A method for constructing a kidney yang deficiency constitution rat model

Abstract

The application discloses a method for constructing a kidney yang deficiency constitution rat model, which comprises the following steps: step 1, selecting adult rats, first feeding the male and female rats in separate cages for 10-30 days, and intervening the rats every day during the feeding process; step 2, after step 1, feeding the male and female rats together, and detecting the female rats every day, if the female rats are pregnant, transferring the female rats to single cages for feeding until all the female rats are pregnant, and persisting in intervening the rats every day during the feeding process and the single cage feeding process of the female rats; step 3, stopping the intervention of the female rats in single cages three days before delivery, and the obtained first generation of rats are the kidney yang deficiency constitution rat model; wherein the intervention comprises intragastric stimulation, low temperature stimulation and fright stimulation. The method is convenient and simple, and the kidney yang deficiency constitution of the obtained first generation of rats meets the requirements.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal model construction, and particularly relates to a method for constructing a kidney yang deficiency constitution rat model. BACKGROUND

[0002] Kidney yang deficiency refers to a syndrome caused by deficiency of kidney yang qi and decline of warming function, and is mainly manifested in soreness and weakness of waist and knees, aversion to cold and cold limbs, mental inactivity, pale tongue with white and thick fur, and weak and powerless pulse. It is the largest proportion of constitution type in Chinese population. Kidney yang deficiency constitution is formed by various factors of congenital and acquired factors, involves multiple system dysfunctions such as immunity, endocrine and nerve, and finally easily appears various pathological changes such as osteoporosis, cartilage degeneration and reduced reproductive capacity, which greatly affects the health and quality of life of residents. Establishing a standard kidney yang deficiency constitution animal model and exploring the nature of kidney yang deficiency constitution are indispensable parts of the goal of healthy China.

[0003] The inventor team has long been engaged in basic and clinical research of kidney yang deficiency syndrome and kidney yang deficiency constitution. Combined with the research experience and domestic related research reports, it is found that the current preparation of kidney yang deficiency syndrome animal model mainly uses traditional Chinese medicine etiology or chemical drugs or surgical modeling method. These modeling methods are simple and stable in results, but at the same time, there are the following problems: ①ignoring the key role of genetic factors in the formation process of syndrome and constitution; ②the dose-effect of traditional Chinese medicine etiology stimulation is difficult to quantitatively evaluate; ③there is no recognized standardized model establishment and evaluation method. In order to solve the above problems, the application provides a method for constructing a kidney yang deficiency constitution rat model and an evaluation standard. SUMMARY

[0004] In order to solve the above technical problems, the purpose of the present application is to provide a method for constructing a kidney yang deficiency constitution rat model.

[0005] In order to achieve the above purpose, the technical scheme of the present application is as follows: a method for constructing a kidney yang deficiency constitution rat model, comprising the following steps,

[0006] Step 1: select adult rats, first separate male and female rats for cage feeding for 10-30 days, and intervene the rats every day during the cage feeding process;

[0007] Step 2: after step 1, mix the male and female rats for feeding, and detect the female rats every day. If the female rats are pregnant, they are transferred to single cage feeding until all the female rats are pregnant. The intervention treatment is insisted on every day during the mixed feeding process and the single cage feeding process of the female rats;

[0008] Step 3: stop the intervention treatment of the female rats in single cage feeding 3 days before delivery, and the obtained first generation of rats are kidney yang deficiency constitution rat models;

[0009] The intervention treatment includes gavage stimulation, low-temperature stimulation and fright stimulation.

[0010] In the step 1, the adult rats are 70-day-old rats.

[0011] In the step 1, the male and female rats are bred for 20 days.

[0012] In the step 1, the number of female rats is twice the number of male rats.

[0013] In the step 2, the pregnancy of the female rats is determined by detecting the vaginal plug of the female rats.

[0014] The gavage stimulation is that each rat is given Phellodendri Chinensis Powder for gavage once a day, and the gavage amount is not more than 0.18 g / kg based on the weight of the rat.

[0015] The low-temperature stimulation is that each rat is placed in 0-6 DEG C for 1.5-2.5 hours per day, preferably, the temperature of the low-temperature stimulation is 4 DEG C, and the time length is 1 hour.

[0016] The fright stimulation is that the rats are frightened by an electronic cat for 0-3 times per day, preferably, the time length of each fright is 1-2 hours.

[0017] The beneficial effects of the present application are:

[0018] ① There is no research on kidney yang deficiency constitution animal model in the industry at present. Traditional Chinese medicine believes that constitution is formed in the prenatal period and is nurtured in the postnatal period. The present scheme is the first to take the essence of traditional Chinese medicine constitution as the core, to take the prenatal endowment combined with postnatal intervention as the means, and to construct the preparation method and evaluation system of the spontaneous kidney yang deficiency constitution animal model.

[0019] ② The common kidney yang deficiency syndrome animal model establishment methods can be divided into three types: animal model simulating traditional Chinese medicine etiology, animal model simulating western medicine etiology and pathology, and animal model combining disease and syndrome. The animal model simulating traditional Chinese medicine etiology is close to the essence of traditional Chinese medicine etiology and pathogenesis, but the model has the problems of instability, inability to form a standard, and difficulty in replication. The animal model simulating western medicine etiology and pathology has the advantages of stability, practicality and strong repeatability. Western medicine modeling and surgical modeling can simulate the kidney yang deficiency rat model, but there are disadvantages of being disconnected with traditional Chinese medicine theory, and sometimes unable to cover the extensive pathological changes of kidney yang deficiency syndrome. The present scheme is more suitable for the modeling method of traditional Chinese medicine constitution characteristics, and constructs the spontaneous kidney yang deficiency constitution animal model based on the essence of traditional Chinese medicine constitution etiology and pathogenesis. Moreover, the intervention means of the present scheme is quantified, and the standard model can be formed and successfully replicated.

[0020] At present, the evaluation system of kidney yang deficiency constitution animal model mainly includes TCM syndrome evaluation and biochemical index evaluation. The TCM syndrome evaluation standard is subjective and will inevitably be mixed with subjective factors such as color fading, and only has qualitative standard without grade standard, so it is difficult to distinguish between mild and severe kidney yang deficiency and will lose part of the data. Through literature review, expert consultation and experimental observation, a spontaneous kidney yang deficiency constitution rat macroscopic score table is designed, which has full coverage, representativeness, quantifiable and repeatable indexes, and reduces data loss. The scheme completely and dynamically monitors the overall macro situation of the mother generation and the first generation of rats, and provides a basis for the research of TCM constitution theory. The endocrine indexes of hypothalamic-pituitary-target gland axis are often used as the standard to evaluate whether the kidney yang deficiency model is successful. The classical endocrine indexes of hypothalamic-pituitary-target gland axis are used as the evaluation standard of the spontaneous kidney yang deficiency constitution rat model in the scheme. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The change graph of the mother rat's anal temperature and body weight in the application;

[0022] Figure 2 The change graph of the mother rat's open field activity in the application;

[0023] Figure 3 The change graph of the first generation rat's body temperature and body weight in the application;

[0024] Figure 4 The change graph of the first generation rat's open field activity distance in the application;

[0025] Figure 5 The change graph of the first generation rat's open field activity speed in the application;

[0026] Figure 6 The change graph of the first generation rat's open field four corner time in the application;

[0027] Figure 7 The change graph of the first generation rat's different age stages of feces water content in the application;

[0028] Figure 8 The change graph of the first generation rat's macroscopic score in the application;

[0029] Figure 9 The ELISA index result graph of the HPA axis related to the 3-month-old rat in the application;

[0030] Figure 10 The ELISA index result graph of the HPG axis related to the 3-month-old rat in the application;

[0031] Figure 11 The ELISA index result graph of the HPG axis related to the 3-month-old rat in the application. DETAILED DESCRIPTION

[0032] The principles and features of the present application are described below in conjunction with the accompanying drawings, in which the examples are used to explain the present application and are not intended to limit the scope of the present application. In the following paragraphs, the present application is described in more detail with reference to the accompanying drawings. The advantages and features of the present application will become more apparent from the following description and claims. It should be noted that the drawings are very simplified and use non-precise proportions only for the purpose of facilitating, clarifying and assisting in the explanation of the embodiments of the present application.

[0033] A method for constructing a kidney yang deficiency constitution rat model, comprising the following steps,

[0034] Step 1: Select adult rats, first separate male and female rats for cage feeding for 10-30 days (preferably 20 days), and intervene and treat the rats every day during the cage feeding process;

[0035] Step 2: After step 1, mix the male and female rats for feeding, and detect the female rats every day, if the female rats are pregnant, transfer them to single cage feeding, until all the female rats are pregnant, and insist on intervening and treating the rats every day during the mixed feeding process and the single cage feeding process of the female rats;

[0036] Step 3: Stop the intervention and treatment of the female rats in single cage feeding 3 days before delivery, and the resulting first generation of rats are kidney yang deficiency constitution rat models;

[0037] Wherein, the intervention and treatment includes gavage stimulation, low temperature stimulation and fright stimulation.

[0038] Wherein, the adult rats selected in step 1 are 70-day-old rats.

[0039] Wherein, the duration of cage feeding of male and female rats in step 1 is 20 days.

[0040] Wherein, the number of female rats in the adult rats selected in step 1 is twice the number of male rats.

[0041] Wherein, the judgment standard for pregnancy of female rats in step 2 is that if the female rats' vaginal plugs are detected, they can be identified as pregnant.

[0042] Wherein, the gavage stimulation is to gavage each rat with Huangbai free decoction granules once a day, and the gavage amount per day does not exceed 0.18g / kg based on the rat body weight.

[0043] Wherein, the low temperature stimulation is to place each rat in 0-6℃ for 1.5-2.5h every day, preferably, the temperature of the low temperature stimulation is 4℃, and the duration is 1h.

[0044] Wherein, the fright stimulation is to use an electronic cat to frighten the rats 0-3 times a day, preferably, the duration of each fright is 1-2h.

[0045] In the determination of the optimal intervention treatment condition, the intervention treatment is carried out by using a three-factor three-level orthogonal experiment, and the orthogonal experiment factor level table is shown in Table 1:

[0046] Table 1: Orthogonal experiment factor level table

[0047]

[0048] Table 2: Orthogonal experiment factor combination table

[0049]

[0050]

[0051] In each of Examples 1-9, 70-day-old adult rats (15, 10 female and 5 male) were selected, and the female and male rats were first housed in separate cages for 20 days, then mixed and housed together, and after the female and male rats were mixed and housed together, the female rats were detected daily (the standard for judging whether a female rat is pregnant is that if a female rat's vaginal plug is detected, it is determined to be pregnant), and if a female rat is pregnant, it is transferred to a single cage for feeding until all female rats are pregnant. In the feeding process, each rat is intervened daily, and the intervention method of each example is shown in Table 2.

[0052] In addition, a blank group and a control group were set up according to the experimental needs, and the specific treatment methods of the blank group and the control group were as follows:

[0053] Blank group: same as Example 1, except that the rats were not intervened during feeding. Control group (i.e., model control group): same as Example 1, same as blank group, except that the rats were given hydrocortisone during feeding.

[0054] Experimental results

[0055] I. Macroscopic traits of the maternal generation

[0056] 1. Changes in anal temperature and body weight of the maternal generation rats

[0057] The average anal temperature and average body weight of the maternal female rats in Examples 1-9, the blank group and the control group at different stages (before modeling, when the female and male rats were housed together, during early pregnancy and late pregnancy, wherein before modeling refers to when the rats were 70 days old, when the female and male rats were housed together refers to when the female and male rats were housed together for feeding, early pregnancy refers to when the rats were detected to be pregnant, and late pregnancy refers to three days before delivery) were analyzed:

[0058] For example, Figure 1As shown, the average anal temperature of the maternal rats in Example 1-Example 9 and the control group was significantly lower than that of the blank group, which can indicate the success of the model construction. The average anal temperature of the maternal rats in Example 1-Example 9, the blank group and the control group increased in the late pregnancy, and the blank group was the highest. Compared with the blank group, the average anal temperature of the maternal rats in Example 2, Example 3, Example 5, Example 6, Example 9 and the control group in the late pregnancy was significantly lower than that of the blank group (P<0.01), and the average anal temperature of the maternal rats in Example 1 in the late pregnancy was also lower than that of the blank group (P<0.05). The average anal temperature of the maternal rats in Example 4, Example 7 and Example 8 in the late pregnancy was lower than that of the blank group, but did not reach statistical significance.

[0059] From Figure 1 It can be seen that the average body weight of the maternal rats in Example 1-Example 9 and the control group at each stage had no significant difference compared with the blank group, and had no statistical significance.

[0060] 2, the open field activity of maternal rats

[0061] (1) The total distance of the maternal open field: from Figure 2 It can be seen that the total distance of the maternal open field of each group was at the baseline, and the total distance of the maternal open field of the control group was significantly lower than that of the blank group (P<0.01). The total distance of the maternal open field of Example 3 and Example 9 was lower than that of the blank group (P<0.05). The total distance of the maternal open field of the remaining examples (Example 1, Example 2, Example 4, Example 5, Example 6, Example 7, Example 8) had a decreasing trend compared with the blank group, but had no statistical significance.

[0062] (2) The speed of the maternal open field: from Figure 2 It can be seen that the speed of the maternal open field of Example 1-Example 9 and the control group had a decreasing trend compared with the blank group, but had no statistical significance.

[0063] (3) The four corner time of the maternal open field: from Figure 2 It can be seen that the four corner time of the maternal open field of Example 1, Example 2, Example 3, Example 5, Example 6, Example 8 and Example 9 and the control group had a significantly increasing trend compared with the blank group (P<0.01). The four corner time of the maternal open field of Example 4 was significantly higher than that of the blank group (P<0.05). The four corner time of the maternal open field of Example 7 had an increasing trend compared with the blank group, but had no statistical significance.

[0064] II. The macroscopic traits of the offspring

[0065] 1. The body temperature trend of the offspring:

[0066] (1) Pre-weaning body temperature:

[0067] ①As Figure 3As shown, the body temperature of the F1 generation female mice (0-21 days) in all examples and the control group showed a decreasing trend compared with the blank group. At 21 days of age, the body temperature of the F1 generation female mice in the control group was lower than that in the blank group (P<0.05). The body temperature of the F1 generation female mice in Examples 3 and 5 showed a decreasing trend compared with the blank group. The body temperature of the F1 generation female mice in Examples 1, 2, 4, 6, 7, 8 and 9 was higher than that in the blank group, but there was no statistical significance.

[0068] ②For example Figure 3 As shown, the body temperature of the first-generation male mice (0-21 days) showed a decreasing trend except for Example 3. At 21 days of age, the body temperature of the first-generation female mice of Examples 3, 6 and 9 was significantly lower than that of the control group (p<0.01). The body temperature of the first-generation female mice of Example 5 was significantly lower than that of the control group (P<0.05). The body temperature of the remaining examples and the control group, except for Example 7, showed a decreasing trend compared with the control group, but none of them were statistically significant (including Example 7).

[0069] (2) Body temperature after weaning

[0070] ①For example Figure 3 As shown, the body temperature of the first-generation female mice (30-90 days) after weaning in Example 3 and the control group was lower than that in the other examples and the blank group. Compared with the blank group, the body temperature of the first-generation female mice in Examples 3, 5 and 6 was significantly lower at 21 days of age (p<0.01), the control group was significantly lower (p<0.05), and the other examples all showed a decrease, but it was not statistically significant.

[0071] ②For example Figure 3 As shown, the birth body temperature of the F1 generation male mice (30-90 days) was lower than that of the control group in Example 3, while the birth body temperature of the F1 generation male mice in the other examples and the control group did not show a significant increasing or decreasing trend compared with the control group. At 21 days of age, the F1 generation male mice in Examples 4, 6, and the control group showed significantly lower body temperatures compared with the control group (p<0.01), while Examples 5 and 7 showed significantly lower body temperatures (p<0.05). The other examples also showed lower body temperatures, but these were not statistically significant.

[0072] 2. Weight trend of F1 generation rats

[0073] (1) As Figure 3As shown, the weight gain trend of the first-generation female mice (0-90 days) in each example and the control group was not significantly different from that in the blank group. At 90 days of age, compared with the blank group, the weight gain of the first-generation female mice in each example and the control group was significantly lower in Example 4 and the control group (p<0.05), while it was lower in Example 3, Example 5 and Example 6, and higher in the other examples, but none of them were statistically significant.

[0074] (2) Figure 3 As shown, the weight gain trend of the first-generation male mice (0-90 days) in each example and the control group was not significantly different from that in the blank group. At 90 days of age, compared with the blank group, the weight gain of each example and the control group was significantly lower in each example and the control group, except for Example 7, which showed an increase but no statistical significance. Example 4 showed a significant decrease (P<0.05), and the remaining examples (Examples 1-3, Example 5, Example 6, Example 8 and Example 9) and the control group all showed significant decreases (p<0.01).

[0075] 3. Open field activity of one-month-old F1 generation rats

[0076] (1) F1 generation female mice

[0077] ① Total distance of the open field

[0078] like Figure 4 As shown, all embodiments and the control group showed a decreasing trend compared with the blank group. Among them, embodiments 1-6, embodiment 9 and the control group showed a significant decrease compared with the blank group (P<0.01). Embodiments 7 and 8 showed a decreasing trend compared with the blank group, but there was no statistical significance.

[0079] ② Total velocity of the open field

[0080] like Figure 5 As shown, all embodiments and the control group showed a decreasing trend compared with the blank group. Among them, embodiments 1-6, embodiment 9 and the control group showed a significant decrease compared with the blank group (P<0.01). Embodiments 7 and 8 showed a decreasing trend compared with the blank group, but there was no statistical significance.

[0081] ③ Total time spent at the four corners

[0082] like Figure 6 As shown, Examples 1, 3, 5, 6, 8, 9 and the control group showed a significant increase compared to the blank group (P<0.01), Examples 2 and 7 showed a significant decrease compared to the blank group (P<0.05), and Example 4 showed a decreasing trend compared to the blank group, but this was not statistically significant.

[0083] (2) First-generation male rats

[0084] ①Total distance in open field

[0085] As shown in Figure 4 , each example showed a decreasing trend compared with the blank group, and examples 1-6, 8 and 9 showed a significant decrease (P<0.01), while example 7 showed a decreasing trend but no statistical significance.

[0086] ②Total speed in open field

[0087] As shown in Figure 5 , each example showed a decreasing trend compared with the blank group, and examples 1-6, 9 and the control group showed a significant decrease (P<0.01), while examples 7 and 8 showed a decreasing trend but no statistical significance.

[0088] ③Total residence time in four corners

[0089] As shown in Figure 6 , each example showed an increasing trend compared with the blank group, and examples 3 and 6 (P<0.05) showed a significant increase, while the remaining examples showed an increasing trend but no statistical significance.

[0090] 4. Activity trend of the F1 generation 3-month-old rats in open field

[0091] (1) F1 female rats

[0092] ①Total distance in open field

[0093] As shown in Figure 4 , each example and the control group showed an increasing trend compared with the blank group, but no statistical significance. The remaining examples and the control group were lower than the blank group, and examples 6 and 7 showed a significant decrease (P<0.01), example 3 showed a significant decrease (P<0.05), and the remaining examples and the control group showed no statistical significance.

[0094] ②Total speed in open field

[0095] As shown in Figure 5 , each example and the control group showed a decreasing trend compared with the blank group, and the control group showed a significant decrease (P<0.05), while the examples showed a decreasing trend but no statistical significance.

[0096] ③Total residence time in four corners

[0097] As shown in Figure 6 , each example and the control group showed an increasing trend compared with the blank group, and examples 2, 3, 5, 6, 9 and the control group showed a significant increase (P<0.01), example 1 showed a significant increase (P<0.05), and the remaining examples showed an increasing trend but no statistical significance.

[0098] (2) First-generation male rats

[0099] ① Total distance of the open field

[0100] like Figure 4 As shown, compared with the blank group, all embodiments showed a decreasing trend. Among them, Embodiment 3 showed a significant decrease (P<0.01), Embodiments 5 and 6 showed a significant decrease (P<0.05), and the remaining embodiments showed a decreasing trend, but there was no statistical significance.

[0101] ② Total velocity of the open field

[0102] like Figure 5 As shown, compared with the blank group, all embodiments showed a decreasing trend, with embodiments 3 and 6 showing a significant decrease (P<0.05), and the remaining embodiments all showed a decreasing trend, and none of them were statistically significant.

[0103] ③ Total time spent at the four corners

[0104] like Figure 6 As shown, each embodiment showed an increasing trend compared to the blank group, but none of them were statistically significant.

[0105] 5. Water content in feces of F1 generation rats

[0106] (1) One-month-old female offspring

[0107] like Figure 7 As shown, all examples and the control group showed an increasing trend compared with the blank group. Among them, Examples 1, 2, 3, 6, 9 and the control group showed significant increases (p<0.01), Example 8 showed a significant increase (p<0.05), and the remaining examples had no statistical significance.

[0108] (2) One-month-old male offspring

[0109] like Figure 7 As shown, compared with the blank group, except for Example 7 which showed a decreasing trend, all other examples showed an increasing trend. Among them, Examples 2, 3 and 6 showed significant increases (p<0.01), while the other examples had no statistical significance.

[0110] (3) Three-month-old female offspring

[0111] like Figure 7 As shown, all examples and the control group showed an increasing trend compared with the blank group. Among them, Example 2 showed a significant increase (p<0.05), Examples 3, 6 and 9 showed significant increases (p<0.01), and the remaining examples and the control group showed no statistical significance.

[0112] (4) Three-month-old male offspring

[0113] As shown in Figure 7 , each embodiment shows an increasing trend compared with the blank group, among which, embodiment 2, embodiment 3, embodiment 6 and embodiment 9 significantly increase (p<0.01), and the rest of the embodiments have no statistical significance.

[0114] 6, offspring macroscopic score table (using the table in the appendix to score the female and male offspring mice macroscopically)

[0115] (1) Female offspring mice

[0116] As shown in Figure 8 , each embodiment and the control group shows an increasing trend compared with the blank group, among which, embodiment 7 and embodiment 8 increase, but have no statistical significance, and the rest of the embodiments and the control group significantly increase (p<0.01).

[0117] (2) Male offspring mice

[0118] As shown in Figure 8 , each embodiment shows an increasing trend compared with the blank group, except that embodiment 7 decreases, among which, embodiment 2, embodiment 3, embodiment 6 and embodiment 9 significantly increase (p<0.01), and the rest of the embodiments have no statistical significance.

[0119] 7, statistical results of orthogonal design (macroscopic score table)

[0120] Low temperature (A) F=64.253, P=0.015 (<0.05); Huangbai free decoction gavage (B) F=64.645, P=0.015 (<0.05); fright (C) F=5.318, P=0.158 (>0.05); it can be considered that the overall mean of low temperature and Huangbai free decoction gavage is not equal. From the F value, the influence of each intervention factor from high to low is B>A>C, combined with professional knowledge, the best combination is A3B3C1, that is, high-dose Huangbai free decoction gavage + 4℃ low temperature environment (among which, the number of fright can be 0 times, or 1-2 times per day).

[0121] III. ELISA index results of HPA axis related in 3-month-old offspring rats

[0122] 1, ACTH

[0123] (1) Female offspring mice

[0124] As shown in Figure 9As shown, compared with the blank group, the plasma ACTH content in female rats in all groups showed a decreasing trend. The model control group, Examples 1, 2, 3, 6, and 9 showed significant decreases (P<0.01), Example 5 showed a marked decrease (P<0.05), and the remaining groups showed a decreasing trend, but without statistical significance. Compared with the model group, except for Examples 4, 7, 8, and the non-intervention group of Example 3 which showed significant increases (P<0.01), the remaining groups showed no statistical significance.

[0125] (2) First-generation male rats

[0126] like Figure 9 As shown, compared with the blank group, the plasma ACTH content in male rats in all groups showed a decreasing trend. The model control group, Examples 1, 2, 3, 5, 6, and 9 showed significant decreases (P<0.01), while the remaining groups showed a decreasing trend, but without statistical significance. Compared with the model group, except for Examples 4, 7, 8, and the non-intervention group of Example 3 which showed significant increases (P<0.01), the remaining groups showed no statistical significance.

[0127] 2. CORT

[0128] (1) F1 generation female mice

[0129] like Figure 9 As shown, compared with the blank group, the plasma CORT content of female rats showed a decreasing trend in all groups except for slightly increased levels in Examples 1, 4, 7, 8, and 3 without intervention. The model control group and Example 2 showed a significant decrease (P<0.01), Example 6 showed a significant decrease (P<0.01), and the remaining groups showed a decreasing trend, but there was no statistical significance.

[0130] (2) First-generation male rats

[0131] like Figure 9 As shown, compared with the blank group, the plasma CORT content of male rats showed a decreasing trend in all groups. The model control group, Examples 2, 3, and 6 showed significant decreases (P<0.01), while the remaining groups showed a decreasing trend, but without statistical significance. Compared with the model control group, Examples 1, 5, 7, 8, and 9 showed significant increases (P<0.01), while the non-intervention groups of Examples 4 and 3 showed significant decreases (P<0.05). The remaining groups showed no statistical significance.

[0132] 3, 5-HT

[0133] (1) F1 generation female mice

[0134] like Figure 9As shown, compared with the blank group, the serum 5-HT content of female rats in each group was reduced, except for Example 3, wherein the model control group, Example 4, Example 7, Example 8, Example 9, Example 3 without intervention group was significantly reduced (P<0.01), and the rest of each group had a decreasing trend, but no statistical significance.

[0135] (2) Sub-first-generation male rats

[0136] As shown, compared with the blank group, the serum 5-HT content of male rats in each group was increased, except for Example 1 and Example 3, wherein the model control group and Example 9 were significantly reduced (P<0.01), and the rest of each group had a decreasing trend, but no statistical significance. Figure 9

[0137] Four, ELISA indicators related to HPG axis of 3-month-old offspring rats

[0138] 1. GnRH

[0139] (1) Sub-first-generation female rats

[0140] As shown, compared with the blank group, the serum GnRH content of female rats in each group was decreased, wherein the model control group, Example 1, Example 2, Example 3, Example 5, Example 6, and Example 9 were significantly lower than the blank group (P<0.01), and the rest of each group had a decreasing trend but no statistical significance. Compared with the model group, Example 4, Example 8, and Example 3 without intervention were significantly increased (P<0.01), Example 7 was significantly increased (P<0.01), and the rest of each group was increased, but no statistical significance. Figure 10 (2) Sub-first-generation male rats

[0141] As shown, compared with the blank group, the serum GnRH content of male rats in each group was decreased, wherein the model control group, Example 2, Example 3, and Example 6 were significantly lower than the blank group (P<0.01), and Example 9 was significantly lower than the blank group (P<0.05), and the rest of each group had a decreasing trend but no statistical significance. Compared with the model control group, except for Example 7, Example 8, and Example 3 without intervention group which were significantly increased (P<0.01), the rest of each group was increased, but no statistical significance.

[0142] Figure 10 2. FSH

[0143] (1) Sub-first-generation female rats

[0144] As shown, compared with the blank group, the serum FSH content of female rats in each group was decreased, wherein the model control group, Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, and Example 9 were significantly lower than the blank group (P<0.01), and the rest of each group had a decreasing trend but no statistical significance. Compared with the model group, Example 4, Example 8, and Example 3 without intervention were significantly increased (P<0.01), and the rest of each group was increased, but no statistical significance.

[0145] (2) Sub-first-generation male rats Figure 10 ​​As shown, compared with the blank group, the serum FSH content of female rats in each group showed a decreasing trend. Among them, the model control group, example 3 and example 9 had statistical significance (P<0.05), and the rest had no statistical significance. Compared with the model control group, the rest showed an increasing trend, but had no statistical significance.

[0146] (2) Sub-generation male rats

[0147] As shown, compared with the blank group, the serum FSH content of female rats in each group showed a decreasing trend. Among them, the model control group, example 3 and example 9 had statistical significance (P<0.05), and the rest had no statistical significance. Compared with the model control group, the rest showed an increasing trend, but had no statistical significance. Figure 10 3、LH

[0148] (1) Sub-generation female rats

[0149] As shown, compared with the blank group, the serum FSH content of female rats in each group showed a decreasing trend. Among them, the model control group, example 3 and example 9 had statistical significance (P<0.05), and the rest had no statistical significance. Compared with the model control group, the rest showed an increasing trend, but had no statistical significance.

[0150] Figure 10 (2) Sub-generation male rats

[0151] As shown, compared with the blank group, the serum FSH content of female rats in each group showed a decreasing trend. Among them, the model control group, example 3 and example 9 had statistical significance (P<0.05), and the rest had no statistical significance. Compared with the model control group, the rest showed an increasing trend, but had no statistical significance.

[0152] 4、E2 Figure 10 As shown, compared with the blank group, the serum FSH content of female rats in each group showed a decreasing trend. Among them, the model control group, example 3 and example 9 had statistical significance (P<0.05), and the rest had no statistical significance. Compared with the model control group, the rest showed an increasing trend, but had no statistical significance.

[0153] 5、PG

[0154] Figure 10 (1) Sub-generation female rats

[0155] As shown, compared with the blank group, the serum FSH content of female rats in each group showed a decreasing trend. Among them, the model control group, example 3 and example 9 had statistical significance (P<0.05), and the rest had no statistical significance. Compared with the model control group, the rest showed an increasing trend, but had no statistical significance.

[0156] (2) Sub-generation male rats

[0157] As shown, compared with the blank group, the serum FSH content of female rats in each group showed a decreasing trend. Among them, the model control group, example 3 and example 9 had statistical significance (P<0.05), and the rest had no statistical significance. Compared with the model control group, the rest showed an increasing trend, but had no statistical significance. Figure 10 ​​As shown, compared with the blank group, the serum Pg content of female rats increased in Examples 1, 2, 4, 5, 6, 7, 8, and 3 without intervention, with significant increases in Examples 6 and 7 (P<0.01), and a significant increase in M3 without intervention. The remaining groups showed a decreasing trend, but none of them were statistically significant.

[0158] (2) First-generation male rats

[0159] like Figure 10 As shown, compared with the control group, FSH levels in all groups of male rats showed a decreasing trend. The non-intervention group in Example 3 showed a significantly lower FSH level than the control group (P<0.05), while all other groups showed a significant decrease (P<0.01), all of which were statistically significant. Compared with the model control group, the non-intervention groups in Examples 1, 2, 4, 7, 8, and 3 showed significantly increased FSH levels (P<0.01), while Examples 3, 5, 6, and 9 showed no significant difference and were not statistically significant.

[0160] V. Results of HPG axis-related ELISA in 3-month-old offspring rats

[0161] 1. TRH

[0162] (1) F1 generation female mice

[0163] like Figure 11 As shown, compared with the control group, the serum TRH levels in female rats increased in all groups except for those in Examples 4 and 3 where no intervention was performed, but none of these increases were statistically significant. Compared with the model group, the levels in Examples 3 and 9 were increased, while those in the other groups were decreased, but none of these increases were statistically significant.

[0164] (2) First-generation male rats

[0165] like Figure 11 As shown, compared with the blank group, the serum TRH content of male rats in all groups was increased, with the model control group, Example 6 and Example 3 showing a significant increase (P<0.05), Example 5, Example 7 and Example 8 showing a significant increase (P<0.01), and the remaining groups showing no statistical significance.

[0166] 2. TSH

[0167] (1) F1 generation female mice

[0168] like Figure 11 As shown, compared with the blank group, the serum TSH levels in the female rats in each group were significantly increased (P<0.01), with an increasing trend in Examples 3 and 9, and decreased in the remaining groups, but without statistical significance. Compared with the model control group, all groups were significantly decreased (P<0.01).

[0169] (2)Sub-one generation male rats

[0170] As shown in Table 2, compared with the blank group, the serum TSH content of male rats in each group except the model control group and the non-intervention group of Example 3 increased, but there was no statistical significance. Figure 11

[0171] 3、T3

[0172] (1)Sub-one generation female rats

[0173] As shown in Table 3, compared with the blank group, the serum T3 content of female rats in each group except Example 4, Example 5, Example 7, and Example 8 increased and had no statistical significance, and the rest were significantly reduced (P<0.01). Compared with the model group, except that Example 1 was significantly increased (P<0.05) and Example 2, Example 4, Example 5, Example 7, and Example 8 were significantly increased (P<0.01), the rest had no statistical significance. Figure 11 (2)Sub-one generation male rats

[0174] As shown in Table 4, compared with the blank group, the serum T3 content of male rats in each group except Example 1, Example 7, and Example 8 decreased, and the model control group, Example 2, Example 3, Example 6, Example 9, and the non-intervention group of Example 3 were significantly reduced (P<0.01), and the rest had no statistical significance.

[0175] Figure 11

[0176] 4、T4

[0177] (1)Sub-one generation female rats

[0178] As shown in Table 5, compared with the blank group, the serum T4 content of female rats in each group increased, but there was no statistical significance. Compared with the model group, each group also had no statistical significance. Figure 11 (2)Sub-one generation male rats

[0179] As shown in Table 6, compared with the blank group, the serum T4 content of male rats in each group except the model control group, Example 3, Example 6, Example 7, Example 8, and Example 9 increased, and the rest showed a decreasing trend, but there was no statistical significance.

[0180] Figure 11

[0181] Appendix

[0182] Spontaneous kidney yang deficiency constitution rat trait score table

[0183]

[0184] ​​​​​

[0185]

[0186] Scoring criteria:

[0187] Total score <7 points: normal constitution;

[0188] Total score 8-17 points: mild kidney yang deficiency constitution exists;

[0189] Total score 18-33 points: moderate kidney yang deficiency constitution exists;

[0190] Total score 34-51 points: severe kidney yang deficiency constitution exists.

[0191] The above only for the preferred embodiments of the present application, and not to the present application in any form of restrictions; for the ordinary skilled in the art can be shown in the drawings and the above described by the present application; however, without departing from the scope of the present application technical solutions, the use of the above disclosed technical content of some change, modification and evolution of the equivalent changes, are equivalent embodiments of the present application; at the same time, according to the essence of the present application of the above examples of any equivalent changes made by the modification and evolution, etc., are still within the scope of the present application technical solutions protection.

Claims

1. A method for constructing a rat model of kidney-yang deficiency, characterized in that: Includes the following steps, Step 1: Select adult rats and separate male and female rats into different cages for 10-30 days. During the separate cage rearing process, intervene and treat the rats every day. Step 2: After step 1, male and female rats are housed together, and the female rats are tested daily. If a female rat becomes pregnant, she is transferred to a single cage until all female rats are pregnant. During the mixed housing process and the single cage housing process, the rats are treated daily. Step 3: The intervention treatment was stopped 3 days before the female rats were housed in individual cages. The offspring rats were the rat model of kidney yang deficiency. The interventions include gavage stimulation, hypothermia stimulation, and fright stimulation. The gavage stimulation was performed by administering Huangbai granules (without decoction) to each rat once a day, and the daily gavage volume was no more than 0.18 g / kg based on the rat's body weight. The low-temperature stimulation involved placing each rat at 0-6℃ for 1.5-2.5 hours per day.

2. The method for constructing a rat model of kidney-yang deficiency constitution according to claim 1, characterized in that, The adult rats selected in step 1 are 70-day-old rats.

3. The method for constructing a rat model of kidney-yang deficiency constitution according to claim 1, characterized in that, In step 1, the male and female rats are housed separately for 20 days.

4. The method for constructing a rat model of kidney-yang deficiency constitution according to claim 1, characterized in that, In step 1, the number of female adult rats selected is twice the number of male adult rats.

5. The method for constructing a rat model of kidney-yang deficiency constitution according to claim 1, characterized in that, In step 2, the criterion for determining pregnancy in female rats is that pregnancy can be confirmed if a vaginal plug is detected in the female rat.

6. The method for constructing a rat model of kidney-yang deficiency constitution according to claim 1, characterized in that, The low-temperature stimulation involved placing each rat at 4°C for 1 hour daily.

7. The method for constructing a rat model of kidney-yang deficiency constitution according to claim 1, characterized in that, The frightening stimulation was achieved by using an electronic cat to frighten the rats 0-3 times a day.

8. The method for constructing a rat model of kidney-yang deficiency constitution according to claim 7, characterized in that, Each intimidation session lasts 1-2 hours.

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