| Objective:Based on the etiology modeling of traditional Chinese medicine,a rat model of spontaneous kidney yang deficiency was constructed,and the optimal model of rats was screened out by using the three-factor and three-level orthogonal design,and finally the corresponding model evaluation system was established.Methods:1.Model construction:(1)The three-factor three-level orthogonal design was used to construct a kidney yang deficiency SD rat model,and the specific conditions(three factors)included: cold environment(1h per day),cold food(phellodendron gastric gavage),fright(ultrasonic drive,20-55 khz / 55-110 khz at a distance of 1 m)(respectively identified A,B,C).The three-factor redesign is divided into three levels,and the cold environment is divided into: 4°C,15°C,26°C according to the temperature;the cold food is divided into: 0 g /kg,1.08 g/kg,2.16 g/kg gavage;Fright is divided into 0 h/d,1 h/d,2 h/d according to the length of time(represented by 1,2,3),a total of 99 SD rats(♀ 77,22 ♂)were included.(2)Grouping: model 1 group(A1B1C1,M1),model 2 group(A1B2C3,M2),model 3 group(A1B3C2,M3),model 4 group(A2B1C3,M4),model 5 group(A2B2C2,M5),model 6 group(A2B3C1,M6),model 7 group(A3B1C2,M7),model 8 group(A3B2C1,M8),model 9 group(A3B3C3,M9);with the healthy control group(K10)and the model control group(C11,Hydrocortisone was administered by gavage),final had a total of 11 groups.(3)Parental generation and offspring generation rats are molded in groups,until three months of age.2.Model evaluationAccording to the industry standard of Classification and Judgment of Traditional Chinese Medicine(ZYYXH / T 157-2009)issued by Chinese Medical Association in 2010 and the Expert Evaluation of Syndrome Diagnosis Standard of Kidney Yang deficiency formulated by Professor Yan Shilin,the following indicators are selected to evaluate the model:(1)To evaluate the renal Yang deficiency traits in maternal rats1 General condition and metabolic: The body temperature(ear temperature,palm temperature,(before/after)and anal temperature)was measured before the mold,before together cage,in the early and late pregnancy;the tendency of rats was measured by the test method of cold and hot plate,and the activity of rats was measured.Body weight and body temperature were measured once weekly during the whole procedure.(2)The degree of Yang deficiency in rats: the tendency of cold and heat was measured by the test method of cold and hot plate,and the activity of rats was measured by the open field test.Finally,the data of body temperature,body weight,metabolic cage,absenteeism and cold and hot plate were included in the statistics.(3)To evaluate the effect of kidney Yang deficiency on kidney reproductive function:pregnancy rate,abortion rate,litter size,and record gestational age,fetal arrest,malformation rate in maternal.(2)Evaluation of renal Yang deficiency traits in offspring generation rats(1)Birth status: Test the birth weight,quantity,general condition,body temperature,etc.,the survival rate of mice since birth(three weeks of age),male and female ratio,etc.(2)Growth and development: The growth and development of the daughter mice(0-21d)were evaluated and recorded,and the body temperature(frontal temperature)and body weight were recorded every three days.(3)General situation and metabolic indicators: 30 days of age,through the cold and cold plate,absenteeism test in the same group,male and female half(n=7),and measured weight,body temperature,metabolic access.Body weight and body temperature were measured once a week after 30 days to 90 days of age.(4)Evaluation of kidney Yang deficiency: 90 days of age,in addition to measuring the relevant indicators of 30 days of age,the physical characteristics of kidney Yang deficiency were observed and recorded and scored.(3)Physiopathological changes of major target organs in the hypothalamic-pituitary-target gland axis of offspring generation rats The thyroid gland,pituitary gland,hypothalamus,right side: adrenal gland,kidney,ovary / testis were taken,and pathological sections were made and observed.(4)Neuroendocrine changes in the hypothalamic-pituitary-target gland axis in offspring generation rats Serum and plasma measurement of 90-day age of offspring generation rats: indicators on the HPT axis: rat thyrotropin-releasing hormone(TRH),rat thyrotropin(TSH),triiodothyronine(T3),and thyroxine(T4),free triiodothyronine(FT3),free thyroxine(FT4);HPA axis indicators:corticotropin-releasing hormone(CRH),adrenocorticotropic hormone(ACTH),cortisol(CORT),serotonin(5-HT);Gonadotropin-releasing hormone(Gn Rh);HPG axis indicators: follicle stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E2),progesterone(P).3.Screening of optimal scheme:(1)Design the evaluation form and score the M1-M9 group(2)Included the score,and obtained the optimal mold-making method in statistically.4.Statistical method:Statistical method: Statistical analysis was performed using SPSS 25 software,and the data were expressed as mean ± standard deviation.When the variance was aligned,the mean differences of the two groups were compared by the Tukey method,and when the variance was not equal,the mean was compared by the Dunnett T3 method,and the difference was less than P <0.05.For multi-factor comparison between each model group,extreme difference analysis and variance analysis of three-level orthogonal table were used to determine the quality of factors at each level,the primary and secondary of analysis factors,and predict the best mold making mode.Results:1.Model construction,a total of 99 75-day-old SD rats.M1-M9,K10 groups chosen rats with obvious kidney Yang deficiency,7 each male and female,140 in total,and there was no surviving offspring in C11(21d)due to pathological damage.2.Model evaluation(1)Renal Yang deficiency traits were evaluated in maternal rats1 General conditions and metabolic:1)Body temperature: Before caged together: M1-M9,the anterior palm temperature(except M3,4,6),the posterior palm temperature(except M1-M6,M9),the anal temperature,and the ear temperature(except M3)is all lower than K10.Late pregnancy: M1-M9,C11 anterior palm temperature,posterior palm temperature,anal temperature,ear temperature were all lower than K10.2)Body weight: Before caged together: C11 and M1-M9 group body weight(except M3,M8)was lower than K10,and third trimester: M1-M9 group(except M7)was lower than K10.3)Metabolic access quantity: Before caged together: C11 and M1-M9,food intake(except M1,M3,M4,M8),water intake(except M1,M3,M4),the amount of stool(except M1-3,M8)were lower than K10;and urine output(except M2)were higher than K10.(2)Kidney Yang deficiency degree:Activity degree,he total open field path of M1-M9 group is lower than K10,the average speed is lower than K10,and the residence time of the four corners is higher than K10.Cold and hot tendency,M1-M9 groups(except M4,M5,M8)are higher than K10.(3)To evaluate the effect of kidney Yang deficiency on kidney reproductive function: no pregnant mice in M1-M9 group had fetal arrest,abortion,dystocia,none of the offspring generation had malformation,the number of offspring was lower than K10.(2)Evaluation of renal Yang deficiency traits in offspring generation rats1 Birth status: The mortality rate of offspring in M1-M9 group was higher than K10,and the birth weight was lower than K10.There was no difference in weight groups,but the individual differences were large.2 Growth and development: M1-M9 group,which was quite different in grasping,crawling and air turning,and each group appeared one week later than the same day of rats in the same group.The M3 group was the most significant.3 General conditions and metabolic(M1-M9 group)1)Body temperature: In rats with spontaneous kidney Yang deficiency in the child generation,the body temperature of female and male rats in the M1-M9 group was lower than that of the K10.By 30 days of age,the anal temperature female mice(except M1)(M5,p<0.01)and male mice(except M1,M2)(M3-6,M8,p<0.01)were lower than K10.2)Body weight: In the rats with spontaneous kidney Yang deficiency,both female and male mice in M1-M9 group were weighed less than K10 after birth.By 30 days of age,body weight of M1-M9 females(except M1)(M3-7,M9,p<0.01)and males(except M1 and 2)(M3-6,M8,p<0.01)were lower than K10.3)Metabolic access quantity: In child generation rats with spontaneous kidney Yang deficiency,female mice M1-M9 group(30-day-old),food intake,water intake and stool volume(except M1)were all lower than K10,and stool water content and urine content were higher than K10.30-day-old M1-M9 males had food intake(except M7),water intake(except M1,M7),stool output(except M2,M7),and urine output(except M1,M7)were lower than K10.(4)Kidney Yang deficiency degree:Degree of activity and the child generation spontaneous kidney Yang deficiency rats,the female mice and males M1-M9 group were consistent with those of the maternal generation(the 30-day-old).Cold and hot tendency:In the offspring rats,female and male rats in the M1-M9 group stayed higher than K10 in the hot plate(30-day-old).(3)Physiathological changes of the main target organs of the hypothalamic-pituitary-target gland axis: in offspring rats,the pathological tissue changes of female 90-day-old M1-M9 group and male mice were slight.(4)Neuroendocrine changes in the hypothalamic-pituitary-target gland axis:1)HPT axis: In offspring rats with spontaneous kidney-Yang deficiency,90-day-old females,T3content(except M4,M5,M7,M8),TSH content(except M3,M9)were lower than K10,T4 content,TRH content(except M4),FT3(M6 most obvious,M4 times),FT4(except M1)were higher than K10,among which M6 was the most obvious.In the 90-day-old M1-M9 males,T3(M1,M7,M8)(M2,M3,M6,M9),T4(M6,M7,M8,M9)were lower than K10,TRH(M3,M5,M6-M8),TSH,FT3(M1,M9),FT4(M3)were higher than K10.2)HPA axis: offspring generation spontaneous kidney Yang deficiency rats,90-day-old M1-M9 females,CRH content,ACTH content(M1,M2,M3,M6,M9)CORT content(M1,M4,M7,M8)(M2 most obvious),5-HT(except M3)(M4,M7-M9 most obvious)were lower than K10.In the 90-day-old M1-M9 males,CRH(except M1-M3,M6,M9),ACTH(M1-M3,M5,M6,M9),CORT(except M1,M4,M7,M8)(M2,M3,M6 most obvious),5-HT(except M1,M3)were all lower than K10.3)HPG axis: In spontaneous renal Yang deficiency rats,90-day-old M1-M9 females,Gn RH content(M1-M3,M5,M6,M9),FSH content(M3,M9),LH content and E2 content were lower than K10,and P content(except M3,M9)(M6,M7)were higher than K10.In the 90-day-old M1-M9 males,Gn RH(M2,M3,M6),FSH,LH(M2,M3,M5)were lower than K10,and P(M1,M5-M8)was higher than K10.3.Statistical results of three-level orthogonal designCold(A)F=36.521,P=0;cold(B)F=61.528,P=0;shock(C)F=3.287,P=0.045.P <0.05,the order is B> A> C;the best the best combination is A1B3C3.Males had cold environment(A)F=25.659,P=0;cold food(B)F=41.48,P=0;P <0.05,startle(C)F=1.351,P=0.267> 0.05.Through comparison,it is concluded that the order of factor importance from large to small is B> A> C;the best combination is A1B3C3.Combined P values the best combination is A1B3C1(2,3).Conclusions:1.Based on the theory of TCM etiology,Child generation rats were successfully constructed as a model of spontaneous kidney Yang deficiency,and its characteristics showed that more inclined to warm environment,decreased anal temperature and body weight,reduced activity.2.Through a three-factor,three-level orthogonal design,optimal molding scheme was selected.For the female mice were AIB3C3,2.16 g / kg,4℃ 1h environment,electronic cat for 2h;for the male mice was A1B3,2.16 g/kg,and 4℃ 1h environmental intervention.3.An evaluation method was established for combining macroscopic traits with microscopic hypothalamic-pituitary-target line axis endocrine parameters in rats with renal Yang deficiency.4.TSH,FT3 / FT4 in the HPT axis,ACTH in the HPA axis,Gn RH,FSH,LH,and E2 in the HPG axis were significantly neuroendocrine disorder. |
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