Identification of oxytocin receptor activating chemical components from traditional Japanese medicines

Abstract

Oxytocin (Oxt) is known to regulate social communication, stress and body weight. The activation of Oxt receptors (OTR) has clinical potential to abate stress disorders and metabolic syndrome. Kamikihito (KKT) is a traditional Japanese medicine used to treat psychological stress-related disorders. We investigated the effects of KKT, its ingredients and chemical components on Oxt neurons and OTR. C-Fos expression was examined after oral and peripheral administration of KKT in rats. Electrophysiological change of Oxt neurons and Oxt release upon application of KKT were measured in rat brain slice. The direct effect of KKT, its ingredients and its chemical components were examined by cytosolic Ca²⁺([Ca²⁺]_i) measurement in Oxt neurons and OTR-expressing HEK293 cells. Both intraperitoneal and oral administration of KKT in rats induced c-Fos expression in neurons of the paraventricular nucleus (PVN) including Oxt neurons. Application of KKT induced activation of Oxt neurons and Oxt release. KKT increased [Ca²⁺]_i in OTR-expressing HEK293 cells, and failed to activate with OTR antagonist. KKT-induced PVN Oxt neuron activation was also attenuated by OTR antagonist. Seven chemical components (rutin, ursolic acid, (Z )-butylidenephtalide, p-cymene, senkunolide, [6]-shogaol, [8]-shogaol) of three ingredients (Zizyphi Fructus, Angelicae Acutilobae Radix, Zingiberis Rhizoma) from KKT had potential to activate OTR. KKT can directly activate PVN Oxt neurons by interacting with OTR. The interaction of seven chemical components from KKT may contribute to activate OTR. Effect of KKT on Oxt neurons and OTR may contribute to the treatment of Oxt related disorders.

Fig. 1

c-Fos expression in the PVN after oral or intraperitoneal (ip) administration of KKT. a, b: c-Fos expression after oral administration of distilled water (a) or KKT (500 mg/kg) in the PVN (b). 3V: third ventricle. c: Number of c-Fos-positive neurons per section in the PVN (n = 6, 6). d–g: Double staining of c-Fos and Oxt after oral distilled water (d, e) or KKT (f, g). e and g indicate the enlarged image of the dotted area in d and f, respectively. Arrows indicate c-Fos and Oxt double-positive neurons. h: The percentage of c-Fos-positive neurons among Oxt neurons (n = 6, 6). Scale bars in a, b, d, f = 100 μm. Scale bars in e, g = 10 μm * p < 0.05, unpaired t-test. i, j: c-Fos expression after ip administration of saline (i) or KKT (500 mg/kg) in the PVN (j). 3V: third ventricle. k: Number of c-Fos-positive neurons per section in the PVN (n = 5, 5). l–o; Double staining of c-Fos and Oxt after ip saline (l, m) or KKT (n, o) administration. m and o indicate the enlarged image of the dotted area in l and n, respectively. Arrows indicate c-Fos and Oxt double-positive neurons. p: The percentage of c-Fos-positive neurons among Oxt neurons (n = 5, 5). Scale bars in i, j, l, n = 100 μm. Scale bars in m, o = 10 μm ** p < 0.01, unpaired t-test.

Fig. 2

Effects of KKT on the activation of PVN Oxt neurons and the release of Oxt. a: Representative recording of membrane potential from an identified Oxt neuron in Oxt-mRFP rat. b: Mean membrane potential (+ SEM) before, during, and 5 min after 500 μg/mL KKT washout (n = 5). c: Mean firing frequency (+ SEM) before (control), during, and 5 min after 500 μg/mL KKT washout (n = 5). **p < 0.01, one-way ANOVA followed by Tukey’s multiple range test. d: Representative cytosolic Ca²⁺ ([Ca²⁺ ]_i) chart upon absence of KKT (KKT0), 5 μg/mL (KKT5) and 50 μg/mL (KKT50) treatment in the single neuron of Oxt. Scale bar = 10 μm. e: Amplitude of ([Ca²⁺ ]_i) in each dose of KKT-responsive neurons in the PVN. *p < 0.05, one-way ANOVA followed by Tukey’s multiple range test. f: Incidence of KKT-responsive neurons in PVN neurons (%). (n = 18). g: Oxt concentration after incubation with or without KKT (500 μg/mL). (n = 9, 9) *p < 0.05, paired t-test.

Fig. 3

Co-localization of Oxt and OTR in the PVN. a–c: Confocal images of Oxt (a), OTR (b) and merged image of a and b (c). 3V: third ventricle. Scale bars = 100 μm. d–f: Enlarged image of the dotted area in a–c, respectively. Scale bars = 10 μm. Arrow heads indicate representative Oxt and OTR double-positive neurons. Arrows indicate representative single Oxt immunoreactive neurons. Asterisk (*) indicates representative single OTR positive neurons.

Fig. 4

The effect of KKT on OTR-expressing HEK293 cells. a–h: Representative confocal image of HEK293 cells with (e–h) or without (a–d) transfection of OTR. Among analysis of 2705 cells from 8 plate, 1248 (46.1 ± 3.6%) cell were found to be transfected. a, e indicate bright images. b, f indicate DAPI nuclear staining. c, g indicate OTR expressing cells. d, h indicate merged images of b and c, f and g, respectively. Scale bars = 10 μm. i–k: Representative [Ca²⁺ ]_i images upon applying 10⁻⁹M Oxt (i), 10⁻⁷ M Oxt (j) and 10⁻⁵ M Oxt (k). l: Mean amplitude of [Ca²⁺ ]_i in each dose of Oxt. (−) indicate absence of Oxt (control). (Controln = 105, 10⁻⁹ MOxt n = 35, 10⁻⁷M Oxt n = 35, 10⁻⁵ M Oxt n = 35). m-p: Representative [Ca²⁺ ]_i images upon applying KKT 0.5 μg/mL (m), 25 μg/mL (n), 50 μg/mL (o) and 75 μg/mL (p). q: Mean of [Ca²⁺ ]_i amplitude in each dose of KKT. 0 indicates the absence of KKT (control). (Control n = 242, 0.5 μg/mL n = 72, 25 μg/mL n = 103, 50 μg/mL n = 33, 75 μg/mL n = 34). **p < 0.01, one-way ANOVA followed by Tukey’s multiple range test.

Fig. 5

The effect of OTR inhibitor on the effect of KKT in HEK293 cells and PVN Oxt neurons. a: Representative change of [Ca²⁺ ]_i image applying with 10⁻⁷M Oxt in HEK293 cells, which was not transfected OTR. b, c: Representative change of [Ca²⁺ ]_i image applied with 10⁻⁷MOxt (b) or 10⁻⁷ M Oxt pre-treatment of 10⁻⁷M H4928 (c) in OTR-transfected HEK293 cells. d: Representative change of [Ca²⁺ ]_i image applying with 50 μg/mL KKT in HEK293 cells, which was not transfected OTR. e, f: Representative change of [Ca²⁺ ]_i image applying with 50 μg/mL KKT (e) or 50 μg/mL KKT pre-treatment of 10⁻⁷M H4928 (f) in OTR transfected HEK293 cells. g, h: Mean of amplitude of [Ca²⁺ ]_i in each cell and treatment. (g; from left bar, n = 91, 104, 49. h; from left bar, n = 97, 103, 138). **p < 0.01, one-way ANOVA followed by Tukey multiple range test. i, j: Representative recording of membrane potential with application of 500 μg/mL KKT with pre-treatment of 10⁻⁷M H4928 in the Oxt neurons in Oxt-mRFP rat. Two from eight neurons exhibited slight action potentials, as shown in i, and six from eight exhibited no action potential, as shown in j. k, l: Mean membrane potential (+ SEM) and Mean firing frequency (+ SEM) before (control), with H4928 and with both H4928 and KKT treatment (n = 8).

Fig. 6

The membrane potential and firing frequency under treatment of Hochuekkito (HE), Shikunshito (SKS) and Ninjinto (NJ). a, d, g: Representative recording of membrane potential under treatment of HE (a), SKS (d) and NJ (g) from an identified Oxt neuron in Oxt-mRFP rat. b, c, e, f, h, i: mean membrane potential (+ SEM) (b, e, h) and mean firing frequency (+ SEM) (c, f, i) before (control), during, and 5 min after 500 μg/mL HE (b, c, n = 5), SKS (e, f, n = 5) and NJ (h, i, n = 5) washout. **p < 0.01, *p < 0.05. one-way ANOVA followed by Tukey’s multiple range test.

Fig. 7

The effect of Zizyphi Fructus (ZF), Angelicae Acutilobae Radix (AR), Zingiberis Rhizome (ZR) on [Ca²⁺ ]_i in PVN Oxt neurons. a, d, g: Representative [Ca²⁺ ]_i image under treatment of 5 μg/mL (5) and 50 μg/mL (50) ZF (a), AR (d) and ZR (g) in Oxt neurons. b, f, i: Mean of amplitude of [Ca²⁺ ]_i under treatment of 5 μg/mL (5) and 50 μg/mL (50) ZF (b, n = 83), AR (f, n = 44) and ZR (i, n = 15) measured in the PVN neurons. 0 indicates the basal control before treatment of each crude drug. *p < 0.05, one-way ANOVA followed by Tukey’s multiple range test. b, e, h: The incidence of each dose of ZF-(c), AR-(f) and ZR-(i) responsive neurons in the PVN neurons. The criteria of responsive neurons were described in detail in the Methods section. j: Representative [Ca²⁺ ]_i image under treatment of 50 μg/mL ZF-AR-ZR mixture (MIX) and 50 μg/mL KKT in PVN Oxt neuron. k: Mean of amplitude of [Ca²⁺ ]_i under treatment of MIX and KKT (n = 16). There were no significant differences. Unpaired t-test.

Fig. 8

The effect of chemical components of Zizyphi Fructus (ZF), Angelicae Acutilobae Radix (AR), Zingiberis Rhizome (ZR) on [Ca²⁺ ]_i in OTR-expressing HEK293 cells. a: Fold of control ((+ )Oxt)) mean ratio under treatment of each chemical components of ZF. (from left bar; n = 244, 274, 35, 35, 35, 35, 245, 208, 98, 35, 105, 35, 65, 34, 35, 33). b: Fold of control ((+ )Oxt)) mean ratio under treatment of each chemical components of AR. (from left bar; n = 208, 103, 35, 68, 70, 68, 65, 65, 67, 65, 68, 69, 102, 33, 34, 35, 31, 35, 34, 32, 68, 60, 99, 102). c: Fold of control ((+ )Oxt)) mean ratio under treatment of each chemical components of ZR. (from left bar: n = 206, 210, 35, 35, 65, 67, 35, 35, 35, 35, 65, 34, 70, 104, 102, 102, 70, 67, 32, 35). Each ratios were normalized by the ratio for amplitude of [Ca²⁺ ]_i under treatment of 10⁻⁵ M Oxt in each experiment. (−) indicates HEK293 cells without OTR expression. (+ ) indicates OTR-expressing HEK293 cells. **p < 0.01, *p < 0.05, unpaired t-test.

Fig. 9

The change of plasma quercetin levels after oral KKT administration and the effects of quercetin on [Ca²⁺ ]_i in OTR-expressing HEK293 cells. a: Plasma quercetin concentration after oral KKT (500 mg/kg) administration at 0, 20, 60 and 120 min (n = 3). b: Typical chromatograms of carvacrol (upper panel) and quercetin (bottom panel) with the respective internal standards thymol and myricetin. c–e: Representative [Ca²⁺ ]_i image under 10⁻⁷M Oxt application (c), 10⁻⁵M quercetin (d) and 10⁻⁸M quercetin (e) in HEK293 cells without OTR transfection. f–h: Representative [Ca²⁺ ]_i image under treatment of 10⁻⁷M Oxt (f), 10⁻⁵M quercetin (g) and 10⁻⁸M quercetin (h) in OTR-transfected HEK293 cells. The bars in figures c–h indicate the term of treatment. i: Mean of amplitude of [Ca²⁺ ]_i under applications of 10⁻⁷ M Oxt (n = 31, 35), 10⁻⁵M quercetin (n = 35, 32) and 10⁻⁸M quercetin (n = 35, 35). **p < 0.01, unpaired t-test.