Traditional Chinese medicine formulates treatment according to body constitution (BC) differentiation. Different constitutions have specific metabolic characteristics and different susceptibility to certain diseases. This study aimed to assess the characteristic genes of gan-shen Yin deficiency constitution in different diseases. Fifty primary liver cancer (PLC) patients, 94 hypertension (HBP) patients, and 100 diabetes mellitus (DM) patients were enrolled and classified into gan-shen Yin deficiency group and non-gan-shen Yin deficiency group according to the body constitution questionnaire to assess the clinical manifestation of patients. The mRNA expressions of 17 genes in PLC patients with gan-shen Yin deficiency were different from those without gan-shen Yin deficiency. However, considering all patients with PLC, HBP, and DM, only MLH3 was significantly lower in gan-shen Yin deficiency group than that in non-gen-shen Yin deficiency. By ROC analysis, the relationship between MLH3 and gan-shen Yin deficiency constitution was confirmed. Treatment of MLH3 (−/− and −/+) mice with Liuweidihuang wan, classical prescriptions for Yin deficiency, partly ameliorates the body constitution of Yin deficiency in MLH3 (−/+) mice, but not in MLH3 (−/−) mice. MLH3 might be one of material bases of gan-shen Yin deficiency constitution.
The foundation of clinical Traditional Chinese Medicine (TCM) practice was dependent on the syndrome differentiation theory but not the concept of disease in modern medicine [
The material basis of TCM syndrome in physiological functions and pathological status had been studied using clinical and animal experiments in recent years. Because characteristics of genes expression, regulation, and production are closely similar to TCM philosophy, the relationship between genes and material basis of TCM syndrome may be a potential research direction.
In our previous study, there are seventeen characteristic mRNA variables in PBMCs of gan-shen Yin deficiency patients with PLC using microarray analysis [
The study participants were men and women over 18 years old and were recruited between January 2013 and February 2015 from the TCM department, endocrinology department, and cardiology department of Changhai Hospital, the endocrinology department and cardiology department of Shanghai Traditional Chinese Medicine Hospital, and Shanghai Shuguang Hospital (Shanghai, China). Diagnosis and staging standards of PLC referred to “the standard of clinical diagnosis and staging of primary liver cancer” (Chinese Society of Liver Cancer, 2001) [
A Chinese medical doctor, Juan Du (female aged 38), with a degree in medicine, a license as a Chinese physician, and more than 7 years of clinical experience, explained the details of the study to the subjects and solicited written informed consent from each subject. Patients who had taken Chinese medicine within the past month were excluded from the study. A total of 244 subjects (50 PLC patients, 94 HBP patients, and 100 DM patients) were recruited. The characteristics of the participants are shown in Table
Distribution of patients in age, gender, and clinical characteristics.
Variables | Gan-shen Yin deficiency | Non-gan-shen Yin deficiency |
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PLC | Age (years) | |||
≤30 | 1 | 0 | ||
31–50 | 14 | 11 |
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51–70 | 10 | 13 | ||
>70 | 0 | 1 | ||
Gender | ||||
Male/female | 24/1 | 22/3 |
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Tumor size | ||||
≤5 cm | 25 | 25 |
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AFP level | ||||
>400 | 16 | 11 |
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Liver cirrhosis | ||||
Yes | 17 | 13 |
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No | 8 | 12 | ||
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HBP | Age (years) | |||
≤40 | 2 | 1 | ||
41–80 | 41 | 47 |
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>80 | 1 | 2 | ||
Gender | ||||
Male/female | 30/14 | 34/16 |
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Duration of HBP (years) | ||||
<5 | 12 | 13 | ||
5–10 | 23 | 24 | ||
11–20 | 6 | 6 |
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>20 | 3 | 7 | ||
Classification of hypertension | ||||
Class I | 5 | 3 | ||
Class II | 28 | 33 |
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Class III | 11 | 14 | ||
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DM | Age (years) | |||
<40 | 8 | 9 | ||
40–60 | 24 | 21 | ||
61–80 | 14 | 16 |
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>80 | 4 | 4 | ||
Gender | ||||
Male/female | 36/14 | 35/15 |
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Duration of DM (years) | ||||
<5 | 24 | 21 | ||
5–10 | 12 | 13 | ||
11–20 | 10 | 12 |
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>20 | 4 | 4 | ||
BMI (Kg/m2) |
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Three types of data were collected including the body constitution questionnaire (BCQ) responses, the stage of the disease, and clinical records of a Chinese medical doctor (CMD). The BC was assessed from the self-reported BCQ responses and by the CMD. Clinical manifestations of gan-shen Yin deficiency and non-gan-shen Yin deficiency were evaluated using the BCQ [
Finally, 25 PLC patients, 44 HBP patients, and 50 DM patients were included in gan-shen Yin deficiency group and another 25 PLC patients, 50 HBP patients, and 50 DM patients were included in non-gan-shen Yin deficiency group according to the BCQ results. The anticoagulated blood (5 ml) was collected from all patients between 6:30 and 7:00 a.m. All the blood samples were used for PBMC isolation.
Seventeen biomarkers were selected and analyzed for discriminate gan-shen Yin deficiency from other constitutions using real-time RT-PCR [
Seven-week-old male MLH3 (
Animal experiments were approved by the Animal Care and Use Committee of Second Military Medicine University and conformed to the Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD, USA).
The levels of E2, T in serum, and cAMP in plasma were determined by ELISA [
Statistical analyses were performed using SPSS version 19.0 (SPSS, Chicago, IL). Data were analyzed when appropriate by using simple descriptive analyses, such as mean ± standard deviation (
The study group was composed of 50 PLC patients, 94 HBP patients, and 100 type 2 diabetics. Of the 244 participants, 119 were categorized as gan-shen Yin deficiency syndrome characterized by soreness and weakness of the lumbar region, night sweat, dizziness, syrigmus, and so on, and others were categorized as non-gan-shen Yin deficiency syndrome according to BCQ. Table
The seventeen characteristic mRNA variables in PBMCs of PLC patients with gan-shen Yin deficiency syndrome were further determined. To explore the genes most closely related to gan-shen Yin deficiency, we measured the transcriptional expression of these 17 mRNA variables in 25 PLC samples, 44 HBP samples and 50 DM samples from gan-shen Yin deficiency groups and 25 PLC samples, 50 HBP samples, and 50 DM samples from non-gan-shen Yin deficiency groups. Of the PLC samples, we found that the transcriptional expressions of
Analysis on the differentially expressed genes associated with gan-shen Yin deficiency in patients with different diseases. (a) Real-time RT-PCR analysis of 17 mRNAs in PLC patients with or without gan-shen Yin deficiency symptoms. (b) Real-time RT-PCR analysis of 17 mRNAs in HBP patients with or without gan-shen Yin deficiency symptoms. (c) Real-time RT-PCR analysis of 17 mRNAs in DM patients with or without gan-shen Yin deficiency symptoms.
Of the HBP samples, we found that the transcriptional expression of MLH3 in the PBMCs of gan-shen Yin deficiency group was significantly lower than that in non-gan-shen Yin deficiency group (Figure
Of the DM samples, we found that the transcriptional expression of MLH3 in the PBMCs of gan-shen Yin deficiency group was significantly lower than that in non-gan-shen Yin deficiency group (Figure
From ROC analysis, we found that 90.0% of the PLC patients, 77.0% of the HBP patients, and 81.0% of the DM patients with gan-shen Yin deficiency syndrome had low expression of MLH3 (Figure
The effects of MLH3 in patients with gan-shen Yin deficiency syndrome were further explored in MLH3 (
MLH3 KO mice were used to validate the status of MLH3 in gan-shen Yin deficiency. (a) ELISA analysis of cAMP in plasma of mice. (b) ELISA analysis of E2 and T in serum of mice.
The theory of syndrome differentiation plays a prominent role in the diagnosis and the treatment decision of TCM. According to the theory of Yin and Yang, the occurrence, development, and changes of disease lie in the imbalance between Yin and Fang. Clinically all kinds of diseases, including pathological changes of complexion, voice, and pulse condition as well as the nature of diseases, can be generalized and analyzed with the theory of Yin and Yang. [
To further validate that these 17 variables were for gan-shen Yin deficiency syndrome in TCM, but not for PLC disease, DM and HBP patients with the same syndrome of gan-shen Yin deficiency were also included in this study. A total of 244 effective cases of in-patients with PLC, DM, and HBP were collected. The objective clinical manifestations of them from traditional four diagnostic methods were collected by BCQ. Of them, 119 patients were classified as gan-shen Yin deficiency group; others were classified as non-gan-shen Yin deficiency group. No difference of basic characteristics, but BMI, between syndromes of gan-shen Yin deficiency and non-gan-shen Yin deficiency was found. The BMI in the gan-shen Yin deficiency group with DM was significantly lower than that in non-gan-shen Yin deficiency group. This finding might be explained that gan-shen Yin deficiency patients always have the symptom of emaciation, compared to non-gan-shen Yin deficiency patients.
By real-time RT-PCR and ROC analysis, only MLH3 expressions were lower in patients with gan-shen Yin deficiency syndrome than that in patients without gan-shen Yin deficiency syndrome among 17 variables. Other 16 variables might be related to PLC. MLH3 is a MutL homolog protein in mammals. Its basic role is in the DNA mismatch repair mechanism, while it has been proposed to play a distinct role in the meiotic recombination mechanism [
MLH3 KO mice were used to validate the effects of MLH3 in vivo. Reduced activity, decreased and yellow urine, and decreased reproductive ability were found in MLH3
In conclusion, results of this study demonstrate that the MLH3 might contribute to the specific manifestation in patients with gen-shen Yin deficiency. We recommend that future studies explore the relationship between MLH3 and gan-shen Yin deficiency syndrome by expanding the type of disease and the number of samples. Moreover, the mechanism of MLH3 in patients should be conducted to further elucidate the effect of MLH3 in gan-shen Yin deficiency syndrome.
The authors declare that they have no conflicts of interest.
Juan Du, Maofeng Zhong, and Dong Liu contributed equally to this work.
This work was supported by the National Natural Science Funds (nos. 81473487, 81273881, and 81202973) and Shanghai Natural Science Funds (no. 14ZR1408400) and by grants from E-Institutes of Shanghai Municipal Education Commission (no. E03008).