| Objective:To study the clinical effect of the method The animal model of spontaneous kidney yang deficiency was established by the method of "screening strengthening traits mating and generation of the same constitution" combined with multiple factors of successive days.The model of kidney yang deficiency constitution was verified from four aspects of animal macroscopic characterization,biological index,pathological section and prescription test,so as to construct an appropriate animal model for TCM constitution research.Methods:1.Screening of cold and heat prone ratsThe rats with a residence time of more than 300 s at 40 ℃ were selected from ordinary SD rats by the differential method of hot and cold plates.2.Animal modeling(1)Impairment of the kidney due to indulgence in sexual activities modeling :The male rats and the estrous female rats were fed in the same cage at the ratio of 1:3 for 1 month,and the female rats in estrus were replaced by a batch of rats within 3 days.(2)Cold food modeling: the parent female rats were confirmed to be pregnant and began to feed Cortex Phellodendri feed(salt Cortex Phellodendri powder:excipients = 1:12.58)until the birth of the first generation.(3)Low temperature modeling: at the same time of cold food modeling,the pregnant rats were raised in the low temperature box(5 ℃-8 ℃)for 2 hours every day.(4)Fear of kidney injury model: at the same time of cold food modeling,pregnant rats were intimidated twice a day with electronic rodent repellent(frequency= 22Hz)for 1 hour each time.3.Generation and breedingCharacter screening: the offspring of each generation were fed to 60 days old,and the offspring with obvious heat tendency were screened by hot and cold plate.Character enhancement: the rats with obvious heat tendency were mated and bred with the constitution,and the rats were intervened until the third generation of offspring was born according to the parental rat modeling method.4.Model evaluation(1)Macroscopic behavior: observe the mental state,hair luster,activity and aggregation of rats in the blank group and their offspring(offspring 1,2,3);detect the physiological indicators such as body temperature(Anal temperature),body weight,food intake,drinking water,urine volume,fecal water content and other physiological indicators of each generation of rats.(2)Biochemical indexes: the serum levels of T3,T4,TSH,TRH,ACTH,CRH,CORT,17-OHCS,t,E2,camp,c GMP,camp / c GMP were detected by ELISA.(3)Histopathology: the pituitary gland,adrenal gland,hypothalamus and kidney of the blank group and the third filial generation rats were embedded in paraffin,and pathological sections were made to observe the changes of cell and tissue structure.(4)The rats in the treatment group were given Yougui Pill solution by gavage(concentration: 0.234g/ml,dose: 1ml / 100g),once a day for 28 days;the rats in the model group were given the same amount of normal saline.The physiological and biochemical indexes with obvious difference between the blank group and the offspring rats were selected,and the differences of related indexes among the blank group,model group and treatment group were compared.5.Statistical methodsThe data were input into spss24.0 statistical software,and the data were analyzed by one-way ANOVA.The differences of various indexes between blank group and offspring rats,between blank group and model group,and between treatment group were compared.Result:1.Screening results of cold and heat prone rats20 male and 20 female rats with obvious heat tendency were selected from the normal SD rats.2.Macro behavioral resultsGeneral situation: compared with the blank group,the offspring rats had the performance of slightly dark eyes,slightly dull reaction,dark coat color,no luster,disorder,fear of cold,reduced activity,like quiet,frequent clustering and so on.Temperature: compared with the blank group,the body temperature of the offspring rats was lower(P = 0.359 > 0.05 for the 1st generation,P = 0.527 > 0.05 for the 2nd generation,and 0.001 < 0.05 for the 3rd generation).Weight: compared with the blank group,the weight of offspring rats decreased(offspring 1: P = 0.000 < 0.01;filial generation 2: P = 0.004 < 0.01;filial generation 3:P = 0.002 < 0.01).Food intake and drinking water consumption: compared with the blank group,the food intake of offspring rats was not significantly different,but the waterconsumption was significantly decreased(offspring 1: P = 0.000 < 0.01;filial generation 2: P = 0.000 < 0.01;filial generation 3: P = 0.000 < 0.01).Urine volume and fecal water content: compared with the blank group,there was no significant difference in the urine volume of offspring rats,but the fecal water content was increased(offspring 1: P = 0.880 > 0.05;offspring 2: P = 0.000 < 0.01;offspring 3: P = 0.006 < 0.01).3.Biochemical index test results(1)Axis index of hphphphphphphpt Axis axis index: compared with rats in the blank group,compared with rats in the blank group,T3 level of offspring rats in offspring rats decreased(offspring: P = 1.00 > 0.05;second generation: P = 1.00 >0.05;second generation: P = 1.00 > 0.05;third generation: P = 0.003 < 0.01;third generation: P = 0.035 < 0.05;second generation: P = 0.000 < 0.01;third generation: P= 0.000 < 0.01;third generation: P = 0.000 < 0.01),TSH level is increased(offspring:P = 1.00 > 0.05;second generation: P = 1.00 > 0.05;second generation: P = 1.2: P =1.00 > 0.05;second generation: P = 0 004 < 0 01),TRH level decreased(F1: P =0.974 > 0.05;2nd generation: P = 0.124 > 0.05;3rd generation: P = 0.029 < 0.05)(2)HPA axis index: compared with the blank group,CRH of offspring rats had no significant change;ACTH was disordered(offspring 1,self generation increased,offspring 3 decreased);CORT decreased(offspring 1: P = 0.624 > 0.05;filial generation 2: P = 1.00 > 0.05;filial generation 3: P = 0.001 < 0.01).(3)Axis index of hphphpg axis: compared with rats in blank group,E2 of offspring rats has no obvious change(1st generation: P = 1.00 > 0.05;2nd generation:P = 1.00 > 0.05;2nd generation: P = 1.00 > 0.05;3rd generation: P = 0.981 > 0.05);t has no obvious change(first generation: P = 1.00 > 0.05;second generation: P =0.935 > 0.05;second generation: P = 0.935 > 0.05;third generation: P = 0.972 > 0.05);17-OHCS is unstable(P = 0.248 > 0.05;second generation: P = 1.248 > 0.05;second generation: P = 1.3: P = 0.981: P = 0.05;third generation: P = 0.981 > 3 generations It decreased:(P = 0.001 < 0.01).(4)Levels of c AMP,c GMP,c AMP / CGM: compared with blank group rats:compared with blank group rats: compared with blank group rats: camp in offspring rats decreased(offspring: P = 0.003 < 0.01;offspring: P = 0.503 < 0.01;second generation: P = 0.503 > 0.05;third generation: P = 0.000 < 0.01;third generation: P =0.000 < 0.01);cggmp is increased in the first generation: P = 1.00 > 0.05;second generation: P = 1.00 > 0.05;third generation: P = 0.007 < 0.01;camp / cggmp is decreased(offspring: P = 0.009 < 0.01;second generation: P = 0.01;second generation: P = 0.503 > 0.05;third generation: P = 0.000 < 0.007 < 0.01;progeny 3: P= 0.0 00<0.01)。4.Pathological sectionCompared with the control group,the proportion of basophils in the pituitary of the third generation rats was significantly reduced,and brown pigmentation was observed in the fascicular zone cells of the adrenal gland,and there was no significant change in the sections of hypothalamus and kidney.5.By Means Of Prescriptions To Speculate The Symptoms(1)Physiological indexes:Compared with the blank group,the body temperature of rats in the model group was lower(P = 0.001 < 0.01),the amount of drinking water was significantly lower(P= 0.000 < 0.01),and the water content of stool was significantly increased(P = 0.001< 0.01).After treatment with Yougui Pill,compared with the model group,rats in the treatment group had higher body temperature(P = 0.000 < 0.01),higher water intake(P = 0.000 < 0.01),and significantly lower fecal water content(P = 0.001 < 0.01),which were close to the level of the blank group.(2)Biochemical indexes:Compared with blank group,T3 decreased(P = 0.003 < 0.01),T4 decreased(P =0.000 < 0.01),TSH increased(P = 0.029 < 0.05).After Yougui Pill treatment,compared with the model group,T3 increased(P = 0.001 < 0.01),T4 increased(P =0.000 < 0.01),TSH decreased(P = 0.034 < 0.05)in the treatment group,which was close to the level of the blank group.Compared with blank group,CORT of model group was significantly decreased(P = 0.001 < 0.01);after treatment with Yougui Pill,CORT of treatment group was increased(P = 0.855)compared with model group,which was close to the level of blank group.Compared with the blank group,camp decreased(P = 0.000 < 0.01),c GMP increased(P = 0.000 < 0.01)and camp / c GMP decreased(P = 0.000 < 0.01).After treatment with Yougui Pill,compared with model group,camp increased(P = 0.000 < 0.01),c GMP decreased(P = 0.011 < 0.05),and camp / c GMP increased(P = 0.019 < 0.05).Conclusion1.The animal model of spontaneous kidney yang deficiency in this experiment accords with the characteristics of Yang deficiency constitution in macroscopic behavior and hypothalamus pituitary target gland axis dysfunction in biological indexes.2.T3,T4,CORT,camp,camp / c GMP,TSH and c GMP may be used as the main indexes for evaluating kidney yang deficiency;among hypothalamus pituitary target gland axis,HPT axis has the closest relationship with kidney yang deficiency.3.It is feasible to establish the animal model of TCM Constitution by "screening,mating,generation and strengthening traits". |
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