Efficacy of Bushen Culuan decoction (补肾促卵方) on ovarian follicle and follicular granulosa cells in mice with premature ovarian insufficiency induced by tripterygium wilfordii polyglycoside

doi:10.19852/j.cnki.jtcm.2022.01.002

Abstract

Abstract:

OBJECTIVE: To investigate the protective efficacy of Bushen Culuan decoction 补肾促卵方, BCD) on ovarian follicle and follicular granulosa cells in mice with premature ovarian insufficiency (POI) induced by tripterygium wilfordii polyglycoside, and to study the potential mechanism underlying the action.

METHODS: Eighty female Balb/c mice were randomly divided into 4 groups (n = 20 each): blank group, model group, Bushen Culuan decoction intervening group (BCD group) and estradiol valerate intervening group (EV group). In the first 14 model establishing d, mice in model group, BCD group and EV group were under Tripterygium wilfordii polyglycoside (TWP) gavage to establish POI models. In the 14-day therapeutic stage, mice in BCD group were taken BCD 18.35 mg·kg^-1·d^-1, mice in EV group were taken EV solution 0.15 mg·kg^-1·d^-1, while mice in blank group and model group were taken normal saline. When the mice accomplished therapy, whole blood was collected for serum hormone including follicle stimulating hormone (FSH), luteal hormone (LH), estradiol (E2), anti-mullerian hormone (AMH) levels and vascular endothelial growth factor (VEGF), bone morphogenetic protein-7 (BMP-7) measurement. Ovarian tissues were harvested for morphologic observation, follicle counting, ovarian follicular graulosa cell apoptosis test and testing BMP-7 and caspase-3 expressions.

RESULTS: The body weights of the mice kept growing stably in the process expect in TWP intervening stage. Compared with model group, BCD group had significantly higher ovarian index, serum E₂, AMH, VEGF, BMP-7 levels and significantly lower FSH level (P < 0.05). Meanwhile the VEGF level in BCD group was higher than in EV group (P < 0.05). Compared with model group, the histopathological damage and GCs apoptosis were mitigated; developing follicle counting, BMP-7 expression were up-regulated, and caspase-3 expression was down-regulated in BCD groups (P < 0.05).

CONCLUSION: BCD treatment could attenuate pathological process in POI ovaries, suppress GC apoptosis, probably through promoting BMP-7 expression and following inhibiting caspase-3 activation.

Key words: primary ovarian insufficiency, tripterygium, granulosa cells, apoptosis, Bushen Culuan decoction

Kun MA, Yuan YUAN, Yanxia CHEN, Bochao YUAN, Kaili WANG, Caidie TIAN, Jiani LI. Efficacy of Bushen Culuan decoction (补肾促卵方) on ovarian follicle and follicular granulosa cells in mice with premature ovarian insufficiency induced by tripterygium wilfordii polyglycoside[J]. Journal of Traditional Chinese Medicine, 2022, 42(1): 23-29.

Figures/Tables 9

Table 1 Body weight comparison among each group (g, $bar{x}$ ± s)

Group	n	Day –14	Day 1	Day 15	Day 29
Blank	20	18.8±0.7	19.3±1.0	19.5±0.7^a	19.9±0.7^c
Model	20	19.0±0.5	19.4±0.6	19.2±0.5	19.4±0.5
BCD	20	19.2±0.6	19.5±0.5	19.3±0.9	19.70±0.7
EV	20	19.1±0.6	19.5±0.6	19.1±0.3^b	19.5±0.4

Table 2 Estrous cycle comparison among each group (χ2)

Group	n	Days –14-–1 Normal	Days 1-14					Days 15-28			Ratio of irregular cycle (%)
Group	n	Days –14-–1 Normal	Normal	Prolonged	Stagnate	No cycle	Normal	Prolonged	Stagnate	No cycle	Ratio of irregular cycle (%)
Blank	20	20^a	19	0	1	0	18	1	1	0	10
Model	20	20	1	6	3	10	5	7	5	3	75
BCD	20	20	2	8	1	9	17	2	0	1	15^b
EV	20	20	1	5	6	8	17	1	1	1	15

Table 3 Gonad index comparison among each group ($bar{x}$ ± s)

Group	n	Ovarian index	Uterine index
Blank	20	0.87±0.35	3.42±1.92
Model	20	0.72±0.30	2.67±0.71
BCD	20	0.97±0.28^a	3.41±2.47
EV	20	0.84±0.38	2.96±1.47

Table 4 Serum hormone comparison among each group ($bar{x}$ ± s)

Group	n	FSH (IU/L)	LH (ng/mL)	E2 (ng/L)	AMH (ng/mL)	VEGF (ng/L)	BMP-7 (pg/mL)
Blank	10	9.26±2.36	2.18±0.29	65.39±9.74^b	14.98±3.12	183.48±29.26	1202.88±139.00
Model	10	11.41±2.39^a	2.38±0.77	56.32±10.91	14.09±2.49	180.36±48.23	1078.86±213.40
BCD	10	9.89±1.35	2.01±0.38	64.13±10.72^c	16.95±3.51^d	228.24±48.90^e	1311.59±230.36^f
EV	10	10.11±1.45	2.21±0.60	57.99±9.06	15.56±3.16	163.64±39.58	1180.40±216.47

Figure 1 Ovaries of each group (HE Staining, ×200) A: blank group; B: model group; C: BCD group; D: EV group. Arrows: apoptotic cells. White arrows: primordial follicles. Yellow arrows: preantral follicles. Red arrows: antral follicles. Blue arrows: corpus luteum. Black arrows: atretic follicles. Blank group: equal amount of normal saline, 14 d; model group: equal amount of normal saline, 14 d; BCD group: Bushen Culuan decoction, 18.35 g·kg-1·d-1, 14 d; EV group: estradiol valerate intervening group, 0.15 mg·kg-1·d-1, 14 d.

Table 5 Unit follicle account comparison among each group ($bar{x}$ ± s)

Group	n	Primordial follicles	Preantral follicles	Antral follicles	Preovulatory follicles	Corpus luteum	Atretic follicles
Blank	10	21.60±7.37	17.30±3.30	3.30±1.57	1.50±0.31	3.80±1.55	1.60±0.34
Model	10	10.10±3.81	11.10±4.31	2.00±1.49	0.60±0.22	2.10±0.99	4.70±0.42
BCD	10	21.70±4.19^a	15.20±3.36^ab	3.60±1.65^a	1.30±0.26	3.50±1.58^a	1.80±0.47^a
EV	10	18.80±3.68	12.80±3.85	2.70±1.16	0.90±0.23	2.40±1.26	2.30±0.60

Figure 2 Comparison of ovarian granulosa cell apoptosis (TUNEL staining, ×400) A: blank group; B: model group; C: BCD group; D: EV group. Arrows: apoptotic cells. Blank group: equal amount of normal saline, 14 d; model group: equal amount of normal saline, 14 d; BCD group: Bushen Culuan decoction, 18.35 g·kg-1·d-1, 14 d; EV group: estradiol valerate intervening group, 0.15 mg kg-1·d-1, 14 d. TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; BCD: Bushen Culuan decoction; EV: estradiol valerate.

Figure 3 Comparison of BMP-7 and caspase-3 expressions by Western blot (n = 3) Blank group: equal amount of normal saline, 14 d; model group: equal amount of normal saline, 14 d; BCD group: Bushen Culuan decoction, 18.35 g·kg-1·d-1, 14 d; EV group: estradiol valerate intervening group, 0.15 mg·kg-1·d-1, 14 d. BMP-7: bone morphogenetic protein-7. aP < 0.05, compared with model group. bP < 0.05, compared with EV group. cP < 0.05, compared with model group.

Table 6 BMP-7 and caspase-3 expressions among each group ($bar{x}$ ± s)

Group	n	BMP-7	Caspase-3
Blank	3	0.520±0.080	0.590±0.030
Model	3	0.410±0.010	0.650±0.010
BCD	3	0.640±0.030^ab	0.540±0.040^c
EV	3	0.440±0.020	0.610±0.040

References 24

[1]	Chen ZJ, Tian QJ, Qiao J, et al. Expert consensus of premature ovarian insufficiency clinical diagnosis and treatment. Zhong Hua Fu Can Ke Za Zhi 2017;52:577-81.
[2]	Webber L, Davies M, Anderson R, et al. ESHRE guideline: management of women with premature ovarian insufficiency. Hum Reprod 2016;31:926-37.
[3]	Hu YL, Zhang JL, Hou XW, Jin L, Zhang Y. Menopausal hormone therapy and risk of ovarian cancer: a Meta-analysis. Zhong Guo Zhong Liu Xue Za Zhi 2016;22:549-53.
[4]	Zhang JQ, Zhang Q, Meng L. Overview of estrogen / progestin replacement therapy and breast cancer occurrence risk. Zhong Guo Fu You Bao Jian 2015;30:6669-72.
[5]	Ye N, Dong XY, Li DH. The progress in apoptotic mechanism of ovarian granulosa cells involved in premature ovarian failure. Shou Du Yi Ke Da Xue Xue Bao 2014;35:379-83.
[6]	Liu LQ, Liu YF, Yang M, et al. Effectiveness of tonifying-kidney and regulating-liver therapy on diminished ovarian reserve: a systematic review and Meta-analysis of randomized controlled trials. J Tradit Chin Med 2020;40:343-54.
[7]	Sun YY, Tan Y, Chen SP. Effectiveness of nourishing Yin and tonifying Yang sequential therapy in combination with Climen on diminished ovarian reserve: a retrospective study. J Tradit Chin Med 2020;40:150-6.
[8]	Kayamori T, Kosaka N, Miyamoto A, Takashi S. The differential pathways of bone morphogenetic protein (BMP)-4 and -7 in the suppression of the bovine granulosa cell apoptosis. Mol Cell Biochem 2009;323:161-8.
[9]	Zhu GQ, Cui YH, Wang QL, Kang YG, Lü YZ, Wang JG. Bone morphogenetic proteins (BMP) 2, 4, 6 and 7 affect ovarian follicu- lar development through regulation of follicle-stimulating horm- one receptor (FSHR) and luteinizing hormone receptor (LHR) expression in goat granulosa cells. J Cell Biol Genet 2013;3:14-21.
[10]	Ma K, Fu FZ, Jiang K, Sun LH, Cai LX. The effectiveness of Tiaojing Zhuluan decoction treating anovulatory infertility. Zhong Hua Zhong Xi Yi Jie He Za Zhi 1998;18:372-3.
[11]	Ma K, Liu YF, He JQ, Li M, Shan J. A multi-center, randomized, double-blind clinical study on Bushen Huoxue in treatment of ovulatory dysfunction caused infertility. Zhong Guo Zhong Yao Za Zhi 2015;40:2911-5.
[12]	Ma K. The effectiveness of Bushen Culuan decoction on ovulation and corpus luteum function in experimental rats. Fujian Zhong Yi Yao Za Zhi 1997;28:3-4.
[13]	Zhao W, Sun GZ. The medication dosage conversion among different animals. Zhong Guo Xu Mu Shou Yi Za Zhi 2010;5:52-3.
[14]	Zhao SG, Chen ZJ. Molecular mechanisms of major reproductive diseases. Sheng Ming Ke Xue Tong Bao 2017;29:43-51.
[15]	Shang YJ, Chen Y, Lu S. Systemic review and Meta-analysis of Bushen Huoxue Chinese medicine in the treatment of premature ovarian failure. Zhong Yi Za Zhi 2018;59:1295-9.
[16]	Luo X, Li X, Cheng J, et al. Systemic review and Meta-analysis of efficacy of acupuncture in the treatment of premature ovarian failure. Zhong Yi Za Zhi 2016;57:1027-32.
[17]	Visser JA, de Jong FH, Laven JSE, Themmen APN. Anti-Mullerian hormone: a new marker for ovarian function. Reproduction 2005;131:1-9.
[18]	Yan DW, Zhou L, Sun ZY. Research progress in establishing animal models of premature ovarian failure and its evaluation indexes. Zhong Hua Yao Li Yu Du Li Xue Za Zhi 2015;29:486-92.
[19]	Grasa P, Sheikh S, Krzys N, et al. Dysregulation of follicle development in a mouse model of premature ovarian insufficiency. Reproduction 2016;152:591-601.
[20]	Luan Y, Edmonds ME, Woodruff TK, Kim SY. Inhibitors of apoptosis protect the ovarian reserve from cyclophosphamide. J Endocrinol 2019;240:243-56.
[21]	Ma M, Chen XY, Li B, Li XT. Melatonin protects premature ovarian insufficiency induced by Tripterygium glycosides: role of SIRT1. Am J Transl Res 2017;9:1580-602.
[22]	Yuan Y. Research progress of influence of bone morphogenetic protein 7 in women reproductive system and its intervention with Traditional Chinese Medicine. Guangzhou Zhong Yi Yao Da Xue Xue Bao 2019;36:297-302.
[23]	Wang WN, Zhang WF, Li DH, et al. Lichong decoction inhibits micro-angiogenesis by reducing the expressions of hypoxia inducible factor-1α and vascular endothelial growth factor in hysteromyoma mouse model. J Tradit Chin Med 2020;40:928-37.
[24]	Shimizu T, Magata F, Abe Y, Miyamoto A. Bone morphogenetic protein 4 (BMP-4) and BMP-7 induce vascular endothelial growth factor expression in bovine granulosa cells. Anim Sci J 2012;83:663-7.

Home

Online First

Author Center

Reviewer Center

Issues

Announcement

About Us