| "Correlation between Formula and Syndrome"is an important logic proposition in the diagnosis and treatment based on differentiation of symptoms and signs and formula-ology.The function and usage of formula not only have correlation with medical compatibility but also compatible with the syndrome which it will have effect on,that is the main effect of the formula depends on a considerable extent on the pathology and the role of targets of medical compatibility.There is a problem of extent of correlation between the formula and syndrome.but until now , No one can really provide systematic and comprehensive scientific evidence for"correlation between formula and syndrome".Based on the background,the research is beginned with invariable association of formula and syndrome in connotation,we systematically explore objective validity of the proposition"Correlation between Formula and Syndrome"by the research methods of correlation and different Formulas for One Syndrome.The research is mainly based on the theory of etiology and pathology,clinical manifestations of the syndromes of spleen-qi deficiency in Chinese medicine and features of etiology and pathology of related disease in western medicine.We use compound factors to replicate rat model of syndromes of spleen-qi deficiency,by observing multiple indicators in the neuro-endocrine-immune system,we get Chinese medical syndromes of the animal model and some biological characteristics;On this basis,we comparatively observe the different and same effects of the three formula-SJZT,CHSGS and CSSJT with invigorating the spleen,dispersing the depressed liver,invigorating the spleen and dispersing the depressed liver to the animal model,and approach the different and same effects of the three formula at the same condition of the syndromes of spleen-qi deficiency,in order to obtain possible relevance and modern connotation of relevance between this three formula and the syndromes of spleen-qi deficiency.Then we observed other effects of SJZT on thyroid function and energy metabolism,hemorheology of the syndromes of spleen-qi deficiency,and comprehended biological foundation of SJZT effect spectrum on the condition of"Formula gearing with Syndrome".Finally,we explored genes expression of the model and SJZT influencing it by applying microarray technology,and deeply approached molecular mechanism of the model and SJZT influencing it.This thesis is divided into two main parts : literature review and experimental research.Literature review is mainly focused on the prospect and existing problem of replication animal model of the syndromes of spleen-qi deficiency;Review of experimental research and clinical application on SJZT;Microarray technology in the field of Chinese medicine and the status of research and application prospects were reviewed.Experiment study is mainly focused on four aspects:the replication and evaluation on animal model of the syndromes of spleen-qi deficiency;the effects of SJZT,CHSGS and CSSJT on the syndromes of spleen-qi deficiency;regulatory effects of SJZT on thyroid axis,energy metabolism,hemorheology of the syndromes of spleen-qi deficiency;Gene expression profiles associated with preliminary exploration of the syndromes of spleen-qi deficiency of rat model and study of the effect of SJZT on the model.Study 1 Establishing the animal model of syndromes of spleen-qi deficiency. Methods:Wistar rats were randomly divided into the control group and model group with syndromes of spleen-qi deficiency.Each group has 40 rats.Model group was made by adopting excess fatigue and out of constant diet.Model group was forced to swim in a plastic barrel filled with a warm-water for 10 minutes in 2:00pm every day and fed every other day (the next day was the fasting diet,the other day was given enough to eat),consecutive 3 weeks.In the first 22 days of modeling,stopping giving the above factors,the rat was fed in natural conditions under observation one week,we killed the rats in the 1,2,3,4 week after modeling respectively.We used phloroglucinol to measure the excretion rate of urinary D-xylose,Using high performance liquid chromatography(HPLC)with electrochemical detection of monoamine neurotransmitters , and serum were measured by radioimmunoassay(RIA)to test the content of GAS,IL-1β,IL-2,IL-6,TNFα;plasma MTL,SS,VIP,NPY,SP,β-EP and ACTH,CORT and hypothalamus CRH.Results:1.Observing appearance and behavior:rats of the spleen-qi deficiency model group show a serial of appearance and behavior change after modeling 2 weeks,the hair was dry chaos and no luster;still,extrados,gather together,excited poor;drowsiness,tired;diet significantly reduced,feces rarefaction;weight decreased significantly.It has the most significant changes in the third week after modeling,it shows no significant improvement after stopping model for one week.2.Gastrointestinal function:the excretion rate of urinary D-xylose of spleen-qi deficiency rats was obviously lower in the second week,it was the lowest level in third week,despite the resumption of its fourth week,it is still significantly lower than the control group.Compared with the control group,serum GAS has no significant changes in the first and second week,It dropped significantly in the third week,It is obviously high in the fourth week;plasma MTL is significantly high in the first and fourth week,whereas it is obviously low in the second and third week;plasma SS is significantly high in the third week,and in the fourth week it resumes at the original level;plasma VIP is obviously increased in its second week,it dropped suddenly in the third week and has no significant difference in the fourth week;plasma NPY is significantly low in the second and third week,it has no significant difference in the fourth week;plasma SP is significantly high in the third week,in the fourth week it is low;plasmaβ-EP has no significant difference in the period of three weeks,significantly lower in the fourth week.3.Immune system:rat thymus weight of spleen-qi deficiency model group in the first to third was significantly lower than the control group,despite in the fourth week the weight is rebounded,but it is still significantly lower than the control group.Spleen weight was significantly lower than the control group in the first to fourth week;thymus index dropped in the first to third week,in the first week it is significantly lower than the control group,in the fourth week it is increased to normal level.Spleen index shows a gradual increase after modeling.Compared with the control group,in the third and fourth week it significantly increased;serum IL-1βsignificantly increased in the first week,it was significantly lower in the third and fourth week;serum IL-2 is significantly low in the third week,in the fourth week it increased significantly ; Serum IL-6 significantly increased in the third week , it significantly low in the fourth week;Serum TNFαsignificantly increased in the third week and it is low in the fourth week.4.Neurotransmitters:Compared with the control group,hippocampus 5-HT of spleen-qi deficiency model group was significantly reduced in the third week,and it was significantly increased in the natural recovery period. hippocampus DA was significantly higher in the first to fourth week;hippocampus NE was significantly lower in the third week;hippocampus HIAA was significantly lower in the third week;hippocampus HVA was significantly lower in the second week;hippocampus DOPAC is decreased gradually in the first to fourth week,in the third week it is significantly low.5.HPA axis:the weight of adrenal gland of spleen-qi deficiency model group in the first and second week is close to the control group,it is significantly lower than the control group in the third and fourth week;adrenal gland index is high in the first to third week,in the fourth week it comes to the normal level.During this period in the third week it is significantly higher.Compared with the control group,hypothalamus CRH is significantly reduced,in the fourth week it increased significantly;plasma ACTH is gradually reduced in the first to third week,in the third week it significantly reduced,and in the fourth week it rebounded;plasma CORT continued to reduce in the first to third week.In the fourth week it raise rapidly,in the first,third,fourth week it was significantly low.Conclusion:The rat model showed some symptoms similar to syndromes of spleen-qi deficiency.It has intestinal absorption dysfunction,gastrointestinal dysfunction,the immune system,neurotransmitters and HPA axis dysfunction.The results suggest the model is consistent with the syndromes of spleen-qi deficiency and there exist gastrointestinal dysfunction and regulatory disorder of neuro-endocrine-immune network in much link.Study 2 Effects of SJZT CSSJT and CHSGS on the syndromes of spleen-qi deficiencyMethods : Wistar rats were randomly divided into two groups : control group(20)and model group(50).The rats are modeled for three weeks according to the methods mentioned above.At the end of three week,10 rats either from control group and model group were killed respectively.In the fourth week,we stopped modeling,the animals were randomly divided into spleen-qi deficiency model group(model),CHSGS,SJZT,CSSJT 4 groups,there is 10 rats in each group.Chinese medicine CHSGS,SJZT,CSSJT was given by 1mL/100g consecutive seven days once a day.Model group and the control group were given equal volume of distilled water.At the end of fourth week we killed the rats,we use phloroglucinol to measure the excretion rate of urinary D-xylose and HPLC with electrochemical detection of monoamine neurotransmitters,and serum were measured by RIA to test GAS,IL-1β,IL-2,IL-6,TNFα,plasma MTL,SS,VIP,ACTH,CORT and pituitary ACTH content;and determination of spleen T lymphocyte proliferation function.Results:1.Gastrointestinal function:Compared with the control group,the excretion rate of urinary D-xylose was significantly low when the model group was modeled for three weeks.the excretion rate of urinary D-xylose was significantly low in the spleen-qi deficiency model group in the fourth week.Compared with spleen-qi deficiency model group,the excretion rate of urinary D-xylose in the SJZT,CSSJT group markedly increased.Especially the SJZT group is better than CSSJT group,CHSGS group has no obvious change.Serum GAS,plasma MTL,VIP in model group in the third week are significantly low,plasma SS is significantly high;after a week's natural recovery,serum GAS,plasma MTL of spleen-qi deficiency model group is significantly high;Plasma SS,VIP is significantly low.Compared with the Spleen-qi deficiency model group,SJZT,CSSJT group can significantly increased serum GAS,and there is no significant difference between the two groups,CHSGS group has no obvious change;plasma MTL of SJZT group is significantly low,CHSGS group and CSSJT group has no obvious change;plasma SS of SJZT group and CSSJT group significantly increase,CHSGS group has no obvious change;plasma VIP of SJZT group significantly increase,CHSGS group significantly reduce,CSSJT group has no significant change.Compared with the CHSGS group,plasma VIP of SJZT group significantly increase.2.Immune system:Compared with the control group,serum IL-1β,IL-2 level of the model group was significantly low in the third week after modeling.Serum IL-6,TNFαlevel is high.After one week's natural recovery,the serum IL-1β,IL-2,IL-6,TNFαlevel of spleen-qi deficiency model group was significantly low.Compared with the Spleen-qi deficiency model group,serum IL-1βof SJZT group,CSSJT group significantly increase,and there are no significant differences between the two groups,CHSGS group has little influence;serum IL-2 of CHSGS group significantly reduce and SJZT group significantly increase,CSSJT group has no significant change;serum IL-6 of SJZT group significantly increased and the other two groups have rising trend;serum TNFαof SJZT group significantly increase,the other two groups have no significant change.spleen T lymphocyte proliferation function of spleen-qi deficiency model group was significantly lower than the control group.Compared with the spleen-qi deficiency model group,CHSGS,CSSJT group significantly lower the absorbance value under 570NM, whereas SJZT group markedly improved the absorbance value,and its role was significantly better than CHSGS group and CSSJT group.3.Neurotransmitters:Compared with the control group,hippocampus 5-HT and NE of the model group is significantly low in the third week after modeling,whereas the hippocampus DA is significantly high.After natural recovery for one week,hippocampus 5-HT and NE of spleen-qi deficiency model group were significantly low,but DA is significantly increased.Compared with the Spleen-qi deficiency model group,hippocampus 5-HT of SJZT group significantly increase,CHSGS group and CSSJT group has a trend to rise it,but it is not as obvious as SJZT group;hippocampus DA of the three groups all obviously reduce.SJZT group is close to the value of the control group,CSSJT group dropped remarkably;hippocampus NE of the three groups significantly increased,the three groups have no significant difference.4.HPA axis:Compared with the control group,pituitary ACTH,plasma ACTH and CORT of the model group are significantly low after modeling for 3 weeks.After One week's natural recovery,pituitary ACTH of spleen-qi deficiency model group tended to decrease,Plasma ACTH and CORT are significantly low.Compared with spleen-qi deficiency model group,the three group has no significant change on pituitary ACTH,plasma ACTH of SJZT group and CSSJT group significantly increased,especially the CSSJT group.But rising value of SJZT group is close to the control group,CHSGS group has no significant change;plasma CORT of the three group obviously increase,especially the CSSJT group.Conclusion : Spleen-qi deficiency model has the abnormal changes of gastrointestinal function,immune system,neurotransmitters,the HPA axis;the three observed formula have different degree regulary effects on the syndromes of spleen-qi deficiency.Among the total,SJZT can obviously decrease plasma MTL,hippocampus DA;and can obviously increase serum GAS,IL-1β,IL-2,IL-6,TNFα,hippocampus 5-HT,NE,plasma SS,VIP,ACTH,CORT,spleen T lymphocyte proliferation function of the syndromes of spleen-qi deficiency;CHSGS can obviously decrease plasma VIP,serum IL-2,hippocampus DA and spleen T lymphocyte proliferation function,and can obviously increase hippocampus NE,plasma CORT of the syndromes of spleen-qi deficiency;CSSJT can obviously increase serum GAS,IL-1β,plasma SS,ACTH,CORT,hippocampus NE,and can obviously decrease hippocampus DA and spleen T lymphocyte proliferation function of the syndromes of spleen-qi deficiency.The results cue SJZT can regulate gastrointestinal dysfunction and neuro-endocrine-immune network abnormalities,and it is better than CHSGS and CSSJT.Compared with CHSGS and CSSJT,SJZT has the major relevance to the syndromes of spleen-qi deficiency.Study 3 Other effects of SJZT on rat model of the syndromes of spleen-qi deficiencyMethods : Wistar rats were randomly divided into two groups : control group(20)and model group(30).The method is mentioned above,at the end of the third week we randomly killed 10 rats from the model group and the control group.We stopped modeling at the end of fourth week.Model group rats were randomly divided into spleen-qi deficiency group(model),SJZT group.Each group had 10 rats.SJZT group was given 1mL/100g for seven consecutive days once a day.Spleen-qi deficiency model group and the control group were given equal volume of distilled water.At the end of four week the rat was killed.We test serum TSH,T3,T4 and plasma TXB2,6-keto-PGF1a;Energy charge of liver tissue can be detected by HPLC,using automatic biochemical analyzer to test LDL,CK,P-AMY;Using colorimetric method to test Na+-K+-ATPase,Ca2+-Mg2+-ATPase,Ca2+-ATPase of liver tissue;To test hemorheology by using blood viscosity equipment.Results:1.Thyroid axis:Compared with the control group,serum TSH,T3 and T4 of the model group are significantly low in the third week.In the fourth week serum TSH,T3,T4 of spleen-qi deficiency model group is still significantly low.Compared with the spleen-qi deficiency model group,serum TSH,T3,T4 of SJZT group were significantly high.2.Energy charge of Liver tissue:Compared with the control group,Liver tissue ATP,ADP,EC of the model group were significantly high.AMP of liver tissue was significantly low in the third week.ATP,ADP,EC of spleen-qi deficiency model group are still significantly high;AMP is still significantly low.Compared with the spleen-qi deficiency model group,AMP of liver tissue in SJZT group was significantly lower,EC increased significantly.3.Enzyme of Serum and liver tissue:Compared with the control group,serum LDH,CK are significantly low,Serum P-AMY is significantly high,and the activity of Na+-K+-ATPase,Ca2+-Mg2+-ATPase,Ca2+-ATPase of liver tissue of the model group is significantly low in the third week;Serum LDH is significantly low,serum P-AMY is still significantly high,serum CK has the trend of rising,and the activity of Na+-K+-ATPase,Ca2+-Mg2+-ATPase,Ca2+-ATPase of liver tissue of spleen-qi deficiency model group remained obviously low in the fourth week.Compared with the spleen-qi deficiency model group,serum LDH of SJZT group obviously increase,and P-AMY reduced,serum CK has no obvious change,the activity of liver tissue Ca2+-Mg2+-ATPase , Ca2+-ATPase of SJZT group is significantly increased,there is no obvious change in Na+-K+-ATPase activity.4.Hemorheology and TXB2,6-keto-PGF1a:Compared with the control group,In the third week the whole blood viscosity and reduced blood viscosity of the model group is significantly high,hematocrit is significantly high,and aggregation index is significantly low,deformation index has no significant change,and plasma TXB2,6-keto-PGF1a and the ratio are significantly high.In the fourth week the whole blood viscosity and reduced blood viscosity of the model group is still significantly high,aggregation index is obviously low,the deformation index and hematocrit have no obvious change,and the TXB2,6-keto-PGF1a of spleen-qi deficiency model group are higher than that in the third week,and the 6-keto-PGF1a is obviously high,the ratio is obviously low.Compared with spleen-qi deficiency model group,at the 150/s,10/s,5/s shear rate the whole blood viscosity of SJZT group decreased obviously.the reduced blood viscosity is decreased obviously at the 150/s shear rate;hematocrit,aggregation index,deformation index of SJZT group have no obvious change.and plasma TXB2,6-keto-PGF1a of SJZT group obviously decreased,its ratio obviously high. Conclusion:Spleen-qi deficiency model exists disorder of thyroid function ,energy metabolism,hemorheology and TXB2,6-Keto-PGF1a abnormalities.SJZT can obviously promote serum TSH,T3,T4,LDH,EC,the activity of Ca2+-Mg2+-ATPase,Ca2+-ATPase of liver tissue,TXB2/6-keto-PGF1a,and can obviously decrease serum P-AMY,AMP of liver tissue,the whole blood viscosity and reduced blood viscosity,plasma TXB2,6-keto-PGF1a of the model group.The results indicate SJZT can regulate disorder of thyroid function and energy metabolism,hemorheology abnormalities of the syndromes of spleen-qi deficiency.Study 4 Exploration of gene expression on the syndromes of spleen-qi deficiency and the effects of SJZTMethods:Wistar rats were randomly divided into two groups:control group(20) and model group(30).The method is mentioned above.At the end of third week we respectively give spleen operation under aseptic conditions.In the fourth week we stopped modelling,model animals were randomly divided into spleen-qi deficiency group(model),SJZT group and each group had 10 rats.SJZT group was fed according to 1mL/100g consecutive seven days once a day.the model group and the control group were given equal volume of distilled water.According to the method mentioned above , we get spleen under aseptic conditions for gene exploration.We extract RNA from spleen,and tag RNA by fluorescence,hybridizate and cleanse,scan chip,collect picture and analyze data;At last we inquest functions of obstained different genes by gene bank of http://www.biorag.org/. Results:Compared with the control group,spleen-qi deficiency model group after modeling for 3 weeks,there are 70 differentially expressed genes,among them,6 genes are upregulation and 64 downregulation,referring to inquest known functional genes as followed:RSK,Mpo,Epx_predicted,Ear11,Rn30000067,Rn30023859,Rn30000254,Hsd17b2,Serpinb10,Serpinb2,Rn30014199,Ppbp,Rn30003138,Rhag,Rn30014371,Ngp_predicted,Arl4,Gnaz,Depdc1_predicted,Gp5,Neu2,Rn30016562.Compared with spleen-qi deficiency model group,SJZT group has 446 differentially expressed genes,among them 201 are upregulation and 245 are downregulation;referring to inquest known functional genes as followed:Map3k1,Pla2g1b,Gprk2l,IL9,Akt2,Pik3r1,Nfkbia,Abcb9,Abcb3,Pnlip,Pnliprp1,Pnliprp2,Gp2,RGD1310132,RGD1565722,Iiig9.Conclusion:the model of the syndromes of spleen-qi deficiency exists abnormal expression of part genes,known functional genes of involved in the process of cytokine regulation function,intracellular signal transduction,enzyme activity,energy metabolism,protein synthesis and so on;SJZT has generally regulate to genes expression of spleen-qi deficiency,the main known functional genes mainly influence MAPK signaling pathway,Glycerophospholipid metabolism,G protein-coupled receptor signaling system,JAK-STAT signaling pathway and several other aspects.The results cue that abnormal expression of several genes is possibly molecular foundation of the syndromes of spleen-qi deficiency,however related formula of treating the syndromes of spleen-qi deficiency have complicate regulate to many genes including abnormal expression genes of the syndromes of spleen-qi deficiency.On the whole,we replicated the model of syndromes of spleen-qi deficiency by adopting excess fatigue and out of constant diet,the model rats show a serial of appearance:debility,tired,decreased food appetite,athrepsy,feces rarefaction of some symptoms similar to the syndromes of spleen-qi deficiency,and exist multisystem functional disorder of gastrointestinal function,neuro-endocrine-immune network,hemorheology,energy metabolism and so on,and also some genes take place abnormal expression.The results cue the syndromes of spleen-qi deficiency are related to syndromes of gastrointestinal dysfunction and regulatory disorder of neuro-endocrine-immune network in much link,based on abnormal expression of some genes.SJZT,CHSGS and CSSJT have some regulation of neuro-endocrine-immune network of the syndromes of spleen-qi deficiency.SJZT can finely regulate on the aspects of gastrointestinal hormones,immune factor, neurotransmitters of the syndromes of spleen-qi deficiency ; CHSGS has regulation on neurotransmitters,and can decrease plasma VIP,serum IL-2,spleen T lymphocyte proliferation function;CSSJT mainly regulate neurotransmitters and HPAA.SJZT can regulate gastrointestinal function and neuro-endocrine-immune network abnormalities,and it is better than CHSGS and CSSJT.Comprehensive estimation the three formula from action spectrum and intension of SJZT,CHSGS and CSSJT treating the syndromes of spleen-qi deficiency,therapeutic effect of SJZT is the best,CSSJT is better and CHSGS is worse.SJZT has the major relevance to the syndromes of spleen-qi deficiency.Spleen-qi deficiency model exists disorder of thyroid function,hemorheology,energy metabolism,enzymology and gene,SJZT can improve above-mentioned abnormality to some extent and has general effects on abnormal gene expression.The results indicate SJZT educes regulation of the syndromes of spleen-qi deficiency at the distinct function latitudes and the different constructional decks of integer-tissue-cell-gene,it shows integrated efficiency of traditional Chinese medicine.The topic relatively overall explored on the syndromes of spleen-qi deficiency and biological foundation of related formula from the angle of different Formulas for One Syndrome.It not only carried out beneficial technology exploration for revealing the important logic proposition of"Correlation between Formula and Syndrome",but also provided objective evidence for recognizing"Correlation between Formula and Syndrome"and its scientific connotation.
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