J Korean Ophthalmol Soc.  2012 Feb;53(2):323-332.

The Antifibrotic Effects of alpha-Tocotrienols in Primary Cultured Orbital Fibroblasts from Thyroid-Associated Ophthalmopathy Patients

• Kim SJ
• Kang HK
• Kang SM

Abstract

PURPOSE
To evaluate the antiproliferative and antifibrotic effects of alpha-tocotrienols in primary cultured orbital fibroblasts from thyroid-associated ophthalmopathy (TAO) patients.
METHODS
Orbital fibroblasts were cultured (5 eyes from TAO patients, 5 eyes from normal patients) and classified into a control group, alpha-tocotrienol group and alpha-tocopherol group. The cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The proliferation of orbital fibroblasts was measured using the Click-iT(TM) assay. The collagen production of the control and alpha-tocotrienol groups was measured using a hydroxyproline assay.
RESULTS
The alpha-tocotrienol and alpha-tocopherol groups showed no cytotoxicity up to 150 microm in orbital fibroblasts from TAO and normal patients. The proliferation of orbital fibroblasts from TAO and normal patients was significantly inhibited with alpha-tocotrienol at 80 microm and 120 microm. The collagen production of orbital fibroblasts from TAO patients was significantly inhibited with alpha-tocotrienol at 120 microm.
CONCLUSIONS
The results from the present study indicate that non-toxic concentrations of alpha-tocotrienol have significant antiproliferative and antifibrotic effects on orbital fibroblasts from TAO patients.

Keyword

alpha-tocopherol; alpha-tocotrienol; Fibrosis; Orbital fibroblast; Thyroid-associated ophthalmopathy

MeSH Terms

alpha-Tocopherol
Cell Survival
Collagen
Eye
Fibroblasts
Fibrosis
Graves Ophthalmopathy
Humans
Hydroxyproline
Orbit
Tetrazolium Salts
Thiazoles
Troleandomycin
Vitamin E
Collagen
Hydroxyproline
Tetrazolium Salts
Thiazoles
Troleandomycin
Vitamin E
alpha-Tocopherol

Figure

• 

  Figure 1 Effect of α-tocotrienol on the viability of orbital fibroblast from TAO (Thyroid associated ophthalmopathy) patients and normal patients. (A) The viability of orbital fibroblasts from TAO patients was not affected by 48 hour incubation with α-tocotrienol concentrations of up to 150 µm. (B) The viability of orbital fibroblasts from normal patients was not affected by 48 hour incubation with α-tocotrienol concentrations of up to 150 µm. The cell viability was determined using MTT assay. Results are expressed as mean ± SD in relative OD units (compared with controls). The experiment was performed 3 times independently.

• 

  Figure 2 Effect of α-tocopherol on the viability of orbital fibroblast from TAO patients and normal patients. (A) The viability of orbital fibroblasts from TAO patients was not affected by 48 hour incubation with α-tocopherol concentrations of up to 150 µm. (B) The viability of orbital fibroblasts from normal patients was not affected by 48 hour incubation with α-tocopherol concentrations of up to 150 µm. The cell viability was determined using MTT assay. Results are expressed as mean ± SD in relative OD units (compared with controls). The experiment was performed 3 times independently.

• 

  Figure 3 Effect of α-tocotrienol on the proliferation of orbital fibroblast from TAO patients and normal patients. M2 shows total newly proliferation cells and M3 shows peak of newly proliferated cells. (A) The total newly proliferation of orbital fibroblasts (M2) from TAO patients was inhibited with α-tocotrienol at 80 µm and 120 µm. (B) The total newly proliferation of orbital fibroblasts(M2) from normal patients was inhibited with α-tocotrienol at 80 µm and 120 µm. (C)The peak of newly proliferation of orbital fibroblasts(M3) from TAO patients was inhibited with α-tocotrienol at 80 µm and 120 µm. (D) The peak of newly proliferation of orbital fibroblasts(M3) from normal patients was inhibited with α-tocotrienol at 80 µm and 120 µm. The cell proliferation was determined using Click-iTTM assay. Results are expressed as mean ± SD (compared with controls). The experiment was performed 3 times independently. The asterisk (*) indicates a statistically significant difference within group (*p < 0.05).

• 

  Figure 4 Effect of α-tocotrienol on the proliferation of orbital fibroblast from TAO patients. The proliferation of orbital fibroblasts from TAO patients was significantly inhibited with α-tocotrienol at 120 µm. (A) Orbital fibroblasts from TAO patients (control). (B) Orbital fibroblasts 2 days after 120 µm α-tocotrienol treatment from TAO patients.

• 

  Figure 5 M2 shows total newly proliferation cells and M3 shows peak of newly proliferated cells. The total newly proliferation of orbital fibroblasts (M2) from TAO patients was inhibited with α-tocotrienol at 120 µm. The peak of newly proliferation of orbital fibroblasts (M3) from TAO patients was inhibited with α-tocotrienol at 120 µm. The cell proliferation was determined using Click-iT™ assay. (A) Result of Facscalibur flow cytometer in from TAO patients (control). (B) Result of Facscalibur flow cytometer 2 days after 120 µm α-tocotrienol treatment from TAO patients.

• 

  Figure 6 Effect of α-tocopherol on the proliferation of orbital fibroblast from TAO patients and normal patients. M2 shows total newly proliferation cells and M3 shows peak of newly proliferated cells. Various dose of α-tocopherol was not affected on the proliferation of orbital fibroblast from TAO patients and normal patients. The cell proliferation was determined using Click-iT™ assay. Results are expressed as mean ± SD (compared with controls). The experiment was performed three times independently.

• 

  Figure 7 Effect of α-tocotrienol on the collagen production of orbital fibroblast from TAO patients. The total collagen production of orbital fibroblasts from TAO patients was inhibited with α-tocotrienol at 120 µm. The hydroxyproline level was determined using hydroxyproline assay. Results are expressed as mean ± SD (compared with controls). The experiment was performed 3 times independently. The asterisk (*) indicates a statistically significant difference within group (*p < 0.05).

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