Research on the effects and preliminary mechanism of action of Shaoyao Gancao granule (芍药甘草颗粒) on hypothalamic-pituitary-adrenal axis and T lymphocytes in stressed alopecia areata mice

doi:10.19852/j.cnki.jtcm.2025.05.005

Abstract

Abstract:

OBJECTIVE: To investigate the effect and mechanism of Shaoyao Gancao granule (SGG, 芍药甘草颗粒) on the hypothalamic-pituitary-adrenal (HPA) axis and immune imbalance status in stressed alopecia areata (AA) mice, and to provide an objective experimental basis for the clinical application of SGG.

METHODS: Seventy female C57BL/6J mice aged 5-7 weeks were randomly divided into two groups: 10 mice in the blank control group and 60 mice in the mock group. The moulding group received topical imiquimod cream in combination with chronic unpredictable mild stress. On day 10, the moulding group was further divided into six groups: Shaoyao Gancao granule low-dose (SGL), Shaoyao Gancao granule medium-dose (SGM), Shaoyao Gancao granule high-dose (SGH), Antalarmin, and compound glycyrrhizin (CG). On day 24, overall and trichoscopic photographs of mice were taken on day 24 of the experiment; behavioral tests were completed; serum corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and cortisol levels were measured by enzyme-linked immunosorbent assay; and T helper cell (Th)1/Th2 and Th17/Treg cell differentiation in peripheral blood T lymphocyte subpopulations was detected by flow cytometry.

RESULTS: The dorsal skin lesions of mice in all SGG groups showed faster hair growth, less dilated skin capillaries, and scaly conditions compared with those in the model group. In the open field test, compared with those of the model group, the moving distance and number of uprights and entries into the central area of the mice in the SGM and SGH groups significantly increased (P < 0.05), while in the forced swimming test, compared with the model group, the rest time of the mice in the SGL, SGM, and SGH groups significantly decreased (P < 0.05). The enzyme-linked immunosorbent assay results showed that, compared with the model group, the mice in the SGH group had significantly reduced CRH levels (P < 0.05), and the ACTH and cortisol levels in the SGM and SGH groups were significantly reduced (P < 0.05). The flow cytometry results showed that, compared with those in the model group, Th2 levels were significantly higher (P < 0.05), Th17 levels were significantly lower (P < 0.05), the Th1/Th2 ratio was significantly lower (P < 0.05), and the Th17/Treg ratio was significantly lower (P < 0.05) in the SGM and SGH groups. The Th1 and Treg cell ratios were reduced in all SGG groups, but the difference was not statistically significant.

CONCLUSION: SGG may exert therapeutic effects in AA by modulating the HPA axis and regulating immune imbalance.

Key words: alopecia areata, stress, hypothalamic-pituitary-gonadal axis, T-lymphocytes, Shaoyao Gancao granule

LYU Shuying, QU Baoquan, LIN Wenjun, YANG Dingquan. Research on the effects and preliminary mechanism of action of Shaoyao Gancao granule (芍药甘草颗粒) on hypothalamic-pituitary-adrenal axis and T lymphocytes in stressed alopecia areata mice[J]. Journal of Traditional Chinese Medicine, 2025, 45(5): 979-986.

Figures/Tables 6

Figure 1 Total ion chromatogram of SGG A: the extracted ion flow chromatogram of the control; B: the extracted ion flow chromatogram of the samples, in which the retention time of paeoniflorin was 4.7 min, that of liquiritin was 5.16 min, and that of glycyrrhizin acid was 7.3 min. SGG: Shaoyao Gancao granule.

Figure 2 Overall and trichoscopic changes of hair in mice A: photographs of the backs of mice at different times in each group; B: trichoscopic photographs (× 30) of the backs of mice at different times in each group. A1-A7: Control group, Model group, SGL group, SGM group, SGH group, Antalarmin group, and CG group dorsal photographs on Day 1. A8-A14: Control group, Model group, SGL group, SGM group, SGH group, Antalarmin group, and CG group dorsal photographs on Day 7. A15-A21: Control group, Model group, SGL group, SGM group, SGH group, Antalarmin group, and CG group dorsal photographs on Day 10. A22-A28: Control group, Model group, SGL group, SGM group, SGH group, Antalarmin group, and CG group dorsal photographs on Day 24. B1-B7: Control group, Model group, SGL group, SGM group, SGH group, Antalarmin group, and CG group dorsal trichoscopic images on Day 1. B8-B14: Control group, Model group, SGL group, SGM group, SGH group, Antalarmin group, and CG group dorsal trichoscopic images on Day 7. B15-B21: Control group, Model group, SGL group, SGM group, SGH group, Antalarmin group, and CG group dorsal trichoscopic images on Day 10. B22-B28: Control group, Model group, SGL group, SGM group, SGH group, Antalarmin group, and CG group dorsal trichoscopic images on Day 24. Control group: no durg, no stress; Model group: IMQ + CUMS; CG group: IMQ + CUMS + CG (20 mg·kg-1·d-1, days 10-24); SGL group: IMQ + CUMS + SGL (1.95 g/kg, days 10-24); SGM group: IMQ + CUMS + SGM (3.9 g/kg, days 10-24); SGH group: IMQ + CUMS + SGH (7.8 g/kg, days 10-24); Antalarmin group: IMQ + CUMS + Antalarmin (20 mg/kg, days 17-24). IMQ: imiquimod; CUMS: chronic unpredictable mild stress: CG: compound glycyrrhizin; SGL: Shaoyao Gancao granule low dose; SGM: Shaoyao Gancao granule medium dose; SGH: Shaoyao Gancao granule high dose.

Table 1 Mouse body weight change graph (g, x - ± s)

Group	n	Day 1	Day 7	Day 10	Day 20	Day 24
Control	10	17.5±0.8	18.6±1.0	18.8±0.9	19.0±1.1	19.4±0.7
Model	10	17.0±0.7	15.3±0.8	18.5±0.9	16.9±0.7	18.0±0.8
SGL	10	17.1±0.8	15.3±0.8	17.9±0.7	17.8±0.8	17.6±0.7
SGM	10	17.1±1.1	15.0±1.1	18.1±1.1	18.3±1.1	18.3±0.7
SGH	10	16.8±0.9	15.4±1.1	17.4±0.7	18.0±0.7	18.3±0.9
Antalarmin	10	17.5±1.0	15.2±1.2	18.2±1.2	17.0±0.9	17.9±1.2
CG	10	17.2±1.1	14.9±0.9	17.6±0.7	17.7±1.2	17.9±0.8
P value	-	0.555	<0.001^a	0.01^b	<0.001^a	<0.001^a

Table 2 OFT and FST of mice ( x - ± s)

Group	n	Moving distance (m)	Upright number	Number of entries into central area	Swimming immobility time (s)
Control	8	54.1±15.9^a	22.9±11.6^a	17.9±4.6^b	82.4±13.9^b
Model	8	33.4±5.8	8.0±4.6	5.5±1.9	135.7±15.8
SGL	8	39.3±8.1	8.5±3.0	9.1±4.2^c	93.8±9.6^b
SGM	8	53.2±5.2^b	17.6±4.7^a	12.1±2.2^b	86.2±25.6^a
SGH	8	52.6±16.1^c	20.6±3.9^b	16.5±2.9^b	88.5±17.3^b
Antalarmin	8	54.4±6.6^b	20.1±7.7^a	14.8±5.8^a	92.1±18.4^b
CG	8	48.5±8.9^a	15.9±4.2^a	13.9±3.4^b	99.1±19.0^a

Table 3 Serum CRH, ACTH, and cortisol levels in mice ( x - ± s)

Group	n	CRH	ACTH	Cortisol
Control	6	75.0±8.4^a	61.2±6.8^c	9.5±1.2^a
Model	6	106.9±10.6	93.1±14.5	15.7±2.4
SGL	6	96.7±10.0	88.8±13.6	13.5±1.9
SGM	6	95.6±10.3	76.7±10.6^b	11.9±2.2^b
SGH	6	92.7±3.5^b	69.0±15.0^b	10.3±1.4^c
Antalarmin	6	88.1±13.9^b	68.1±15.6^b	10.9±2.5^c
CG	6	87.6±13.9^b	71.1±14.4^b	10.7±2.4^c

Figure 3 T cell differentiation of mice in each group A: differentiation of mouse peripheral blood Th1 cells; B: differentiation of mouse peripheral blood Th2 cells; C: mouse peripheral blood Th1/Th2 cell ratio; D: differentiation of mouse peripheral blood Th17 cells; E: differentiation of mouse peripheral blood Treg cells; F: mouse peripheral blood Th17/Treg cell ratio. Control group: no durg, no stress; Model group: IMQ + CUMS; CG group: IMQ + CUMS + CG (20 mg·kg-1·d-1, days 10-24); SGL group: IMQ + CUMS + SGL (1.95 g/kg, days 10-24); SGM group: IMQ + CUMS + SGM (3.9 g/kg, days 10-24); SGH group: IMQ + CUMS + SGH (7.8 g/kg, days 10-24); Antalarmin group: IMQ + CUMS + Antalarmin (20 mg/kg, days 17-24). IMQ: imiquimod; CUMS: chronic unpredictable mild stress: CG: compound glycyrrhizin; SGL: Shaoyao Gancao granule low dose; SGM: Shaoyao Gancao granule medium dose; SGH: Shaoyao Gancao granule high dose. Comparisons between multiple groups used one-way analysis of variance analysis. Data are presented as mean ± standard deviation (n = 6). Compared with the model group, aP < 0.05, bP < 0.001, cP < 0.01.

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