IFN-α-2b Reduces Postoperative Arthrofibrosis in Rats by Inhibiting Fibroblast Proliferation and Migration through STAT1/p21 Signaling Pathway

Objective To investigate the effect of IFN-α-2b in preventing postoperative arthrofibrosis in rats, its antiproliferation effect on fibroblasts in vitro, and its molecular mechanism. Methods The rat model of arthrofibrosis was established and treated with different concentrations of drugs. Knee specimens were collected for histological and immunohistochemical staining to observe the effect of IFN-α-2b on arthrofibrosis in rats. The biological information was further mined according to the database data, and the possible regulatory mechanism of IFN-α-2b on fibroblasts was analyzed. The inhibitory effect of IFN-α-2b on fibroblast proliferation and migration in vitro was detected by cell counting kit-8 (CCK-8), immunofluorescence analysis, cell cycle test, EdU assay, wound healing test, and Transwell method, and the analysis results were verified by Western blotting method. Results The test results of rat knee joint specimens showed that IFN-α-2b significantly inhibited the degree of fibrosis after knee joint surgery, the number of fibroblasts in the operation area was less than that of the control group, and the expression of collagen and proliferation-related proteins decreased. In vitro experimental results show that IFN-α-2b can inhibit the proliferation and migration of fibroblasts. According to the results of database analysis, it is suggested that the STAT1/P21 pathway may be involved, and it has been verified and confirmed by Western blotting and other related methods. Conclusion IFN-α-2b can reduce surgery-induced arthrofibrosis by inhibiting fibroblast proliferation and migration, which may be related to the regulation of STAT1/p21 signaling pathway.


Introduction
Arthrofibrosis is a common complication in joints after trauma and surgery, which is characterized by the production of excessive fibrous scar tissue in joints [1][2][3]. Previous studies have shown that arthrofibrosis is related to the excessive proliferation of fibroblasts in the surgical area. The excessive proliferated fibroblasts will migrate to the area after surgery, secreting excessive extracellular matrix (ECM) and collagen deposition, which eventually leads to arthrofibrosis [4,5]. As the overhyperplasia of fibrosis tissue causes pain and limits the normal range of joint activity, it can seriously affect the patient's postoperative life [6,7].
In recent years, many strategies have been adopted, such as all-round movements of the knee joint after anesthesia, arthroscopic cleaning of joint cavity, and local drug treatment to reduce the formation of postoperative arthrofibrosis [8][9][10]. Other studies have reported that the best treatment for arthrofibrosis is early identification and intervention [11]. Therefore, the early application of drugs to prevent arthrofibrosis deserves attention. But, although some achievements have been made in the research of locally applied drugs, there are still considerable limitations before clinical trials due to the side effects of the above drugs or the effect of the route of administration.
IFN-α-2b is a kind of cytokine with many biological activities such as antiviral, antitumor, antiproliferation, and immunomodulation. It has good drug tolerance in the body, even in high-dose application [12]. Previous studies have shown that IFN-α-2b can treat some fibroproliferative diseases (such as hypertrophic scar and keloid) [13]. In addition, it also plays an active preventive role in scar formation after glaucoma filtration surgery and scar formation after cleft palate surgery [14][15][16]. It can not only inhibit the proliferation of fibroblasts but also reduce the formation of collagen, thus preventing the formation of fibrosis. However, there is no research on IFN-α-2b for arthrofibrosis in the existing literature. Based on the above research background, we choose IFN-α-2b on rats for the prevention of postoperative arthrofibrosis and the treatment of fibroblasts in vitro in order to explore its effect of preventing knee arthrofibrosis and the possible mechanism involved.

Animals.
The animal research in this experiment was approved by the Animal Research Committee of Yangzhou University. All rats received strict care. A total of 36 SD male rats weighing about 300 g were selected and randomly divided into 3 groups (12 in each group).

Establishment of Arthrofibrosis Model.
After the rats were successfully anesthetized, the knee joint was opened through the medial approach of the patella, and the medial and lateral femoral condyles were fully exposed. The cortical bone of about 4 * 4 mm was excised until the cancellous bone was exposed, and the articular cartilage was intact; then, the wound was covered with saline or IFN-α-2b gauze for 10 minutes. After hemostasis, the gauze was removed to suture the joint capsule and skin. After the operation, antibiotics were applied for 3 consecutive days (intramuscularly, 50 mg/kg), and saline or corresponding concentration of IFN-α-2b 50 μl was injected locally in the operation knee joint 3 times a week [17].

Histological
Analysis. The experiment ended 4 weeks after surgery. Six rats were randomly selected from each group, and knee joint specimens were collected for histological analysis. The knee joint specimens were fixed with 4% paraformaldehyde for 48 hours, fully decalcified in ethylenediamine tetra acetic acid (EDTA), and embedded in paraffin. 4 μm serial sections were deparaffinized to water, and hematoxylin-eosin (HE) staining was performed to detect the degree of fibrosis and the number of fibroblasts, and Masson's trichrome staining was used to observe the collagen production. The collagen was stained with picric acid-Sirius red, and the type I collagen and type III collagen were observed and distinguished under a polarized light The results of optical density analysis suggest that IFN-α-2b can effectively reduce the production of collagen. * Compared with the control group. # Comparison between the two groups of IFN-α-2b medications, P < 0:05 (n = 6).      5 Mediators of Inflammation online software to analyze raw data and identify differentially expressed genes (DEGs). P < 0:05 and jlogFCj ≥ 1 were used as cut-off criteria to obtain DEGs.

Enrichment Analyses of DEGs and PPI Network.
The protein-protein interaction (PPI) network was established by STRING database (http://string-db.org) [18]. In this research, the interaction with high confidence > 0:7 was statistically significant. In order to explore more biological information related to DEGs and obtain more comprehensive gene and protein function, we conducted Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis through Metascape (http://metascape.org) [19]. GO enrichment analysis includes biological processes (BP), cell component (CC), and molecular function (MF). In addition, we also performed TRRUST transcriptional regulatory network analysis [20].
2.10. Cell Cycle Analysis. Fibroblasts were treated with IFNα-2b of 5,000 U/ml for 48 hours and then operated according to the instructions of Cell Cycle Testing Kit (Beyotime, Shanghai, China). Cells were collected, centrifuged at 2,000 r/min for 5 minutes, washed with precooled PBS, fixed   Mediators of Inflammation with 70% absolute ethanol, and fixed overnight at 4°C. After centrifugation again, cells were resuspended with 500 μl propidium iodide staining solution prepared in advance, incubated at room temperature for 30 minutes in the dark, and then, flow cytometry was performed. Finally, the distribution of cells in different periods was calculated by ModFit LT software.

Statistical Analysis.
All data in this study were analyzed by SPSS 19.0 statistical software. The data were expressed as mean ± standard deviation. Data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's test for comparison between groups. P < 0:05 was considered statistically significant.

IFN-α-2b Improves Postoperative Arthrofibrosis in Rats.
According to the HE staining results, the number of scar

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Mediators of Inflammation fibroblasts and dense fibrous tissue around the knee operation area in the IFN-α-2b group decreased, the tissue structure was sparse, and the degree of fibrosis was improved (Figures 1(a) and 1(b)); the number of fibroblasts decreased with the increase of IFN-α-2b concentration (Figure 1(c)). Masson's staining was used to evaluate the degree of collagen synthesis in arthrofibrosis after local administration of IFNα-2b. The staining results showed that collagen synthesis in arthrofibrosis decreased after application of IFN-α-2b, especially in the high-dose application group (Figure 1(d)); the results of optical density analysis also suggest that IFN-α-2b can effectively reduce the production of collagen (Figure 1(e)). Immunohistochemical staining showed that the expression of type I collagen in the IFN-α-2b treatment group was lower than that in the normal saline application group (Figures 2(a) and 2(b)). The same was true for the expression of type III collagen (Figures 2(c) and 2(d)). Sirius red staining showed that type I collagen fibers (red or yellow) and type III collagen fibers (green) decreased in the IFN-α-2b treatment group (Figure 2(e)). Similarly, immunohistochemical staining of proliferation-and migration-related proteins PCNA (Figure 2(f)) and α-SMA (Figure 2(h)) showed that the expression of the IFN-α-2b-treated group was significantly lower than that of the control group (Figures 2(g) and 2(i)). In conclusion, the above in vivo experimental results suggest that IFN-α-2b has the potential to improve arthrofibrosis in rats.

DEG Acquisition and PPI Network
Relationship, GO, KEGG, and TRRUST Enrichment Analyses. The volcano map shows the distribution of 28,553 expressed genes (Figure 3(a)). Among them, according to jlogFCj ≥ 1 and P < 0:05 as the cut-off criteria, 78 DEGs were extracted, and the interaction relationship is shown in Figure 3(b). In order to obtain more biological information, we used the online database (Metascape) to perform GO and KEGG enrichment analyses. DEGs are divided into three functional groups: biological processes (BP), molecular functions (MF), and cellular components (CC). GO analysis shows that changes in biological processes are significantly enriched in defense response to virus, negative regulation of viral process, response to type I interferon, positive regulation of type I interferon production, and other immune response and transcriptional regulation. As for cellular components, DEGs are abundant in the perinuclear region of cytoplasm. Changes in molecular functions are mainly concentrated in 2 ′ -5 ′ -oligoadenylate synthetase activity, RNA helicase activity, nuclearoside-triphosphatase activity, ubiquitin-protein transferase activity, protein homodimerization activity, ubiquitin-like protein ligase binding, nuclear receptor binding, and protein phosphatase binding aspect (Figure 3(c)). KEGG pathway analysis showed that DEGs play a key role in hepatitis C/B, RIG-I-like receptor signaling pathway, and TNF signaling pathway (Figure 3(d)). The TRRUST database can provide information on how to regulate these interactions, and analysis suggests that DEG transcriptional regulation is mainly enriched in genes such as STAT1, IRF1, BRCA1, and RELA (Figure 3(e)). The protein-protein interaction (PPI) network results showed 22 hub genes and 125 connections (Figure 3(f)), including the STAT1 gene. Through the above analysis, we speculate that the role played by IFN-α-2b on fibroblasts is mainly regulated by STAT1, and P21, as a downstream molecule of STAT1, can regulate cell proliferation. Therefore, we verified the STAT1/P21 pathway in vitro.
3.3. IFN-α-2b Inhibits Fibroblast Proliferation. CCK-8 assay showed that the activity of fibroblasts decreased with the increase of IFN-α-2b concentration and the prolongation of treatment time. The results showed that IFN-α-2b inhibited the activity of fibroblasts in a time-and concentrationdependent manner (Figure 4(a)). Flow cytometry analysis showed that the proportion of IFN-α-2b-treated cells increased significantly in the S phase, suggesting that the cell cycle may be blocked in the S phase (Figure 4(b)). Fluorescence microscopy analysis showed that with the increase of IFN-α-2b concentration, the fluorescence intensity of type I collagen and α-SMA gradually decreased, suggesting that IFN-α-2b reduces the formation of extracellular matrix (Figure 4(c)).
The analysis of EdU experimental results showed that as the concentration of IFN-α-2b increased, EdU-positive fibroblasts decreased, the statistical results showed a significant difference, and the fludarabine group partially reversed the trend (Figures 5(a) and 5(b)). In addition, Western blot results showed that the expression levels of proliferationrelated proteins PCNA, cyclin A, and type I collagen also decreased with the increase of IFN-α-2b concentration (Figures 5(c) and 5(d)), while STAT1 and P21 were activated with the application of IFN-α-2b. This is because the expression levels of p-STAT1/STAT1 and P21 gradually increased (Figures 5(e) and 5(f)). With the addition of fludarabine, the above phenomenon reversed the blocking expression trend to a certain extent.

IFN-α-2b Inhibits Fibroblast Migration.
After treatment with IFN-α-2b, the migration of fibroblasts was inhibited. Compared with the control group, the wound healing rate of the IFN-α-2b group decreased in a dose-dependent manner (Figures 6(a) and 6(b)). The results of Transwell migration experiment showed that the concentration of IFN-α-2b increased. As the concentration of IFN-α-2b increases, the number of cells on the bottom membrane of the migration chamber gradually decreases (Figures 6(c) and 6(d)). Western blot showed that the expression level of α-SMA in the IFN-α-2b application group showed a downward trend, and the fludarabine application group also saw a partial reversal of the inhibition trend (Figures 6(e) and 6(f)). In summary, the above experimental results show that IFN-α-2b can inhibit the proliferation and migration of fibroblasts, and the STAT1/P21 signaling pathway may be involved in the regulation.

Crosstalk between STAT1/P21 and TGFβ/Smad
Signaling Pathways. Our team before published an article on IFN-α-2b treatment of epidural postoperative adhesion, which showed that IFN-α-2b can play a role by inhibiting TGFβ/ Smad signal pathway [17]. The relevant signaling pathways 13 Mediators of Inflammation involved in this manuscript are experimentally verified by us after screening the network database. The results showed that IFN-α-2b could inhibit proliferation and fibrosis after knee surgery through STAT1/P21 pathway. For these two signaling pathways in the study of the relationship, we conducted the experiment verification. Here, we added Smad3-specific inhibitor (SIS3) and STAT1-specific inhibitor fludarabine for prestimulation [21,22], and the results showed that STAT1 phosphorylation was activated by IFN-α-2b application and Smad7 expression was increased (which inhibited Smad3 phosphorylation). In addition, Smad3 phosphorylation and type I collagen expression were significantly decreased, which could be reversed by fludarabine pretreatment. The activation of p-STAT1 and the expression of Samd7 decreased, the phosphorylation of Smad3 increased, and the expression of type I collagen increased. However, the phosphorylation level of Smad3 was significantly reduced in the group treated with SIS3 in advance, although the expression of type 1 collagen also showed a downward trend, but there was no statistical difference, and the activated expression levels of Smad7 and p-STAT1 did not change significantly (Figures 7(a) and 7(b)). According to the experimental results, we inferred that there is crosstalk between STAT1/P21 and TGFβ/Smad signaling pathways, which may be due to the phosphorylation and activation of STAT1, which stimulates the increase of Smad7 and then plays a role in inhibiting the activation of Smad3.

Discussion
As mentioned earlier, excessive proliferation of fibroblasts and excessive secretion of extracellular matrix (ECM) in the surgical area can lead to fibrosis [10], while collagen is one of the important components of extracellular matrix, which can promote cell proliferation and migration [23]. Fibroblasts are transformed into myofibroblasts during fibrosis, resulting in excessive synthesis and deposition of extracellular matrix proteins [24,25]. The expression of α-SMA is considered to be a specific marker of myofibroblasts [26]. Clinical studies have reported that the expression of α-SMA increased significantly in joint fibrosis specimens [27], and the high expression of α-SMA is also closely related to the proliferation and migration of fibroblasts [28,29]. In this study, both Masson's staining and Sirius red staining showed a decrease in collagen deposition in the IFN-α-2b treatment group. The results of in vitro experiments also suggested that the expression of collagen I and α-SMA decreased in the IFN-α-2b-treated group.
A number of studies have reported that IFN-α-2b can activate STAT1, resulting in the phosphorylation of STAT1 [30,31]. STAT1 is a member of STAT protein family. In response to cytokines and growth factors, STAT family members are phosphorylated by receptor-related kinases and then form homodimers or heterodimers, which are transferred to the nucleus as transcriptional activators. It has been reported that the mice, struck off the STAT1 gene, are more likely to have chemically induced lung and liver fibrosis [32]. In the skin model, the expression of collagen and α-SMA in the granulation tissue around the wound of mice with fibroblast STAT1 gene defects increased, and the perivascular fibrosis increased significantly. These results indicated that STAT1 plays a role in tissue repair [33]. As a key element of IFN-α-2b signal transduction, the function of STAT1 is mainly determined by its phosphorylation state. As an active form of STAT1, p-STAT1 has been shown to inhibit tumor growth by regulating cell cycles [34]. As a regulator downstream of STAT1, p21 is the first identified cyclin-dependent kinase inhibitor (CKI) protein member. P21 can bind to several compounds of cyclins and cyclindependent kinases (CDK), such as cyclin A/CDK2, cyclin E/CDK2, cyclin D1/CDK4, and cyclin D2/CDK4 [35,36]. The increased expression of p21 can reduce the expression of cyclin, thus inducing cell cycle stagnation and inhibiting cell proliferation [37], which is consistent with our experimental results. In this study, with the increase of IFN-α-2b concentration, the expression of p-stat1 and p21 increased, while cell proliferation and migration were inhibited. The results of the pretreatment group with STAT1-specific inhibitor (fludarabine) partially reversed this trend. This means that STAT1/p21signaling pathway is involved in the antiproliferation and migration inhibition of IFN-α-2b. In this study, we explored the relationship between STAT1/ P21 and TGFβ/Smad signaling pathways. After all, the TGFβ/Smad signaling pathway plays a crucial role in the formation of fibrosis. The results suggest that there is crosstalk between the two signal pathways; that is, the activation of Stat1 can promote the increase of the expression of Smad7, thus preventing the expression of Smad3, which is similar to some previous studies [38,39].
During our experiment, there were no delayed wound healing, epidermal necrosis, surgical incision infection, and death. However, we did not explore the application of larger dose and time, nor did we screen the optimal application concentration of drugs, which is also the deficiency of this experiment. In addition, the formation mechanism of fibrosis is very complex. In addition to the proliferation and migration of fibroblasts, inflammatory response is also involved in the formation of arthrofibrosis [40]. Interestingly, STAT1 acts as a transcription factor in regulating proinflammatory and anti-inflammatory responses, making STAT1 an attractive anti-inflammatory target [41]. However, these were not measured in this study. We will continue to refine these explorations in future research. We are looking forward to more comprehensive and in-depth research and application.

Conclusion
In conclusion, this study firstly verified that IFN-α-2b can reduce surgery-induced arthrofibrosis by inhibiting fibroblast proliferation and migration, which may be related to the regulation of STAT1/p21 signaling pathway. Our study provides a new therapeutic target for intervention in arthrofibrosis.