Effect of Astragalus-hawthorn on ovarian reproductive function and inflammatory mechanism of action in rats with polycystic ovary syndrome

TIAN Xiu-xiu, LI Mei-lin, GONG Yu-hang, MA Xi-ya, ZHANG Xue-juan

1.Shanxi University of Traditional Chinese Medicine, Taiyuan 030024, China

2.School of Public Health, Shanxi Medical University, Taiyuan 030000, China

3.Shanxi Children's Hospital, Taiyuan 030013, China

Keywords:

ABSTRACT Objective: To investigate the effects of prophylactic administration of Astragalus-Hawthorn on ovarian reproductive function and inflammatory mechanism in rats with polycystic ovary syndrome (PCOS).Methods: Fifty 21-day-old female rats were randomly divided into five groups of 10 rats each: Normal group, Model group, Astragalus group, Hawthorn group and Astragalus-Hawthorn group.All groups, except the normal group, were fed a high-fat model diet.Each treatment group received astragalus, hawthorn and an astragalus-hawthorn solution orally during the molding period for an intervention period of 15 weeks.The estrous cycle of the rats in each group was observed under the microscope from week 8, the interference rate was calculated, changes in ovarian tissues were observed by HE staining and the levels of sex hormones, proinflammatory and anti-inflammatory factors in the serum of the rats were measured by ELISA.Results: Rate of estrous cycle disorders were more frequent in the model group than in the normal group.(P＜0.01); ovarian tissue was polycystic, with few corpora lutea and sinusoids and numerous follicular ovarian cysts ; Luteinising hormone/follicle-stimulating hormone (LH/FSH), testosterone (T), interleukin 6 (IL-6), interleukin 17A(IL-17A) and tumour necrosis factor (TNF-α) levels were significantly higher in the model group than in the normal group; interleukin 10 (IL-10) was significantly lower than in the normal group (P＜0.05).Compared to the model group, rats in the Astragalus and hawthorn groups had a lower rate of estrous cycle disorders (P＜0.01); the number of cystic follicles in the ovarian tissue decreased; the number of mature follicles and corpus luteum increased; LH/FSH, T, IL-6 and IL-17A levels were significantly lower in the Astragalus group, hawthorn group and the Astragalus and hawthorn groups; TNF-α levels were significantly lower in the astragalus and hawthorn groups; IL-10 levels were significantly higher in the hawthorn and astragalus hawthorn groups (P＜0.05).IL-10 levels were significantly higher in the astragalus and hawthorn groups (P＜0.05).Compared to the astragalus group, T levels were significantly lower in the astragalus and hawthorn groups and IL-10 levels were significantly higher in the astragalus and hawthorn groups (P＜0.05).Compared to the hawthorn group, levels of T and IL-17A were significantly lower in the astragalus-hawthorn group (P＜0.05).Conclusion:Astragalus-Hawthorn prophylactic administration can improve ovarian reproductive function in rats with PCOS, and the mechanism of action may be related to reducing the levels of proinflammatory factors IL-6, IL-17A and TNF-α, increasing the levels of anti-inflammatory factor IL-10, and reducing the inflammatory state.

1.Introduction

Polycystic ovary syndrome (PCOS) is a common metabolic endocrine disorder and reproductive disorder disease in women,currently showing a popular trend, the incidence of about 6%,with excessive androgens, irregular ovulation and polycystic ovaries as the main manifestations, but also manifested as type 2 diabetes, insulin resistance and cardiovascular disease and a series of metabolic complications[1,2,3].The pathogenesis of the disease has not been fully elucidated, and the onset time is as early as puberty, or even accompanied by life, and there is currently no cure,which seriously affects the physical and mental health of women of childbearing age[4].

Modern studies have shown that PCOS patients have ovulation dysfunction, with low pregnancy rate and high abortion rate, which has become the most important cause of infertility in women of reproductive age.Early intervention and treatment are of great significance to improve the reproductive function of PCOS patients.At present, traditional Chinese medicine has unique advantages in preventing and treating the occurrence and development of PCOS diseases[5].Astragalus originated in Shennong Herbal Classics.Modern pharmacological studies have found that quercetin, the active ingredient in Astragalus, can improve the inflammatory microenvironment[6].Animal experimental studies have shown that Astragalus can play a regulatory role in the expression of antioxidant genes in ovarian tissue, and further effectively improve the reproductive ability of aging ovarian mice[7].The main components of traditional Chinese medicine hawthorn are flavonoids, organic acids, etc., which have a variety of pharmacological effects such as anti-inflammatory, hypoglycemic and lipid-lowering[8].Therefore, this experiment intends to establish a PCOS rat model through high-fat feed feeding, and explore the effect of astragalushawthorn preventive administration on the reproductive function and inflammatory mechanism of ovary in PCOS rats, so as to provide an experimental reference for the clinical drug prevention and treatment of PCOS.

2.Materials and methods

2.1 Materials

2.1.1 Animals

Fifty 21-day-old SPF-level SD rats, females, with laboratory animal production certificate No.: SCXK (Beijing) 2019-0010, were provided by Spaf (Beijing) Biotechnology Co., LTD., and raised in the Experimental Center of Shanxi Academy of Traditional Chinese Medicine.The indoor temperature of the experimental animal room was kept at (28±1.5) C, the indoor humidity was kept at (40±5) %,the indoor environment was ventilated, the light and dark alternated between day and night (12-12 h), the diet and drinking were free,and the experiment was started after 1 week of adaptive feeding.The experimental process follows animal ethics, and the animal experiment ethics approval number is NO:201911048.

2.1.2 Animal Feeds

The energy components of ordinary animal feed were 12.11% fat,22.47% protein and 65.42% carbon water, which were derived from Huafukang rat and mouse maintenance feed (1022).The energy composition of animal high-fat feed is 60% fat, 20% protein, and 20% carbon water, which comes from Parker Biology (M10160).

2.1.3 Drugs

Astragalus formula granules (specification 1 g=10 g tablets,drug batch number: 2006003C, Sanjiu Pharmaceutical Co., Ltd.),hawthorn formula granules (specification 1 g=6.7 g tablets, drug batch number: 1906002s, Sanjiu Pharmaceutical Co., Ltd.).

2.1.4 Reagents and instruments

LH, FSH, T, TNF-α, IL-6, IL-17A, IL-10 ELISA kit (batch number:ML064293, ML059034, ML024321, M1022281,M1037365, M1003298, M1002894), Shanghai Enzyme Linked Biotechnology Co., Ltd.; Chloral hydrate solution, methylene Blue, 0.9% normal saline (batch number: 10003460, 100023444,10002734), Sinopsin Chemical Reagent Co., Ltd.; HE Dyeing Set(Lot number: G1003), Servicebio; Enzyme labeling instrument(RT-6100, 450nm wavelength), Rayto; Slide (G6004), Servicebio;Pathological Microtome (RM2016), Shanghai Leica Instrument Co.,Ltd.;Table high speed refrigerated centrifuge (Model: TGL16M),Shanghai Luxiang Instrument Co., Ltd.;Dehydrator (Donatello),DIAPATH; Electron microscope, Nikon, Japan.

2.2 Methods

2.2.1 Grouping, modeling and administration of animals

Fifty 21-day-old SPF-level SD rats, female, were selected and grouped into groups of 10 according to the random number table method, and divided into 5 groups, which were Normal group,Model group, Astragalus group, Hawthorn group and Astragalus-Hawthorn group.The rats in normal group were fed ordinary animal feed, and the rats in model group and each drug administration group were fed high-fat animal feed.The high-fat induced PCOS rat model was established for 15 weeks.During the experiment,normal group and model group were given distilled water, and the solutions of Astragalus, Hawthorn and Astragalus-Hawthorn in each administration group were prepared according to the concentration of astragalus 0.6 g/mL, hawthorn 0.3 g/mL, and astragalus-hawthorn(3:1) 0.6 mL/g, respectively.The rats in each administration group were given interventional administration by gavage according to 0.3 g/kg of astragalus and 0.97 g/kg of hawthorn (calculated by referring to the body surface area of rats and adult females and the results of pre-experimental efficacy).Elevated levels of androgens in the serum of rats and disturbance of anovulation in the estrous cycle were taken as indicators of successful modeling of PCOS model rats[9].

2.2.2 Estrous cycle of rats in each group

Starting from the 8th week of modelling, vaginal smears were taken at 8:00 every day for three consecutive estrous cycles in each group of rats.It was confirmed that the duration of a complete estrous cycle(pre-estrus, estrus, post-estrus and inter-estrus) in normal rats was roughly 4-5 d[10].If the duration is 6 d, the cycle is disorganised,or there is a prolonged stay of a certain period, the rat is considered to have a disturbed estrous cycle[11].The estrous cycle duration of each rat in each group was recorded and summarized, and the estrous cycle disturbance rate of rats in each group was calculated according to the number of rats in each group (only)/the total number of rats in each group (only) ×100%.

2.2.3 Sample collection

After 15 weeks, the rats in each group were fasted for 12 h,administered peritoneal anesthesia with 10% chloral hydrate solution,and blood was taken from the inferior vena cava.The extracted serum samples were placed for 2 h, then placed in a centrifuge at 3 000 r/min, centrifuged for 15 min, and the upper serum was taken.The rats were killed immediately, the adipose tissue was dissected quickly,and one side of the ovarian tissue was separated and then fixed in 4% paraformaldehyde solution, and stored at -80 ℃ for subsequent observation.

2.2.4 Ovarian tissue of rats in each group

The left ovary placed in 4% paraformaldehyde solution was fixed, and the paraffin-embedded ovarian tissue was processed by sectioning using a microtome, air-dried vertically, deparaffinized,rehydrated, stained with eosin hematoxylin ( HE) and sealed, and photographed and observed under a microscope on the rat ovarian tissue slides.

2.2.5 Determination of serum sex hormone related indexes of rats in each group

Serum samples taken from the inferior vena cava were assayed with an ELISA kit to determine the levels of FSH, LH, and T in the serum of each group of rats, and the ratio of LH/FSH was calculated.

2.2.6 Determination of serum inflammatory factors related indexes in each group

Serum samples taken from the inferior vena cava were assayed with ELISA kits to determine the levels of pro-inflammatory cytokines IL-6, IL-17A, TNF-α and anti-inflammatory cytokine IL-10 in the serum of each group of rats.

2.2.7 Statistical methods

The data were statistically analyzed using the software SPSS 25.0.Percentage (%) was used to express count data, and comparisons between groups were made using the χ2test, and Fisher’s exact probability method was used if more than 20 % of T ＜ 5 or at least 1 T ＜ 1.Mean ± standard deviation indicated the measurement data,and comparisons between multiple groups were performed using ANOVA one-way analysis of variance, and comparisons between two were performed using the LSD test.P ＜0.05 was considered to be statistically significant.

3 Results

3.1 Changes of estrous cycle of rats in each group

Compared with the normal group, the proportion of rats with estrous cycle disturbance in the model group was significantly increased (P ＜ 0.01).Compared with model group, the proportion of estrous cycle disturbance in Astragalus and Hawthorn group was significantly decreased (P ＜ 0.01).There was no significant difference between Astragalus group and Hawthorn group and Astragalus-Hawthorn group (P ＞ 0.05).(Table 1)

Tab 1 Disturbance rate of estrous cycle of rats in each group (n=10)

3.2 Morphological changes of ovarian tissue in each group

The cross-section of the ovaries of rats in the normal group showed regular histological morphology, dense granule cell layers,abundant corpus luteum and healthy follicles, and a small number of atresia and cystic follicles.A smaller number of corpus luteum and sinus follicles with many follicular ovarian cysts were observed in the model group, with pronounced cystic changes in the ovaries.Compared with the model group, rat follicles in each administration group were well developed, abundant, the number of granule cell layers increased, the number of cystic follicles decreased, the number of mature follicles increased and the number of corpus luteum increased.Among them, rats in the astragalus-hawthorn group showed that the number of cystic follicles in ovarian tissue decreased significantly, the number of mature follicles and the number of corpus luteum increased significantly, and the improvement effect of cysticness of rat ovarian tissue in astragalus group and hawthorn group was slightly worse than that in the combined administration group of astragalus hawthorn.( Fig.1)

3.3 Changes of serum sex hormones LH/FSH and T levels in rats in each group

Compared with the normal group, the LH/FSH and T levels of rats in the model group were significantly increased (P＜0.05).Compared with the model group, the LH/FSH and T levels of rats in Astragalus group, hawthorn group and Astragalus-hawthorn group were significantly reduced (P＜0.05).Compared with Astragalus and Hawthorn groups, the T level was significantly reduced (P＜0.05)in the Astragalus hawthorn group, and there was no significant difference in LH/FSH levels between Astragalus and Hawthorn and Astragalus-Hawthorn (P＞0.05).(Table 2)

Fig 1 Ovarian tissue morphology of rats in each group (HE×40)

Tab 2 Changes in the levels of sex hormones LH/FSH and T of rats in each group(±s, n=10)

Tab 2 Changes in the levels of sex hormones LH/FSH and T of rats in each group(±s, n=10)

*P＜0.05,when compared with the normal group.#P ＜ 0.05,when compared with the model group.ΔP＜0.05 ,when compared with the astragalus group.▲P＜0.05,when compared with the hawthorn group.

Group T(ng/dL) LH/FSH Normal group 16.11±0.74 0.77±0.03 Model group 18.45±0.35* 2.02±0.09*Astragalus group 14.21±0.27# 1.25±0.16#Hawthorn group 15.05±1.36# 1.41±0.07#Astragalus-Hawthorn group 12.3±0.39#Δ▲ 1.15±0.16#F 7.225 11.256 P 0.003 ＜0.001

3.4 Serum levels of IL-6, IL-17A and TNF-α in rats in each group

Compared with the normal group, the levels of IL-6, IL-17A and TNF-α in rats in the model group were significantly increased(P＜0.05).Compared with the model group, the levels of IL-6 and IL-17A in the Astragalus group, hawthorn group and Astragalushawthorn group were significantly reduced (P＜0.05), and the levels of TNF-α in rats in astragalus group and astragalus-hawthorn group were significantly reduced (P＜0.05).Compared with the astragalus group, there were no significant differences in the levels of IL-6, IL-17A and TNF-α in the hawthorn group and the hawthorn group(P＞0.05).Compared with the hawthorn group, the level of IL-17A in rats in the astragalus-hawthorn group was significantly reduced(P＜0.05).(Table 3)

Tab 3 Changes of serum levels of IL-6, IL-17A and TNF-α in each group(±s,n=10)

Tab 3 Changes of serum levels of IL-6, IL-17A and TNF-α in each group(±s,n=10)

*P＜0.05,when compared with the normal group.#P ＜ 0.05,when compared with the model group.▲P＜0.05,when compared with the hawthorn group.

Group IL-6(pg/mL) IL-17A(pg/mL) TNF- (pg/mL)Normal group 82.03±7.83 13.67±2.85 137.51±21.95 Model group 116.84±9.78* 27.21±0.85* 206.56±26.58*Astragalus group 94.43±11.40# 18.71±1.69# 172.73±17.46#Hawthorn group 102.33±9.63# 19.79±0.53# 179.07±27.23 Astragalus-Hawthorn group 92.27±7.98# 16.39±2.07#▲153.90±22.97#F 11.38 23.707 7.438 P 0.001 ＜0.001 ＜0.001

3.5 Serum IL-10 levels varied in rats in each group

The IL-10 level in rats in the model group was significantly reduced compared to the normal group (P＜0.05).The level of IL-10 in rats in hawthorn group and astragalus-hawthorn group was significantly higher compared with the model group (P＜0.05).Compared with the astragalus group, the IL-10 of rats in the astragalus-hawthorn group was significantly elevated (P＜0.05).Compared with the hawthorn group, there was no significant difference in IL-10 between the astragalus group and the hawthorn group (P＞0.05).(Table 4)

Tab 4 Changes of serum IL-10 level in each group (±s, n=10)

Tab 4 Changes of serum IL-10 level in each group (±s, n=10)

*p＜0.05,when compared with the normal group.#p ＜ 0.05,when compared with the model group.Δp＜0.05 ,when compared with the astragalus group.

Group IL-10(pg/mL)Normal group 53.77±2.07 Model group 31.45±3.72*Astragalus group 41.64±41.64 Hawthorn group 43.96±43.96#Astragalus-Hawthorn group 47.87±3.01#Δ F 23.442 P＜0.001

4.Discussion

The name of PCOS does not appear in TCM medical texts, but it is similar to the clinical manifestations of diseases such as “infertility”and “oligomenorrhea”, The occurrence of PCOS disease is attributed to the dysfunction of the reproductive axis of “kidney-tianxi-chong ren-uterus”, It is believed that the etiology of the disease is based on the false standard, mainly kidney deficiency, spleen deficiency and internal organ deficiency, mixed with sputum dampness, blood stasis and other solid pathological products[12].Fat people have an unruly diet, like to eat fat and thick and greasy products, damage the temper for a long time, qi deficiency and phlegm, just like the cloud of“Medical Realm”: “Fat people are wet, their phlegm is easy to form,and there are also people who stagnate blood due to wetness”.The“Fetal Birth Guide” records that “those who hold phlegm, sputum and saliva coagulate,......for menstrual amenorrhea, for childless disease”.The spleen is the innate foundation, which can transport water and moisture, and if the temper is deficient, it is unfavorable,wet and aggregated into phlegm, phlegm is not transported, the flow of qi and blood is blocked, hindered in the chongren and uterus,affecting the metaplasia and nourishment of kidney essence, and then leading to water disorders, it is difficult to fertilize and conceive[13].Therefore, in this study, the traditional Chinese medicine astragalus and hawthorn were selected for preventive administration, and PCOS was treated by exerting the effect of strengthening the spleen and invigorating qi, breaking stasis and dissolving phlegm, reflecting the idea of “Prevention and treatment of disease”.Traditional Chinese medicine believes that the spleen is healthy by rising, and the application of the Chinese medicine astragalus is sweet and warm,which is good at entering the spleen and lungs, replenishing the qi of the spleen and lungs, and nourishing the blood and stagnating[14].The traditional Chinese medicine hawthorn acid is sweet and warm,awakens the spleen and eliminates food, breaks stasis and dissolves phlegm[15].The combined application of the two drugs gives full play to the effect of tonifying and purging, and achieves the function of treating both symptoms and root causes.

Polycystic ovary syndrome is a common endocrine metabolic disease in women of childbearing age, and it is also a reproductive disorder disease, which seriously affects women's reproductive health and is a frontier hot spot in the current research on ovarian reproductive function[16].With the in-depth study of PCOS disease, more and more scholars realize that PCOS is in a lowgrade chronic inflammatory state, and inflammatory cytokines interact with each other, which plays an important role in managing ovarian reproductive function[17].The presence of highly expressed inflammatory factors in PCOS, which regulate the follicular and luteal phases of normal ovarian activity, aggravate hyperandrogenemia and accelerate the apoptosis of follicles[5,18].A large number of studies have shown that abnormal expression of inflammatory cytokines, resulting in immune dysfunction or dysregulation of immune-related factors, can lead to the occurrence of PCOS, which can directly affect follicle development or ovulation disorders, leading to low reproductive function and even infertility[19].Therefore, this experiment explored the effect on the reproductive function of the ovaries of PCOS rats by measuring the changes of inflammation-related factors in rats.

In this experiment, the number of rats with estrous cycle disorder in the model group increased, the disorder rate increased significantly,the ovarian tissue underwent cystic changes, a small number of corpus luteum and sinus follicles with many follicular ovarian cysts were visible under microscopy, and the levels of sex hormones LH/FSH and T increased significantly, indicating that rats with high-lipid-induced PCOS did have ovarian reproductive function disorders.It is consistent with the research conclusion concluded by Jing Yanlin et al.[20] that female reproductive rats have follicle cyst development under the induction of high-fat diet, which interferes with ovulation and then affects the reproductive function of rats.After interventional gavage administration of rats with astragalus, hawthorn and astragalus-hawthorn aqueous solution, it was found that the number of rats with estrus cycle disorder in each administration group decreased, the disorder rate was significantly reduced, the number of cystic follicles in ovarian tissue decreased,the number of mature follicles and corpus luteum increased, and the levels of sex hormones LH/FSH and T were significantly reduced,among which the number of mature follicles and corpus luteum in the astragalus-hawthorn group increased more obviously, and the T level decreased more significantly than that in the dosing group alone (P＜0.05).It was suggested that preventive administration of astragalus and hawthorn could improve the ovarian reproductive function of PCOS rats, but the improvement effect of astragalus and hawthorn combined was more prominent.

Many studies have shown a strong link between PCOS and ovarian reproductive function and chronic low-grade inflammation throughout the body[21].In this experiment, the levels of proinflammatory factors IL-6, IL-17A and TNF-α in PCOS model rats increased significantly, and the levels of anti-inflammatory factor IL-10 were significantly reduced (P＜0.05), indicating that the PCOS model rats had abnormal expression of inflammatory factors.The ovarian tissue of PCOS patients is exposed to inflammatory infiltration for a long time, and its reproductive capacity is reduced,which is caused by the elevated level of IL-6 in serum, which activates inflammatory cells and releases more inflammatory factors[22].Yan Dongmei[23] also confirmed that PCOS patients showed ovarian hyporeactivity, decreased function, and poor ovulation promotion effect, which may be related to the high expression of pro-inflammatory factor IL-17A.The regulation of ovarian function is inseparable from the participation of TNF-α, which is related to FSH concentration and increased T level, which can induce the proliferation of follicular membrane cells and regulate follicular development, ovulation and xanthinization[24].In PCOS,the decreased level of IL-10, an anti-inflammatory factor involved in inflammation and immunosuppression, can directly stimulate the production of inflammatory factors, enhance the activity of inflammatory cells, and exacerbate the inflammatory state of ovarian reproductive function.After interventional administration of Astragalus, Hawthorn, and Astragalus hawthorn aqueous solutions,the levels of IL-6, IL-17A and TNF-αwere decreased in different degrees, and IL-10 was increased in different degrees compared with the model group.The results of this study showed that the level of IL-17A in the astragalus-hawthorn group was more significantly lower than that in the hawthorn group, and the level of IL-10 was more significant than that in the hawthorn drug group (P＜0.05), indicating that astragalus-hawthorn has anti-inflammatory effects, and also indicates that astragalus-hawthorn can exert a protective effect on the reproductive function of the ovaries of PCOS rats by reducing inflammation.

In summary, the preventive administration of astragalus-hawthorn can improve the ovarian reproductive function of PCOS rats, and its mechanism of action may be related to reducing the levels of proinflammatory factors IL-6, IL-17A, TNF-α, increasing the level of anti-inflammatory factors IL-10, and reducing inflammatory states.This study provides guidance and experimental basis for the clinical use of PCOS, but the treatment of diseases by traditional Chinese medicine has the characteristics of multiple pathways-targets at a deeper molecular level, and the mechanism of action of PCOS ovarian reproductive function can be further explored.

Authors' contribution

Zhang Xuejuan: Experimental design; Tian Xiuxiu: experiment execution, statistical analysis and document writing; Li Meilin, Gong Yuhang: Experiment and index detection; Mahia: Some additional information; Zhang Xuejuan: Thesis guidance and proofreading.

All authors declare no conflict of interest