WANG Shengchun,JIANG Yiming,QIU Lirong,SU Meng

WANG Shengchun,Department of Acupuncture Physiotherapy Rehabilitation,Shandong Provincial Hospital Affiliated to Shandong First Medical University,Jinan 250021,China

JIANG Yiming,QIU Lirong,SU Meng,Specialty of acupuncture and massage,Traditional Chinese Medicine of Shandong University,Jinan 250014,China

Abstract OBJECTIVE:To investigate the efficacy of needling acupoins of Guanyuan (CV4),Sanyinjiao (SP6),Zusanli (ST36),Pishu (BL20),Shenshu (BL23),Zigong(EX-CA1)on the expression of p38 mitogen-activated protein kinase (p38MAPK) in ovarian tissue in rats with premature ovarian failure induced by cyclophosphamide,and to study the underlying mechanism.METHODS:Forty specific pathogen free female Sprague-Dawley rats were randomly divided into the blank group,the model group,the acupuncture group,the Western Medicine group and the Western Medicine combined with acupuncture group.Except the blank group,the rest of the rats were given with cyclophosphamide for 14 d to establish premature ovarian failure model.No intervention was conducted in the blank group and model group;the acupuncture group was given with acupuncture daily;the Western Medicine group was given with estradiol valerate (0.09 mg/kg) by intragastrical gavage daily;the combination group was given with acupuncture combined with estradiol valerate (0.09 mg/kg) daily.Each group was intervened in continuously for 14 d.After the last treatment,the levels of estradiol (E2),follicle stimulating hormone (FSH) and luteinizing hormone(LH)were detected by enzyme-linked immunosorbent assay,then the ovarian tissue was dissected.Western blot was used to detect the expression of p38MAPK protein.RESULTS:Compared with the blank group,E2 in the serum of rats in the model group was significantly decreased(P<0.05),FSH and LH were significantly increased (P <0.05),and the expression of p38MAPK protein in the ovarian tissue of the rats was significantly increased (P <0.05).Compared with the model group,E2 in the serum of the acupuncture group,Western Medicine group and the combination group were significantly increased(P<0.05),FSH and LH levels were significantly decreased(P<0.05),and the expression of p38MAPK protein in the ovarian tissue of the rats was significantly decreased(P<0.05).However,there was no statistically significant difference between the Western Medicine group and the acupuncture group(P>0.05).CONCLUSIONS:Acupuncture has the same effect as estrogen in interfering POF caused by cyclophosphamide,and its mechanism may be related to inhibiting the expression of p38MAPK protein in ovarian tissue and affecting the activation of p38MAPK signaling pathway.

Keywords:acupuncture;premature ovarian insufficiency;p38 mitogen-activated protein kinases;signal transduction

INTRODUCTION

Premature ovarian failure1(POF)refers to the phenomenon that women's ovary function decline or even fail before the age of 40.The symptoms are secondary amenorrhea,infertility,accompanied by a series of symptoms of low estrogen,such as menstrual disorders,hot flashes and night sweats,insomnia,forgetfulness,osteoporosis and other perimenopausal symptoms.2As a common and incurable disease in gynecology,POF's incidence has reached 1%-3% in China.3In recent years,premature ovarian failure has gradually showed a younger trend,which has seriously decreased patients' quality of life.4As the main embodiment of ovarian function,ovarian granulosa cells can directly lead to a decline in ovarian reserve function,which caused premature ovarian failure.The p38MAPK signal transduction pathway belongs to the mitogen activated protein kinase (MAPK) signal transduction pathway family,which mainly participates in signal transmission of inflammation,stress and injury,and P38 is involved in the development of granulosa cells at various stages and plays an important role in the regulation of inflammatory response and apoptosis.5-7Early clinical studies have improved that acupuncture is effective in treating patients with premature ovarian failure.Therefore,this study was aimed to investigate the efficacy of needling acupoints of Guanyuan (CV4),Sanyinjiao (SP6),Zusanli (ST36),Pishu (BL20),Shenshu(BL23),Zigong (EX-CA1) on the expression of p38MAPK in ovarian tissue in rats with premature ovarian failure induced by cyclophosphamide,and to study the underlying mechanism.

MATERIALS AND METHODS

Animals

Forty specific pathogen free 8-week-old unmated female Sprague-Dawley (SD) rats weight (198 ± 22) g were used.All experimental animals were purchased from the Experimental Animal Center of Shandong Provincial Hospital.The animal certificate number was SCXK (Lu) 2019-0001.During the experiment,they were all kept in Shandong Provincial Experimental Animal Center.The breeding environment:under the conditions of room temperature 20-25 ℃,humidity 50%-60%,rats could eat and drink freely and were kept in single cage.

Grouping

Forty female SD rats were randomly divided into the blank group,the model group,the acupuncture group,the Western Medicine group and the combination group,with 8 rats in each group.Rats in all groups were conventionally fed for 3 d to adapt to the environment.

Chemicals

Cyclophosphamide (Hengrui Pharmaceutical Company Limited,Jiangsu,China H32020857),estradiol valerate (Shengdi Pharmaceutical Company Limited,Jiangsu,China),rabbit anti-rat phosphorylated P38 monoclonal antibody (Abcam Company,Cambridge,UK) and corresponding secondary antibody (Boster Company,Wuhan,China),BCA protein concentration determination kit(Biyuntian Biotechnology Company Limited,Shanghai,China),β-Actin internal reference antibody (Shanghai Shengong,Shanghai,China),rat luteinizing hormone (LH),rat estradiol (E2),rat follicle-stimulating hormone (FSH),and enzyme-linked immunosorbent assay (ELISA kits) (Elerat Biotechnology Company,Beijing,China).Disposable sterile acupuncture needles (Huatuo brand 0.25 mm ×25 mm,Suzhou medical supplies Company Limited),fluorescent microscope (Olympus,Tokyo,Japan),electrophoresis apparatus and electrophoresis tank(bio-rad,CA,USA),4 ℃ cryogenic centrifuge(5702REppendorf,Hamburg,,Germany).

Rat model of premature ovarian failure

In this study,POF rat model was established by intraperitoneal injection of cyclophosphamide (CTX) according to the method used by Fu Xiafei and He Yuli.8,9The model group,the acupuncture group,the Western Medicine group and the combination group were all injected with cyclophosphamide intraperitoneally to make chemotherapy-induced premature ovarian failure rats.The dose of cyclophosphamide was calculated according to the weight of the rats.The first dose of 50 mg/kg CTX solution was injected intraperitoneally,and then the intraperitoneal injection of 8 mg·kg－1·d－1was continued for 14 d.At the beginning of modeling,the estrous cycle of rats was analyzed by observing vaginal exfoliated cell smears every day.The emergence of estrous cycle disorders indicated that the modeling was successful.SD rats in the blank group were injected intraperitoneally with a corresponding dose of 0.9%sodium chloride solution.

Acupuncture point positioning

According to"Experimental acupuncture and moxibustion experimental guidance".10

Guanyuan(CV4):25 mm below the umbilicus;

Sanyinjiao(SP6):10 mm on the tip of the hind ankle;

Zusanli (ST36):the lower part of the hind knee is about 5 mm below the fibula head;

Pishu(BL20):12th thoracic vertebra inferior lateral intercostal space;

Shenshu(BL23):the second lumbar lower both sides;Zigong (EX-CA1):35 mm below the umbilicus and 25 mm laterally.

Experiment protocol

After the successful modeling,in the model acupuncture group,0.25 mm×25 mm Huatuo brand acupuncture needles(Suzhou Medical Supplies Company,Suzhou,China)were used to acupuncture rats'Guanyuan(CV4),Sanyinjiao (SP6,double),Zusanli (ST36,double),Pishu (BL20,double),Shenshu (BL23,double),Zigong (EX-CA1,double),and direct or flat acupuncture points 3-5 mm for 20 min once every day.The 5-days is a course of treatment,each course of rest for 2 d,continuous for 14 d.In the Western Medicine group,estradiol valerate (0.09 mg/kg) was given by intragastrical gavage once every day.In the combination group,acupuncture combined with Western Medicine was given,and the methods of intervention were the same as above.The rats in blank group and control group were grasping and fixation by the same method.Each group took 5 d as a course of intervention,and rested for 2 d between each course of intervention,with a total intervention of 14 d.

Specimen collection

After the experiment,all the rats were in narcotism by pentobarbital sodium,and then the bilateral ovaries of the rats were removed after the abdominal skin was disinfected with 75% ethanol.The fascia and adipose tissue attached to the ovarian tissue were completely stripped,and the rat ovarian tissue was rinsed and wiped with normal saline,and then stored at low temperature for subsequent work such as protein extraction and concentration determination.

Index detection

(a) Levels of E2,FSH and LH in serum of rats.After anesthesia,blood samples were collected from the abdominal aorta of the rats and detected ELISA.After centrifugation,the serum samples were diluted and standard holes,blank holes and sample holes to be measured were set on the cladding plate.After the sample was added and sealed,the plate was incubated in temperature.After the washing and enzyme were added,the plate was incubated and washed again in temperature.After the addition of color developing agent,the color was displayed in darkness for 15 min,and finally the termination reaction was performed by adding the termination solution.The absorbance (OD value) was measured within 15 min after adding the termination solution,and the sample concentration was calculated according to the OD value.

(b)Western blot assay.Total protein was extracted from ovarian tissues of each group,then was determined by Bicinchoinic Acid (BCA) method.After 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation,the electrotransfer was transferred to the nitro fiber membrane,p38MAPK primary antibody was added and incubated overnight at 4 ℃,then secondary antibody was added dropwise,which was enhanced chemiluminescence in the dark room,pressed to the film and exposed.The gel image processing system was used to scan the X-ray film in grayscale.After scanning the image,Image-Pro-Plus 6.0 software was used to analyze the band grayscale,made semi-quantitative comparison with internal reference-actin.

Statistical analysis

SPSS 25.0 software (International Business Machines Corporation) was used for statistical analysis.The mean ± standard deviation (±s) was used to represent the measurement data.The overall mean comparison between multiple groups was analyzed by single factor analysis of variance.Further multiple comparisons were analyzed by least significant difference test.Dunnett's method is used to analyze data when the variance is uneven.P-value <0.05 was considered statistically significant.

RESULTS

Levels of E2,FSH and LH in serum of rats

The levels of E2,FSH and LH in the serum of rats in each group were detected by ELISA.The results showed that compared with the blank group,the levels of E2 in the serum of rats in the other groups decreased significantly (P <0.05),and the levels of FSH and LH increased significantly (P <0.05).Compared with the model group,the levels of E2 in the serum of rats in the Western Medicine group,acupuncture group and the combination group were significantly increased(P<0.05),and the levels of FSH and LH were significantly decreased (P <0.05).Among them,there was no statistically significant difference in the indicators between the Western Medicine group and the acupuncture group (P >0.05).The combination group compared with the Western Medicine group or the acupuncture group had statistically significant differences in each index(P<0.05)(Table 1).

P38 protein expression in rat ovarian tissue

Western Blot was used to detect the expression of p38MAPK protein in the ovarian tissue of rats in each group.The results of Western blot showed that compared with the blank group,the expression of p38MAPK protein in the ovarian tissue of the other groups was significantly decreased (P <0.05).Compared with the model group,p38MAPK protein expres-sion in the acupuncture group,Western Medicine group,and combination group was significantly increased (P <0.05).Among them,the p38MAPK protein expression in the combination group and the acupuncture group was significantly lower than the Western Medicine group (P <0.05).Compared with the Western Medicine group,the p38MAPK protein expression of the acupuncture group was lower than that of the Western Medicine group,but the difference was notstatistically significant(P>0.05)(Table2,Figure1).

Figure 1 p38/β-Actin protein expression in each group

Table 1 Levels of E2,FSH and LH in serum of rats(±s)

Table 1 Levels of E2,FSH and LH in serum of rats(±s)

Notes:group 1:the blank group without any intervention;group 2:the model group,only the premature ovarian failure model is established without any intervention;group 3:the Western Medicine group,which is treated with acupuncture for 14 d;group 4:the acupuncture group,which is given with estradiol valerate(0.09 mg/kg)for 14 d;group 5:the combination group,which is given with acupuncture combined with estradiol valerate(0.09 mg/kg)for 14 d.E2:estradiol;FSH:follicle stimulating hormone;LH:luteinizing hormone.Compared with the blank group,aP<0.05;compared with the model group,bP<0.05;compared with the medicine group,cP<0.05.

Table 2 P38 protein expression in rat ovarian tissue(±s)

Table 2 P38 protein expression in rat ovarian tissue(±s)

Notes:group 1:the blank group without any intervention;group 2:the model group,only the premature ovarian failure model is established without any intervention;group 3:the Western Medicine group,which is treated with acupuncture for 14 d;group 4:the acupuncture group,which is given with estradiol valerate (0.09 mg/kg) for 14 d;group 5:the combination group,which is given with acupuncture combined with estradiol valerate (0.09 mg/kg) for 14 d.p-p38MAPK:phospho-p38-mitogen activated protein kinase.Compared with the blank group,aP<0.05;compared with the model group,bP<0.05;compared with the medicine group,cP<0.05.

DISCUSSION

With the rapid development of society,the prevalence of premature ovarian failure is increasing year by year under high stress in modern women,and the age of the disease is gradually becoming younger.The prevalence rate among women before the age of 40 ranged from 1% to 3.8%,and among women before the age of 30 was 0.1%,11which seriously affected the physical and mental health of women and caused huge burden and pain to patients and their families.Therefore,the problem of women's POF needs to be resolved urgently.According to the clinical symptoms of premature ovarian failure,it can be classified into traditional Chinese medicine "Amenorrhea","Infertility","Peri-menopause syndrome".The site of POF is mainly in the uterus,and the main pathogenesis is Decoy early exhaustion,kidney essence deficiency,at the same time,closely related to the liver,spleen.According to the previous clinical experience,Guanyuan (CV4),Sanyinjiao(SP6),Zusanli (ST36),Pishu (BL20),Shenshu(BL23),Zigong (EX-CA1)point were selected to strengthen kidneyQiand regulateYinandYang.Guanyuan (CV4) is on conception vessel which is called "the sea ofYinpulse",and its deep part is the uterus,so Guanyuan (CV4) is the most important point in the treatment of gynecological diseases,which has the merit of cultivating yuan and fixing the root and coordinating chong and conception vessel;Zusanli(ST36)12can transport spleen and stomach,invigorateQiand blood;Sanyinjiao (SP6),the intersection point of the liver,spleen and kidney,can regulate and nourish the liver,spleen and kidney combining with Pishu(BL20) and Shenshu (BL23).13Zigong (EX-CA1) can regulate menstruation,relieve pain,regulateQiand raiseYang,which is helpful to accelerate follicle rupture and ovulation,and improve the curative effect of treating ovarian diseases.14

At present,it is believed that the causes of premature ovarian failure are complex and the pathogenesis is unclear,and it is related to genetics,immunity,infection,metabolism,iatrogenic and environmental factors,among them,the decline in estrogen is closely related to premature ovarian failure.In addition,studies have found15that no matter what the reasons are,POF is accompanied by the breakdown of ovarian granulosa cells,which indicates that the abnormality of granulosa cells is directly related to POF.Its proliferation,differentiation and apoptosis have direct effects on follicular development,maturation,ovulation and steroid hormones (E2,FSH,LH,etc.).The apoptosis of granulosa cells induces the apoptosis of oocytes,and the excessive atresia of oocytes leads to the decrease of ovarianreserve function,which leads to POF.16-20Mitogen activated protein kinase (MAPK) is an important transmembrane signal transduction pathway which plays an important role in the proliferation and differentiation of ovarian granulosa cells,and p38MAPK is a classic cellular pathway that can be activated by stress factors such as pro-inflammatory cytokines and toxins,and participates in a variety of biological responses,such as inflammatory response,cell proliferation and apoptosis,tissue differentiation,tumorigenesis,reproductive physiology.21-25P38MAPK is the main form of its pathway.Through phosphorylation,the transcriptional expression of genes involved in granulosa cell response is activated.Long-term phosphorylation will accelerate the apoptosis of ovarian granulosa cell,disrupt hormone level,decrease ovarian function,and result in POF.

The traditional treatment method for POF is mainly Western Medicine,but its treatment effect is not very ideal,and it can easily cause many adverse reactions.After the patient stops taking the medicine,the disease will recur.The application of acupuncture therapy to the treatment of patients with POF has the advantages of good treatment effect and small adverse reactions.26Guo27found that the patient's perimenopausal symptoms improved,and FSH,LH,and E2 returned to normal levels,and the ovarian function improved by the acupuncture treatment of "Tiaorentongdu".There are relatively few studies on the mechanism of acupuncture for POF.Some scholars have found that acupuncture can regulate hormone levels in POF rats,up-regulate the PI3K/Akt signal pathway and reduce apoptosis of granulosa cells.This may be one of the mechanisms of acupuncture for POF.25We found that p38MAPK,a classic signaling pathway,is involved in the apoptosis of ovarian granulosa cells,and the decline and apoptosis of granulosa cell function can trigger follicular atresia,which is closely related to POF.Studies have shown that cyclophosphamide (CTX) can cause different degrees of damage to granulosa cells in the ovarian follicles,change the ovarian microenvironment and cause premature ovarian failure.28,29The ovary is the main target organ for female reproductive toxicity of CTX.30CTX crosses DNA and inhibits DNA synthesis,which has the most obvious effect on S phase,causing the synthesis of DNA,RNA and proteins to be inhibited,and the gonad tissue Permanent injury.31Therefore,we propose a hypothesis that the mechanism of acupuncture treatment of premature ovarian failure may be related to the p38MAPK signaling pathway.In this study,in order to further explore the mechanism of acupuncture treatment for premature ovarian failure,we applied cyclophosphamide to prepare rats of chemotherapy-induced premature ovarian failure and used acupuncture,Western Medicine (estradiol valerate) and combined acupuncture to conduct intervention,and further verified the hypothesis by observing the expression of p38MAPK protein in the ovarian tissue of rats with different intervention methods.

In conclusion,the results showed that acupuncture can improve the E2,FSH and LH level of rats with premature ovarian failure,and promote the recovery of ovarian function,which has the same effect as estrogen (estradiol valerate).At the same time,acupuncture can inhibit the expression of p38MAPK protein.The mechanism of acupuncture treatment on premature ovarian failure may be related to inhibiting the expression of p38MAPK protein,thereby inhibiting the activation of p38MAPK signaling pathway and reducing the apoptosis of granulosa cells.