Study on the mechanism of effect of Daodi Tongguan Decoction on rats with premature ovarian failure

LONG Yun-xia, ZHONG Yi-hui, WU Yuan-yuan, CHEN Qiu-jin, DU Li, ZHENG Hong, LI Yin-ying, LUO Zhi-juan✉

1.Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning 530011, China

2.Liuzhou Maternity and Child Healthcare Hospital, Liuzhou 545001, China

Keywords:

ABSTRACT Objective: To investigate the mechanism of the effect of Daodi Tongguan Decoction on ovarian function in rats with premature ovarian failure.Methods: Forty SD female rats with regular estrous cycle were randomly divided into the blank group, the model group, the positive group (Bujiale 0.099 mg/kg), the Daodigitongguan decoction high-dose group (1.4 g/100 g) and the Daodigitongguan decoction medium-dose group (0.7 g/100 g).The model group,positive group, Daodi Tongguan Decoction high-dose group and Daodi Tongguan decoction medium-dose group were given Tripterygium wilfordii polyglycoside tablets suspension(50 mg/kg) by continuous gavage for 14 days to establish a rat model of premature ovarian failure.After the modeling, each group was given the corresponding intervention agent for 28 consecutive days.After the intervention, serum and ovarian tissue were collected from rats in each group.The morphological structure of rat ovarian tissue was observed after HE staining.Serum AMH level was determined by ELISA.The expressions of Bcl-2, Bax and VEGF in ovarian tissue were detected by IHC.The apoptosis of granulosa cells in ovary was observed by TUNEL.Results: Compared to the normal group, the growth follicles in the model group were reduced and the atretic follicles were increased.Serum AMH level was significantly decreased (P<0.01).The expression of Bax protein in ovarian tissue was increased (P<0.01).The protein expressions of Bcl-2 and VEGF were decreased (P<0.01, P<0.05), and the apoptosis rate of follicular granulosa cells in ovarian tissue was increased (P<0.01).Compared to the molding group, the serum AMH level in the TCM dose groups was increased to varying degrees, and the change of the AMH in high-dose group was the most significant (P<0.05).The expression of Bax protein in ovarian tissue decreased (P<0.05), while the expression of Bcl-2 and VEGF protein increased (P<0.05, P<0.05).The apoptosis rate of follicular granulosa cells in ovarian tissue was decreased in the high dose group (P<0.05).Conclusion: Daodi Tongguan Decoction can improve the histological morphology of ovary and relieve ovarian injury, and its mechanism may be related to improving the sex hormone level of POF model rats, reducing the expression level of Bax protein in ovarian tissue, increasing the expression level of Bcl-2 and VEGF protein in ovarian tissue, and reducing the apoptosis rate of follicular granulosa cells in ovarian tissue.

1.Introduction

Premature ovarian failure is a disease characterized by amenorrhea, low estrogen levels, elevated gonadotropin levels,and lack of mature follicles in women prior to age 40[1].There is no clear cause of POF, and its influencing factors may include genetic factors, autoimmune disease factors, iatrogenic factors,viral infection factors, environmental factors, etc.[2].With the gradual intensification of social pressure, the incidence of female POF also increases year by year, about 1% of females are affected by this disease, and the incidence population tends to be younger and younger[3].At present, there are limited treatment methods,mainly hormone replacement therapy, which has obvious side effects and may increase the risk of breast cancer, stroke and gallbladder disease in women[4].Traditional Chinese medicine has great advantages in the treatment of this disease, and the side effects are less.Daodi Tongguan Decoction is the clinical prescription of Professor Luo Zhijuan, a famous Chinese medicine in Guangxi.It is composed of 10 after-taste traditional Chinese medicines, such as Astragalus, cassia branch, poria coia, Radix scrophulariae, panax notoginseng and Radix sanguinae.It has the effect of beneficial qi and invigorating spleen, clearing heat and dampness, promoting blood circulation and removing blood stasis.Previous studies[5] have confirmed that this formula can improve pelvic blood flow, reduce local microcirculation disorders, accelerate the repair of damaged tissues, and has obvious effect on tubal infertility.Moreover, this formula is treated by enema, which has no gastrointestinal reaction compared with oral administration.Damaged ovarian blood vessels will lead to poor ovarian blood circulation, resulting in impaired follicular development and ovulation, and increased follicular atresia,which may be one of the reasons for the occurrence of POF[6].On the basis of previous studies, this experiment explored the efficacy and mechanism of Daodi Tongguan Decoction in the treatment of premature ovarian failure, providing experimental data support for the follow-up development of this prescription.

2.Materials and methods

2.1 Material

2.1.1 Animal

SD rat, female, 7-8 weeks old, weighing 180-200 g, purchased from Hunan Slyke Jingda Experimental Animal Co., LTD.[Conformity number: 430727211100682662].The rats were raised in SPF animal laboratory of Guangxi University of Traditional Chinese Medicine.After adaptive feeding (7 d), the changes of the rats in two estrous cycles (10 d) were observed by smears of the cells shed from the vagina, and 40 rats with regular estrous cycles were screened for subsequent experiments.

1.1.2 Drug

Daodi Tongguan Decoction formula: soil poria, scrophulariae,astragalus, cassia twig, panax notoginseng, Radix sanguinae and other drugs, a total of 186 g, through water decoction concentrated to the raw drug volume of 3.5, 7.0 g/mL.Tripterygium wilfordii polyglycoside tablets (Hunan Qianjin Zhili Pharmaceutical Co.,LTD., Sinopharm approval number: Z43020138) Bujiale (Bayer Pharmaceutical Healthcare Co., LTD., Sinopharm approval number:J20171038) and above drugs are provided by the First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine.

2.1.3 Reagent

HE kit (Solebault Company, item No.G1120-100); FSH kit, E2 kit,AMH kit (item number: YT003287-96T, YT003296-96T, YT002952-96T); Bax primary antibody (item AF0120), Bcl-2 primary antibody(item AF6139) and VEGF primary antibody (item AF5131) were purchased from Affinity Biological Company.DAPI dyeing solution(Bude Company, AR1177); tunel Kit (Biyuntian Company, Red Light, article number C1089, Green light, article number C1086)

2.1.4 Instrument and equipment

High speed refrigerated centrifuge, Thermo, model FRESCO 21; ASP300S dehydrator, Leica Microsystems Limited; Model RM2255 Automatic rotary microtome, Leica Microsystem Co.,LTD., Microplate Incubator, Mio, China, model MT70-2; Enzyme label Instrument, Beijing Prang New Technology Co., LTD., Model DNM-9602; Optical microscope, OLYMPUS, Japan, model BX43;Imaging Systems, OLYMPUS, Japan; Model UC90.

2.2 Methods

2.2.1 Grouping and molding method

A total of 40 screened rats with normal estrous cycles were randomly divided into 5 groups (8 rats in each group): the blank group, the model group, the positive group, the Daodigitongguan decoction high-dose group and the Daodigitongguan decoction medium-dose group.Except the blank group, all the other groups were given tripterygium glycoside tablets at a dose of 50 mg/kg for 14 consecutive days to establish a rat model of premature ovarian failure [7].At the same time, from the fourth day of modeling,vaginal exudation smear was performed every day until the end of modeling.If the estrous cycle of rats is disorganized or prolonged(6 d) or stays in a certain period for a long time, modeling is considered successful[8].

2.2.2 Drug administration

After the successful establishment of the rat model of premature ovarian failure, the dosage was calculated according to the formula of converting the body surface area used by the previous research group[9], and the dosage of each treatment group was calculated and administered separately.The positive drug group was given 0.099 mg/kg by gavage, while Daodi Tongguan Decoction high-dose and medium-dose groups were given 1.4 g/100 g and 0.7 g/100 g by enema, respectively.Rats in blank group and model group were given normal saline 2 mL/d by enema for 28 d.

2.2.3 Draw materials

After the last administration, the rats were fasted, and isoflurane was given to the rats the next day, after which the rats were completely anesthetized, abdominal aorta blood was taken,serum was separated, and divided into EP tubes and stored in the refrigerator at -20 ℃ for future use.Both ovaries were separated and the excess tissue was stripped off.One ovarian tissue was placed in the refrigerator at -80 ℃ for reserve, and the other ovarian tissue was completely immersed in 4% paraformaldehyde solution to fix the reserve section.

2.2.4 Observation of ovarian histomorphology

The ovarian tissue was fixed completely, paraffin embedded, sliced,baked and stained by HE.The morphological structure of the ovarian tissue was observed under the electron microscope, and the follicles at all levels were counted.

2.2.5 Serum AMH level was detected

Serum AMH levels were determined by ELISA.The above operations are carried out in accordance with the reagent instructions.

2.2.6 Expression of Bax, Bcl-2 and VEGF protein in ovarian tissue

Paraffin samples of ovarian tissue were taken, sliced, sliced,baked, dewaxed, antigen repair, sealing, antibody incubation, DAB color development and hematoxylin restaining, and then observed under microscope after sealing.Image J image processing software was used to calculate the optical density of each image.Statistical analysis was made according to the percentage of the total area of positive cells in the field of visual field.

2.2.7 The apoptosis of ovarian granulosa cells was detected by TUNEL staining

Paraffin sections of ovarian tissue were taken and stained according to the instructions of the test kit.Apoptosis was observed after staining.Apoptosis rate =(number of apoptotic cells/total number of apoptotic cells)×100%.

2.2.8 Statistical processing

Statistical analysis was conducted by Graphpad Prism6.0 software.The measurement data were expressed as mean ± standard deviation(±s).P<0.05 was considered as statistically significant difference, and P<0.01 was considered as extremely significant difference.

3.Results

3.1 Changes of general condition and estrous cycle in rats

Compared with the rats in the blank group, the rats in the modeling period had dull fur color, ordinary appetite, slow movement, more sensitive to the surrounding things, preferring to shrink corners and showing strong stress.During the modeling period, vaginal smear observation showed that the estrous period of the rats in the modeling group was shortened or disappeared, and the interestrous period was prolonged.Most of the rats were in the preestrous and interestrous period.(See Figure 1 for the picture of vaginal shedding cells at different stages of the estrous cycle of rats).

Fig 1 Estrous cycle of rats(HE, ×200)

3.2 Changes of ovarian histomorphology induced by Daodi Tongguan Decoction in POF rats

Compared with blank group, the number of growing follicles and corpus luteum in ovary in model group was reduced, the number of atretic follicles was increased, the number of granulosa cell layers in growing follicles was reduced, and the arrangement was disordered,which showed nuclear contraction and reduced blood vessels in ovary.Compared with model group, the number of growing follicles and corpus luteum in ovary of rats in positive group, Daodi Tongguan decoction high-dose and medium-dose groups increased,the number of atretic follicles was less, the granulosa cells in the follicles were full, neatly arranged and well developed, and the blood supply in the ovary was richer.(See Figure 2).

3.3 Effect of Daodi Tongguan Decoction on serum AMH in POF rats

Compared with blank group, AMH values in all groups were significantly decreased (P<0.01).Compared with the model group,AMH in the treatment group was up-regulated to varying degrees,especially in the Daodi Tongguan decoction high-dose group(P<0.01), but there was no difference between the model group, the positive group and the Chinese medicine medium-dose group (P>0.05).(See Table 1).

Fig 2 Ovarian tissue morphology of rats in each group after administration(HE, ×200)

Tab 1 Effect of Daodi Tongguan Decoction on serum AMH of POF rats(n=8, ±s)

Tab 1 Effect of Daodi Tongguan Decoction on serum AMH of POF rats(n=8, ±s)

Note: Compared with blank group, **P<0.01; Compared with model group,##P<0.01.

Blank group AMH(pg/mL)Model group 4 261±513.4 Positive group 2 731±568.7**Daodi Tongguan Decoction high dose group 3 146±328.0**Daodi Tongguan Decoction medium dose group 3 582±483.5**##Blank group 3 023±344.2**F 27.04

3.4 Effect of Daodi Tongguan Decoction on expression of VEGF, Bax and bcl-2 protein in ovarian tissue of POF rats

Bax, Bcl-2 and VEGF were all expressed in ovarian granulosa cells,follicles and luteal cells.Compared with blank group, the expression of Bax protein in model group was increased (P<0.01), while the expression of Bcl-2 and VEGF protein was decreased (P<0.01,P<0.05).Compared with model group, the protein expression of Bax in positive group and Daoditongguan decoction high-dose group was decreased (P< 0.01, P<0.05), while the protein expressions of Bcl-2 and VEGF in Daoditongguan decoction high-dose group were increased (P<0.05, P<0.05).(See Figure 3 and Table 2).

3.5 Effect of Daodi Tongguan Decoction on apoptosis of ovarian granulosa cells in POF rats

Compared with blank group, the apoptosis level of follicular granulosa cells in model group was significantly increased (P<0.01).Compared with the model group, the granulosa cell apoptosis level of follicles in the high-dose and positive groups of Daodigitongguan decoction was decreased (P<0.05), while the apoptosis level of granulosa cells in the medium-dose group of Daodigitongguan decoction was decreased, but the difference was not statistically significant (P>0.05).(See Figure 4 and Table 3).

Fig 3 Effect of Daodi Tongguan Decoction on Bax, Bcl-2, VEGF protein in ovarian tissue of POF rats (IHC, ×200)

Tab 2 Effect of Daodi Tongguan Tang on the average integrated absorbance of VEGF, Bax, and Bcl-2 protein expression in ovarian tissue of POF rats(n=8, ±s)

Tab 2 Effect of Daodi Tongguan Tang on the average integrated absorbance of VEGF, Bax, and Bcl-2 protein expression in ovarian tissue of POF rats(n=8, ±s)

Note: Compared with blank group, *P<0.05, **P<0.01; Compared with model group, #P<0.05, ##P<0.01.

Group Bax protein Bcl-2 protein VEGF protein Blank group 0.052 93±0.007 044 0.203 2±0.051 51 0.214 7±0.065 69 Model group 0.494 1±0.026 22** 0.001 211±0.000 136 9** 0.044 37±0.000 479 7*Positive group 0.021 75±0.014 91## 0.087 92±0.024 83* 0.119 5±0.010 69 Daodi Tongguan Decoction high dose group 0.130 4±0.129 2# 0.144 7±0.009 818# 0.175 2±0.025 17#Daodi Tongguan Decoction medium dose group 0.215 7±0.090 06 0.0247 2±0.003 614** 0.084 14±0.009 147 F 13.93 20.65 9.098

Fig 4 Effect of Daodi Tongguan Decoction on apoptosis of ovarian granulosa cells in POF rats (tunel staining, ×200)

Tab 3 Comparison of apoptosis rate of ovarian granulosa cells of rats in each group(n=8, ±s)

Tab 3 Comparison of apoptosis rate of ovarian granulosa cells of rats in each group(n=8, ±s)

Note: Compared with blank group, *P<0.05, **P<0.01; Compared with model group, #P<0.05.

Group Apoptosis rate /%Blank group 3.6±1.140 Model group 54±16.32**Positive group 27.2±9.550*#Daodi Tongguan Decoction high dose group 31.4±13.13**#Daodi Tongguan Decoction medium dose group 38.8±10.66**

4.Discussion

At present, there are many methods to establish POF animal models, such as chemotherapy drug modeling, radiation damage modeling, autoimmune damage modeling, tripterygium glycoside tablets modeling, etc[10].In this experiment, tripterygium wilfordii polyglycoside tablet modeling method, which is widely used in China, was selected in this study.This method is simple and has high repeatability, and the induced model can highly simulate the disease state of women with premature ovarian failure caused by tripterygium polyglycoside tablet treatment in clinic[11].In this experiment, the level of AMH was generally decreased in rats after the intervention of tripterygium wilfordii polyglycoside tablets.Unlike FSH and E2, which fluctuate greatly with the menstrual cycle, AMH has little variability with the menstrual cycle[12].At present, among the methods commonly used to detect ovarian reserve function, such as sex hormone detection, sinus follicle count,cytokine and ovarian volume measurement, some studies have shown that AMH may be one of the most accurate detection methods[13], so AMH was selected as the evaluation of sex hormone level in POF model rats in this experiment.Based on the estrous cycle of rats, morphological changes of ovaries and changes in AMH level, it can be concluded that the establishment of POF model is successful.In ancient Chinese medicine books, there is no such disease as premature ovarian failure, which can be classified into TCM diseases such as “blood dry”, “premature water break”, “infertility” and“dirty dryness” according to clinical symptoms of POF[14].Women are based on blood, Qi and blood are full, and kidney essence is sufficient, which can moisten Chong Ren and nourish cell palace.Otherwise, Qi and blood deficiency cannot nourish kidney essence,and the woman will be exhausted, have tunnel obstruction, have bad shape and no children, leading to the occurrence of POF[15].The main movement of the spleen is the acquired origin and the source of Qi and blood biochemistry.If the spleen is not healthy and the movement is abnormal, the menstrual blood is not generated, the cell palace is not nourished, the spleen is not healthy and the blood is easy to be wet and endogenous, resulting in the movement of the cell palace Qi and blood is not smooth, resulting in amenorrhea,infertility and other diseases.In most areas of Lingnan, the climate is humid and hot all year long, and dampness is easy to trap the spleen, so the residents in this area suffer from frequent diseases of the spleen and stomach[16].According to the guiding principle of “all diseases are caused by the failure of the spleen and stomach” and the simultaneous treatment of different diseases, doctors in Lingnan often use drugs with the effects of clearing heat and dampness,invigorating qi and invigorating the spleen in the treatment of many diseases, so as to soothe the spleen and stomach and soothe the body.Inspired by the above academic thoughts and combined with the previous research results, we carried out a study on the feasibility of Daodi Tongguan Decoction in the treatment of POF.Daodi Tongguan Decoction is a gynecological clinical prescription created by Lingnan physician Professor Luo Zhijuan.Most of the medicinal materials of the whole prescription are from Lingnan area.The whole prescription has the effect of beneficial qi and spleen, clearing heat and dampness, promoting blood circulation and removing blood stasis.Prescription with astragalus, cassia branch Qi and spleen,in view of Lingnan climate is more hot and humid, spleen easy for dampness, with soil poria, Qianjin pull and other drugs clearing heat and dampness, and to POF disease course is longer, traditional Chinese medicine has “long illness will be blood stasis” said, then combined with notoginseng, big blood vine to promote blood stasis,so as to achieve the effect of strengthening the spleen and stomach,clearing heat and dampness, blood stasis.“The spleen and stomach and the blood is born” “the woman’s emphasis on blood, blood can construct essence, fetal pregnancy is into”, normal function of the spleen and stomach, the way to get Qi and blood filled, women can have normal menstruation, fetal pregnancy.

Previous experiments have confirmed the correlation between VEGF, Bax and Bcl-2 proteins and the occurrence of POF.VEGF is involved in the generation of follicular membrane vessels[17],which are the channels through which follicles absorb nutrients and hormones needed for their growth from the blood[18].Wu Jianfei[19]et al.found that a certain concentration of exogenous VEGF could improve the vitality of granulosic cells and reduce the apoptosis rate.Both Bax and Bcl-2 were expressed in ovarian tissue, and high Bax and/or low Bcl-2 expression and increased ratio of both were detected in atretic follicles[20].Many experiments have confirmed that decreased VEGF protein expression in ovary of POF model rats,increased VEGF protein expression, may promote the expression of Bcl-2 protein, reduce the expression of Bax protein, and reduce the occurrence of atretic follicles[21,22].In this experiment, compared with the model group, the pathological morphology of the ovarian tissue of rats in the TCM treatment group was improved to varying degrees, and the serum AMH value was increased to varying degrees, and there was a certain positive correlation with the concentration of the drug used.According to the above results, we speculated that Daodi Tongguan Decoction can regulate hormone levels, improve internal environment and relieve ovarian injury.Compared with blank group, the expression of Bax protein in ovarian tissue and granulosa cell apoptosis rate in follicle of rats in model group increased, while the expression of VEGF and Bcl-2 protein decreased.Except for special pathological conditions such as wound healing and tumor, most organs will not actively reshape the vascular system.However, ovary is special, and its vascular system will undergo changes in angiogenesis and increased vascular permeability along with the changes of ovulation cycle[23].VEGF plays an important role in the construction of new blood vessels,and the number of new blood vessels determines the nutritional environment of tissues[24].The decreased expression of VEGF suggested that tripterygium glycosides might block the formation of some new blood vessels in the ovary, reduce the nutrient supply channels in the ovary, prematurely abort the development of follicles, and force the increase of atretic follicles, leading to the occurrence of POF.Secondly, decreased expression of VEGF may break the regulatory balance between Bax and Bcl-2, initiate the proapoptotic mechanism, and further aggravate the occurrence of atretic follicles.According to the results of this experiment, compared with the model group, the expression of VEGF and Bcl-2 increased and the expression of Bax decreased in the Daodi Tongguan decoction treatment group, indicating that Daodi Tongguan Decoction can promote ovarian angiogenesis, improve the nutritional environment of the ovary and inhibit the expression of pro-apoptotic protein.

In summary, the results of this study indicate that Daodi Tongguan Decoction can improve the serum hormone levels and ovarian tissue structure of POF rats, reduce the apoptosis of ovarian granulosa cells,and slow down the process of POF.The specific mechanism may be related to regulating the expression of VEGF, Bcl-2 and Bax proteins in ovarian tissue of POF rats, promoting the repair of damaged ovarian tissue and reducing the apoptosis of ovarian granulosa cells.This study provides a new idea for the treatment of POF, and also provides theoretical support for expanding the application of Daodi Tongguan Decoction.

Author’s contribution:

Long Yun-xia: experimental design and operation, data analysis,paper writing; Luo Zhi-juan, Zhong Yi-hui, Wu Yuan-yuan:Designed the experiment and gave guidance to the paper; Chen Qiujin, Du Li, Zheng Hong, Li Yin-ying: Participated in the collection of experimental data.

All authors declare no conflict of interest.