妇科实寒症与虚寒症代谢组学及证候形成过程中相关网络调控的比较研究
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摘要
目的：寒证为八纲之一，妇科寒证是临床较为多见的病证之一。妇科寒证有实寒证、虚寒证之分，两者的病机、临床表现和治法用药各不相同。临床鉴别实寒证、虚寒证主要以患者主诉为依据，但常有主观因素。对于两证客观指标的变化，学者们应用病理学、分子生物学等方法进行了大量研究，发现实寒证、虚寒证机体神经内分泌、免疫、细胞因子及体内微量元素含量都较正常有不同程度的变化，但尚缺乏两证的对照研究，且未发现可以区分两者的特异性指标。因此，实寒证、虚寒证的生物学本质之间究竟有无差异及其差异点至今仍未明确。“证”是中医辨证论治的根本，也是中医精华所在和发展中医学的关键。“证”本质一直是中医基础理论研究的核心内容，阐明中医“证”本质是实现中医现代化必须解决的关键问题。
本课题从临床研究和动物实验两个层次，以代谢组学为研究手段，比较实寒证、虚寒证痛经患者尿液代谢产物的异同，寻找两证的差异性物质；创建妇科虚寒证大鼠模型；以妇科实寒证、虚寒证模型大鼠为研究对象，从机体神经内分泌免疫系统及能量代谢角度，进一步阐释临床代谢组学结果，探究妇科实寒证、虚寒证发生过程中机体多个系统的变化规律，比较不同证在发生过程中机体调控机制的异同，明确两者的生物学本质特点，促进中医诊断学和中医妇科学的发展。
方法：
1以实寒证、虚寒证痛经患者及正常对照组为研究对象，选择N-甲基-N-三甲基-硅基三氟乙酰胺（MSTFA）作为衍生化试剂对各组月经周期第2d（MC2）尿液代谢产物进行气相色谱-飞行时间质谱（GC-TOFMS）检测，得出各组总离子流色谱图（TIC），结合非监督的主成分分析（PCA）进行多维统计分析，寻找实寒组、虚寒组及正常组的差异代谢产物。放射免疫分析法（RIA）检测上述三组MC2血清生殖内分泌激素，包括卵泡刺激素（FSH）、黄体生成素（LH）、雌二醇（E_2）、孕酮（P）及睾酮（T）；化学法检测三组MC_2、MC_7空腹血糖（FPG）、血脂及尿糖。
2模拟妇科虚寒证发生的病因，依据“寒邪伤阳”的中医理论，采用延长冰箱冷冻加冰水浸泡时间方法创建妇科虚寒证大鼠模型。将SD雌性大鼠按随机数字表法分为正常组、模型组和治疗组，每组各10只。模型组、治疗组置于0℃~1℃冰水中并放入4℃冰箱中，2次/d，20min/次，共30d。正常组常规饲养，治疗组造模同时灌胃金匮温经汤，其余两组灌胃等体积蒸馏水。造模结束后，择动情间期处死大鼠，观察各组大鼠一般情况、肛温、体重及子宫、卵巢形态；检测血清FSH、LH、E2、P、T，血浆皮质酮（CORT），血清白介素2（IL-2），血浆cAMP、cGMP、cAMP/cGMP及胸腺、脾脏、子宫、卵巢指数。
3以妇科实寒证、虚寒证模型大鼠为研究对象，比较两证形成过程中神经-内分泌-免疫（NEI）调控的异同。采用冰水浸泡方法创建实寒证大鼠模型；采用延长冰箱冷冻加冰水浸泡时间方法创建虚寒证大鼠模型；分别在造模不同时间点（3d为一个时间单位）检测下丘脑-垂体-肾上腺皮质（HPA）轴、下丘脑-垂体-甲状腺（HPT）轴、下丘脑-垂体-卵巢轴（HPOA）血液相关激素及免疫系统相关指标的变化。血液T淋巴细胞亚群采用流式细胞仪检测；血清干扰素-γ（IFN-γ）和白介素4（IL-4）采用酶联免疫吸附法（ELISA）检测；血浆促肾上腺皮质激素（ACTH）、CORT，血清总三碘甲状腺原氨酸（T_3）、总甲状腺素（T4），血清生殖内分泌激素（FSH、LH、E_2、T、P）及下丘脑促肾上腺皮质激素释放激素（CRH）、促甲状腺激素释放激素（TRH）、促性腺激素释放激素（GnRH）均采用RIA检测。子宫雌激素受体α（ERα）表达采用免疫组化法。
4以妇科实寒证、虚寒证模型大鼠为研究对象，比较实寒证、虚寒证形成过程中机体能量代谢的异同。分别在造模不同时间点（3d为一个时间单位）检测组织线粒体能量代谢及氧化损伤相关指标的变化情况。肝脏三磷酸腺苷（ATP）酶、琥珀酸脱氢酶（SDH）及乳酸脱氢酶（LDH）活性采用化学比色法，肝脏ATP含量采用高效液相法，肝糖原（Gn）含量采用蒽酮法；子宫超氧化物歧化酶（SOD）活力采用比色法，丙二醛（MDA）含量采用硫代巴比妥法；子宫解偶联蛋白2（UCP_2）表达采用免疫组化法。
结果：
1实寒组与正常组比较，差异代谢产物有葡萄糖、果糖、木糖醇、半乳糖、异柠檬酸、马尿酸、丙氨酸、甘氨酸8种；其中实寒组升高的有葡萄糖、果糖、木糖醇、半乳糖、异柠檬酸、马尿酸；降低的有丙氨酸、甘氨酸。虚寒组与正常组比较，差异代谢产物有果糖、麦芽糖、阿拉伯呋喃糖、异柠檬酸、乳酸、马尿酸、甘氨酸、丝氨酸、苏氨酸、软脂酸、硬脂酸、原藻醇12种；其中虚寒组升高的只有乳酸，其余均降低。实寒组与虚寒组之间差异代谢产物有葡萄糖、果糖、异柠檬酸、乳酸、甘氨酸、原藻醇、马尿酸7种，其中虚寒组升高的只有乳酸，其余均降低。
实寒组、虚寒组、正常组MC2生殖内分泌激素（FSH、LH、E2、P、T）组间比较差异均无统计学意义。MC2、MC7空腹血糖（FBG）及血脂指标（TC、TG、LDL-C）组间比较差异均无统计学意义。实寒组有两例样本MC2尿糖（±），其余尿糖均为（－）。
2模型组大鼠外观差，被毛无光泽，耳、爪紫暗；精神萎靡、蜷缩少动、反应迟钝、喜扎堆，体重减轻，肛温降低，便溏；动情周期延长、间期延长；血清FSH、LH、E_2、P、IL-2及血浆CORT、cAMP/cGMP均降低，血浆cGMP升高；胸腺、脾脏、子宫、卵巢指数均降低；与正常组比较差异均有统计学意义。子宫、卵巢形态异常。治疗组较模型组上述指标均有不同程度改善。
3实寒组大鼠表现为HPA、HPT亢进，下丘脑CRH、TRH含量上升，血浆ACTH和CORT，血清T_3、T_4均升高；后期下丘脑CRH、TRH含量、血浆ACTH、CORT，血清T_3稍下降逐渐趋于稳定。免疫系统，前期CD_4＋/CD~＋_8、Th_1/Th_2降低，后期CD~＋_4/CD~＋_8、Th_1/Th_2回升。虚寒组大鼠前期HPA、HPT亢进，下丘脑CRH、TRH含量，血浆ACTH、CORT，血清T3、T4均升高；后期下丘脑CRH、TRH含量、血浆ACTH、CORT，血清T＋＋3下降；Th_1/Th_2、CD_4/CD_8下降。
实寒组、虚寒组均表现为HPOA抑制。实寒组大鼠血清FSH下降明显，15d与0d比较差异有统计学意义；血清T升高，9d与0d比较差异有统计学意义；其余指标均有下降趋势，但与0d比较差异均无统计学意义；子宫内膜ERα表达增强，12d、15d与0d比较差异均有统计学意义。虚寒组大鼠下丘脑GnRH含量及血清FSH、LH、E_2、P均下降，27d、30d下丘脑GnRH含量及血清FSH、E_2与0d比较差异均有统计学意义，30d血清LH、P与0d比较差异均有统计学意义；子宫内膜ERα表达前期有升高趋势后期明显降低，27d、30d与0d比较差异有统计学意义。
4实寒组大鼠肝脏Na~+-K~+-ATP酶、Ca~(~(2+))-Mg~(2+)-ATP酶、SDH活性、ATP含量增加；Na+-K+-ATP酶9d、12d与0d比较差异均有统计学意义；Ca~(2+)-Mg~(2+)-ATP酶15d与0d比较差异有统计学意义；SDH活性、ATP含量12d与0d比较差异均有统计学意义；子宫UCP_2表达、血清FBG、FFA先升后降，UCP_2表达及FBG6d与0d比较差异均有统计学意义，FFA9d与0d比较差异有统计学意义；肝Gn含量先降后升，6d与0d比较差异有统计学意义；血清INS、GLU、肝脏LDH活性、子宫SOD活性、MDA含量各时间点与0d比较差异均无统计学意义。
虚寒组大鼠肝脏Na~+-K~+-ATP酶先升后降，Ca~(2+)-Mg~(2+)-ATP酶、SDH活性及ATP含量前期略有升高中后期明显降低，27d、30d与0d比较差异均有统计学意义；子宫SOD活性降低，24d、27d、30d与0d比较差异均有统计学意义；肝脏LDH活性、子宫UCP_2及MDA含量升高，24d、27d、30d与0d比较差异均有统计学意义；FBG、FFA、GLU均先升后降，肝Gn含量以降低为主，FBG、GLU、肝Gn含量27d、30d与0d比较差异均有统计学意义；INS各时间点与0d比较差异均无统计学意义。
结论：
1实寒证痛经患者、虚寒证痛经患者、正常对照组尿液的差异代谢物质涉及到糖代谢、氨基酸代谢、脂酸代谢等代谢途径以及肠道菌群的紊乱。实寒组与虚寒组的差异主要为糖代谢。
2采用延长冰箱冷冻加冰水浸泡时间方法创建的大鼠模型成功模拟了妇科虚寒证的病因及致病特点，结合临床症状，经实验室检测及药物反证，提示该模型创建成功，可用于妇科虚寒证的研究。
3NEI调控变化是寒证发生的机制之一，也是机体表现出实寒、虚寒不同证候的机制之一。实寒组、虚寒组前期均以亢进为主，实寒组逐渐适应建立新平衡。虚寒组最终转为衰竭导致机体免疫低下及病理性损伤。
4寒证的发生与线粒体能量代谢及氧化损伤有关，其损伤程度是机体表现出实寒、虚寒不同证候的机制之一。实寒组线粒体损伤较轻微；虚寒组线粒体损伤明显，能量代谢障碍。
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 (E_2), progesterone (P),testosterone (T) at MC_2in 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℃~1℃ice 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, E_2, 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 (T_3), thyroxine (T_4), FSH, LH, E_2, 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, E_2, P, and T) of excess-cold group, asthenia-cold group and the normal group at MC_2all 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 MC_2, 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, E_2, 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 T_3were slightlydecreased and gradually tend to be stable. Immune systems, at early daysCD_4/CD_8, Th_1/Th_2was reduced, later period CD_4/CD_8, Th_1/Th_2picked 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 T_3and T_4were all increased; later periodhypothalamus CRH and TRH content, plasma ACTH and CORT, serum T_3were all decreased. Immune system, CD_4/CD_8, Th_1/Th_2was 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, E_2, and P in asthenia-cold group was decreased significantly;27d and30dof GnRH, FSH, and E_2had 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, Ca~(2+)-Mg~(2+)-ATP enzymes, SDH activity, and ATP content.9d,12dof Na~+_-K~+--ATP enzymes had significant difference compared with0d;15dof Ca~(2+)-Mg~(2+)-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 UCP_2and 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 Ca~(2+)-Mg~(2+)-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.
本课题从临床研究和动物实验两个层次，以代谢组学为研究手段，比较实寒证、虚寒证痛经患者尿液代谢产物的异同，寻找两证的差异性物质；创建妇科虚寒证大鼠模型；以妇科实寒证、虚寒证模型大鼠为研究对象，从机体神经内分泌免疫系统及能量代谢角度，进一步阐释临床代谢组学结果，探究妇科实寒证、虚寒证发生过程中机体多个系统的变化规律，比较不同证在发生过程中机体调控机制的异同，明确两者的生物学本质特点，促进中医诊断学和中医妇科学的发展。
方法：
1以实寒证、虚寒证痛经患者及正常对照组为研究对象，选择N-甲基-N-三甲基-硅基三氟乙酰胺（MSTFA）作为衍生化试剂对各组月经周期第2d（MC2）尿液代谢产物进行气相色谱-飞行时间质谱（GC-TOFMS）检测，得出各组总离子流色谱图（TIC），结合非监督的主成分分析（PCA）进行多维统计分析，寻找实寒组、虚寒组及正常组的差异代谢产物。放射免疫分析法（RIA）检测上述三组MC2血清生殖内分泌激素，包括卵泡刺激素（FSH）、黄体生成素（LH）、雌二醇（E_2）、孕酮（P）及睾酮（T）；化学法检测三组MC_2、MC_7空腹血糖（FPG）、血脂及尿糖。
2模拟妇科虚寒证发生的病因，依据“寒邪伤阳”的中医理论，采用延长冰箱冷冻加冰水浸泡时间方法创建妇科虚寒证大鼠模型。将SD雌性大鼠按随机数字表法分为正常组、模型组和治疗组，每组各10只。模型组、治疗组置于0℃~1℃冰水中并放入4℃冰箱中，2次/d，20min/次，共30d。正常组常规饲养，治疗组造模同时灌胃金匮温经汤，其余两组灌胃等体积蒸馏水。造模结束后，择动情间期处死大鼠，观察各组大鼠一般情况、肛温、体重及子宫、卵巢形态；检测血清FSH、LH、E2、P、T，血浆皮质酮（CORT），血清白介素2（IL-2），血浆cAMP、cGMP、cAMP/cGMP及胸腺、脾脏、子宫、卵巢指数。
3以妇科实寒证、虚寒证模型大鼠为研究对象，比较两证形成过程中神经-内分泌-免疫（NEI）调控的异同。采用冰水浸泡方法创建实寒证大鼠模型；采用延长冰箱冷冻加冰水浸泡时间方法创建虚寒证大鼠模型；分别在造模不同时间点（3d为一个时间单位）检测下丘脑-垂体-肾上腺皮质（HPA）轴、下丘脑-垂体-甲状腺（HPT）轴、下丘脑-垂体-卵巢轴（HPOA）血液相关激素及免疫系统相关指标的变化。血液T淋巴细胞亚群采用流式细胞仪检测；血清干扰素-γ（IFN-γ）和白介素4（IL-4）采用酶联免疫吸附法（ELISA）检测；血浆促肾上腺皮质激素（ACTH）、CORT，血清总三碘甲状腺原氨酸（T_3）、总甲状腺素（T4），血清生殖内分泌激素（FSH、LH、E_2、T、P）及下丘脑促肾上腺皮质激素释放激素（CRH）、促甲状腺激素释放激素（TRH）、促性腺激素释放激素（GnRH）均采用RIA检测。子宫雌激素受体α（ERα）表达采用免疫组化法。
4以妇科实寒证、虚寒证模型大鼠为研究对象，比较实寒证、虚寒证形成过程中机体能量代谢的异同。分别在造模不同时间点（3d为一个时间单位）检测组织线粒体能量代谢及氧化损伤相关指标的变化情况。肝脏三磷酸腺苷（ATP）酶、琥珀酸脱氢酶（SDH）及乳酸脱氢酶（LDH）活性采用化学比色法，肝脏ATP含量采用高效液相法，肝糖原（Gn）含量采用蒽酮法；子宫超氧化物歧化酶（SOD）活力采用比色法，丙二醛（MDA）含量采用硫代巴比妥法；子宫解偶联蛋白2（UCP_2）表达采用免疫组化法。
结果：
1实寒组与正常组比较，差异代谢产物有葡萄糖、果糖、木糖醇、半乳糖、异柠檬酸、马尿酸、丙氨酸、甘氨酸8种；其中实寒组升高的有葡萄糖、果糖、木糖醇、半乳糖、异柠檬酸、马尿酸；降低的有丙氨酸、甘氨酸。虚寒组与正常组比较，差异代谢产物有果糖、麦芽糖、阿拉伯呋喃糖、异柠檬酸、乳酸、马尿酸、甘氨酸、丝氨酸、苏氨酸、软脂酸、硬脂酸、原藻醇12种；其中虚寒组升高的只有乳酸，其余均降低。实寒组与虚寒组之间差异代谢产物有葡萄糖、果糖、异柠檬酸、乳酸、甘氨酸、原藻醇、马尿酸7种，其中虚寒组升高的只有乳酸，其余均降低。
实寒组、虚寒组、正常组MC2生殖内分泌激素（FSH、LH、E2、P、T）组间比较差异均无统计学意义。MC2、MC7空腹血糖（FBG）及血脂指标（TC、TG、LDL-C）组间比较差异均无统计学意义。实寒组有两例样本MC2尿糖（±），其余尿糖均为（－）。
2模型组大鼠外观差，被毛无光泽，耳、爪紫暗；精神萎靡、蜷缩少动、反应迟钝、喜扎堆，体重减轻，肛温降低，便溏；动情周期延长、间期延长；血清FSH、LH、E_2、P、IL-2及血浆CORT、cAMP/cGMP均降低，血浆cGMP升高；胸腺、脾脏、子宫、卵巢指数均降低；与正常组比较差异均有统计学意义。子宫、卵巢形态异常。治疗组较模型组上述指标均有不同程度改善。
3实寒组大鼠表现为HPA、HPT亢进，下丘脑CRH、TRH含量上升，血浆ACTH和CORT，血清T_3、T_4均升高；后期下丘脑CRH、TRH含量、血浆ACTH、CORT，血清T_3稍下降逐渐趋于稳定。免疫系统，前期CD_4＋/CD~＋_8、Th_1/Th_2降低，后期CD~＋_4/CD~＋_8、Th_1/Th_2回升。虚寒组大鼠前期HPA、HPT亢进，下丘脑CRH、TRH含量，血浆ACTH、CORT，血清T3、T4均升高；后期下丘脑CRH、TRH含量、血浆ACTH、CORT，血清T＋＋3下降；Th_1/Th_2、CD_4/CD_8下降。
实寒组、虚寒组均表现为HPOA抑制。实寒组大鼠血清FSH下降明显，15d与0d比较差异有统计学意义；血清T升高，9d与0d比较差异有统计学意义；其余指标均有下降趋势，但与0d比较差异均无统计学意义；子宫内膜ERα表达增强，12d、15d与0d比较差异均有统计学意义。虚寒组大鼠下丘脑GnRH含量及血清FSH、LH、E_2、P均下降，27d、30d下丘脑GnRH含量及血清FSH、E_2与0d比较差异均有统计学意义，30d血清LH、P与0d比较差异均有统计学意义；子宫内膜ERα表达前期有升高趋势后期明显降低，27d、30d与0d比较差异有统计学意义。
4实寒组大鼠肝脏Na~+-K~+-ATP酶、Ca~(~(2+))-Mg~(2+)-ATP酶、SDH活性、ATP含量增加；Na+-K+-ATP酶9d、12d与0d比较差异均有统计学意义；Ca~(2+)-Mg~(2+)-ATP酶15d与0d比较差异有统计学意义；SDH活性、ATP含量12d与0d比较差异均有统计学意义；子宫UCP_2表达、血清FBG、FFA先升后降，UCP_2表达及FBG6d与0d比较差异均有统计学意义，FFA9d与0d比较差异有统计学意义；肝Gn含量先降后升，6d与0d比较差异有统计学意义；血清INS、GLU、肝脏LDH活性、子宫SOD活性、MDA含量各时间点与0d比较差异均无统计学意义。
虚寒组大鼠肝脏Na~+-K~+-ATP酶先升后降，Ca~(2+)-Mg~(2+)-ATP酶、SDH活性及ATP含量前期略有升高中后期明显降低，27d、30d与0d比较差异均有统计学意义；子宫SOD活性降低，24d、27d、30d与0d比较差异均有统计学意义；肝脏LDH活性、子宫UCP_2及MDA含量升高，24d、27d、30d与0d比较差异均有统计学意义；FBG、FFA、GLU均先升后降，肝Gn含量以降低为主，FBG、GLU、肝Gn含量27d、30d与0d比较差异均有统计学意义；INS各时间点与0d比较差异均无统计学意义。
结论：
1实寒证痛经患者、虚寒证痛经患者、正常对照组尿液的差异代谢物质涉及到糖代谢、氨基酸代谢、脂酸代谢等代谢途径以及肠道菌群的紊乱。实寒组与虚寒组的差异主要为糖代谢。
2采用延长冰箱冷冻加冰水浸泡时间方法创建的大鼠模型成功模拟了妇科虚寒证的病因及致病特点，结合临床症状，经实验室检测及药物反证，提示该模型创建成功，可用于妇科虚寒证的研究。
3NEI调控变化是寒证发生的机制之一，也是机体表现出实寒、虚寒不同证候的机制之一。实寒组、虚寒组前期均以亢进为主，实寒组逐渐适应建立新平衡。虚寒组最终转为衰竭导致机体免疫低下及病理性损伤。
4寒证的发生与线粒体能量代谢及氧化损伤有关，其损伤程度是机体表现出实寒、虚寒不同证候的机制之一。实寒组线粒体损伤较轻微；虚寒组线粒体损伤明显，能量代谢障碍。
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 (E_2), progesterone (P),testosterone (T) at MC_2in 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℃~1℃ice 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, E_2, 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 (T_3), thyroxine (T_4), FSH, LH, E_2, 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, E_2, P, and T) of excess-cold group, asthenia-cold group and the normal group at MC_2all 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 MC_2, 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, E_2, 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 T_3were slightlydecreased and gradually tend to be stable. Immune systems, at early daysCD_4/CD_8, Th_1/Th_2was reduced, later period CD_4/CD_8, Th_1/Th_2picked 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 T_3and T_4were all increased; later periodhypothalamus CRH and TRH content, plasma ACTH and CORT, serum T_3were all decreased. Immune system, CD_4/CD_8, Th_1/Th_2was 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, E_2, and P in asthenia-cold group was decreased significantly;27d and30dof GnRH, FSH, and E_2had 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, Ca~(2+)-Mg~(2+)-ATP enzymes, SDH activity, and ATP content.9d,12dof Na~+_-K~+--ATP enzymes had significant difference compared with0d;15dof Ca~(2+)-Mg~(2+)-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 UCP_2and 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 Ca~(2+)-Mg~(2+)-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.
引文
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