Study On Establishment Of Kidney-yang Deficiency Constitution Rat Model And Regulatory Mechanism Of Energy Metablism Based On Theroy Of Natural Endowment

Objective: This study was to preliminarily establish the model rat of kidneyyang deficiency constitution based on the TCM theory of kidney-yang deficiency and constitution.It was proved that the kidney-yang deficiency constitution model was successfully established through evaluation of the mitochondrial energy metabolic standard of brown adipose tissue(BAT)and research on regulatory mechanism between hypothalamus and brown adipose tissue.Thus a feasible experimental animal model was provided for research on kidney yang deficiency and constitutions of tranditional Chinese medicine.Method:(1)A simple cole-heat behavior equipment was made according to the principle of Xiao’s animal thermal behavior intelligent detection system,Using the model,the old female and male rats which were yang deficiency were filtered out based on theory of "feeling cold while being yang-deficiency".The yang-deficiency rats were mated.while being pregnant,maternal rats were molded by "Fright impairing kidney-qi","cold impairing yang" and Cortex Phellodendri intragastric administration until offsprings were born.Thus kidney yang deficiency constition rat model was preliminarily established.(2)A simple cole-heat behavior equipment was used to compare the remaining rate on 40℃ board and birth weight between model group and control group to initially provide a proof for the successful establishment.(3)Transmission electron microscope was used to observe the mitochondrial ultrastructure in BAT to study the physiological structure of energy matabolism.(4)ELISA method was used to measure the density of ATP and western blot method to mesure density of complex-1,2,3,4,5 in BAT to research on the capacity of energy expenditure.(5)ELISA method was used to measure the density of iNOS and western blot method was used to mesure expression of Fis-1,Drp-1,LC3 a,LC3b and Opa-1 in BAT to reasearch on the mitochondrial health state of BAT.(6)RT-PCR method was used to measure the expression of PGC-1α/AMPK-α2/UCP-1 in BAT and the expression of NPY/NPY1 R and POMC/MC4 R in hypothalamus to research on the upstream regulatory mechanism of hypothalamus on the energy expenditure of BAT.Result:(1)The birth weights of KYD group were significantly lower than control group(P<0.01).The remaining rate on 40℃ board of male rats in KYD group showed a slightly rise compared to male rats in control group.(2)Compared to maternal group and control gro up,both male and female rats in KYD group showed remarkable weak mitochondrial ultrastructure with serious swelling state and more disordered cracked mitochondrial crista.(3)Compared to maternal group,rats in KYD group had lower density of ATP(P<0.05),lower expression of complex-1(P<0.01),complex-4(P<0.001).Besides,male rats in KYD group had lower expression of complex-1 and complex-4(P<0.01).Female rats in KYD group had lower density of ATP(P<0.05),lower expression of complex-1(P<0.05)and lower expression of complex-4(P<0.01).Compared to male rats in control group,male rats in KYD group had quite lower expression of complex-1(P<0.001).Compared to female rats in control group,female rats in KYD group had lower expression of complex-1(P<0.01).(4)Compared to maternal group,rats in KYD group had higher density of iNOS(P<0.05),higher expression of Fis-1/Drp-1(P<0.05),LC3a/LC3b(P<0.05)and quite lower expression of Opa-1(P<0.0001).Besides,male rats in KYD group had higher density of iNOS(P<0.01)and quite lower expression of Opa-1(P<0.001),while female rats in KYD group had lower expression of Opa-1(P<0.01).Compared to male rats in control group,male rats in KYD group had higher density of iNOS(P<0.05)and lower expression of Opa-1(P<0.05).Compared to female rats in control group,female rats in KYD group had lower expression of Opa-1 and Drp-1(P<0.01).(5)Compared to maternal group,rats in KYD group showed higher expression of NPY(P<0.05)in hypothalamus.Compared to male rats in control group,male rats in KYD group showed quite lower expression of NPY(P<0.01)and PGC-1α.Compared to female rats in control group,female rats in KYD group showed higher expression of UCP-1(P<0.01).Conclusion:(1)Rats in KYD group showed lower density of ATP,higher remaining rates on 40℃ board and lower birth weight,which kept in accordance with clinic manifestation of kidney yang deficiency constitution and provided a proof that the model had been established primerily.(2)Rats in KYD group showed abnomal expression of respiratory chain enzymes and complex-1 might be the key indicator to evaluate the low energy metablism lever of kidney-yang deficiency constitution model rats.(3)As a target organ for upstream regulation of mitochondrial energy metabolism,hypothalamus restrained the energy metabolism of rats in KYD group,which made the energy metabolism level of model rats show a declining trend and proved the successful extablishment of model from a microcosmic angle.(4)It was concluded that the homeostasis unbalance of mitochondrial fission and fussion in brown tissue could be pathological characteristic indicating the kidneyyang deficiency constitution model had been built successfully.(5)Male and female rats in KYD group showed differences in kidney yang deficiency characters and energy metabolic regulatory factors.It needed further research to make it certain whether gonadal hormone concentration promoted model establishment or not.