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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 4  |  Page : 189-197

Caulis sargentodoxae prescription inhibits angiogenesis-related cytokines in a rat endometriosis model


1 Department of Gynecology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China
2 Key Laboratory of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai 200032, China

Date of Submission31-Oct-2017
Date of Web Publication7-Feb-2018

Correspondence Address:
Zhao-Gui Sun
Key Laboratory of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai 200032
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.224911

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  Abstract 


Background: To determine the efficacy of the Caulis Sargentodoxae prescription, which is an empirical formula of Chinese herbs and has definite curative effects on endometriosis.
Methods: The Caulis Sargentodoxae prescription on the growth of ectopic endometria was studied with a rat endometriosis (EMS) model. The EMS model was established by autoplastic transplantation. To study the curative effects of Chinese medicine on EMS in comparison with western medicine, gestrinone and an angiogenesis inhibitor were introduced. The rats were randomly divided into seven groups: normal group, model group, ovariectomized group, gestrinone (western medicine) group, Caulis Sargentodoxae prescription (Chinese medicine) group, apatinib (inhibitor) group, and combination (Chinese medicine + inhibitor) group. After administration for 21 days, the growth inhibitory rates of ectopic endometria in the treatment groups were evaluated, and the levels of vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) were detected by ELISA in the serum and peritoneal fluid, as well as in the ectopic endometrial tissues by real-time polymerase chain reaction and Western blotting.
Results: The growth inhibitory rates of ectopic endometria in the treatment groups were significantly higher (P < 0.05). In the Caulis Sargentodoxae prescription group, the levels of angiogenesis-related factors, including VEGF and VEGFR2, were reduced in the serum and peritoneal fluid compared with the model group (P < 0.05). In addition, the positive expression of VEGF and VEGFR2 in ectopic endometria significantly decreased in the Caulis Sargentodoxae prescription group both at mRNA and protein levels.
Conclusions: VEGF and VEGFR2 levels in the serum and peritoneal fluid can be used as a clinical reference for endometriotic pathogenesis and treatment, and the Caulis Sargentodoxae prescription has reliable therapeutic effects on EMS for its target-action ability to decrease angiogenesis.

Keywords: Angiogenesis; Caulis Sargentodoxae Prescription; Endometriosis; Rat Model


How to cite this article:
Zhuang MF, Cao Y, Shi Y, Yu L, Niu YN, Zhang TT, Sun ZG. Caulis sargentodoxae prescription inhibits angiogenesis-related cytokines in a rat endometriosis model. Reprod Dev Med 2017;1:189-97

How to cite this URL:
Zhuang MF, Cao Y, Shi Y, Yu L, Niu YN, Zhang TT, Sun ZG. Caulis sargentodoxae prescription inhibits angiogenesis-related cytokines in a rat endometriosis model. Reprod Dev Med [serial online] 2017 [cited 2018 Aug 17];1:189-97. Available from: http://www.repdevmed.org/text.asp?2017/1/4/189/224911




  Introduction Top


Endometriosis (EMS) is a chronic inflammatory disease, characterized by implantation and growth of endometrial tissue outside of the uterine cavity.[1] Studies have shown that the growth of ectopic endometrial tissue outside of the uterine cavity requires the nutritional support of new blood vessels. Vascular endothelial growth factor (VEGF) is the most important angiogenic factor.[2] Studies have confirmed that the expression of VEGF is maintained at high levels in the uterus and ectopic endometrial lesions of patients with EMS.[3],[4] VEGF receptor-2 (VEGFR2) is a transmembrane protein kinase, which is one of the specific membrane receptors of VEGF. A clinical study by Cho et al.[5] showed that the levels of VEGF and its receptor VEGFR2 in the peritoneal fluid of patients with EMS are higher than those in the fluid from control patients. Therefore, it is necessary to focus on angiogenesis in the pathogenesis of EMS.

The Caulis Sargentodoxae prescription is an empirical formula of Chinese herbs created by Deying Dai, a famous expert of traditional Chinese medicine in Shanghai. After decades of clinical studies, it was found that Dai's formula had definite curative effects on EMS, with a total clinical effective rate of ~90% on EMS,[6],[7],[8] 90.63% on menstrual fever, and 96.25% on dysmenorrhea. It also effectively prevented endometrial recurrence [9] after surgery and significantly improved the quality of life of EMS patients with dysmenorrhea and chronic pelvic pain.[10]

In this study, we chose Caulis Sargentodoxae prescription, whose clinical efficacy in the treatment of EMS has been recognized, to study its mechanism of action and effects on the volume of ectopic endometria, as well as on the levels of various angiogenesis-related factors in the peritoneal microenvironment of an EMS rat model. Through this research, we hope to explain the molecular mechanism of Caulis Sargentodoxae prescription on EMS via angiogenesis and to determine the holistic efficacy of a traditional Chinese medicine prescription.


  Methods Top


Animals

Sprague–Dawley rats (aged 6–8 weeks, Shanghai B&K Universal Limited, China) weighing 160–200 g were used for experiments. They were housed under specific pathogen-free conditions in an air-conditioned room with 12-h light/12-h dark cycles and at 22–24°C with 55%–65% relative humidity. Rats had free access to food and water. All experiments were carried out in accordance with the guidelines for the water. The use of laboratory animals was approved by the Ethics Committee of the Shanghai Institute of Planned Parenthood Research (SIPPR). All procedures were performed in accordance with the guidelines established by the Ethics Committee of the SIPPR.

Chemicals and reagents

Chinese traditional herbs were denoted in Latin and recorded in Ancient Chinese classical works. The herbs were as follows: Sargentodoxa cuneata (Oliv.) Rehd. et Wils. recorded in “Bencao tujing”; Typha angustifolia L. recorded in “Shennong bencaojing”; Ostrea gigas Thunberg, oyster in English, recorded in “Shennong bencaojing”; Corydalis Corydalis yanhusuo W. T. Wang recorded in “Leigong paozhilun”; Paeonia suffruticosa Andr. recorded in “Shennong bencaojing”; Prunus persica Batsch, peach kernel in English, recorded in “Shennong bencaojing”; and Cyperus rotundus L. recorded in “Mingyi bielu”. The decoction was prepared by the pharmacy at the Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, which is affiliated with the Shanghai University of Traditional Chinese Medicine, according to a conventional preparation method. It was equivalent to the traditional Chinese medicine concentration of 2.75 g/mL.

Gestrinone active pharmaceutical ingredient (API) and apatinib API were purchased from Fengzhulin Chemexpress Co., Ltd. (Wuhan, China). Rat VEGF and theVEGFR2 ELISA kit were purchased from X-Y Biotechnology (Shanghai, China). The rabbit anti-VEGFR2 monoclonal antibody (cat. no. 9698) was purchased from Cell Signaling Technology, Inc., (Boston, MA, USA). The rabbit anti-VEGF polyclonal antibody (cat. no. 19003-1-AP) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (cat. no. 10494-1-AP) were purchased from Proteintech Group, Inc. (Rosemont, IL, USA).

Establishment of the rat model

According to the method of Jones,[11] the EMS rat model was established under sterile conditions. The rats underwent a vertical abdominal incision after being anesthetized with 3% pelltobarbitalum natricum. A section on the left side of the uterus was removed and immediately placed into physiologic saline. The endometrium was separated from the myometrium and cut into 0.5 cm × 0.5 cm fragments. The uterine endometrial segments were sutured onto the peritoneum near blood vessels. The incision was closed and disinfected, and the animals were allowed to recover from anesthesia.

Medicine administration groups and sample collection

At 21 days after the surgery, the rats underwent a second laparotomy to check the growth of the ectopic endometrial tissue. The volume (length × width × height) was measured with an electronic digital caliper. Rats with ectopic tissue volumes larger than 20 mm3 were randomly divided into groups for further experiments [Figure 1]. To compare the different effects between Chinese medicine and western medicine and angiogenesis inhibitors, we had set up seven groups: normal group, model group, ovariectomized group, gestrinone (western medicine) group, Caulis Sargentodoxae prescription (Chinese medicine) group, apatinib (inhibitor) group, and combination (Chinese medicine + inhibitor) group. The normal group consisted of five rats, and rats in the other six groups received different treatments as listed in [Table 1].
Figure 1: Ectopic endometria growing on the intraperitoneal wall of rats. (a) Ectopic endometria growing on the abdominal wall of rats at second laparotomy; (b) a vesicular growth of ectopic endometria; (c) a nodular growth of ectopic endometria. The arrowhead refers to the ectopic endometrium on the intraperitoneal wall of EMS rat model.

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Table 1: Regimens of medicine administration to rats

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After the administration of medicines for 21 days, 10 mL of phosphate-buffered saline (PBS) buffer was injected into the rat abdominal cavity of rats after anesthesia. Gently shook the rats for 10 min. The rat abdominal wall was punctured with a blunt needle, and the peritoneal fluid was extracted with a 10 mL syringe. Then, the abdominal wall was cut longitudinally, and the blood was extracted into a 5 ml syringe with a needle from the abdominal aorta. The peritoneal fluid was extracted about 5–8 mL, and the blood was extracted about 3–5 mL. Rats were then sacrificed and dissected. The supernatant of the peritoneal fluid and serum samples were harvested and stored at −80°C. Ectopic and eutopic endometrias were collected. Half of the tissues were fixed in 4% neutral paraformaldehyde for sectioning and histological analysis, whereas the other half were frozen in liquid nitrogen for molecular analyses.

Enzyme-linked immunosorbent assay

Peritoneal fluid and serum samples were collected and stored at 2–8°C for no more than 5 days. Thereafter, the cytokine levels of VEGF and VEGFR2 were measured according to the manufacturer's instructions. Standard curves were prepared, and the peritoneal fluid and serum samples were diluted 1:5 with the diluent buffer as directed by the manufacturer's instructions. Antibody incubation and washing steps were performed according to the routine protocols. After the trimethoprim color development, the absorbance at A450 was recorded on a model ELx-800 ELISA microplate reader (BIO-TEK Instruments Inc.).

Immunohistochemistry

Tissues fixed in 4% neutral paraformaldehyde were subjected to washing, dehydration, waxing, and embedding and then cut into serial 5 μm thick sections. After the sections were dewaxed in xylene and rehydrated, antigen retrieval was carried out by boiling in 10 mmol/L sodium citrate buffer (pH 6.0) in a microwave twice for 5 min. After washing with PBS (0.01 mmol/L, pH 7.4) thrice for 5 min, sections were immersed in 3% H2O2 solution for 10 min to inactivate the endogenous peroxidase. Thereafter, 10% serum was used to block nonspecific-binding sites, and in the experimental groups, sections were incubated overnight with primary VEGF and VEGFR2 antibodies (diluted 1∶100) or the negative control. Biotinylated goat anti-rabbit IgG and streptavidin-labeled horseradish peroxidase (HRP) (Proteintech Group, Inc.,) were then applied to react with the primary antibodies, and the DAB color solution was added to develop the immune complex as brown precipitates. After the sections were stained with hematoxylin solution and mounted using a general protocol, each slide was observed and recorded under a microscope. The integrated absorbance (IA) values of VEGF and VEGFR2 in the ectopic endometria were measured by an Image-Pro Plus system (Media Cybernetics, Maryland, USA).

Quantification of mRNA

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by reverse-transcription to cDNA (1 g/L) with reverse transcriptase. Gene-specific primers were designed using Primer Express Software v2.0 (ABI, Waltham, Mass, USA). Primer sequences are shown in [Table 2]. The mRNA levels were detected by real-time polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA) using 2× Taq PCR Master Mix (TIANGEN, Beijing, China). All reactions were performed in triplicate, and the thermal cycling conditions were as follows: 5 min at 94°C, followed by 38 cycles at 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s. GAPDH was used to normalize target mRNA levels.
Table 2: Primer sequences used for quantitative RT-PCR in this study

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Western blotting analyses

Total proteins were extracted from cells (Beyotime Biotechnology Research Institute). In each experimental group, 20 μg of proteins was loaded per lane and subjected to 10% SDS/PAGE. The separated protein bands were transferred to polyvinylidene difluoride membranes, and the membranes were blocked for 1 h in 5% bovine serum albumin (BSA) buffer and then incubated with VEGF and VEGFR2 monoclonal antibodies in blocking buffer (diluted 1∶1,000 in 5% BSA) for 90 min at 37°C. After washing thrice with PBST, the membranes were incubated with an HRP-conjugated secondary antibody (diluted 1∶4,000 with 5% BSA) for 60 min at 37°C. The primary GAPDH antibody was diluted 1∶2,000 with 5% BSA and served as an internal control. The membranes were washed thrice with PBST, and photographs were taken immediately after color development using a Bio-Rad Gel Imaging System (Hercules, CA, USA). The relative levels of proteins were semi-quantitatively determined using ImageJ analysis software (ImageJ2x 2.1.4.7, National Institutes of Health, Maryland, Baltimore, USA). The target protein concentration of each sample was determined as a gray value ratio relative to GAPDH.

Statistical analysis

Data were presented as means ± standard deviation. SPSS 18.0 software (Statistical Product and Service Solutions, IBM Corp., Armonk, NY, USA) was used for statistical analysis. The statistical significance among three or more groups was determined by one-wayANOVA analysis. Pairwise comparisons between group means were conducted using the least significant difference method. Two-tailed P < 0.05 was considered to be statistically significant.


  Results Top


Effect of Chinese medicines on the body weight of an EMS rat model

There was no significant difference in the body weight among the groups before treatment (P > 0.05). Compared with the data before treatment, there was no significant decrease in body weight after treatment. The body weight of rats in the ovariectomized group increased after treatment, and the difference was statistically significant compared with the model group (P < 0.05) [Table 3] and [Figure 2].
Figure 2: The body weight of EMS rat models. A: normal control group; B: model group; C: ovariectomized group; D: gestrinone group; E: Caulis Sargentodoxae prescription group; F: apatinib group; G: combination group. In the bar charts, *P < 0.05 compared with the model group. EMS: Endometriosis.

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Table 3: The body weight of rats before and after (x¯ ± s)

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Effects of Chinese medicines on the volume and growth inhibitory rates of ectopic endometria in an EMS rat model

The growth inhibition rate of the ectopic endometria (%) was calculated as follows: (volume before treatment − volume after treatment)/volume before treatment × 100%. There was no significant difference in the volume of ectopic endometrial tissue before treatment (P > 0.05). The volume of the ectopic endometrium in each group was reduced by varying degrees after treatment. The volume of the ectopic endometrium after treatment in the other groups was significantly lower, and the growth inhibition rate in other groups was higher than that in the model group (P < 0.05). Specifically, the growth inhibition rate of the ectopic endometrium in the Caulis Sargentodoxae prescription group was higher than that in the apatinib group though lower than in the gestrinone group. However, the growth inhibition rate of the combination group was higher than that of the inhibitor group [Table 4] and [Figure 3].
Figure 3: The volume and growth inhibitory rates of ectopic endometria in rats with EMS. (a) The volume of ectopic endometria in rats with EMS before and after treatment; (b) the growth inhibitory rates of ectopic endometria in rats with EMS. A: model group; B: ovariectomized group; C: gestrinone group; D: Caulis Sargentodoxae prescription group; E: apatinib group; F: combination group. In the bar charts, *P < 0.05 compared with the model group. EMS: Endometriosis.

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Table 4: The volume and growth inhibitory rates of ectopic endometria before and after treatment (x¯ ± s)

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Effects of Chinese medicines on the levels of VEGF and VEGFR2 in the serum and peritoneal fluid in rats with EMS

After treatment, there were significant differences in the levels of VEGF and VEGFR2 in the serum and peritoneal fluid of rats in each group (P < 0.05). The levels of VEGF and VEGFR2 in the serum and peritoneal fluid in the model group were significantly higher than that in the normal group (P < 0.05), and their levels in the peritoneal fluid were higher than that in the serum. There was a pronounced decline in the levels of VEGF and VEGFR2 in the serum and peritoneal fluid in all administration groups compared with the model group (P < 0.05). In comparison with the model group, the level of VEGF in the serum and peritoneal fluid in the Caulis Sargentodoxae prescription group, apatinib group, and combination group sharply decreased (P < 0.05). Except for the ovariectomized group and gestrinone group, the level of VEGFR2 in the serum and peritoneal fluid in other groups was significantly lower than that in the model group (P < 0.05). In addition, the levels of VEGF and VEGFR2 in the serum and peritoneal fluid in the Caulis Sargentodoxae prescription group were lower than those in the gestrinone group, which were also much lower in the apatinib group [Table 5], [Table 6] and [Figure 4].
Figure 4: The levels of VEGF and VEGFR2 in the serum and peritoneal fluid in each group by ELISA assay. (a) The levels of VEGF in the serum and peritoneal fluid in each group; (b) the levels of VEGFR2 in the serum and peritoneal fluid in each group. A: model group; B: ovariectomized group; C: gestrinone group; D: Caulis Sargentodoxae prescription group; E: apatinib group; F: combination group. In the bar charts, *P < 0.05 compared with the model group. P < 0.05 compared with the normal control group. VEGF: Vascular endothelial growth factor; VEGFR2: Vascular endothelial growth factor receptor-2.

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Table 5: The content of VEGF in the serum and peritoneal fluid (x¯ ± s)

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Table 6: The content of VEGFR2 in the serum and peritoneal fluid (x¯ ± s)

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Effect on the expression of VEGF and VEGFR2 in the ectopic endometrium of rats with EMS

The results of the immunohistochemistry experiment [Figure 5] showed that the positive expression of VEGF in the ectopic endometrial tissue in model group was strong but that it was reduced to varying degrees in all administration groups. The positive expression of VEGF in the ectopic endometrium was significantly decreased in the Caulis Sargentodoxae prescription group and combination group. Furthermore, the positive expression of VEGFR2 in the ectopic endometrium in the model group was intense [Figure 6]. The area and intensity of the positive expression signal of VEGFR2 in each administration group were decreased compared with the model group. In addition, the positive expression of VEGFR2 in the ectopic endometrium in the Caulis Sargentodoxae prescription group, apatinib group, and combination group was clearly decreased, and the positive expression of VEGFR2 was found only in a few interstitial cells in the apatinib group and combination group. The IA values of VEGF and VEGFR2 in the ectopic endometrium were significantly different between the treatment groups and the model group (P < 0.01) [Table 7].
Figure 5: The localization of VEGF in ectopic endometrial tissues of rats in each group by IHC after DAB staining. (a) Model group, ×20; (b-h) model group, ovariectomized group, gestrinone group, Caulis Sargentodoxae prescription group, apatinib group, combination group, and negative control, ×40. VEGF: Vascular endothelial growth factor; IHC: Immunohistochemistry. The arrowheads refer to the positive expression of VEGF in the ectopic endometrium.

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Figure 6: The localization of VEGFR2 in ectopic endometrial tissues of rats in each group by IHC after DAB staining. (a) Model group, ×20; (b-h) model group, ovariectomized group, gestrinone group, Caulis Sargentodoxae prescription group, apatinib group, combination group, and negative control, ×40. VEGFR2: Vascular endothelial growth factor receptor-2; IHC: Immunohistochemistry. The arrowheads refer to the positive expression of VEGFR2 in the ectopic endometrium.

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Table 7: The IA values of VEGF and VEGFR2 in the ectopic endometrium (x¯ ± s)

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The relative expression of VEGFR2 in the ectopic endometrium after treatment was assessed by real-time quantitative PCR and western blotting at mRNA and protein levels, respectively [Figure 7]. When the relative protein level was semi-quantitatively determined, statistical analyses were performed. The difference between groups at the mRNA level by one-way variance analysis did not reach a significant level (P > 0.05), but the relative protein level of VEGFR2 in the ectopic endometrium after treatment was statistically different between groups (P < 0.05). The mRNA and protein expression of VEGFR2 in the ectopic endometrium in the model group was high. The relative expression of mRNA of VEGFR2 in the ectopic endometrium in the Caulis Sargentodoxae prescription group, apatinib group, and combination group was lower than that in the model group (P < 0.05) [Figure 7]a. Compared with the model group, the relative protein level of VEGFR2 in ectopic endometria in the other groups was markedly decreased, except for the gestrinone group (P < 0.05) [Figure 7]b.
Figure 7: The mRNA and protein amount of VEGFR2 in ectopic endometrial tissues of rats in each group. (a) The mRNA levels of VEGFR2 in ectopic endometria as assessed by real-time PCR; (b) semi-quantitative analysis of the relative protein levels of VEGFR2; (c) the protein bands of VEGFR2 as presented by Western blotting. A: model group; B: ovariectomized group; C: gestrinone group; D: Caulis Sargentodoxae prescription group; E: apatinib group; F: combination group. In the bar charts, *P < 0.05 compared with the model group. VEGFR2: Vascular endothelial growth factor receptor-2; PCR: Polymerase chain reaction.

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  Discussion Top


Several clinical reports [12],[13],[14],[15] have shown that the Caulis Sargentodoxae prescription has good curative effects in the treatment of EMS because it can relieve dysmenorrhea and chronic pelvic pain and improve the clinical symptoms and quality of life of patients with postoperative recurrence. Furthermore, its side effects and adverse reactions are mild. Our previous results from animal experiments [16],[17],[18],[19],[20] showed that the Caulis Sargentodoxae prescription can inhibit the growth of ectopic endometrial lesions in rats, reduce the expression of matrix metalloproteinases and adhesion molecules in eutopic and ectopic endometria of rats with EMS, decrease the hydrolysis of the extracellular matrix by the ectopic endometrium, and inhibit the expression of VEGF and its receptor fetal liver kinase-1 in ectopic endometria. Our in vitro experiments [21] also showed that the Caulis Sargentodoxae prescription can inhibit the growth of endometrial cells in rats. Although we previously performed research on the Caulis Sargentodoxae prescription in the treatment of EMS, we did not define the inhibitory effects of the Caulis Sargentodoxae prescription from dynamical and comparative perspectives, especially with regard to angiogenesis in the pathology of EMS. In this study, since the traditional Chinese medicine treatment was not only compared with progesterone but also compared with the VEGFR2 receptor inhibitor as the control, the ability of the Caulis Sargentodoxae prescription to inhibit EMS via angiogenesis was deduced. The association between the levels of VEGF and VEGFR2 in the serum and peritoneal fluid and their expression changes in the endometrial tissues were found so that VEGF/VEGFR levels could be used as potential biomarkers for EMS from the serum or peritoneal fluid and furthermore contribute to the mechanistic analysis of the shedding of the membrane receptor VEGFR2 as previously reported.[22],[23]

The results of this study showed that the growth inhibition rate of the ectopic endometrium was significantly higher (reaching about 58.4%) in the Caulis Sargentodoxae prescription group compared with the model group. The body weights of rats with EMS in the Caulis Sargentodoxae prescription group increased after treatment as expected. No obvious abnormality was found, other than the broken uterine ends indicative of a state of edema. Therefore, we thought that the Caulis Sargentodoxae prescription had a significant inhibitory effect on the growth of ectopic endometria but no other obvious side effects in rats with EMS.

The experimental results showed that the levels of VEGF and VEGFR2 in the serum and peritoneal fluid of the Caulis Sargentodoxae prescription group were significantly reduced. Furthermore, the expression of the two factors in the ectopic endometrium decreased, both at the mRNA and protein levels. When combined with the VEGFR inhibitor, the effect of apatinib was effectively replenished, suggesting that the Caulis Sargentodoxae prescription can effectively inhibit angiogenesis by downregulating the levels of VEGF and VEGFR2 in the ectopic endometria, with concomitant decreases in the serum and peritoneal fluid. With regard to the inhibition of angiogenesis in rats with EMS, the Caulis Sargentodoxae prescription and VEGFR inhibitor apatinib both had significant effects though gestrinone did not directly act on VEGFR2 to regulate the VEGF/VEGFR2 signaling pathway as in the above experiments. Because VEGF can stimulate angiogenesis under both physiological and pathological conditions [24],[25] and via VEGFR2,[26],[27] the VEGFR inhibitor Apatinib may affect the physiological function of the body. However, we found that the multiple holistic effects of the Caulis Sargentodoxae prescription can inhibit angiogenesis, specifically in the ectopic endometria without significant side effects. Because of the association between the levels of VEGF/VEGFR2 in the serum and peritoneal fluid and their expression in endometrial tissues, the decreased levels of VEGF/VEGFR2 in the serum and peritoneal fluid may reflect inhibited angiogenesis in endometria.

In recent years, research on inhibitors to VEGFR has provided new ideas for the treatment of EMS. In the experiment, we choose the angiogenesis-targeted therapy of apatinib as control. At the same time, we set up the combination group (Chinese medicine + apatinib), not only to further explain the molecular mechanism involved in the pathogenesis of EMS, but also to provide new ideas for the treatment of EMS. In the experiment, we choose the angiogenesis-targeted therapy of apatinib as control. At the same time, we set up the combination group (Chinese medicine + apatinib) not only to further explain the molecular mechanism involved in the pathogenesis of EMS but also to evaluate the curative effect of traditional Chinese medicine more objectively from the angle of combination of Chinese and western medicine. Apatinib is a kind of molecular-targeted antitumor drugs, which belongs to the small molecule of selective tyrosine kinase inhibitor against VEGFR. It can inhibit VEGFR tyrosinase activity, block signal transduction after the binding of VEGF to the receptor, and inhibit angiogenesis to treat tumors. It has now entered Phase III clinical trial.[28] In this experiment, our results showed that the growth inhibition rates to ectopic endometria in the apatinib group and the combination group were higher. The levels of VEGF and VEGFR2 in serum and peritoneal fluid were significantly decreased, and the expressions of VEGF and VEGFR2 (especially VEGFR2) in ectopic endometria were reduced. This suggested that Apatinib can reduce the levels of VEGF and VEGFR2 in serum and peritoneal fluid in EMS rat models, decrease the expression of VEGFR2 in ectopic endometria, and lower the combination between VEGF and VEGFR2, thus play a role in inhibiting angiogenesis and reducing the nutritional support for the growth of ectopic endometria, eventually causing the ectopic endometria to grow slowly or turn to atrophy.

In conclusion, the Caulis Sargentodoxae prescription has verified therapeutic effects in reducing ectopic endometrial volumes in rats with EMS by a mechanism that inhibits angiogenesis in ectopic endometria and with paralleled drops in the levels of VEGF and VEGFR2 in the serum and peritoneal fluid, suggesting that VEGF and VEGFR2 levels in the serum and peritoneal fluid can be used as biomarkers of endometrial angiogenesis, which can improve treatment.

Financial support and sponsorship

This work was supported by The Natural Science Foundation of China (grant no. 81373684).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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