Spleen Deficiency Rat Energy Metabolism And Neuroendocrine Changes In Experimental Research

There are two types of syndromes about spleen yang deficiency syndrome, one is yang deficiency syndrome whose symptoms include chill, cold limbs, low body temperature, the other is spleen Qi deficiency syndrome which involves with symptoms as weakness, anorexia, weight loss, abdominal pain, diarrhea, lassitude,etc. the above are based on the theory of traditional Chinese medicine while its biological base is disorder of digestion and absorption system, in a word, spleen yang deficiency syndrome include two types of syndromes, one is low ability of heat generating, the other is disorder of digestion and absorption system.1 This thesis systemically discusses the development of spleen yang deficiency syndrome.At first, the research shows the development of the theory, experimental research and neuroendocrine research of spleen yang deficiency syndrome, and focuses on the present status, deficiency and future to systemically indicate the traits on energy metabolism especially on heat generating about UCPs; meanwhile, summarizes the regulating features of digestion, absorption,feed and metabolism about Ghrelin, which are the foundations for this research.2 The original consider about designing the researchBased on creating a new spleen yang deficiency syndrome model, aiming at the traits during the energy metabolism and exploring the key mechanism in spleen yang deficiency syndrome. To discuss the main factors of disorder of spleen function and low heat generating based on modern systemic biological integration theory.2.1 Setting up rat model with spleen yang deficiency syndromeIncising BAT, high fatty feeding and cold stimulating every other day to create a reliable spleen yang deficiency syndrome rat model, which emphasizing the traits of reinforce " destroying yang" by incising BAT to decrease the resource of heat generating, which is an innovation in model creating field.2.2 Research on energy metabolism in spleen yang deficiency syndromeSpleen yang deficiency syndrome includes chill, low body temperature symptoms, which is similar with the symptoms caused by low energy metabolism and basal metabolism rate. In modern biochemistry, during the process of energy metabolism always companied with ATP change which can directly supply energy. Many biologic mole and substance take part in this process of energy metabolism.Mitochondrion is main site of cell energy metabolism. the content of UCP is about 6%-14%of total content of inner membrane of mitochondrion which has close relationship with heat generating.Uncoupling proteins(UCPs) is a protein family which are involved with heat generating function.Physiologic research has shown it can decrease mitochondrial membrane potential and uncouple the process of oxidation and phosphorylation, in this way, it can inhibit synthesis of ADP even ATP and the result is to make energy to emit by way of heat. During the process, UCPs generates heat but not energy, and it also has close relationship with energy metabolism and is key to maintain body temperature. Since spleen yang deficiency syndrome is involved with low ability of heat generating, then, it should have a relationship with UCPs expression. This thesis will explain the relationship between UCP and spleen yang deficiency syndrome.2.3 Research on neuroendocrine in rats with spleen yang deficiency syndromeThe latest modern biology research shows brain-intestinal, gastric-intestinal hormone and intestinal nervous system controlling the stomach and intestine. Spleen yang deficiency syndrome has a close relationship with neuroendocrine based on main digestion system and multi organs'disorder. Ghrelin and CCK is familiar with regulating the food in-intake and energy metabolism and hopefully to become a bridge between function of digestive system and energy metabolism status.3 Conclusions about experimental researches3.1 Research on isoproterenol (ISO) inducing heat generating in rats with spleen yang deficiency syndrome3.1.1 Objective:Creating spleen yang deficiency syndrome rat model by removing interscapular brown adipose tissue, high fatty diet, and coldness stimulating every other day, then, injecting ISO as external stimulating resource and observing the sensibility to heat generating and ISO dose-effect relationship with spleen yang deficient rats.3.1.2 Methods:Removing interscapular brown adipose tissue, high fatty diet, and coldness stimulating every other day in rats with reference to evaluation on spleen yang deficiency syndrome rat model referencing the process of emergence and development of spleen yang deficiency syndrome according to the theory of traditional Chinese medicine, observing the rats' behavior, setting up spleen yang deficiency syndrome rat model. then, under 28℃circumstance, injecting ISO and recording the rectal temperature,the temperature of chest and rear leg in spleen yang deficiency syndrome rats, and exploring the relationship between ISO dose and the temperature of each part.3.1.3 Results:the relationship between ISO dose and effect:effective dose 50(EDso) of the rectal temperature peak and the area under curve is 0.28 mg/kg and 0.106 mg/kg, ED50 of the temperature peak of chest and the area under curve is 0.066 mg/kg and 0.070 mg/kg, ED50 of the temperature of real leg peak and the area under curve is 0.071 mg/kg and 0.172 mg/kg. The changing tendency of the rectal temperature, the temperature of rear leg and chest is direct ratio.3.1.4 Conclusions:①totally, spleen yang deficiency syndrome model rats is not that sensible to external heat generating stimulating resource. Low ability of heat generating is probably one of the reasons that rats'slivering and chilling.drugs can improve symptoms of low ability of heat generating which makes an example for how Acotinum Lizhong decoction acts on rats with spleen yang deficiency syndrome.②the tendency of ISO dose-effect of heat generating is direct ratio, but the appropriate dose is not easy to fix due to the change of temperature of circumstance,noise, and light factors etc.3.2 Experimental research on the relationship between brown adipose tissue and UCP1 in spleen yang deficiency syndrome3.2.1 Objective:Exploring the relationship and mechanism about brown adipose tissue, (uncoupling protein 1,UCP1),high fatty diet and Chinese herbal decoction during the process of energy metabolism.3.2.2 Methods:the rats are divided into four groups, and each 16 cases.①control group:fed with ordinary food, d70-d98 ig. With 0.9% NaCl 10 ml/kg/d. d98 testing the ability of ISO deducing heat generating and observing variety curve of the rectal temperature and its peak within two hours and recording the area under the curve.②yang deficiency syndrome group:the same as the control group's except removing brown adipose tissue on d42③spleen yang deficiency syndrome group:the same as the yang deficiency syndrome group's except of being fed with high fatty diet (including 83% ordinary diet,15% triglycerides,2% cholesterol)from d49 to d98 and placed in 19℃circumstance every other day.④Chinese herbal decoction group:the same as the spleen yang deficiency syndrome group except of ig.5ml/kg/d with Aconitum Lizhong decoction and recording weight every week. Recording the index of the weight growth rate, the peak of temperature and the area under the curve of temperature. Using brown adipose tissue to test the content of UCP1.3.2.3 Results:①weight growth rate compared with control group, in yang deficiency syndrome group weight growth rate got lower (P<0.01), compared with yang deficiency syndrome group, weight growth rate in spleen yang deficiency syndrome group got lower(P<0.01), compared with spleen yang deficiency syndrome group, weight growth rate got higher in Chinese herbal decoction group (P<0.01).②temperature peak of heat generating compared with control group, in yang deficiency syndrome group temperature peak of heat generating got lower (P<0.01), compared with yang deficiency syndrome group, temperature peak of heat generating in spleen yang deficiency syndrome group got higher(P<0.01), compared with spleen yang deficiency syndrome group, temperature peak of heat generating got higher in Chinese herbal decoction group (P<0.01). Variety between weight growth rate and temperature peak of heat generating shows direct ratio.③area under the tempreature curve of heat generatingCompared with control group, in yang deficiency syndrome group, area under the tempreature curve of heat generating decreasing (P<0.01), compared with yang deficiency syndrome group, area under the tempreature curve of heat generating in spleen yang deficiency syndrome group increasing (P＜0.01), compared with spleen yang deficiency syndrome group, area under the tempreature curve of heat generating increasing in Chinese herbal decoction group (P<0.01). Variety between weight growth rate and area under the tempreature curve of heat generating is direct ratio.Compared with yang deficiency syndrom group and spleen yang deficiency syndrome group, Chinese herbal decoction group upregulates content of UCP1 and difference has high significance (P<0.01).3.2.4 Conclusions:①Chinese herbal decoction with function of heat generating and tonifying spleen is inclined to make high fatty diet turn into brown adipose tissue and advance its activity.②Chinese herbal decoction with function of warming middle energizer and tonifying spleen can upgrade level and expression of UCP1 in brown adipose tissue, and then make brown adipose tissue decomposed by the mechanism of uncoupling with UCP1, finally dispelling coldness from the bodies in the rats with yang deficiency syndrome.3.3 Interrelationship between energy charge and UCP3 in mitochondrion of skeletal muscle in rats with spleen yang deficiency syndrome3.3.1 Objective:Exploring effect of UCP3 and muscle energy charge after administrated Aconitum Lizhong decoction in rats with spleen yang deficiency syndrome.3.3.2 Methods:Rats are divided into four groups, and each 18 cases. All rats in four groups fed with ordinary food and water for 42 days, then chosen randomly three groups to mock the spleen yang deficiency syndrome, then the left as control group, to the control group always ordinary diet and water. For the three groups:①to choose randomly one group to remove brown adipose tissue(BAT) on d42 and then fed with ordinary diet and water, this group called yang deficiency syndrome group.②the other two groups, also, removed BAT on d42 but both of them are fed with high fatty diet (including 83% ordinary diet,15% triglycerides,2% cholesterol) and placed in the circumstance of 19℃every other day. When on d82, to choose randomly one of groups to ig. Aconitum Lizhong decoction according to the standard of 5ml/kg/d each rat, so this group called Chinese herbal decoction group while the other is called spleen yang deficiency syndrome group. On d98 using the rats blood and skeletal muscle to test indications.3.3.3 Results:①energy charge in Chinese herbal decoction group is lower than those in other three groups, which indicates content of ATP is also lower than those in other three groups and compared with control group, difference has highly significance (P<0.01); compared with yang deficiency syndrome group, difference has significance (P<0.05);compared with spleen yang deficiency syndrome group,difference has significance (P<0.05).compared with spleen yang deficiency group, chinese herbal decoction group upgrade the expresson of UCP3 (P<0.01) and acitivity of ATPase (P<0.05)②compared with control group, energy charge in spleen yang deficiency syndrome is lower and difference has significance (P<0.05); compared with yang deficiency syndrome group, energy charge is also lower but there is no significance (P>0.05).compared with control group, activity of ATPase of myosin raises in spleen yang dificency group (P<0.01)③compared with control group,energy charge in yang deficiency syndrome group is lower but there is no significance (P>0.05)3.3.4 Conclusions:The research shows that the mechanism of dysfunctional heat generating lies in the unbalance between heat generating and energy generating, Aconitum Lizhong decoction increases the content of muscle UCP3 and activity of ATPase of myosin,then inhibits synthesis of ATP (energy generating) to maintain the body temperature by the mechanism of uncoupling of UCP3 finally.3.4 Experimental research on the relationship between UCP3 and blood sugar, triglyceride, total cholesterol in rats with spleen yang deficiency syndrome3.4.1 Objective:Exploring the relationship between UCP3 and blood sugar, triglyceride, total cholesterol after administrated Aconitum Lizhong decoction in rats with spleen yang deficiency syndrome.3.4.2 Methods:The rats are divided into four groups, and each 16 cases.①control group:fed with ordinary food and water, d70-d98 ig. with 0.9% NaCl 10 ml/kg/d.②yang deficiency syndrome group:the same as the control group's except removing brown adipose tissue on d42③pleen yang deficiency syndrome group:the same as the yang deficiency syndrome group's except that fed with high fatty diet (including 83% ordinary diet,15% triglycerides,2% cholesterol)from d49 to d98 and placed in circumstance of 28℃and 19℃in turn.④Chinese herbal decoction group:the same as the spleen yang deficiency syndrome group's except of ig.5ml/kg/d with Aconitum Lizhong decoction. To draw blood from left common carotid artery in all the rats in the four groups on d98 and then to test the content of blood sugar, triglyceride, total cholesterol. Finally to test the content of UCP3 of skeletal muscle by Immunohistochemistry technology.3.4.3 Results:①content of blood sugar compared with control group,content of blood sugar is lower than that in yang deficiency syndrome group (P<0.01); compared with yang deficiency syndrome group, content of blood sugar in spleen yang deficiency syndrome group is higher (P<0.01);compared with yang deficiency syndrome group, content of blood sugar in Chinese herbal decoction group is higher (P<0.01).②content of triglyceride compared with control group,content of triglyceride is lower than that in yang deficiency syndrome group (P<0.01); compared with yang deficiency syndrome group, content of triglyceride in spleen yang deficiency syndrome group is higher (P<0.01); compared with yang deficiency syndrome group, content of triglyceride in Chinese herbal decoction group is higher (P＜0.01).③content of total cholesterol compared with control group, content of total cholesterol in both spleen yang deficiency syndrome group and Chinese herbal decoction group is higher (P<0.01).④expression of UCP3 cytomorphology in each groupⅠ. expression of UCP3 cytomorphology in control group under microscope observing muscle cell fiber arranging regular and in muscle cell fiber is full of yellow (means positive) parts.Ⅱ. expression of UCP3 cytomorphology in yang deficiency syndrome group under microscope observing muscle cell fiber with yellow parts of muscle cell fiber decreases when compared with that in control group.Ⅲ. expression of UCP3 cytomorphology in spleen yang deficiency syndrome group under microscope observing muscle cell fiber with yellow parts of muscle cell fiber decreases when compared with that in control group.Ⅳ. expression of UCP3 cytomorphology in Chinese herbal decoction group under microscope observing muscle cell fiber with yellow parts of muscle cell fiber are more than that in both spleen yang deficiency syndrom and yang deficiency syndrom group.⑤compare of relative content of UCP3 in each group compared with control group, relative content of UCP3 is higher in Chinese herbal decoction group, spleen yang deficiency syndrom group and yang deficiency syndrom group,difference has high significance(P<0.01); after administrating Chinese herbal decoction, relative content of UCP3 increases in Chinese herbal decoction group and difference has high significance when compared with yang deficiency syndrome group and spleen yang deficiency syndrome group(P<0.01).3.4.4 Conclusions:①Chinese herbal decoction increase content of UCP3, and UCP3 alleviates metabolism of fatty acid, which lower content of triglyceride and increase use of glucose and alleviates its metabolism and then cause its levels lower.②fed with high fatty diet in spleen yang deficiency syndrome group cause content of triglyceride rise, which lead to increase free fatty acid (FFA), but due to competitive with glucose, level of FFA can inhibit use of glucose,which cause level of blood sugar to rise.③UCP1 mainly takes effect in brown adipose tissue and UCP3 act in muscle cell fiber, both of them have high specificity and have slight effect on metabolism of cholesterol, besides, high fatty diet increases level of cholesterol, which cause level of cholesterol to rise,and it is the reason that level of cholesterol in control group is higher than that in both spleen yang deficiency syndrome group and Chinese herbal decoction group.④Chinese herbal decoction raises content of UCP3 and then by the mechanism of uncoupling of UCP3 to improv heat generating, which in turn causing content of blood sugar, triglyceride and total cholesterol to rise, so the mechanism of uncoupling of UCP3 is main and key in this process of metabolism.3.5 Experimental research on effects on Ghrelin after administrated Acotinum Lizhong decoction in rats with spleen yang deficiency syndrome3.5.1 Objective:Exploring effects on Ghrelin after administrated Chinese herbal decoction in rats with spleen yang deficiency syndrome and its physiologic mechanism.3.5.2 Methods:The rats are divided into four groups, and each 16 cases.①control group:fed with ordinary food and water, d70-d98 ig. with 0.9% NaCl 10 ml/kg/d. On d98 prohibited to take in food for 12 hours, and then to draw blood from caudal vein and to add EDTA-Na2 and Aprotinin Protein inhibitor to the blood, then to test level of serum Ghrelin. ②yang deficiency syndrome group:the same as the control group's except removing brown adipose tissue on d42..③spleen yang deficiency syndrome group:the same as the yang deficiency syndrome group's except that fed with high fatty diet (including 83% ordinary diet,15% triglycerides,2% cholesterol)from d49 to d98 and placed in circumstance of 28℃and 19℃in turn.④Chinese herbal decoction group:the same as the spleen yang deficiency syndrome group's except of ig.5ml/kg/d with Aconitum Lizhong decoction.3.5.3 Results:①compared with control group, weight growth rate in yang deficiency syndrome group is lower (P<0.01); compared with spleen yang deficiency syndrom group,weith growth rate in Chinese herbal decoction group is higher (P<0.01) difference has high significance.②compared with control group, in spleen yang deficiency syndrome group, the content of Ghrelin decreases, and difference has high significance (P<0.01); while compared with spleen yang deficiency syndrome group, the content of Ghrelin in Chinese herbal decoction group rises, difference high significance (P<0.01). and is close to that of control group (P>0.05).3.5.4 Conclusions:①mechanismⅠ:Aconitum Lizhong decoction can raise level of Ghrelin, and then Ghrelin regulates energy metabolism,alleviates gastric acid secretion,raises food intake and growth,which thus can alleviates the symptoms of anorexia, weight loss of spleen yang deficiency syndrome.②mechanismⅡ:Aconitum Lizhong decoction raises level of Ghrelin, and the latter can alleviate disorder of gastrointestinal motility, which also alleviates the symptoms of lack of appetite, weight loss of spleen yang deficiency syndrome.4 Summarization for the experimental researches4.1 The traits about energy metabolism in spleen yang deficiency syndromeWith the results of above experiences and by multi-factors analysis, here are some infers:(1) level of energy metabolism lowering, and energy substance such as glucose lowering, ATP and body temperature are also decreasing. (2) Regulating in integration, organ and cell or mole to adapt the changing circumstance in the status of low energy metabolism under the control of neuroendocrineimmune network. And by pre-shivering tone, shivering generating heat and non-shivering generating heat with UCP1 in BAT or UCP3 in skeletal muscle.4.2 Two aspects about how Aconitum Lizhong decoction interfering spleen yang deficiency syndrome. (1) elevating the level of energy metabolism to warm spleen yang since it can raise expression of UCP1 of BAT,UCP3 of skeletal muscle and activity of ATPase in myosin. (2) the expression of Ghrelin can regulate digestive and absorption function and energy metabolism to return the normal function of spleen.5 Innovations of the research5.1 Based on traditional theory of spleen yang deficiency syndrome, and considering both disorder of energy metabolism and disorder of gastric and intestinal function. Firstly remodeling spleen yang deficiency syndrome rat model with removing brown adipose tissue, feeding high fatty food. Coldness stimulating every other day with multi-factors. It takes a long time to create the model and totally considers the course of happening and development of spleen yang deficiency syndrome, for above reasons, the model is reliable and practicable.5.2 Ghrelin, UCPs and ATPase are chosen as main indexes, for Ghrelin which is discussed about its physiologic mechanism and the way of regulation from angle of theory of neuroendocrine, which is a new angle deserving further research. For UCPs, CUP1 and CUP3 are related with synthesis of ATP and energy charge, which are viewed as innovations in this research too.5.3 Ghrelin is chosen to be research on its mechanism and the methods on spleen yang deficiency syndrome, which is also a creative way or angle for the research.