Ixeris dentata Extract Increases Salivary Secretion through the Regulation of Endoplasmic Reticulum Stress in a Diabetes-Induced Xerostomia Rat Model

Abstract

This study aimed to investigate the molecular mechanism of diabetes mellitus (DM)-induced dry mouth and an application of natural products from Ixeris dentata (IXD), a recently suggested regulator of amylase secretion in salivary cells. Vehicle-treated or diabetic rats were orally treated with either water or an IXD extract for 10 days to observe the effect on salivary flow. We found that the IXD extract increased aquaporin 5 (AQP5) and alpha-amylase protein expression in the submandibular gland along with salivary flow rate. Similarly, the IXD extract and its purified compound increased amylase secretion in high glucose-exposed human salivary gland cells. Furthermore, increased endoplasmic reticulum stress response in the submandibular gland of diabetic rats was inhibited by treatment with the IXD extract, suggesting that IXD extract treatment improves the ER environment by increasing the protein folding capacity. Thus, pharmacological treatment with the IXD extract is suggested to relieve DM-induced dry mouth symptoms.

Keywords: Ixeris dentata; amylase; aquaporin 5; diabetes-associated xerostomia; endoplasmic reticulum stress; saliva secretion.

Figure 1

The effect of the different grades of ethanol on extraction of IXD. Purified compounds, (A) Ixerin M; (B) Ixerin F; (C) epiisolipidiol-3-β-d-glucopyranoside (8-EI-3-G) in the IXD extract found by extraction with different grades of ethanol and 100% methanol; (D,E) effect of different grades of ethanol on extraction of IXD and measurement of amylase secretion in HSG cells exposed to high glucose concentrations; (D) amylase secretion measurement using cell lysates; (E) amylase secretion measurement using cell cultured medium.

Figure 2

The IXD extract increases amylase synthesis and secretion in human salivary gland cells exposed to high glucose levels. (A,B) Amylase activity was measured in cell lysates (A) and cultured medium (B) of HSG cells exposed to high glucose concentrations with or without the IXD extract for 1, 2, 3, 5, and 7 days. Values are represented as mean ± SEM (n = 3, p < 0.05). * indicates significant differences with the IXD-untreated cell condition; (C,D) amylase expression in cell lines exposed to high glucose concentrations treated with or without the IXD extract for the indicated time period; (C) amylase protein expression was analysed using cell lysates; (D) amylase protein expression was analysed using cell cultured medium; (E,F) HSG cells were exposed to high glucose concentrations (40 mM) for 1, 2, 3, 5, and 7 days in the presence or absence of Ixerin M, a purified IXD component, and amylase expression was measured in cell lysates (E) and cell medium (F); (G,H) amylase protein expression was analysed in cell lysates or cultured medium in the presence or absence of Ixerin F and (I,J) epiisolipidiol-3-β-d-glucopyranoside (8-EI-3-G).

Figure 3

The effects of the IXD extract on salivary parameters. (A) Total salivary volume (mL) collected at 10 min after pilocarpine injection; (B) the total salivary flow rate was measured in μL/min; (C) measurement of glucose concentration in saliva; (D) measurement of total protein concentration in saliva (mg/mL). Values are represented as mean ± SEM (n = 10 per group, p < 0.05). * indicates significant differences compared to vehicle-treated control rats and # indicates significant differences compared to STZ-induced diabetic rats; (E) haematoxylin and eosin staining showing the morphological appearance of the submandibular glands of control and diabetic rats. Red arrowheads show variable degrees of vacuolization of the connective tissue stroma. Black arrows show slightly dilated and pyknotic nuclei in striated ducts. Characters showing in pictures such as A, C, S and G denotes for acinar cells, connective tissue stroma, striated ducts and granular convoluted tubules respectively. Magnification: 20×; Scale bar: 100 μm.

Figure 4

Expression of α-amylase in the submandibular gland and saliva. (A) Amylase expression in submandibular gland tissue lysates; (B) amylase expression in saliva; (C) paraffin-embedded submandibular gland tissues were sectioned at 5 μm thickness, and immunostaining was performed using an amylase antibody. Brownish red colours indicate immunolocalization of α-amylase in submandibular gland cells. Magnification: 40×; Scale bar: 100 μm.

Figure 5

Expression of AQP5 and NHE1 in the submandibular gland. (A) Western blot analysis showing AQP5 protein expression in submandibular gland tissue lysates. The blot identified two bands of AQP5 (27 kDa and 31 kDa) revealing non-glycosylated and glycosylated forms of the AQP5 protein respectively; (B) the expression of AQP5 (1:100) was performed on submandibular gland tissue sections of either water or IXD extract-treated control or diabetic rats. Brownish red colour indicates the positive expression of AQP5. Magnification: 40×; Scale bar: 100 μm; (C) immunohistochemical detection of NHE1 in submandibular gland tissue from normal and diabetic rats treated with water or the IXD extract. Yellow arrow heads show the expression of NHE1 in duct cells, whereas black arrow heads indicate acinar expression. Magnification: 40×; Scale bar: 100 μm.

Figure 6

The IXD extract regulates the ER stress response in submandibular gland of diabetic rats. (A) Western blot analysis was performed to demonstrate the ER stress response using GRP78, CHOP, ATF6α, p-eIF2α, total eIF2α, p-PERK, total PERK, p-IRE1α, total IRE1α, and sXBP-1 antibodies. An equal concentration of protein (30 μg) was loaded in each lane of the gels. Beta-actin was used as a loading control. The molecular weight of each protein is indicated in kDa; (B) immunostaining of GRP78; (C) immunostaining of CHOP in the submandibular gland. Magnification: 40×; Scale bar: 100 μm; (D) expression of secreted GRP78 in saliva in the indicated groups. Coomassie Brilliant Blue (CBB) staining was performed as a control for equal protein loading.

Figure 7

The IXD extract regulates ER folding environment. (A) Western blot analysis showing the expression of PDI and ERO1α in submandibular gland lysates; (B) immunostaining of PDI in the submandibular gland. Magnification 40×; Scale bar: 100 μm; (C) submandibular gland lysates were immunoprecipitated with anti-amylase or AQP5 antibody and immunoblotted with the anti-PDI (upper), anti-amylase or anti-AQP5 antibody (lower); (D) TBARS assay was performed to measure lipid peroxidation level in vehicle or IXD-treated submandibular gland homogenates. Values are expressed as percentage of control, (n = 10 per group, p < 0.05). * indicates significant differences compared to vehicle-treated control rats and # indicates significant differences compared to STZ-induced diabetic control rats; E: Detection of salivary carbonyls using Western blotting. The molecular weight (MW) is indicated in kDa. Red rectangular box shows the high protein carbonylation pattern in the saliva of STZ-induced diabetic rats.

Figure 8

The proposed mechanism of an IXD extract against diabetes-induced xerostomia. The schematic diagram shows diabetes-associated ER stress and oxidative stress, subsequently, altering the protein expression of AQP5 and NHE1 in the submandibular gland. This may cause salivary gland dysfunction with reduction in salivary secretion leading to xerostomia. This graphic shows that IXD extract increases salivary secretion through the regulation of ER stress and prevents from xerostomia.