HU Xijiao,CHENG Yinglong,KANG Huanan,LI Shuoxi,WANG Yawen,LIU Jinzhe,SUN Yiming,LIU Li

HU Xijiao,Department of obstetrics and gynecology,the Second Affiliated Hospital of Heilongjiang University of Chinese Medicine,Heilongjiang 150001,China

CHENG Yinglong,Deapartment of Hysteroscopy chamber,the First Affiliated Hospital of Heilongjiang University of Chinese Medicine,Harbin 150040,China

KANG Huanan,College of pharmacy,Heilongjiang University of Chinese Medicine,Harbin 150040,China

LI Shuoxi,Midwifery Teaching and Research Section,Heilongjiang University of Chinese Medicine Jiamusi College,Harbin 150040,China

WANG Yawen,The department of obstetrics and gynecology,the First Affiliated Hospital of Henan University of Chinese Medicine,Henan 450000,China

LIU Jinzhe,Department of Chinese Pharmacy,School of Pharmacy,Guizhou University of Chinese Medicine,Guizhou 550000,China

SUN Yiming,Andrology department Heilongjiang Academy of Traditional Chinese Medicine,Harbin 150036,China

LIU Li,Endoscopy room,the First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine,Hysteroscopy Department,Harbin 150040,China

Abstract OBJECTIVE: To explore the effect of electroacupuncture(EA) on rats with chronic fallopian tube inflammation and its potential mechanisms.METHODS: Thirty-six female Sprague-Dawley rats were divided into Control,Model and EA groups.The pathological morphology of the fallopian tubes was observed by hematoxylin-eosin (HE) and Masson staining.The results of transforming growth factor-β1(TGF-β1),P38 mitogen-activated protein kinase (MAPK),phosphorylation (p)-p38MAPK in rat oviduct tissues were detected by immunohistochemistry.Results of P38MAPK,p-P38MAPK and TGF-β1 in rat oviduct tissues were detected by immunofluorescence.The expression level of p38MAPK,p-P38MAPK,TGF-β1 protein in rats was detected by Western blot.Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect mRNA expression levels of TGF-β1.RESULTS: It found that collagen fibers counts decreased significantly in EA group compared to Model group.The phosphorylation of P38MAPK in EA group was significantly reduced compared to Model group.Theserum TGF-β1 expressions in EA group increased decreased significantly.CONCLUSION: Electroacupuncture was able to attenuate chronic salpingitis through down-regulating TGF-β1/MAPK signaling pathway.

Keywords: electroacupuncture;salpingitis;transforming growth factor beta1;mitogen-activated protein kinase

1.INTRODUCTION

Female infertility is becoming an important and growing point with the development of modern society and tubal factor infertility due to pelvic inflammatory disease (PID)and salpingitis causing adhesion is the most common reason for female infertility.1,2Tube damage is mainly manifested by fibrotic agglutination,fibrotic phimosis,and fallopian tube obstruction.Antibiotics and enzyme drug are the common drug therapy for Chronic salpingitis.3And physical therapy,Interventional therapy andin vitrofertilization and embryo transfer (IVF-ET)are also becoming the important methods of treating Chronic salpingitis.4However,chronic salpingitis is still a complication due to adverse reactions,such as tubal pregnancy,adhesion after adhesion,and there is no satisfactory drug therapy at present.5Therefore,it is necessary to find the effective treatment for chronic salpingitis.

Acupuncture is one of the characteristic therapies of Traditional Chinese Medicine.Acupuncture had been used to treat infertility extensively,including ovulatory dysfunction,IVF-ET.6,7It could treat the inflammation of the fallopian tube and even the entire pelvic inflammatory disease.Also,acupuncture could increase the rate of pregnancy and decrease the rate of abortion without any obvious adverse reactions,but with strong operability,and easy acceptance by patients.8,9This method effectively improved the microcirculation and hemorheological properties of the pelvic tissues,reduced inflammatory reactions,promoted the regeneration and repaired of diseased tissues,and achieved the role of dredging the fallopian tubes.10The main principle of treatment of tubal inflammatory infertility was to mediate the fallopian tube and its surrounding blood circulatory system through external effects and the body's blood reflex function,so that inflammation gradually disappeared and strengthened the function of the fallopian tube tissue itself.11

It was shown that the correlation between electroacupuncture regulation and transforming growth factorβ1 (TGF-β1)/ mitogen-activated protein kinase (MAPK)related signal pathways mainly focused on antiinflammation and analgesia,anti-injury,such as regulation of gastrointestinal function,and prevention and treatment of cardiovascular,kidney,lung diseases.12-16However,little research had been done on the connection among electroacupuncture (EA),TGFβ1/MAPK pathway,and chronic salpingitis.Therefore,we did the experiments and analyzed the efficacy and mechanisms of electroacupuncture therapy related to the TGF-β1/MAPK signal pathway to help in the clinical treatment of chronic salpingitis.

2.MATERIALS AND METHODS

2.1.Experiment animals

Female Sprague-Dawley rats (n=36,8 weeks old,specific pathogen free grade,weight 180-200 g) were purchased from Experimental Animal Center of Heilongjiang University of Chinese Medicine and Pharmacy.The license number for animal manufacture was SCXK (HEI) 2017-004.Rats were treated in accordance with guidelines of Experimental Animal Management and Use Guide.Nanjing University of Chinese Medicine Biosafety and Animal Research Committees approved all experimental protocols.

2.2.Preparation of animal model of chronic salpingitis

After the rats were fed for one week,the experiment would start formally and the model would be prepared.On the first day of the experiment,all the other five groups except the normal group were subjected to surgical modelling: first,the rats were anesthetized by intraperitoneal injection with 3% Sodium pentobarbital at a dose of 0.1 mL/100 g body weight,and the rats were placed on the operating table after the anesthesia was successfully finished,prepared skins on both kidney areas on the back of the rats,and performed routine disinfection with iodophor.Making a 10 mm transverse incision near the spine in the kidney area near the costal border,and then opening the abdominal cavity layer by layer.Gently peeling off other tissues in the abdominal cavity to fully expose the unilateral uterus,fallopian tubes and ovaries,and injecting 0.1 mL of Staphylococcus aureus suspension (concentration: 4 × 109/ mL)into the fallopian tube-ovary direction of the rats.During the bacterial process,attention should be paid to prevent the bacterial fluid from flowing back and avoided mechanical damage to the fallopian tubes.After the operation,the abdomen was closed and sutured layer by layer,and the operation area was disinfected again with iodophor.The operation method of the contralateral fallopian tube was the same as before.

2.3.Acupuncture method

The limbs and head of the rat were bound to a self-made fixed net with nylon strapping.After routine disinfection of the acupoints with alcohol,the rat was acupunctured with 0.13 × 2.5 cm Andy brand disposable sterile acupuncture.After the acupuncture got the breath,immediately adjusted the depth to prevent the needle from falling off,and then connected the electronic acupuncture device,selected the density wave,adjusted the output frequency to 2 Hz,and the intensity to 1 V.The stimulus intensity was that the rat's body trembles slightly and could keep quiet.It was advisable to keep the needle for 20 min,1 time/d,for 28 d of continuous acupuncture.

2.4.Animals and treatment

The rats were divided into the Control group (n=12),the Model group (n=12) and the EA group (n=12).(a)Control group: no treatment.(b) Model group: the same amount of distilled water was given to the stomach by gavage,once a day;each group was given continuous intervention for 4 weeks,and the non-acupuncture group was given the same degree of grasping and binding for the same time in the acupuncture group.(c) EA group:acupuncture at Guanyuan (CY4),Zusanli (ST36),Sanyinjiao (SP6),and Shenshu (BL23) points were connected to an electronic acupuncture device,kept the needle for 20 min,1 time/d,and gave an equal amount of distilled water to the stomach.

2.5.Hematoxylin-eosin and masson staining

The fallopian tube tissue sections were stained by hematoxylin and eosin and Masson Staining to observe the change of histopathology and fibrosis.

2.6.Enzyme-linked immunosorbent assay (ELISA) kit

The expression of TGF-β1 in serum was detected by ELISA kit which were performed according to the manufacturer's specifications.

2.7.Immunohistochemistry and immunofluorescence

The expression levels of TGF-β1,P38MAPK,p-P38MAPK in rats fallopian tube tissue were tested by immunohistochemistry.The expression levels of P38MAPK,p-P38MAPK,and TGF-β1 in rats fallopian tube tissues were tested by Immunofluorescence.

2.8.Western blot analysis

Proteins from fallopian tube tissues were separated in 4%-20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes.Remove the PVDF membrane,add the TGF-β1 (21898-1-AP,Proteintech,Wuhan,China),p38 (14064-1-AP,Proteintech,Wuhan,China),p-P38 (P00176-1,BOSTER,Wuhan,China) primary antibodies were incubated overnight at 4 ℃.Add the secondary antibody and incubated for 1.5 h on a shaker at room temperature.Place the PVDF film on the fresh-keeping film,mixed the two luminescent reagents A and B in the ECL kit,mixed them and added to the surface of the film,moved them into the gel imaging analyzer,exposed and developed in the chemical photosensitive mode.

2.9.Real-time quantitative polymerase chain reaction(RT-qPCR)

mRNA expression of TGF-β1 was measured using RTqPCR which were carried out as the following program:15 s at 95 ℃ for cDNA denaturation,45 s at 60 ℃ for annealing and extension,followed by 40 repeat cycles.And the Primers were showed in Table 1.GAPDH was used as a reference gene and comparison in expression between groups was made using the 2-ΔΔCtmethod.

Table 1 Primers sequences

2.10.Statistical analysis

Statistical analysis was performed using SPSS software(vers 25.0,IBM Corp.,Armonk,NY,USA).Data was presented as mean ± standard deviation.Groups were compared using one-way analysis of variance.If the variances were even,Tukey Honestly Significant Difference test was used to compare the two groups;otherwise,Duunett-T3 test was used when the variances were uneven;P＜ 0.05 was considered to be statistically significant.

3.RESULTS

3.1.EA improved the pathological changes of chronic salpingitis

HE staining showed that the morphology and structure of the fallopian tubes of rats were normal,without inflammatory cell aggregation or infiltration.While,in model group,a large number of inflammatory cells could be gathered and infiltrated in the oviduct lumen of rats,mucosal epithelial cells fall off,oviduct tissue cells were arranged in disorder,cell adhesion,hyperemia,cell body swelling,and some cell nuclei were also accompanied by swelling,which indicated that the model was successfully prepared.And the structure of the fallopian tube was improved compared with the model group(Figure 1A-1C)).Then Masson staining were used to observer the collagen deposition in oviduct tissue.It was only a small amount of collagen deposition under the mucosa of the fallopian tube tissue in the normal group and Masson staining could show a slight blue.During the formation of tubal fibrosis,collagen deposited on the mucosa and submucosa gradually increased,and the area and degree of blue staining increased.Compared with the normal group,the blue staining area of the model group is larger,and the degree of blue staining was deep,and the degree of fibrosis was obviously aggravated,indicating that the model preparation was successful;after treatment,the electroacupuncture group was compared with the model group.The blue-stained area was reduced and the degree of fibrosis was significantly reduced,suggesting that electroacupuncture could improve the degree of fibrosis of the fallopian tube in rats(Figure 1D-1E).Besides,the Collagen area were counted which indicated that compared with the control group,there were a large amount of blue collagen fiber deposition in the fallopian tube tissue of the model group.In the EA group,collagen fiber deposition was significantly reduced compared with the model group (P＜ 0.01,Figure 1G).

3.2.EA improved the expression of TGF-β1

TGF-β1 played an important role in the development of salpingitis.Compared with the normal group,the fluorescence expression intensity of TGF-β1 increased in the model group (P＜ 0.01).And compared with the model group,the expression level of TGF-β1 was significantly decreased in the EA group (P＜ 0.01,Figure 2A-2I).Compared with the normal group,it was observed the high expression TGF-β1 in Immunohistochemical image and the average optical density of TGF-β1 in the fallopian tube tissue of the model groupwas obviously improved (P＜ 0.01) which were reduced in EA group (P＜ 0.01,Figure 2J-2M).And the expression of TGF-β1 in serum and oviduct were also indicated that compared with normal group,TGF-β1 levels were significantly increased in model group but the EA significantly alleviated the changed (P＜ 0.01,Figure 2N-2P).Besides,RT-qPCR results showed TGFβ1 mRNA expression was improved in model group and decreased in EA group (P＜ 0.01,Figure 2Q).EA also down-regulated the expression level of TGF-β1,interleukin 17 (IL-17),connective tissue growth factor(CTGF),fibronectin (FN),α-smooth muscle actin (α-SMA) and collagen,type I (COL-1) and up-regulated IL-10 and matrix metalloprotein-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1) .

Figure 1 Effect of EA on the pathological changes in oviduct

3.3.EA regulated the p38MPAK signaling way

Immunofluorescence technique was used to analysis the expression of P38 and p-P38 which was indicated that the fluorescence expression intensity of p-P38,increased in the model group compared with the normal group.However,compared with the model group,the expression level of p-P38 was significantly decreased in the EA group (Figure 3A-3I).Similarly,immunohistochemical analysis also showed that p-P38 level had high expression in model group but low expression in EA group (P＜ 0.01).While the average optical density of p38 in the fallopian tube tissue of the model group had no significant difference suggesting that the fallopian tube tissue of each group the total amount of p38 protein did not change significantly (P＞ 0.05,Figure 3J-3Q).And according to the WB results (Figure 3R),the relative production of p-P38 was obviously higher in model group than normal group which were also reduced after the treatment of EA (Figure 3S).

Figure 2 Effect of EA on the expressions of TGF-β1

Figure 3 EA regulated the p38 signaling way

4.DISCUSSION

Acupuncture therapy had been long used in the treatment of chronic salpingitis in clinical because it was conductive to shortening in-hospital therapy.17Chronic salpingitis as the major cause of blocked fallopian tubes always manifested as the fallopian tube adhesion,fibrosis,and even sclerosis which finally leaded to the infertility.18In this study,chronic salpingitis model rats showed oviduct tissue cells were arranged in disorder,mucosal epithelial cells fall off and so on,but EA could obviously alleviate the pathological changes.Besides,Inflammatory cytokines also played an important role in the development of chronic salpingitis.19EA could reduce the expression of proinflammatory cytokines TGF-β1,which could promote the occurrence and development of inflammation,adhesion and fibrosis in the process of chronic salpingitis.20Therefore,EA could regulate the inflammatory response to improve the chronic salpingitis.

On the other hand,fibrosis of oviduct was another pathological character of chronic salpingitis.21Masson staining showed the degree of fibrosis was obviously aggravated in chronic salpingitis rats and EA treatment significantly reduced the degree of oviduct fibrosis.TGF-β1 could also promote the fibrosis of oviduct by increasing the synthesis of ECM protein,and was essential in the process of tissue repair.22,23TGF-β1 had strong synthesis and secretion ability which increased the synthesis of ECM.24The reduction of degradation leaded to the deposition of a large amount of ECM,which intensified the progress of tubal fibrosis.

According to the research showed TGF-β1 could regulated the p38MAPK signaling way which was one of the most important members of the MAPK family and was known as the most classic inflammation pathway.25,26Our study indicated EA could downregulate level of p-P38MAPK and TGF-β1.Besides,Tsukadaet al27found that p38MAPK signaling way also regulated with the expression of TGF independently liver fibrosis.The evidenced supported that EA therapy reduced the expression of TGF-β1 to improve the oviduct fibrosis by mediating the P38 signaling way.

In summary,EA could alleviate chronic salpingitis by improving inflammatory response and oviduct fibrosis by regulating the P38MAPK signaling way.However,our research was limited to the TGF-β1/p38MAPK pathway and more potential mechanisms of EA in the treatment of salpingitis should be further explored in the future.