MiR-146a Reduces Fibrosis after Glaucoma Filtration Surgery in Rats

MiR-146a Reduces Fibrosis after Glaucoma Filtration Surgery in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article MiR-146a Reduces Fibrosis after Glaucoma Filtration Surgery in Rats Ruiqi Han, Huimin Zhong, Yang Zhang, Huan Yu, Yumeng Zhang, Shouyue Huang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3883641/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2024 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Purpose: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-β1 in rat Tenon’s capsule fibroblasts. Methods: Cultured rat Tenon's capsule fibroblasts were treated with TGF-β1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon’s capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-β1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1β，TNF-α，SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. Results: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-β1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1β，TNF-α，SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. Conclusions: MiR-146a effectively reduced TGF-β1-induced fibrosis in rat Tenon’s capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS. fibrosis glaucoma glaucoma filtration surgery miR-146a rat Tenon’s fibroblasts TGF Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Glaucoma filtration surgery (GFS) plays a crucial role in the treatment of glaucoma by reducing intraocular pressure (IOP) via redirecting the flow of aqueous humor towards the subconjunctival area, resulting in the formation of a bleb. The main challenge of this surgical procedure is scarring of the filtering bleb within the subconjunctival region, which reduces treatment effectiveness. Although extensive prospective randomized studies have demonstrated the efficacy of mitomycin C (MMC) or 5-fluorouracil (5-FU) in preventing scarring and improving GFS outcomes, their clinical use is limited owing to potential risks to vision. 1–3 Therefore, there is an urgent need for innovative antifibrotic medications that can effectively prevent post-surgery scarring. Transforming growth factor beta (TGF-β) is a cytokine that is crucial for wound healing. 4 TGF-β stimulates transformation of fibroblasts into myofibroblasts, which are primarily responsible for producing fibrous tissues. 5 The formation of scars at the sclerostomy site or bleb connective tissue negatively affects the outcome of filtration surgery because of the abnormal extracellular matrix production and excessive proliferation. 6,7 Tissue fibrosis stems from the accumulation of the extracellular matrix linked to the persistence of myofibroblasts. 8 Activated rat Tenon’s fibroblasts generate collagen, and TGF-β 5 regulates this process in an autocrine loop by activating TGF-β receptors. 9 Consequently, reduction of collagen expression and suppressing the activation of Tenon’s fibroblasts are crucial for developing treatments for postsurgical scarring. Several studies have recently highlighted the vital roles that microRNAs (miRNAs) play in organ fibrosis, 10 proposing a novel approach for regulating fibrotic processes. 11 By base pairing with corresponding 3′-untranslated regions, these small, 22-nucleotide RNAs, known as miRNAs, modify the expression of genes by preventing translation or promoting miRNA degradation. In addition to controlling cell division, apoptosis, and proliferation, miRNAs participate in the developmental and metabolic processes. 10,11 We employed miRNA microarray analysis to identify the miRNAs involved in bleb scarring and found that nine miRNAs were upregulated in activated rat Tenon’s fibroblasts, whereas seven miRNAs were downregulated. Among these miRNAs, miR-146a was significantly up-regulated in TGF-β1-activated rat Tenon’s fibroblasts. Previous studies have shown that miR-146a specifically targets a group of mRNAs encoding profibrotic proteins in various tissues, showing promising effects in treating fibrotic conditions in the kidneys 12,13 , liver 14 , heart 15 , and other organs. Notably, Smad4 was identified as one of the miR-146a target genes. 16–18 Furthermore, Smad4 deletion attenuated the progression of renal fibrosis by preventing the development of the collagen matrix in obstructive nephropathy and inhibiting the production of collagen I by fibroblasts induced by TGF-β1. 19 TGF-β activates SMAD4, which then transmits signals to regulate downstream physiological processes. Disruption of Smad4 decreased renal Smad7 mRNA expression, promoted renal inflammation dependent on NF-κB signaling, and attenuated the suppressive influence of TGF-β1 on macrophages in vivo and in vitro during an inflammatory response induced by interleukin-1β. 20 Collectively, these findings suggest that SMAD4 could play a substantial role in mediating numerous functions of TGF-β1 in fibrogenesis and inflammation. 20 Postsurgical scarring is an irreversible condition with therapeutic challenges similar to those of organ fibrosis. Identifying concomitant changes in miRNA expression levels suggests the possibility of shared antifibrotic strategies. Postoperative scar development can be treated using miRNA therapy, which effectively treats fibrosis in other organs. Based on previous data about functional significance of miR-146a in the fibrosis of various organs and its involvement in the SMAD4 pathway regulation, we postulated that miR-146a is associated with the growth of rat Tenon’s fibroblasts and fibrosis resistance. Guided by relevant bioinformatics data and miRNA expression profiles, we investigated the effects of miR-146a on the biological features of rat Tenon’s fibroblasts and potential mechanisms of its antifibrotic action. Currently, the role of miR-146a in rat Tenon’s fibroblasts remains unknown. Our findings enrich understanding of the properties of rat Tenon’s fibroblasts and offer a novel approach for treating bleb scarring following GFS. Materials and Methods Cell Cultures and Chemicals Two 5-week-old SD rats were selected for weighing purposes. Intraperitoneal administration of chloral hydrate 10% anesthesia (0.35 mL/100 g) was performed. The eye area was then treated with proparacaine hydrochloride, a local anesthetic, followed by disinfection using iodine volt. Under the microscope, the subconjunctival Tenon's capsule tissue was excised. After immersing the tissue in a sterile phosphate buffered solution (PBS) containing double antibodies (100IU/ml Penicillin-Streptomycin) for 30 minutes, it was transferred to an ultra-clean platform. The tissue underwent two rounds of cleansing with PBS and was subsequently sliced into 1-2mm implants using ophthalmic scissors. Finally, the implants were arranged in a sterile petri dish measuring 6cm with a gap ranging from 0.5 to 1.5cm between them. Following a ten-minute interval, each implant received a droplet of Dulbecco's modified eagle media (DMEM) and incubated at 37°C with 5% CO2 overnight. On the following day, Dulbecco's modified eagle media (DMEM) supplemented with 10% fetal bovine serum (FBS) was added at a volume of 4mL per dish. By replacing the cell medium every 3–4 days, it is possible for the inoculated cells to reach maximum capacity within a culture dish within a period of approximately10 to14 days. Rat Model of GFS This trial was approved by the Ethics Committee of the Ruijin Hospital in Shanghai. The Association for Research in Vision and Ophthalmology (ARVO) guidelines were followed for conducting all the experiments. Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Zhejiang, China) supplied mature male SD rats that weighed around 250 g. The animals were maintained under a 12-h light/dark cycle. Initially, rats received intraperitoneal injections of xylazine (10 mg/kg; Sigma–Aldrich, St. Louis, MO) and ketamine hydrochloride (25 mg/kg; Sigma–Aldrich) to induce a basic sedation state. A drop of 0.5% proparacaine hydrochloride (Tianlong, Suzhou, China) was applied for anesthetizing the eyes being operated on. The left eyes of the rats underwent GFS, as previously reported by Sherwood et al 2004 21 . A conjunctival flap, generated by conjunctival incision and a straightforward dissection of the underlying Tenon’s capsule, was positioned 3–5 mm behind the limbus. Subsequently, a 25-G needle was carefully inserted into the anterior chamber to prevent puncture of the iridal blood vessels and to build a full-thickness scleral tunnel. Rats that had any hyphae were excluded. Viscoelastic fluid was injected into the needle to uphold the anterior chamber. Subsequently, a beveled 30-G micro cannula (Qiu Jin, Shanghai, China) was inserted via the scleral tunnel. After securing the microcannula with limbus fixation, the Tenon’s capsule and conjunctiva were sealed with a monofilament nylon suture (10 − 0, 0.1 metric). The animals with eyes that showed cannula slippage or dislocation were excluded. Rats were anesthetized by inhalation of isoflurane (2–4%; Sigma-Aldrich) prior to IOP measurements. 5-FU, MMC (Kyowa Hakko Kirin Co., Ltd., Shizuoko, Japan), normal saline(NS), miR-146a mimics, and miR-146a inhibitor were administered individually to rats with surgically repaired eyes. Wet the cellulose sponge with MMC (0.4 mg/mL) and 5-FU(25mg/mL), respectively. Subsequently, a syringe was used to irrigate the treated region with 2 mL of 0.9% sodium chloride. The untreated fellow eyes were maintained under controlled conditions. A minimum of six eyes were included in each group. We used lentiviral vectors (LV) to transfect suitable cells with miR-146a according to the manufacturer's guidelines. GenePharma (Shanghai, China) prepared and identified miR-146a LV carrying green fluorescent protein (GFP). Both negative control lentivirus (NC-LV) and recombinant miR-146a -LV were generated and subsequently titrated to 1 × 10 9 transfection units/mL. On postoperative day 1, a subconjunctival injection with a sterile microinjector was performed near the filtering bleb. The expression of miR-146a was assessed by routine examination of enucleated ocular tissues. A total of 48 rats (48 eyes) were divided into eight random groups of six rats each with stable IOP (7–20 mm Hg): ( 1 ) no surgery—the group without any treatment; ( 2 ) sham surgery—this group underwent simple conjunctiva cutting as treatment; ( 3 ) MMC—in this positive control group, GFS was performed using a cotton pad with 0.4 mg/mL MMC intraoperatively for approximately 3 min; ( 4 ) 5-FU—in this positive control group, GFS was performed using a cotton pad with 25mg/mL 5-FU intraoperatively for approximately 3 min; ( 5 ) surgery + NS—the negative control group was subconjunctivally injected with 25 µL NS on day 1; ( 6 ) miR-146a mimics—on day 1 following surgery, this experimental group received a subconjunctival injection of miR-146a mimics (25 µL); ( 7 ) miR-146a inhibitor—on day 1 following surgery, this experimental group received a subconjunctival injection of miR-146a inhibitor (25 µL);( 8 ) surgery + no tube: This group underwent surgery without the use of a tube, which resulted in the formation of a complete scleral tunnel through the surgical incision of the conjunctiva. The eyes of the operated rats were closely monitored from postoperative day 1 to day 28 (D1–D28). During this time, we observed and documented the IOP, filtration of blebs, and any subsequent complications. After the surgery, we stained D28 samples with hematoxylin and eosin (HE) and Masson trichrome, as well as using conventional immunohistochemical, immunofluorescent, and real-time PCR approaches. Histopathological Analysis All rats were euthanized via forced air embolism following deep general anesthesia and their eyes were excised. The number and dispersion of myofibroblasts and the degree of fibrosis were estimated by Masson staining, HE staining, and immunostaining for α-smooth muscle actin (α-SMA). The primary antibodies were as follows:Vimentin(1:50 dilution; selleckchem, A5862), Cytokeratin(1:50 dilution; selleckchem, A5991), COL1A1 (1:50 dilution; ABclonal, A1352), α-SMA (1:50 dilution; ABclonal, A17910) Quantitative RT-PCR TRIzol (Invitrogen, Wuhan, China) was used to obtain total tissue RNA. Subsequently, a spectrophotometer (Leng Guang, Shanghai, China) was used for quantitative and qualitative analysis of the extracted RNA. We generated cDNAs from 10 ng RNA samples, which were subsequently used for quantitative RT-PCR with the SYBR Green Expression Master Mix (Applied Biosystems, Inc., Foster City, CA, USA). Each experiment was conducted three times. The ΔΔCT technique (2 −ΔΔCt ) was employed to determine the differences in the relative RNA expression levels between the control and treatment groups. Western Blot Analysis The samples were washed three times with phosphate-buffered saline at 4 ° and then extracted in cold RIPA lysis buffer (strong) composed of 1 mmol/L EDTA, 1% Na 3 VO 4 , 5 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride, Other components included 2.5 mmol/L sodium pyrophosphate, 150 mmol/L NaCl, 1% Triton X-100, and 20 mmol/L Tris (pH 7.5). The supernatant was centrifuged at 16,099 × g for 10 min, quantified, and finally used for western blot analysis. Immunoreactive proteins were observed on autoradiograph films using chemiluminescence detection reagents (ECL; GE Healthcare, Laurel, MD, USA). Monoclonal antibodies against fibronectin (FN), collagen Iα, α-SMA, SMAD4, NF-κB p65 subunit, IL-1β, and COX2 were obtained from Cell Signaling Technologies and GE Healthcare; β-actin (Sigma-Aldrich Corp., St. Louis, MO, USA) was used as loading control. The primary antibodies were as follows:β-actin (1:1000 dilution; ABclonal, AC026), TNF-α (1:1000 dilution;Abcam, ab205587), IL-1β (1:500 dilution; Abcam, ab205924), α-SMA (1:1000 dilution; ABclonal, A17910), SMAD4(1:1000 dilution; ABclonal, A21487), NF-KB p65 (1:1000 dilution; ABclonal, A2547), Fibronectin (1:1000 dilution; Abways, CY9537), TGFβ1(1:1000 dilution; ABclonal, A2124), COL1A1 (1:1000 dilution; ABclonal, A1352) and COX2(1:1000 dilution; Abways, CY3818) Statistical Analysis Each experiment was performed in triplicate. The results are presented as the mean ± standard error of the mean. The Student’s t -test was used to statistically analyze the results and compare the differences between the treated and blank groups. Differences between multiple groups were analyzed using one-way analysis of variance. Differences were considered statistically significant if P < 0.05. Results Expression of miRNA146a Increased in TGF-β1-Stimulated Rat tenon Fibroblasts After 4–7 days, cells with unusual shapes, such as spindle-shaped and stellate cells, appeared around the tissue of the adhering rat tenon. Following passage, the cells moved to the surrounding area and were confluent for at least 90% of the time (Fig. 1 A). Fibroblasts were identified by immunofluorescence staining. Most fibroblasts had blue-stained nuclei; however, the cytoplasm glowed red when stained with vimentin specific antibody but cytokeratin staining was negative(Fig. 1 B). Next, to identify miRNAs involved in scarring following filtration surgery, rat Tenon’s fibroblasts were treated with TGF-β1, and their miRNA expression profiles were compared to those in the untreated primary rat Tenon’s fibroblasts (control group) from the same passages (Fig. 1 C-E). Out of the 507 distinct miRNAs displayed on the microarrays, 16 had significantly changed expression in rat Tenon’s fibroblasts upon stimulation with TGF-β1. Expression levels of nine of these miRNAs were more than 2-fold higher, whereas for seven of them, expression levels were more than 2-fold lower than those in control cells (Fig. 1 C,D). Expression pattern cluster analysis was used to create a heat map representation of miRNAs that showed a 2-fold or greater change in expression in activated rat Tenon’s fibroblasts following 24 h stimulation with TGF-β1(Fig. 1 D). miR microarray analysis demonstrates a substantial (~ 120-fold) increase in miR-146a expression in TGF-β1-stimulated rat Tenon’s fibroblasts compared to that in the untreated fibroblasts (Fig. 1 F). Administration of miR-146a Mimics Enhances the Persistence of Filtering Blebs Following GFS in Rats Following GFS, there were no obvious congestive, cataract and other complications in anterior segment in each group (Fig. 2 A) and there was no significant change in IOP between the treatment groups. Consequently, the IOP of these two groups was not much lower than that in the blank group on postoperative D21 (Fig. 2 B) ( P > 0.05). A functional filtering bleb is crucial for regulating IOP after GFS. Therefore, we investigated the influence of seven different treatments on the durability of filtering blebs following GFS. The outcomes of the bleb survival analysis revealed a significant effect of treatment on the survival rates in the surgery alone, miR-146a mimics, MMC, and 5-FU groups ( P < 0.001). Post-GFS, rats in the surgical “no tube” group experienced a steep decline in filtering blebs, with a paucity of such blebs observed at D14. The persistence of filtering blebs in the mimics, MMC, and 5-FU groups was notably greater in comparison to that in the surgery + NS group. The degradation of filtering blebs in the 5-FU group started on D14 and finished by D28. In contrast, filtering blebs in the MMC group survived noticeably longer than those in the 5-FU group. The degradation of filtering blebs in the MMC group commenced on D14, and 50% of the blebs were still present at D28. Filtering blebs disappeared in the miR-146a mimics group on D21, but 30% of them still persisted at D28. Compared to the observations in the surgery plus NS group, therapy with miR-146a mimics significantly enhanced bleb survival ( P < 0.001). Altogether, these results indicate that treatment with miR-146a mimics enhanced filtering bleb survival following GFS, and its benefits seem to be as substantial as 5-FU (Fig. 2 C). In Vivo Transfection of miR-146a Regulates Fibrosis On day 1 postoperatively, eye operation sites were subjected to a subconjunctival injection of miR-146a-LV (25 µL). Masson and HE staining of samples was performed on postoperative D14 and D28. The collagen development between the conjunctiva and sclera of the operated area dramatically reduced in the experimental group transfected with miR-146a -mimics, as shown by Masson trichrome staining. We observed some collagen production in the positive control group. Collagen was heavily deposited in the groups that underwent a single surgery and in the negative control group. To ascertain the extent of fibrosis and collagen deposition, we performed α-SMA immunohistochemistry staining. In the blank group, α-SMA was not expressed between the conjunctiva and sclera. However, under the blebs, α-SMA was strongly expressed in the groups that underwent a single surgery and in the negative control group. α-SMA was extensively expressed at the surgery site, but modestly expressed in the positive control group. Moreover, α-SMA expression was infrequent in the experimental group. At D28, the eyes treated with miR-146a had significantly less collagen deposition than the operated eyes in other groups. Based on these findings, we inferred that transfection with miR-146a mimics dramatically suppressed the expression of α-SMA mRNA in the rat model of GFS (Fig. 3 ). Western blot analysis revealed significant reductions in α-SMA, FN, TNFα, IL-1β, Col1A1, and SMAD4 levels in the surgery + NS, negative control, positive control, experimental, and blank groups. Furthermore, the degree of these reductions positively correlated with miR-146a expression (Fig. 4 A-C). TGF-β1 Promotes Proliferation of Rat Tenon’s Fibroblasts It has been shown previously that TGF-β1 strongly promotes fibroblast proliferation. To examine its impact on rat Tenon’s fibroblasts, TGF-β1 was applied to these cells at a concentration of 10 ng/mL for 0, 6, 12, or 24 h. The western blot assay findings indicated a time-dependent increase in cell proliferation, which was further supported by the production of two indicators of cell fibrosis, TGF-β1 and collagen Iα (Fig. 5 A-C). We then examined the amounts of FN, collagen Iα, α-SMA, SMAD4, NF-κB p65, IL-1β, and COX2 in the Tenon’s tissues in both normal participants and those treated with TGF-β1. Western blot analysis and immunofluorescence revealed that all the molecules that may activate rat Tenon’s fibroblasts had substantially greater proteins expression levels in TGF-β1-treated patients than in control subjects (Fig. 5 D,E,G,H). Moreover, TGF-β1 therapy increased miR-146a expression (Fig. 5 F). Impact of miR-146a Mimics on Rat Tenon’s Fibroblasts Stimulated with TGF-β1 Using western blotting, we next investigated the impact of miR-146a mimics on rat Tenon’s fibroblasts with respect to expression of fibrotic markers FN, collagen Iα, and α-SMA. Stimulation with TGF-β1 augmented expression levels of FN, collagen Iα, and α-SMA, and these increases were reversed by miR-146a mimics (30 nM, Fig. 6 A-H). To examine the role of miR-146a in TGF-β1-induced myofibroblast transdifferentiation, rat Tenon’s fibroblasts were treated with miRNA mimics and inhibitors (Fig. 6 A-C). Following transfection, inhibitors of miR-146a enhanced fibrosis expression, whereas miR-146a mimics had an opposite effect(Fig. 6 D-G). These findings suggested that TGF-β1-induced myofibroblast transdifferentiation may be impacted via miR-146a expression modulation. MiR-146a Regulates Fibrosis by Targeting SMAD4 Transfection with a miR-146a mimic specifically targets the 3′-untranslated region of Smad4 mRNAs in various cells. SMAD4 protein level decreased significantly after transfection with miR-146a mimics, compared with that in cells treated with control mimics (Fig. 7A,B). Furthermore, SMAD4 expression increased significantly upon miR-146a expression inhibition compared with that in the presence of control inhibitors. We also transfected rat Tenon’s fibroblasts with small interfering RNA against SMAD4 and found that the decreased SMAD4 expression reduced miR-146a expression (Fig. 7C). After transfection with miR-146a mimics and SMAD4-siRNA, the levels of both Smad4 and α-SMA proteins were significantly reduced compared to those treated with TGFβ1. Conversely, the expression levels of Smad4 and α-SMA showed a noticeable increase when using miR-146a-inhibitor + SMAD4-siRNA instead of incorporating miR-146a analogs and SMAD4-siRNA(Fig. 7D). Discussion GFS is a surgical intervention typically performed when conservative treatment outcomes are unfavorable. However, most patients develop bleb scarring following GFS, which is the primary cause of surgery failure. Notably, the intra- and postoperative application of chemotherapeutic drugs 5-FU and MMC significantly reduce the occurrence of postoperative bleb scarring. However, the systemic use of these drugs can lead to cellular damage, bleb leakage, consistently low IOP, corneal scarring, scleritis, and endophthalmitis. Therefore, innovative therapeutic strategies are being developed to enhance the success rate of GFS by suppressing scar formation and inhibiting fibroblast proliferation within the subconjunctival tissue. In previous studies, researchers experimented with various biological, chemical and physical approaches to suppress fibroblast proliferation and scar tissue formation, including the inhibition of TGF-β1, vascular endothelial growth factor, 22 Rho-associated protein kinase, 23 , as well as photodynamic therapy, 24 and others. Although some of these treatments had remarkable results and improved GFS outcomes in the standard rat model, only few of them were successful in large-scale prospective clinical trials. Thus, so far, none of these strategies have replaced MMC or 5-FU use in clinical practice. MiRNAs are crucial regulators of fibrotic processes, and promising target candidates for treating fibrosis in various organs. 25 Tenon’s fibroblasts, which are the primary fibrotic cells in the Tenon’s tissue, contribute to bleb scarring in rodent models after they transdifferentiate into myofibroblast-like cells. In vitro assessments have revealed that the number of myofibroblasts decrease when miR-146a is directly targeted. 26 In the present study, we used LV-facilitated transfection for gene delivery in vivo. We performed RT-PCR and western blot analysis to detect the expression of the transfected genes and assess alterations in their protein levels, respectively. In rats that underwent GFS, miR-146a was successfully transfected when LV concentrate was injected into the subconjunctiva adjacent to the filtering bleb. The resulting up-regulation of miR-146a expression inhibited the formation of new collagen in the surgical site by suppressing SMAD4 signaling pathway. We propose that miR-146a effectively reduces collagen deposition when delivered exogenously, thereby suppressing bleb scarring in rats undergoing GFS. To the best of our knowledge, this is the first time that in vitro and in vivo studies have collectively confirmed that delivering miR-146a can efficiently prevent scarring of the subconjunctival tissue in rats subjected to GFS. Hence, our novel therapeutic approach could be employed in glaucoma surgery in the near future. Previous studies have established various strategies to explore the functional role of miR-146a in fibrosis and to identify miR-146a target genes. 16,27–29 However, these studies have primarily employed in vitro experimental designs. For instance, Gordon et al. 15 utilized human and mouse cardiac microvascular endothelial cells to demonstrate that miR-146a negatively regulates NF-κB and Col1α1 mRNA expression. Luna et al. 30 showed that rats treated with miR-146a had a sustained reduction in IOP, without any observable signs of inflammation or other adverse effects. Sun et al. 26 found that the expression of FN, collagen Iα, and α-SMA protein induced by TGF-β1 treatment was reduced upon the introduction of miR-146a mimics. Additionally, SMAD4 protein levels were significantly decreased in response to miR-146a mimics. 26 Zhang et al. 31 and Kim et al. 32 found that miR-146a expression is enhanced in response to various inflammatory stimuli, and anti-inflammatory effects can be achieved by regulating miR-146a expression. Based on these results, we developed a rat model of GFS and tested gene therapy to prevent scarring. Usually, these strategies require long-term transgene expression in subconjunctival tissues. We fulfilled this prerequisite condition by using a single subconjunctival injection of a concentrated preparation of LVs harboring miR-146a in our rat model. Although 28 days was not sufficient for the experiment to be considered long-term, transfected GFP gene was abundantly expressed and visualized in vivo in the enucleated tissues obtained from the operated sites. Although identifying a therapeutic transgene is challenging, we cannot downplay the clinical advantages of gene therapy in hindering bleb scarring. This study had several limitations. As previous studies have not performed LV-mediated miR-146a transfection into rats that had undergone GFS, we had to determine the transfection duration based on the characteristics of the fibrotic process: after undergoing GFS, fibroblast proliferation increased in rats. The conjunctival wound healing in these rats takes place within 7–14 days post-surgery. Additionally, we were unable to determine the optimal frequency and dosage for subconjunctival injections. Prior studies have shown that in vivo LV-mediated transfection has low efficiency, indicating a need for further evaluation of whether gene therapy is a suitable method. In this study, we used a high titer LV preparation and administered it in the undiluted form to maximize transfection levels. However, factors such as the selection of promoters, quality of vector preparation, and viral dose must be carefully considered in the future studies. Moreover, we presumed that vector particles were not shed in tears or aqueous humor. These assumptions are valid for short observation periods; however, future studies should explore whether time-dependent changes affect transfection efficiency. Conclusion our study is the first one to describe LV-mediated transfection of microRNA for gene delivery in an animal model of GFS. Drawing on our collective research findings, we believe that modulating miR-146a levels could mitigate bleb scarring following GFS. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interest. Funding This work was supported by the National Natural Science Foundation of China [NO. 82070953, NO. 82371048, NO.82000885], and the Shanghai Science and Technology Committee Project Foundation [NO. 21Y11909700]; The funding sources had no involvement all through the study. Authors' contributions Ruiqi Han performed the in vitro experiments and image quantification. Huimin Zhong and Yang Zhang performed the HE and Masson's trichrome staining as-says. Yumeng Zhang and Huan Yu conducted the in vivo experiments. Shouyue Huang helped perform the experiments and analyzed the data. Yisheng Zhong, Shouyue Huang, and Zijian Yang organized the work and revised the manuscript, which was written by Ruiqi Han. All authors have read and approved the final manuscript. Acknowledgements Funding: National Natural Science Foundation of China [NO. 82070953, NO. 82371048, NO.82000885], and the Shanghai Science and Technology Committee Project Foundation [NO. 21Y11909700]. Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Knapp A, Heuer DK, Stern GA, Driebe WT. Serious corneal complications of glaucoma filtering surgery with postoperative 5-fluorouracil. Am J Ophthalmol. 1987;103(2):183–7. Rubinfeld RS, Pfister RR, Stein RM, et al. Serious complications of topical mitomycin-C after pterygium surgery. Ophthalmology. 1992;99(11):1647–54. Shapiro MS, Thoft RA, Friend J, Parrish RK, Gressel MG. 5-Fluorouracil toxicity to the ocular surface epithelium. Investig Ophthalmol Vis Sci. 1985;26(4):580–3. Shah M, Foreman DM, Ferguson MW. 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Role of microRNA-146a in regulation of fibrosis in orbital fibroblasts from patients with Graves' orbitopathy. Br J Ophthalmol. 2018;102(3):407–14. Su Y-L, Wang X, Mann M, et al. Myeloid cell-targeted miR-146a mimic inhibits NF-κB-driven inflammation and leukemia progression in vivo. Blood. 2020;135(3):167–80. Qiu M, Li T, Wang B, Gong H, Huang T. miR-146a-5p Regulated Cell Proliferation and Apoptosis by Targeting SMAD3 and SMAD4. Protein Pept Lett. 2020;27(5):411–8. Liu X, Zhang K, Wang L, et al. Fluid shear stress-induced down-regulation of miR-146a-5p inhibits osteoblast apoptosis via targeting SMAD4. Physiol Res. 2022;71(6):835–48. Luna C, Parker M, Challa P, Gonzalez P. Long-Term Decrease of Intraocular Pressure in Rats by Viral Delivery of miR-146a. Translational Vis Sci Technol. 2021;10(8):14. Zhang J, Li P, Zhao G, et al. Mesenchymal stem cell-derived extracellular vesicles protect retina in a mouse model of retinitis pigmentosa by anti-inflammation through miR-146a-Nr4a3 axis. Stem Cell Res Ther. 2022;13(1):394. Kim SJ, Russell AE, Wang W, et al. miR-146a Dysregulates Energy Metabolism During Neuroinflammation. J Neuroimmune Pharmacology: Official J Soc NeuroImmune Pharmacol. 2022;17(1–2):228–41. Cite Share Download PDF Status: Published Journal Publication published 07 May, 2024 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 26 Jan, 2024 Reviewers invited by journal 25 Jan, 2024 Editor assigned by journal 24 Jan, 2024 First submitted to journal 22 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3883641","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":269530032,"identity":"49503aff-f19c-4594-b01b-1919f55e86aa","order_by":0,"name":"Ruiqi Han","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBACAyjJY9/M//EBAw+SIEEtBuwMxiCKWC0gBj+DmQSUj1+LOfvZY595CqxlzJkZ0ip/yPxJbGBv3ibBUHMHpxbLnrzk2TwG6TyWzQzHbvPwGCQ28Bwrk2A49gy3ww7kGDPzGBzmYTjM2HabAaRFIsdMgrHhMG4t59/AtDCzFf4AaZF/Q0DLDagtBofZ2BjADpPgIaTljTHjHKBfJJt5mKV5eIyN23jSii0SjuFzWI4xw5s/1vb8/GcYP/7skZPtZz+88caHGtxaoIAZQjH2MDCwgRgJhDTAtTD8IKx0FIyCUTAKRh4AAPDjSBWZ0rj/AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7379-8135","institution":"Ruijin Hospital: Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital","correspondingAuthor":true,"prefix":"","firstName":"Ruiqi","middleName":"","lastName":"Han","suffix":""},{"id":269530033,"identity":"f8fee118-a375-4522-98b4-47daf8d34507","order_by":1,"name":"Huimin Zhong","email":"","orcid":"","institution":"Shanghai First People's Hospital: Shanghai Jiaotong University First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Zhong","suffix":""},{"id":269530034,"identity":"8d212e32-5f09-4f34-90dd-c05b05b943ed","order_by":2,"name":"Yang Zhang","email":"","orcid":"","institution":"Ruijin Hospital: Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Zhang","suffix":""},{"id":269530035,"identity":"48d0e9c7-a394-46aa-a6f5-422c513851d8","order_by":3,"name":"Huan Yu","email":"","orcid":"","institution":"Ruijin Hospital: Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Yu","suffix":""},{"id":269530036,"identity":"f26264eb-86a3-494d-8084-69bb763317fa","order_by":4,"name":"Yumeng Zhang","email":"","orcid":"","institution":"Ruijin Hospital: Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yumeng","middleName":"","lastName":"Zhang","suffix":""},{"id":269530037,"identity":"a0617f3b-f241-4041-9a56-02877d47e3dd","order_by":5,"name":"Shouyue Huang","email":"","orcid":"","institution":"Ruijin Hospital: Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shouyue","middleName":"","lastName":"Huang","suffix":""},{"id":269530038,"identity":"4c8e4acc-be05-4652-acf3-a884a28ae6f7","order_by":6,"name":"Zijian Yang","email":"","orcid":"","institution":"Ruijin Hospital: Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zijian","middleName":"","lastName":"Yang","suffix":""},{"id":269530039,"identity":"8ce66aa6-2513-44a0-87a9-192b3fa1caf2","order_by":7,"name":"Yisheng Zhong","email":"","orcid":"https://orcid.org/0000-0001-5604-6591","institution":"Ruijin Hospital: Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yisheng","middleName":"","lastName":"Zhong","suffix":""}],"badges":[],"createdAt":"2024-01-21 05:28:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3883641/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3883641/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-024-05170-2","type":"published","date":"2024-05-08T00:46:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50390000,"identity":"c09ee324-828b-42b9-8ed3-a58aa4c83d88","added_by":"auto","created_at":"2024-01-30 18:39:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":635519,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of \u003cem\u003emiR-146a\u003c/em\u003e is obviously increased in TGF-β1-stimulated rat Tenon’s fibroblasts. After 4–7 days, the cells moved out of the adhering rat Tenon’s tissue, and following passages, achieved confluence of 90% or more. Images were captured at a magnification of 100× (A). Staining of rat Tenon’s fibroblasts with antibodies against vimentin, cytokeratin, and fibroblast surface protein. Blue indicates nuclei staining with 4′,6-diamidino-2-phenylindole (DAPI); red indicates vimentin staining. Cytokeratin staining is negative. Each image was captured at 400× magnification (B). \u0026nbsp;The volcano plot shows differentially expressed miRNAs. Red, blue, and gray indicate up-regulated, down-regulated, and unchanged miRNAs, respectively. Fold changes were used to express the values in relation to the corresponding miRNA levels in quiescent primary rat Tenon’s fibroblasts (C). Expression pattern cluster analysis was used to create a heat map representation of miRNAs (D). miR microarray analysis demonstrates a substantial (~120-fold) increase in \u003cem\u003emiR-146a\u003c/em\u003e expression in TGF-β1-stimulated rat Tenon’s fibroblasts compared to that in the untreated fibroblasts (E). Quantitative real-time RT-PCR reveals a significant increase in \u003cem\u003emiR-146a\u003c/em\u003e expression (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) in rat Tenon’s fibroblasts stimulated with TGF-β1, thereby confirming microarray data (F). After 3 days, the transfection rate of lentivirus reached 90%, as indicated by green fluorescent protein expression. Images were captured at 100× magnification(G). Rat Tenon’s fibroblasts treated with \u003cem\u003emiR-146a\u003c/em\u003e mimics showed a substantial rise in miR-146a expression compared to that in controls or in TGF-β1-treated cells (****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). An inhibitor of \u003cem\u003emiR-146a\u003c/em\u003e had opposite effects. There was no significant difference between group mimics and group TGF -β1 (H).\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3883641/v1/125544152fb558f36147e28e.jpg"},{"id":50389998,"identity":"ef683ad7-5d55-41c4-82ec-82ad8f1f5610","added_by":"auto","created_at":"2024-01-30 18:39:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":291498,"visible":true,"origin":"","legend":"\u003cp\u003eMiR-146a mimics enhanced filtering bleb survival following GFS. Administration of \u003cem\u003emiR-146a\u003c/em\u003e mimics enhances the persistence of filtering blebs following GFS in rats. Development of filtration blebs in no surgery, sham surgery, MMC, 5-FU, surgery + NS, \u003cem\u003emiR-146a\u003c/em\u003e mimics, \u003cem\u003emiR-146a\u003c/em\u003e inhibitor, and surgery + no tube eyes groups at D7, D14, D21, and D28 (A). IOP scores on D1- D28 in the no surgery, sham surgery, MMC, 5-FU, surgery + NS, \u003cem\u003emiR-146a\u003c/em\u003e mimics, \u003cem\u003emiR-146a\u003c/em\u003e inhibitor, and surgery + no tube groups post GFS (B). Persistence of filtering blebs in no surgery, sham surgery, MMC, 5-FU, surgery + NS, \u003cem\u003emiR-146a\u003c/em\u003emimics, \u003cem\u003emiR-146a\u003c/em\u003e inhibitor, and surgery + no tube groups following GFS(C).\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3883641/v1/712540590d9249f3ae6f4f75.jpg"},{"id":50390001,"identity":"7061dbe1-3557-4b8c-928b-5929d98c03b6","added_by":"auto","created_at":"2024-01-30 18:39:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":803787,"visible":true,"origin":"","legend":"\u003cp\u003eMiR-146a mimics reduced subconjunctival fibrosis after GFS surgery. Analysis of the conjunctiva and sclera within the bleb region of the eight experimental groups using HE, Masson trichrome, and α-SMA immunohistochemistry staining (×100).\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3883641/v1/6e60fd086e6ef2c52ec46388.jpg"},{"id":50390002,"identity":"2184e77b-3d89-4844-ad7f-a1a6407ccb1b","added_by":"auto","created_at":"2024-01-30 18:39:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":305637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMiR-146a inhibits fibrosis in vivo.\u003c/em\u003e Polymerase chain reaction analysis of changes in miR-146a expression after injections of NS, 5-FU, MMC, or transfection with \u003cem\u003emiR-146a\u003c/em\u003e mimics and inhibitors(A). Representative western blot images of α-SMA, FN, TNFα, IL-1β, Col1A1, and SMAD4 protein levels normalized by β-actin levels(B). Blue (DAPI) and red staining indicate nuclei and α-SMA expression, respectively(C). Bar scale: 100 μm; magnification: ×20.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3883641/v1/bcace66f0429b910722da89d.jpg"},{"id":50391205,"identity":"1a37a7a9-8fc8-4fe8-8eda-257374092bf5","added_by":"auto","created_at":"2024-01-30 18:47:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":660888,"visible":true,"origin":"","legend":"\u003cp\u003eTGF-β1 Promotes Proliferation of Rat Tenon’s Fibroblasts. TGF-β1 stimulates the development of rat Tenon’s fibroblasts. Rat Tenon’s capsule fibroblastswere treated with 10 ng/mL TGF-β1 for 0, 6, 12, or 24 h(A). Western blot assays was performed to determine the expression of cell fibrosis markers TGF-β1 and collagen Iα related to cell proliferation. Compared to controls, **** P \u0026lt; 0.001(A-C). Treatment of rat Tenon’sfibroblasts with TGF-β1 for 24 h led to a significant rise in \u003cem\u003emiR-146a \u003c/em\u003eexpression compared to that in control (untreated) cells; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01(F). Staining of TGF-β1-treated and untreated (control) rat Tenon’s fibroblast cells with antibodies against α-SMA, collagen Iα, and fibronectin protein. Red and blue indicate α-SMA and nuclear staining(g), Nuclei were identified with DAPI (blue) and collagen Iα-positive staining (green)(H) ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Every image was captured at 400× magnification. \u0026nbsp;Extended treatment with TGF-β1 led to elevated expression of COX2, SMAD4, NF-kB p65, α-SMA, collagen Iα, TGF-β1, IL-1β, and total FN(D,E). The information in the columns presents the average relative density ratio ± standard deviation of three distinct experiments, all adjusted to the β-actin concentration within the same specimen. Statistical significance of differences between treated and untreated cells is illustrated as follows: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3883641/v1/8cf69c89c5da4988ae63ca38.jpg"},{"id":50391204,"identity":"0f7c854c-3e12-4e8f-a78c-cdf84fa4e2ae","added_by":"auto","created_at":"2024-01-30 18:47:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":418706,"visible":true,"origin":"","legend":"\u003cp\u003eMiR-146a inhibits TGF-β1-induced myofibroblast transdifferentiation . Representative western blot images of α-SMA, FN, Col1A1 protein levels normalized by β-actin levels(A-H). Nuclei were stained using 4′,6-diamidino-2-phenylendole (DAPI) and visualized ×20 magnification. Bar scale: 100 μm. Immunofluorescence staining for α-SMA (red) and collagen Iα (green) (×20 magnification. Bar scale: 100 μm) (I,J). \u003cem\u003emiR-146a\u003c/em\u003e mimics and inhibitors, respectively, decreased and increased expression of α-SMA and collagen Iα in control and TGF-β1-treated fibroblasts of rat Tenon’s capsule. The information in the columns shows the average relative density ratio ± standard deviation of three distinct experiments, all adjusted to the β-actin concentration within the same specimen. Statistical significance of differences between treated and untreated cells is shown as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3883641/v1/9d098d3be2b96278add18614.jpg"},{"id":50390003,"identity":"99f6f5b6-3074-4930-8b2b-56bd644e8545","added_by":"auto","created_at":"2024-01-30 18:39:20","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":198612,"visible":true,"origin":"","legend":"\u003cp\u003eMiR-146a Regulates Fibrosis by Targeting SMAD4. Representative western blot images showing SMAD4 protein level normalized by β-actin protein level(A,B). Polymerase chain reaction analysis of changes in miR-146a expression after transfection with siRNA against \u003cem\u003eSMAD4\u003c/em\u003e(C). Representative western blot images showing SMAD4 protein level normalized by β-actin protein level(D).\u003c/p\u003e","description":"","filename":"floatimage9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3883641/v1/0fd54d29af016c6d4baacf0a.jpg"},{"id":56128018,"identity":"234ef88a-e269-4d1c-97f1-0d0c906b7129","added_by":"auto","created_at":"2024-05-09 00:46:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1525524,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3883641/v1/d5a6a546-7697-4448-b06f-2bf87d09ff10.pdf"}],"financialInterests":"","formattedTitle":"MiR-146a Reduces Fibrosis after Glaucoma Filtration Surgery in Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlaucoma filtration surgery (GFS) plays a crucial role in the treatment of glaucoma by reducing intraocular pressure (IOP) via redirecting the flow of aqueous humor towards the subconjunctival area, resulting in the formation of a bleb. The main challenge of this surgical procedure is scarring of the filtering bleb within the subconjunctival region, which reduces treatment effectiveness. Although extensive prospective randomized studies have demonstrated the efficacy of mitomycin C (MMC) or 5-fluorouracil (5-FU) in preventing scarring and improving GFS outcomes, their clinical use is limited owing to potential risks to vision.\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e Therefore, there is an urgent need for innovative antifibrotic medications that can effectively prevent post-surgery scarring.\u003c/p\u003e \u003cp\u003eTransforming growth factor beta (TGF-β) is a cytokine that is crucial for wound healing.\u003csup\u003e4\u003c/sup\u003e TGF-β stimulates transformation of fibroblasts into myofibroblasts, which are primarily responsible for producing fibrous tissues.\u003csup\u003e5\u003c/sup\u003e The formation of scars at the sclerostomy site or bleb connective tissue negatively affects the outcome of filtration surgery because of the abnormal extracellular matrix production and excessive proliferation.\u003csup\u003e6,7\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTissue fibrosis stems from the accumulation of the extracellular matrix linked to the persistence of myofibroblasts.\u003csup\u003e8\u003c/sup\u003e Activated rat Tenon\u0026rsquo;s fibroblasts generate collagen, and TGF-β\u003csup\u003e5\u003c/sup\u003e regulates this process in an autocrine loop by activating TGF-β receptors.\u003csup\u003e9\u003c/sup\u003e Consequently, reduction of collagen expression and suppressing the activation of Tenon\u0026rsquo;s fibroblasts are crucial for developing treatments for postsurgical scarring. Several studies have recently highlighted the vital roles that microRNAs (miRNAs) play in organ fibrosis,\u003csup\u003e10\u003c/sup\u003e proposing a novel approach for regulating fibrotic processes.\u003csup\u003e11\u003c/sup\u003e By base pairing with corresponding 3\u0026prime;-untranslated regions, these small, 22-nucleotide RNAs, known as miRNAs, modify the expression of genes by preventing translation or promoting miRNA degradation. In addition to controlling cell division, apoptosis, and proliferation, miRNAs participate in the developmental and metabolic processes.\u003csup\u003e10,11\u003c/sup\u003e We employed miRNA microarray analysis to identify the miRNAs involved in bleb scarring and found that nine miRNAs were upregulated in activated rat Tenon\u0026rsquo;s fibroblasts, whereas seven miRNAs were downregulated. Among these miRNAs, \u003cem\u003emiR-146a\u003c/em\u003e was significantly up-regulated in TGF-β1-activated rat Tenon\u0026rsquo;s fibroblasts. Previous studies have shown that \u003cem\u003emiR-146a\u003c/em\u003e specifically targets a group of mRNAs encoding profibrotic proteins in various tissues, showing promising effects in treating fibrotic conditions in the kidneys\u003csup\u003e12,13\u003c/sup\u003e, liver\u003csup\u003e14\u003c/sup\u003e, heart\u003csup\u003e15\u003c/sup\u003e, and other organs. Notably, \u003cem\u003eSmad4\u003c/em\u003e was identified as one of the \u003cem\u003emiR-146a\u003c/em\u003e target genes.\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e Furthermore, \u003cem\u003eSmad4\u003c/em\u003e deletion attenuated the progression of renal fibrosis by preventing the development of the collagen matrix in obstructive nephropathy and inhibiting the production of collagen I by fibroblasts induced by TGF-β1. \u003csup\u003e19\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTGF-β activates SMAD4, which then transmits signals to regulate downstream physiological processes. Disruption of \u003cem\u003eSmad4\u003c/em\u003e decreased renal \u003cem\u003eSmad7\u003c/em\u003e mRNA expression, promoted renal inflammation dependent on NF-κB signaling, and attenuated the suppressive influence of TGF-β1 on macrophages in vivo and in vitro during an inflammatory response induced by interleukin-1β.\u003csup\u003e20\u003c/sup\u003e Collectively, these findings suggest that SMAD4 could play a substantial role in mediating numerous functions of TGF-β1 in fibrogenesis and inflammation.\u003csup\u003e20\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ePostsurgical scarring is an irreversible condition with therapeutic challenges similar to those of organ fibrosis. Identifying concomitant changes in miRNA expression levels suggests the possibility of shared antifibrotic strategies. Postoperative scar development can be treated using miRNA therapy, which effectively treats fibrosis in other organs. Based on previous data about functional significance of \u003cem\u003emiR-146a\u003c/em\u003e in the fibrosis of various organs and its involvement in the SMAD4 pathway regulation, we postulated that \u003cem\u003emiR-146a\u003c/em\u003e is associated with the growth of rat Tenon\u0026rsquo;s fibroblasts and fibrosis resistance. Guided by relevant bioinformatics data and miRNA expression profiles, we investigated the effects of \u003cem\u003emiR-146a\u003c/em\u003e on the biological features of rat Tenon\u0026rsquo;s fibroblasts and potential mechanisms of its antifibrotic action. Currently, the role of \u003cem\u003emiR-146a\u003c/em\u003e in rat Tenon\u0026rsquo;s fibroblasts remains unknown. Our findings enrich understanding of the properties of rat Tenon\u0026rsquo;s fibroblasts and offer a novel approach for treating bleb scarring following GFS.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Cultures and Chemicals\u003c/h2\u003e \u003cp\u003eTwo 5-week-old SD rats were selected for weighing purposes. Intraperitoneal administration of chloral hydrate 10% anesthesia (0.35 mL/100 g) was performed. The eye area was then treated with proparacaine hydrochloride, a local anesthetic, followed by disinfection using iodine volt. Under the microscope, the subconjunctival Tenon's capsule tissue was excised. After immersing the tissue in a sterile phosphate buffered solution (PBS) containing double antibodies (100IU/ml Penicillin-Streptomycin) for 30 minutes, it was transferred to an ultra-clean platform. The tissue underwent two rounds of cleansing with PBS and was subsequently sliced into 1-2mm implants using ophthalmic scissors. Finally, the implants were arranged in a sterile petri dish measuring 6cm with a gap ranging from 0.5 to 1.5cm between them. Following a ten-minute interval, each implant received a droplet of Dulbecco's modified eagle media (DMEM) and incubated at 37\u0026deg;C with 5% CO2 overnight. On the following day, Dulbecco's modified eagle media (DMEM) supplemented with 10% fetal bovine serum (FBS) was added at a volume of 4mL per dish. By replacing the cell medium every 3\u0026ndash;4 days, it is possible for the inoculated cells to reach maximum capacity within a culture dish within a period of approximately10 to14 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRat Model of GFS\u003c/h2\u003e \u003cp\u003e This trial was approved by the Ethics Committee of the Ruijin Hospital in Shanghai. The Association for Research in Vision and Ophthalmology (ARVO) guidelines were followed for conducting all the experiments. Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Zhejiang, China) supplied mature male SD rats that weighed around 250 g. The animals were maintained under a 12-h light/dark cycle. Initially, rats received intraperitoneal injections of xylazine (10 mg/kg; Sigma\u0026ndash;Aldrich, St. Louis, MO) and ketamine hydrochloride (25 mg/kg; Sigma\u0026ndash;Aldrich) to induce a basic sedation state. A drop of 0.5% proparacaine hydrochloride (Tianlong, Suzhou, China) was applied for anesthetizing the eyes being operated on. The left eyes of the rats underwent GFS, as previously reported by Sherwood et al 2004\u003csup\u003e21\u003c/sup\u003e. A conjunctival flap, generated by conjunctival incision and a straightforward dissection of the underlying Tenon\u0026rsquo;s capsule, was positioned 3\u0026ndash;5 mm behind the limbus. Subsequently, a 25-G needle was carefully inserted into the anterior chamber to prevent puncture of the iridal blood vessels and to build a full-thickness scleral tunnel. Rats that had any hyphae were excluded. Viscoelastic fluid was injected into the needle to uphold the anterior chamber. Subsequently, a beveled 30-G micro cannula (Qiu Jin, Shanghai, China) was inserted via the scleral tunnel. After securing the microcannula with limbus fixation, the Tenon\u0026rsquo;s capsule and conjunctiva were sealed with a monofilament nylon suture (10\u0026thinsp;\u0026minus;\u0026thinsp;0, 0.1 metric). The animals with eyes that showed cannula slippage or dislocation were excluded. Rats were anesthetized by inhalation of isoflurane (2\u0026ndash;4%; Sigma-Aldrich) prior to IOP measurements. 5-FU, MMC (Kyowa Hakko Kirin Co., Ltd., Shizuoko, Japan), normal saline(NS), \u003cem\u003emiR-146a\u003c/em\u003e mimics, and \u003cem\u003emiR-146a\u003c/em\u003e inhibitor were administered individually to rats with surgically repaired eyes. Wet the cellulose sponge with MMC (0.4 mg/mL) and 5-FU(25mg/mL), respectively. Subsequently, a syringe was used to irrigate the treated region with 2 mL of 0.9% sodium chloride. The untreated fellow eyes were maintained under controlled conditions. A minimum of six eyes were included in each group. We used lentiviral vectors (LV) to transfect suitable cells with \u003cem\u003emiR-146a\u003c/em\u003e according to the manufacturer's guidelines. GenePharma (Shanghai, China) prepared and identified \u003cem\u003emiR-146a\u003c/em\u003e LV carrying green fluorescent protein (GFP). Both negative control lentivirus (NC-LV) and recombinant \u003cem\u003emiR-146a\u003c/em\u003e-LV were generated and subsequently titrated to 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e transfection units/mL. On postoperative day 1, a subconjunctival injection with a sterile microinjector was performed near the filtering bleb. The expression of \u003cem\u003emiR-146a\u003c/em\u003e was assessed by routine examination of enucleated ocular tissues. A total of 48 rats (48 eyes) were divided into eight random groups of six rats each with stable IOP (7\u0026ndash;20 mm Hg): (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) no surgery\u0026mdash;the group without any treatment; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) sham surgery\u0026mdash;this group underwent simple conjunctiva cutting as treatment; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) MMC\u0026mdash;in this positive control group, GFS was performed using a cotton pad with 0.4 mg/mL MMC intraoperatively for approximately 3 min; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) 5-FU\u0026mdash;in this positive control group, GFS was performed using a cotton pad with 25mg/mL 5-FU intraoperatively for approximately 3 min; (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) surgery\u0026thinsp;+\u0026thinsp;NS\u0026mdash;the negative control group was subconjunctivally injected with 25 \u0026micro;L NS on day 1; (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) \u003cem\u003emiR-146a\u003c/em\u003e mimics\u0026mdash;on day 1 following surgery, this experimental group received a subconjunctival injection of \u003cem\u003emiR-146a\u003c/em\u003e mimics (25 \u0026micro;L); (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) \u003cem\u003emiR-146a\u003c/em\u003e inhibitor\u0026mdash;on day 1 following surgery, this experimental group received a subconjunctival injection of \u003cem\u003emiR-146a\u003c/em\u003e inhibitor (25 \u0026micro;L);(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) surgery\u0026thinsp;+\u0026thinsp;no tube: This group underwent surgery without the use of a tube, which resulted in the formation of a complete scleral tunnel through the surgical incision of the conjunctiva.\u003c/p\u003e \u003cp\u003eThe eyes of the operated rats were closely monitored from postoperative day 1 to day 28 (D1\u0026ndash;D28). During this time, we observed and documented the IOP, filtration of blebs, and any subsequent complications. After the surgery, we stained D28 samples with hematoxylin and eosin (HE) and Masson trichrome, as well as using conventional immunohistochemical, immunofluorescent, and real-time PCR approaches.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Analysis\u003c/h2\u003e \u003cp\u003eAll rats were euthanized via forced air embolism following deep general anesthesia and their eyes were excised. The number and dispersion of myofibroblasts and the degree of fibrosis were estimated by Masson staining, HE staining, and immunostaining for α-smooth muscle actin (α-SMA). The primary antibodies were as follows:Vimentin(1:50 dilution; selleckchem, A5862), Cytokeratin(1:50 dilution; selleckchem, A5991), COL1A1 (1:50 dilution; ABclonal, A1352), α-SMA (1:50 dilution; ABclonal, A17910)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR\u003c/h2\u003e \u003cp\u003eTRIzol (Invitrogen, Wuhan, China) was used to obtain total tissue RNA. Subsequently, a spectrophotometer (Leng Guang, Shanghai, China) was used for quantitative and qualitative analysis of the extracted RNA. We generated cDNAs from 10 ng RNA samples, which were subsequently used for quantitative RT-PCR with the SYBR Green Expression Master Mix (Applied Biosystems, Inc., Foster City, CA, USA). Each experiment was conducted three times. The ΔΔCT technique (2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e) was employed to determine the differences in the relative RNA expression levels between the control and treatment groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eThe samples were washed three times with phosphate-buffered saline at 4 \u0026deg; and then extracted in cold RIPA lysis buffer (strong) composed of 1 mmol/L EDTA, 1% Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 5 \u0026micro;g/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride, Other components included 2.5 mmol/L sodium pyrophosphate, 150 mmol/L NaCl, 1% Triton X-100, and 20 mmol/L Tris (pH 7.5). The supernatant was centrifuged at 16,099 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min, quantified, and finally used for western blot analysis.\u003c/p\u003e \u003cp\u003eImmunoreactive proteins were observed on autoradiograph films using chemiluminescence detection reagents (ECL; GE Healthcare, Laurel, MD, USA). Monoclonal antibodies against fibronectin (FN), collagen Iα, α-SMA, SMAD4, NF-κB p65 subunit, IL-1β, and COX2 were obtained from Cell Signaling Technologies and GE Healthcare; β-actin (Sigma-Aldrich Corp., St. Louis, MO, USA) was used as loading control. The primary antibodies were as follows:β-actin (1:1000 dilution; ABclonal, AC026), TNF-α (1:1000 dilution;Abcam, ab205587), IL-1β (1:500 dilution; Abcam, ab205924), α-SMA (1:1000 dilution; ABclonal, A17910), SMAD4(1:1000 dilution; ABclonal, A21487), NF-KB p65 (1:1000 dilution; ABclonal, A2547), Fibronectin (1:1000 dilution; Abways, CY9537), TGFβ1(1:1000 dilution; ABclonal, A2124), COL1A1 (1:1000 dilution; ABclonal, A1352) and COX2(1:1000 dilution; Abways, CY3818)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eEach experiment was performed in triplicate. The results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. The Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to statistically analyze the results and compare the differences between the treated and blank groups. Differences between multiple groups were analyzed using one-way analysis of variance. Differences were considered statistically significant if \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExpression of\u003c/strong\u003e \u003cstrong\u003emiRNA146a\u003c/strong\u003e \u003cstrong\u003eIncreased in TGF-\u0026beta;1-Stimulated Rat tenon Fibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 4\u0026ndash;7 days, cells with unusual shapes, such as spindle-shaped and stellate cells, appeared around the tissue of the adhering rat tenon. Following passage, the cells moved to the surrounding area and were confluent for at least 90% of the time (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Fibroblasts were identified by immunofluorescence staining. Most fibroblasts had blue-stained nuclei; however, the cytoplasm glowed red when stained with vimentin specific antibody but cytokeratin staining was negative(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eNext, to identify miRNAs involved in scarring following filtration surgery, rat Tenon\u0026rsquo;s fibroblasts were treated with TGF-\u0026beta;1, and their miRNA expression profiles were compared to those in the untreated primary rat Tenon\u0026rsquo;s fibroblasts (control group) from the same passages (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC-E). Out of the 507 distinct miRNAs displayed on the microarrays, 16 had significantly changed expression in rat Tenon\u0026rsquo;s fibroblasts upon stimulation with TGF-\u0026beta;1. Expression levels of nine of these miRNAs were more than 2-fold higher, whereas for seven of them, expression levels were more than 2-fold lower than those in control cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC,D). Expression pattern cluster analysis was used to create a heat map representation of miRNAs that showed a 2-fold or greater change in expression in activated rat Tenon\u0026rsquo;s fibroblasts following 24 h stimulation with TGF-\u0026beta;1(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). miR microarray analysis demonstrates a substantial (~\u0026thinsp;120-fold) increase in miR-146a expression in TGF-\u0026beta;1-stimulated rat Tenon\u0026rsquo;s fibroblasts compared to that in the untreated fibroblasts (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdministration of\u003c/strong\u003e \u003cstrong\u003emiR-146a\u003c/strong\u003e \u003cstrong\u003eMimics Enhances the Persistence of Filtering Blebs Following GFS in Rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing GFS, there were no obvious congestive, cataract and other complications in anterior segment in each group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) and there was no significant change in IOP between the treatment groups. Consequently, the IOP of these two groups was not much lower than that in the blank group on postoperative D21 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eA functional filtering bleb is crucial for regulating IOP after GFS. Therefore, we investigated the influence of seven different treatments on the durability of filtering blebs following GFS. The outcomes of the bleb survival analysis revealed a significant effect of treatment on the survival rates in the surgery alone, \u003cem\u003emiR-146a\u003c/em\u003e mimics, MMC, and 5-FU groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Post-GFS, rats in the surgical \u0026ldquo;no tube\u0026rdquo; group experienced a steep decline in filtering blebs, with a paucity of such blebs observed at D14. The persistence of filtering blebs in the mimics, MMC, and 5-FU groups was notably greater in comparison to that in the surgery\u0026thinsp;+\u0026thinsp;NS group. The degradation of filtering blebs in the 5-FU group started on D14 and finished by D28. In contrast, filtering blebs in the MMC group survived noticeably longer than those in the 5-FU group. The degradation of filtering blebs in the MMC group commenced on D14, and 50% of the blebs were still present at D28. Filtering blebs disappeared in the \u003cem\u003emiR-146a\u003c/em\u003e mimics group on D21, but 30% of them still persisted at D28. Compared to the observations in the surgery plus NS group, therapy with \u003cem\u003emiR-146a\u003c/em\u003e mimics significantly enhanced bleb survival (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Altogether, these results indicate that treatment with \u003cem\u003emiR-146a\u003c/em\u003e mimics enhanced filtering bleb survival following GFS, and its benefits seem to be as substantial as 5-FU (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eIn Vivo Transfection of miR-146a Regulates Fibrosis\u003c/h2\u003e\n \u003cp\u003eOn day 1 postoperatively, eye operation sites were subjected to a subconjunctival injection of miR-146a-LV (25 \u0026micro;L). Masson and HE staining of samples was performed on postoperative D14 and D28. The collagen development between the conjunctiva and sclera of the operated area dramatically reduced in the experimental group transfected with \u003cem\u003emiR-146a\u003c/em\u003e-mimics, as shown by Masson trichrome staining. We observed some collagen production in the positive control group. Collagen was heavily deposited in the groups that underwent a single surgery and in the negative control group. To ascertain the extent of fibrosis and collagen deposition, we performed \u0026alpha;-SMA immunohistochemistry staining. In the blank group, \u0026alpha;-SMA was not expressed between the conjunctiva and sclera. However, under the blebs, \u0026alpha;-SMA was strongly expressed in the groups that underwent a single surgery and in the negative control group. \u0026alpha;-SMA was extensively expressed at the surgery site, but modestly expressed in the positive control group. Moreover, \u0026alpha;-SMA expression was infrequent in the experimental group. At D28, the eyes treated with \u003cem\u003emiR-146a\u003c/em\u003e had significantly less collagen deposition than the operated eyes in other groups. Based on these findings, we inferred that transfection with \u003cem\u003emiR-146a\u003c/em\u003e mimics dramatically suppressed the expression of \u0026alpha;-SMA mRNA in the rat model of GFS (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eWestern blot analysis revealed significant reductions in \u0026alpha;-SMA, FN, TNF\u0026alpha;, IL-1\u0026beta;, Col1A1, and SMAD4 levels in the surgery\u0026thinsp;+\u0026thinsp;NS, negative control, positive control, experimental, and blank groups. Furthermore, the degree of these reductions positively correlated with \u003cem\u003emiR-146a\u003c/em\u003e expression (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-C).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eTGF-\u0026beta;1 Promotes Proliferation of Rat Tenon\u0026rsquo;s Fibroblasts\u003c/h2\u003e\n \u003cp\u003eIt has been shown previously that TGF-\u0026beta;1 strongly promotes fibroblast proliferation. To examine its impact on rat Tenon\u0026rsquo;s fibroblasts, TGF-\u0026beta;1 was applied to these cells at a concentration of 10 ng/mL for 0, 6, 12, or 24 h. The western blot assay findings indicated a time-dependent increase in cell proliferation, which was further supported by the production of two indicators of cell fibrosis, TGF-\u0026beta;1 and collagen I\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-C).\u003c/p\u003e\n \u003cp\u003eWe then examined the amounts of FN, collagen I\u0026alpha;, \u0026alpha;-SMA, SMAD4, NF-\u0026kappa;B p65, IL-1\u0026beta;, and COX2 in the Tenon\u0026rsquo;s tissues in both normal participants and those treated with TGF-\u0026beta;1. Western blot analysis and immunofluorescence revealed that all the molecules that may activate rat Tenon\u0026rsquo;s fibroblasts had substantially greater proteins expression levels in TGF-\u0026beta;1-treated patients than in control subjects (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD,E,G,H). Moreover, TGF-\u0026beta;1 therapy increased \u003cem\u003emiR-146a\u003c/em\u003e expression (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eImpact of\u003c/strong\u003e \u003cstrong\u003emiR-146a\u003c/strong\u003e \u003cstrong\u003eMimics on Rat Tenon\u0026rsquo;s Fibroblasts Stimulated with TGF-\u0026beta;1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eUsing western blotting, we next investigated the impact of \u003cem\u003emiR-146a\u003c/em\u003e mimics on rat Tenon\u0026rsquo;s fibroblasts with respect to expression of fibrotic markers FN, collagen I\u0026alpha;, and \u0026alpha;-SMA. Stimulation with TGF-\u0026beta;1 augmented expression levels of FN, collagen I\u0026alpha;, and \u0026alpha;-SMA, and these increases were reversed by miR-146a mimics (30 nM, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-H).\u003c/p\u003e\n \u003cp\u003eTo examine the role of \u003cem\u003emiR-146a\u003c/em\u003e in TGF-\u0026beta;1-induced myofibroblast transdifferentiation, rat Tenon\u0026rsquo;s fibroblasts were treated with miRNA mimics and inhibitors (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). Following transfection, inhibitors of \u003cem\u003emiR-146a\u003c/em\u003e enhanced fibrosis expression, whereas \u003cem\u003emiR-146a\u003c/em\u003e mimics had an opposite effect(Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD-G). These findings suggested that TGF-\u0026beta;1-induced myofibroblast transdifferentiation may be impacted via \u003cem\u003emiR-146a\u003c/em\u003e expression modulation.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMiR-146a\u003c/strong\u003e \u003cstrong\u003eRegulates Fibrosis by Targeting SMAD4\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTransfection with a \u003cem\u003emiR-146a\u003c/em\u003e mimic specifically targets the 3\u0026prime;-untranslated region of \u003cem\u003eSmad4\u003c/em\u003e mRNAs in various cells. SMAD4 protein level decreased significantly after transfection with \u003cem\u003emiR-146a\u003c/em\u003e mimics, compared with that in cells treated with control mimics (Fig. 7A,B). Furthermore, SMAD4 expression increased significantly upon \u003cem\u003emiR-146a\u003c/em\u003e expression inhibition compared with that in the presence of control inhibitors. We also transfected rat Tenon\u0026rsquo;s fibroblasts with small interfering RNA against \u003cem\u003eSMAD4\u003c/em\u003e and found that the decreased SMAD4 expression reduced \u003cem\u003emiR-146a\u003c/em\u003e expression (Fig. 7C).\u003c/p\u003e\n \u003cp\u003eAfter transfection with miR-146a mimics and SMAD4-siRNA, the levels of both Smad4 and \u0026alpha;-SMA proteins were significantly reduced compared to those treated with TGF\u0026beta;1. Conversely, the expression levels of Smad4 and \u0026alpha;-SMA showed a noticeable increase when using miR-146a-inhibitor\u0026thinsp;+\u0026thinsp;SMAD4-siRNA instead of incorporating miR-146a analogs and SMAD4-siRNA(Fig. 7D).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGFS is a surgical intervention typically performed when conservative treatment outcomes are unfavorable. However, most patients develop bleb scarring following GFS, which is the primary cause of surgery failure. Notably, the intra- and postoperative application of chemotherapeutic drugs 5-FU and MMC significantly reduce the occurrence of postoperative bleb scarring. However, the systemic use of these drugs can lead to cellular damage, bleb leakage, consistently low IOP, corneal scarring, scleritis, and endophthalmitis. Therefore, innovative therapeutic strategies are being developed to enhance the success rate of GFS by suppressing scar formation and inhibiting fibroblast proliferation within the subconjunctival tissue. In previous studies, researchers experimented with various biological, chemical and physical approaches to suppress fibroblast proliferation and scar tissue formation, including the inhibition of TGF-β1, vascular endothelial growth factor,\u003csup\u003e22\u003c/sup\u003e Rho-associated protein kinase,\u003csup\u003e23\u003c/sup\u003e, as well as photodynamic therapy,\u003csup\u003e24\u003c/sup\u003e and others. Although some of these treatments had remarkable results and improved GFS outcomes in the standard rat model, only few of them were successful in large-scale prospective clinical trials. Thus, so far, none of these strategies have replaced MMC or 5-FU use in clinical practice. MiRNAs are crucial regulators of fibrotic processes, and promising target candidates for treating fibrosis in various organs.\u003csup\u003e25\u003c/sup\u003e Tenon\u0026rsquo;s fibroblasts, which are the primary fibrotic cells in the Tenon\u0026rsquo;s tissue, contribute to bleb scarring in rodent models after they transdifferentiate into myofibroblast-like cells. In vitro assessments have revealed that the number of myofibroblasts decrease when \u003cem\u003emiR-146a\u003c/em\u003e is directly targeted.\u003csup\u003e26\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the present study, we used LV-facilitated transfection for gene delivery in vivo. We performed RT-PCR and western blot analysis to detect the expression of the transfected genes and assess alterations in their protein levels, respectively. In rats that underwent GFS, \u003cem\u003emiR-146a\u003c/em\u003e was successfully transfected when LV concentrate was injected into the subconjunctiva adjacent to the filtering bleb. The resulting up-regulation of \u003cem\u003emiR-146a\u003c/em\u003e expression inhibited the formation of new collagen in the surgical site by suppressing SMAD4 signaling pathway. We propose that \u003cem\u003emiR-146a\u003c/em\u003e effectively reduces collagen deposition when delivered exogenously, thereby suppressing bleb scarring in rats undergoing GFS. To the best of our knowledge, this is the first time that in vitro and in vivo studies have collectively confirmed that delivering miR-146a can efficiently prevent scarring of the subconjunctival tissue in rats subjected to GFS. Hence, our novel therapeutic approach could be employed in glaucoma surgery in the near future. Previous studies have established various strategies to explore the functional role of \u003cem\u003emiR-146a\u003c/em\u003e in fibrosis and to identify \u003cem\u003emiR-146a\u003c/em\u003e target genes.\u003csup\u003e16,27\u0026ndash;29\u003c/sup\u003e However, these studies have primarily employed in vitro experimental designs. For instance, Gordon et al.\u003csup\u003e15\u003c/sup\u003e utilized human and mouse cardiac microvascular endothelial cells to demonstrate that \u003cem\u003emiR-146a\u003c/em\u003e negatively regulates NF-κB and \u003cem\u003eCol1α1\u003c/em\u003e mRNA expression. Luna et al.\u003csup\u003e30\u003c/sup\u003e showed that rats treated with \u003cem\u003emiR-146a\u003c/em\u003e had a sustained reduction in IOP, without any observable signs of inflammation or other adverse effects. Sun et al.\u003csup\u003e26\u003c/sup\u003e found that the expression of FN, collagen Iα, and α-SMA protein induced by TGF-β1 treatment was reduced upon the introduction of miR-146a mimics. Additionally, SMAD4 protein levels were significantly decreased in response to \u003cem\u003emiR-146a\u003c/em\u003e mimics.\u003csup\u003e26\u003c/sup\u003e Zhang et al. \u003csup\u003e31\u003c/sup\u003e and Kim et al.\u003csup\u003e32\u003c/sup\u003e found that \u003cem\u003emiR-146a\u003c/em\u003e expression is enhanced in response to various inflammatory stimuli, and anti-inflammatory effects can be achieved by regulating \u003cem\u003emiR-146a\u003c/em\u003e expression. Based on these results, we developed a rat model of GFS and tested gene therapy to prevent scarring. Usually, these strategies require long-term transgene expression in subconjunctival tissues. We fulfilled this prerequisite condition by using a single subconjunctival injection of a concentrated preparation of LVs harboring \u003cem\u003emiR-146a\u003c/em\u003e in our rat model. Although 28 days was not sufficient for the experiment to be considered long-term, transfected GFP gene was abundantly expressed and visualized in vivo in the enucleated tissues obtained from the operated sites. Although identifying a therapeutic transgene is challenging, we cannot downplay the clinical advantages of gene therapy in hindering bleb scarring.\u003c/p\u003e \u003cp\u003eThis study had several limitations. As previous studies have not performed LV-mediated \u003cem\u003emiR-146a\u003c/em\u003e transfection into rats that had undergone GFS, we had to determine the transfection duration based on the characteristics of the fibrotic process: after undergoing GFS, fibroblast proliferation increased in rats. The conjunctival wound healing in these rats takes place within 7\u0026ndash;14 days post-surgery. Additionally, we were unable to determine the optimal frequency and dosage for subconjunctival injections. Prior studies have shown that in vivo LV-mediated transfection has low efficiency, indicating a need for further evaluation of whether gene therapy is a suitable method. In this study, we used a high titer LV preparation and administered it in the undiluted form to maximize transfection levels. However, factors such as the selection of promoters, quality of vector preparation, and viral dose must be carefully considered in the future studies. Moreover, we presumed that vector particles were not shed in tears or aqueous humor. These assumptions are valid for short observation periods; however, future studies should explore whether time-dependent changes affect transfection efficiency.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eour study is the first one to describe LV-mediated transfection of microRNA for gene delivery in an animal model of GFS. Drawing on our collective research findings, we believe that modulating \u003cem\u003emiR-146a\u003c/em\u003e levels could mitigate bleb scarring following GFS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e Consent for publication\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNot applicable. \u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e Competing interests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e Funding\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [NO. 82070953, NO. 82371048, NO.82000885], and the Shanghai Science and Technology Committee Project Foundation [NO. 21Y11909700]; The funding sources had no involvement all through the study. \u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e Authors\u0026apos; contributions\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eRuiqi Han performed the in vitro experiments and image quantification. Huimin Zhong and Yang Zhang performed the HE and Masson\u0026apos;s trichrome staining as-says. Yumeng Zhang and Huan Yu conducted the in vivo experiments. Shouyue Huang helped perform the experiments and analyzed the data. Yisheng Zhong, Shouyue Huang, and Zijian Yang organized the work and revised the manuscript, which was written by Ruiqi Han. All authors have read and approved the final manuscript. \u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e Acknowledgements\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eFunding: National Natural Science Foundation of China [NO. 82070953, NO. 82371048, NO.82000885], and the Shanghai Science and Technology Committee Project Foundation [NO. 21Y11909700].\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e Availability of data and material\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKnapp A, Heuer DK, Stern GA, Driebe WT. Serious corneal complications of glaucoma filtering surgery with postoperative 5-fluorouracil. Am J Ophthalmol. 1987;103(2):183\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRubinfeld RS, Pfister RR, Stein RM, et al. 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Front Immunol. 2020;11:623693.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou Y, Li S, Li Z, Song D, Zhang S, Yao Q. MiR-146a attenuates liver fibrosis by inhibiting transforming growth factor-β1 mediated epithelial-mesenchymal transition in hepatocytes. Cell Signal. 2019;58:1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng B, Chen S, Gordon AD, Chakrabarti S. miR-146a mediates inflammatory changes and fibrosis in the heart in diabetes. J Mol Cell Cardiol. 2017;105:70\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Q, Cai R, Tang G, Zhang W, Pang W. MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes. J Anim Sci Biotechnol. 2021;12(1):12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuang W, Zheng L, Xu X, et al. Dysregulation of the miR-146a-Smad4 axis impairs osteogenesis of bone mesenchymal stem cells under inflammation. Bone Res. 2017;5:17037.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Huang J, Dai L, et al. miR-146a, an IL-1β responsive miRNA, induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4. Arthritis Res Therapy. 2012;14(2):R75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLan HY. Diverse roles of TGF-β/Smads in renal fibrosis and inflammation. Int J Biol Sci. 2011;7(7):1056\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng X-M, Huang XR, Xiao J, et al. Disruption of Smad4 impairs TGF-β/Smad3 and Smad7 transcriptional regulation during renal inflammation and fibrosis in vivo and in vitro. Kidney Int. 2012;81(3):266\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSherwood MB, Esson DW, Neelakantan A, Samuelson DA. 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Physiol Res. 2022;71(6):835\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuna C, Parker M, Challa P, Gonzalez P. Long-Term Decrease of Intraocular Pressure in Rats by Viral Delivery of miR-146a. Translational Vis Sci Technol. 2021;10(8):14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Li P, Zhao G, et al. Mesenchymal stem cell-derived extracellular vesicles protect retina in a mouse model of retinitis pigmentosa by anti-inflammation through miR-146a-Nr4a3 axis. Stem Cell Res Ther. 2022;13(1):394.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SJ, Russell AE, Wang W, et al. miR-146a Dysregulates Energy Metabolism During Neuroinflammation. J Neuroimmune Pharmacology: Official J Soc NeuroImmune Pharmacol. 2022;17(1\u0026ndash;2):228\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"fibrosis, glaucoma, glaucoma filtration surgery, miR-146a, rat Tenon’s fibroblasts, TGF","lastPublishedDoi":"10.21203/rs.3.rs-3883641/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3883641/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose: \u003c/strong\u003eTo explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-β1 in rat Tenon’s capsule fibroblasts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Cultured rat Tenon's capsule fibroblasts were treated with TGF-β1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon’s capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-β1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1β，TNF-α，SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-β1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1β，TNF-α，SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eMiR-146a effectively reduced TGF-β1-induced fibrosis in rat Tenon’s capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.\u003c/p\u003e","manuscriptTitle":"MiR-146a Reduces Fibrosis after Glaucoma Filtration Surgery in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-30 18:39:15","doi":"10.21203/rs.3.rs-3883641/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-01-26T14:57:49+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-26T01:12:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-24T07:09:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2024-01-22T07:02:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b83e2eff-1a75-41bb-b759-ac78c240338f","owner":[],"postedDate":"January 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-09T00:46:03+00:00","versionOfRecord":{"articleIdentity":"rs-3883641","link":"https://doi.org/10.1186/s12967-024-05170-2","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2024-05-08 00:46:02","publishedOnDateReadable":"May 8th, 2024"},"versionCreatedAt":"2024-01-30 18:39:15","video":"","vorDoi":"10.1186/s12967-024-05170-2","vorDoiUrl":"https://doi.org/10.1186/s12967-024-05170-2","workflowStages":[]},"version":"v1","identity":"rs-3883641","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3883641","identity":"rs-3883641","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}