The study has been approved by the Ethics Committee of Zhejiang Hospital [No. 2018 (9K)] and all patients signed the written informed consent.

A total of 73 perimenopausal patients with abdominal obesity who received treatment in the Acupuncture Department of Zhejiang Hospital from December 2017 to January 2019 were recruited in this study. The diagnostic criteria for perimenopausal period were formulated according to the chapter of “Menopausal Syndrome” in the 8th edition of “Obstetrics and Gynecology,” combined with “Chinese Obstetrics and Gynecology.” The details are as follows: (1) 40–60 years old; (2) Clinical manifestations of menstrual disorders for more than 3 months, or amenorrhea within a year, etc.; (3) One or more of the following possible related symptoms, such as typical symptoms of unstable vasomotor function, psychoneurological symptoms (anxiety, tension, depression, irritability, etc.), physical symptoms (dry skin, muscle, and bone pain, etc.) and symptoms of urogenital atrophy (vaginal dryness, pain during intercourse, leakage of urine, repeated urinary tract infections, etc.); (4) Imaging examination to exclude organic diseases such as uterus and ovaries. The standards for abdominal obesity were formulated with reference to the “Guidelines for the Prevention and Control of Overweight and Obesity in Chinese Adults”: waist circumference ≥ 80 cm, and (or) WHtR ≥ 0.50, and (or) WHR ≥ 0.78. The general information of the patients including age and height was recorded (Table 1).

The inclusion criteria of patients in this study were as follows: (1) patients were diagnosed with perimenopausal and abdominal obesity; (2) patients had not received weight-loss treatment in the past 3 months. The exclusion criteria of patients in this study were as follows: (1) patients with secondary obesity caused by hypothalamic syndrome, pituitary tumor, Cushing’s syndrome, hypothyroidism, insulinoma, or polycystic ovary syndrome; (2) patients who were diagnosed with uterine or ovarian malformations, have undergone uterine or ovarian resection, or in pregnancy or breastfeeding; (3) patients with severe diseases such as hyperthyroidism, diabetes, psychosis, malignant tumors, heart, liver, and kidney diseases, and hematopoietic system diseases.

The patients were randomly divided into two groups: the Control group (n = 36) and the Electroacupuncture group (n = 37). The patients in the Control group received healthy lifestyle education, while those in the Electroacupuncture group received electroacupuncture treatment combined with dietary intervention for 8 weeks. Before and after treatment, the stool and blood of the patients were collected for subsequent analyses, and the weight, height, waist circumference and hip circumference of the patients were recorded to calculate waist-height ratio (WHtR), waist to hip ratio (WHR), and body mass index (BMI). Here, the method of measuring waist circumference recommended by the World Health Organization is adopted: the subject takes off his shoes, hat and coat, fasts, and stands upright, with feet being separated by 25–30 cm. The measurement position is at the midpoint of the line between the horizontal anterior superior iliac crest and the inferior edge of the twelfth rib.

The main acupuncture meridians are Renmai, Stomach, Spleen, and Bladder Meridians. The acupoints are Zhongwan, Qihai, Guanyuan, Tianshu, Daheng, Pishu, Shenshu, and Dachangshu. The acupoint combinations in the study were as follows: Sanyinjiao and Inner Court were combined for the patients who belonged to the gastric-heat stagnation type; Yinlingquan and Fenglong were combined for the patients who pertained to the phlegm dampness Neisheng type; Hegu and Taichong were combined for the patients who appeared liver stagnation; Zusanli and Taibai were combined for the patients with spleen deficiency; and Mingmen and Shenmai were combined for the patients who presented symptoms of spleen and kidney yang.

Regarding the treatment, 0.25 mm × 40 mm (190,030) and 0.30 mm × 50 mm (190,185) disposable acupuncture needles (Huatuo, Suzhou, China) as well as an SDZ-V electronic acupuncture instrument (Huatuo, Suzhou, China) were employed. 1.5 inch acupuncture needles were used at the Backshu acupoints and four-limb acupoints. For the Backshu acupoints, the needle was inserted obliquely at 45° into the spine at 1-inch depth, while for the four-limb acupoints, the needle was inserted straight at 0.8–1.0-inch depth (0.3–0.5-inch depth for Taibai, Taichong, and Neiting). 2.0 inch acupuncture needles were inserted at the acupoints on the abdomen at 2-inch depth. Electroacupuncture was applied to Tianshu, Abdomen Jie, Daheng, and Meridian, and the waveform was a dense wave with a frequency of 2/100Hz. The needle was retained for 30 min. The acupuncture acupoints without electroacupuncture treatment were slightly lifted and twisted3 times every 10 min. Patients in the Electroacupuncture group received electroacupuncture treatment every 2 days (3 times a week) for 8 weeks.

Patients in the Electroacupuncture group were given a strict low-sugar, low-fat, low-salt, and high-fiber diet. The diet contained staple foods, meat, and vegetables (each accounting for one third of the total dietary ingestion), fruits, and vegetables (no less than 250 g), and mineral supplements. Besides, the patients drunk 2,000–2,500 ml of water and no more than 20 g of liqueur, ingested less than 6 g of salt per day, and only ate fish products twice a week. Here, dietary intervention was implemented on the basis of menopausal health management strategies (Zhang, 2015).

Before and after treatment, the weight (kg), height (cm), waist circumference (cm), and hip circumference (cm) of the patients were recorded. Then WHtR, WHR, and BMI were calculated using the following formulae: WHtR = waist/height; WHR = waist/hip; BMI = weight/height².

Before and after treatment, the serum of all the patients was analyzed using an Automatic biochemical analyzer (BS-280; mindray, Shenzhen, China) to evaluate the levels of serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein cholesterol (HDL-C), fasting insulin (FINS), and fasting blood glucose (FBG). Besides, Homeostasis Model Assessment-insulin resistance (HOMA-IR) was calculated according to the formula: HOMA-IR = FBG × FINS/22.5.

The intestinal flora in the patients was detected by 16s rRNA sequencing technology according to the previous publication (Di Segni et al., 2018). In brief, after the stool samples were collected, the RNA in the samples was extracted and subsequently subjected to polymerase chain reaction (PCR) (16s rRNA forward primer: 5′-GTGCCAGCMGCCGCGG-3′; 16s rRNA reversed primer: 5′-CCGTCAATTCMTTTRAGTTT-3′). Then the PCR product was harvested and sequenced through Illumina system. Meantime, the Clean Tags of the PCR product were obtained for the Operational Taxonomic Units (OTU) analysis.

In line with the clustering results of OTU, the representative sequence of each OTU was annotated to obtain the corresponding species annotation information and species abundance distribution. Then, the species abundance in the sample and the species differences between samples were obtained through the visual statistical analysis of OTUs, and the Venn and Partial Least Squares Discrimination Analysis (PLS-DA) diagrams were plotted. Next, the information was subjected to Alpha diversity analysis to acquire the community structure information with differences in the sample. The Linear discriminant analysis (LDA) effect size (LEfSe) statistical analysis was applied to further investigate the differences in community structure among samples, and to identify different species via testing the species composition and community results of the grouped samples.

All data involved in this study except for those from intestinal flora analysis were tested the normality of the variable distribution and analyzed by student’s t-test. Statistical data were presented as mean ± standard deviation. The data were recognized as statistically significant when P < 0.05. These analyses were performed under SPSS 20.0 software.

Given that there was no statistical difference in the general information (cases, age, and height) (Table 1) of the two groups of patients, the patients in the two groups received different treatments. The physical indexes (weight, waist circumference, hip circumference, WHtR, WHR, and BMI) (Table 2) of patients in the two groups before and after treatment were documented. As shown in Table 2, in the Electroacupuncture group, the weight (P < 0.01), waist circumference (P < 0.01), hip circumference (P < 0.01), BMI (P < 0.01), WHtR (P < 0.01), and WHR (P < 0.01) of the patients were decreased after treatment when compared with those before treatment, while the patients in the Control group after treatment showed a larger waist circumference (P < 0.01) and a higher WHR (P < 0.01) than before treatment. Additionally, the waist circumference (P < 0.01), WHtR (P < 0.01), and WHR (P < 0.01) of patients in the Electroacupuncture group were notably lower than those in the Control group, which indicated that electroacupuncture combined with diet treatment might ameliorate abdominal obesity in perimenopausal patients.

Then the levels of TG, TC, LDL, HDL-C, FINS, FBG, and HOMA-IR of the patients in the two groups before and after treatment were evaluated. As illustrated in Table 3, in the Control group, the LDL level of patients after treatment was elevated (P < 0.05) as compared with that before treatment. Moreover, the TG (P < 0.01) and LDL levels (P < 0.01) of patients in the Electroacupuncture group were markedly lower than those in the Control group, which proved that electroacupuncture combined with diet treatment ameliorated the TG and LDL levels of perimenopausal patients with abdominal obesity.

During the analysis of intestinal flora, the information in the Electroacupuncture group before and after treatment was named Z1 team and Z2 team, respectively, and the information in the Control group before and after treatment was called D1 team and D2 team, respectively. As depicted in Figure 1, in the Electroacupuncture group, the number of OTUs that existed both before and after treatment was 579, and that of specific OTUs was reduced from 111 to 53 after treatment. Meanwhile, in the Control group (Figure 2), the number of OTUs that existed both before and after treatment was 775, and that of specific OTUs was decreased to 64 after treatment. These discoveries indicated that electroacupuncture combined with diet treatment decreased the abundance of the intestinal flora in the patients.

In addition, according to the PLS-DA analysis based on OTU determination (Figure 3), the samples belonging to the same group (the Electroacupuncture group or the Control group) in each team (Z1 and Z2, or D1 and D2) were almost identical to each other; the structure site of the flora sample in each team was relatively low; the distance between the samples of team Z (the Electroacupuncture group) and team D (the Control group) was far; and the difference in bacterial community structure between the two groups was relatively significant. All these results verified that the grouping results were reasonable, accompanied by satisfactory efficiency of the classification model.

The phylum-level and genus-level changes of the intestinal flora in patients before and after treatment were exhibited in Figures 4, 5, respectively. As delineated in Figure 4 and Table 4, at the phylum level, the most abundant flora in both groups before (D1 and Z1) and after (D2 and Z2) treatment were Firmicutes, Bacteroidetes, and Proteobacteria. However, difference appeared at the genus-level, as shown in Figure 5 and Table 5. Although the most dominant flora in both groups before (D1 and Z1) and after (D2 and Z2) treatment were Bacteroides, Prevotella, Faecalibacterium, and Roseburia, the Enterococcus level was decreased (P < 0.05) and the Enterobacter level was increased in Z2 group, when compared with the Z1 group (P < 0.05), while the Blautia level was lessened (P < 0.05) and the Enterobacter level was augmented in D2 group, when compared with the D1 group (P < 0.05).

The Alpha diversity of intestinal flora was evaluated. As shown in Figure 6 and Table 6, although there was no statistically difference between Z1 and Z2 groups, the observed species, chao, ace, and shannon indexes in the Electroacupuncture group after treatment (Z2) showed an upward trend, while the simpson index presented a downward trend, signifying that the species abundance in the Electroacupuncture group after treatment was on the rise. Meanwhile, despite no statistically difference between D1 and D2 groups, the same observed indexes as above in D2 group showed a downward trend, while the simpson index was on the contrary, manifesting that the species abundance in the Control group was inhibited. These results indicated that electroacupuncture combined with diet treatment improved the species diversity of intestinal flora in patients.

The species difference of intestinal flora in the Electroacupuncture group was analyzed. As profiled in Figure 7, the abundance of Enterococcus, Enterococcaceae, Escherichia, Enterobacteriaceae, and Enterobacteriales was reduced yet that of Klebsiella and Kosakonia was enhanced after treatment, which demonstrated that electroacupuncture combined with diet treatment increased the species differences of intestinal flora in patients, and Klebsiella and Kosakonia were instrumental in the treatment of abdominal obesity in perimenopausal patients.