Construction Of Kidney-Yang Deficiency Constitution Animal Model And Study On Mitochondrial Energy Metabolism

Objective:The animal model of the Kidney-Yang deficiency is construct through the method of complex model of the cause and the pathogenesis.The animal model of Kidney-Yang deficiency constitution was successfully constructed and evaluated by macroscopical behavior and molecular biological indexes.The relationship between Kidney-Yang deficiency constitution and mitochondrial energy metabolism was studied by mitochondrial enzyme index and mitochondrial weight sequencing method.Methods:1.Parent selection: 130 female and 90 male SD rats were selected by cold-heat plate selection.The rats whose cold-heat board score was more than 300 s and the scale score greater than 10 were included in the parent group.2.The model method: Yang deficiency pregnant rats model in low temperature environment,fear of kidney injury model,salt cedar gavage model,normal pregnant rats normal saline gavage.Model-making time: after picking up Yin suppository,pregnant rats begin to model and normal group is fed until the offspring is born.3.Post-model evaluation: the birth weight and number of offspring were observed.8 female rats,8 male rats and 8 normal rats were randomly selected to observe the general condition,body weight,anal temperature and so on,to carry out open-field experiment to observe the time of piling up and the number of particles in feces,etc.The serum samples were collected and detected by ELISA method,the main indexes were as follows: c AMP,c GMP.4.Mitochondrial enzyme assay: 8 male rats were randomly selected from the model group and 8 rats in the normal group.The serum samples were collected and detected by ELISA method.The main indexes were mitochondrial ATP enzyme,Complex I,complex Ⅲ,Complex Ⅴ,Na+K+-ATPase,and the main indexes were as follows: mitochondrial ATP enzyme,Complex I,complex Ⅲ,Complex Ⅴ,Na+K+-ATPase.5.Mitochondrial weight sequencing: three blood samples from female rats in model group and three blood samples from normal group were randomly selected to isolate white blood cells and complete mitochondrial genome sequencing was carried out by DNA resequencing technique.Results:1.The parents selected 85 female rats and 41 male rats.2.The body weight of newborn mice in the quantitative model group was significantly lower than that in the normal group(P < 0.05).There were 23 rats in the normal group and 65 rats in the model group(1 dead).3.Model evaluation:Macro-behavioral results :(1)General situation: parent rats: the male and female rats were in good mental state,normal activity,quick response,bright eyes and bright fur before modeling.In the model group,the mental state was fine,the reaction was slow and quiet,the tongue color was dark,the hair color was dark and disordered.(2)Body weight and anal temperature: the body weight of the model group was significantly higher than that of the normal group,and the anal temperature of the model group was lower than that of the normal group.(3)The amount of drinking water in the model group was lower than that in the normal group.(4)The number of grains in the stool of the model group was lower than that in the normal group.(5)Compared with the female rats in the normal group,the time in the model group was longer than that in the normal group,The urine volume of male rats in the model group was significantly higher than that in the normal groupMicroscopic detection:compared with the normal group,the levels of serum c AMP and c GMP in the model group were higher than those in the normal group,and those in the model group were significantly higher than those in the control group(P< 0.01).The ratio of c AMP/c GMP decreased significantly(P < 0.01).4.Results of mitochondrial energy metabolism related enzymes: Complex Ⅰ,Na K-ATPase in model group was significantly lower than that in normal group;Complex Ⅲ in normal group was lower than that in model group.5.Mitochondrial DNA resequencing results: SNV results showed that there were 99 mutation sites,including 21 missense mutations and 72 silent mutations,and the other 6 software did not give the mutation type.They were related to gene:mt-Rnr1,Trnw,mt-Co1,mt-Co2,mt-Atp6,mt-Nd4 respectively.Conclusion:1.The rat model of Kidney-Yang deficiency constitution was constructed based on the successive days compound modeling method.It is feasible to establish the rat model by macroscopical behavioral evaluation and microcosmic molecular biological evaluation model.2.The level of mitochondria-related enzymes in Kidney-Yang deficiency rat model was lower than that in normal group,which indicated that the metabolic ability and productivity of Kidney-Yang deficiency constitution were decreased,which was consistent with the clinical manifestation of cold fear and cold fear in Kidney-Yang deficiency constitution group.3.The screening of mitochondrial mutation sites in Kidney-Yang deficiency animal model demonstrated to a certain extent that Kidney-Yang deficiency constitution is related to mitochondrial mutation.After the subsequent experimental demonstration,it can provide the evaluation basis for the animal model of Kidney-Yang deficiency constitution in traditional Chinese medicine.To provide a target for clinical treatment.