OU-YANG Yun, PENG Jun, TAN Han-Yu, LI Wen-Juan, WU Quan-Long, PENG Qing-Hua. Effect of Flos Buddlejae Granules on Apoptosis Factors Bax, Bcl-2, Fas and FasL in Lacrimal Gland Cells of Castrated Male Rabbits[J]. Digital Chinese Medicine, 2018, 1(2): 115-121.
Citation:
OU-YANG Yun, PENG Jun, TAN Han-Yu, LI Wen-Juan, WU Quan-Long, PENG Qing-Hua. Effect of Flos Buddlejae Granules on Apoptosis Factors Bax, Bcl-2, Fas and FasL in Lacrimal Gland Cells of Castrated Male Rabbits[J]. Digital Chinese Medicine, 2018, 1(2): 115-121.
OU-YANG Yun, PENG Jun, TAN Han-Yu, LI Wen-Juan, WU Quan-Long, PENG Qing-Hua. Effect of Flos Buddlejae Granules on Apoptosis Factors Bax, Bcl-2, Fas and FasL in Lacrimal Gland Cells of Castrated Male Rabbits[J]. Digital Chinese Medicine, 2018, 1(2): 115-121.
Citation:
OU-YANG Yun, PENG Jun, TAN Han-Yu, LI Wen-Juan, WU Quan-Long, PENG Qing-Hua. Effect of Flos Buddlejae Granules on Apoptosis Factors Bax, Bcl-2, Fas and FasL in Lacrimal Gland Cells of Castrated Male Rabbits[J]. Digital Chinese Medicine, 2018, 1(2): 115-121.
Effect of Flos Buddlejae Granules on Apoptosis Factors Bax, Bcl-2, Fas and FasL in Lacrimal Gland Cells of Castrated Male Rabbits
Han-Yu TAN, lecturer, Ph.D.. Research direction: ophthalmology of integrated medicine treatment
Qing-Hua PENG, Professor Grade Ⅱ, chief physician. Research direction: integrated medicine treatment for ocular disease, glaucoma and ocular fundus pathology. E-mail: pqh410007@126.com
ObjectiveTo observe the effect of different concentration Flos Buddlejae (Mi Meng Hua, 密蒙花) granules on apoptosis factors of lacrimal gland cells of castrated male rabbits, and study the treatment effects of Flos Buddlejae (Mi Meng Hua, 密蒙花) granules in the dry eye model of castrated male rabbits.
Methods Flos Buddlejae (Mi Meng Hua, 密蒙花) raw material was made into granules. Thirty healthy adult New Zealand rabbits were randomly divided into five groups, six rabbits in each group. Group A: blank group, Group B: model group, Group C: Flos Buddlejae (Mi Meng Hua, 密蒙花) granule group, Group D: placebo group, Group E: testosterone group. Except for group A, all rabbits underwent removal of bilateral testis and epididymis. Rabbits in group C were administered Flos Buddlejae (Mi Meng Hua, 密蒙花) granules (100 mg/kg), three times per day; rabbits in group D were administered normal saline, three times per day. Rabbits in group E were injected with testosterone propionate (0.5 mL/kg) in the thigh muscle, every 3 days. All rabbits were tested by Schirmer I test (SIT) and tear film break-up time (BUT) before operation and 4 weeks after operation. After 4 weeks, all rabbits were sacrificed by air embolism and clipping of the lacrimal gland. Apoptosis factors, including Bax, Bcl-2, Fas and FasL of lacrimal gland cells were characterized by immunohistochemistry, and resulting data were statistically analyzed.
Results(1) Comparison of SIT and BUT before and after operation: There were statistically significant differences between groups B and D (P < 0.01), but not among other groups (P > 0.05). (2) Comparison of apoptosis factors Bax, Bcl-2 Fas and FasL: In a comparison of groups B and D, there was no statistically significant difference after operation (P > 0.05). In a comparison of the other groups, there were statistically significant differences (P < 0.01). In comparisons among A, C and E groups, there were no statistically significant differences (P > 0.05).
Conclusion Compared with androgen, Flos Buddlejae (Mi Meng Hua, 密蒙花) granules caused similar but slightly weaker depression of Bax, Fas and FasL, and increased expression of Bcl-2.
Dry eye, also known as keratoconjunctivitis sicca, encompasses a variety of diseases characterized by abnormal tear quality or quantity due to any reason, or changes of tear film stability caused by abnormal dynamics; it is accompanied by ocular discomfort and/or ocular surface tissue disease [1]. Dry eye is a common ocular surface disease. Recent epidemiological and clinical studies have found a higher incidence of the disease than previously thought.
In previous clinical studies and animal experiments, we confirmed that Flos Buddlejae (Mi Meng Hua, 密蒙花) has a good curative effect on dry eye. Herbs used in the experiments, such as Flos Buddlejae (Mi Meng Hua, 密蒙花), Chinese Wolfberry (Gou Qi Zi, 枸杞子), and Chrysanthemum (Ju Hua, 菊花), were prepared by modern technology. To further explore the molecular mechanism of Flos Buddlejae (Mi Meng Hua, 密蒙花) effects, we investigated the effects of Flos Buddlejae (Mi Meng Hua, 密蒙花) Granules (BOG) on apoptosis factors, such as Bax, Bcl-2, Fas and FasL, in lacrimal gland cells that were isolated from castrated male rabbits who had undergone androgen reduction.
2.
Materials and Methods
2.1
Animals
Thirty adult male New Zealand white rabbits, body weight between 1.5-2.0 kg (from the Experimental Animal Center of Hunan University of Chinese Medicine, Laboratory animal quality certificate No. SCXK (Xiang) 2009-0012), were selected for this study.
2.2
Equipment
Slit lamp microscope, hand-held direct ophthalmoscope, GB11241-89 constant temperature water bath box, Leica Paraffin section machine, micro camera, computer image analysis system, common surgical knife, surgical scissors, etc. were used in this study. All analyses were performed by the Laboratory of Morphology and Laboratory of Ophthalmology in Hunan University of Chinese Medicine.
2.3
Drugs and reagents
2.3.1
Drugs
The following drugs were used in this study: testosterone propionate for injection (1 mL/25 mg; Tianjin Tianyao Pharmaceutical Co., Ltd. [National Drug Standards No. (NDSN) H12020531]; benzylpenicillin sodium for injection (400, 000 units; Guangzhou Baiyun Mountain Tianxin Pharmaceutical Ltd. (NDSN H444022446)); sodium chloride for injection (500 mL/4.5 g; Jiangsu Yabang Shengyuan Pharmaceutical Co., Ltd. (NDSN H32024531); chloral hydrate (analytical pure; 250 g; Tianjin Kermel Chemical Reagent Co., Ltd.); sterile water for injection (500 mL; Shanghai Treeful Jinshan Pharmaceutical Co., Ltd. (NDSNH31021935)); and Schirmer test, fluorescein sodium eye detection test paper (Tianjin Jingming New Technology Development Co., Ltd.).
Flos Buddlejae (Mi Meng Hua, 密蒙花) granule [consisting of Flos Buddlejae (Mi Meng Hua, 密蒙花), Chinese Wolfberry (Gou Qi Zi, 枸杞子), Chrysanthemum (Ju Hua, 菊花) and other drugs] was prepared with modern technology by the Department of Medicine of Hunan University of Chinese Medicine.
2.3.2
Reagents
The following reagents were used in this study: Harris reagent and 3% peracetic acid (both from Beijing Golden Bridge Biotech Corp.); Rabbit anti-rabbit Bax antibody (200 μg/mL), Rabbit anti-rabbit Fas monoclonal antibody (200 μg/mL), Rabbit anti-rabbit FasL monoclonal antibody (200 μg/mL), streptavidin- biotin complex (SABC immunological staining reagent), 3, 3-diaminobenzidine (DAB chromogenic agent) (all from Wuhan Bostr Biological Engineering Co., Ltd.).
2.4
Experimental method
2.4.1
Animal grouping
Thirty healthy adult New Zealand rabbits were randomly divided into five groups, six rabbits per group. Group A: blank group, Group B: model group, Group C: Flos Buddlejae granule group, Group D: placebo group, Group E: testosterone group.
2.4.2
Establishment of animal model of dry eye
Except for rabbits in the normal control group, all rabbits were treated by bilateral testicular and epididymis resection, as in prior studies [2, 3].
2.4.3
Postoperative administration
Rabbits in group A and B were not given any medicine. From the third day after operation, rabbits in group C underwent intragastric administration of Flos Buddlejae (Mi Meng Hua, 密蒙花) granules (100 mg/kg), three times per day; rabbits in group D underwent intragastric administration of saline, three times perday; rabbits in group E were injected with testosterone propionate (0.5 mL/kg) in the thigh muscles, once every 3 days.
2.5
Dry eye routine inspection method
Each test was completed by the same experimental personnel. Schirmer I test (SIT) and tear film break-up time (BUT) determinations were performed for all rabbits, 1 day before and 4 weeks after the operation.
2.6
Specimen collection and processing
After 4 weeks, all rabbits were scarified by air embolism. Lacrimal glands of both eyes were removed, and specimens were fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin and sectioned.
2.7
Parameter assay
Excised lacrimal gland tissuesunderwent eosin staining and immunohistochemical staining. Inflammatory factors of lacrimal gland cells were then photographed at 400× with an optical microscope.
2.8
Statistics
All experimental data were processed by SPSS16.0 statistical software. Measurement data were expressed by mean ± standard deviation (SD). First, the normal distribution and homogeneity of variance were tested; if these were satisfied, variance analysis was used for multiple groups comparison. If the data did not satisfy the normality and homogeneity of variance, nonparametric multiple comparisons were used. Enumerated data were analyzed by using chi-squared tests; other homogeneity of variance data were analyzed with rank-sum tests. P < 0.05 was considered to be statistically significant.
3.
Results
3.1
Dry eye routine examination
Comparison of SIT and BUT values before and after operation in each group is shown in Table 1. Compared with preoperative values, SIT and BUT in groups B and D were significantly reduced (P < 0.01). Compared with group A, the SIT and BUT values of groups B and D significantly decreased after operation (P < 0.01). Compared with group B, SIT and BUT significantly increased in groups C and E (P < 0.01).Compared with group D, SIT and BUT significantly increased in groups C and E (P < 0.01).
Table
1.
Schirmer I test (SIT) and tear break-up time (BUT) values of rabbits before and after the operation (x±SD, n=12)
Group
SIT (mm)
BUT (s)
Before
After
Before
After
A
14.75±2.26
14.50±1.45
14.33±2.42
14.08±1.88
B
14.83±2.04
7.75±1.54*△
14.42±1.38
6.75±1.66*△
C
14.77±1.68
14.27±1.14▲○
14.67±1.89
14.30±1.38▲○
D
14.67±1.92
7.50±1.62*△
14.25±2.42
6.83±1.70*△
E
14.83±1.59
14.33±1.15▲○
14.75±2.22
14.50±1.24▲○
Note: Compared with this group before operation, *P < 0.01; Compared with group A after treatment, △P < 0.01; Compared with group B after treatment, ▲P < 0.01; Compared with group D after treatment, ○P < 0.01.
3.2
Expression of Bax, Bcl-2, Fas and FasL of lacrimal gland cells in each rabbit after the operation
Compared with group A, expression of Bax, Fas and FasL in groups B and D increased significantly; however, Bcl-2 significantly decreased (P < 0.01). Compared with group B, expression of Bax, Fas and FasL in groups C and E decreased significantly; however, Bcl-2 significantly increased (P < 0.01). Compared with group D, expression of Bax, Fas and FasL in group E decreased significantly; however, Bcl-2 significantly increased (P < 0.01).
3.3
Observation of apoptosis factors Bax, Bcl-2, Fas and FasL
The expression of Bax, Bcl-2, Fas and FasL after operation in each group is shown in Figs. 1, 2, 3, and 4. Group A: clear lacrimal gland structure; no expression of Bax, Bcl-2, Fas, or FasL. Group B: lacrimal gland structure is blurred; Bax, Fas and FasL are abundantly expressed in cell membrane and cell pulp, shown as brown and yellow particles; Bcl-2 expression is minimal. Group C: clear lacrimal gland structure, minimal expression of Bax, Fas and FasL; Bcl-2 is expressed at high levels in cell membrane and cytoplasm, shown as brown and yellow particles. Group D: lacrimal gland structure is blurred; Bax, Fas and FasL are abundantly expressed in cell membrane and cell pulp, shown as brown and yellow particles; Bcl-2 expression is minimal. Group E: clear lacrimal gland structure, minimal expression of Bax, Fas and FasL; no expression of Bcl-2.
Figure
1.
Bax expression in each group after the operation (400×)
The 2007 International Seminar on Dry Eye provided a new definition of dry eye: a multifactorial disease involving both tears and the ocular surface, which could cause eye discomfort, visual disturbance, and instability of tear film, resulting in ocular surface damage. The disease is accompanied by inflammation of the ocular surface and increased tear film permeability [4]. Compared with the traditional definition of dry eye, this new definition emphasizes the important role of inflammation in the pathogenesis of dry eye disease. Although the initial cause of dry eye varies, inflammation ultimately becomes a key factor in the pathogenesis of dry eye. Regulation of neurotransmitters, apoptosis factors, and sex hormones is also involved in the pathogenesis of dry eye. When dry eye and apoptosis of the ocular surface epithelium of the lacrimal gland increase, expression of apoptosis factors (e.g., Bax, Fas and FasL) and proinflammatory cytokines (e.g., IL-1 and TNF-alpha) increasing in the ocular surface and tears, thus activating the apoptosis pathway [5].
In recent years, the sex hormone androgen has been shown to play an especially important role in tear secretion. Androgen exerts an immunosuppressive effect by stimulating the synthesis of TGF-beta to reduce TNF-alpha and IL-1 beta levels in the lacrimal gland, effectively protecting the lacrimal gland from inflammation and degeneration, as well as accelerating the metabolism and regulating the morphology and secretion functions of the lacrimal and meibomian glands.
Dry eye belongs to "Bai She Zheng" of traditional Chinese medicine, also called "Gan She Hun Hua Zheng" "Shen Shui Jiang Ku Zheng" or "Shen Qi Ku Cui". Generally, dry eye is caused by liver and kidney deficiency, yin and blood loss, and lack of nourishment. Flos Buddlejae (Mi Meng Hua, 密蒙花) granule includes Flos Buddlejae (Mi Meng Hua, 密蒙花), Chinese Wolfberry (Gou Qi Zi, 枸杞子), and Chrysanthemum (Ju Hua, 菊花). Flos Buddlejae (Mi Meng Hua, 密蒙花) is sweet, slightly cold, and enters the liver meridian, thereby reducing heat-fire, nourishing liver, and removing nebula to improve eyesight [6].It is a monarch drug used to treat conditions such as red eye, blurred vision, eye dryness, and cloudy vision due to liver deficiency [7, 8]. Chinese Wolfberry (Gou Qi Zi, 枸杞子) is a minister drug that has effects of nourishing liver and kidney, nourishing yin, and improving vision. Chrysanthemum (Ju Hua, 菊花) is sweet and bitter, slightly cold, and enters the lung and liver meridians; it is an assistant and guide drug for suppressing hyperactive liver, reducing heat, and improving eyesight. According to modern research, the active ingredients of Flos Buddlejae (Mi Meng Hua, 密蒙花)are flavonoids [9]; some flavonoids have androgen-like effects that can be used to treat certain diseases induced by the reduction of sex hormone levels, such as bone loss and osteoporosis [10, 11].
We have confirmed in previous experimental studies of Flos Buddlejae (Mi Meng Hua, 密蒙花) that the main components of Flos Buddlejae (Mi Meng Hua, 密蒙花) extract can exert an androgen-like effect, such as reducing the occurrence of dry eye in rats after the reduction of androgen levels, inhibiting apoptosis of lacrimal gland cells, and maintaining the stability and volume of basic secretions of the lacrimal gland [12-19].
The present experiment studied the effects of Flos Buddlejae (Mi Meng Hua, 密蒙花) granules on the secretion of tears, BUT, and expression of apoptosis factors Bax, Bcl-2, Fas and FasL in lacrimal gland cells. This study confirmed that Buddleja Officinalis granules had effects similar to androgen in rabbits: inhibition of apoptosis factors Bax, Fas and FasL, up regulation of Bcl-2, and reduction of apoptosis of lacrimal gland cells induced by androgen deficiency. However, these effects were weaker than observed during androgen treatment.
Currently, dry eye is treated mainly by drugs. Clinically the commonly used drugs are artificial tears, which can mainly relieve eye discomfort, but cannot change the fundamental pathological state of dry eye. Our research supports the use of a new natural drug for the treatment of dry eye disease; this approach avoids the side effects caused by long-term use of androgen, which has been widely used for many years.
Acknowledgements
We thank for the funding support from the National Natural Science Foundation of China (NSFC) (No. 30772824 and No. 81574031); 225 Project of High-Level Medical Talents of Hunan Province, Research Project of Science and Technology Department of Hunan Province (No. 2015SF2016-6); Research Project of Hunan Provincial Development and Reform Commission (No. [2014]658); Major Project of Changsha Science and Technology Plan (K1501014-31); Construction Project of Key Discipline of Chinese Ophthalmology of State Administration of Traditional Chinese Medicine; Construction Project of Key Discipline of Ophthalmology and Otolaryngology of Chinese Medicine of Hunan Province.
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Table
1.
Schirmer I test (SIT) and tear break-up time (BUT) values of rabbits before and after the operation (x±SD, n=12)
Group
SIT (mm)
BUT (s)
Before
After
Before
After
A
14.75±2.26
14.50±1.45
14.33±2.42
14.08±1.88
B
14.83±2.04
7.75±1.54*△
14.42±1.38
6.75±1.66*△
C
14.77±1.68
14.27±1.14▲○
14.67±1.89
14.30±1.38▲○
D
14.67±1.92
7.50±1.62*△
14.25±2.42
6.83±1.70*△
E
14.83±1.59
14.33±1.15▲○
14.75±2.22
14.50±1.24▲○
Note: Compared with this group before operation, *P < 0.01; Compared with group A after treatment, △P < 0.01; Compared with group B after treatment, ▲P < 0.01; Compared with group D after treatment, ○P < 0.01.
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