Combined acupuncture and herb treatment improves intestinal flora inabdominally obese subjects based on 16s rRNA sequencing: a randomized controlled trial

Abstract

OBJECTIVE:

To investigate Acupuncture-herb therapy modulates gut microbiota in abdominal obesity.

METHODS:

A randomized controlled trial was designed in accordance with standard protocols. Abdominally obese subjects were randomized into four groups: A2 (Control): double placebo, A1 (Needle): press needle + placebo herb, A3 (herb): herbal medicine (Huatan Lishi Fang化痰利湿方) + placebo needle, A4 (Combination): press needle + herbal medicine. After 12 weeks of treatment, groups were relabeled B1-B4. Weight, waist circumference, and body mass index were measured monthly. Gut microbiota was analyzed via 16S rRNA sequencing for diversity and abundance.

RESULTS:

Combined needle-herb therapy significantly reduced waist circumference (P < 0.05). All treatments altered gut microbiota composition. The combination group showed significant changes in diversity (Chao1, Shannon, Simpson; P < 0.05). Needle therapy increased Bacteroidia; herbs reduced Lachnospiraceae and Megamonas. All results were significant (P < 0.05).

CONCLUSION:

Combined needle-herb treatment modulated 25 key gut flora across multiple taxonomic levels in abdominal obesity. It reduced Firmicutes and Bacteroidota. Bacteroidota, Actinobacteriota, and Prevo-tellaceae may suppress obesity, whereas Proteobacteria, Lachnospiraceae, and Megamonas may promote it. The combination specifically altered Bacteroidaceae, Lachno-spiraceae, Bacteroidia, and Megamonas.

1. INTRODUCTION

Abdominal obesity, or central obesity, is linked to metabolic diseases in a harmful cycle that reduces both quality of life and life expectancy. Due to changing lifestyles, its global incidence is rising year by year, affecting people at younger ages and posing a growing health threat worldwide.¹ A herbal treatment with ginseng, atractylodes, and lotus leaf was more effective at reducing weight, blood sugar, and symptom scores in obese type 2 diabetes patients than Western Medicine after three months.² Electroacupuncture reduced weight, waist circumference, hip circumference, body mass index, waist to hip ratio in perimenopausal women with abdominal obesity, combined acupuncture and herbal treatment increased gut flora diversity and was more effective than the control. In patients with phlegm-dampness type polycystic ovary syndrome and abdominal obesity, it also reduced waist circumference, waist-hip ratio, and body mass index (BMI), while improving hormone levels and promoting ovulation Recent studies on Traditional Chinese Medicine (TCM) have explored its mechanisms for treating abdominal obesity. TCM has shown promising results by regulating the body's internal environment, reducing acidosis, and shortening the anion gap.³ regulating glucose and lipid metabolism and inhibiting insulin resistance, improving the regulation of adipokine sensitive protein B gene. Wang and regulating the structure of intestinal flora to promote the production of beneficial bacteria and inhibit harmful bacteria.^4-6 Gut flora has become a major research focus in obesity studies. Recent findings show that obesity is often associated with changes in gut bacteria, which influence eating behavior through brain regulation and affect various body systems. Herbal medicine has been shown to reduce abdominal obesity by modifying gut flora, improving insulin sensitivity, inhibiting fat synthesis, and lowering blood triglycerides and cholesterol—ultimately helping regulate blood sugar and lipids.^7-10 It also alleviates the dysfunction of intestinal flora, inflammatory response, and obesity in high-fat diet-induced obese mice by reg ulating the intestinal flora function.¹¹ A combined treatment using needle-patch and herb was clinically proven to reduce abdominal obesity. This study confirmed its efficacy and used 16s rRNA sequencing of gut flora before and after treatment to explore how the therapy works.

2. METHODS

2.1. Study design

This multi-center, triple-blind, randomized trial (ChiCTR2100053216) investigated the effect of acupuncture-herb combination on abdominal obesity and gut microbiota. Approved by the ethics committee of Beijing University of Chinese Medicine (2020BZYLL0702), the study followed Good Clinical Practice guidelines, included independent monitoring (herb Safety Monitoring Board), and used trained staff and standard operating procedure to ensure quality. All participants provided written consent.

2.2. Participants and interventions

This study enrolled 92 abdominally obese adults meeting Chinese criteria (BMI ≥ 24; waist: men ≥ 90 cm, women ≥ 80 cm). After one dropout, 91 were randomized into four 12-week intervention groups: control (placebo + sham needle), needle alone, herbal medicine alone, or combination (needle + herbs). All treatments used standardized protocols. Post-intervention, groups were relabeled B1-B4. Herbal medicine was standardized granules (Poly Pharm, batch 7062642, Ganzhou, China).

Interventions: herb/Placebo: the active herbal formula contained: Huajuhong (Exocarpium Citri Tomentosae); Fuling (Poria); Shanzha (Fructus Crataegus Pinnatifidae); Heye (Folium Nelumbinis). Placebo contained starch and flavoring. Both were taken daily dissolved in warm water. Needle/Sham: press needles (Riyi Company, Beijing, China) or sham needles (0.22 mm × 2.0 mm) were applied at Zusanli (ST36), Sanyinjiao (SP6), and Fenglong (ST40) bilaterally, replaced every 5 d.

Procedures: after 3 months, gut microbiota (within and between groups) were analyzed. A WeChat group was used for communication, instruction, and data collection (waist circumference, weight, stool samples). All participants received tutorials and repeated guidance to ensure accuracy. Participants were randomized 1∶1∶1∶1 into four groups (A1-A4) using center-stratified statistical analysis system generated codes with block sizes of 4 or 8. Study products were prepackaged and labeled according to group assignment in a concealed three-blind procedure (participants, investigators, and statisticians blinded).

Observation indexes: primary outcomes: weight, waist, BMI. Exploratory: 16s rRNA sequencing for flora alpha/beta diversity, structure, and functional prediction.

Stool sample collection: Stool samples were collected pre-and post-treatment. Subjects self-sampled using provided kits. Samples were stored at -80°C and sent for 16s rRNA sequencing.

2.3. 16s rRNA sequencing analysis and statistical methods

16S rRNA sequencing was performed on NovaSeq6000 (HuDa genomics institute, Beijing, China) DNA was amplified, libraries prepared, and data processed for operational taxonomic unit clustering, diversity analysis, and taxonomic classification. Statistical comparisons used t-tests or Mann-Whitney tests in SPSS statistics 26.0 (IBM Corp., Armonk, NY, USA). A P-value of less than 0.05 is considered statistically significant. For comparisons between two groups, data conforming to a normal distribution were described as mean ± standard deviation ($\overline{x}\pm s$).

3. RESULTS

3.1. Analysis of basic demographic indicators

Table 1 presents no significant differences in gender, age, or group size were found at baseline (P > 0.05), indicating comparability. The flow of this randomized controlled trial is illustrated in Figure 1.

Table 1.

Age and gender demographics of the four groups ($\overline{x}\pm s$)

Group	n	Gender (n)		Age (years)
		Male	Female
A1	23	10	13	29±9
A2	23	6	17	25±7
A3	22	6	16	32±13
A4	23	2	21	29±6
total	91	24	67	28±9

Notes: A1 (Needle): press needle + placebo herb; A2 (Control): double placebo; A3 (herb): herb + placebo needle; A4 (press needle and herb combination). Statistical comparisons used t-tests or Mann-Whitney tests. P < 0.05.

Figure 1. CONSORT flow diagram of participants in the randomized controlled trial.

CONSORT: Consolidated Standards of Reporting Trials; RCT: randomized controlled trial; ITT: intention-to-treat; PP: per-protocol; OA: outcomes assessment; BMI: body mass index.

3.2. Analysis of efficacy indexes

Table 2 presents the outcome of this study. Post-intervention, waist circumference, weight, and BMI improved in all active treatment groups (A1, A3, A4). The combination group (A4) showed significant waist reduction (P < 0.05), demonstrating effective obesity treatment.

Table 2.

Efficacy indicators before and after treatment ($\overline{x}\pm s$)

Group	n	Waist	Weight	Body max index
A1	23	90.5±7.5	72.2±9.1	26.5±1.9
B1		86.0±8.0	71.2±9.0	26.2±2.6
A2	23	93.4±9.7	79.2±16.7	27.7±3.8
B2		88.4±12.5	75.7±17.1	26.3±3.6
A3	22	92.0±9.5	74.6±10.9	26.7±2.5
B3		83.1±19.1	72.6±10.3	25.0±2.4
A4	23	90.2±7.7	74.7±11.3	27.3±3.6
B4		83.3±9.4a	71.7±11.0	26.1±3.7

Notes: A1 (Needle): press needle + placebo herb; A2 (Control): double placebo; A3 (herb): herb + placebo needle; A4 (press needle and herb combination). After 12 weeks of treatment, groups were relabeled B1-B4. Statistical comparisons used t-tests or Mann-Whitney tests. Intra group comparison: ^aP < 0.05.

3.3. Regulation of outcount of intestinal flora

Fecal 16S rRNA sequencing revealed total operational taxonomic units (OUTs) increased from 7152 pre-treatment to 7828 post-treatment. Shared OTUs decreased from 702 to 628, while unique OTUs and their proportions rose significantly across all groups (e.g., A4: 34.0% to 43.0%). Needle and herb groups showed marked OTU differences (P < 0.05), indicating enhanced microbial richness and diversity.

3.4. Species structure analysis

Phylum level: At the phylum level, the gut flora mainly consisted of Firmicutes, Bacteroidota, Proteobacteria, Actinobacteriota, and Verrucomicrobiota. Before treatment, the combination group showed increased Firmicutes and Verrucomicrobiota, and decreased Bacteroidota, Proteobacteria, and Actinobacteriota. After treatment, the combination group had higher Firmicutes, Actinobacteriota, and Verrucomicrobiota, and lower Bacteroidota and Proteobacteria. A significant change in Firmicutes was observed in the press needle group after treatment. All results were statistically significant.

Class level: the top 10 gut flora classes were analyzed. The main classes included Clostridia, Bacteroidia, Gammaproteobacteria, Negativicutes, and Actinobacteria. Before treatment, the combination group showed increased Clostridia and decreased levels of the other four classes. Significant differences were observed pre-treatment: Clostridia in the combination group (P < 0.05) and Bacteroidia in the herbal group (P < 0.05). After treatment, Actinobacteria and Negativicutes increased in the combination group, while the other three decreased. Significant changes in Actinobacteria occurred in both the press needle and herbal groups after treatment. All results were statistically significant.

Order level: at the order level, the top 10 gut flora mainly included Bacteroidales, Lachnospirales, Veillonellales-Selenomonadales, Oscillospirales, and Enterobacterales. Before treatment, the combination group showed increased Lachnospirales and Oscillospirales, and decreased Bacteroidales, Veillonellales-Selenomonadales, and Entero-bacterales. After treatment, the combination group had higher Oscillospirales and Enterobacterales, and lower Bacteroidales, Lachnospirales, and Veillonellales-Selenomonadales. A significant change in Veillonellales-Selenomonadales was observed in the combination group after treatment. All results were statistically significant.

Family level: at the family level, the main gut bacteria included Lachnospiraceae, Enterobacteriaceae, Bacter-oidaceae, Ruminococcaceae, and Prevotellaceae. Before treatment, the combination group showed increased Lachnospiraceae, Bacteroidaceae, Ruminococcaceae, and Prevotellaceae, but decreased Enterobacteriaceae. After treatment, the combination group had higher Rumin-ococcaceae, lower Lachnospiraceae, Bacter-oidaceae, and Prevotellaceae, and unchanged Entero-bacteriaceae. Significant differences were found in Ruminococcaceae (press needle group) and Bacteroidaceae (combination group). All results were statistically significant.

Genus level: at the genus level, the main gut bacteria included Bacteroides, Prevotella, Escherichia-Shigella, Bifidobacterium, and Megamonas. Before treatment, the combination group showed increased Bacteroides and Megamonas, and decreased Prevotella, Escherichia-Shigella, and Bifidobacterium. After treatment, the com-bination group had higher Bacteroides, Bifidobacterium, and Megamonas, and lower Prevotella and Escherichia-Shigella. All reported results were statistically significant.

3.5. Regulation of alpha diversity in intestinal flora

Alpha diversity analysis, including Chao1, Shannon, and Simpson, is used to assess the species diversity in individual samples. The index reflecting community richness is primarily Chao1, while Shannon and Simpson are more reflective of the community diversity. The higher the Shannon index, the more diverse the community. Meanwhile, the larger the Simpson index, the lower the community diversity. Show that alpha diversity varies by comparison within each group. Three treatment groups corresponding to the analyses Chao1, Shannon, and Simpson, showed a decrease in the species abundance after Huatan Lishi prescription and press needle treatments. There was a statistically significant difference in Chao1, Shannon, and Simpson indices before and after the treatment between the needle-herb combination groups (P < 0.05). Although there was no statistical difference between the control group and the three treatment groups, the Simpson index increased after the treatment, suggesting a decrease in species diversity in the control group compared to before the treatment. These results suggest that the combination of Huatan Lishi prescription and press needle treatments improved the species composition of the gut flora and increased their abundance in the subjects.

3.6. Regulation of beta diversity of intestinal flora

The principal coordinates analysis (PCoA) was based on the distance matrix to find potential principal components affecting the differences in the composition of sample communities. There are some differences in the diversity of the intestinal flora of the eight groups when PC1 (27.89%) and PC2 (15.33%) form the horizontal and vertical coordinouts at the OTU levels, respectively. The dispersion of the flora of the control and the three treatment groups is known to be better. While the four groups exhibit differences among them, the three treatments groups show differences before and after the treatment.

3.7. Moderating effect of differences in intestinal flora species

The results of the species analysis of intestinal flora differences in subjects before and after the treatment were compared and a total of seven differential bacteria were screened by the LEfSe analysis. The relative abundance of Firmicutes and Actinobacteriota increased after treatment in the press needle group. After the treatment of the control group, the relative abundance of Oscillospirales, Ruminococcaceae, Eubacterium faecium, and Negativicutes decreased. The relative abundance of Oscillospirales, Ruminococcaceae, Eubacterium faecium, and Negativicutes decreased after the treatment in the Traditional Chinese Medicine group. The relative abundance of Oscillospirales, Ruminococcaceae, Euba-cterium faecium decreased after the needle and herb combination group treatment. Oscillospirales, Rumin-ococcaceae, Eubacterium faecium, Negativicutes, and Bacteroidaceae may be responsible for abdominal obesity, and Actinomycetes may help interfere with abdominal obesity in perimenopausal subjects. These results indicate that the combination of needle and herb treatment improved the species structure of intestinal flora.

4. DISCUSSION

In this study, analysis of gut flora showed that the needle-herb combination improved the 25 dominant intestinal flora across phylum, order, family, and genus levels in individuals with abdominal obesity. The Firmicutes/ Bacteroidota ratio decreased after treatment, suggesting that this change contributed to weight loss.¹²

Furthermore, analyses of OTU-based species richness, alpha and beta diversity, and LEfSe revealed that the needle-herb combination reduced overall species abundance but increased diversity. It significantly decreased Firmicutes and increased Bacteroidota, lowering their ratio. Treatment also reduced Oscillospirales, Ruminococcaceae, and Eubacterium faecium. Specifically, needle therapy increased Bacteroidales and Bacteroidaceae, while the herbal prescription decreased Lachnospirales and Megamonas in abdominally obese subjects. The distinct features of these two interventions offer a basis for further study. PCoA indicates that both the Huatan Lishi prescription and press needle therapy help regulate lipid metabolism, microbial abundance, and diversity. LEfSe analysis suggests that the combined treatment may reshape gut flora by upregulating the abundance and diversity of Bacillus faecium, Bacteroidaceae, and Actinomycetes.

Previous studies show that TCM improves gut microbiota in obesity not only through antimicrobial effects, but also by reducing leptin, regulating hypothalamic neuropeptides, and suppressing white fat production. These actions enhance gut flora and immune response. TCM compounds also offer multi-target therapeutic advantages. Baicalin¹³ and puerarin¹⁴ reduces free fatty acids and inflammation by improving insulin sensitivity and regulating lipid metabolism. Poria cocos polysaccharide and lutein ¹⁵ act on gut flora, enhances peroxisome proliferator-activated receptor gamma expression, inhibits fat cell differentiation, reduces lipopolysaccharide release to protect the intestinal barrier, and promotes weight loss in obesity.¹⁶ increased beneficial bacteria like Bacteroidia, Bifidobacterium, and Lactobacillus, raised the Firmicutes/ Bacteroidetes ratio, and reduced thick-walled bacteria in obese mice.

Acupuncture was shown to reduce abdominal obesity by enhancing pancreatic beta-cell function and insulin production in a study on gut microbiota in obese individuals,¹⁷ enhances glucose use, stimulates organs, and balances glucose-lipid metabolism.¹⁸ Another study found that acupuncture inhibits obesity by modulating hypothalamic pro-opiomelanocortin/Sirtuin1 (SIRT1), activating SIRT1/nuclear factor-kappa B signaling, suppressing inflammation, and improving gut environment.¹⁹ Acupuncture reduced weight by improving leptin resistance and upregulating leptin receptor isoform B expression.²⁰ This aligns with prior research. A clinical study by Koliada involving 61 subjects found a higher Firmicutes/Bacteroidota ratio in obese individuals through macrogenomic analysis.²¹ Probiotics reduced body fat in overweight children in a randomized placebo-controlled trial.²² Seventy obese patients with type II diabetes were treated with the combination of gigagliptin-metformin or glimepiride-metformin combination therapy for 24 weeks and showed a decrease in tumoral bacteria and Clostridium bacteria, with significant differences between treatments.²³ Forty-one obese volunteers who ate one portion of whole-grain pasta daily for 12 weeks and maintained their habitual dietary treatment for four weeks found lower plasma resist in and higher fecal abundance in the subgroup of hyperglycemic volunteers. The mechanisms by which Faecobacter regulates immunity in the colon and reduces intestinal toxin accumulation while increasing lipopolysaccharide may inhibit obesity.²⁴

Mechanism studies found that Actinomycetes contain Bifidobacteria, a probiotic bacteria, and acidify the gut environment to produce vitamins and amino acids that reduce inflammatory responses and protect the intestinal mucosal barrier. Bacterium faecium affects the carbohydrate uptake using polysaccharides, regulates the nutrient uptake, and inhibits obesity formation.²⁵ Animal studies showed an increase in the number of Prevotella, which contains succinic acid, in high-fiber fed mice, possibly preventing abdominal obesity by promoting increased glycogen storage and improved glucose metabolism in individuals.²⁶

In summary, this study demonstrates that combining needle therapy with a herbal formula significantly enhances gut flora diversity and regulates the intestinal environment, leading to reduced abdominal obesity. The treatment resulted in notable decreases in weight, BMI, and waist circumference. Additionally, the natural, food-derived ingredients and non-invasive needle technique ensure ease of use, minimal side effects, and high patient acceptance, supporting its potential for long-term obesity management.

Several limitations in this clinical study should be addressed: (a) High-throughput sequencing provides limited resolution; future work could use metagenomic sequencing to identify flora at the species or strain level for better mechanistic insight. (b) Long-term follow-up data are currently lacking. Further research is needed to explore how acupuncture-herbal combination therapy modulates gut microbiota for weight loss, positioning the gut flora as a novel target for preventing abdominal obesity and related metabolic disorders.

In conclusion, combined needle-herb therapy alleviates abdominal obesity and modulates gut microbiota, reducing F/B ratio and enriching beneficial bacteria. These changes offer novel targets for TCM treatment and support personalized medicine, though deeper mechanistic research remains needed.

Funding Statement

Supported by National Key R&D Program of China: Clinical Evaluation Research on Intervention Technologies for Abdominal Obesity; the Role of Gut Microbiota in the Effects of Combined Acupuncture and Medication on Abdominal Obesity: an Exploration with 16S rRNA Technology (Project Code: 2019YFC1710102)