Silybin inhibits fibrosis after glaucoma filtration surgery in rabbits by promoting fibroblast autophagy

doi:10.12122/j.issn.1673-4254.2026.03.20

Abstract

Abstract:

Objective To investigate the effect of silybin in inhibiting fibrosis after glaucoma filtration surgery and the underlying mechanism. Methods Twenty-five healthy rabbits undergoing unilateral trabeculectomy on the left eye were randomly assigned into 5 groups for treatment with subconjunctival injections of sterilized water (control) or 50, 100, 200, and 250 μmol/L silybin for 7 consecutive days. Postoperative intraocular pressure (IOP) and filtration bleb morphology (Krofeld classification) were monitored. On day 28, tissues samples were harvested from the operated eyes for HE staining, Masson's trichrome staining, and immunofluorescence detection of fibronectin and collagen I. In cultured rabbit Tenon's capsule fibroblasts with TGF‑β1-induced fibrosis, the effects of silybin on autophagy and apoptosis were analyzed using Western blotting (LC3II/LC3I ratio and p62 expression) and flow cytometry. Results From postoperative day 7 to day 21, the rabbits with silybin treatment showed significantly lower IOP than those in the control group, and in the 200 and 250 μmol/L silybin groups, IOP reduction was maintained up to postoperative day 28. From postoperative day 14 to day 21, silybin dose-dependently increased the formation rate of functional filtering blebs, the 200 and 250 μmol/L silybin groups maintained more than 40% functional filtering blebs from postoperative day 14 to day 28. HE staining revealed obviously lessened inflammatory cell infiltration in silybin treatment groups compared with that in the control group. Masson's trichrome staining showed progressively reduced fibroblast numbers and collagen deposition in the silybin groups. Immunofluorescence staining confirmed that silybin dose-dependently reduced fibronectin and collagen I-positive cells. In cultured rabbit Tenon's capsule fibroblasts, silybin treatment effectively reversed TGF-β1-induced fibroblast fibrotic phenotype, increased the LC3II/LC3I ratio, decreased p62 expression, and promoted apoptosis of the fibroblasts. Conclusion Silybin significantly inhibits postoperative fibrosis in rabbit models of glaucoma likely by activating cellular autophagy and inducing apoptosis to reduce fibroblast activation.

Key words: silybin, glaucoma filtration, fibrosis, autophagy, apoptosis, rabbit Tenons capsule fibroblasts

Yuyang ZHANG, Ying SHEN, Xinyu TONG, Yukui DUAN, Yunna LUO, Wenqi GUO. Silybin inhibits fibrosis after glaucoma filtration surgery in rabbits by promoting fibroblast autophagy[J]. Journal of Southern Medical University, 2026, 46(3): 666-674.

Figures/Tables 11

Fig.1 Test results of intraocular pressure of the rabbits in the experimental groups and control group. *P<0.05, **P<0.01, ***P<0.001 vs Control group (n=15).

Tab.2 Comparison of the number of functional filtration blebs between experimental groups and control group (n, %)

Surgery time (day)	Control	Silybin (μmol/L)
Surgery time (day)	Control	50	100	200	250
1	5 (100%)	5 (100%)	5 (100%)	5 (100%)	5 (100%)
7	5 (100%)	5 (100%)	5 (100%)	5 (100%)	5 (100%)
14	0 (0%)	1 (20%)	3 (60%)*	5 (100%)***	5 (100%)***
21	0 (0%)	1 (20%)	2 (40%)	4 (80%)***	5 (100%)***
28	0 (0%)	0 (0%)	0 (0%)	2 (40%)	4 (80%)***

Fig.2 HE staining results in the experimental groups and control group (Original magnification: ×40). The yellow arrow indicates the scleral side.

Fig.3 Masson staining results in the experimental groups and control group (×40). The yellow arrow points to the scleral side.

Fig.4 Immunofluorescence staining for fibronectin and collagen I in each group (×40). The nuclei were stained by DAPI (blue under ultraviolet excitation), and green fluorescence indicates positive expression. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001 vs Control group.

Fig.5 Morphology of cultured rabbit Tenons capsule fibroblasts (RYTF). Green arrow indicates significant fibrosis characteristics of rabbit Tenon's capsule cells (×40).

Fig.6 Immunofluorescence identification of RYTF (×400). Cell nuclei were stained blue with DAPI (A). RYTF showed a positive reaction for vimentin with red staining labeled with CY3 (B), while negative staining for keratin with green staining labeled with 488 (C).

Fig.7 Morphological observation of TGF-β1-induced rabbit Tenons capsular fibroblasts treated with silybin (×40). A: Control group. B: TGF-β1 treatment group. C: TGF-β1 and 200 μmol/L silybin treatment group. Green arrows indicate significant fibrosis characteristics of rabbit Tenon's capsule cells.

Fig.8 Western blotting for detecting autophagy markers in each group. ***P<0.001 vs Control; #P<0.05, ##P<0.01,###P<0.001vs Control.

Fig.9 Cell apoptosis detected by flow cytometry. ***P<0.001 vs Control; ##P<0.01, ###P<0.001 vs Control.

References 33

[1]	Guo L, Wang N, Chen J, et al. Cellular senescence and glaucoma[J]. Exp Gerontol, 2025, 202: 112718. doi：10.1016/j.exger.2025.112718
[2]	Tham Y, Li X, Wong TY, et al. Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis[J]. Ophthalmology, 2014,121(11):2081-90. doi：10.1016/j.ophtha.2014.05.013
[3]	Boland MV, Ervin AM, Friedman DS, et al. Comparative effectiveness of treatments for open-angle glaucoma: a systematic review for the U.S. preventive services task force[J]. Ann Intern Med, 2013, 158(4): 271-9. doi：10.7326/0003-4819-158-4-201302190-00008
[4]	Gedde SJ, Schiffman JC, Feuer WJ, et al. Treatment outcomes in the tube versus trabeculectomy (TVT) study after five years of follow-up[J]. Am J Ophthalmol, 2012, 153(5): 789-803.e2. doi：10.1016/j.ajo.2011.10.026
[5]	Lai C, Shao SC, Chen YH, et al. Trabeculectomy with antimetabolite agents for normal tension glaucoma: a systematic review and meta-analysis[J]. Front Med, 2022, 9: 932232. doi：10.3389/fmed.2022.932232
[6]	苗晓晴, 黄祖烽, 陈玄之, 等. 两种质量浓度丝裂霉素C应用于青光眼手术中的效果比较[J]. 广东医学院学报, 2016, 34(4): 419-21. doi：10.3969/j.issn.1005-4057.2016.04.024
[7]	Li WB, Qu XN, Kang XP, et al. Silibinin eliminates mitochondrial ROS and restores autophagy through IL6ST/JAK2/STAT3 signaling pathway to protect cardiomyocytes from doxorubicin-induced injury[J]. Eur J Pharmacol, 2022, 929: 175153. doi：10.1016/j.ejphar.2022.175153
[8]	Chen HQ, Wang XJ, Wang MH, et al. Behavioral and neurochemical deficits in aging rats with increased neonatal iron intake: silibinin's neuroprotection by maintaining redox balance[J]. Front Aging Neurosci, 2015, 7: 206. doi：10.3389/fnagi.2015.00206
[9]	Liu B, Liu WW, Liu PW, et al. Silibinin alleviates the learning and memory defects in overtrained rats accompanying reduced neuronal apoptosis and senescence[J]. Neurochem Res, 2019, 44(8): 1818-29. doi：10.1007/s11064-019-02816-2
[10]	Gillessen A, Schmidt HHJ. Silymarin as supportive treatment in liver diseases: a narrative review[J]. Adv Ther, 2020, 37(4): 1279-301. doi：10.1007/s12325-020-01251-y
[11]	Ma ZC, Zang WW, Wang HG, et al. Silibinin enhances anti-renal fibrosis effect of MK-521 via downregulation of TGF-β signaling pathway[J]. Hum Cell, 2020, 33(2): 330-6. doi：10.1007/s13577-019-00314-9
[12]	Liu K, Zhou SJ, Liu JY, et al. Silibinin attenuates high-fat diet-induced renal fibrosis of diabetic nephropathy[J]. Drug Des Dev Ther, 2019, 13: 3117-26. doi：10.2147/dddt.s209981
[13]	Shen Y, Zhao HX, Wang ZG, et al. Silibinin declines blue light-induced apoptosis and inflammation through MEK/ERK/CREB of retinal ganglion cells[J]. Artif Cells Nanomed Biotechnol, 2019, 47(1): 4059-65. doi：10.1080/21691401.2019.1671430
[14]	邰雪, 申颖, 赵海霞, 等. 雷帕霉素在兔眼滤过性手术后的抗瘢痕作用[J]. 南方医科大学学报, 2020, 40(9): 1346-52. doi：10.12122/j.issn.1673-4254.2020.09.19
[15]	Wotke J, Homolka P, Vasku J, et al. Histopathology image analysis in two long-term animal experiments with helical flow total artificial heart[J]. Artif Organs, 2016, 40(12): 1137-45. doi：10.1111/aor.12689
[16]	Kim JS, Han NK, Kim SH, et al. Silibinin attenuates radiation-induced intestinal fibrosis and reverses epithelial-to-mesenchymal transition[J]. Oncotarget, 2017, 8(41): 69386-97. doi：10.18632/oncotarget.20624
[17]	Ali SA, Saifi MA, Godugu C, et al. Silibinin alleviates silica-induced pulmonary fibrosis: Potential role in modulating inflam-mation and epithelial-mesenchymal transition[J]. Phytother Res, 2021, 35(9): 5290-304. doi：10.1002/ptr.7210
[18]	Zhang F, Liu K, Cao MD, et al. Rosiglitazone treatment prevents postoperative fibrosis in a rabbit model of glaucoma filtration surgery[J]. Invest Ophthalmol Vis Sci, 2019, 60(7): 2743. doi：10.1167/iovs.18-26526
[19]	Hu R, Wang XJ, Chen S, et al. Qingguang'an-induced autophagy in TFs inhibits scar formation: a follow-up in vivo mechanistic investigation[J]. J Tradit Complementary Med, 2024, 14(2): 173-81. doi：10.1016/j.jtcme.2023.10.002
[20]	Chen YH, Liang CM, Chen CL, et al. Silibinin inhibits myofibroblast transdifferentiation in human tenon fibroblasts and reduces fibrosis in a rabbit trabeculectomy model[J]. Acta Ophthalmol, 2013, 91(7): e506-15. doi：10.1111/aos.12160
[21]	Wang XH, Chen KP, Yao YH, et al. TGFβ1-induced fibrotic responses of conjunctival fibroblasts through the Wnt/β‑catenin/CRYAB signaling pathway[J]. Am J Pathol, 2024, 194(9): 1764-79. doi：10.1016/j.ajpath.2024.05.002
[22]	Chen YY, Chen YR, Li CM, et al. Transcription factor SP1 drives TGF‑β1-induced transformation of human tenon's capsule fibro-blasts by transcription activation-mediated SNAI2 upregulation[J]. Int Ophthalmol, 2025, 45(1): 243. doi：10.1007/s10792-025-03581-8
[23]	Wu XP, Liang J, Liu JF, et al. Silibinin attenuates TGF-β2-induced fibrogenic changes in human trabecular meshwork cells by targeting JAK2/STAT3 and PI3K/AKT signaling pathways[J]. Exp Eye Res, 2024, 244: 109939. doi：10.1016/j.exer.2024.109939
[24]	Gupta A, Kafetzis KN, Tagalakis AD, et al. RNA therapeutics in ophthalmology-translation to clinical trials[J]. Exp Eye Res, 2021, 205: 108482. doi：10.1016/j.exer.2021.108482
[25]	Cao JJ, Han FF, Kou ZY, et al. Effect of SB431542 on autophagy and epithelial mesenchymal transition in retinal pigment epithelial cells induced by high glucose[J]. Chin J Ophthalmol, 2025,61(3):202-10.
[26]	Yamoto M, Alganabi M, Chusilp S, et al. Lysosomal overloading and necrotizing enterocolitis[J]. Pediatr Surg Int, 2020, 36(10): 1157-65. doi：10.1007/s00383-020-04724-x
[27]	Shang JN, Yu CG, Li R, et al. The nonautophagic functions of autophagy-related proteins[J]. Autophagy, 2024, 20(4): 720-34. doi：10.1080/15548627.2023.2254664
[28]	Wu NX, Chen LB, Yan D, et al. Trehalose attenuates TGF‑β1-induced fibrosis of hSCFs by activating autophagy[J]. Mol Cell Biochem, 2020, 470(1): 175-88. doi：10.1007/s11010-020-03760-4
[29]	Zhang YL, Zhang YQ, Lin HL, et al. Epigallocatechin-3-gallate increases autophagic activity attenuating TGF‑β1-induced transfo-rmation of human Tenon's fibroblasts[J]. Exp Eye Res, 2021, 204: 108447. doi：10.1016/j.exer.2021.108447
[30]	Huang X, Zhang JP, Yao J, et al. Phase separation of p62: roles and regulations in autophagy[J]. Trends Cell Biol, 2025, 35(10): 854-65. doi：10.1016/j.tcb.2025.01.010
[31]	Aredia F, Guamán Ortiz LM, Giansanti V, et al. Autophagy and cancer[J]. Cells, 2012, 1(3): 520-34. doi：10.3390/cells1030520
[32]	Ji Y, Li L, Ma YX, et al. Quercetin inhibits growth of hepatocellular carcinoma by apoptosis induction in part via autophagy stimulation in mice[J]. J Nutr Biochem, 2019, 69: 108-19. doi：10.1016/j.jnutbio.2019.03.018
[33]	朱晓敏, 钟颖, 黄娜娜, 等. 基于Akt/mTOR通路研究地榆皂苷Ⅱ诱导肝癌细胞凋亡和自噬作用机制[J]. 中草药, 2024, 55(11): 3726-34. doi：10.7501/j.issn.0253-2670.2024.11.015