Silibinin inhibits myofibroblast transdifferentiation in human tenon fibroblasts and reduces fibrosis in a rabbit trabeculectomy model

Purpose: To investigate the effect of silibinin in myofibroblast transdifferentiation and in animal trabeculectomy models. Methods: The effect of silibinin on the expression of α-smooth muscle actin (α-SMA) and vimentin in response to transforming growth factor-β1 (TGF-β1) was determined in human tenon fibroblasts (HTFs). Cell migration and collagen contraction arrays were used to demonstrate the functionality of silibinin-modulated HTFs. ELISA analysis was used to determine the effect of silibinin on the release of type 1 collagen and connective tissue growth factor (CTGF). The effect of silibinin on the activation of the TGF-β receptor–related pathway was evaluated by Western blotting. A rabbit model of trabeculectomy was established to assess the effect of silibinin in vivo. Results: TGF-β1 elevated the expression of α-SMA and vimentin in HTFs; this elevation was inhibited by silibinin. TGF-β1 increased cell migration and collagen contraction of HTFs, which were also suppressed by silibinin. The production of both CTGF and type 1 collagen in TGF-β1-treated HTFs was inhibited by silibinin. The effects of silibinin on TGF-β1-stimulated HTFs were mediated via the down-regulation of TGF-β receptor–related SMAD signalling pathways. In the rabbit model of trabeculectomy, silibinin increased the period of decreasing intraocular pressure after trabeculectomy and reduced the production of collagen and α-SMA at the site of blebs in vivo. Conclusion: Silibinin inhibited the TGF-β receptor–related signalling pathway in TGF-β-treated HTFs and several of the downstream events associated with myofibroblast transdifferentiation. Silibinin also improved the outcome of trabeculectomies by reducing the fibrotic response in the bleb tissue in vivo. Glaucoma is a leading cause of irreversible blindness in the world and presents as a disease with optic nerve atrophy (Thylefors et al. 1995; Quigley 1996). Although elevated intraocular pressure (IOP) is not the only reason for optic damage, the aim of current therapy is to lower IOP by medical, laser or surgical treatments (Caprioli & Varma 2011), by which glaucoma has been proven to be arrested (Kass et al. 2002; Ederer et al. 2004). The most popular surgical treatment for glaucoma is trabeculectomy, which creates a direct pathway from the anterior chamber to the subconjunctival space (Razeghinejad et al. 2012). Lowering IOP can be achieved by increasing aqueous humour drainage through this artificial route. However, the success of trabeculectomy is related to an unpredicted factor: fibrosis in the subconjunctival space. The scar formation occludes the drainage pathway and decreases the absorption of the aqueous humour (Broadway & Chang 2001). To resolve this, several modifications have been studied, such as the adjunctive use of mitomycin C (Chen et al. 1990; Fontana et al. 2006) or 5-fluorouracil (Heuer et al. 1984; Cunliffe & Longstaff 1993), or the implantation of antifibrosis materials. Until now, no satisfactory methods have been developed to resolve this issue. Transforming growth factor-β (TGF-β) plays an important role in mediating the normal process of wound healing and postoperative scarring in multiple tissues (Branton & Kopp 1999; Ihn 2002; Akhmetshina et al. 2012). Three isoforms, including TGF-β2, which is present in the aqueous humour, provide similar responses in cultured fibroblasts to convert them to myofibroblasts. This specialized form of fibroblasts is present in abundance in fibrotic lesions, which can synthesize the extracellular matrix (Krieg et al. 2007). The expression of α-smooth muscle actin (α-SMA) and its incorporation into stress fibres can subsequently increase cell contractility and promote wound closure (Sappino et al. 1990). Myofibroblasts are critical in normal wound healing and disappear in the late stages of trabeculectomy, such as bleb formation. However, they persist in fibrotic disease and contribute to excess scarring (Desmouliere et al. 1995; Phan 2002; Shin & Minn 2004), such as the encapsulated blebs in trabeculectomy (Classen et al. 1996). Therefore, the reduction in the adverse effects of TGF-β or the inhibition of myofibroblast transdifferentiation could be a therapeutic strategy to increase the success rate of trabeculectomies. Silibinin is the major component of the silymarin complex, which is extracted from milk thistle. It is widely used for hepatitis, liver cirrhosis, liver diseases associated with alcohol consumption and environmental toxin exposure (Pradhan & Girish 2006). Silibinin has been shown to have antioxidant effects, oestrogenic activity, modulatory effects on drug transporters (P-glycoprotein) and specific action on DNA expression via the suppression of nuclear factor (NF)-κB (Gazak et al. 2007). It exerts its anticancer effects through multiple molecular mechanisms that block all stages of carcinogenesis – initiation, promotion and progression – thus verifying its possible use as a preventive and therapeutic agent in the treatment for more advanced and aggressive forms of cancer (Kren & Walterova 2005; Ramasamy & Agarwal 2008). Finally, although there have been studies showing that silibinin potentially arrests hepatic fibrosis (Trappoliere et al. 2009; El-Lakkany et al. 2012), its antifibrotic effect in trabeculectomies is still lacking. Here, we report that silibinin prevented TGF-β1-induced myofibroblast transdifferentiation in human tenon fibroblasts (HTFs) on a structural and functional level. Furthermore, the inhibition of α-SMA expression and collagen production by silibinin was observed in a rabbit model of trabeculectomy, thereby improving the surgical outcome. This evidence suggests that silibinin may thus provide a novel approach to counteract postoperative scarring in glaucoma surgery. Small tenon samples were biopsied during the standard intraocular procedures. The regulations of the Declaration of Helsinki were followed, and the procedures were approved by the institutional ethics committee. Primary HTFs were obtained as an expansion culture of the human tenon explants and propagated in Dulbecco’s modified Eagle’s medium (DMEM-F12; Gibco, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS; Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in the logarithmic growth phase. For all experiments, cells from passages 3 to 6 were used. All experiments were performed at least three times with similar results. The cell viability test was based on the ready-to-use cell viability reagent 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1; Roche Diagnostics, Indianapolis, IN, USA). After treatment for 24 hr with various concentrations of silibinin (Sigma-Aldrich) in serum-free medium, 10 μl WST-1 reagent was added to the medium in each well. The cells were incubated in a humidified atmosphere at 37 °C in 5% CO2/95% air for 1 hr, the multititre plate was shaken thoroughly for 1 min, and the absorbance was read at 450 nm. The background absorbance was measured in wells containing only the dye solution and culture medium. Cell viability data were obtained from at least three experiments with at least six wells at each concentration in separate 96-well plates. The mean optical density values corresponding to the untreated controls were defined as 100%. The results were expressed as the percentage of the optical density of treated cells relative to that of untreated controls. Cytotoxicity was assessed by measuring the release of lactate dehydrogenase from the cells (LDH activity), which was measured using the LDH Cytotoxicity Detection kit (Clontech, Mountain View, CA, Canada). A total of 4 × 104 cells were plated in triplicate and incubated at 5% CO2, 37 °C for 24 hr. Untreated cells were used as the low control and cells treated with lysis solution served as the high control. Cytotoxicity was determined after 24 hr of compound treatment. Following 30 min of incubation at room temperature, the absorbance at 450 nm was measured to determine LDH activity. The percentage of cytotoxicity was calculated as the ratio of the test sample absorbance to the high control absorbance. For Western blot analysis, the cells were treated with 10 ng/ml TGF-β1 (Pepro Tech, New York, NY, USA) for 24 hr in the absence and presence of silibinin at 12.5 or 25 μm. Treated and untreated cells were washed with PBS, harvested by scraping and centrifuged at 1000 × g. The cell pellets were resuspended and sonicated in cold lysis buffer (EDTA-free PRO-PREPTM Protein Extraction The were centrifuged at × for 10 min, and the concentration in the was determined with the kit USA). were by 10% and to were with 5% milk for 1 hr at room and incubated at room for min with of NY, α-SMA TGF-β or TGF-β USA). were washed and incubated with USA) for 1 hr at room temperature, and was using an USA). The cells were treated with 10 ng/ml TGF-β1 for hr in the absence or presence of 25 silibinin The cells were with and with vimentin CA, USA). were with The were using a was measured with a modified chamber as et al. cells were at a density of × in the chamber with an The lower chamber was with medium containing 10 ng/ml After a the were washed with PBS, with cold for 10 min and with for The of cells was by were cells were for 24 and with 10 ng/ml TGF-β1 with or silibinin for The collagen in the test samples was with and with at to convert collagen to were after and ELISA was performed with the human type collagen kit USA). CTGF was with a CTGF ELISA kit (Pepro Tech, New York, NY, USA). were with CTGF in buffer at 4 The were with bovine serum for 1 hr at room and washed with Cell culture were added to each and incubated for 1 hr at 37 The were washed with and incubated for 1 hr at 37 °C with CTGF in buffer containing 4 and The were washed with and incubated for 1 hr at 37 °C with The were developed with in buffer and and the optical density was read at nm. was used as a collagen matrix contraction using a modified from that by and & type collagen (Sigma-Aldrich) was in and at 4 °C The wells of a plate were with to block cells were resuspended at a density of 1 × in The cell was with of 3 collagen (Sigma-Aldrich) and serum-free containing U/ml penicillin and 100 μg/ml and μl 1 The was in a plate and incubated at 37 °C for 1 hr to The collagen were from the of the wells after and the were in 1 containing 10% After 24 the medium was the were washed with serum-free and incubated at 37 °C for 6 in serum-free containing 10 ng/ml TGF-β1 with or silibinin. The medium was cells were used to the The of each matrix was and measured with an at 1 and The percentage of contraction was calculated as at at × trabeculectomy on of New and 3 was performed on the All the and were performed The were with an of a of and 10 A was also After an the a measured was in the of the at the the and After a × 3 was A × was by The was by with at the Finally, the wound was by a of at the in a After the surgical subconjunctival with or silibinin was to the and control The rabbit were by procedures including for the presence of bleb elevation and measuring The IOP was by an in the The IOP was the as the and on the after surgery. of three was as the IOP was after the the bleb to be in The of the was as the All procedures were approved by the and of the and were in with the for in and for the of in and tissues at the site of the trabeculectomies were and for Western blot analysis and after surgery. was performed and after surgery. were in 10% solution for 24 at the and in of containing the and were at a of on and The were in in alcohol and with and and For tissue were in for 10 min to activity. was performed by each at 100 °C for 30 min in 10 buffer containing After three of min each in the samples were with bovine serum in for 1 hr and incubated with α-SMA as the at 4 After three of min each in the were incubated with CA, USA) for min and incubated with as the for 30 After three was developed with at room were by analysis of a was multiple of were performed with the The data are as is of at least three All were and evaluated at the of The WST-1 was used to determine and the LDH was used to test cytotoxicity as a of shown in The of silibinin on was observed at concentrations of μm. The cytotoxicity of silibinin on HTFs was observed at concentrations of μm. Silibinin concentrations of 12.5 and 25 were for the The cell viability and cytotoxicity of silibinin on human tenon The cell viability was determined by the WST-1 and is shown in The cytotoxicity of silibinin was observed by the LDH and is shown in Cells were incubated with silibinin for 24 hr in from to μm. All data are expressed as the To the effect of silibinin on TGF-β1-induced α-SMA and vimentin expression in HTFs, we treated HTFs with TGF-β1 with or silibinin. can be in HTFs with TGF-β1 increased α-SMA which could be by silibinin in a is an and as a of with the control and the TGF-β1 the expression of vimentin in HTFs with a more and silibinin the effect the of HTFs was after TGF-β1 and silibinin also the The HTFs in the control and the and A cell was with after TGF-β1 which was by silibinin The effect of silibinin on the expression of muscle actin (α-SMA) and vimentin in transforming growth factor The effect of silibinin on the expression of α-SMA was by Western blot and is shown in were from HTFs treated for 24 hr with silibinin. of α-SMA expression was treatment with Silibinin suppressed the of α-SMA expression in a The effect of silibinin on vimentin expression was determined by and is shown in The HTFs were with silibinin for 24 hr, and HTFs were with TGF-β1 for hr. Cells were with and with were with with the control and the TGF-β1 vimentin expression and Silibinin inhibited the effect of TGF-β1 data for α-SMA expression are as and responses that are and the migration to the wound site plays a critical role and is by To assess the effect of silibinin on TGF-β1-induced cell migration of HTFs, we used a modified chamber is in HTFs treated with TGF-β1 increased cell However, both 12.5 and 25 silibinin reduced the TGF-β1-induced The effects of silibinin on TGF-β1-induced cell migration and collagen contraction in HTFs. of the silibinin effect on TGF-β1-induced cell migration is shown in HTFs were treated with TGF-β1 in the absence and presence of 12.5 or 25 and migration was with a modified chamber Silibinin suppressed the TGF-β1-induced cell The effect of silibinin on TGF-β1-induced collagen contraction is shown in HTFs were treated with TGF-β1 in the absence or presence of 12.5 or 25 silibinin. or expansion was determined as a percentage of the Silibinin suppressed the TGF-β1-induced collagen The data are as and responses that are and The contraction of collagen has been to the of the matrix during and tissue To the effect of silibinin on TGF-β1-induced collagen we measured the of the with collagen containing HTFs incubated with TGF-β1 in the absence or presence of silibinin. with the by HTFs the after was reduced to However, the percentage of reduction was the most in the presence of also that the contraction in response to TGF-β1 was by the presence of silibinin in a CTGF and type 1 collagen are downstream To the effect of silibinin on TGF-β1-induced CTGF and type 1 collagen production in HTFs, the production from HTFs with TGF-β1 with or silibinin was for HTFs with TGF-β1 of CTGF and type 1 collagen to be into the medium with the untreated control However, the of 12.5 or 25 silibinin a inhibition in the release of both CTGF and type 1 collagen in The effect of silibinin on release of connective tissue growth factor and type 1 collagen in HTFs. with TGF-β1 the release of CTGF and type 1 collagen from HTFs into the medium, as determined by The HTFs were with 12.5 or 25 silibinin for hr, and HTFs were with TGF-β1 for 24 hr. Silibinin the production of CTGF and type 1 The data are as and responses that are and The of TGF-β the formation of containing each of TGF-β 1 and TGF-β which to the of and and the activity. To the effect of silibinin on the of TGF-β HTFs were treated with TGF-β1 with or and Western blot analysis with and TGF-β was HTFs treated with TGF-β1 in an increase in the ratio of the forms of TGF-β However, the elevated ratio of was inhibited by of the cells with silibinin The effect of silibinin on of TGF-β receptor–related signalling pathways. The expression and of TGF-β are shown in and of and are shown in HTFs were with 12.5 or 25 silibinin for 24 hr. Cells were treated with TGF-β1 for min, and cell were with and to and Protein was with a with the control the and increased after TGF-β1 and after 12.5 or 25 silibinin treatment. In the ratio of not after silibinin treatment. data for the are as responses that are of the downstream from the TGF-β is To the effect of silibinin on the of HTFs were treated with TGF-β1 with or and Western blot analysis with and total and was TGF-β1 treatment for HTFs in an increase in the of the forms of and that and The was inhibited by the of the cells with was not To the antifibrosis effect of silibinin also in an animal model and the we the agent with or silibinin into the space the surgical wound in the rabbit model of a in the results of glaucoma IOP was measured on the to the effect of silibinin on IOP after glaucoma surgery. the there was no in the IOP the In both the reduced IOP by the was maintained at a lower in the period after the as with the IOP from the the surgery. However, the IOP in the in the silibinin with silibinin could be maintained at a lower after and as with the IOP from the control The effects of silibinin on the IOP and bleb of rabbit models. The IOP in the period are shown in In with the IOP from the IOP was in the period in both The silibinin with silibinin maintained IOP at low on and after as with the control A bleb in the silibinin and control is shown in The bleb in the silibinin was that in the control responses that are silibinin an to bleb of and with silibinin an of results are in a The silibinin a bleb the control via the test and were used to the of collagen and α-SMA production in the subconjunctival The expression of collagen on and and α-SMA on in the site treated with silibinin was with silibinin the tissues on were for analysis by Western in the that the α-SMA expression in the bleb tissues was by a silibinin The effect of silibinin on bleb of bleb tissue from control and treatment with of the rabbit trabeculectomy models. was used to collagen in with the control on and silibinin the collagen with a in the bleb tissue on and of α-SMA expression in the bleb tissue on are in the control and the silibinin A effect of silibinin on α-SMA expression was which was also by the Western blot analysis The of the with are shown in the of each In glaucoma is to the healing process in the surgical wound healing is to bleb and fibrosis in both the subconjunctival space and drainage pathway is also to increase the surgical success Until now, scar formation has been as the major reason for in glaucoma surgery. In this we that the myofibroblast transdifferentiation of HTFs by TGF-β1 was inhibited by silibinin. the TGF-β1-induced cell collagen and production of type 1 collagen and CTGF were all by silibinin. The mechanisms were mediated through the of TGF-β and downstream The antifibrotic effect of silibinin was also present in the in trabeculectomy which to the of the fibrosis is to be a in the healing process of a normal wound and is by scarring (Chen & the a role by into the wound and extracellular Myofibroblasts are present in abundance fibrotic and can also be observed in fibrotic such as liver or fibrosis (Desmouliere et al. 1995; Phan several growth are in fibrotic and TGF-β is to as a et al. Therefore, to the effect of silibinin on TGF-β1-induced myofibroblast transdifferentiation in HTFs, we assessed the expression of the myofibroblast α-SMA and that silibinin TGF-β1-induced vimentin expression in HTFs. This was with a on hepatic which that silibinin reduced the and the expression of extracellular matrix and the growth factor (Trappoliere et al. The TGF-β signalling pathway including cell and TGF-β is also to to synthesize extracellular and increase the production of its downstream CTGF et al. Therefore, to the of silibinin on the TGF-β1-induced myofibroblast transdifferentiation of HTFs, we measured cell collagen and type 1 collagen and CTGF production in HTFs. All of the results that silibinin the TGF-β1-induced functional In TGF-β to wound as observed by elevated TGF-β in human et al. and increased expression of TGF-β in after (Chen et al. the of TGF-β and CTGF have been to increase in blebs in and growth are to in bleb scarring that et al. 2004). Here, we that silibinin can the response to TGF-β and the production of CTGF in HTFs, thus its as an antifibrotic agent in glaucoma The of TGF-β are mediated by several signalling of which is the SMAD TGF-β to a of TGF-β type and TGF-β type both of which & 2012). the and are by the TGF-β to the SMAD and into the The to to the of specific It has also been shown that the SMAD pathway is for the of such as type collagen and CTGF & et al. In silibinin suppressed the of TGF-β and its downstream in the TGF-β signalling including the of the and the release of type 1 collagen and CTGF in HTFs. The and collagen in HTFs were by silibinin. The signalling pathway has also been to be a in fibrotic such as et al. an for TGF-β 1 was to TGF-β action by the down-regulation of in and in and was to be a novel therapy for and scarring et al. Therefore, which the TGF-β effect by the may also be a agent for bleb scarring in glaucoma Silibinin is the major of silymarin extracted from milk which is widely used in liver has been in the of of its and through a of molecular mechanisms & Agarwal 2008). normal wound three are the the and the phase. of can fibrotic In the and are and various are such as and can the factor which and the expression of various in the silibinin has been to this response (Gazak et al. 2007). The results in extracellular matrix production and Silibinin has also been to to and its activation and associated signalling via et al. wound fibroblasts to which are by the wound in is mediated through a which the of and the activation of and et al. In silibinin to fibrotic by several silibinin been shown to and growth in hepatic cells (Trappoliere et al. TGF-β is a major factor in and we the effect of silibinin on fibrotic responses in responses could be by silibinin via the signalling pathway in HTFs, which is with report in and fibroblasts et al. scar formation is a process to multiple It was not that silibinin could be used in trabeculectomies was to have a effects For this we an animal to assess the effect of silibinin on trabeculectomies. Silibinin treatment IOP during trabeculectomies as to treatment silibinin. Furthermore, collagen and α-SMA expression in the site at with the of silibinin in This lower fibrotic in the subconjunctival site after the silibinin and scar formation may the success rate of in and in we can that a adjunctive treatment with silibinin in may the scarring in blebs via the effect of silibinin on This was a to the evidence that can be used to the success rate of by of the antifibrotic effect of silibinin. However, several are silibinin can be used in the concentrations of silibinin used in the were by the results of responses in possible adverse effects with similar of silibinin in be although the in of rabbit provide evidence that is with the results we obtained in is by several growth or which not via TGF-β signalling pathways. It is of to which signalling the and are in the of blebs and the effect of silibinin on pathways. In we that silibinin myofibroblast cell migration and tissue in which are the events in Silibinin also the effects of TGF-β in both in culture and in an in rabbit model of All of may be the of the down-regulation of the TGF-β receptor–related signalling pathway by silibinin. on we that silibinin downstream events that are associated with tissue TGF-β is the major factor of the effect of silibinin on TGF-β its as a therapeutic as an adjunctive agent to the rate of glaucoma This was by from the and

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