Study on the Intervention Effect of Qi Gong Wan Prescription on Patients with Phlegm-Dampness Syndrome of Polycystic Ovary Syndrome Based on Intestinal Flora

Abstract

Objective: This was a population-based cohort study, to compare the type and structure of intestinal flora in patients with polycystic ovary syndrome (PCOS) with phlegm-dampness syndrome, nonphlegm-dampness syndrome, and normal population. Besides, changes in the intestinal flora and the clinical curative effects of Jiawei Qi Gong Wan on phlegm-dampness syndrome in patients with polycystic ovary syndrome (PCOS) were evaluated. Patients and Methods. A total of 22 patients with PCOS with phlegm-dampness syndrome, 21 PCOS patients with nonphlegm-dampness syndrome, and 20 control volunteers were selected for this study. The general index, sex hormone index, fasting blood glucose (FPG), and serum fasting insulin (FINS) were determined in each of the groups. The intestinal flora of each group was determined by the 16s rDNA high-throughput sequencing technique. Besides, the PCOS with phlegm-dampness syndrome group was treated with Jiawei Qi Gong Wan, and the changes in TCM syndrome, sex hormone index, BMI, WHR, FPG, FINS, HOMA-IR, and intestinal flora were determined before and after treatment.

Results: PCOS patients with phlegm-dampness syndrome showed characteristics of obesity and insulin resistance. There were several differences in both structure and function of the intestinal flora between PCOS patients with phlegm-dampness syndrome, PCOS patients with nonphlegm-dampness syndrome, and the control group. An imbalance in the intestinal flora may be a key factor in the pathogenesis of PCOS with phlegm-dampness syndrome and also has a specific influence on glucose and lipid metabolism, obesity, and the menstrual cycle in PCOS patients with phlegm-dampness syndrome. Besides, the imbalance is associated with a decrease in the number of butyrate-producing bacteria, an increase in the number of lipopolysaccharide-producing bacteria, and an increase in proinflammatory bacteria. The intestinal flora in PCOS patients with phlegm-dampness syndrome was found to be linked to obesity, impaired glucose tolerance, and hyperandrogenemia. Treatment with Jiawei Qi Gong Wan was found to increase the diversity of intestinal flora, increase the number of intestinal probiotics, and improve the structure and functional genes of intestinal flora which improved the state of insulin resistance, regulated endocrine metabolism, and improved the overall symptoms.

Conclusions: Intestinal flora imbalance is a key factor in the pathogenesis of PCOS with phlegm-dampness syndrome. Besides, Jiawei Qi Gong Wan improves insulin resistance which is linked to the status of the intestinal flora in PCOS patients with phlegm-dampness syndrome.

Figure 1

Dilution curve. Abscissa is the number of sequencing strips randomly selected from a sample, and the ordinate is the number of OTU that can be constructed based on the number of sequencing strips, and it is used to reflect the sequencing depth; different samples are represented by curves of different colors; with the expansion of sequencing quantity, the curve tends to flatten, indicating that the sequencing data are sufficient, and further sequencing does not increase species diversity.

Figure 2

Box diagram showing the differences between groups of α diversity index. Horizontal axis: A.Q: PCOS with phlegm-dampness syndrome group intestinal flora; B: PCOS with nonphlegm-dampness syndrome group intestinal flora; C: control group intestinal flora. Vertical axis: α diversity index.

Figure 3

PCoA analysis of β diversity for weighted UniFrac indexes. The abscissa represents one principal component, the ordinate represents another principal component, and the percentage represents the contribution value of the principal component to the sample difference; each point in the figure represents a sample, and the samples of the same group are represented by the same color. The more similar in terms of colony composition, the closer the distance in the picture.

Figure 4

Each point in the graph represents a sample, the distance between the points indicates the degree of difference, and the samples of the same group are represented by the same color. When stress is less than 0.2, it shows that NMDS can accurately reflect the degree of difference between samples.

Figure 5

The ordinate is the rank of the distance between the samples, and the abscissa refers to the result between the two groups, and the other two are the results within the respective groups. The R value is between (−1, 1), and the R value is greater than 0, indicating that the differences between the groups are significant. The R value is less than 0, indicating that the difference within the group is greater than the difference between the groups. The credibility of the statistical analysis is expressed by P value, and P < 0.05 indicates significant differences.

Figure 6

Venn plot diagram of the OTU distribution in the three groups. Each circle in the figure represents one (group) sample, and the numbers of the circle and the circle overlap represent the number of OTUs shared between the samples (group). The numbers without overlap represent the unique OTUs of the sample (group). A.Q: intestinal flora of PCOS with phlegm-dampness syndrome group; B: intestinal flora of PCOS with nonphlegm-dampness syndrome group; C: intestinal flora of the control group.

Figure 7

The abscissa is the group name; the ordinate represents the relative abundance. (a) Histograms of relative abundance of species at the phyla levels (top10) of the three groups; (b) histogram showing species relative abundance (top10) at the family level in the three groups; (c) histograms showing the relative abundance of species at the level of the genera (top 10) in the three groups; (d) histograms showing the relative abundance of species at the level of the genera (top30) in the three groups.

Figure 8

LDA value distribution histogram shows the species whose LDA score is greater than the set value (the default setting is 4), that is, the biomarker with statistical differences between groups. Species with significantly different abundances in different groups are shown, and the length of the histogram represents the magnitude of the impact of the different species (i.e., the LDA score). In the evolutionary branch diagram, the circle radiating from the inside to the outside represents the classification level from the door to the genera (or species). Each small circle at a different classification level represents a classification at that level, and the diameter of the small circle is proportional to the relative abundance. Coloring principle: the species with no significant difference is uniformly colored yellow, and the different species biomarkers follow the group for coloring. The red node indicates that the microbial group plays an important role in the red group, and the green node indicates that it plays an important role in the microbial groups. If a group is missing in the picture, it means that there are no significant differences in species in this group, and the group is missing. The names of the species are indicated by their English letters in the legend on the right.

Figure 9

Spearman correlation analysis between environmental factors and intestinal flora: red represents positive correlation, and blue represents a negative correlation. Vertical is the environmental factor, horizontal is species information, and the corresponding value in the middle heat map is the spearman correlation coefficient r which is between −1 and 1; r < 0 is a negative correlation, r > 0 is a positive correlation, and ^∗significance level at P value <0.05 and ^∗∗P value <0.01.

Figure 10

In the CCA ordination chart, environmental factors are generally expressed by arrows, and the length of the arrow line represents the degree of correlation between an environmental factor and community distribution and species distribution. The longer the arrowhead is, the greater the correlation, and vice versa. The angle between the arrow line and the sorting axis represents the correlation between an environmental factor and the sorting axis, the smaller the angle, the higher the correlation, and vice versa. When the angle between environmental factors is acute, it means that there is a positive correlation between the two environmental factors, and a negative correlation when the angle is obtuse.

Figure 11

T_test analysis of functional differences between PCOS with phlegm-dampness group and PCOS with nonphlegm-dampness group in the second level.

Figure 12

Horizontal axis: A.Q: PCOS with phlegm-dampness syndrome group: intestinal flora before treatment; A.H: intestinal flora of PCOS with phlegm-dampness syndrome group after treatment. Vertical axis: alpha diversity index.

Figure 13

β diversity PCoA analysis of weighted UniFrac before and after treatment.

Figure 14

(a) Histogram of species relative abundance at the phylum level before and after treatment (top10); (b) histogram of species relative abundance at the genus level before and after treatment (top10).

Figure 15

Histogram and evolutionary branching diagram of LDA value distribution of sample flora before and after treatment.

Figure 16

T-test analysis of differential function at level 2 before and after treatment.