Study On Mitochondria Oxidate Damage And Changes Of Mitochondrial DNA And Genetic Expression In Spleen-deficiency Syndrome

Purposes: The spleen provides the materiel basis of the acquired constitution,is the source of growth and development, and occupies an important positionin the course of life activity. For a long time, spleen-deficiency syndromehas been a hot subject people have been studying. Mitochondria is an organellefor energy transform and "the power factory" of cells of every system ofbody. The energy ATP from mitochondria is always in the centric position ofevery aspects of vital movement of every system. The functional status ofmitochondria directly decides the cellular life condition, which decides theexistence condition of tissue, organ, system and whole body. So mitochondriais a typical and internal contact index, also is a marked index of cell biologyof spleen asthenia syndrome. In 1987 my tutor took the lead to put forwardthat the spleen has close relation with cell mitochondria. In recent years,the extensive development of the studies for animal models showing syndromeof TCM and the upsurge of molecular biology have laid a theoretical foundationfor further probe into the essence of "insufficiency of the spleen". Nowmany researches proved that the damage of free radicle is one of the patho-baseof Spleen-deficiency Syndrome. The emerging of oxygen free radical caninfluence the respiratory function of mitochondria and harm to mtDNA.In order to explore the molecular biology mechanisms of the relationshipbetween Spleen-deficiency Syndrome and mitochondria, the mechanism ofmitochondrial oxidative damage, the mechanism of Chinese herbs ofreplenishing spleen and the changes of mtDNA and its expression wereinvestigated.Methods: The research consists of two parts: literature review andexperimental study. In literature review, we made systematic summary onresearch progress of TCM and western medicine of spleen, Spleen-deficiency, Spleen-deficiency Syndrome, mitochondria, co-relation of Spleen-deficiencyand mitochondria. The experimental study consists of two parts: firstly, 60SD rats were randomly denied into 6 groups: normal control group, model controlgroup, sijunzitang group, dangguibuxuetang group, huangqisijunzi group,dangguibuxue-sijunzitang group, 10 rats in each group. The rats in the controlgroup were fed normally and pleased with normal saline every day. While therats in the model group were fed with half full and perfuse with XiaochengqiTang(60g/kg) every day to build animal model of Deficiency of Spleen-energy.The period of building animal model is about 15 days. Then the drugadministration was began and the time is about 30 days. The general condition,the changes of SOD,GSH-Px,MDA in liver and skeletal muscle tissue, the levelof membrane potential in liver and spleen tissue were measured and observed.Secondly, selecting 40 patients of splenasthenic syndrome and 10 patients ofdamp-heat stagnating in the spleen syndrome, the mutation of mtDNA ATP6 geneorder and the change of gene expressions compared with 8 health adults weredetected.Results:1. The model groups had the typical symptoms of splenic asthenia, but the rectaltemperature had no changes. The drug administration groups can relieve thesesyndromes.2. Compared with normal control group, the lever of SOD,GSH-Px in liver andskeletal muscle tissue was decreased (P＜0.01). Every drug treatmental groupscan enhance the lever of SOD,GSH-Px compared with model group (P＜0.05或P＜0.01).The elevated order: dangguibuxue-sijunzitang group＞huangqisijunzitang group (P＜0.05)＞sijunzitang group (P＜0.05)＞dangguibuxuetang group (P＜0.05).3. Compared with normal control group, the lever of MDA in liver and skeletalmuscle tissue was increased (P＜0.01).Every drug treatmental groups candecrease the lever of MDA compared with model group (P＜0.05或P＜0.01).Thereducing order: dangguibuxue-sijunzitang group＞huangqisijunzitang group(P＜0.05)＞sijunzitang group (P＜0.05)＞dangguibuxuetang group (P＜0.05).4. Compared with normal control group, the lever of membrane potential in liverand spleen tissue was decreased (P＜0.01).Every drug treatmental groups canenhance the lever of membrane potential compared with model group (P＜0.05或P＜0.01).The elevated order: dangguibuxue-sijunzitang group＞ huangqisijunzitang group (P＜0.05)＞sijunzitang group (P＜0.05)＞dangguibuxuetang group (P＜0.05).5. The total mutation rate of the splenasthenic syndrome group is 95%, thesplenogastric hygropyrexia group is 100%. The total mutation rate of thedeficiency of spleen-QI group is 80%. Among the total, the samesense mutationrate is 33%, the absence rate is 20% and the insertion mutation rate is 13%.The total mutation rate of the deficient QI failing to control blood groupis 100%. Among the total, the samesense mutation rate is 58%, the absence rateis 0 and the insertion mutation rate is 17%.The total mutation rate of theasthenic splenoyin group is 100%. Among the total, the samesense mutation rateis 0, the absence rate is 25% and the insertion mutation rate is 25%.The totalmutation rate of the insufficiency of spleen-YANG group is 100%. Among thetotal, the samesense mutation rate is 11%, the absence rate is 0 and theinsertion mutation rate is 0.â‘ In deficiency of spleen-QI group: There are a 8550T and a 8599Cinsertion mutations which can induce the change of open-reading frame. Theincidence rates are both 6.7%. There are a A8555T, a C8563T, a C8575G, a A8566G,a A8567G point mutations which can change coding of amino acids. The incidencerates are all 6.7%.There are a A8564, a A8566 and a C8560 deletion mutationswhich can induce the change of open-reading frame. The incidence rates areall 6.7%.â‘¡In the QI failing to control blood group: There are two 8570T insertionmutations which can induce the change of open-reading frame. The incidencerates are both 17%.There are two A8567G and one C8552G point mutations whichcan change coding of amino acids. The incidence rates are 17% and 8.3%.â‘¢In asthenic splenoyin group: There are 4 patients. One is 8608C insertionmutation. One is C8599 deletion mutation. Those mutations can induce the changeof open-reading frame. The other two are C8649A and T8602C point mutationswhich can change coding of amino acids.â‘£In insufficiency of spleen-YANG group: There are 4 C9196T and 4 C9199Tpoint mutations which made amino acids to become nonsense codon. The incidencerates are both 44%.⑤In splenogastric hygropyrexia group: There are 3 C9197T, 1 C9200T and2 G8885A point mutations which can change coding of amino acids. The incidencerates are 30%,10% and 20%. 6. The fouorescent growth curve of health adults group is positive obviously.Compared with health adults group, the fouorescent growth curve ofsplenasthenic syndrome group and splenogastric hygropyrexia group are weak(P＜0.01). Compared with insufficiency of spleen-YANG group, the fouorescentgrowth curve of splenasthenic syndrome group is weak (P＜0.01). The fouorescentgrowth curve of splenasthenic syndrome group is weak which has no statisticsmeaning compared with splenogastric hygropyrexia group (P＞0.05). Thefouorescent growth curve of insufficiency of spleen-YANG group isweak, compared with splenogastric hygropyrexia group (P＜0.01).Conclusions:1. Irregular eating in combination with bitter drugs for purgation andqi-promoting method is an ideal method for building animal models of spleeninsufficiency.2. Model of spleen deficiency has the disorder of oxidation/anti-oxidation.So splenasthenic syndrome has close relationship with the oxidative damageof mitochondria.3. The mitochondrial membrane potential in the tissues of splenasthenicsyndrome is decreasing. So the damage of mitochondrial function exists in themodel of splenasthenic syndrome. Splenasthenic syndrome has closerelationship with the mitochondrial membrane potential.4. The nourishing both qi and blood method is better than the invigorate thespleen and vital energy method in enhancing the lever of SOD, GSH-Px,mitochondrial membrane potential and decreasing the lever of MDA. Sonourishing the blood can enhance the effect of invigorate vital energy.5. The point mutation, deletion, insertion of gene exists in splenasthenicsyndrome. Those changes induce the damage of mtDNA and the decreasing of geneexpression. Then the proteinums which have no function or disfunction willinfluence the activity of F₀F₁-ATPase directly. Finally it will effect theenergy metabolism of cells and effect the provision of energy of cells.