A CRH PVN Construction and application of a mouse model of liver depression-type breast cancer induced by neuronal activation

Abstract

The present invention discloses a CRH PVN The invention relates to a method for constructing a mouse model of liver depression-type breast cancer with neuronal activation and its application, and belongs to the technical field of animal model construction. The method comprises the following steps: (1) injecting 140-160 nl of liquid containing both rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry into the PVN brain region of CRH-IRES-Cre mice, wherein the titers of the two viruses are 2.4-2.6×10 12 pfu; (2) 25-30 days after virus injection, all mice were implanted with breast cancer cells, and CRH was obtained 38-42 days after breast cancer cell inoculation. PVN The method for constructing a mouse model of liver depression type breast cancer with neuron activation is simple to operate and has good repeatability, and can overcome the disadvantage of large individual differences in animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and particularly relates to a CRH PVN Construction method and application of a neuron-activated liver-depression type breast cancer mouse model. Background Art

[0002] Breast cancer is the most common malignant tumor in women and one of the three most common cancers worldwide. The lifetime risk of breast cancer in women is as high as 8%-10%, posing a serious threat to women's health. Psychological factors are a key factor in the development and progression of breast cancer. The progression of breast cancer can increase the psychological burden on patients, further exacerbating tumor growth and creating a vicious cycle. Breast cancer growth caused by psychological stress bears strong similarities to liver depression-type breast cancer in Traditional Chinese Medicine (TCM), suggesting that breast cancer treatment should prioritize soothing the liver and relieving depression.

[0003] To further explore the nature of liver depression promoting breast cancer growth and identify effective traditional Chinese medicine treatments, it is essential to replicate animal models of liver depression-induced breast cancer. Ideally, the replication method for this syndrome-related animal model should be simple, objective, stable, and reproducible, with the etiology, manifestations, and efficacy consistent with Traditional Chinese Medicine theory.

[0004] However, due to various technical difficulties, there is currently a lack of an animal model that can simulate liver depression-induced breast cancer. Furthermore, current animal models used in liver depression research suffer from complex procedures, poor reproducibility, large individual variability, long modeling times, and high animal mortality rates, making them inappropriate for direct application in animal models simulating induced breast cancer. Therefore, there is an urgent need in the field to develop a stable, reproducible, and easy-to-use animal model that can simulate the pathological development and progression of breast cancer associated with liver depression in humans. Currently, there are no reports of a mouse model for liver depression-induced breast cancer.

[0005] Due to the limitations of medical ethics, many clinical studies on liver depression syndrome are difficult to carry out, and sample tissues from patients with liver depression syndrome are even more difficult to obtain. Patients' non-cooperation also adds a lot of difficulties to the clinical research of liver depression syndrome. Therefore, it is necessary to find a substitute for human clinical research. Animal models of traditional Chinese medicine syndromes provide a way to solve this problem due to the sufficient number of animals, strong operability, and reliable models. They have been widely used in modern research in traditional Chinese medicine. Because the emotional changes such as depression and low mood manifested by liver depression syndrome are very similar to the main symptoms of depression (low mood, loss of interest), traditional Chinese medicine researchers borrowed the animal model of depression in Western medicine and combined it with the etiology and pathogenesis theory of liver depression syndrome in traditional Chinese medicine to produce an animal model of liver depression syndrome with traditional Chinese medicine characteristics. Currently, there are mainly 5 types of relatively mature animal models of liver depression syndrome in traditional Chinese medicine syndrome:

[0006] (1) Chronic stress modeling

[0007] The chronic unpredictable stress (CUS) model uses a variety of chronic stimuli to create a model. Although animals exhibit symptoms of liver depression syndrome, such as anhedonia and decreased desire for survival, the high number of stimuli and the intensity of these stimuli lead to high mortality, making it unsuitable for animal research on liver depression. Therefore, Wilmer et al. improved on this model and proposed the chronic unpredictable mild stress (CUMS) model. This model uses a variety of chronic, unpredictable, low-intensity stress events, including fasting, water deprivation for three days, hanging from an empty bottle, restraint, ice water swimming, heat exposure, horizontal shock, foreign body stimulation, tail clamping, and overnight illumination. Rodents are exposed to a series of repeated, unpredictable mild stimuli over a period of three weeks to three months, inducing anhedonia and depressive behaviors, such as weight loss, decreased sugar consumption, and decreased desire for survival.

[0008] This model incorporates a high number of stressors with low intensity, resulting in a highly effective and long-lasting stimulation. It better simulates the conditions in which people experience liver depression, often caused by long-term exposure to various adverse physical and mental stimuli in daily life. Furthermore, it is effective with antidepressants and resembles clinical treatment scenarios. This model is valuable for studying the etiology, pathogenesis, and therapeutic mechanisms of depression and is currently one of the most widely used models. However, its operation is complex and can result in significant individual variability.

[0009] (2) Drug modeling method

[0010] ① Carbon tetrachloride (CCl) acute poisoning method

[0011] Frustrated emotions and anger damage the liver, leading to liver dysfunction and overpowering the spleen. Alternatively, improper diet and excessive fatigue can damage the spleen, leading to spleen dysfunction, dampness stagnation, and liver failure to regulate and release qi. This can lead to "liver depression and spleen deficiency syndrome," characterized by depression, poor appetite, abdominal distension, fatigue, intestinal rumbling, loss of breath, and flank pain. CCl is a recognized liver poison. Acute CCl poisoning can cause neurological symptoms (such as headache, dizziness, confusion, convulsions, hyperreflexia, drowsiness, coma, and respiratory muscle paralysis) and digestive symptoms (hepatomegaly, impaired liver function, nausea, vomiting, abdominal pain, and diarrhea). In 1979, Hunan Medical College pioneered the use of acute CCl poisoning to establish an animal model of liver depression and spleen deficiency in mice and rats. A study selected rats and gave them a subcutaneous injection of CC peanut oil solution (0.005mlg) once on the 1st and 4th day respectively. The rats showed symptoms of "liver depression and spleen deficiency": dry hair, lack of struggle, decreased appetite, weight loss, soft and light-colored stools; accompanied by liver lesions, such as significant liver enlargement, liver cell necrosis, and abnormal liver function. This model has opened up a new example for the study of animal models of liver depression.

[0012] ② Artemisia argyi injection method

[0013] Artemisia argyi (Mugwort leaf) is pungent, warm, and bitter in nature. It enters the liver, spleen, and kidney meridians, and has the effects of warming the meridians to stop bleeding, dispelling cold and relieving pain; it can also be used externally to dispel dampness and relieve itching. Artemisia argyi oil is a volatile oil found in dried or fresh leaves, the main active ingredients of which are terpinenol-4 and α-terpineol. Studies have shown that artemisia argyi oil has adrenaline-like effects and has a stimulating effect on the central nervous system (CNS). Excessive intake can cause toxic hepatitis. In 1979, researchers at Hunan Medical College administered 6.4g / kg of artemisia argyi oil intraperitoneally to wild-type mice twice a day. After 2-3 months of continuous injections, the mice developed manic symptoms characteristic of liver depression: increased activity, constant fighting, and difficulty grasping. Mice were given intraperitoneal injection of mugwort leaf injection (0.6 ml, once a day), and rats were given intraperitoneal injection of mugwort leaf injection (2 ml, twice a day). After continuous medication for 40 days, the mice showed symptoms of liver depression18, and pathological changes in the liver occurred, such as increased CAMP and CAMP / CGMP in the liver, cerebral cortex and thalamus.

[0014] ③Adrenal subcutaneous injection

[0015] The liver governs the flow of qi, maintaining the ascending, descending, entering, and exiting movements of the body's qi. If various causes lead to liver dysfunction, qi flow is compromised. Qi is the leader of blood; when qi flows, blood circulates; when qi stagnates, blood stagnates, resulting in the syndrome of "qi stagnation and blood stasis." The subcutaneous adrenal injection model is a representative example. Xiao Chun et al. replicated a rat model of endometriosis with liver stagnation using surgical transplantation and 1% adrenal subcutaneous injection (0.15 ml per rat, once weekly for four weeks). This animal model exhibits clinical symptoms associated with liver stagnation and pathological changes in the adrenal glands, and has a short treatment cycle, but requires high surgical skills and a low animal survival rate.

[0016] ④ Polyinosinic-polycytidyl acid (PolyI:C) intraperitoneal injection

[0017] Clinical studies have found that after high-dose interferon alpha (IFN-a) is used to treat liver infections or some malignant tumors, up to 40%-50% of patients experience symptoms of liver depression, such as depression or anxiety, loss of appetite, and abnormal behavior. PolyI:C is a double-stranded RNA composed of inosinic and cytidylic acid molecules that can induce the production of tumor necrosis factor-a (TNF-a) in human and animal cells. Huang Weiliang et al. used a high dose of PolyIC (3 mg / kg) to induce a mouse model of liver depression. Behavioral testing revealed a significant decrease in activity in the open field test (OFT), a significant decrease in sucrose consumption in the sucrose preference test (SPT), and prolonged immobility in the tail suspended test (TST) and forced swimming test (FST).

[0018] The above drug modeling methods simulate the manifestations of liver depression syndrome to a certain extent. However, due to the large side effects of the drugs, affecting more organs, and being quite different from the disease patterns of clinical liver depression patients, they are not the preferred modeling methods for the animal model of liver depression syndrome in traditional Chinese medicine.

[0019] (3) Emotional stimulation modeling

[0020] The primary cause of liver depression is emotional imbalance. Emotional distress and unfulfilled emotional desires lead to a dysfunctional liver qi system, resulting in liver qi stagnation and liver depression. Therefore, many researchers have developed several animal models of liver depression, focusing on the emotional pathogenesis of liver depression, including tail-pinching, provoking fighting, single-cage feeding with a neck yoke, and restraint. Xu Huiren et al. used tail-pinching to induce fighting in rats, causing them to become enraged. After three days of continuous stimulation, the rats developed symptoms of liver depression, including decreased biting, physical fatigue, and even decreased appetite, weight loss, and body mass. Chen Xiaoye et al. applied a homemade neck yoke to rats, restricting their daily behaviors such as grooming and itching. Seven to ten days after modeling, the rats showed signs of sluggishness, dull fur, fatigue, and decreased appetite, even dark purple nails and scales on their tails. Qiao Mingqi and Lü Zhiping et al. used thin strips of medical bandage to restrain rats' limbs, restricting their movement and making walking difficult. After one week of restraint, the rats developed symptoms of liver qi stagnation: decreased activity, dull eyes, decreased appetite, and avoidance of irritants. Peng Cheng et al. used a method of restraint plus water immersion, immersing the rats below the xiphoid process in water and restricting their movement, forcing them to maintain a standing position in the water, to achieve a model of liver qi stagnation.

[0021] This modeling method aligns with the pathological process of emotional imbalance leading to liver dysfunction, resulting in an agitated state in the modeled animals. However, current research focuses on the liver qi stagnation syndrome, which primarily manifests as emotional suppression and melancholy rather than anger. This is inconsistent with the clinically observed pathological mechanism of liver qi dysfunction leading to unfulfilled desires and unfulfilled aspirations.

[0022] (4) Orphanage or maternal separation (MS)

[0023] Separating animals accustomed to group housing and raising them in solitary housing for long periods of time can lead to symptoms of liver qi stagnation, such as low mood and a reluctance to eat. The MS model is a form of early life stress, and by repeatedly separating pups from their mothers, it causes lasting changes in the pups' physiology and behavior. A few months after modeling, pups can develop symptoms of liver qi stagnation, such as depression and melancholy. This model, with its simple preparation and operation, is primarily used to study the effects of early stress on the physiological, pathological, and behavioral changes of pups in adulthood. However, due to the relatively small number of children with liver qi stagnation in clinical practice and the large number of cases of solitary housing, it has certain limitations.

[0024] (5) Combining multiple modeling methods

[0025] To better achieve a liver depression model, two or more of the above modeling methods can be combined. Cui Xiangyang et al. used a liver depression rat model induced by tail clamping and epinephrine injection and found that the blood estradiol (E2) and progesterone (P) of the model rats were significantly reduced, and prolactin (PRL) was significantly increased. Zhu Xiaoxia et al. used the CUMS combined with solitary care method to replicate the liver depression rat model and explore the mechanism of action of Xiaoyaosan in improving liver depression. This modeling method is based on many years of research on liver depression. The drugs used for modeling are also selected through multiple screenings of some hormones with obvious correlation with liver depression and combined with some more mature modeling methods. However, whether the increase in these indicators can represent the essence of liver depression requires a lot of experimental research. Summary of the Invention

[0026] One of the purposes of the present invention is to provide a CRH PVN A method for constructing a neuron-activated liver depression-type breast cancer mouse model, the method comprising the following steps:

[0027] (1) 140-160 nl of liquid containing rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry was injected into the PVN brain region of CRH-IRES-Cre mice. The viral titers of rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry were both 2.4-2.6×10 12 pfu;

[0028] (2) 25-30 days after virus injection, all mice were implanted with breast cancer cells. CRH was obtained 38-42 days after breast cancer cell inoculation. PVN A mouse model of liver-depression breast cancer with neuronal activation.

[0029] Preferably, in step (1), the PVN brain region of the mouse is injected with 150 nl of liquid containing both rAAV-CRH-CRE-WPRE-hGHpolyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry.

[0030] More preferably, the virus titers of rAAV-CRH-CRE-WPRE-hGHpolyA and chemical genetically activated virus rAAV-hM3D(Gq)-mCherry in step (1) are both 2.5×10 12 pfu.

[0031] More preferably, 28 days after the virus injection in step (2), all mice are implanted with breast cancer cells.

[0032] More preferably, the specific process of implanting breast cancer cells in step (2) is as follows:

[0033] The breast cancer cell Py230 pellet was suspended in PBS solution and then mixed with Matrigel to make a density of 6-8×10 6 A cell suspension of 100 cells / mL was inoculated in situ into the fourth papillary fat pad on the right side of the mouse, with 0.08-0.12 mL injected into each mouse.

[0034] More preferably, the density of breast cancer cells Py230 in the cell suspension in step (2) is 7×10 6 pieces / mL.

[0035] More preferably, in step (2), each mouse is injected with 0.1 ml.

[0036] More preferably, CRH can be obtained 40 days after the breast cancer cells are inoculated in step (2). PVN A mouse model of liver-depression breast cancer with neuronal activation.

[0037] A second object of the present invention is to provide an application of the mouse model constructed by the above construction method in drug screening for liver depression type breast cancer.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The construction method of the present invention is simple to operate and has good repeatability, and can overcome the disadvantage of large individual differences in animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the central CRH in Example 1 PVN Liver depression phenotypes associated with neuronal activation, including: A. Experimental design diagram; B. Sugar preference test results for each group of mice; C. Open field test results for each group of mice; D. Tail suspension test results for each group of mice, n = 6 / group; Ctrl: control group; CRH+: central CRH PVN Neuronal activation group; CRH+XYS: central CRH PVN Neuronal activation+Xiaoyaosan group; ** indicates P < 0.01.

[0041] Figure 2 is the central CRH in Example 1 PVNNeuronal activation-induced breast cancer with liver depression syndrome, including A. Representative tumor image; B. Tumor growth curves of mice in each group; C. Comparison of tumor weights among mice in each group; D. Comparison of Ki67 expression in tumor tissues among mice in each group, n = 6 / group; Ctrl: control group; CRH+: central CRH PVN Neuronal activation group; CRH+XYS: central CRH PVN Neuron activation + Xiaoyaosan group; * indicates P < 0.05, ** indicates P < 0.01.

[0042] Figure 3 is the central CRH in Example 2 PVN Comparison of the effects of neurons and CUMS on the induction of liver depression phenotypes, including: A. Sugar preference test for each group of mice; B. Open field test for each group of mice; C. Tail suspension test for each group of mice. n = 6 / group; Ctrl: control group; CRH+: central CRH PVN Neuronal activation group; CUMS: emotional stress group; CUMS+CRH-: emotional stress + central CRH PVN Neuronal inhibition group; * indicates P < 0.05, ** indicates P < 0.01.

[0043] Figure 4 is the central CRH in Example 2 PVN Results of tumor growth testing in mice regulated by neurons, including: A. Representative tumor image; B. Tumor growth curves for each group of mice; C. Comparison of tumor weights for each group of mice; D. Comparison of Ki67 expression in tumor tissues for each group of mice, n = 6 / group; Ctrl: control group; CRH+: central CRH PVN Neuronal activation group; CUMS: emotional stress group; CUMS+CRH-: emotional stress + central CRH PVN Neuronal inhibition group; * indicates P < 0.05, ** indicates P < 0.01. DETAILED DESCRIPTION

[0044] Example 1

[0045] SPF clean grade CRH-IRES-Cre mice, female, 8 weeks old. After 1 week of adaptive feeding, the control group (Ctrl), central CRH PVN Neuronal activation group (CRH+) and central CRH PVN Neuronal activation + Xiaoyaosan group (CRH + XYS). In the PVN brain region of mice in the Ctrl group, 150 nl of liquid containing a virus titer of 2.5 × 10 12pfu rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetics blank vector virus rAAV-DIO-mCherry were injected into the PVN brain region of mice in the CRH+ group and CRH+XYS group. The virus titer in the liquid was 2.5×10 12 pfu rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetically activated virus rAAV-hM3D(Gq)-mCherry. Four weeks after virus injection, all mice were implanted with breast cancer cells. Specifically, Py230 breast cancer cells in the logarithmic growth phase were taken. When the cells were well attached to the wall and growing, the cells were digested and centrifuged. The cell pellet was suspended in PBS solution and mixed with Matrigel at a density of 7×10 6 A cell suspension of 100 cells / mL was inoculated in situ into the fourth papillary fat pad on the right side of all mice, with 0.1 mL injected per mouse. After tumor implantation, all mice were treated with clozapine N-oxide (CNO). In addition, mice in the CRH+XYS group were treated with Xiaoyaosan (6.5 g / kg), and the remaining mice were treated with the same volume of normal saline. The tumor diameter of the mice was measured every 5 days, and the tumor volume of the mice was calculated. Behavioral tests were performed 40 days after tumor implantation to evaluate the depressive state of mice in each group. Mouse breast cancer cells Py230 were purchased from the American Type Culture Collection (ATCC), and the specific protocol is as follows:

[0046] (1) Animal grouping:

[0047] SPF clean grade CRH-IRES-Cre mice, female, 8 weeks old. After 1 week of adaptive feeding, they were divided into groups using a simple random allocation method, with 6 mice in each group. They were control group (Ctrl), central CRH PVN Neuronal activation group (CRH+) and central CRH PVN Neuronal activation + Xiaoyaosan group (CRH+XYS).

[0048] (2) Stereotaxic injection:

[0049] After anesthetizing the mouse with isoflurane, shave the hair on the mouse's head. Fix the mouse's head on a brain stereotaxic instrument, observe the position of the mouse's head front, back, left, and right to reach the level with the naked eye, and observe whether the mouse is in good respiratory condition. After the head is fixed, apply erythromycin eye ointment to the mouse's eyes, disinfect the head with iodine, cut the mouse's scalp, and then use a cotton swab dipped in normal saline to wipe off the surface tissue of the skull to fully expose the Bregma point. Level the head according to the Bregma point and Lambda point to ensure that the two points are arranged in a straight line and the depth difference between the two points does not exceed 0.05mm. Then, with the Bregma point as the center, measure the depth of the left (AP 0, ML-2.0) and right (AP 0, ML+2.0) points to ensure that the depth difference between the two points does not exceed 0.05mm. After leveling the head, the microelectrode was positioned over the target brain region. A skull drill was used to gently abrade the skull in the PVN (ML: ±0.3; AP: -0.6; DV: -4.5). When the dura mater appeared, it was carefully punctured with a medical syringe needle. 50 nl of virus was drawn up using the microelectrode and injected into the PVN at a rate of 10 nl / min. After virus injection, the microelectrode was left in the PVN for 15 minutes before being slowly removed to prevent the virus from leaking out of the brain as the needle was removed. After the microelectrode was removed, the skull was cleaned with saline, and the skin of the mouse's head was sutured with 6-0 sutures and disinfected with iodine. After surgery, the mouse was placed on a heating pad and returned to its home cage after awakening.

[0050] (3) Specific virus injection:

[0051] The PVN of mice in the Ctrl group were injected with 150 nl of sterile PBS containing a virus titer of 2.5 × 10 12 pfu rAAV-CRH-CRE-WPRE-hGH polyA (BrainVTA, PT-0588) and chemical genetics blank vector virus rAAV-DIO-mCherry (BrainVTA, PT-0115); 150 nl of liquid was injected into the PVN brain region of mice in the CRH+ group and CRH+XYS group, and the virus titer was 2.5×10 12 pfu of rAAV-CRH-CRE-WPRE-hGH polyA (BrainVTA, PT-0588) and chemically genetically activated virus rAAV-hM3D(Gq)-mCherry (BrainVTA, PT-0019).

[0052] (4) Tumor cell implantation and subsequent intervention:

[0053] Four weeks after virus injection, all mice were implanted with breast cancer cells. Py230 breast cancer cells in the logarithmic growth phase were taken and digested and centrifuged when the cells were well attached to the wall. The cell pellet was suspended in PBS solution and mixed with Matrigel at a ratio of 1:1 to prepare a 7×10 6 A cell suspension of 1000 cells / mL was inoculated in situ into the fourth papillary fat pad on the right side of all mice, with 0.1 mL injected per mouse. After tumor implantation, all mice were treated with clozapine N-oxide (CNO), specifically by intraperitoneal injection of 1.0 mg / kg CNO per day, and 1.25 mg / kg CNO was added to the drinking water of the mice. The use period lasted until the day of sampling (42 days after tumor inoculation). In addition, mice in the CRH+XYS group were given Xiaoyaosan (6.5 g / kg) for treatment, specifically by gavage, once a day, for a period until the day of sampling (42 days after tumor inoculation). The remaining mice were treated with the same volume of normal saline. The tumor diameter of the mice was measured every 5 days, and the tumor volume of the mice was calculated. After 40 days, behavioral tests were performed to assess the depressive state of mice in each group. Mouse breast cancer cells Py230 were purchased from the American Type Culture Collection (ATCC).

[0054] (5) Assessment of Liver Depression Syndrome:

[0055] ① Sugar water preference experiment: 40 days after tumor implantation, the mice were first housed individually and trained to drink sucrose water within the next 48 hours. During this training phase, we provided two drinking options: one bottle contained 2% sucrose solution, and the other bottle was pure water. To ensure the fairness of the results, we exchanged the positions of the two bottles of liquid 24 hours after the start of training. After the training was completed, a 16-hour water deprivation treatment (but not fasting) was implemented, followed by a 4-hour test. After the first 2 hours of the test, the positions of the two bottles were exchanged again. When the test was completed, we recorded the consumption of sucrose water and pure water. Sugar water preference (%) = sugar water consumption ÷ (distilled water consumption + sugar water consumption) × 100%.

[0056] ② Open field test: 41 days after tumor implantation, the animals were placed in the center of a 50 cm × 50 cm × 45 cm experimental chamber. After acclimation for 10 minutes, the distance traveled by the mice within 5 minutes was recorded using a video camera system. Behavioral videos were analyzed using Smart 3.0 behavioral software.

[0057] Tail suspension test: 42 days after tumor implantation, mice were suspended upside down with tape fixed to their tails for 6 minutes, with the tail tip approximately 30 cm above the ground. A video camera was used to record the total immobility time (the mice gave up struggling and remained completely motionless) over the 6-minute period. Behavioral videos were analyzed using Smart 3.0 behavioral software.

[0058] Mice exhibit liver depression, characterized by one or more of the following phenotypes:

[0059] ① The preference for sugar water (%) was significantly lower than that in the control group, P < 0.05; the preference for sugar water increased after Xiaoyaosan intervention, P < 0.05;

[0060] ② The movement distance in the open field test decreased, P<0.05; the movement distance increased after Xiaoyaosan intervention, P<0.05;

[0061] ③ The immobility time in the tail suspension test was prolonged, P<0.05; the immobility time was shortened after Xiaoyaosan intervention, P<0.05.

[0062] result:

[0063] (1) Central CRH PVN Neuronal induction of liver depression phenotype

[0064] To clarify the central CRH PVN To investigate the causal relationship between neurons and liver depression in mice, we used a chemical genetic approach to activate central CRH in breast cancer mice. PVN Neurons, and given Xiaoyaosan intervention treatment to observe central CRH PVN Can neuronal activation induce liver depression phenotype? Figure 1 A). The results of the sucrose preference experiment showed that the mice in the CRH+XYS group had a significantly higher preference for sucrose than those in the CRH+ group (P<0.01, Figure 1 B), and there was no significant difference with the Ctrl group ( Figure 1 B) The results of the open field test showed that the distance explored by mice in the CRH+XYS group to the surrounding environment was significantly higher than that in the CRH+ group (P<0.01, Figure 1 C), and there was no significant difference with the Ctrl group ( Figure 1 C). The results of the tail suspension test showed that the proportion of mice in the CRH+XYS group showing a state of "behavioral despair" when facing stress was significantly lower than that in the CRH+ group (P < 0.01, Figure 1 D), and there was no significant difference with the Ctrl group ( Figure 1 D).

[0065] (2) Central CRH PVN Neuron-induced liver depression-induced breast cancer

[0066] By observing the growth volume of breast cancer in each group of mice, we found that the tumor volume of the CRH+XYS group was significantly lower than that of the CRH+ group (P < 0.05, Figure 2 AB), and there was no significant difference between the mice in the Ctrl group ( Figure 2 AB). After 40 days, the tumor-bearing mice were sampled and the tumor weight was measured. The results also showed that the tumor weight of the CRH+XYS group mice was significantly smaller than that of the CRH+ group mice (P < 0.01, Figure 2 C), and there was no significant difference between the mice in the Ctrl group. Immunohistochemistry results also showed that the level of Ki-67 in the tumor tissues of the mice in the CRH+XYS group was significantly lower than that in the CRH+ group (P < 0.05, Figure 2 D), and there was no significant difference between the mice in the Ctrl group ( Figure 2 D) The above results show that Xiaoyaosan can relieve the central CRH PVN Neuronal activation induces the progression of tumor malignant growth.

[0067] Example 2

[0068] The CUMS liver depression syndrome modeling process is as follows:

[0069] SPF clean grade CRH-IRES-Cre mice, female, 8 weeks old. After 1 week of adaptive feeding, they were divided into groups using a simple random allocation method, with 6 mice in each group. They were control group (Ctrl), central CRH PVN Neuronal activation group (CRH+), emotional stress group (CUMS) and emotional stress + central CRH PVN Neuronal inhibition group (CUMS+CRH-). 150 nl of liquid containing virus with a titer of 2.5×10 12 pfu of rAAV-CRH-CRE-WPRE-hGH polyA (BrainVTA, PT-0588) and chemical genetics blank vector virus rAAV-DIO-mCherry (BrainVTA, PT-0115); 150 nl of liquid containing both viruses with a titer of 2.5×10 12 pfu rAAV-CRH-CRE-WPRE-hGH polyA (BrainVTA, PT-0588) and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry (BrainVTA, PT-0019) were injected into the PVN brain region of CUMS+CRH- mice. The fluid contained 150 nl of virus with a titer of 2.5×10 12pfu rAAV-CRH-CRE-WPRE-hGHpolyA (BrainVTA, PT-0588) and chemical genetic inhibition virus rAAV-hM4D(Gi)-mCherry (BrainVTA, PT-0020). Five days after virus injection, mice in the CUMS group and CUMS+CRH- group were intervened with the CUMS protocol. After 4 weeks of CUMS intervention, all mice were implanted with breast cancer cells, namely: breast cancer Py230 cells in the logarithmic growth phase were taken, and when the cells were in good adhesion and growth state, the cells were digested and centrifuged. The cell pellet was suspended in PBS solution and mixed with Matrigel 1:1 to make a density of 7×10 6 A cell suspension of 1000 cells / mL was inoculated in situ into the fourth nipple fat pad on the right side of all mice, with 0.1 mL injected per mouse. After tumor implantation, CUMS was continued to be used to intervene in the CUMS group and CUMS+CRH-group mice. At the same time, all mice were given CNO treatment. The tumor diameter of the mice was measured every 5 days, and the tumor volume of the mice was calculated. Behavioral tests were performed 40 days after tumor implantation to evaluate the depressive state of mice in each group. Mouse breast cancer cells Py230 were purchased from the American Type Culture Collection (ATCC).

[0070] The above-mentioned chronic unpredictable mild stress (CUMS) intervention process is as follows:

[0071] Each day, the animals were randomly exposed to two different chronic unpredictable mild stress (CUMS) stimuli, including restraint, cage tilt, bedding removal, wet bedding, food and water deprivation, ice water swimming, stroboscopic light, and day / night reversal. The same stimulation could not be given on two consecutive days (see Table 3.1-1 for specific operating methods).

[0072] Table 3.1-1 CUMS specific operation methods

[0073]

[0074] (1) Central CRH PVN Comparison of the effects of neurons and CUMS on inducing liver depression phenotype

[0075] Chemical genetics to regulate central CRH PVN The mice with neuronal activity were tested for depressive-like behaviors (including sugar preference test, open field test and tail suspension test 40 days after tumor implantation). The results showed that compared with the Ctrl group, the CRH+ group mice showed a decreased proportion of sucrose intake (P < 0.01, Figure 3 A), exploration of the surrounding environment was reduced (P < 0.01, Figure 3 B) and showed a state of "behavioral despair" when facing stress (P < 0.01, Figure 3 C). Compared with the CUMS group, there were no significant differences in the behavioral indicators of mice in the CRH+ group ( Figure 3 AC). On the other hand, the sucrose intake ratio of mice in the CUMS+CRH- group was significantly higher than that in the CUMS group (P<0.01, Figure 3 A) Exploration of the surrounding environment (P<0.05, Figure 3 B) increased, and the proportion of those who showed "behavioral despair" when facing pressure decreased (P < 0.01, Figure 3 C). Compared with the Ctrl group, there were no significant differences in the behavioral indicators of mice in the CUMS+CRH- group ( Figure 3 AC). The above results show that central CRH PVN Neuronal activation can induce liver depression phenotype in mice, and there is no significant difference between the CUMS group and the control group; while inhibition of central CRH PVN Neuronal function can alleviate the liver depression induced by CUMS. Therefore, central CRH PVN Neuron is one of the key neurons that induce liver depression in mice.

[0076] (2) Central CRH PVN Comparison of the effects of neurons and CUMS in inducing breast cancer phenotypes with liver depression syndrome

[0077] Subsequently, we dynamically observed the growth of breast cancer in each group of mice. Around the 5th day after inoculation of mouse-derived breast cancer cells, tumor nodules could be measured subcutaneously in each group of tumor-bearing mice. Tumor volume was measured every 5 days thereafter, and tumor growth curves were drawn based on the tumor volume of each breast cancer mouse at different times ( Figure 4 AB). The results showed that around 30 days after inoculation, the tumor volume of mice in the CRH+ group began to be significantly larger than that of the Ctrl group (P < 0.01, Table 1); on the contrary, around 30 days after inoculation, the tumor volume of mice in the CUMS+CRH- group began to be significantly smaller than that of the CUMS group (P < 0.05, Table 1). After 40 days, samples were collected from the tumor-bearing mice and the tumor weights were weighed. The results also showed that the tumor weights of mice in the CRH+ group were significantly larger than those of mice in the Ctrl group (P < 0.01, Figure 4 C), while the tumor weight of mice in the CUMS+CRH- group was significantly smaller than that in the CUMS group (P < 0.01, Figure 4 C). In addition, immunohistochemistry results showed that the level of Ki-67 in tumor tissues of mice in the CRH+ group was significantly higher than that in the Ctrl group (P < 0.05, Figure 4 D), while the Ki-67 level in tumor tissues of mice in the CUMS+CRH- group was significantly lower than that in the CUMS group (P < 0.05, Figure 4D) The above results show that central CRH PVN Neuronal activation can not only induce emotional stress in mice, but also promote the malignant growth of breast cancer; central CRH PVN Neuronal inhibition can mitigate the effects of emotional stress on breast cancer growth. Therefore, these findings support the role of central CRH PVN This provides strong evidence that neurons play an important role in the malignant growth of emotional stress-induced breast cancer.

[0078] Table 1 Central CRH PVN Effects of neurons on tumor volume in mice

[0079]

[0080]

[0081] Note: Shapiro-Wilk test, P < 0.05 on the 5th, 10th and 35th days did not meet the normal distribution, and M (P 25 , P 75 ). The Kruskal-Wallis test showed no statistically significant difference between the groups on day 5 (H = 2.397, P = 0.494 > 0.05). Significant differences were observed on days 10 (H = 15.614, P = 0.001 < 0.01) and 35 (H = 16.267, P = 0.001 < 0.01). Multiple comparisons were further analyzed using the Bonferroni test. Data on days 15, 20, 25, 30, and 40 showed a normal distribution (P > 0.05) and are reported as x ± s. Homogeneity of variance was tested on days 15, 20, 30, and 40 (P > 0.05). One-way analysis of variance was performed, and multiple comparisons were performed using the Tukey test. Homogeneity of variance test: P < 0.05 on day 25, indicating that homogeneity of variance was not met. Welch's ANOVA analysis was used, and Dunnett's T3 test was used for multiple comparisons. P1 represents the comparison with the Ctrl group; P2 represents the comparison with the CUMS group.

[0082] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A CRH PVN The method for constructing a neuron-activated liver depression-type breast cancer mouse model is characterized by: The construction method comprises the following steps: (1) 140-160 nl of liquid containing rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry was injected into the PVN brain region of CRH-IRES-Cre mice. The viral titers of rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry were both 2.4-2.6×10 12 pfu; (2) 25-30 days after virus injection, all mice were implanted with breast cancer cells. CRH was obtained 38-42 days after breast cancer cell inoculation. PVN A mouse model of liver-depression breast cancer with neuronal activation.

2. The construction method according to claim 1, characterized in that In step (1), 150 nl of liquid containing both rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry was injected into the PVN brain region of the mouse.

3. The construction method according to claim 2, characterized in that The viral titers of rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry in step (1) were both 2.5×10 12 pfu.

4. The construction method according to claim 3, characterized in that 28 days after the virus injection in step (2), all mice were implanted with breast cancer cells.

5. The construction method according to claim 4, characterized in that The specific process of breast cancer cell implantation in step (2) is as follows: The breast cancer cell Py230 pellet was suspended in PBS solution and then mixed with Matrigel to make a density of 6-8×10 6 A cell suspension of 100 cells / mL was inoculated in situ into the fourth papillary fat pad on the right side of the mouse, with 0.08-0.12 mL injected into each mouse.

6. The construction method according to claim 5, characterized in that: The density of breast cancer cells Py230 in the cell suspension in step (2) is 7×10 6 pieces / mL.

7. The construction method according to claim 6, characterized in that: In step (2), each mouse was injected with 0.1 ml.

8. The construction method according to claim 7, characterized in that: CRH can be obtained 40 days after the breast cancer cells are inoculated in step (2). PVN A mouse model of liver-depression breast cancer with neuronal activation.

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