Effect of Wenyang Jieyu Decoction on HPA Axis in Depression Model Rats
OBJECTIVE: To study the effect of Wenyang Jieyu Decoction on HPA axis in rats with chronic stress depression, and to explore its antidepressant mechanism. METHODS: Male Wistar rats were randomly divided into 6 groups. The normal control group and the model control group were intragastrically administered with 0.9% sodium chloride solution. The warm, medium and low doses of Wenyang Jieyu Decoction were administered by Wenyang Jieyu Decoction. The agent (8, 6, 4g/kg) and the positive drug control group were intragastrically administered with fluoxetine hydrochloride (3.33mg/kg). Except the normal control group, the other groups received chronic unpredictable mild stimulation (CUMS) stimulation. And solitary 28d. The behavioral evaluation of each group of rats was carried out by forced swimming test (FST), tail suspension experiment (TST) and open field experiment (OFT). q PCR was used to detect the expression of CRH m RNA and hippocampal GR m RNA in rat hypothalamus. The content of ACTH and CORT in the blood of rats was detected by immunoassay. The morphology of hippocampus was observed by HE staining. RESULTS: Wenyang Jieyu Decoction could reduce the FST and TST immobility time and increase the number of OFT crossings (P<0.05). It could reduce the levels of CRH, GR, ACTH and CORT caused by chronic stress (P< 0.05) and can reduce hippocampal tissue damage. Conclusion: Wenyang Jieyu Decoction can improve the stress and depression behavior of CUMS rats and the damage of high glucocorticoids to the brain during chronic stress. The mechanism may be related to the regulation of HPA axis.
Key words: Wenyang Jieyu Decoction; depression; hypothalamic-pituitary-adrenal axis
As a special psychiatric disorder, depression has a wide range of symptoms, including lack of pleasure, helplessness, sadness and despair, and is thought to be related to the environment (such as stress or trauma) and genetic factors. When the body is under stress, the body regulates the sympathetic-adrenal medullary system and the hypothalamic pituitary gonadal axis (HPA) to stabilize and restore the homeostasis. A certain degree of stress is on the body. Beneficial, but long-term stress can cause depression. The HPA axis is an important endocrine axis and a hub for the neuroendocrine network, which plays a vital role in maintaining the balance of the body's environment. Studies have shown that the occurrence of depression is inextricably linked to the disorder of HPA axis function. Chinese medicine has a good therapeutic effect on depression through warming up kidney yang, regulating qi and relieving depression, and has received more and more attention in the treatment of depression. This study established a classic chronic unpredictable mild stress (CUMS) depression rat model, studied the effect of Wenyang Jieyu Decoction on the HPA axis of depression model rats, and explored the mechanism of antidepressant.
Materials and Methods
1. Materials 180 SPF male Wistar rats, initial body mass 150-180 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., certificate number. Fluoxetine hydrochloride dispersible tablets were purchased from Eli Lilly, 20mg, batch number: 4089A; cortisol (CORT) and adrenocorticotropic hormone (ACTH) radioimmunoassay kits were purchased from Beijing Huaying Biotechnology Research Institute; The total RNA miniprep kit was purchased from Aisijin Biotechnology Co., Ltd.; the PCR primer was designed and synthesized by Bioengineering Biotechnology (Shanghai) Co., Ltd.; 2×Easy Taq PCR Super Mix, Trans Script TM Green Two-Step q RT- PCR Super Mix and Trans DNA Marker I were purchased from Beijing Quanjin Biotechnology Co., Ltd.; other experimental related reagents were provided by the Central Laboratory of Shanxi Integrated Traditional Chinese and Western Medicine Hospital.
2. Preparation of Wenyang Jieyu Decoction by Wenyang Jieyu Decoction: 15g of Guizhi and Fuzi, 10g of Zhigancao, 30g of ginger and ebony, and 5 jujube; the above drugs are weighed according to the proportion, and the medicine is added. The weight is 10 times and the 70% ethanol solution is extracted 3 times, the first time is 1.5h, the second and third times are respectively 45min, the extracts are combined, the ethanol is recovered under reduced pressure, concentrated to a thick paste, and dried to a dry paste under reduced pressure. Smash into fine powder and pass through a 80 mesh sieve for use.
3. Grouping, modeling and administration Wistar rats were first raised for 7 days to adapt to the environment. The last day was performed by Supermaze video tracking analysis system for Open-field behavioral analysis. 180 healthy male rats with similar scores were selected. The quality was randomly divided into 6 groups of 30 each. The rats in the blank control group and the model control group were intragastrically administered with 0.9% sodium chloride solution; the high, middle and low doses of Wenyang Jieyu Decoction were respectively administered with Wenyang Jieyu Decoction concentrated decoction 8, 6, 4g/kg (equivalent Yusheng drugs 18.6, 27.9, 37.2g / kg); positive drug control group was administered fluoxetine hydrochloride (3.33mg / kg). The gastric perfusion capacity of each group was 0.2m L/10g, and it was administered for 28 days. Except the normal control group, the other groups were treated with CUMS combined with orphan stimulation. Use clip tail (1min), noise (1h), flash (1h), ice water swimming (4°C, 5min), wet padding (100m L, 24h), tilting cage (30°, 24h), day and night upside down, Fasting and water-free 9 kinds of mild stimuli were randomly arranged at 9:00 and 16:00 daily for stimulation for 28 days. On the 29th day, behavioral evaluation, material selection and index determination were performed.
4. Rat behavioral evaluation The following behavioral experimental instruments used in this experiment were purchased from Shanghai Xinsoft Information Technology Co. , Ltd. , forced swim test (FST): rats were swimming for 6 minutes, and accumulated within 4 minutes after recording. Movement time; tail suspension test (TST): rat tail suspension 6min, cumulative immobility time within 4min after recording; open field test (OFT): record within 5min of the open box Determine the number of movements and the number of grooming.
5. Determination of serum CORT and ACTH levels After behavioral evaluation, 20 rats in each group were tested for serum CORT and ACTH levels. The rat was quickly taken from the abdominal aorta, placed in a centrifuge tube, centrifuged at 4 000 r/min for 10 min at 4 ° C, serum was taken, and stored at -20 ° C for testing. The content of CORT and ACTH was determined by radioimmunoassay.
6. Rat hypothalamic corticotropin-releasing hormone (CRH) m RNA and hippocampus glucocorticoid receptor (GR) m RNA expression level after taking blood, each group takes 10 large Rats were quickly opened in the ice tray to remove the brain and the brain hypothalamus and hippocampus were removed and stored at -70 °C. Total RNA from hypothalamus and hippocampus was extracted and reverse transcribed into cDNA by RT-PCR. The expression of CRH m RNA in the hypothalamus and the expression of GR m RNA in hippocampus were detected by q-PCR. Design primers based on the rat gene sequence in Genbank, CRH primer: upstream: 5'-AGC CGC CCA TCT CTC TGG ATC TC-3', downstream: 5'-TCC CGA TAA TCT CCA TCA GTT-3'; GR primer: upstream :5'-GAT TGA ACC CGA GGT GTT GTA TGC-3', downstream: 5'-CGG CAA ATG CCA TGA GAA ACA TCC-3'; β-actin Primer: Upstream: 5'-GAG ACC TTC AAC ACC CCA GC -3', downstream: 5'-ATG TCA CGC ACG ATT TCC C-3'. The Ct value was read and the relative expression level of CRH m RNA was calculated using 2-ΔΔCt.
7. Rat hippocampal hematoxylin-eosin (HE) staining After behavioral evaluation, 10 rats in each group were subjected to HE staining in hippocampus. Rat brain tissue was fixed by 4% paraformaldehyde heart perfusion method, and the brain was craniotomy, paraffin embedded, and coronal section. Hippocampal tissue sections were taken for HE staining, and pathological changes of hippocampus were observed under microscope.
8. Statistical Methods Statistical processing of all data was performed on the SPSS 19.0 software package. Measurement data were expressed as x-±s, paired t-test was used for comparison within the group, and analysis of variance was used for comparison between groups. The composition data was used for the count data, and the χ2 test was used for comparison between groups. The difference was statistically significant at P<0.05.
discuss
Depression is a multi-factor mental illness associated with HPA axis dysfunction, and drugs targeting the HPA axis can play a role in the treatment of depression. In recent years, the use of traditional Chinese medicine Wenyang Jieyu theory to treat depression has been reported frequently. The theory of traditional Chinese medicine emphasizes that "yang is active", and kidney yang is the root of Yuanyang, which dominates the body's yang, and the overall clinical manifestation of depression is mainly "low motion" such as low mood, slow thinking, and decreased initiative. Insufficiency is closely related to the onset of depression. "Su Wen, angry and heavenly theory" said: "If you are yang, if you lose the life of the sky and the day, you will lose your life, so the heavenly movement will be lightened by the sun", "the yang is the essence, the essence is the god, the softness is the reinforcement." ". It emphasizes that human life activities depend on the promotion of yang and the warming effect, so that the yin and yang of the human body are relatively balanced and coordinated. Wenyang Jieyu Decoction was made by Zheng Qin'an "Medical Law Yuantong", which was supplemented by Danhua. In the middle of the party, "the aconite is hot, can complement the true yang in the ridge, and the true yang is the kind of the sinister fire. "Buy Jun Huo also", at the same time help Xinyang, Zhongwen spleen yang; Guizhi, ginger Xintong Xinyang, Shuganqi, Wenganyang, liver supplement; Zhigancao, jujube succulent, Hexin Ganyang The meaning of sour yin and yin, at the same time, "the licorice supplement has a fire to return to the roots of the wonderful", ginger, jujube warm in the coke, help the spleen yang, ebony lead medicine into the liver, warming liver yang, so that the liver can be yang When you raise your hair, you can gather the liver and raise the liver and blood to soften the liver, and combine the physiological characteristics of the liver with the body and the yang. The combination of various medicines, Fushenyang, Bugan Yang, warm heart yang, Yunpiyang, storage of micro-yang in the kidney, volts in the fire, in line with the pathogenesis of depression.
The CUMS animal model constructed in this study was able to show similar behavioral and hormonal changes as those with depression. The effects of FST, TST and OFT on the behavior of rats were evaluated by FST, TST and OFT. The results showed that the FST and TST in the model control group were prolonged, and the number of OFT crossings was reduced, which was consistent with the results of Cheng RF and other studies. The behavioral delay of CUMS rats was improved after Yangjieyu decoction decoction, suggesting that the Wenyang Jieyu formula used in this study can relieve the symptoms of emergency depression in CUMS rats.
This study also examined the expression and secretion levels of HPA-related hormones. The HPA axis consists of the hypothalamus, pituitary and adrenal cortex and its secreted CRH, ACTH and CORT. When the body is under stress, the HPA axis is in a state of hyperactivity. CORT plays a role in regulating the body's response to stress. CORT acts on the hypothalamus and hippocampal GR at the same time, down-regulating the secretion of CRH, thereby down-regulating the overall hormone level of the HPA axis and restoring it to the basal state. Under long-term chronic stress, excessive CORT continues to stimulate GR, which reduces the ability of GR to bind to CORT, and the negative feedback regulation capacity decreases, resulting in continued excitation of the HPA axis. In addition, sustained high levels of CORT can cause damage to hippocampal neurons. Studies have shown that Wenyang Jieyu Decoction can down-regulate the levels of CRH, ACTH and CORT, and reduce the damage of high-glucocorticoids on the brain during stress, suggesting that this prescription may exert antidepressant effects by regulating the HPA axis. In addition, the prescription can restore the expression level of GR in hippocampus of depressed rats, suggesting that the prescription can stabilize the HPA axis negative feedback regulation function by protecting the hippocampus, which is consistent with the results of Yue Guangxin et al. As an important part of the emotional regulation center, impaired tissue function may lead to depression. In this study, HE staining results of hippocampus showed that Wenyang Jieyu Decoction concentrate can protect hippocampal tissue while lowering HPA axis hormone levels. Therefore, the improvement of depressive symptoms by Wenyang Jieyu Decoction may be related to the protection of hippocampal tissue negative feedback regulation of HPA axis and down-regulation of HPA axis hormone levels. The mechanism remains to be further studied.
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