Comparative Study Of Gynecological Excess-cold Syndrome And Asthenia-cold Syndrome Based On Metabolomics And Correlated Network During Formation Process Of Syndrome

Objective: Cold syndrome is one of the eight principal syndromes andcold syndrome of gynecology included excess-cold syndrome and asthenia-cold syndrome which have different pathogenesis, clinical manifestation,treatment and drug with each other is more common disease in clinical.Excess-cold syndrome and asthenia-cold syndrome are identified according topatient's chief complaint in clinical, but often with subjective factor.Extensive researches had been applications with cell biology, molecularbiology methods and so on to search the objective indicators of identifying thetwo syndromes. The results showed that both excess-cold syndrome andasthenia-cold syndrome had different changes in neuroendocrine, immune,cytokines and their trace elements content compared with normal. Controlstudy between excess-cold syndrome and asthenia-cold syndrome is stillasthenia. And specificity index distinguish excess-cold syndrome andasthenia-cold syndrome have not discovered. Therefore, problems such aswhether excess-cold syndrome or asthenia-cold syndrome has the samebiology essence and their disparate points are still unknown."Syndrome" isfoundation of treatment based on syndrome in TCM and also the essence andkey point to the development of TCM. Study on the essence of "syndrome" isthe core content of the basic theory of TCM. To expound the Chinese"syndrome" is the important turning point; it is also the key problem that mustbe solved in modernization of TCM.This study was made with the clinical research and animal experiment.Clinical research was studied with Metabolism to research the similarities anddifferences of dysmenorrheal patients' urine metabolites to look for variabilitymaterial between excess-cold syndrome and asthenia-cold syndrome. Animal experiment was researched with rat model of gynecology excess-coldsyndrome and asthenia-cold syndrome from the neuroendocrine immunologyand energy metabolism points to verification clinical metabolomics results,probe the multiple system changes regularity in the development of disease,detect the similarities and differences about organism regulatory mechanism indifferent syndromes, expound the similarities and differences about biologyessential with gynecology excess-cold syndrome and asthenia-cold syndrome,promote diagnosis and gynecology of TCM.Methods:1Studied with excess-cold syndrome and asthenia-cold syndrome ofdysmenorrheal patients and the normal group, N-Methyl–N-(Trimethylsilyl)Tri-fluoroacetamide (MSTFA) was choosed as derivatization reagent, detectedurine metabolites at the second day in menstrual cycle (MC2) of the threegroups using gas chromatography-time of flight mass spectrometer (GC-TOFMS), drawn the total ion chromatogram(TIC) of each group, combinationwith unsupervised principal component analysis (PCA) for the multi-dimensional statistical analysis to look for variability material of the threegroups. Serum reproductive hormones including follicular stimulatinghormone (FSH), luteinizing hormone (LH), estradiol (E₂), progesterone (P),testosterone (T) at MC₂in excess-cold group, asthenia-cold group, and thenormal group were detected with radioimmunoassay (RIA). Fasting bloodglucose (FPG), blood lipid and glucose in urine were detected with chemicalmethod of excess-cold group, asthenia-cold group at MC2and MC7.2Simulation the etiology of gynecology asthenia-cold syndrome, basedon "Pathogenic Cold Impairing Yang" TCM theory, rat model of gynecologyasthenia-cold syndrome was made by extending the time of ice waterimmersion and refrigerator. Female SD rats were randomly divided intonormal group, model group and treatment group with10rats in each group.Model group and treatment group were placed in0℃¹â„ƒice water andplaced in4℃refrigerator,2times/day,20min/time, a total of30days.Temperatures were controlled at10℃. Body temperature and estrous cycle of rats were measured daily. Normal group was feeding in normal. Treatmentgroup was gavaged with JinGuiWenJingtang from the first day of modeling.The other two groups were gavaged with equal volume of distilled water. Ratswere killed when diestrus after modeling, Rats general situation, temperature,weight, and were observed. Uterine and ovarian morphology were observed,the correlative indices including serum FSH, LH, E₂, T, and P, plasmacorticosterone (CORT), serum interleukin-2(IL-2), plasma cAMP, cGMP,cAMP/cGMP ratio, and thymus, spleen, uterus, ovary index were detected.3Studied with rat model of gynecology excess-cold syndrome andasthenia-cold syndrome to comparing the similarities and differences inneuroendocrine immune (NEI) network regulation during the formationprocess of excess-cold syndrome and asthenia-cold syndrome. Rat model ofgynecology excess-cold syndrome was induced by ice water immersion. Ratmodel of gynecology asthenia-cold syndrome was made by extending the timeof ice water immersion and refrigerator. Detected blood hormone and immunesystem related indexes of hypothalamus-pituitary-adrenal cortex (HPA) axis,hypothalamus-pituitary-thyroid (HPT) axis, and hypothalamus-pituitary-ovaryaxis (HPOA) at different time points in model progress (3d is a time unit). Tlymphocyte subpopulations was detected with flow cytometer, seruminterferon-γ (IFN-γ) and interleukin-4(IL-4) was detected with enzyme linkedimmunosorbent assay (ELISA); plasma adrenocorticotropin hormone (ACTH),CORT, triiodothyronine (T₃), thyroxine (T₄), FSH, LH, E₂, P, T, andhypothalamic corticotropin-releasing hormone (CRH), thyrotropin releasinghormone (TRH), gonadotropin releasing hormone (GnRH) were detected byRIA. Uterine estrogen receptor (ERα) expressions were observed by immuno-histochemistry.4Studied with rat model of gynecology excess-cold syndrome andasthenia-cold syndrome to comparing the similarities and differences inenergy metabolism during the formation progress of excess-cold syndromeand asthenia-cold syndrome. Detected correlated index about energymetabolism and oxidative damage of mitochondria at different time points in model progress (3d is a time unit). Liver adenosine triphosphate (ATP)enzyme, succinate dehydrogenase (SDH), and lactate dehydrogenase (LDH)activity were detected with chemical colorimetry, liver ATP content wasdetected with high-performance liquid chromatography, liver glycogen(Gn)was detected with anthrone colorimetry; uterine superoxide dismutase (SOD)activity was detected with colorimetry, malondialdehyde (MDA) content wasdetected with penthiobarbital, expression of uterine uncoupling protein2(UCP2) was observed by immunohistochemistry.Results:1Compared with the normal group, eight variability materials inexcess-cold group were detected. They were glucose, fructose, xylitol,galactose, citric acid, hippuric acid, alanine, and glycine. Glucose, fructose,xylitol, galactose, citric acid, and hippuric acid in excess-cold group werehigher than in the normal group; alanine and glycine in excess-cold groupwere lower than in the normal group. Compared with the normal group,twelve variability materials in asthenia-cold group were detected. They werefructose, maltose, arbinofuranose, isocitric acid, lactic acid, hippuric acid,glycine, serine, threonine, palmitic acid, octadecanoic acid, and erythritol.Among the total materials only lactic acid in asthenia-cold group was higherthan in the normal group; the rest materials in asthenia-cold group were alllower than in the normal group. Compared with excess-cold group, sevenvariability materials in asthenia-cold group were detected. They were glucose,fructose, isocitric acid, lactic acid, glycine, erythritol, and hippuric acid.Among the total materials only lactic acid in asthenia-cold group was higherthan in excess-cold group; the rest materials in asthenia-cold group were alllower than in excess-cold group.Reproductive endocrine hormones (FSH, LH, E₂, P, and T) of excess-cold group, asthenia-cold group and the normal group at MC₂all had nosignificant difference between each group comparisons. Fasting blood glucose(FBG) and blood lipid index (TC, TG, and LDL-C) all had no significantdifference between each group comparisons at MC2or MC7. Two Excess-cold syndromes of dysmenorrheal patients had urine glucose (±) at MC₂, others alldisplayed (-).2Compared with the normal group, after30days, model rat appearedchill, hunched, lazy and cyanosis in tail and claw, weight lost, bodytemperature decreased significantly, loose stools; estrous cycle and diestrusprolonged. Rats were apathetic, unresponsive, crowding together. Serum FSH,LH, E₂, P, IL-2and plasma CORT, cAMP/cGMP ratio were all significantlydecreased; plasma cGMP content was significantly increased; thymus, spleen,uterus, ovary indexes were decreased significantly; the above indicators allhad significant difference compared with the normal group. Uterus andovarian morphologic were abnormalities. Treatment group compared with themodel group, the above-mentioned indicators were improved.3Rats of excess-cold group at early days displayed hyperfunction ofHPA and HPT, hypothalamus CRH and TRH content, plasma ACTH andCORT, serum T3, and T4were all increased, later period hypothalamus CRHand TRH content, plasma ACTH, CORT, and serum T₃were slightlydecreased and gradually tend to be stable. Immune systems, at early daysCD₄/CD₈, Th₁/Th₂was reduced, later period CD₄/CD₈, Th₁/Th₂picked up andwas stability. Rats of asthenia-cold group at early days displayedhyperfunction of HPA and HPT, hypothalamus CRH and TRH content,plasma ACTH and CORT, serum T₃and T₄were all increased; later periodhypothalamus CRH and TRH content, plasma ACTH and CORT, serum T₃were all decreased. Immune system, CD₄/CD₈, Th₁/Th₂was continuingreduced all the time.Both excess-cold group and asthenia-cold group showed inhabitedHPOA. Serum FSH in excess-cold group was obviously reduced,15dhad significant difference compared with0d; serum T was increased,9dhad significant difference compared with0d; GnRH content inhypothalamus, serum FSH, LH, and P showed downward trend; there hadnot statistically significant between group comparisons; endometrialexpression of ERα were enhanced,12d and15d both had significant difference compared with0d. GnRH content in hypothalamus, serum FSH,LH, E₂, and P in asthenia-cold group was decreased significantly;27d and30dof GnRH, FSH, and E₂had significant difference compared with0d.30dof LH and P had significant difference compared with0d. Endometrialexpression of ERα was first increased, but had not obviously and thenobviously decreased;27d and30d had significant difference comparedwith0d.4Rats of excess-cold group displayed increased liver Na⁺_-K⁺_-ATPenzymes, Ca²⁺-Mg²⁺-ATP enzymes, SDH activity, and ATP content.9d,12dof Na⁺_-K⁺--ATP enzymes had significant difference compared with0d;15dof Ca²⁺-Mg²⁺-ATP enzymes had significant difference compared with0d;12d of SDH activity and ATP content had significant difference comparedwith0d; uterine expression of UCP2, serum FBG, and FFA at early days wasincreased, later fell back;6d of expression of UCP₂and FBG had significantdifference compared with0d;9d of FFA had significant differencecompared with0d; Liver Gn at early days was decreased, later increased,6dhad significant difference compared with0d. Serum INS, GLU, liver LDHactivity, uterine SOD activity, and MDA content had no obviously change.Rats of asthenia-cold group displayed elevated liver Na⁺--K⁺_-ATPenzymes, slightly elevated Ca²⁺-Mg²⁺-ATP enzymes, SDH activity, and ATPcontent in the early stage and had significantly decreased since midanaphase,27d and30d of above indexes had significant difference compared with0d;uterine SOD activity was decreased;24d,27d, and30d had significantdifference compared with0d; liver LDH activity, uterine UCP2, and MDAcontent were increased;24d,27d, and30d of above indexes had significantdifference compared with0d; FBG, FFA, GLU, and liver Gn content wereincreased first and then decreased;27d and30d of FBG, GLU and liver Gncontent were lower than that of0d and there was significant differencecompared with0d; INS was not significantly decreased.Conclusions:1Excess-cold syndrome and asthenia-cold syndrome of dysmenorrheal patients and the normal group suggested significant difference in urinemetabolites. These variability materials are related to several metabolicpathways such as glycolmetabolism, amino acid metabolism, fatty acidmetabolism and disturbance of enterobacteria. Excess-cold group andasthenia-cold group mainly discrepancy is glycometabolism.2We have successfully copied gynecology asthenia-cold syndrome ratmodel by extending the time of ice water immersion and refrigerator bysimulation of gynecology etiology, combination gynecology symptoms. Thismodel is successfully created combined with laboratory tests and drugevidence, can be used for the study of gynecological asthenia-cold syndrome.3NEI regulation is one of the mechanisms in the process of coldsyndrome, and one of the mechanisms which the body has the difference ofexcess-cold syndrome and asthenia-cold syndrome. Rats of gynecologyexcess-cold syndrome and asthenia-cold syndrome at early stage mainlydisplayed hyper function. Excess-cold group was gradually adapt to the highreaction state and established a new balance in model progress; asthenia-coldgroup at early stage displayed hyper function, but failure at the end, eventuallyleading to low immune function or pathological damage.4Energy metabolism and oxidative damage of mitochondria is the one ofthe mechanisms in the process of cold syndrome. The degree of energymetabolism and oxidative damage is the mechanism which the body has thedifference of excess-cold syndrome and asthenia-cold syndrome. Rats ofgynecology excess-cold syndrome have minor injured with mitochondria. Ratsof asthenia-cold group have obviously injured with mitochondria and haveenergy dysbolismus.