Comparison between Traditional Chinese Medicine Constitution and Blood Biochemical Markers Associated with Left and Right Mammary Hyperplasia in Rural Areas of Southwest China.

Background: Hyperplasia of mammary glands (HMG) is the breast disease with the highest clinical incidence. Many traditional Chinese medicine (TCM) doctors suggest that the treatment of HMG should be based on the left and right breast pain difference. However, these views are based on case reports, and an objective basis has not been established for treatment according to left-side and right-side differences.
Methods: We enrolled 150 patients who met the clinical diagnostic criteria of HMG. The incidence bias was determined according to the score difference between bilateral breast pain and mass in patients with HMG. A left group, right group, and bilateral group were included, and TCM constitution was investigated in each group. Blood biochemical indicators were measured for 120 fasting patients. We conducted a network pharmacology study of the key herb qingpi and chenpi, which are used by TCM doctors to treat different lateral HMG.
Results: In patients with biased onset of HMG, the results showed that the frequency and constitution score of stagnant blood in the L group were higher than those of the R group, and the frequency and constitution score of phlegm-dampness in the R group were higher than those of the L group. Both the L and R groups had high proportion of stagnant Qi. The results indicated that the concentration of coagulation factor VIII (FVIII) was higher in the L group than that in the R group, and the concentration of lipoprotein a (Lp-α) was higher in the R group than that in the L group. The results showed that sinensetin and neohesperidin contained in qingpi might interfere with platelet activation, thrombogenesis, prolactin signaling pathway, and atherosclerosis process, in removing "blood stasis" and eventually treating the left-leaning group of HMG patients. Sitosterol and citromitin contained in chenpi could regulate lipid metabolism by interfering with regulation of lipolysis in adipocytes, salivary secretion, estrogen signaling pathway, and thyroid hormone signaling pathway. Chenpi could eliminate "phlegm turbidity" and treat HMG patients in the right-leaning group. Conclusions: We preliminarily confirmed that the clinical pathogenesis of HMG is not a left-right equilibrium and TCM constitution, coagulation function, and lipid metabolism may be used as the objective basis for the difference between the left and right in HMG pathogenesis. For left-sided HMG patients, the doctor can consider qingpi, herb of activating blood and removing stasis, to treat HMG. However, for right-sided HMG, we think doctors should apply herb of activating Qi and eliminating phlegm, such as chenpi.

1. Introduction

Hyperplasia of mammary gland (HMG), also known as breast dysplasia, is a benign breast disease caused by abnormal development and degeneration of breast tissue that is not a result of inflammation or tumor [1]. The occurrence of this disease is affected by reproductive risk factors such as diet, emotion, nonpregnancy, and lack of breastfeeding [2]. The incidence of HMG is high in young and middle-aged women [3, 4], accounting for about 70% of breast diseases in middle-aged women [5]. The incidence of the disease is on the rise yearly [6], particularly among young individuals. Kader et al. [7] found that the risk of breast cancer in women with atypical breast hyperplasia was about four times higher than the risk for women without hyperplasia, and the risk of breast ducts and breast lobules was also increased. Treatment of breast hyperplasia and its progression is an effective method to prevent breast cancer [8].

The theory of “left blood and right Qi” and “left stasis and right phlegm” has been a part of TCM since ancient times. It is believed that the human body is not completely balanced and symmetrical, and there are physiological and pathological differences between the left and the right side. In ancient Chinese medical books, many patients are described as having diseases that exhibited left- and right-side differences. By continuous accumulation of experience in treating diseases, TCM doctors have summed the differences for many side-dependent diseases and the corresponding differential treatments. The “Suwen” [9] stated that the redness of the left cheek was caused by liver-related heat diseases, whereas the redness of the right cheek was caused by lung-related heat diseases. “ZhangshiYitong” [10] indicated that the left flank and rib pain was caused by blood stasis, and the right flank and rib pain was caused by phlegm and turbidity. In “DanxiXinfa” [11], it was asserted that prescriptions should be used to promote blood circulation and remove blood stasis for hemiplegia on the left side, and prescriptions should be used to reduce phlegm and promote Qi for hemiplegia on the right side.

Modern medicine proposes that human embryos show a high degree of internal asymmetry in their left and right axes in the early stages of development [12]. This left-right axis asymmetry occurs both in paired and unpaired organs [13]. There are left- and right-sided differences in many diseases, and the epidemiological, clinical, and histological parameters, and prognosis of different sides varies, including varicocele [14], inguinal hernia [15], colon cancer [16-18], ischemic stroke, and carotid atherosclerosis [19]. For example, Naess et al. [20] found that the infarction rate of the left cerebral hemisphere was significantly higher than that of the right cerebral hemisphere. Fink et al. [21] found that patients with left-sided stroke had better prognosis than right-sided stroke after 3 months in a cohort of 384 patients with ischemic stroke treated with intravenous thrombolysis. Previously, the National Institutes of Health Stroke Scale (NIHSS) also proposed that lateral factors should be taken into account when evaluating stroke prognosis [22]. Clinical statistics have found that varicoceles are more common on the left side than on the right side. The anatomical explanation is that varicoceles are caused by the dysfunction of the varicocele valve. Physiological anatomical asymmetry makes blood more likely to accumulate in the left blood vessels [14], which is consistent with the view of TCM that blood stasis mostly occurs in the left side of the human body. TCM suggests that ischemic stroke is caused by the mutual accumulation of phlegm and blood stasis, blocking the cerebral collaterals. Blood stasis is more serious than phlegm in the patients with left stroke, so the program of promoting blood circulation and removing stasis should be used. Phlegm is more serious than blood stasis in the patients with right stroke, and the program of activating Qi and eliminating phlegm. Modern medicine uses thrombolytic therapy for left cerebral stroke patients and has achieved better prognosis than in right cerebral stroke. It also demonstrates the rationality of the theory of “left stasis and right phlegm” from a clinical perspective. This “modern medicine-left cerebral apoplexy-thrombolysis-good prognosis” and “TCM-left stasis-symptomatic treatment-promoting blood circulation and removing blood stasis” are essentially the same.

Similarly, TCM doctors contend that the incidence of HMG is also different between the left and the right. Patients with different breast hyperplasia have different scores of symptoms and signs in the left and right breasts. Some symptoms are more serious on the left side, some are more serious on the right side, and some are similar on both sides. Lu and Jinmo [23], Qiaxi and Ruiting [24], and Fan and Liu [25] reported that the lesions of the left breast with severe disease lay more stress on blood stasis, and the treatment should attach importance to removing blood stasis and promoting blood circulation, and the treatment with Citri Reticulatae Pericarpium Viride (qingpi) has significant effects. The disease of the right breast is serious, and its pathological changes are more important to Qi stagnation and sputum coagulation, so we should focus on regulating Qi and resolving phlegm, and the treatment with Citrus reticulata (chenpi) has significant effects. These case reports suggest that there may be differences in pathogenesis between left and right HMG and selection of key herbs for treatment. However, no systematic investigation or statistical studies have been performed, and no specific evaluation indices have been found to objectively confirm the reported differences.

TCM constitution is formed on the basis of innate endowment and acquired nourishment of human body. Physical constitution is an individualized physical condition that predisposes people to certain diseases [26]. Influenced by natural and social environment, physical constitution changes dynamically with physiological and pathological states in the process of growth [27, 28]. Therefore, physical constitution is adjustable. Furthermore, TCM constitution is focused on the integrity of the person. The physiological and pathological state of the patient in the past year does not fluctuate due to the mood and diet state in a short period of time, so TCM constitution has stability. This degree of stability and relative adjustability gives the theory of constitution recognition a significant advantage in the study of chronic diseases. The occurrence and development of chronic diseases are closely related to biased constitution [29-31]. There are differences in the applicable regulation methods for patients with different constitutions [32-34].

Therefore, with the constitution theory of TCM as the starting point, we measured blood biochemical indices of HMG patients to assess the differences in the constitutions on different sides, and to guide individualized precise treatment [32, 35].

2. Material and Methods

2.1. Participants

We enrolled 150 patients with HMG who were admitted to Traditional Chinese Medicine Hospital Dianjiang Chongqing from August 2018 to February 2019. The inclusion criteria were the following: (1) Patients met the diagnostic criteria of HMG. In China, TCM and modern medicine adopt the same diagnostic criteria for HMG [36, 37]. (2) Patients were less than 56 years of age. (3) Patients did not receive treatment related to HMG within three months before enrollment. (4) Patients provided informed consent. Exclusion criteria were the following: (1) Patients with physical hyperplasia of the breast during puberty, pregnancy, and lactation. (2) Patients with benign and malignant breast tumors and inflammatory diseases. (3) Patients with serious primary diseases of the liver, heart, lung, kidney, and blood system. (4) Patients with serious systemic diseases or mental disorders. Our study was certified on May 9, 2018, and the Ethics Committee of Traditional Chinese Medicine Hospital Dianjiang Chongqing approved the study (ZY201802097).

2.2. Data Collection

The investigators collected the basic clinical information of patients and recorded the pain scores of bilateral mammary glands. The patients were examined by color ultrasonography of breast, and the data of breast gland thickness, breast duct, breast mass, blood flow changes, and bilateral axillary lymph nodes were recorded. The breast pain and lumps grading quantitative criteria (Supplementary Table 1) were completed according to the pain score of the patient's bilateral mammary glands and the mass data from ultrasound detection. The bias of the onset of HMG was determined by referring to the method of Lu and Huang Mei [38], that is, the difference of the total scores of symptoms and signs between the left and right breasts in the same breast patient was ≥9 points. We think patient belongs to the left group, when left breast score higher than the score of 9 points or more right breast, and vice versa. If the difference between the scores of the two breasts is less than 9 points, it is considered to be bilateral group. If one side of the breast did not meet the diagnostic criteria for breast hyperplasia, that is, no breast pain, no breast mass, and no abnormal hyperplasia observed by color Doppler examination, the score of HMG on that side was designated as 0. There were 53 left group, 40 right group, and 57 bilateral group patients.

According to the standard of Classification and Determination of TCM Constitutions [39, 40], 150 patients were evaluated for their TCM constitutions, and the types of constitutions were determined. The TCM constitutions survey scale was completed (Supplementary Table 2), and the TCM constitutions data of the patients were collected. 120 patients fasted and provided blood samples (elbow venous blood) to measure blood biochemical indicators, including blood coagulation routine, blood lipid, renal function, and liver function.

2.3. Analysis of Chinese Medicine Compounds and Their Targets

On the basis of the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (https://old.tcmsp-e.com/tcmsp.php) [41], we collected the identified compounds and related targets in qingpi and chenpi, and the compounds were screened with the basic threshold (oral bioavailability (OB)≥30%, drug-likeness (DL) > 0.16, intestinal epithelial permeability (Caco-2) >−0.04) [42, 43]. We used the “Clusterprofiler” and “org.hs.eg.Db” package to complete Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for the herb-related targets, based on R.studio.

2.4. Data Statistics

Descriptive statistics for baseline characteristics, nine TCM constitution scores, and blood index values were provided. Continuous variables, normally distributed variables, non-normally distributed variables, and categorical variables were described by means, standard deviations (SDs) and medians, interquartile ranges (IQRs), and frequencies and percentages, respectively. Between-group differences were reported using the one-way analysis of variance (ANOVA), chi-square (χ2) test, fisher's exact test or Kruskal–Wallis H test, as appropriate, and 95% confidence intervals (CIs) were calculated. If the main effect of ANOVA or Kruskal–Wallis H test was statistically significant, further post hoc two-by-two comparisons were performed, with the multiple comparison method used being Student Newman Keuls method for ANOVA and Holm's sequential Bonferroni procedure for Kruskal–Wallis H test.

All analyses were performed using R (version 4.0.5). All statistical tests were two-sided, and P < 0.05 was considered as statistically significant.

3. Results

3.1. Age

The average age of the 150 patients with HMG was 39.66 ± 9.86 years (range 19–55 years). The age of HMG patients included in this study was divided according to the “Tiangui cycle” of TCM, with age division every 7 years. Figure 1 shows that there were differences in the distribution of 150 HMG patients in different age groups (P < 0.01), and many patients were in the “six-seven” stage. Table 1 shows that there was no difference in the proportion and age of each group.

Figure 1

Tiangui cycle of age distribution in 150 patients (cases). Chi-square (χ2) test; overall comparison of 6 age groups; P < 0.01. Two-seven stage: 14–20 years old; three-seven stage: 21–27 years old; four-seven stage: 28–34 years old; five-seven stage: 35–41 years old; six-seven stage: 42 to 48 years old; seven-seven: 49–55 years old.

Table 1

Age distribution in patients (x ± s).

Group	Case (%)	Age (years)
L	53 (35.33)	40.21 ± 9.51
R	40 (26.67)	38.27 ± 10.65
D	57 (38.00)	40.12 ± 9.68
P value	0.206	0.586

Chi-square (χ2) test, one-way analysis of variance (ANOVA). L, left; R, right; D, bilateral.

3.2. Basic Breast Data of Patients

Table 2 lists the basic information of the breasts of patients with HMG. Of the 150 patients in the study, three patients were diagnosed with HMG in only one breast, and no HMG was found in the other breast. Therefore, 297 breasts were included, and 57 patients were identified as bilateral (D), 40 patients as right-sided (R), and 53 patients as left-sided group (L). In group L, pain and mass scores were significantly higher in HMG patients in the left breast than in the right breast (P < 0.05). In group R, HMG patients had significantly higher pain and mass scores in the right breast than in the left breast (P < 0.05). In group D, there was no statistically significant difference in pain and mass scores between the left and right breasts of HMG patients.

Table 2

The distribution of breast data (x ± s).

Group	Case	Breast	Breast pain and lump score (score)		Thickness of breast glands (cm)
			Left breast	Right breast	Left breast	Right breast
L	53	104^	33.42 ± 6.78	18.74 ± 6.78a	1.20 ± 0.49	1.17 ± 0.43
R	40	79∗	17.55 ± 5.06	31.57 ± 5.52a	1.27 ± 0.50	1.25 ± 0.52
D	57	114	28.37 ± 6.34	28.21 ± 5.78	1.15 ± 0.35	1.16 ± 0.37

Comparison of left and right breasts in the same group was performed by the paired T test. aIn the same group, there was a difference between the right breast and the left breast, P < 0.05. ^51 patients had hyperplasia on both sides of the breast, and 2 patients had hyperplasia only on the left side of the breast. There was no pain or mass on the right side of the breast, and no pathological hyperplasia was found by color ultrasound examination.There were 39 patients with bilateral hyperplasia, and 1 patient had hyperplasia of only the right breast, without pain or mass of the left breast, and no pathological hyperplasia was found in the breast color ultrasound examination.

As shown in Tables 3 and 4, axillary lymph nodes were commonly seen in patients with breast hyperplasia, whereas breast ducts were rarely dilated. There were no significant differences in the detection of lymph nodes and breast ducts between the groups.

Table 3

Node detection in three groups.

Group	Total breast	Breasts with lymph nodes visible
		Left breast	Right breast
L (N = 53)	104	3.77% (2/53)	3.92% (2/51)
R (N = 40)	79	15.00% (6/40)	5.13% (2/39)
D (N = 57)	114	14.04% (8/57)	7.02% (4/57)
P value	—	0.117#	0.900#

#Fisher's exact test.

Table 4

Distribution of pathological status of patients' breast ducts.

Group	Total breast	Breasts with ducts dilated
		Left breast	Right breast
L (N = 53)	104	3.77% (2/53)	3.92% (2/51)
R (N = 40)	79	2.50% (1/40)	2.56% (1/39)
D (N = 57)	114	5.26% (3/57)	5.26% (3/57)
P value	—	0.884#	0.879#

#Fisher's exact test.

3.3. Overall Distribution of TCM Constitution of HMG Patients

Compound constitution means that two or more constitutions are detected in the same person. Single constitution means that only one constitution is detected. According to the results of TCM constitutions, 134 patients had compound constitutions and 16 patients had single constitutions. Among the patients with single constitution, there were 12 patients with single balanced constitution, 3 patients with single stagnant blood constitution, and 1 patient with single stagnant qi constitution. Figure 2 shows the proportions of compound constitution and single constitution.

Figure 2

Patient compound constitution and single constitution and detection status.

The most detected constitution was stagnant Qi constitution (117 patients), followed by 102 patients with stagnant blood constitution, and 10 patients with stagnant blood constitution. Figure 3 shows the proportion of nine TCM constitutions detected.

Figure 3

Percent of TCM constitution.

3.4. TCM Constitution Distribution and Score of Three Groups

Table 5 shows the frequency and percentage of each TCM constitution of HMG patients in L, R, and D groups. The frequency of stagnant blood, phlegm-dampness, and stagnant Qi in the three groups varied, and the overall difference was statistically significant (P < 0.05). As shown in Table 6, the stagnant blood, phlegm-dampness, and stagnant Qi constitution scores of the three groups were not identical, and the overall difference was statistically significant (P < 0.05). The focus was on comparing the L group and R group with laterality of HMG. Results showed that the frequency and constitution score of stagnant blood in the L group were higher than those of the R group, and the frequency and constitution score of phlegm-dampness in the R group were higher than those of the L group. Both the L group and the R group had high proportion of stagnant Qi, where the L group was 86.79% and the R group was 70.00%. Taking into account the two types of constitutions with the highest frequency and proportion in L and R groups, HMG patients may have the characteristics of “group L-stagnant Qi-stagnant blood” and “group R-stagnant Qi-phlegm-dampness.” In HMG patients with biased onset, the constitution score may have the characteristics of severe blood stasis in the L group and severe phlegm-dampness in the R group.

Table 5

Distribution of TCM constitution types of patients with HMG, n (%).

TCM constitution	L (N = 53)	R (N = 40)	D (N = 57)	P value
Balanced	9 (16.98)	9 (22.50)	13 (22.81)	0.712
Qi-deficient	25 (47.17)	9 (22.50)	21 (36.84)	0.050
Yang-deficient	20 (37.74)	11 (27.50)	25 (43.86)	0.260
Yin-deficient	15 (28.30)	8 (20.00)	12 (21.05)	0.564
Phlegm-dampness	11 (20.75)b	28 (70.00)a	17 (29.82)b	<0.001
Damp-heat	15 (28.30)	16 (40.00)	18 (31.58)	0.480
Stagnant blood	43 (81.13)a	17 (42.50)b	42 (73.68)a	<0.001
Stagnant qi	46 (86.79)a	28 (70.00)b	43 (75.44)b	0.002
Inherited special	4 (7.55)	1 (2.50)	5 (8.77)	0.514#

#Fisher's exact test. For each indicator, if the overall comparison among the three groups was statistically significant (P < 0.05), further pairwise comparisons were performed. The same letter indicates that the difference is not statistically significant, and the different letters indicate that the difference is statistically significant. Because the patients had complex constitution, the total number of constitution types (511) exceeded the total number of patients (150).

Table 6

TCM physique scores of patients.

TCM constitution	L (N = 53)	R (N = 40)	D (N = 57)	P value
Balanced	56.00 (41.00; 75.00)	69.00 (53.00; 72.75)	63.00 (50.00; 72.00)	0.311
Qi-deficient	25.00 (16.00; 38.00)	22.00 (15.25; 28.75)	22.00 (16.00; 38.00)	0.414
Yang-deficient	25.00 (11.00; 43.00)	18.00 [11.00; 33.00]	25.00 (11.00; 43.00)	0.322
Yin-deficient	22.00 (13.00; 31.00)	14.50 (9.00; 25.00)	19.00 (9.00; 28.00)	0.490
Phlegm-dampness	22.00 (13.00; 28.00)b	39.50 (25.00; 44.75)a	22.00 (16.00; 34.00)b	<0.001
Damp-heat	21.00 (8.00; 33.00)	25.00 (13.00; 38.00)	25.00 (13.00; 33.00)	0.299
Stagnant blood	41.74 ± 14.22a	29.40 ± 16.07b	40.86 ± 15.29a	<0.001
Stagnant Qi	46.06 ± 14.60a	37.77 ± 16.58b	40.72 ± 15.68b	0.034
Inherited special	7.00 (0.00; 21.00)	5.50 (0.00; 14.00)	7.00 (0.00; 18.00)	0.548

#Fisher's exact test. For each indicator, if the overall comparison among the three groups was statistically significant (P < 0.05), further pairwise comparisons were performed. The same letter indicates that the difference is not statistically significant, and the different letters indicate that the difference is statistically significant.

3.5. Blood Biochemical Indicators

Table 7 shows the test results of blood indices of HMG patients in the three groups. There were differences in the measurement of coagulation factor VIII (FVIII), lipoprotein a (Lp-α), serum creatinine (SCr), uric acid (Uric), cystatin C (Cys C), and β 2-microglobulin (β2MG) among the three groups (P < 0.05). Considering that the measurements of SCr, URIC, Cys C, and β2MG in all groups were within the normal physiological range, and the influence of renal function-related indices on the pathogenesis of HMG may not be as good as that of lipid metabolism indices and coagulation-related indices. Therefore, we focused on the FVIII and Lp-α of HMG patients in L and R groups. Table 7 shows that the concentration of FVIII was higher in the L group than in the R group, and the concentration of Lp-α in the R group was higher than in the L group.

Table 7

HMG patient blood indicators.

	Biochemical indicators	L	R	D	P value
		(N = 41)	(N = 30)	(N = 50)
Routine blood test	WBC (109/L)	5.53 (4.97; 6.57)	6.02 (4.84; 7.49)	5.58 (4.74; 6.84)	0.741
	RBC (1012/L)	4.40 ± 0.50	4.35 ± 0.49	4.22 ± 0.43	0.171
	HB (g/L)	130.00 (123.00; 138.00)	129.00 (120.00; 134.75)	128.50 (120.50; 133.75)	0.854
	PLT (109/L)	210.00 (190.00; 251.00)	219.00 (185.00; 254.00)	209.50 (180.00; 261.75)	0.932
	NEUT (109/L)	66.64 ± 11.59	61.53 ± 11.91	65.20 ± 7.96	0.116
	LYM (109/L)	25.07 ± 9.65	29.30 ± 10.28	25.60 ± 8.05	0.126
	MONO (109/L)	5.86 ± 2.00	6.53 ± 2.15	6.38 ± 1.42	0.248
	EO (109/L)	1.30 (0.70; 2.70)	1.45 (0.83; 1.98)	1.30 (0.70; 2.38)	0.973
	BASO (109/L)	0.50 (0.40; 0.60)	0.50 (0.32; 0.70)	0.40 (0.30; 0.60)	0.813
	HCT (%)	39.70 (37.20; 41.90)	40.05 (36.92; 41.58)	39.55 (37.28; 41.27)	0.752
Routine coagulation tests	PT (s)	10.60 (10.20; 10.80)	10.60 (10.10; 10.90)	10.50 (10.20; 10.95)	0.924
	APTT (s)	25.20 (23.20; 27.70)	26.15 (25.13; 27.32)	25.90 (24.00; 27.50)	0.442
	Fib (g/L)	2.76 (2.44; 3.24)	2.83 (2.40; 3.33)	2.53 (2.21; 3.52)	0.752
	TT (s)	17.35 ± 1.15	17.22 ± 1.20	17.77 ± 0.96	0.059
	FVIII (IU/dL)	155.90 (114.80; 177.00)a	115.05(95.33; 134.20)c	132.00 (112.10; 155.15)b	0.001
Renal function tests	Urea (mmol/L)	4.44 ± 1.26	4.45 ± 1.20	4.62 ± 1.35	0.757
	SCr (umol/L)	59.00 (50.00; 62.00)a	53.60 (49.38; 56.75)b	59.00 (54.00; 64.50)a	0.02
	URIC (umol/L)	236.00 (209.00; 280.00)b	251.50 (209.75; 292.75)ab	274.00 (237.25; 310.25)a	0.04
	Cys C (mg/L)	0.84 (0.80; 0.88)a	0.78 (0.74; 0.84)b	0.86 (0.79; 0.94)a	0.023
	β2MG (mg/L)	1.31 (1.23; 1.58)ab	1.21 (1.12; 1.36)b	1.42 (1.26; 1.61)a	0.022
Blood lipids tests	TG (mmol/L)	1.18 (0.82; 1.51)	1.35 (0.92; 1.58)	1.17 (0.85; 1.76)	0.905
	TC (mmol/L)	4.19 ± 0.68	4.68 ± 1.06	4.40 ± 1.04	0.105
	HDL (mmol/L)	1.42 (1.12; 1.70)	1.64 (1.39; 1.84)	1.50 (1.24; 1.73)	0.196
	LDL (mmol/L)	2.63 ± 0.67	3.05 ± 1.05	2.83 ± 0.91	0.140
	APOA1 (g/L)	1.45 ± 0.21	1.56 ± 0.23	1.52 ± 0.25	0.134
	APOB (g/L)	0.82 ± 0.20	0.93 ± 0.31	0.87 ± 0.25	0.210
	Lp-α (nmol/L)	18.00 (5.00; 50.40)b	57.75 (8.27; 127.25)a	11.50 (7.20; 37.50)b	0.032
Liver function tests	TP (g/L)	75.20 ± 5.81	75.20 ± 5.87	73.89 ± 4.74	0.427
	ALB (g/L)	47.09 ± 4.11	47.58 ± 3.78	45.92 ± 3.14	0.111
	GLB (g/L)	28.40 (25.60; 30.60)	26.85 (25.23; 29.60)	28.00 (26.42; 30.28)	0.626
	A/G	1.70 ± 0.24	1.75 ± 0.26	1.67 ± 0.25	0.351
	ALT (U/L)	13.20 (10.40; 16.20)	15.85 (12.27; 20.15)	14.50 (11.53; 19.77)	0.066
	AST (U/L)	17.40 (14.70; 19.90)	18.55 (14.90; 22.92)	19.20 (15.95; 23.20)	0.165
	AST/ALT	1.29 (1.07; 1.62)	1.19 (0.90; 1.43)	1.22 (1.04; 1.47)	0.315
	TBIL (umol/L)	8.90 (7.30; 12.50)	9.60 (6.58; 11.80)	8.35 (6.23; 12.47)	0.753
	DBIL (umol/L)	3.50 (2.90; 4.70)	3.55 (3.02; 4.65)	3.40 (2.62; 4.47)	0.703

One-way analysis of variance (ANOVA), Kruskal–Wallis H test. For each indicator, if the overall comparison among the three groups was statistically significant (P < 0.05), further pairwise comparisons were performed. The same letter indicates that the difference is not statistically significant, and the different letters indicate that the difference is statistically significant.

3.6. Network Pharmacology of Qingpi and Chenpi

According to the network pharmacology inclusion conditions we set, five natural compounds were obtained from qingpi and chenpi each. Naringenin, 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl) chroman-4-one, and nobiletin are the common compounds of qingpi and chenpi. In addition, the qingpi contains sinensetin and neohesperidin, while chenpi contains sitosterol and citromitin. On the basis of KEGG enrichment analysis (Figure 4(a)) and the targets of qingpi compounds, we found the following results in KEGG enrichment analysis for qingpi: hepatitis B, platelet activation, fluid shear stress and atherosclerosis, prolactin signaling pathway, and aldosterone synthesis and secretion. Chenpi, a herb for regulating Qi and resolving phlegm, is associated with the following pathways (Figure 4(b)): neuroactive ligand-receptor interaction, PI3K-Akt signaling pathway, regulation of lipolysis in adipocytes, salivary secretion, estrogen signaling pathway, and thyroid hormone signaling pathway.

Figure 4

KEGG enrichment analysis of herbal targets (a) Qingpi in KEGG enrichment analysis; (b) Chenpi in KEGG enrichment analysis. The abscissa represents the degree of association between the pathway and the target, the ordinate represents the name of the pathway, the size of the dot represents the number of genes, and the color represents the P value.

4. Discussion

TCM believes that Tiangui is a special subtle substance that promotes human growth, development, and reproduction, and the female “Tiangui cycle” divides women's physiological stages with every 7 years as an age span. The number of HMG patients in the “six-seven” stage (42–48 years old) is the largest, which is basically consistent with the HMG-prone populations announced by various institutions. The theory of “Tiangui” holds that when a woman reaches the age of 14, she enters the “two-seven” stage. At this stage, Tiangui is produced, women gradually establish a menstrual cycle, female breasts are plump, secondary sexual characteristics become more obvious, and women initially have fertility. After the age of 42, women enter the “six-seven” stage, Tiangui gradually weakens, female reproductive function weakens, and women appear fatigued and develop weakness, lackluster complexion, hair whitening, menstrual disorders, and other symptoms of Qi and blood deficiency, which is similar to the condition “perimenopause syndrome” used in modern medicine. At this stage, the level of sex hormones in the patient is disordered, and the periodical hyperplasia and involution of the mammary glands are unbalanced. Therefore, it is seen that “six-seven” is the age group with the highest risk of HMG. After the age of 49, women enter the “seven-seven” stage, and Tiangui is depleted. Most women undergo menopause at this age, their breasts sag, their bodies age, and they lose their fertility. It can be seen that the “Tiangui” cycle theory of TCM is more in line with the fluctuations of female reproductive function, endocrine, and hormone levels throughout the reproductive life. Therefore, we propose to use the “Tiangui cycle,” that is, to divide the incidence of women's diseases in the interval of 7 years, rather than the commonly used 10 years old, which may have more clinical value.

The results of our study suggest that HMG patients are mainly composed of compound constitution, and a small number of patients have single constitution. We consider that this result may be related to sample source. All study patients were from the rural areas in southwestern China. This region is hot in summer and cold in winter, and it has high humidity all year. The topography is mainly mountainous and hilly, and the diet is complex. Combined with the complex climate conditions, many factors complicate the pathogenesis of HMG. Therefore, the TCM constitution of most HMG patients in this region is compound constitution. China has a vast territory, a large latitude span, and a huge difference in climate between the north and the south. The results obtained by the epidemiological information collected by research teams in different regions of local HMG patients may be different. For example, in the TCM constitution of HMG patients in Beijing published by a research team in northern China, the constitution of Yang and Yin deficiency is more obvious than that of southern patients [44-46]. We think that this may be related to the cold and relatively dry climate in northern China because cold can damage the Yang Qi of the human body, causing patients to have a yang deficiency constitution, while a dry climate can damage the body fluids of the human body, causing patients to have a Yin deficiency constitution. The results of the study on the constitution of HMG patients in Guangdong Province published by the Southern China team suggest that the frequency of phlegm-dampness and damp-heat patients is higher than that in the North [47-49], which may be related to the damp-heat climate in Guangdong. By comparing the differences in TCM constitution of HMG patients in different regions, we suggest that Chinese doctors should consider geographical, climate, and other objective conditions when formulating consensus on HMG diagnosis and treatment and adjust treatment plans and TCM constitution conditioning methods accordingly.

TCM doctors generally assert that obstruction of breast meridians caused by Qi stagnation, phlegm coagulation, and blood stasis are the core pathological links. The TCM constitution determination of HMG patients indicated that, 117 of the 150 patients had stagnant Qi constitution. As the TCM constitution with the highest detection rate, the stagnant Qi constitution was the primary risk factor for the occurrence of HMG, which agreed with the research of Jansen and Muenz [50]. HMG patients are generally characterized by nervousness, irritability, and restlessness. Therefore, the key to the prevention and treatment of breast diseases is to formulate therapeutic strategies from an etiological point of view, regulate emotions, smooth the human body Qi, and ensure the smooth circulation of breast Qi, blood, and body fluids.

Our findings suggest that HMG patients might have the characteristics of “group L-stagnant Qi-stagnant blood” and “group R-stagnant Qi-phlegm-dampness.” There may be serious blood stasis in the L group and serious phlegm-dampness in the R group. Blood stasis and phlegm turbidity block breast ducts and are the core link between pain and mass in HMG patients. Blood stasis and phlegm turbid reflect that breast has a phenomenon that the tissue exists obstacle of blood circulation and lymphatic circulation.

The concentration of FVIII in the L group was higher than the concentration in the R group, and Lp-α in the R group was significantly higher than the concentration in the L group. As one of the objective indicators of blood stasis, coagulation factor is the guarantee of local microcirculation. Previous reverse validation of drug efficacy showed that lipid metabolic disorder is the basis of phlegm turbidities, and increased serum lipid content can be used as an indicator for microscopic syndrome differentiation of phlegm turbidities [51]. Lp-α might reflect the essence of phlegm in the R group.

Therefore, stagnant blood constitution and blood coagulation indicator represented by FVIII were significantly correlated with the incidence of left-leaning HMG patients. Phlegm-dampness constitution and lipid metabolism index represented by Lp-α were significantly correlated with the incidence of right-leaning HMG patients.

The plant sources of qingpi and chenpi are the same. However, due to the differences in fruit maturity, harvest time, storage period, and processing method of qingpi and chenpi, there are differences in the types and contents of their chemical components [52, 53]. According to our network pharmacology inclusion conditions for qingpi and chenpi, the natural compounds contained in each of them are analyzed. The qingpi contains sinensetin and neohesperidin, while the chenpi contains sitosterol and citromitin.

The representative compound of qingpi, neohesperidin, a flavonoid glycoside with a strong bitter taste [54], is also one of the most important natural flavonoid glycosides, with strong anti-inflammatory, anticancer, and cardiovascular protective effects [55]. Relevant research results show that neohesperidin can significantly inhibit the proliferation of breast cancer (MCF-7 cells) in a dose-dependent manner [56]. Sinensetin has been shown to significantly inhibit breast cancer resistance protein (ABCG2 or BCRP), thereby improving the clinical efficacy of breast cancer [57]. Both of these natural compounds have a well-defined targeting effect on the mammary gland. In addition, sinensetin possesses vasodilatory activity [58], strong antiangiogenic activity, and low toxicity [59], and a well-defined antiplatelet adhesion activity [60]. The results of KEGG enrichment analysis suggested that the action pathways of qingpi compounds include platelet activation, fluid shear stress and atherosclerosis, prolactin signaling pathway, and aldosterone synthesis and secretion. Activation of the platelet activation pathway [61] can significantly affect coagulation time and thrombogenesis, which may be the main mechanism of the effect of qingpi to achieve therapeutic goals in the L group. Fluid shear stress and atherosclerosis are pathways related to blood viscosity [62], and qingpi can influence this pathway to remove blood stagnation. The aldosterone synthesis and secretion pathway can significantly affect platelet aggregation, fibrinolysis, and thrombus formation by way of halocorticoid receptor [63]. Therefore, sinensetin and neohesperidin contained in the qingpi may promote blood circulation and remove blood stasis in patients with left-sided HMG by acting on these pathways.

The representative compound in chenpi, sitosterol, whose chemical structure is similar to mammalian cell-derived cholesterol [64], is a special phytochemical that only exists in plants [65]. Many in vitro and in vivo studies have proven that sitosterol has antianxiety, lipid-lowering, and hepatoprotective effects [64]. It has been reported that intake of a diet rich in sitosterol can reduce the risk of cancer by 20% [66], especially in in vitro experiments, and sitosterol is effective in human lung cancer (A549 cells) [67], colon cancer (HT116 cells) [68], and breast cancer (MCF-7 cells) [69] and have antiproliferative effect. It has also been reported that citromitin may be the core compound of the core TCM in expectorants [70]. The results of KEGG enrichment analysis indicated that the action pathways of chenpi compounds include estrogen signaling pathway, thyroid hormone signaling pathway, and regulation of lipolysis of adipocytes. Abnormal secretion of estrogen can constantly stimulate the mammary gland tissue and induce hyperplasia, which is the most important factor in the pathogenesis of HMG [71]. The estrogen signaling pathway is related mainly to lipid metabolism, and the regulation of lipolysis in adipocytes is of significance for the decomposition and redistribution of fats [72]. The thyroid hormone signaling pathway can affect brown adipose tissue function; activation of this pathway increases fatty acid oxidation and mitochondrial biogenesis, which regulates lipid content and distribution [73]. Elevated lipid is not only the main characteristic of phlegm but also is the biochemical basis of phlegm [51]. The effect of chenpi on regulating blood lipid can eliminate phlegm. Therefore, sitosterol and citromitin contained in chenpi may be conducive to regulate Qi and resolve phlegm in patients with right-sided HMG through the action of these targeted pathways.

According to TCM constitution, blood lipid and coagulation index of HMG patients as well as the network pharmacological analysis of qingpi and chenpi comprehensively, we believe that it is advantageous to adopt targeted therapeutic strategies according to the differences between the left and right sides for the patients with HMG.

5. Conclusions

We confirmed that the clinical pathogenesis of HMG is not a left-right equilibrium and found that TCM constitution, coagulation function, and lipid metabolism might be used as objective bases for the pathogenic difference between left and right HMG. The TCM constitutions of HMG patients with the left disease tendency may be more inclined to stagnant Qi constitution and stagnant blood constitution, and the blood tests indicated that the concentration of FVIII was higher than the concentration in the right group of HMG patients. The clinical effect could be satisfied by drugs such as qingpi to promote Qi and blood circulation and remove blood stasis. The results showed that sinensetin and neohesperidin contained in the qingpi might interfere with platelet activation, thrombogenesis, prolactin signaling pathway and atherosclerosis process, remove blood stasis, and achieve the purpose of treating the left-leaning group of HMG patients. The TCM constitutions of HMG patients with right-leaning disease may be more inclined to stagnant Qi constitution and phlegm-dampness constitution, and the blood tests showed that the concentration of Lp-α was higher than that of left-leaning group of HMG patients. Clinical treatment with activating Qi and eliminating phlegm drugs such as chenpi could achieve satisfactory curative effect. Sitosterol and citromitin contained in chenpi could regulate lipid metabolism by interfering with regulation of lipolysis in adipocytes, salivary secretion, estrogen signaling pathway, and thyroid hormone signaling pathway. Chenpi could eliminate phlegm turbidity and treat HMG patients in the right-leaning group.

The clinical characteristics of lopsided HMG are closely related to the TCM theory of “left blood and right Qi,” which agrees with the theory of “left blood stasis and right phlegm.” Therefore, in the treatment of HMG, we might give full consideration to the affected and take a targeted approach by using qingpi for patients with a left slant and chenpi for patients with a right slant.

6. Study Limitations

The limitations of this study are that all the included cases were from rural areas in southwest China, and the sample size was relatively small due to regional limitations. In order to make the results of this study more universally applicable, multicenter, and large-scale, epidemiological investigation of TCM constitution and blood biochemical indices of patients with unilateral HMG will be actively sought in the future. In addition, our research group will further design clinical randomized controlled trials to test the difference in efficacy of qingpi and chenpi in the treatment of HMG in different partial groups and to test their effects on patients' pain, mass score, and blood biochemical indices.