Preparation method of stable rat model for primary dysmenorrhea with syndrome of cold coagulation and blood stasis

Abstract

The invention relates to a preparation method of a stable rat model for primary dysmenorrhea with syndrome of cold coagulation and blood stasis, belongs to the technical field of animal model construction, and particularly relates to a construction method of a rat model for primary dysmenorrhea according with the pathogenesis characteristics of cold coagulation and blood stasis in traditional Chinese medicine. The model simulates the pathological process of cold evil invasion and uterus stasis and combines a modern medical hormone intervention means to realize the construction of a'disease and syndrome combined 'animal model, and is mainly used for the anti-dysmenorrhea mechanism research and drug screening of traditional Chinese medicine compound, acupuncture and other therapies. The adopted preparation method comprises the steps that 0.4 mg of estradiol benzoate is subcutaneously injected to each rat every day for 12 days, the injection dosage of the first day and the injection dosage of the last day are increased to 2 times, the rats are placed in the environment of-20 DEG C after injection is completed, epinephrine is injected twice every day from the 6th day of molding, and 2 U of oxytocin is injected to each rat intraperitoneal cavity 24 hours after the epinephrine is injected for the last time. The animal model obtained through the method is stable in phenotype and good in repeatability, compared with pure cold or pure irritation, more remarkable pain behaviors and biochemical changes can be obtained in a short period through combination with epinephrine, and pharmacodynamic screening is facilitated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of animal model construction, and particularly relates to a construction method of a primary dysmenorrhea (PD) rat model conforming to the pathogenesis characteristics of the TCM (Traditional Chinese Medicine) syndrome of 'cold coagulating blood stasis'. The model simulates the pathological process of invasion of cold pathogen and obstruction of the uterus, and combines modern medical hormone intervention means to realize the construction of an animal model combining disease and syndrome, and is mainly used for the research on the anti-dysmenorrhea mechanism of traditional Chinese medicine compound, acupuncture and other therapies and drug screening. BACKGROUND

[0002] 1. Disease definition and clinical significance:

[0003] Primary dysmenorrhea (PD) refers to periodic menstrual abdominal pain without pelvic organ lesions, and the incidence rate is as high as 50%-90%. PD is classified into 'dysmenorrhea' and 'irregular menstruation' in TCM, and is considered to be caused by invasion of cold pathogen, improper emotions and disorder of blood circulation. The 'cold coagulating blood stasis' syndrome is a typical syndrome of PD, and is clinically manifested as severe pain, warm and pressing, dark red menstrual blood with clots, and cold aversion. The syndrome may be accompanied by more complex blood flow obstruction and inflammatory damage process on the basis of PD. Modern research has also found that cold stimulation can significantly affect uterine blood flow and prostaglandin secretion, further aggravating the pain response, reflecting the pathogenesis characteristics of TCM 'cold coagulation causing stasis and stasis causing pain'.

[0004] 2. TCM theoretical basis and PD mechanism of cold coagulating blood stasis syndrome

[0005] The pathological nature of cold coagulating blood stasis primary dysmenorrhea (CCBS-PD) includes:

[0006] Abnormal blood rheology: abnormal blood flow resistance and perfusion, increased whole blood viscosity and plasma viscosity, and 'thick, sticky, coagulation and aggregation' state;

[0007] Under the background of cold stress, the experimental model observed significant enhancement of inflammatory signals, activation of the immune system, rhythm regulation disorder and imbalance of stress response and other pathological characteristics. Multi-omics analysis shows that multiple key signaling pathways closely related to uterine function regulation are activated, including but not limited to: PI3K-Akt signaling pathway (involved in cell survival and inflammatory mediator release); HIF-1 signaling pathway (regulating hypoxic response and angiogenesis); cGMP-PKG signaling pathway (affecting smooth muscle relaxation and rhythm conduction); Rap1 signaling pathway (regulating cell adhesion and barrier function). The synergistic activation of the above signaling pathways exacerbates the abnormal contraction of uterine smooth muscle, promotes the occurrence and development of uterine spasm pain, and constitutes an important molecular mechanism basis of cold coagulating blood stasis type primary dysmenorrhea. SUMMARY

[0009] Technical problems to be solved:

[0010] The systemic stress and cardiovascular risks caused by the modeling with adrenaline are solved, such as increased heart rate, blood pressure fluctuations, arrhythmia, stress-induced hyperglycemia, and death risk, specifically, irreversible tissue damage and high mortality rate of experimental animals caused by extreme cold or long-term ice bath stimulation; no other operations are performed within two hours before and after the injection of adrenaline.

[0011] In addition, the problems of 1. separation of disease and syndrome, i.e. the existing animal model cannot simultaneously simulate the macroscopic signs and microscopic pathology of cold coagulation and blood stasis syndrome, leading to the disconnection between the model and the clinic; 2. narrow evaluation dimension, i.e. lack of a multi-level verification system integrating behavior, hemorheology, tissue morphology and molecular biology; 3. poor drug efficacy prediction, i.e. low response rate of the model to the TCM treatment of warming the meridians and promoting blood circulation, which cannot meet the practical needs of new drug research and development of traditional Chinese medicine, are solved.

[0012] Technical solutions

[0013] A cold stimulation combined with benzoic acid estradiol, adrenaline and oxytocin is used to establish a cold coagulation and blood stasis primary dysmenorrhea rat model, which takes 12 days. The rats are subcutaneously injected with benzoic acid estradiol 0.4 mg per rat per day, and the injection dose is doubled on the first and last days. After the injection is completed, the rats are placed in a-20 ℃ environment. Adrenaline is injected twice a day from the 6th day of modeling. Twenty-four hours after the last injection of adrenaline, the rats are injected with oxytocin 2 U per rat in the abdominal cavity, and the number of writhing reactions and writhing latency of the rats within 30 min are observed. The color Doppler ultrasound instrument is used to observe the hemodynamic changes of the uterine artery, the blood viscosity is detected by the hemorheology and coagulation indexes, the uterine tissue pathology is observed, and the expression of progesterone receptor (PR) in uterine tissue is detected by immunohistochemical method. At the same time, the levels of SOD, MDA and COX2 in plasma and the level of prostaglandin F2α (PGF2α) in uterine tissue are measured. On this basis, the uterine tissue samples are collected for transcriptomics and metabolomics detection, combined with bioinformatics methods to screen differential genes and differential metabolites, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment is performed, and according to the results of metabolomics detection, partial least squares discriminant analysis (PLS-DA) method is used for statistical analysis.

[0014] Advantages

[0015] Adrenaline, cold environment and estrogen are used, and the advantages of using adrenaline for modeling are particularly used:

[0016] The pathogenesis chain of "cold → vasoconstriction → blood stasis" is as follows: adrenaline mainly causes vasoconstriction and reduces perfusion of the uterus and surrounding tissues through α receptors, and superimposed hypothermia is more likely to form "cold blood stasis" construct validity and face validity.

[0017] The phenotype is stable and has good reproducibility: regular administration (such as 0.3 mg / kg twice a day from the 6th day) can form a relatively controllable microcirculation and coagulation / fibrinolysis imbalance, and behavior (number of writhing and latency), uterine electrophysiology, hemorheology, and histology are easier to repeat. Compared with simple cold or simple irritation, the combination of adrenaline can usually obtain more significant pain behavior and biochemical changes in a shorter period, which is convenient for pharmacodynamic screening.

[0018] Specifically described as follows:

[0019] 1. Disease and syndrome combination, strong specificity, precise simulation of core pathology of cold blood stasis syndrome

[0020] Triple pathology synchronously activates cold pathogenesis: -20 ℃ exposure directly simulates the pathogenesis of "external cold invasion", inducing rats to shiver and curl up; blood stasis is formed: adrenaline is injected twice a day to cause α receptor stimulation, leading to persistent spasm of microvessels; dysmenorrhea occurs: oxytocin (2 U / each) triggers uterine tetanic contraction, simulating "pain due to obstruction". The combination of cold and blood stasis induces two major elements, constructing a composite animal model consistent with TCM syndrome differentiation, with strong clinical reproducibility and stable physiological characteristics.

[0021] 2. Hormone fluctuation design consistent with clinical practice

[0022] Benzoic acid estradiol is doubled at the beginning and end (0.4 mg / each × 10 days + 0.8 mg / each for the first and last two days):

[0023] Simulate the "steep rise and steep fall" characteristics of female menstrual cycle estrogen.

[0024] Induce down-regulation of uterine progesterone receptors (PR).

[0025] 3. Multi-layer verification, high model reliability

[0026] Through the observation of tongue, hemorheology, uterine artery ultrasound, uterine tissue pathology and immunohistochemistry, ELISA and other indicators, the success rate and stability of the model are verified, and the typical "cold blood stasis" type of menstrual pain pathological characteristics is shown.

[0027] 4. Clear mechanism of inflammation and oxidative stress

[0028] Biochemical tests showed that the model animals had systemic oxidative stress (decreased SOD and increased MDA) and local inflammatory activation (increased COX2 and PGF2α levels), which verified the high fitting of this model to the pathophysiological mechanism of "cold coagulation and blood stasis" pain.

[0029] 5. Clear molecular basis

[0030] Through transcriptome and metabolome joint analysis, it was found that the primary dysmenorrhea model of cold coagulation and blood stasis showed more complex pathological mechanisms at the molecular pathway level, and the pathogenesis was more in line with the TCM essence of "cold stagnation and blood stasis", which clearly embodied the molecular mechanism path of "cold coagulation leading to blood stasis → inflammatory activation → energy metabolism disorder and endocrine disorder", and systematically revealed the core characteristics of primary dysmenorrhea of cold coagulation and blood stasis, providing an ideal experimental model and theoretical basis for TCM syndrome typing, drug screening and mechanism research.

[0031] 6. Wide platform applicability

[0032] This model is suitable for the intervention mechanism research of traditional Chinese medicine compounds, single compounds and western medicine, and can also be used for TCM syndrome modeling, biomarker screening, new drug efficacy evaluation and other research directions, which has wide application prospect and popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The figure is a schematic diagram of the weight change of the rat.

[0034] Figure 2 The figure is a schematic diagram of the rat tongue.

[0035] Figure 3 The figure is the result of rat abdominal ultrasound.

[0036] Figure 4 The figure is the result of blood rheology.

[0037] Figure 5 The figure is the result of rat uterine HE staining.

[0038] Figure 6 The figure is the result of rat uterine tissue progesterone receptor (PR) immunohistochemistry.

[0039] Figure 7 The figure is the content of rat blood COX2, SOD and MDA.

[0040] Figure 8 The figure is the content of rat uterine PGF2α.

[0041] Figure 9 The figure is the transcriptome result of primary dysmenorrhea.

[0042] Figure 10 The figure is the transcriptome result of primary dysmenorrhea of cold coagulation and blood stasis.

[0043] Figure 11 Metabolomics results of primary dysmenorrhea.

[0044] Figure 12 Metabolomics results of primary dysmenorrhea with blood stasis due to cold accumulation (CCBS-PD). DETAILED DESCRIPTION

[0045] EXAMPLE

[0046] Primary dysmenorrhea with blood stasis due to cold accumulation (CCBS-PD) group: CCBS-PD rat model was established by cold stimulation combined with estradiol benzoate, adrenaline and oxytocin for a total of 12 days. Rats were purchased from Nanjing Kaisijia Biological Technology Co., Ltd.

SYXK (Su) 2021-0025

SYXK (Su) 2016-2018

[0047] Primary dysmenorrhea (PD) group: PD rat model was prepared using estradiol benzoate and oxytocin. Rats were subcutaneously injected with estradiol benzoate 0.4 mg per day, with the first and last day injection doses increased to 2 times. Oxytocin 2 U per rat was injected intraperitoneally before sample collection.

[0048] RESULTS

[0049] 1. Body weight change: During the modeling period, the body weight of the blank group rats gradually increased over time, and the overall body weight of the PD group also increased slightly, while the body weight of the primary dysmenorrhea with blood stasis due to cold accumulation (CCBS-PD) group rats decreased significantly from the 4th day of modeling and maintained a low level in the late modeling period, which was significantly lower than the other two groups. This indicates that cold and blood stasis multi-factor intervention can inhibit the normal weight gain of rats, which is consistent with the characteristics of metabolic slowing and reduced food intake exhibited by cold accumulation and blood stasis syndrome (see Figure 1 ).

[0050] 2. Writhing reaction: The rats in the PD group and the CCBS-PD group showed obvious writhing reaction after oxytocin injection, manifested as abdominal contraction, straightening of hind limbs, body twisting, and more severe in the CCBS-PD group. Statistical analysis showed that the rats in the CCBS-PD group had shorter writhing latency, more writhing times in the same time, and more severe dysmenorrhea compared with the PD group (Table 1).

[0051] Table 1

[0052]

[0053] 3. Tongue image changes: At the end of modeling, the tongue images of the three groups of rats were observed. The rats in the blank group had pale red tongue, smooth and moist tongue surface; the rats in the PD group had slightly darker tongue than normal, slightly reddish tongue body; the rats in the CCBS-PD group had obviously dark red tongue, some appeared ecchymosis, dry tongue surface, suggesting the existence of blood stasis and typical tongue changes of cold coagulation causing microcirculation disorder Figure 2 ).

[0054] Ultrasound changes: Compared with the blank group, the PI, RI and MV of the uterine artery of the rats in the PD group and the CCBS-PD group were increased, and the increase was more obvious in the cold coagulation and blood stasis primary dysmenorrhea group. It is suggested that the cold coagulation and blood stasis state can increase the local blood flow resistance and perfusion of the uterus, forming the pathological basis of blood flow disturbance of "cold coagulation and blood stasis" Figure 3 ).

[0055] Hemorheology: The whole blood low shear relative viscosity, whole blood high shear relative viscosity and plasma viscosity of the rats in the CCBS-PD model group were significantly higher than those in the blank group and the PD group. Statistical analysis showed that compared with the blank group, the above indexes in the PD group increased slightly but had no significant difference, while the CCBS-PD group increased significantly Figure 4 ).

[0056] HE staining changes of uterine tissue: HE staining results showed that the endometrial structure of the blank group was complete, the gland shape was regular, the arrangement was uniform, there was no obvious edema or inflammatory cell infiltration, the endometrial epithelial layer was clear, and there was no shedding or degeneration change; the endometrial structure of the PD group was slightly disordered, the number of glands in the local area increased, the arrangement was irregular, individual glands appeared mild dilation, inflammatory cells in some areas were slightly infiltrated, and there was a certain degree of inflammatory reaction; while the CCBS-PD group showed obvious pathological damage, including endometrial shedding, gland disorder, interstitial edema and a large number of inflammatory cell infiltration Figure 5 ).

[0057] 4. Immunohistochemistry: The results of immunohistochemical detection showed that the progesterone receptor (PR) of the uterine tissue of the blank group was strongly positive, mainly distributed in the periglandular area; the PR expression of the PD group decreased, the color intensity decreased; and the PR expression of the cold coagulation and blood stasis primary dysmenorrhea group further decreasedFigure 6 )。

[0058] 5. Plasma COX2, SOD, MDA content: compared with the blank group, the COX2 and MDA levels in the PD group were significantly increased, and the SOD level was significantly decreased. The COX2 and MDA levels in the CCBS-PD group were further increased, and the SOD level was further decreased. Figure 7 ).

[0059] 6. Uterine tissue PGF2α content: compared with the blank group, the PGF2α in the uterine tissue of the PD group and the CCBS-PD group was significantly increased. Figure 8 )。

[0060] 7. Omics results:

[0061] 7.1 Transcriptomics:

[0062] The KEGG pathways of the PD group and the blank group were significantly enriched in inflammation and immune-related pathways: NF-kappa B signaling pathway (NF-κB, nuclear factor kappa B signaling pathway), PI3K-Akt signaling pathway (PI3K-Akt, phosphatidylinositol 3-kinase / protein kinase B signaling pathway), Cytokine-cytokine receptor interaction (cytokine-cytokine receptor interaction); uterine contraction regulation-related pathways: Platelet activation (platelet activation), Vascular smooth muscle contraction (vascular smooth muscle contraction); cell adhesion and exogenous infection response-related pathways: Cell adhesion and molecul (cell adhesion and molecules), ECM-receptor interaction (extracellular matrix-receptor interaction), see Figure 9 .

[0063] CCBS-PD significantly up-regulated the hypoxia, energy, and stress-related pathways: HIF-1 signaling pathway, cGMP-PKG signaling pathway; cell-matrix remodeling pathways: ECM-receptor interaction, Focal adhesion; signal transduction enhancement pathways: Rap1 signaling pathway, etc. It is suggested that the primary dysmenorrhea of CCBS has cell-matrix abnormalities, hypoxic stimulation, and metabolic regulation disorders, and the mechanism is more complex. Figure 10 .

[0064] 7.2 Metabolomics:

[0065] The main separation was between the PD group and the CON group in component 1 (26.1%), and component 2 (27.8%) supplemented part of the intra-group differences; the confidence ellipses were almost non-overlapping, suggesting that the metabolic / molecular fingerprints were significantly different. The KEGG pathway enrichment analysis results of the PD group and the CON group showed that the Purine metabolism, Pantothenate and CoA biosynthesis, Valine. leucine and isoleucine biosynthesis, etc. pathways were significantly enriched. The above pathways are closely related to energy metabolism and amino acid synthesis. In the state of dysmenorrhea, the uterine tissue appears energy metabolism disorder and amino acid metabolic reprogramming phenomenon, see Figure 11 .

[0066] The separation between the CCBS-PD group and the CON group was more significant, and component 1 contributed more (38%). The confidence ellipse of CCBS-PD was narrow and long, and the intra-group dispersion was low, suggesting that the molecular pattern corresponding to the CCBS-PD type was more stable. The differential metabolites of the CCBS-PD group and the CON group were significantly enriched in the Arachidonic acid metabolism, Pantothenate and CoA biosynthesis, Citrate cycle, Glyoxylate and dicarboxylate metabolism, etc. metabolic pathways. It is particularly noteworthy that the Arachidonic acid metabolism pathway is significantly enriched, suggesting that CCBS stimulation can further enhance prostaglandin synthesis by activating this pathway, exacerbating uterine contraction and inflammatory stress response.

[0067] Summary The animal model obtained by using the method of the present application has stable phenotype and good repeatability. Compared with cold stimulation or irritation stimulation alone, the combination of adrenaline can generally obtain more significant pain behavior and biochemical changes in a shorter period, which is convenient for pharmacodynamic screening.

Claims

1. A method for preparing a stable rat model of primary dysmenorrhea due to cold-induced blood stasis, comprising subcutaneously injecting 0.4 mg / rat of estradiol benzoate daily for 12 days, with the dose doubled on the first and last days; after injection, the rats are placed in an environment of -20 ℃, characterized in that: Starting from the 6th day of modeling, rats were injected with adrenaline twice a day. 24 hours after the last adrenaline injection, oxytocin 2U / rat was injected intraperitoneally.

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