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 min, it was transferred to an ultra-clean platform. The tissue underwent two rounds of cleansing with PBS and was subsequently sliced into 1–2 mm implants using ophthalmic scissors. Finally, the implants were arranged in a sterile petri dish measuring 6 cm with a gap ranging from 0.5 to 1.5 cm 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.

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(25 mg/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 × 109 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 25 mg/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.

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).

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.

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% Na3VO4, 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).

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.