 3 per group

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qRT-PCR analysis of tissue

Gene expression of GLS1 in tissue specimens from the rotator cuff tear site of Types 1 and 3 were compared. GLS1 expression was significantly higher in Type 3 compared with in Type 1 (Fig. 3).

Fig. 3
figure 3

Real-time polymerase chain reaction (RT-PCR) showed significantly higher expression of GLS1 in Type 3 (p < 0.05).　n = 6 per group

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In vitro experiments

Cell proliferation assay

The WST assay showed that the proliferation of cells cultured in the presence of 2.5 µM GLS1 inhibitor (G-I): C968 for 48 h was significantly higher than that of the control cells (p < 0.05). The relative fold changes in cell proliferation are shown in Fig. 4. As C968 concentrations > 10 µM had toxic effects, we used a C968 dose of 2.5 µM. The relative proliferation of cultured cells in the four experimental groups is shown in Fig. 5. Cell viability was significantly decreased by IL-1β loading and increased by C968 treatment (p < 0.05).

Fig. 4
figure 4

In vitro proliferation of rotator cuff-derived cells assessed using a water-soluble tetrazolium salt (WST)-based assay. Cells were cultured in DMEM at seven different G-I (C968) concentrations (0, 1.25, 2.5, 5, 10, 20, and 40 µM) for 48 h. The 2.5 µM G-I (C968) for 48 h was significantly higher than that of control cells (p < 0.05). G-I (C968) concentrations > 10 µM showed toxic effects. n = 12

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Fig. 5
figure 5

The relative proliferation of cells cultured in four experimental groups. IL-1β non-loading group and G-I (C968) treated group show higher cell viability. n = 12 per group

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Glutaminase enzyme activity

Glutaminase enzyme activity was significantly increased in IL-1β loading and decreased by G-I (C968) treatment (Fig. 6).

Fig. 6
figure 6

Glutaminase enzyme activity was significantly increased in IL-1β loading group and decreased by G-I (C968) treated group. n = 6 per group

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qRT-PCR analysis of rotator cuff derived cells

The mRNA expressions of GLS1, IL-6, and p16 were significantly decreased by G-I (C968) with and without IL-1β (Fig. 7). The mRNA expressions of p21 was significantly decreased by G-I and IL-1β. The mRNA expression of the tendon markers MKX and SCX was significantly decreased by IL-1β, and significantly increased by G-I (C968) under IL-1β loading (Fig. 7). COL1 was significantly increased by G-I without IL-1β and COL3 was significantly decreased by G-I without IL-1β. COL3/COL1 ratio was significantly decreased by G-I without IL-1β.

Fig. 7
figure 7

Real-time polymerase chain reaction (RT-PCR) showed significantly lower expression of GLS1 and p16, p21, IL-6 in G-I (C968) treated group (p < 0.05). The expression of MKX and SCX were significantly lower in IL-1β(+)/G-I(-) than IL-1β(-)/G-I(-), and higher in IL-1β(+)/G-I(+) than IL-1β(+)/G-I(-) (p < 0.05). The expression of COL 1 was significantly increased by G-I without IL-1β and COL3 was significantly decreased by G-I without IL-1β. COL3/COL1 ratio was significantly decreased by G-I without IL-1β (p < 0.05). n = 12 per group

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Immunofluorescence staining of p16

Staining for p16 was semi-quantified as the ratio of red-stained p16 to blue-stained DAPI. The staining rate was significantly higher in IL-1β(+)/G-I(-) than IL-1β(-)/G-I(-), and lower in IL-1β(+)/G-I(+) than IL-1β(+)/G-I(-) (Fig. 8).

Fig. 8
figure 8

Staining for p16 was semi-quantified as the ratio of red-stained p16 to blue staining of 4’,6-diamidio-2-phenylindole. The staining rate was significantly higher in IL-1β(+)/G-I(-) than IL-1β(-)/G-I(-), and lower in IL-1β(+)/G-I(+) than IL-1β(+)/G-I(-) (p < 0.05). n = 6 per group

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Immunofluorescence staining of SCX

SCX staining was semi-quantified as the ratio of green-stained SCX to blue-stained DAPI. The staining rate was significantly higher in G-I (C968) treated group than G-I (C968) non-treated group, and significantly higher in IL-1β(-)/G-I(-) than IL-1β(+)/G-I(-) (Fig. 9).

Fig. 9
figure 9

Staining for SCX was semi-quantified as the ratio of green-stained SCX to blue staining of 4’,6-diamidio-2-phenylindole. The staining rate was significantly higher in G-I (C968) treated group than G-I (C968) non-treated group, and significantly higher in IL-1β(-)/G-I(-) than IL-1β(+)/G-I(-) (p < 0.05). n = 6 per group

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Discussion

Risk factors for retear after arthroscopic rotator cuff repair include tear size, patient age, and fatty infiltration [23,24,25,26]. Takeuchi et al., evaluated the relationship between a retear and stump classification in the suture-bridge and double-row repair techniques for RCT, and reported that the independent predictors of a retear were the stump classification Type 3 (Odds ratio: 4.71), global fatty degeneration index (Odds ratio: 3.87), and anteroposterior tear size (Odds ratio: 1.07) in the suture bridge technique; and in the double-row technique, the independent predictors of retear were stump classification Type 3 (Odds ratio: 7.82), and age (Odds ratio: 1.22) [27]. Thus, stump classification is more closely related to retears than to fatty degeneration or tear size. In our study, GLS1 expression was increased in Type 3 tissues, suggesting a potential association with retears.

In the GLS1 inhibitor administration experiment, we loaded the medium with IL-1β with two main considerations. Firstly, rotator cuff-derived cells of Stump Type 3 exhibit an upregulation of inflammatory cytokines, including that of IL-1β [18]. In the multivariate discriminant analysis of synovial fluid from glenohumeral joint in patients with RCT and other shoulder lesions, the most influential determining factor of whether RCT patients were separable from the non-RCT group, was IL-1β [28]. By subjecting the rotator cuff-derived cells to IL-1β stimulation, we aimed to simulate the inflammatory environment associated with RCT. Secondly, IL-1β has been implicated in the upregulation of the aging marker p16, which is linked with intervertebral disc degeneration [29, 30]. By introducing IL-1β to the culture medium, we sought to induce cellular senescence and degenerative changes in the rotator cuff-derived cells. Our results demonstrated that IL-1β stimulation led to a significant increase in p16 expression, indicative of cellular senescence in rotator cuff-derived cells. However, co-treatment with a GLS1 inhibitor resulted in the reduction of p16 expression than that by IL-1β alone. This suggested that GLS1 inhibitor attenuated IL-1β-induced cellular senescence and its associated degenerative effects. p21 is also known as a senescence marker, and it has been reported that its expression is suppressed by IL-1β [31, 32]. In this study, the expression of p21 was reduced by IL-1β. Moreover, the expression of p21 was also decreased by the GLS1 inhibitor. These findings suggest that GLS1 inhibitors may possess anti-aging properties.

Several studies have investigated the role of GLS1 in inflammation. Gao et al.., have reported a relationship between GLS1 and neuroinflammation [33]. They observed elevated GLS1 expression and accumulation of pro-inflammatory exosomes in rat brains, 72 h after focal cerebral ischemia; and that treatment with a GLS1 inhibitor reversed the inflammatory response and exosome release. They found that the administration of an exosome secretion inhibitor had anti-inflammatory effects similar to those of a GLS1 inhibitor, suggesting that GLS1-mediated exosome secretion played an important role forming a neuroinflammatory microenvironment. Jiang et al., evaluated the role of exosomes in regulating glutamine metabolism in the treatment of osteoarthritis (OA) and found that IL-6 and GLS1 expression was elevated in a mouse OA model, and exosomes extracted from the bone marrow mesenchymal stem cells of the mouse OA model reduced them [34]. Although exosomes have different effects on inflammation depending on the cells from which they are secreted, GLS1 expression increases under conditions of inflammation, and GLS1 inhibitors can be expected to suppress this inflammation. In this study, although there was no significant increase in GLS1 expression by IL-1β loading, the significantly lower IL-6 expression in the GLS1 inhibitor-treated group suggested an anti-inflammatory effect of the GLS1 inhibitors.

To the best of our knowledge, this is the first study to evaluate the effects of GLS1 inhibitors on rotator cuff-derived cells. Spang et al.., reported that scleraxis mRNA (Scx) levels were significantly decreased in cultured plantaris tendon-derived cells treated with glutamate [35]. SCX and Mohawk (MKX) are essential for differentiation of mesenchymal stem cells towards tendon progenitor cells and tenocytes [36, 37]. These markers are also involved in tendon repair and are thought to alter homeostasis and influence tendon maintenance and remodeling by regulating the expression of downstream tendon-related and matrix molecular genes [38,39,40]. Mueller et al.., reported that tenocyte cultures in an inflammation-induced environment showed strong downregulation of SCX expression [41]. Thus, glutamate and tendon markers associated with tendon repair are thought to be related, and the expression of tendon markers may decrease in an inflammatory environment. And indeed, in our study, the expression of SCX and MKX was decreased in the IL-1β loading group, while they significantly increased in the GLS1 inhibitor treated group compared to the non-treated group, suggesting that GLS1 inhibitor could have regenerative effects on the rotator cuff. The type of collagen is important; type I collagen is thought to be responsible for the mechanical strength of tendon tissue, and type III collagen plays an important role in the healing process, but is thinner and more extensible than type I collagen fibers, and its presence may cause weakening of the tensile strength of the tissue [42]. Shinohara et al. reported that in Stump type 3 RCT, which is particularly fragile, the expression of COL3 was increased and the expression of COL1 was decreased. In this study, GLS1 inhibitor increased COL1 and decreased COL3 in the non-IL-1β loading group, suggesting that GLS1 inhibitor may increase rotator cuff strength under non-inflammation condition.

This study had some limitations. First, the sample size was small. Therefore, further studies with larger sample sizes are required to confirm these results. Additionally, although including Stump type 2 would provide a more comprehensive comparison of trends and degrees among all stump types, the limited availability of samples restricted our analysis to only type 1 and type 3. Future studies will aim to incorporate type 2 samples to offer a more complete understanding of the relationship between stump classification and GLS1 expression. Second, we had no information regarding the administration of steroid injections to the patients prior to the study period; however, we were able to confirm that none of the patients had received steroid injections within one month prior to surgery. Steroid administration can affect underlying biological events in the rotator cuff. Third, our findings are based on in vitro analyses without in vivo animal model validation. This limits the physiological relevance and depth of our findings. While our in vitro results suggest a potential role for GLS1 inhibitor in tissue repair and regeneration, in vivo studies are necessary to confirm these effects and to evaluate the therapeutic potential of GLS1 inhibitor in a more complex and physiologically relevant environment. Future research will focus on incorporating in vivo animal models to validate our in vitro findings and to explore the broader implications of GLS1 inhibitor in tissue repair and regeneration.

Our study is among the first to evaluate the relationship between GLS1 expression and Stump classification in patients with rotator cuff tears and the effect of GLS1 inhibitors on rotator cuff-derived cells. Histological evaluation revealed a higher expression of GLS1 in Stump Type 3. Administration of the GLS1 inhibitor decreased inflammation and aging markers, while increasing cell viability and tendon markers in rotator cuff-derived cells. GLS1 inhibitors may have anti-inflammatory effects against synovitis in rotator cuff tears, prevent age-related degeneration of the rotator cuff, and promote remodeling after rotator cuff repair.

Data availability

All of the data are contained within the article.

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Acknowledgements

The authors would like to give a special thanks to M Nagata, K Tanaka and M Yasuda (Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine) for their expert technical assistance. The authors would like to thank Editage (www.editage.com) for English language editing.

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Authors and Affiliations

  1. Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650–0017, Japan

    Tatsuo Kato, Yutaka Mifune, Atsuyuki Inui, Hanako Nishimoto, Shintaro Mukohara, Tomoya Yoshikawa, Issei Shinohara, Takahiro Furukawa, Shuya Tanaka, Masaya Kusunose, Yuichi Hoshino, Takehiko Matsushita, Tomoyuki Matsumoto & Ryosuke Kuroda

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  1. Tatsuo Kato
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  2. Yutaka Mifune
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Contributions

TK, AI, YM, HN, and IS contributed to the study design. TK, TF, and MK contributed to the data acquisition and analysis. TK and YM drafted the article. TK, SM, TY, IS, FT, MK, ST, YH, and TM contributed to the interpretation of the data. YM, TM, and RK critically revised the article. All authors gave final approval of the version to be submitted for publication and agreed to be held accountable for all aspects of the work.

Corresponding author

Correspondence to Yutaka Mifune.

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Ethics approval and consent to participate

The Kobe University Graduate School of Medicine Ethics Committee approved this study (No. B200273). Each author certifies that all investigations were conducted in conformity with ethical principles. All study participants provided informed consent. All procedures performed in this study were in accordance with the ethical standards of the Institutional Review Board of Kobe University Graduate School of Medicine, the 1964 Declaration of Helsinki and its subsequent amendments, or comparable ethical standards.

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The authors declare no competing interests.

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Kato, T., Mifune, Y., Inui, A. et al. Effects of glutaminase 1 inhibitor on rotator cuff derived cells: a preliminary report in 12 patients. BMC Musculoskelet Disord 26, 935 (2025). https://doi.org/10.1186/s12891-025-09105-w

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Keywords

BMC Musculoskeletal Disorders

ISSN: 1471-2474

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