Abstract
OBJECTIVE:
To investigate the mechanism of Yin deficiency syndrome (YDS) by analyzing the liver metabolomic characteristics of three different Yin deficiency rat models
METHODS:
Following the TCM etiology, for clinical features and pathological manifestations of modern medicine, three kinds of animal models of Yin deficiency were induced and replicated. Totally 48 Sprague-Dawley (SD) male rats were randomly divided into blank group, irritation induced model group, Fuzi-Ganjiang induced model group, and thyroxine-reserpine induced model group. After successful development of model, the ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was carried out to detect metabolites in each group. The metabolites of rat liver were analyzed for the characteristics of their biomarkers. The pathway enrichment analysis and metabolic network construction were performed through various online databases including Metabolite Biology Role, Human Metabolome Database, MetaboAnalyst, and Kyoto Encyclopedia of Genes and Genomes.
RESULTS:
The SD rats in the experimental group showed symptoms like less weight gain, reduced diet and water intake, high body temperature, increased liver and kidney indexes, and abnormal liver and kidney tissue morphology. Moreover, the rats showed high increased levels of serum cyclic adenosine monophosphate, estradiol, alanine transaminase, and aspartate aminotransferase and decreased levels of cyclic guanosinc monophosphate and testosterone. We found four key interrelated metabolic pathways in the liver tissue metabolomics, including the biosynthesis of pantothenic acid and coenzyme A, and metabolism of alpha-linolenic acid metabolism, glycerophospholipid metabolism, and sphingolipid.
CONCLUSION:
The liver and kidney YDS is closely related to the biosynthesis of pantothenic acid and CoA and abnormal metabolism of α-linolenic acid, glycerophospholipid, and sphingolipid in SD rats.
Keywords: molecular biology, integrative medicine, Yin deficiency syndrome , syndrome complex
1. INTRODUCTION
In Traditional Chinese Medicine (TCM), the theory of Yin and Yang is often used for to explaining the changes in human tissue structure, physiological functions, and pathological conditions. This theory is also used for the diagnosis and treatment of various diseases.1 In recent years, TCM has carried out multi- dimensional and multi-system research on the experimental and clinical aspects of Yin deficiency syndrome (YDS), and has made great progress in this field.2 YDS is one of the main pathogenesis of the clinical syndromes in TCM, that is responsible for Yin deficiency in various tissues, such as lungs, liver, and kidneys.3 The lung heat disturbs the Yin and leads to lung failure. Similarly, a dysfunction of liver drainage leads to abnormal operation of Qi in the Viscera and Meridian which also disturb liver Yin and and leads to its failure. Kidneys are one of the main organs in the body, which governs Yin and Yang. The failure of kidney essence causes the kidneys to lose moisten, endogenous dryness, and heat, which ultimately lead to damage to the kidney Yin.4 To date, most of the research on syndrome types is carried out from clinical and experimental aspects; nevertheless, there are many research reports on its essence. However, most studies are based on the combination of disease and syndrome, with focus towards unveiling the mechanism of the disease rather than the cause and association of the syndrome types. It only reflects some aspect of the nature. Advances in the experimental and scientific research by using animal models for depicting the cause, relationship, and impact of a certain diseases and their treatment have opened new avenues in the medical practices. Nowadays, most of the experiments in scientific research are carried out by using animal models, and their role in guiding medical practice is becoming increasingly prominent.5 Studies have shown that animal models of YDS mainly involve the long-term irritation method of TCM, warm-drying drugs, thyroid hormones, and reserpine of Western Medicine.6 Therefore, in the present study, we selected three different modeling methods, including the irritation method, TCM method, and Western Medicine method, for establishing animal model of Yin deficiency to study the theoretical basis and pathological mechanism of TCM, which has far-reaching significance to improve the clinical diagnosis of TCM.
Metabolomics studies uses modern analytical approaches for quantitatively and qualitatively analysis of all relatively low-molecular-weight metabolites of a certain organism or a cell within a particular physiological period. It associated systems biology and precision medicine for early diagnosis of a disease.7,⇓-9 Since its beginning, metabonomics has been widely used in various fields, such as disease diagnosis, plants, microorganisms, drug toxicity evaluation, and modernization of Chinese medicine.10 Metabolomics studies use various analytical techniques, such as nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), liquid-nuclear magnetic-mass spectrometry (LNM/MS), electrophoresis-mass spectrometry (CE-MS), inductively coupled plasma-mass spectrometry (ICP-MS), and several other combined technologies.11,⇓,⇓-14 These techniques are used for analyzing the blood, urine, tissue, cerebrospinal fluid, hair, saliva, feces, exhaled air samples.15,⇓,⇓-18 These techniques offer the advantages of small sample loss, high throughput of sample processing, easy detection of metabolites and large amount of information of detection results.19
In recent years, there have been a lot of achievements in TCM syndrome standardization, where metabolomics has become an emerging and promising technology.20 Metabolomics has the characteristics of holistic and dynamic nature, and is consistent with the holistic view of TCM and the features of syndrome differentiation and treatment. The difference of endogenous metabolite content can detect the changes of the internal environment after the pathogenic factors act on the body, which is an objective reflection of the pathological changes in a certain stage of the whole course of the disease.21
This study was aimed to utilize the potential of metabolomics tools for studying the model of YDS in rats. We analyzed the changes of endogenous metabolites in Yin deficiency model rats by different modeling methods and unveiled the related metabolic pathways. The findings of this study provides the experimental basis for clinical diagnosis and treatment of YDS.
2. METHODS
2.1. Experimental materials
Ordinary rat maintenance feed was purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (Hunan, China). Pungent and hot Chinese herbs, including Fuzi (Radix Aconiti Lateralis Preparata) was purchased from Jiangxi Zhangshu Tianqitang Traditional Chinese Medicine Pieces Co., Ltd. (Jiangxi, China), Ganjiang (Rhizoma Zingiberis) was purchased from Jiangxi Zhangshu Tianqitang Traditional Chinese Medicine Pieces Co., Ltd. (Jiangxi, China). The drug was identified by Jiangxi Zhangshu Tianqitang Traditional Chinese Medicine Pieces Co., Ltd. according to"China Pharmacopoeia 2015 Edition One" standard implementation. Levothyroxine sodium tablets were purchased from Shenzhen Zhonglian Pharmaceutical Co., Ltd. (Shenzhen, China). The compound reserpine tablets were purchased from Jiangsu Changjiang Pharma-ceutical Co., Ltd. (Jiangsu, China). The estradiol (E2) and testosterone (T) enzyme-linked immunosorbent assay (ELISA) kits was purchased from Wuhan Huamei Biotech Co., Ltd. (Wuhan, China). The cyclic adenosine monophosphate (cAMP), cyclic guanosine mono-phos-phate (cGMP), aspartate aminotransferase (AST) and alanine transaminase (ALT) ELISA kits were purchased from Nanjing Jiancheng Institute of Biological Engin-eering (Nanjing, China). High performance liquid chro-matography (HPLC) grade acetonitrile and methanol were purchased from Supelco (Hannover, Germany).
2.2. Apparatus
The main apparatus used in the study included, Rotary evaporator (Buchi, RE-220 SE, Flawil, Switzerland); Multifunctional microplate reader (Tecan, Spark 10M, Männedorf, Switzerland); SL8R desktop high-speed ref-rigerated centrifuge (Thermo Fisher, Waltham, MA, USA); Agilent MassHunter B.06 (Agilent Technologies, Santa Clara, CA, USA); Mass Profiler Professional (Agilent Technologies, Santa Clara, CA, USA), and Agi-lent 6538A ultrahigh-performance liquid chromate-graphy-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) (Agilent Technologies, Santa Clara, CA, USA).
2.3. Experimental animals grouping and modeling
The study included 48 Sprague-Dawley (SD) male rats, weighing 180-200 g, specific pathogen free grade, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. [license No. SCXK (Xiang) 2016-0002]. The rats were housed in a specific pathogen-free breeding room [temperature: (20 ± 2) ℃; humidity: 60% ± 5%; 12 h light-dark cycle]. All of the rats were provided with free access to tap water. After 7 d of adaptive feeding, the rats were randomly divided into a blank group and a Yin-deficiency model group. The model group was further divided into three groups: anger-inducing intervention group, Fuzi-Ganjiang inter-vention group, and thyroxine-reserpine intervention group. Each group was fed with ordinary feed, and the squirrel cage was cleaned and replaced once a day.
Referring to the research of Wang et al,22 in the irritation-induced model group, the tail was clamped with an elastomeric clip at a distance of 1 cm from the root of the rat's tail for a duration of 5 min/d. Likewise, referring to the research of Yang et al,23 in the Fuzi-Ganjiang induced model group, the Fuzi (Radix Aconiti Lateralis Preparata) was first decocted for 1 h, and then the Ganjiang (Rhizoma Zingiberis) was decocted for 20 min, and the decoction was concentrated to form Fuzi (Radix Aconiti Lateralis Preparata): Ganjiang (Rhizoma Zingiberis) (1:1) concentrate, containing 1.5 g of crude drug/milliliter, with daily gavage with pungent and hot Chinese herbs the dose of 10 mL·kg-1·d-1. Referring to the study of Fan et al,24 in the thyroxine-reserpine induced model group, 15 mg of levothyroxine sodium and 1.5 mg of reserpine were added to 300 mL of distilled water to make a thyroxine-reserpine suspension. A 10 mL·kg-1·d-1 of thyroxine-reserpine suspension was gavaged daily; the blank group was gavaged with saline daily at a dose of 10 mL·kg-1·d-1.
2.4. Experimental animals observation and evaluation
2.4.1. General observation
(a) Physical signs: the body appearance and behavioral status of each group of rats were recorded every week, and the scoring was carried out according to the method used by Shang et al.25 (b) Weight, body temperature, drinking water, and diet status: the weight, body temperature, diet, and water consumption by rats were observed at regular time points every week (Table 1).
Table 1.
Scoring criteria for appearance behavior of rats
| Observation index | Index score | ||
|---|---|---|---|
| Skin hair | Wither and easy to fall off 2 | Not shiny or fluffy 1 | Supple and shiny 0 |
| Behavioral activity | Active and frighten 2 | Hyperactivity 1 | Moderate activity 0 |
| Urinary status | Yellow and frequent urine 2 | Light yellow urine 1 | Normal urine 0 |
| Stool state | Dry stool 2 | Hard stool 1 | Normal stool 0 |
2.4.2. Biochemical index detection
On the 43rd day of modeling, blood samples were collected from the abdominal aorta of each rats and centrifuged for 15 min at 3000 rpm. The supernatant was used for the preparation of serum samples, and determination of cAMP, cGMP, E2, T, AST and ALT levels, in each group to evaluate the establishment of liver and kidney Yin deficiency models.
2.4.3. Measurement of liver and kidney organ index
After 6 weeks of modeling, the rats were anesthetized with sodium pentobarbital, and the liver and bilateral kidneys were taken out. The morphological differences in all groups were observed, and the excess tissues were stripped off. The bloodstains were washed with normal saline, and the excess water was absorbed by the filter paper and weighed. For determination of weight of the organs containing water, the gross weight of the kidneys was considered as the sum of both sides, and we calculated the liver and kidney organ indexes.
2.4.4. Histopathological observation
After dissection, the liver and kidney tissues were collected for analysis. The samples were fixed, dehydrated, waxed, embedded in paraffin, sliced and baked, dewaxed to water, stained, dehydrated, transparent, and stained by the hematoxylin-eosin (HE) staining method.
3. METABOLOMIES ANALYSIS
3.1. Sample preparation
A 200 g liver sample of each rat was taken and cut into pieces, and then homogenized with magnetic beads in 1.5 mL pre-cooled water-methanol solution (water: methanol = 20∶80) for 20 min. The samples were centrifuged at 15 000 rpm for 15 min at 4 ℃. The supernatant was taken and concentrated by vacuum centrifugation for 3 h, reconstituted with 200 μL of methanol, vortexed for 1 min, centrifuged at 15 000 rpm for 15 min at 4 ℃. A 150 μL supernatant was collected for UHPLC-Q/TOF-MS analysis.
3.2. Chromatographic conditions
Chromatographic separation was carried out by the UHPLC-Q-TOF-MS method that used the chroma-tographic column adopts ZORBAX Extend-C18 (2.1 mm × 100 mm, 3.5 μm). For analysis, a sample volume of 6 μL, the sample room temperature of 4 ℃, column temperature of 35 ℃, and the mobile phase of 0.1% formic acid-water (solvent A) and 0.1% formic acid-acetonitrile (solvent B) were used. The initial mobile phase was 2% B, then changed to 51.5% B in 8 min, 71.3% B in 6 min, 81.2% B in 1 min, 100% B in 3 min, 98% B in 1 min, and 98% B for 2 min. Flow rate was maintained at 0.4 mL/min.
3.3. Mass spectrometry conditions
The dual electrospray ion source (Dual ESI) used the capillary voltages of 4000 V and 3500 V in positive and negative ion source modes respectively. The mass spectrometry was carried out according to these conditions: temperature of drying gas: 350 ℃; volume flow of drying gas: 10 L/min; mass scanning range: 50-1200 m/z; spray chamber pressure was 35 psig; fragment voltage and vertebral foramen voltages were 120 V and 60 V, respectively. The instrument supporting reference liquid passes through the automatic transmission inlet at a speed of 5 μL/min for real-time calibration with reference ions m/z 121.05973 and m/z 922.09798.
3.4. Metabolic profiling
Mass Hunter was used to qualitatively analyze the data and process. Principal component analysis (PCA) on the difference compounds was performed by Mass Profiler Professional (MPP; v12.1). The screening condition is fold change (FC) > 2, and the metabolites obtained were passed through groups nonparametric test, where metabolites with P < 0.05 were possible biomarkers.
3.5. Biomarker screening and identification
Through principal component analysis, using t-test (P < 0.05) and VIP value (VIP > 1) for preliminary screening of potential biomarkers, matching compounds in the Metlin database, obtaining the biomarker name or molecular formula, chemical structure, secondary fragment characteristics under UHPLC-Q-TOF/MS conditions were compared and verified with HMDB for determination of biomarkers.
3.6. Bioinformatics analysis
We introduced the potential biomarkers into the Metabo Analyst database (https://www.metaboanalyst.ca) for metabolic transmission enrichment analysis to obtain significantly disturbed metabolic transmission, and then combine with KEGG (https://www.kegg.jp/) and other relevant information from online databases to reconstruct the disturbed metabolic network of the YDS model.
3.7. Statistical analysis
IBM SPSS Statistics 23 software (IBM Corp., Armonk, NY, USA) was used for analysis of the experimental data, and data was analyzed through analysis of variance. The count data was expressed as mean ± standard deviation ($\bar{x}\pm s$) when α = 0.05, and the data was considered statistically significant at P < 0.05.
4. RESULTS
4.1. General observation
4.1.1. Characteristics of YDS rats
After 6 weeks of model building, compared with the blank group, the rats in the model group showed symptoms of Yin deficiency, including a thin body, dull coat color, hair loss, restlessness, dry stool, and yellow urine. The scores in the 5th and 6th weeks of Fuzi-Ganjiang intervention group and thyroxine-reserpine intervention group were significantly higher than the blank group (P < 0.01) (Table 2).
Table 2.
Comparison of physical sign scores of rats in each group ($\bar{x}\pm s$)
| Group | n | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 |
|---|---|---|---|---|---|---|---|
| Blank | 12 | 0.0±0.0 | 0.1±0.3 | 0.2±0.4 | 0.3±0.5 | 0.2±0.4 | 0.3±0.5 |
| Irritation induced model | 12 | 0.4±0.5 | 0.4±0.8 | 0.8±1.0 | 1.5±1.7a | 2.5±1.7b | 3.8±1.6b |
| Fuzi-Ganjiang induced model | 12 | 0.5±0.7 | 0.7±0.8 | 1.2±1.5 | 1.2±1.3 | 1.8±1.3b | 3.3±1.0b |
| Thyroxine-reserpine induced model | 12 | 0.3±0.5 | 0.4±1.0 | 1.0±1.4 | 1.3±1.3a | 2.7±1.1b | 2.5±1.1b |
Notes: blank: saline gavage treatment; irritation induced model: tail clamping intervention, saline gavage treatment; Fuzi-Ganjiang induced model: Fuzi-Ganjiang suspension gavage treatment; thyroxine-reserpine induced model: thyroxine-reserpine suspension gavage treatment, the above gavage dose are 10 mL·kg-1·d-1. Compared with blank group, aP < 0.05, bP < 0.01.
4.1.2. Weight, body temperature, drinking water, diet status
Compared with the blank group, the rats in the model group showed lower body weight, however, the change was not significant (P > 0.05). The body temperature of the two groups showed dramatic fluctuation, and the body temperature of the model group was higher than that of the blank group after four weeks. The change was obvious (P < 0.01), and as a whole, the water intake and diet of rats in each group increased first and then decreased. The water intake of the model group was lower than that of the blank group. In the second week (P < 0.05) and the 6th week (P < 0.01), the change was significant. The diet in the model group was lower than the blank group; however, slightly higher than the blank group in the 4th week, and the difference was statistically lower than the blank group in the 1st week (P < 0.05), and significantly different than in the 5th week, and the 6th week (P < 0.01) (Tables 3-5).
Table 5.
Comparison of changes in diet in rats of each group (g, $\bar{x}\pm s$)
| Group | n | Week 0 | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 |
|---|---|---|---|---|---|---|---|---|
| Blank | 12 | 16.0±0.6 | 26.7±1.9 | 34.7±8.9 | 31.2±1.3 | 32.3±3.9 | 32.5±1.7 | 28.6±0.9 |
| Irritation induced model | 12 | 16.5±0.5 | 25.6±1.9 | 29.1±1.3 | 26.6±3.7 | 30.5±1.6 | 28.8±2.3b | 25.4±2.2b |
| Fuzi-Ganjiang induced model | 12 | 15.3±1.2 | 24.7±1.7a | 30.2±1.5 | 30.2±2.1 | 32.2±2.4 | 30.1±0.7b | 25.9±2.2b |
| Thyroxine-reserpine induced model | 12 | 13.8±1.3a | 24.8±3.3a | 34.2±2.7 | 37.3±4.7b | 41.5±1.9b | 33.5±5.1 | 31.0±1.7b |
Notes: blank: saline gavage treatment; irritation induced model: tail clamping intervention, saline gavage treatment; Fuzi-Ganjiang induced model: Fuzi-Ganjiang suspension gavage treatment; thyroxine-reserpine induced model: thyroxine-reserpine suspension gavage treatment, the above gavage dose are 10 mL·kg-1·d-1. Compared with blank group, aP < 0.05, bP < 0.01.
Table 3.
Comparison of changes in body temperature in rats of each group (℃, $\bar{x}\pm s$)
| Group | n | Week 0 | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 |
|---|---|---|---|---|---|---|---|---|
| Blank | 12 | 36.4±0.6 | 35.8±0.7 | 36.4±0.8 | 36.4±0.5 | 36.4±0.5 | 36.0±0.6 | 36.1±0.5 |
| Irritation induced model | 12 | 36.2±1.0 | 36.2±0.8 | 36.5±0.8 | 36.7±0.6 | 37.0±0.5a | 36.7±0.7a | 36.5±0.6 |
| Fuzi-Ganjiang induced model | 12 | 36.8±1.1 | 36.2±0.6 | 35.8±0.7 | 36.4±0.6 | 36.5±0.4 | 36.7±0.5a | 36.4±0.5 |
| Thyroxine-reserpine induced model | 12 | 35.7±1.2 | 36.3±0.6 | 36.2±0.7 | 37.0±0.6a | 36.8±0.5 | 36.5±0.5b | 36.3±0.4 |
Notes: blank: saline gavage treatment; irritation induced model: tail clamping intervention, saline gavage treatment; Fuzi-Ganjiang induced model: Fuzi-Ganjiang suspension gavage treatment; thyroxine-reserpine induced model: thyroxine-reserpine suspension gavage treatment, the above gavage dose are 10 mL·kg-1·d-1. Compared with blank group, aP < 0.01, bP < 0.05.
Table 4.
Comparison of changes in water consumption in rats of each group (mL, $\bar{x}\pm s$)
| Group | n | Week 0 | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 |
|---|---|---|---|---|---|---|---|---|
| Blank | 12 | 33.2±3.7 | 48.1±7.7 | 54.2±4.9 | 54.2±4.9 | 61.9±9.1 | 59.2±3.1 | 61.2±7.0 |
| Irritation induced model | 12 | 33.3±4.5 | 39.7±4.6b | 49.9±5.8b | 41.3±2.9a | 56.7±8.2 | 47.2±3.2a | 53.1±6.1b |
| Fuzi-Ganjiang induced model | 12 | 31.5±4.2 | 45.6±3.0 | 49.0±3.5b | 50.0±2.0 | 59.2±4.0 | 56.5±7.5 | 52.2±4.1a |
| Thyroxine-reserpine induced model | 12 | 28.6±1.7a | 42.8±7.1 | 50.4±4.5 | 56.3±7.5 | 66.4±8.4 | 64.0±8.1 | 50.6±7.0a |
Notes: blank: saline gavage treatment; irritation induced model: tail clamping intervention, saline gavage treatment; Fuzi-Ganjiang induced model: Fuzi-Ganjiang suspension gavage treatment; thyroxine-reserpine induced model: thyroxine-reserpine suspension gavage treatment, the above gavage dose are 10 mL·kg-1·d-1. Compared with blank group, aP < 0.01, bP < 0.05.
4.2. Biochemical index detection
The changes in cAMP and cGMP levels in serum and their ratios were closely related to the judgment of Yin deficiency and Yang deficiency syndromes.26 The E2 and T hormone levels and their ratios reflect the rise and fall of kidney Qi.27 The ALT and AST levels reflect a liver damage.28 The results (Table 6) showed that: (a) Compared with the blank group, the cAMP level of the model group was significantly increased (P < 0.01), whereas the cGMP level was significantly decreased (P < 0.01), and their ratios of the two increased. (b) Compared with the blank group, in comparison, the E2 level of the model group significantly increased (P < 0.05), whereas the T level decreased, and their ratios of the two increased. (c) Compared with the blank group, the ALT level of the model group significantly increased (P < 0.01). The level of AST was significantly increased (P < 0.01). All these results suggest the successful development of Yin-deficiency model (Table 6).
Table 6.
Comparison of Serum cAMP, cGMP, cAMP/cGMP, E2, T, E2/T, ALT, AST Levels in Rats of Each Group ($\bar{x}\pm s$)
| Group | n | cAMP (nmol/L) |
cGMP (nmol/L) |
cAMP /cGMP (nmol/L) |
E2 (ng/mL) |
T (ng/mL) |
E2/T (ng/mL) |
ALT (μ/L) |
AST (μ/L) |
|---|---|---|---|---|---|---|---|---|---|
| Blank | 12 | 39.5±8.1 | 38.7±11.4 | 0.9±0.4 | 89.2±16.3 | 9.0±5.0 | 13.6±8.8 | 32.4±8.0 | 133.7±36.3 |
| Irritation induced Model | 12 | 32.7±12.1 | 37.3±14.8 | 1.2±0.4 | 91.2±19.4 | 7.6±3.8 | 17.2±12.3 | 53.1±8.5b | 184.6±52.3b |
| Fuzi-Ganjiang induced model | 12 | 53.8±14.5a | 18.8±4.0a | 3.1±1.3a | 114.3±26.0b | 8.2±5.2 | 19.7±13.0 | 61.4±15.6a | 196.6±61.3a |
| Thyroxine-reserpine induced model | 12 | 52.1±8.8a | 15.3±3.2a | 3.5±0.8a | 127.6±30.1a | 6.7±1.5 | 26.9±18.0 | 76.0±39.0a | 209.8±54.3a |
Notes: cAMP: cyclic adenosine monophosphate; cGMP: cyclic guanosine monophosphate; E2: estradiol; T: testosterone; ALT: alanine transaminase; AST: aspartate aminotransferase; blank: saline gavage treatment; irritation induced model: tail clamping intervention, saline gavage treatment; Fuzi-Ganjiang induced model: Fuzi-Ganjiang suspension gavage treatment; thyroxine-reserpine induced model: thyroxine-reserpine suspension gavage treatment, the above gavage dose are 10 mL·kg-1·d-1. Compared with blank group, aP < 0.01, bP < 0.05.
4.3. Measurement of liver and kidney organ index
The organ coefficient of experimental animals (organ weight / body weight × 100%) can reflects the functional status of the experimental animals and the possibility of histopathological changes. The liver index and kidney indexes of the model group were larger than those of the blank group, and the liver index was statistically different (P < 0.01) (Table 7).
Table 7.
Comparison of liver index and kidney index of rats in each group (%, $\bar{x}\pm s$)
| Group | Liver index | Kidney index |
|---|---|---|
| Blank | 2.58±0.09 | 0.65±0.04 |
| Irritation induced model | 2.58±0.13 | 0.66±0.03 |
| Fuzi-Ganjiang induced model | 2.89±0.37a | 0.70±0.07 |
| Thyroxine-reserpine induced model | 3.03±0.23a | 0.82±0.07a |
Notes: blank: saline gavage treatment; irritation induced model: tail clamping intervention, saline gavage treatment; Fuzi-Ganjiang induced model: Fuzi-Ganjiang suspension gavage treatment; thyroxine-reserpine induced model: thyroxine-reserpine suspension gavage treatment, the above gavage dose are 10 mL·kg-1·d-1. Compared with blank group, aP < 0.01.
4.4. Histopathological observation
The results of HE staining of liver and kidney tissues in each group are shown in Figure 1. The liver cells of the blank group are arranged regularly, with nuclei mostly in the middle, and there is no degeneration and necrosis of liver cells. In the irritation induced model group, some hepatocytes were necrosis, while other hepatocytes had vacuolar degeneration and a small amount of inflammatory cell infiltration and bile duct hyperplasia around the portal area. A part of the cytoplasm was increased in eosinophilia, and the nuclei were pyknotic or fragmented. In the Fuzi-Ganjiang induced model group, a small amount of hepatocyte necrosis, increased eosinophilia, a small amount of inflammatory cell infiltration, and bile duct hyperplasia around the portal area of hepatocyte vacuolar degeneration were found. In the thyroxine-reserpine induced model group, a small amount of hepatocyte necrosis, increased eosinophilia, a small amount of inflammatory cell infiltration, and bile duct hyperplasia around the portal area of hepatocyte vacuolar degeneration were found (Figure 1).
Figure 1. Histopathological changes of rat liver and kidney in each group (HE staining).
A: blank group of liver tissue (× 100); B: blank group of liver tissue (× 400); C: irritation induced model group of liver tissue (× 100); D: irritation induced model group of liver tissue (× 400); E: Fuzi-Ganjiang induced model group of liver tissue (× 100); F: Fuzi-Ganjiang induced model group of liver tissue (× 400); G: thyroxine-reserpine induced model group of liver tissue (× 100); H: thyroxine-reserpine induced model group of liver tissue (× 400); I: blank group of kidney tissue (× 100); J: blank group of kidney tissue (× 400); K: Irritation induced model group of kidney tissue (× 100); L: irritation induced model group of kidney tissue (× 400); M: Fuzi-Ganjiang induced model group of kidney tissue (× 100); N: Fuzi-Ganjiang induced model group of kidney tissue (× 400); O: thyroxine-reserpine induced model group of kidney tissue (× 100); P: thyroxine-reserpine induced model group of kidney tissue (× 400); (Scale bar indicates 100 μm, × 100; scale bar indicates 50 μm, × 400). HE: hematoxylin-eosin.
The kidney cells of the blank group were arranged neatly, and these did not show any degeneration or necrosis. A part of the renal tubular epithelial cells in the irritation induced model group was swollen and necrotic, whereas a part of the renal tubular lumen was filled with eosinophilic protein fluid and formed a cast. In the Fuzi-Ganjiang induced model group. A small portion of renal tubular epithelium was swollen and cytoplasmic was slightly stained. A little bit of renal tubular lumen was filled with eosinophilic protein solution that formed a cast, and some renal tubular epithelial cells died and fell off. In the thyroxine-reserpine induced model group, a small amount of renal tubular epithelium was swollen, and the cytoplasm was loose and slightly stained, and some renal tubular epithelial cells died and fell off (Figure 1).
4.5. Metabolic profile
Figure 2 shows the PCA graph of rat liver tissue metabolism. In the blank group and the Yin deficiency model group, the scatter plots of the samples in the PCA were clustered into one type in the positive and negative ion modes. The results of the two groups suggested a clear separated trend, indicating that the metabolic level in the model rats fluctuated significantly and deviated from the normal metabolism (Figure 2).
Figure 2. PCA scores of liver tissue samples of each group of animals.
A: positive ion mode; B: negative ion mode; 1: blank group; 2: irritation induced model group; 3: Fuzi-Ganjiang induced model group; 4: thyroxine-reserpine induced model group.
4.6. Metabolites and metabolic pathways
The biomarkers related to the Yin deficiency model are combined with the online databases such as HMDB, Metabo Analyst, KEGG, etc., and the relevant biomarkers were identified. The results of liver tissue metabolism are shown in Table 8 and supplementary Figure 1. According to the Impact > 0.1, the key meta-bolic pathways screened included glycerol-phos-pholipid metabolism, α-linolenic acid metabolism, sphingolipid metabolism and biosynthesis of pantothenic acid and CoA biosynthesis, and the results are shown in Table 10. The enrichment analysis of metabolic pathways is shown in Figure 3. Where the circles in the figure are the key pathways in the metabolism of liver tissue in rats with Yin deficiency. The dark and light colors of the circles represent the P-value, the darker P-value is small, while the lighter color P-value is large (Tables 8, 9; Figure 3).
Table 8.
Different endogenous compounds in rat liver
| No. | HMDB ID | Retention time(min) |
m/z | Formula | Biomarkers name |
|---|---|---|---|---|---|
| 1 | HMDB0000254 | 1.047 | 117.0192 | C4H6O4 | Succinic acid |
| 2 | HMDB0006355 | 0.662 | 175.024 | C6H8O6 | D-Glucurono-6,3-lactone |
| 3 | HMDB0032387 | 4.722 | 187.0973 | C9H16O4 | (+/-)-Methyl 5-acetoxyhexanoate |
| 4 | HMDB0012204 | 1.931 | 218.1037 | C10H13N5O | Cis-zeatin |
| 5 | HMDB0000767 | 0.628 | 243.062 | C9H12N2O6 | Pseudouridine |
| 6 | HMDB0002186 | 16.312 | 253.2176 | C16H30O2 | Hypogeic acid |
| 7 | HMDB0003040 | 0.662 | 267.0727 | C10H12N4O5 | Arabinosylhypoxanthine |
| 8 | HMDB0031067 | 11.668 | 269.2483 | C17H3402 | (S)-14-Methylhexadecanoic acid |
| 9 | HMDB0010734 | 11.971 | 271.2283 | C16H32O3 | (R)-3-Hydroxy-hexadecanoic acid |
| 10 | HMDB0003426 | 2.666 | 277.1223 | C11H22N2O4S | Pantetheine |
| 11 | HMDB0001388 | 9.348 | 277.2179 | C18H30O2 | Alpha-Linolenic acid |
| 12 | HMDB0030430 | 9.916 | 279.2334 | C18H32O2 | Linalylcaprylate |
| 13 | HMDB0000207 | 17.749 | 281.2483 | C18H34O2 | Oleic acid |
| 14 | HMDB0000299 | 1.564 | 283.0683 | C10H12N4O6 | Xanthosine |
| 15 | HMDB0000827 | 19.001 | 283.2642 | C18H36O2 | Stearic acid |
| 16 | HMDB0000594 | 2.766 | 293.1146 | C14H18N2O5 | gamma-Glutamylphenylalanine |
| 17 | HMDB0037396 | 17.614 | 299.2581 | C18H36O3 | xi-10-Hydroxyoctadecanoic acid |
| 18 | HMDB0001999 | 9.234 | 301.219 | C20H30O2 | Eicosapentaenoic acid |
| 19 | HMDB0006036 | 17.614 | 303.2323 | C20H32O2 | Mesterolone |
| 20 | HMDB0002925 | 10.885 | 305.2479 | C20H34O2 | 8,11,14-Eicosatrienoic acid |
| 21 | HMDB0061650 | 12.355 | 313.2386 | C18H34O4 | 9,10-Epoxystearic acid |
| 22 | HMDB0006048 | 16.146 | 315.2337 | C21H32O2 | Bolasterone |
| 23 | HMDB0005998 | 11.318 | 319.2279 | C20H32O3 | 20-Hydroxyeicosatetraenoic acid |
| 24 | HMDB0032143 | 17.597 | 331.2637 | C22H36O2 | Palaudine |
| 25 | HMDB0035676 | 9.816 | 337.2388 | C20H34O4 | 1-Hydroxy-1-phenyl-3-hexadecanone |
| 26 | HMDB0002995 | 17.598 | 340.2854 | C20H39NO3 | 12-Keto-tetrahydro-leukotriene B4 |
| 27 | HMDB0013308 | 0.946 | 346.0556 | C10H14N5O7P | Stearoylglycine |
| 28 | HMDB0014096 | 0.678 | 350.1076 | C13H21NO10 | 5'-Hydroxypiroxicam |
| 29 | HMDB0000785 | 7.026 | 351.218 | C20H32O5 | N-Acetyl-7-O-acetylneuraminic acid |
| 30 | HMDB0035338 | 1.948 | 357.0895 | C11H23N2O7PS | Sterebin B |
| 31 | HMDB0001416 | 4.687 | 359.1258 | C18H20N2O6 | Pantetheine 4'-phosphate |
| 32 | HMDB0006045 | 14.225 | 360.2553 | C22H35NO3 | Dityrosine |
| 33 | HMDB0005096 | 16.495 | 403.1591 | C25H24O5 | N-Arachidonoyl glycine |
| 34 | HMDB0030785 | 6.676 | 423.2748 | C24H40O6 | Mammeigin |
| 35 | HMDB0013192 | 16.697 | 429.1753 | C20H30O10 | 3a,7b,21-Trihydroxy-5b-cholanoic acid |
| 36 | HMDB0032622 | 8.48 | 435.2774 | C25H40O6 | Phenethylrutinoside |
| 37 | HMDB0029949 | 18.985 | 437.2665 | C21H43O7P | Pangamic acid |
| 38 | HMDB0007850 | 6.559 | 448.3074 | C26H43NO5 | LysoPA(0:0/18:0) |
| 39 | HMDB0000708 | 9.883 | 452.2795 | C28H39NO4 | Glycoursodeoxycholic acid |
| 40 | HMDB0011473 | 5.64 | 514.285 | C26H45NO7S | LysoPE(0:0/16:0) |
| 41 | HMDB0000036 | 1.046 | 611.1452 | C20H32N6O12S2 | Taurocholic acid |
| 42 | HMDB0003337 | 19.252 | 766.5397 | C43H78NO8P | Oxidized glutathione |
| 43 | HMDB0007949 | 17.597 | 331.2637 | C22H36O2 | PC(15:0/20:4(5Z,8Z,11Z,14Z)) |
| 44 | HMDB0004226 | 0.527 | 130.0854 | C6H11NO2 | N4-Acetylaminobutanal |
| 45 | HMDB0031160 | 0.749 | 137.0453 | C5H12S2 | 1-Pentanesulfenothioic acid |
| 46 | HMDB0031210 | 3.397 | 159.0284 | C6H6O5 | Zymonic acid |
| 47 | HMDB0002052 | 5.121 | 201.0392 | C8H8O6 | Maleylacetoacetic acid |
| 48 | HMDB0011175 | 1.928 | 220.117 | C10H13N5O | Leucylproline |
| 49 | HMDB0032318 | 2.119 | 229.1538 | C11H20N2O3 | Hexanal octane-1,3-diol acetal |
| 50 | HMDB0028932 | 15.964 | 229.216 | C14H28O2 | Leucyl-Isoleucine |
| 51 | HMDB0035358 | 3.588 | 245.1858 | C12H24N2O3 | Ketosantalic acid |
| 52 | HMDB0000086 | 7.902 | 251.1637 | C15H22O3 | Glycerophosphocholine |
| 53 | HMDB0011171 | 10.05 | 258.1094 | C8H20NO6P | gamma-Glutamylleucine |
| 54 | HMDB0034495 | 2.484 | 261.1438 | C11H20N2O6 | 6,10,14-Trimethyl-5,9,13-pentadecatrien-2-one |
| 55 | HMDB0030964 | 9.916 | 263.2365 | C18H30O | Linolenelaidic acid |
| 56 | HMDB0062656 | 15.697 | 279.232 | C18H30O2 | Linoleamide |
| 57 | HMDB0061864 | 15.038 | 280.2632 | C18H33NO | Dihomolinoleic acid |
| 58 | HMDB0002117 | 16.69 | 281.2472 | C18H32O2 | Oleamide |
| 59 | HMDB0006221 | 16.478 | 282.2789 | C18H35NO | 13-cis Retinol |
| 60 | HMDB0002100 | 1.549 | 285.082 | C10H12N4O6 | Palmitoylethanolamide |
| 61 | HMDB0000269 | 19.11 | 285.2783 | C18H36O2 | Sphinganine |
| 62 | HMDB0002177 | 10.267 | 287.2366 | C20H30O | Cis-8,11,14,17-Eicosatetraenoic acid |
| 63 | HMDB0029826 | 7.425 | 300.2892 | C18H37NO2 | Hallacridone |
| 64 | HMDB0004610 | 7.225 | 318.2999 | C18H39NO3 | Phytosphingosine |
| 65 | HMDB0012252 | 7.731 | 324.2892 | C20H37NO2 | Linoleoylethanolamide |
| 66 | HMDB0002183 | 7.093 | 329.2471 | C22H32O2 | Docosahexaenoic acid |
| 67 | HMDB0032476 | 19.304 | 341.3041 | C21H40O3 | Polyoxyethylene (600) monoricinoleate |
| No. | HMDB ID | Retention time(min) |
m/z | Formula | Biomarkers Name |
| 68 | HMDB0002007 | 9.795 | 357.2784 | C24H36O2 | Tetracosahexaenoic acid |
| 69 | HMDB0034031 | 16.595 | 357.3002 | C21H40O4 | 3-(2-Heptenyloxy)-2-hydroxypropyl undecanoate |
| 70 | HMDB0000476 | 14.22 | 362.2687 | C22H35NO3 | 3-Oxo-4,6-choladienoic acid |
| 71 | HMDB0013627 | 6.838 | 371.2574 | C24H34O3 | Cervonoylethanolamide |
| 72 | HMDB0012866 | 8.513 | 373.2734 | C24H36O3 | 9'-Carboxy-alpha-chromanol |
| 73 | HMDB0006898 | 8.494 | 391.2835 | C24H38O4 | Chenodeoxyglycocholic acid |
| 74 | HMDB0000331 | 8.265 | 450.3206 | C26H43NO5 | 3a,7b,12a-Trihydroxyoxocholanyl-Glycine |
| 75 | HMDB0011475 | 5.672 | 466.3154 | C26H43NO6 | LysoPE (0:0/18:1(11Z)) |
| 76 | HMDB0011129 | 11.104 | 480.3083 | C23H46NO7P | LysoPE (0:0/18:0) |
| 77 | HMDB0010395 | 9.73 | 482.3234 | C23H48NO7P | LysoPC (20:4(5Z,8Z,11Z,14Z)) |
| 78 | HMDB0008177 | 6.612 | 516.2981 | C26H45NO7P | PC (18:3(6Z,9Z,12Z)/20:2(11Z,14Z)) |
Figure 3. Enrichment diagram of key metabolic pathways in rats liver metabolism with Yin deficiency.
Table 9.
Analysis of key metabolic pathways in rat liver tissue metabolism
| No. | Pathway Name | Total | Hits | Raw p | Holm adjust | FDR p | Impact |
|---|---|---|---|---|---|---|---|
| 1 | Biosynthesis of unsaturated fatty acids | 36 | 6 | 0.000099 | 0.008348 | 0.008348 | 0 |
| 2 | Glycerophospholipid metabolism | 36 | 4 | 0.0076 | 0.63076 | 0.31918 | 0.29983 |
| 3 | alpha-Linolenic acid metabolism | 13 | 2 | 0.032911 | 1 | 0.92151 | 0.33333 |
| 4 | Arachidonic acid metabolism | 36 | 3 | 0.044951 | 1 | 0.94396 | 0.0212 |
| 5 | Pantothenate and CoA biosynthesis | 19 | 2 | 0.06634 | 1 | 1 | 0.27857 |
| 6 | Sphingolipid metabolism | 21 | 2 | 0.079235 | 1 | 1 | 0.15822 |
| 7 | Linoleic acid metabolism | 5 | 1 | 0.10783 | 1 | 1 | 0 |
| 8 | Taurine and hypotaurine metabolism | 8 | 1 | 0.16702 | 1 | 1 | 0 |
| 9 | Ascorbate and aldarate metabolism | 10 | 1 | 0.20434 | 1 | 1 | 0 |
| 10 | Butanoate metabolism | 15 | 1 | 0.29068 | 1 | 1 | 0 |
| 11 | Glycerolipid metabolism | 16 | 1 | 0.30683 | 1 | 1 | 0.01246 |
| 12 | Ether lipid metabolism | 20 | 1 | 0.3679 | 1 | 1 | 0 |
| 13 | Citrate cycle (TCA cycle) | 20 | 1 | 0.3679 | 1 | 1 | 0.03273 |
| 14 | Propanoate metabolism | 23 | 1 | 0.41025 | 1 | 1 | 0 |
| 15 | Alanine, aspartate and glutamate metabolism | 28 | 1 | 0.47478 | 1 | 1 | 0 |
| 16 | Phosphatidylinositol signaling system | 28 | 1 | 0.47478 | 1 | 1 | 0.00152 |
| 17 | Glutathione metabolism | 28 | 1 | 0.47478 | 1 | 1 | 0.02698 |
| 18 | Arginine and proline metabolism | 38 | 1 | 0.58393 | 1 | 1 | 0.01212 |
| 19 | Tyrosine metabolism | 42 | 1 | 0.62112 | 1 | 1 | 0.05581 |
| 20 | Primary bile acid biosynthesis | 46 | 1 | 0.65508 | 1 | 1 | 0.02285 |
| 21 | Purine metabolism | 66 | 1 | 0.78516 | 1 | 1 | 0 |
Notes: total: total number of compounds in the pathway; hits: actually matched number from the user uploaded data; raw p: original P value calculated from the enrichment analysis; holm p: P value adjusted by holm-bonferroni method; FDR p: P value adjusted using false discovery rate; impact: pathway impact value calculated from pathway topology analysis. FDR: false discovery rate.
Through metabolomics analysis of liver tissue of rats with Yin deficiency, we found that the different compounds mainly play a role in the biosynthesis of pantothenic acid and CoA, α-linolenic acid metabolism, glycerophospholipid metabolism, and sphingolipid metabolism pathways. The relationship between the metabolic pathways is shown in the Figure 4.
Figure 4. Metabolism diagram of liver tissues in rats with Yin deficiency.
A: biosynthesis of pantothenic acid and CoA; B: α-linolenic acid metabolism; C: glycero-phospholipid metabolism; D: sphingolipid metabolism.
5. DISCUSSION
The pathological mechanism of the Yin-deficiency rat model is consistent with the clinical theory of Yin-deficiency "dry heat injure Yin". TCM believes that "anger hurts the liver" and "violent anger hurts Yin", which means that it hurts the Yin and blood of the liver. Therefore, we are using the method of prolonged anger to establish an animal model of Yin deficiency is more consistent with the etiological theory of TCM and the characteristics of the seven emotions causing the disease.29 Based on the basic theories of TCM, such as "liver and kidney are of the same origin" and "Yin deficiency leads to heat", the long-term use of pungent and hot Chinese herbs can easily lead to internal heat and loss of Yin fluid, which will damage the Yin of liver and kidney in the long run. The model prepared by this method is more in line with the basic theories of TCM in terms of drug counter-evidence and the detection of various indicators. At the same time, the animal models established to by using western medicine based on their pathological manifestations have the advantages of stability, practicality and reproducibility in terms of symptoms, signs and physicochemical indicators.30
In terms of general observation, the results of sign score showed that the model group score was significantly increased. The model of Yin deficiency with dry mouth and throat is mostly prone to drinking more; however, there are also cases where the body fluid is not deficient, and the animals are thirsty but do not want to drink water, suggesting that their drinking habit is reduced.31 The results of the present study showed that the rats with the deficiency symptoms of thinner had less drinking habit and diet. Besides, they also showed high temperature and restlessness, those are similar to the patients with YDS, manifested as body weight loss thirsty without drinking desire and five upset fever.32 Many studies have concluded that elevated cAMP or elevated cAMP/ cGMP is the law of Yin deficiency in the body, and the law of sex hormones related to kidney and Yin deficiency is elevated, and the elevated E2 and E2/T ratio, ALT and AST levels elevated are the pattern of liver Yin deficiency.33,⇓,⇓,⇓,⇓-38 In the experimental results, the contents of cAMP, E2, ALT, and AST increased, while the contents of cGMP and T decreased, indicating that the rats had liver damage, weak kidney Qi and Yin deficiency. Evaluation of the model found that the liver and kidney tissues of the model group were abnormal, with liver index and kidney index were higher than those of the blank group. The HE staining showed that the model group had liver and kidney histopathological changes, indicating the development of YDS model. It will cause the damage to the liver and kidney tissues to a certain extent. This experiment combined with the model group's symptoms and signs observation, biochemical index detection and model evaluation results, suggesting that the Yin deficiency model was successfully established.
A number of biomarkers of high relevance are involved in the metabolic results of liver tissues. For example, n-arachidonic acid glycine (NA-Gly), an amino acid derivative of the arachidonic acid and a fatty amide lipid molecule, is present in low levels in the liver.39 However, NA-Gly stimulates the production of reactive oxygen species (ROS) and the release of cytochrome C in liver mitochondria, both of which are closely associated with apoptosis.40,41 LysoPC [14Z)] is a lysophospholipid (LPC) which functions in lipid signaling by acting on the lysophospholipid receptor (LPL-R) and is mainly metabolized in the liver, where impaired metabolism of LPC may signal liver inflammation.42 In hepatitis B virus-associated hepatocellular cancer serum metabolism assay, LPC was identified among the metabolites associated with hepatocellular carcinoma.43 Linoleamide, an endogenous lipid, is an important metabolic marker of hepatic lipid metabolism and the aging process.44
5.1. Alpha-linolenic acid metabolism
ALA as one of the main components of fatty acids, has anti-inflammatory and anti-allergic, anti-thrombotic, and anti-tumor effects as well as lipid-lowering and liver-protecting roles.45,46 In a study, the pathology of liver and kidney tissues showed that ALA significantly reduced liver and kidney damage caused by the organic mercury and reduced lipid peroxidation.47 Another study has shown that ALA promoted the expression of osteogenesis-related genes and proteins (β-catenin, RUNX2, and osterix), and promoted the differentiation of osteoblasts.48 The metabolomics of liver and kidney tissues in polycystic kidney rats studies showed that: ALA and linoleic acid metabolism are the key metabolic pathways.49
5.2. Glycerophospholipid metabolism
Glycerophospholipids are the most abundant phospholipids in the body. These are the main biomarkers in the serum of rats with liver injury. These are an important part of the liver cells, bile, biomembranes and membrane surface active substances. Glycerophospholipids are involved in the recognition and signal transduction of proteins by cell membranes.50 Yuan et al 51 showed that the mechanism of Huanglian-Jiedu Decoction in the treatment of mice with ulcerative colitis mainly regulated the metabolism of arachidonic acid and glycerophospholipid metabolism. Zhang et al 52 found that the glycerophospholipid metabolism pathway may be involved in the biological metabolism of hepatitis B virus-related liver fibrosis. Zhao et al 53 suggested that the mechanism of white peony root in blood deficiency liver depression syndrome model rats may be associated with the regulation of multiple pathways such as linoleic acid metabolism, α-linoleic acid metabolism, and glycerophospholipid metabolism.
5.3. Sphingolipid metabolism
Sphingolipids are compounds with a sphingosine skeleton. The main biologically active molecules are ceramide and its metabolites, sphingosine and its metabolites, and glycosylated derivatives.54 Sphingo-lipids and their metabolites constitute the main components of the eukaryotic cell membranes, and participate in the regulation of cell proliferation, differentiation, aging, apoptosis, and other processes, suggesting that these allow the cells to perform their own biological functions. Abnormal sphingolipid metabolism often leads to cell growth disorders and causes different diseases.55 Sphingolipids are key to regulating the homeostasis of liver cells and have significant effects on liver regeneration and liver diseases. The activation of sphingolipid metabolism and the production and accumulation of ceramide and sphingosine play an important role in the process of liver aging. Sphingolipids and sphingosine metabolism not only play a role in the treatment of cancer, inflammation, and metabolic diseases but are also developing into a key factor in the process of kidney aging and kidney diseases.49,56,⇓,⇓ -59
5.4. Biosynthesis of pantothenic acid and CoA
Pantothenic acid is a water-soluble vitamin B5, which can not only increases the biosynthesis of glutathione and slows down the cell damage and apoptosis, but also biosynthesizes CoA and acyl carrier protein, which are closely related to the body's protein, sugar, fat, and amino acid metabolism as well as liver biotransformation in the body.60,61 In sepsis-induced acute kidney injury experiments,62 we found that several changes in the renal cortex metabolic pathways, including the metabolism of taurine and hypotaurine, and the biosynthesis of pantothenic acid and CoA. In the metabonomics research experiment on the aging changes of mouse kidney tissue,63 we found that the Sijunzi Decoction can resist aging by regulating the metabolic pathways of pantothenic acid and biosynthesis of CoA, amino acid metabolism, and others. A study have also shown that pantothenic acid and CoA biosynthesis can be potential therapeutic targets for the treatment of diabetic nephropathy.64 The metabolomics research of liver cirrhosis, it mainly involves sugar metabolism, amino acid metabolism, and biosynthesis of pantothenic acid and CoA.65
In summary, this study is based on metabolomics that explored the mechanism of YDS. The YDS mechanism of action may be through intervention in the biosynthesis of pantothenic acid and CoA, and metabolism of α-linolenic acid, glycerophospholipid, and sphingolipid. These pathway play the role of "reducing dryness and damaging Yin", thereby causing damage to the liver and kidneys of rats, and further cause the physique of the rats to develop in the direction of Yin deficiency. From the perspective of metabolomics, this study provides a scientific basis for in-dpeth study of YDS.
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