Apigenin, a Single Active Component of Herbal Extract, Alleviates Xerostomia via ERα-Mediated Upregulation of AQP5 Activation

Abstract

Xerostomia is a common symptom in menopausal women, suggesting the role of sex steroids in disease development. Shreds of literature had reported the potential use of herbal extracts to relieve xerostomia. However, a cocktail of multiple components in herbal extract makes it difficult to understand the exact mechanism of action. Aquaporin5 (AQP5), the specific aquaporin expressed in salivary glands, plays an important role in salivary secretion as a downstream of estrogen signaling. In this study, we aimed to unravel a single active herbal component as a therapeutic for xerostomia and investigate its mechanism of action. The effects of apigenin (flavonoid), dauricine (alkaloids), protopine (alkaloids), and lentinan (polysaccharides) on AQP5 transcription were screened in vitro. Only apigenin robustly induced AQP5 transcription and expression, and this effect was even robust compared to the effect of estradiol (E2, a positive control). Overexpression of estrogen receptor α (ERα) in the human salivary gland cell line (HSG) upregulated the AQP5 transcription and expression and the knockdown ERα reversed this effect, suggesting the role of ERα signaling on AQP5 activation in HSG cells. Docking results showed apigenin-specific binding sites in ERα. We further analyzed the therapeutic effect of apigenin on ovariectomized mice as a xerostomia model. The saliva secretion in the xerostomia group was reduced to one-third of the sham group, whereas the apigenin or E2 treatment for 12 weeks reversed this effect. Meanwhile, the water consumption in the xerostomia group was augmented obviously compared to the sham group, whereas the water consumption in the apigenin and E2 group was declined to the level of the sham group. Immunohistochemistry of submandibular glands revealed the downregulation of AQP5 expression in xerostomia mice compared to control. Apigenin, or E2 treatment, upregulated AQP5 expression in xerostomia mice. In conclusion, apigenin, a single active component of herbal extract, upregulated AQP5 expression in HSG cells via activation of ERα signaling and restored saliva flow rates in OVX mice. These results revealed apigenin as a single active component of herbal extract with the potential to treat xerostomia.

Keywords: apigenin; aquaporin5 (AQP5); estrogen receptor α (ERα); ovariectomized (OVX) mice; xerostomia.

FIGURE 1

Natural product apigenin upregulates AQP5 transcription. (A) Structure of apigenin. (B) Luciferase reporter pGL3/AQP5 promoter (AQP5p) plasmid. (C) Relative luciferase activity showing the AQP5p activation induced by apigenin in HSG cells. (D) Apigenin activated the AQP5p in dose dependence. Data are presented as the mean ± SD, n = 3. Significant effect of the treatment, *p < 0.05, **p < 0.01.

FIGURE 2

Apigenin upregulates AQP5 transcription and expression. (A) mRNA relative expression level (AQP5/β-actin) showing the AQP5 transcription in HSG cells by qPCR. (B) Apigenin upregulates AQP5 expression. Representative Western blot image showing endogenous AQP5 protein expression in HSG cells. (C) Immunofluorescence staining showing the AQP5 protein level and localization in HSG cells. Bar scale, 50 μm. Data are presented as the mean ± SD, n = 3. Significant effect of the treatment, *p < 0.05, **p < 0.01.

FIGURE 3

Apigenin upregulates the transcription and translation of AQP5 through the ERα pathway. (A) Scheme showing AQP5 promoter sequence with ERα binding sites. (B) ChIP results showed that ERα occupancy was apparently increased at ERE3 in the presence of apigenin. (C) Relative luciferase activity (the indication of AQP5 expression) in ERα-overexpressed and -knocked down with or without 1 μM apigenin in HSG cells. (D) Western blot analysis showing the effect of apigenin treatment on AQP5 protein expression in ERα-overexpressed and -knocked down HSG cells. Data are presented as the mean ± SD, n = 3. Significant effect of the treatment, *p < 0.05, **p < 0.01.

FIGURE 4

Molecular docking shows specific binding of apigenin to ERα within the E2-ligand binding domain. Images showing the binding site of apigenin (A), E2 (B), and lentinan (C) in ERα. (D) Interaction of amino acid residues located in the ligand binding domain (LBD) of ERα. (E) Binding energy of apigenin, E2, and lentinan with ERα.

FIGURE 5

(A) Ovariectomized operation. (B) Scheme of animal experiment and treatment.

FIGURE 6

Apigenin ameliorates the impairment of saliva secretion and reduced the water consumption rate in OVX mice. (A) Salivary secretion index before ovariectomy and after 4, 8, and 12 weeks of treatments. (B) Salivary secretion index after 12 weeks of treatment. (C) Water consumption rate before ovariectomy and after 4, 8, and 12 weeks of treatments. (D) Water consumption rate after 12 weeks of treatment. Data are presented as the mean ± SD, n = 6. Significant effect of the treatment, *p < 0.05, **p < 0.01.

FIGURE 7

Apigenin treatment upregulates AQP5 expression in the submandibular gland of OVX mice. (A) Histological images (H&E stained) and immunohistochemistry images of AQP5 of mice submandibular gland tissues. Arrows, acinar cells. Triangles, ducts cells. (B) Quantitative analysis of immunohistochemistry images. (C) AQP5 protein expression in the submandibular gland lysate. Data are presented as the mean ± SD, n = 6 mice per group. Significant effect of the treatment, *p < 0.05, **p < 0.01.