The potential of Zishen Yutai pills to facilitate endometrial recovery and restore fertility after induced abortion in rats

Figures & data

Table 1. Detailed information about the components of ZYP.

No	Latin name	English name	Chinese name
01	Cuscuta Chinensis Lam.	Cuscutae Semen	Tusizi
02	Panax ginseng C. A. Mey.	Ginseng Radix et Rhizoma	Renshen
03	Amomum villosum Lour.	Amomi Fructus	Sharen
04	Rehmannia glutinosa (Gaertn.) DC.	Rehmanniae Radix Praeparata	Shudihuang
05	Taxillus chinensis (DC.) Danser	Taxilli Herba	Sangjisheng
06	Asini Corii Colla	Asini Corii Colla	Ejiao
07	Polygonum multiflorum Thunb.	Polygoni multiflori Radix Praeparata	Zhiheshouwu
08	Artemisia argyi H. Lév. & Vaniot	Artemisiae argyi Folium	Aiye
09	Morinda officinalis How.	Morindae officinalis Radix	Bajitian
10	Atractylodes macrocephala Koidz.	Atractylodis macrocephalae Rhizoma	Baizhu
11	Codonopsis pilosula (Franch.) Nannf.	Codonopsis Radix	Dangshen
12	Cervus nippon Temminck	Cervi Cornu Degelatinatum	Lujiaoshuang
13	Lycium barbarum L.	Lycii Fructus	Gouqizi
14	Dipsacus asper Wall. ex DC.	Dipsaci Radix	Xuduan
15	Eucommia ulmoides Oliv.	Eucommiae Cortex	Duzhong

Figure 1. Experimental grouping schedule. IA: intragastrically administration; NS: normal saline; ZYP: Zishen Yutai Pill.

Table 2. The detailed information about the primers.

Gene name	Primer name	Sequence (5′→3′)
VEGF	Forward Primer	5′-GCA CTG GAC CCT GGC TTT ACT-3′
	Reverse Primer	5′-ACT TCA CCA CTT CAT GGG CTTT CTG-3′
ER	Forward Primer	5′-TCT GGA GTG TGC CTG GTT GGA G-3′
	Reverse Primer	5′-GCG GAA TCG ACT TGA CGT AGC C-3′
TGF-β	Forward Primer	5′-AAC ACA GCA GAG TGG TTG TC-3′
	Reverse Primer	5′-TGT CCA GGC TCC AGA TGT A-3′
MMP-9	Forward Primer	5′-CTC CTG GTG CTC CTG GCT CTA G-3′
	Reverse Primer	5′-GCT GTG TGT CCG TGA GGT TGG-3′
LIF	Forward Primer	5′-GTC AAC TGG CTC AAC TCA ACG-3′
	Reverse Primer	5′-CTG GCA GCC CAA CTT CTT C-3′
HB-EGF	Forward Primer	5′-AGG ACT ACT GCA TCC ACG GAG AG-3′
	Reverse Primer	5′-CTA CAG CCA CCA CAG CCA AGA C-3′
GAPDH	Forward Primer	5′-GGC AAG TTC AAC GGC ACA G-3′
	Reverse Primer	5′-GCC AGT AGA CTC CAC GAC AT-3′

Figure 2. The morphological changes of rat endometrium in three groups. (*p < 0.05, **p < 0.01) (a) The morphological changes of the endometrium with Haematoxylin and Eosin Staining (×200). “→” indicates the endometrium structure, and “▲” represents endometrial glands. (b) Comparisons of endometrial thickness (mm) and endometrial gland numbers per unit area. Bar = 200 μm; Con: control; ZYP: Zishen Yutai Pill.

Figure 3. The mRNA and protein expression levels of ER and VEGF of rat endometrium in three groups. (*p < 0.05, **p < 0.01) (a) The mRNA expression levels of ER and VEGF in rat endometrium. (b) The representative images of western blot for ER and VEGF in rat endometrium. (c) The protein expression levels of ER and VEGF in rat endometrium. Con: control; ZYP: Zishen Yutai Pill; VEGF: vascular endothelial growth factor; ER: oestrogen receptors.

Figure 4. The fibrosis of rat endometrium in three groups. (*p < 0.05, **p < 0.01) (a) The fibrosis of the endometrium with Masson staining (×100). (b) Comparisons of endometrial fibrotic areas ratio: the fibrosis of endometrium in the normal group and the ZYP group was lower than that in the control group at all three time points after induced abortion (*p < 0.05; Figure 4(b)). Bar = 200 μm; Con: control; ZYP: Zishen Yutai Pill.

Figure 5. The mRNA and protein expression levels of MMP-9 and TGF-β of rat endometrium in three groups. (*p < 0.05, **p < 0.01) (a) The mRNA expression levels of MMP-9 and TGF-β in rat endometrium. (b) The representative images of western blot for MMP-9 and TGF-β in rat endometrium. (c) The protein expression level of MMP-9 and TGF-β in rat endometrium. Con: control; ZYP: Zishen Yutai Pill; TGF-β: transforming growth factor; MMP-9: matrix metalloproteinase 9.

Figure 6. The morphology and number of pinopodes of rat endometrium during the window of implantation. (*p < 0.05, **p < 0.01) (a) The SEM images of pinopodes of rat endometrium in three groups. (A) (C) (E) pictures are of lower magnification, Bar = 10 μm; white squares are highly magnified regions which were shown in (B) (D) (F), Bar = 5 μm. The triangle represents the pinopodes; Black arrowheads indicate the boundary between cells; Asterisks signify area dense in microvilli. (b)The number of pinopodes of tat endometrium in three groups. Con: control; ZYP: Zishen Yutai Pill; SEM: scanning electron microscopy.

Figure 7. The mRNA and protein expression levels of LIF and HB-EGF at the second oestrous cycle. (*p < 0.05, **p < 0.01) (a) The mRNA expression levels of LIF and HB-EGF in rat endometrium. (b) The representative images of western blot for LIF and HB-EGF in rat endometrium. (c) Comparison of the protein expression levels of LIF and HB-EGF in rat endometrium. Con: control; ZYP: Zishen Yutai Pill; LIF: leukaemia inhibitory factor; HB-EGF: heparin-binding epidermal growth factor.

Figure 8. The expression and localization of LIF and HB-EGF in rat endometrium. (*p < 0.05, **p < 0.01) (a) The expression and localization of LIF in rat endometrium. (b) The expression and localization of HB-EGF in rat endometrium. (c) The fluorescence intensity of LIF and HB-EGF of rat endometrium in three groups. Bar = 200 μm; Con: control; ZYP: Zishen Yutai Pill; LIF: leukaemia inhibitory factor; HB-EGF: heparin-binding epidermal growth factor.

Figure 9. The number of pups of rats in three groups (*p < 0.05, **p < 0.01). Con: control; ZYP: Zishen Yutai Pill.