Metformin prevents peritendinous fibrosis by inhibiting transforming growth factor-β signaling

Injury-induced peritendinous adhesion is a critical clinical problem that leads to tendon function impairment. Therefore, it is very urgent to explore potential approaches to attenuate peritendinous adhesion formation. Recently, several studies have demonstrated the biological effect of metformin in inhibiting multiple tissue fibrosis. In this study, we performed in vitro and in vivo experiments to examine whether metformin prevents injury-induced peritendinous fibrosis. We found that tendon injury induced severe fibrosis formation in rats. However, orally administered metformin significantly alleviated the fibrosis based on macroscopic and histological evaluation. Peritendinous tissue from metformin-treated rats also showed decreased expression of fibrotic genes including col1a1, col3a1, and α-smooth muscle actin (α-SMA), and inhibition of transforming growth factor (TGF)-β1 signaling. The cell counting kit (CCK)-8, flow cytometry, and 5-ethynyl-2′-deoxyuridine (EdU) staining analyses showed that treatment of NIH/3T3 fibroblasts with metformin significantly inhibited excessive cell proliferation and promoted cell apoptosis. Metformin treatment also inhibited the expression of fibrotic genes and decreased the phosphorylation of smad2/3 and extracellular signal-regulated kinase (ERK) 1/2. Furthermore, blocking AMP-activated protein kinase (AMPK) signaling abolished the inhibitory effect of metformin on fibrosis. Our findings indicate that metformin has a protective role against peritendinous tissue fibrosis and suggest its clinical use could be a promising therapeutic approach.


INTRODUCTION
Peritendinous tissue adhesion after tendon injury is an important orthopedic condition that causes gliding dysfunction as well as pain and requires complex surgical intervention [1]. Presently, multiple strategies including drugs, physical barriers, and physiotherapy are used to prevent peritendinous tissue adhesion [2][3][4][5]. Researchers have explored various pharmaceutical approaches, especially nonsteroidal anti-inflammatory drugs (NSAIDs), to prevent adhesion formation [6]. However, the efficiency of these drugs is suboptimal and can lead to increased side effects.
A key mediator in the pathogenesis of tendon adhesion is transforming growth factor (TGF)-β1, which is an important factor in the pathogenesis of tendon adhesion [7,8]. TGF-β1 promotes fibroblast to myofibroblast differentiation (FMD) and myofibroblast proliferation by mediating various signaling pathways, including the canonical TGF-β/Smad2/3 and noncanonical mitogenactivated protein kinase (MAPK) pathways [9]. In the past decades, metformin has become one of the most extensively used oral anti-hyperglycemic medicines for patients with type 2 diabetes. Recent studies have revealed additional biological functions of metformin including a potential protective role in preventing tissue fibrosis in the kidney, lung, heart, and liver [9][10][11][12]. However, whether metformin attenuates injury-induced peritendinous adhesion remains unknown.
In the present study, we sought to answer the following questions: (1) whether metformin inhibits peritendinous tissue fibrosis induced by injury and (2) whether metformin inhibits TGF-β1 signaling activation.

Metformin alleviates injury-induced tendon adhesion in vivo
We first established a peritendinous adhesion rat model by surgery. Three weeks later, all the rats were euthanized and tendons were examined. All the rats survived the experimental period. The gross observation showed thick adhesion tissue around the tendons in the control group, whereas this effect significantly decreased in the metformin-treated group ( Figure 1A). The gross adhesion grading scores of the peritendinous tissues were significantly lower in metformin-treated group (p = 0.005, Figure 1B). No significant difference in maximal tensile strength was observed between the control and metformintreated groups (p = 0.173, Figure 1B).
Histological evaluation using hematoxylin and eosin (H&E) staining showed that rats in the control group exhibited more extensive proliferation of fibroblasts and infiltration of inflammatory cells than the metformin-treated rats did ( Figure 1C). Sirius-red staining showed massive collagen deposition after tendon injury ( Figure 1D). The Sirius-red staining revealed that oral administration of metformin reduced collagen deposition ( Figure 1D). Histological adhesion scoring of tendons also showed more extensive adhesion in the control group (p = 0.002, Figure 1E). Histological tendon healing scores showed no significant difference between control and metformintreated rats (p = 0.667, Figure 1E). We further measured the hydroxyproline (Hyp) content, which is a major constituent of collagen, to quantify the extent of adhesion. Rats in the control group showed significantly higher levels of Hyp than the metformin-treated rats did (268 and 184 μg/mL, respectively, p = 0.011, Figure 1F). Taken together, our data suggest a protective role for metformin in alleviating injuryinduced exacerbation of peritendinous adhesion in vivo.

Metformin inhibits proliferation and TGF-β1 signaling pathway in vivo
Excessive cell proliferation, persistent myofibroblast activation, and extensive extracellular matrix (ECM) protein synthesis are vital for tendon adhesion formation. Therefore, we demonstrated whether metformin could inhibit proliferation and the TGF-β1 signaling pathway in vivo. The Ki67 staining revealed that metformin treatment significantly decreased excessive cell proliferation (Figure 2A).
Smooth muscle actin (SMA) is a marker of myofibroblasts, and activation of myofibroblasts has been shown to promote fibrogenesis. Immunohistochemistry (IHC) for SMA showed intensive positive staining in tendon tissue sections of injured rats, whereas the number of positively stained cells was reduced after metformin treatment ( Figure 2B). To evaluate the effect of metformin on fibrotic gene expression, we performed reverse transcription-polymerase chain reaction (RT-PCR) and western blotting in tendon tissues.
The mRNA expression of col1a1, col3a1, and SMA decreased in metformin-treated rats compared with that in the controls ( Figure 2C). Western blotting also showed significantly reduced protein levels of col1a1, col3a1, and SMA in the treatment group ( Figure 2D). Furthermore, we evaluated the effect of metformin on the TGF-β1 signaling pathway. In the metformin-treated group, the protein level of phosphorylated-AMP-activated protein kinase (p-AMPK) was significantly elevated. Rats treated with metformin showed significantly decreased phosphorylation levels of smad2/3 and extracellular signal-regulated kinase (ERK)1/2, which indicates that metformin inhibited the SMAD and MAPK pathways in injured peritendinous tissues ( Figure 2E).

Metformin induces progressive apoptosis and inhibits proliferation in vitro
Excessive fibroblast proliferation is a vital pathological characteristic of peritendinous tissue fibrosis. The cell counting kit (CCK)-8 showed that metformin decreased the TGF-β1-induced fibroblast viability ( Figure 3A). Flow cytometric analysis of cell apoptosis showed that TGF-β1 decreased fibroblast apoptosis, while metformin alone or in combination with TGF-β1 promoted apoptosis in NIH/3T3 fibroblasts ( Figure 3B).
The 5-ethynyl-2′-deoxyuridine (EdU) staining also revealed that TGF-β1 induced fibroblast proliferation, while cotreatment with metformin inhibited this effect ( Figure  4A). As shown by the cell cycle analysis, the number of G0/G1 NIH/3T3 fibroblasts increased after metformin treatment ( Figure 4B). The number of S and G2/M phase cells increased after TGF-β1 treatment while the effect was abolished by treatment with metformin. Taken together, our data showed that metformin inhibited excessive cell proliferation and promoted apoptosis of NIH/3T3 cells.

Metformin decreases TGF-β1-induced adhesion in vitro
We sought to determine whether metformin decreased TGF-β1-induced peritendinous fibrosis in vitro.
Immunofluorescence (IF) staining of NIH/3T3 fibroblasts showed that treatment with 2 ng/mL TGF-β for 24 h increased the expression of SMA. However, treatment with 5 mM metformin decreased the expression of SMA and inhibited the increased expression of SMA induced by TGF-β1 ( Figure 5A).

Metformin decreases TGF-β1-induced fibrosis by AMPK activation-dependent mechanism
Our results showed that metformin activated the phosphorylation of AMPK in vitro and in vivo.
Furthermore, we demonstrated that metformin inhibited TGF-β1-induced peritendinous tissue fibrosis through AMPK activation. First, we evaluated whether activation of AMPK signaling pathway inhibited peritendinous fibrosis formation. Real-time PCR showed that fibroblasts treated with 1mM 5-aminoimidazole-4-carboxamide1β-D-ribofuranoside (AICAR), a well-known AMPK activator, exhibited a significant downregulation of mRNA and protein levels of fibrotic genes ( Figure 6A and 6B). Then we inhibited AMPK by pretreated with compound C before TGF-β1 or metformin treatment or both.
The real-time PCR and western blotting showed that compound C abolished the metformin-induced downregulation of col1a1, col3a1, and SMA ( Figure 7A). Further, we transfected fibroblasts with small interfering RNA (siRNA) targeting AMPK or negative control. The results showed that pre-transfection of fibroblast with AMPK siRNA blocked the inhibitory effect of metformin on fibrosis ( Figure 8A and 8B). Furthermore, the metformininduced inhibition of the activation of canonical (SMAD) and noncanonical (MAPK) TGF-β1 signaling pathways was abolished by compound C ( Figure 7B) or AMPK siRNA ( Figure 8C). These in vitro studies suggested that metformin might inhibit TGF-β1-induced peritendinous tissue fibrosis by activating AMPK signaling.

DISCUSSION
Peritendinous tissue adhesion, characterized by excessive peritendinous ECM deposition and subsequent tendon dysfunction, is a major clinical complication of tendon injury. Multiple strategies to address this condition, including physical anti-adhesion barriers and drugs, have been extensively studied [2][3][4]13]. However, the effect of these approaches remains uncertain and, therefore, an effective measure to inhibit tendon adhesion at key locations is required.
Although metformin has been used as an oral antidiabetic drug for decades, recent studies have uncovered its novel biological effects including antitumor and antifibrotic activities [9,10,12,[14][15][16]. In the present study, we demonstrated that metformin alleviated peritendinous tissue fibrosis lesions after tendon injury. First, we showed that metformin inhibited abnormally activated proliferation in vitro and in vivo. Activation of TGF-β1 signaling has previously been shown to promote fibrosis in multiple tissues [17,18]. in vitro studies have also demonstrated that TGF-β1 strongly promoted cell proliferation and inhibited apoptosis [9,19]. Our previous studies have reported the activation of proliferation in TGF-β1-stimulated fibroblasts and adhesive tendon tissue [20,21]. However, TGF-β1-treated NIH/3T3 fibroblasts exposed to metformin showed decreased cell viability, and metformin treatment both promoted fibroblast apoptosis and inhibited proliferation. Li showed that TGF-β1 treatment resulted in reduced apoptosis and increased proliferation of HFL-1 cells, whereas metformin cotreatment abolished these effects [9]. It has been established that the cell cycle is inhibited mainly by G0/G1 arrest [14,22,23]. Our results also showed that metformin promoted G0/G1 arrest of TGF-β1-treated fibroblasts.
Second, we showed that metformin exhibited a potent antifibrotic effect by inhibiting the expression of fibrotic markers such as col1a1 and col3a1. Recent studies have shown that myofibroblasts, the activated form of fibroblasts, are the major source of excessive ECM The western blot analysis showed that metformin treatment significantly inhibited canonical (SMAD) and noncanonical (MAPK) TGF-β signaling pathways. Data are expressed as means ± SEM. * P < 0.05; ** P < 0.01. www.impactjournals.com/oncotarget deposition in multiple tissue fibrosis [17,24]. In animal models of aldosterone and salt-induced cardiac fibrosis, metformin treatment significantly reduced the expression of fibrotic genes in the heart [11]. We found that metformin inhibited the increased expression of α-SMA in activated fibroblasts. Cavaglieri recently reported that metformin significantly reduced macrophage infiltration, expression of inflammation markers, and deposition of ECM, thereby reducing interstitial fibrotic lesions in a mouse with unilateral ureteral obstruction [15].
Finally, our study revealed that metformin inhibited TGF-β1-induced peritendinous adhesion by activating the AMPK signaling pathway. Smad2/3 and ERK1/2 have previously been reported to be important downstream mediators of TGF-β1 signaling [7,25]. The ERK1/2 pathway was initially considered a growth-promoting pathway, mediating the transition from the G1 to the S phase and DNA synthesis during the S phase [26]. A previous study has also shown that activation of ERK1/2 and smad2/3 mediated collagen synthesis [25]. Li found that oral metformin treatment inhibited the phosphorylation of both smad2/3 and ERK1/2, and attenuated gefitinibinduced pulmonary fibrosis [9]. Studies have also shown that metformin exerted a protective effect against tumor progression by inhibiting TGF-β1 signaling [27,28].
We have demonstrated that persistent activation of canonical and noncanonical TGF-β1 signaling promoted peritendinous tissue adhesion formation in tendon-injured   animal models. Our previous study showed that the continuous local release of celecoxib significantly inhibited the activation of smad2/3 and ERK1/2, which prevented tendon adhesion [6]. Nevertheless, local celecoxib treatment was shown to slightly impair tendon healing [6]. The results of the present study showed that metformin effectively inhibited the in vitro and in vivo phosphorylation of smad2/3 and ERK1/2. In addition, it is noteworthy that the indexes for tendon healing show no significantly difference between control and metformin-treated rats.
The AMPK pathway is the best known metforminmediated pathway. Metformin reduces the ATP/AMP ratio by suppressing mitochondrial respiratory-chain complex I. Excessive AMP binds and induces a conformational change in AMPK. Liver kinase B1 (LKB1) phosphorylates AMPK and further activates AMPK [29]. We conducted in vitro studies to determine whether AMPK activation was required for the metformin-induced inhibition of TGF-β1-mediated fibrosis. Satriano observed a reduction in AMPK activity in the renal fibrotic tissue and showed that the induction of AMPK activity by metformin or AICAR ameliorated the severity of kidney fibrosis [30]. Several studies showed that the addition of an AMPK inhibitor (compound C) abolished the effect of metformin in decreasing fibrotic gene expression induced by TGF-β1 [9,10,14]. Cui showed that synthesized siRNA targeting the AMPKα1 subunit decreased the inhibitory effect of hepatocyte growth factor (HGF) on the TGF-β1-induced transition of tendon fibroblasts to myofibroblasts. However, Xiao found that metformin was directly bound to TGF-β1 and inhibited the binding of TGF-β1 to its receptor [31,32]. In the present study, exposure to compound C abolished the inhibitory effect of metformin on TGF-β1-treated NIH/3T3 fibroblasts. These different results could be attributed to differences in the in vitro treatment methods and experimental conditions. Therefore, further in vivo studies are needed to determine whether metformin inhibits tendon adhesion by activating the AMPK signaling pathway.   In summary, recent studies have reported that metformin is an effective inhibitor of processes induced by TGF-β1 signaling. Our study provides evidence that metformin alleviated injury-induced peritendinous adhesion by inhibiting TGF-β1-induced excessive fibroblast proliferation, transition into myofibroblasts, and ECM deposition. Considering that adhesion is a common complication after tendon injury and results in various degrees of dysfunction, these findings indicate the clinical usefulness of metformin as a treatment targeting TGF-β1.

Tendon injury model and treatment
Sprague-Dawley (SD) rats were purchased from the Shanghai Laboratory Animal Company (SLAC, Shanghai, China). All animal experimental procedures were approved by the Animal Care Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital. Rats were housed in ventilated microisolator cages under a 12-h light and dark cycle with free access to food and water. For the tendon injury surgery, 8-week-old male SD rats were anesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 50 mg/kg. An incision was made medially over the plantar skin, the superficial flexor tendon was resected, and then the deep flexor tendon was exposed and transected. We repaired the deep flexor tendon using a 6-0 prolene suture (Ethicon, Edinburgh, UK) and subsequently sutured the skin. After surgery, the rats were randomly assigned to the control and metformintreated groups. The metformin-treated and control groups were orally administered 200 mg·kg -1 ·day -1 metformin and phosphate-buffered saline (PBS), respectively. Three weeks after tendon injury, the rats were euthanized and the tendon tissues were collected for analysis.
Macroscopic evaluation of adhesion, maximal strength, and Hyp content were performed by two independent researchers according to previously reported methods [6,9,13]. The severity of peritendinous adhesions, was scored as follows: grade 1, no adhesion formation; grade 2, adhesion could be separated by blunt dissection; grade 3, sharp dissection was needed to separate no more than 50% of adhesive tissues; grade 4, sharp dissection was required to separate 51-97.5% of adhesion tissues; and grade 5, sharp dissection was required to separate > 97.5% of adhesion tissues.

Proliferation and apoptosis assays
NIH/3T3 fibroblasts were seeded at a density of 2 × 10 3 /well into 96-well plates. The cells were cultured for 24 h and then treated with TGF-β1 (2 ng/mL), metformin (5 mM), or both for 24, 48, 72, and 96 h. Cell proliferation was assessed using a CCK8 (Dojindo, Shanghai, China) after a 3-h incubation at 37°C and the absorbance was measured at a wavelength of 450 nm. EdU immunostaining was used to assess cell proliferation as previously reported. For the cell cycle analysis, cells were treated with TGF-β1, metformin, or both for 48h, followed by trypsinization and then they were fixed in 70% ethanol overnight. The fixed cells were stained with propidium iodide (PI) (50 μg/mL) for 30 minutes at room temperature [14]. For the apoptosis analysis, cells were harvested using trypsin, washed three times with PBS, and then resuspended. Then, 5 μL each of Annexin V-fluorescein isothiocyanate (FITC) and PI were added to 100 μL of the cell suspension and incubated for 30 min at room temperature. Cell apoptosis and cell cycle were analyzed using flow cytometry (Beckman Coulter, Brea, CA, USA).

RNA isolation and real-time PCR
The mRNA expression of col1a1, col3a1, and α-SMA was analyzed using real-time PCR. Total RNA was extracted from the peritendinous tissues or cell lysates using Trizol (Invitrogen, USA) according to the manufacturer's instructions. Total RNA was reversetranscribed into cDNA using oligo-dT primers (Promega, USA). The real-time PCR analysis was carried out using the SYBR Green Premix Ex Taq (Takara, Japan) using a Light Cycler 480 (Roche, Switzerland). Relative mRNA expression levels were calculated using comparative Ct values. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference for normalization. www.impactjournals.com/oncotarget

IHC
For the IHC and IF of α-SMA, tendon sections and cells were incubated with the primary antibody for α-SMA in PBS solution containing 1% bovine serum albumin (BSA) overnight at 4°C. Then, the cells or peritendinous tissue sections were washed three times with PBS and incubated with secondary antibodies for 30 min at room temperature. For nuclear staining, the slides were mounted with medium with 4′, 6-diamidino-2-phenylindole (DAPI).

Statistical analysis
All experiments were performed at least three times, and the data are described as the means ± standard error of mean (SEM). Statistical analyses were performed using the two-tailed Student's t-test. Two or more groups were compared using a one-way analysis of variance (ANOVA). All the statistical analyses were conducted using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). A P-value < 0.05 was considered statistically significant.