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Dendrobium candidum extract increases the expression of aquaporin-5 in labial glands from patients with Sjogren's syndrome.

ABSTRACT

This study aimed to investigate the mechanism of Dendrobium candidum extract in promoting expression of aquaporin-5 for treatment of Sjogren's syndrome (SS). Sixteen patients with SS suffered from deficient secretion of saliva due to an autoimmune destruction of salivary glands leading to dry mouth symptoms (xerostomia). However, glandular dysfunction also occurred without destruction. Based upon its abnormal distribution in SS salivary glands, a potential role of the water channel protein aquaporin-5 (AQP-5) in the pathogenesis of SS was proposed. After oral administration of D. candidum extracted liquid (DCEL) for 1 week, saliva and salivary gland biopsies from labial glands of patients were collected and examined by employing immunoreactivity and immunohistochemistry techniques. Results showed that salivary secretion increased by about 65% in patients treated with DCEL as compared with the control group. Higher labeling indices (percentage of acinus area immunoreactive for AQP-5) in the biopsies were found in SS patients who had taken DCEL This study demonstrated that D. candidum would regulate the expression of AQP-5 in labial glands of SS patients and thereby promoted secretion of saliva to improve dry mouth symptoms.

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ARTICLE INFO

Keywords:

Dendrobium candidum

Mechanisms

Aquaporin-5 (AQP-5)

Sjogren's syndrome (SS)

Labial gland

Herb medicine

Introduction

Dendrobium candidum is a herbal medicine that has been used in China and some Southeast Asian countries clinically for maintaining tonicity of the stomach and promoting body fluid production (Zheng 2005). It can also relieve symptoms such as throat dryness and thirst with blurred vision for recovery from sickness (Ding et al. 1998). A clinical report showed that recipes with D. candidum as the main ingredient can promote body fluid production and enhance recovery from Sjogren's syndrome (SS) with regard to chewing, swallowing and speaking (Liu et al. 2005). Another study showed that D. candidum recipes improved gastric fluid excretion in 30 clinical cases resulting in an efficiency rate of 83.40% (Liu and Pan 2002). Some experimental studies have shown that D. candidum reduced the inhibitory effect of atropine on salivary gland secretion and enhanced saliva secretion in normal rabbits (Xu et al. 1995; Wei 2005). However, studies on the expression of aquaporin-5 (AQP-5) in labial glands from Sjogren's syndrome patients have not been reported so far. Hence the present investigation was undertaken and biopsy specimens obtained from labial glands were tested for AQP-5 expression.

Sjogren's syndrome (SS) is a chronic autoimmune disorder of the exocrine glands with associated lymphocytic infiltrates of the affected glands. The clinical manifestations are dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia). Dryness of the mouth and eyes results from involvement of the salivary and lacrimal glands (Fox 2005; Nagler 2004). AQP-5 is a water channel protein considered to play an important role in water movement across the plasma membrane. This channel is the major pathway for regulating water permeability in acinar cells, a critical property of the plasma membrane which determines the flow rate and ionic composition of secreted saliva (Krane et al. 2001). In experimental situations, AQP-5 deficiency in mice caused an approximately. 65% decrease in salivary secretion rate compared to wild-type mice (Krane et al. 2001). Abnormal distribution of AQP-5 in salivary glands (Steinfeld et al. 2001) and lacrimal glands (Tsubota et al. 2001) is likely to contribute to the deficiency of fluid secretion, which is a defining feature of SS.

We hypothesized that the efficacy of D. candidum in nourishing the stomach and promoting secretion of body fluids was the consequence of an increased expression of AQP-5 in Sjogren's syndrome patients. Improvement of SS patients was observed by administering the extract of D. candidum (EDC) orally, validating this hypothesis. The expression of AQP-5 in labial glands from Sjogren's syndrome patients has been investigated. The result showed that D. candidum can promote the expression of AQP-5 and hereby we deduced that the expression of AQP-5 could be one of the mechanisms for alleviating SS.

Materials and methods

Materials and drug preparation

10 kg of dried material of D. candidum (batch no: 20061201-2) was collected in Yunnan, China, and authenticated by experts in the Department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. Live samples and voucher specimens are kept in the specimen room and the greenhouse of China Pharmaceutical University. All materials were rinsed with double distilled water to remove surface contaminants. The material was then sheared into fine segments and marinated in double distilled water for intenerating. Subsequently the material was extracted by blending in an extractor for 1 h (Guo 2003). The blending process was repeated. The suspension was collected and concentrated in a ratio of 0.5 g/ml into D. candidum extracted liquid (DCEL). The suspension was cooled and aliquots of 5 ml/tube were prepared for future use.

For quality analysis, the powder of fresh D. candidum was carefully extracted with chloroform and methanol, respectively, in Soxhlet's System (Yin et al. 2008). The volume of each solvent was 20 times of the weight of sample. Each extract was concentrated to 10 ml before examination with HPLC. To protect the column, the chloroform extract was diluted 1:10-fold and filtered through a 0.45-[micro]m cellulose-acetate membrane (F-0139, Sigma, US) before injection.

Methods

Experimental grouping and detection of saliva flow volume

After obtaining informed consent, female patients (n = 16) who fulfilled the criteria of primary SS were randomly divided into a control group (n = 8) and a D. candidum treatment (n = 8) group. One week after oral administration of DCEL (0.5 g/5 ml, three times daily), saliva was collected for 5 min in graduated test tubes to determine the volume. The test tube containing saliva was allowed to stand in the vertical position for 24 h to allow sedimentation to occur. The volume of the saliva (ml) was recorded according to the mark on the graduated test tube. All subjects did not take any medicines containing antibody throughout the course of the study.

Drug treatment and collection of the salivary gland biopsies

Salivary gland biopsies from the above 16 female volunteers were collected after oral administration of DCEL and divided into eight groups that consisted of drug-treated and control groups. The samples were washed rapidly in saline to remove blood and fixed in 4% para-formaldehyde for 12 h. Then they were put into a 30% sucrose solution and kept at 40 [degrees]C overnight. Subsequently biopsy specimens obtained from labial glands were tested for AQP-5 expression and immunoreactivity using an AQP-5 ELISA kit and immunohistochemistry.

Immuno-histochemical staining

The dehydrated salivary gland biopsy was cut into 6 [micro]m sections and incubated at 37 [degrees]C overnight. It was then reacted with 3% oxidized methyl hydrogen peroxide solution for 10 min to eliminate endogenous peroxidase activity. It was washed with double distilled water and then with 0.01 M PBS for 5 min. The process was repeated three times. The sections were allowed to cool before blocking with 20% goat serum in PBS for 15 min. Sections were then incubated at 37 [degrees]C for 1 h and then overnight at 4[degrees]C with a polyclonal antibody against AQP-5 raised in a rabbit (1:200). After washing with PBS, sections were incubated for 30 min in biotiny-lated secondary antibody (1:400 goat anti-rabbit) before washing and incubated for 30 min at 37 [degrees]C in streptavidin-biotin peroxidase complex (1:400) before washing with 0.01 M PBS for 5 min and developing using DAB kit. DAB was added and incubation carried out for 4-8 min. It was washed with water and then with 1% methyl alcohol for decolorization, dehydrated until transparent, and then covered with neutral gum. The control group with primary antibody omitted was regarded as the negative control. The group without receiving any treatment was regarded as blank. Appearance of yellow brown granules was determined as positive results. The primary and secondary antibodies as well as the staining kit were purchased from Santa Cruz Biotechnology, USA. All steps were treated with DNase.

Quality analysis

For quality analysis, the external standard method using a series of mixed solutions with sample of chloroform, methanol or [H.sub.2]O extract was examined. A reversed-phase column (Xbridge C18, 5 [micro]m, 250 mm x 4.6 mm i.d., Thermo, USA) was used and the mobile phase conditions employed were as follows: (1) for chloroform or [H.sub.2]O extract, acetonitrile (A) and 0.05% sodium dodecyl sulfate (SDS) in 0.1% acetic acid (B) using a gradient program of 80-60% (B) in the first 5 min, 60% (B) in 5-30 min and 60-40% (B) in the last 20 min and (2) for methanol extract, 60-40% (B) in 5 min. and 40% (B) in 5-20 min. The flow rate was 0.9ml/min. DAD detector was set at 254 nm for obtaining chromatograms with the maximum number of peaks.

Extraction and analysis of D. candidum polysaccharides (DP)

D. candidum stems were pre-extracted with 200 ml acetone to remove colored materials. The plant residue was extracted twice with boiling water (2 x 400 ml), 3 h each time. The combined aqueous extract was centrifuged to remove water-insoluble materials and precipitated three times with ethanol (70-90% gradient). The precipitate was dissolved and lyophilized to obtain a polysaccharide fraction.

The phenol-sulfuric acid method (Cuesta et al. 2003; Saravanan and Jayachandran 2008) was used to quantify the DP with modifications. Briefly, the sugar containing solution (0.6 ml) and 5% phenol solution (0.3 ml) were added to screw cap tubes (13 mm x 100 mm) which were capped and vortex-mixed. Then 1.5 ml of concentrated sulfuric acid (98%) was added slowly down the side of tube to the liquid surface. Tubes were then closed, vortex-stirred for 5 s and incubated for 30 min at room temperature. All tubes were allowed to cool down to room temperature before the absorbance was read at 490 nm using distilled water as blank in a Lambda 35 UV/visible spectrophotometer (Perkin-Elmer, USA). Glucose (Sigma) was used as the standard.

For monosaccharide analysis, DP was dissolved in 2 M trifluo-roacetic acid (2ml). and then hydrolyzed at 120[degrees]C for 3 h. After cooling, the solution was neutralized with methanol and concentrated for TLC analysis (CMC-Na, silica gel, Sigma). TLC analysis was developed using chlorofornrethyl acetate:[H.sub.2]O (4:1:5, v/v) as the mobile phase. The spots on chromatograms were detected with sulfuric acid.

Effect of D. candidum polysaccharides (DP) on SS mouse model

A method using an autoallergic mouse model was followed (Wang et al. 2007). The water intake of SS model mice was monitored throughout the experiment. C57BL/6 mice were randomly divided into three groups: control, model and treatment (model + DP). Mice were treated with a volume of 0.2 ml per mouse. Based on our preliminary experiments, mice in the treatment group were treated with DP at a dose of 20 mg/ml. Control and SS model group were treated with an equivalent volume of saline every day.

Statistical analysis

The biopsies were stained using an immuno-histochemical method and the intensity of grey staining was analyzed by computer-assisted imaging analysis system. All data were expressed as mean [+ or -] standard deviation (SD) of three independent experiments and analyzed by one-way ANOVA with SPSS15.0 (SPSS, Inc., Chicago, IL). Differences with a p value of or smaller than 0.05 were considered statistically significant.

Results

The variation in saliva secretion

A conspicuous difference in saliva secretion between patients with and without D. candidum treatment was observed. Salivary secretion increased by about 65% in patients treated with D. candidum (2.5 [+ or -] 0.53 ml) as compared with control (1.49 [+ or -] 0.56 ml), p < 0.05. The results are shown in Fig. 1.

[FIGURE 1 OMITTED]

Increased expression of AQP-5 was observed by immuno-histochemical staining

Stable and clear staining at various locations of the labial gland biopsies was observed when the concentration of AQP-5 was 1:200. AQP-5 appeared mainly at the apical membrane of mucous glands, basolateral membrane and basal membrane of serous acini. There was intense staining in the intercalated duct, striated duct, and secretory duct of the labial gland (Fig. 2A). In control (Fig. 2B) and treated (Fig. 2C) groups, AQP-5 was distributed at the basal membrane and obviously reduced at the apical membrane. In healthy volunteers, AQP-5 immunoreactivity was localized at both apical and basolateral membranes of the acinar cells. In contrast, biopsies from labial glands of primary SS patients demonstrate decreased AQP-5 immunoreactivity at apical membranes and increased immunoreactivity at the basolateral membranes of the acinar cells. D. candidum treatment restored the distribution of AQP-5 to normal.

[FIGURE 2 OMITTED]

Imaging analysis

Immuno-histochemical staining and the intensity of grey staining were analyzed by a computer-assisted imaging analysis system. Results indicated that the drug-treated group shows a significant difference when compared with the control group (Fig. 3). The expression of AQP-5 in labial glands increased by about 35% after D. candidum treatment (p < 0.05).

[FIGURE 3 OMITTED]

Quality analysis

With gradient-programmed elution, extraction with chloroform, methanol and water were examined respectively for quality control (Fig. 4).

[FIGURE 4 OMITTED]

Effect of D. candidum polysaccharides on the water intake of SS mouse model

Calibration curve was carried out with high sensitivity and consistency as shown in Fig. 5. The results showed 95% polysaccharides in D. candidum, consisting of three kinds of monosaccharide: glucose, mannose and galactose (data not shown). As shown in Fig. 6, water intake in the control mice gradually increased, and so did the treatment group. However, the model group had a higher water intake compared to the other two groups. DP treatment significantly decreased the water intake compared with the model, suggesting that DP could reverse the dry mouth condition in the SS model.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Discussion

D. candidum is a valuable herbal medicine for maintaining the tonicity of the stomach and promoting production of body fluids. It can also relieve symptoms such as throat dryness and thirst. Although D. candidum has been used in combination with other herbs to treat SS, this study was the first to furnish direct evidence for the therapeutic efficacy of a single herb, D. candidum alone, on SS. Previous studies have shown that D. candidum can improve the symptoms of weakness in hyperthyroid mice and reduce the inhibitory effect of atropine on salivary gland secretion in rabbits (Zhang et al. 2007). These experiments have demonstrated that D. candidum can promote production of body fluids to mitigate mouth dryness, eye dryness and thirst. However, its mechanism has not been elucidated. This study revealed that some symptoms of SS could be related to deficient expression of AQP-5 and the symptoms can be alleviated by D. candidum. Some factors could affect saliva secretion from SS patients such as individual difference. In this study, saliva secretion in the control group was much lower than that of the drug-treated group. No precise definition of reduction of saliva secretion has been established. Ericsson and Hardwick (1978) reported that the normal range of saliva secretion activated by paraffin in healthy adults was 1.0-3.0 ml/min.

Although there are few studies on the mechanism of thirst in SS, evidence shows that thirst and eye dryness are not the only consequences of glandular damage (Tucci et al. 2005). Other factors including genes, antibody and soluble gene would also lead to the dysfunction of glands (Tucci et al. 2005). Recently, the role of water channel protein in water transport, especially in the salivary gland, has become popular in various research areas. More than 10 water channel proteins have been discovered and they play various important roles according to the structure (Takata et al. 2004). These proteins promote transmembrane movement of water to attain an osmotic gradient rapidly and selectively. Salivary secretion takes place in the gland acini. However, the role of water channel protein in the process of secretion still remains unknown. Recently, a bioactive compound was isolated from another Dendrobium species (Fig. 7) (Zhang et al. 2007) and experimental results showed that it had an effect on the dry eye symptom of SS. We hypothesize that the bioactive compounds could increase the expression of AQP-5 and restore the distribution of AQP-5 in lacrimal glands and corneal epithelia by inhibiting the release of cytokines (1L-1, IL-6, and TNF-[alpha]) mediated by activation of mitogen-activated protein kinase (MAPK) signaling pathways and production of matrix metalloproteinase-9 (MMP-9), ultimately leading to increased saliva and tear secretion. Presently, this study is still in progress to verify' this hypothesis.

[FIGURE 7 OMITTED]

For quality analysis of D. candidum, a series of polarity gradients have been designed. Two single and symmetrical peaks from the chloroform extract at the retention time of 60-40% mobile phase (B) are shown in Fig. 4A. It indicated that two kinds of alkaloids might be present in the sample (Guo 1991). In another extraction, two peaks at the retention time of 60% and 60-40% mobile phase (B) are shown Fig. 4B. It indicates that two kinds of quaternary ammonium hydroxides were present in the sample. Finally, after water extraction, a small peak was obtained (Fig. 4C), it implied that certain unknown compound similar to polysaccharides was present in the sample (Liu and Zhang 1998).

We have demonstrated that the distribution of mucous alveoli is greater than serous acini, which are mainly located at the basal membrane of acinic cells. It was suggested that AQP-5 is implicated in mucous hydration and dissolution, but it remains to be corroborated. Raina et al. (1995) cloned AQP-5 from the rat salivary gland to find a water channel protein from the apical membrane of the gland. The cloned AQP-5 has the same characteristic of water transport as other members of the aquaporin family. It is similar to AQP-2 in having protein kinase A mediated phosphorylation in the cell loops to generate rapidly a "gated" effect. The present results indicated that D. candidum has a higher efficacy for SS than the control group. Analysis using student's r-test revealed that saliva secretion of the D. candidum-treated group was significantly higher (p < 0.01). It implies that D. candidum can promote saliva secretion to relieve the symptom of dry mouth. Results of the experiment using the SS mouse model confirmed the results of the aforementioned investigation on SS patients and disclosed that the active principle in D. candidum extract capable of alleviating SS is a polysaccharide. In conclusion, it is shown in this study that D. candidum upregulates the expression of AQP-5 in labial glands of SS patients and enhances saliva secretion to alleviate xerostomia.

Acknowledgements

This work was supported by grants (Project codes 10208016 and 10400168) from the University of Hong Kong. We appreciate the help of Prof. WANG Zhengtao, Department of Pharmacognosy, China Pharmaceutical University in providing samples and authentication of plant resources by using morphological and histological methods.

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Lin Xiao (a), (1), Tzi Bun Ng (b), (1), Yi-Bin Feng (c), Tong Yao (c), Jack Ho Wong (c), Ren-Min Yao (c), Lei Li (c), Fei-Zhi Mo (c), Yin Xiao (c), Pang-Chui Shaw (d), Ze-Min Li (e), Stephen Cho Wing Sze (c), *, Kalin Yanbo Zhang (c), **

(a) Department of Oral & Maxillofacial Surgery, Central Hospital of Filling, Chongqing China

(b) School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong. China

(c) School of Chinese Medicine. The University of Hong Kong Pokfulam. Hong Kong. China

(d) Department of Biochemistry. The Chinese University of Hong Kong. Shatin. NT. Hong Kong China

(e) Pharmacy. Dalian Rail Hospital, Dalian, China

Abbreviations: APQ, protein aquaporin; DAB. 3,3'-diaminobenzidine; DCEL. Dendrobium candidum extracted liquid; HP, Helicobacter pylori: IgC, immunoglobulin G: IL interleukin; MAPK. mitogen-activated protein kinase: MMP-9. matrix metalloproteinase-9; NO. nitric oxide: NOD, non-obese diabetic: PBS. phosphate buffer saline; PKA, protein kinase A; TNF, tumor necrosis factor; SS. Sjogren's syndrome.

* Corresponding author. Tel.: +852 25890430; fax: +852 28725476.

** Corresponding author. Tel.: +852 25890481; fax: +852 28725476.

E-mail addresses: stephens@hku.hk (S.CW. Sze). ybzhang@hku.hk (K.Y. Zhang). (1) These authors contributed equally to this manuscript.

doi:10.1016/j.phymed.2010.05.002
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Author:Xiao, Lin; Ng, Tzi Bun; Feng, Yi-Bin; Yao, Tong; Wong, Jack Ho; Yao, Ren-Min; Li, Lei; Mo, Fei-Zhi;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Geographic Code:9CHIN
Date:Jan 15, 2011
Words:3897
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