Xu-Tong Guo, Ji-Dong An, Jian-Qiang Mei

Emergency department of Hebei Hospital of traditional Chinese Medicine

Keywords:Saikosaponin A Depression Th17 / Treg cell Cytokines

ABSTRACT Objective: To investigate the Effect of saikosaponin A on Treg and Th17 immune balance in depressive rats. Methods: The rat depression model was established with reference to the Katz method, and the rats were randomly divided into control group, model group, western medicine group, and saikosaponin A group. The western medicine group was given 1.2 mg/kg/d of fluoxetine, and the saikosaponin A group was given 25 mg/kg/d of saikosaponin A, while the control group and model group were given the same volume of normal saline. The evaluation of depression in Rats was analyzed by Openfield-test and sugar water preference test. Flow cytometry was used to detect the expression of Th17 and Treg cells. And the expression of IL-17, IL-23, TNF-α, IL-10, TGF-βwere detected by enzyme-linked immunosorbent assay (ELISA). Results: Compared with the control group, the horizontal exercise score, vertical exercise score, and sugar preference of the model group decreased significantly (P ＜0.05). Compared with the model group, the above indicators were significantly increased in the western medicine group and saikosaponin A group (P ＜0.05). Flow cytometry showed that compared with the control group, the Th17 cells, Th17 / Treg cell ratio in model group increased significantly, whereas the Treg cells decreased significantly (P ＜0.05). Compared with the model group, The Th17 cells and Th17 / Treg ratio in western medicine group and saikosaponin A group decreased, while the Treg cells increased significantly (P ＜0.05). ELISA showed that compared with control group, the serum levels of IL-17, IL-23 and TNF-αin model group increased, while the levels of IL-10 and TGF-β decreased (P ＜0.05). Compared with model group, the levels of IL-17, IL-23 and TNF-αdecreased, while the levels of IL-10 and TGF-β increased in western medicine group and saikosaponin A group (P ＜0.05). Conclusion: Saikosaponin A can reduce the degree of depression by regulating the imbalance of Th17 / Treg cells and the secretion of inflammatory cytokines in depressed rats.

1. Introduction

luding emotional anxiety, sleep disorders, lack of pleasure and repeated Dutch act. According to the statistics of the World Health Organization, by 2020, depression will become the second largest disease after heart disease, and its harm has attracted more and more attention [2]. Because the onset of depression involves many systems, targets and small molecules, the pathogenesis has not been fully elucidated, most antidepressant drugs have poor therapeutic effect, toxic side effects, clinical diagnosis and treatment are very difficult [3]. Bupleurum chinense is derived from the dried roots of Bupleurum chinense and Bupleurum angustifolia. Its main effective components include saikosaponin a and D. It has the functions of soothing the liver, relieving depression, evacuating fever, anti inflammation and protecting the liver. It is a commonly used antidepressant Chinese medicine in clinic [4]. Modern pharmacological research shows that Bupleurum chinese can achieve antidepressant effect by regulating the expression of monoamine neurotransmitter, nerve growth factor, acetylcholine transferase, anti apoptosis of nerve cells and anti injury of immune inflammation [5-6], but the specific effect of saikosaponin a on the immune regulation of depressed patients is not clear. In this study, we studied the immune regulation mechanism of saikosaponin a on depression from the perspective of its effect on Treg and Th17 immune balance in depression rats.

2. Materials and methods

2.1 Experimental animals

86 SPF male SD rats, 7 weeks old, weighing 200-220g, were purchased from Beijing weitonglihua Co., Ltd. [license No.: scxk (Beijing) 2016-0003], and raised in a well ventilated SPF level barrier room with room temperature of 25 ± 1 ℃, relative humidity of 55 ± 5%, during which free diet and activities were carried out. After the rats with horizontal movement times and vertical movement times less than 30 or more than 120 were removed by open field test, they were randomly divided into control group, model group, western medicine group and saikosaponin a group with 20 rats in each group.

2.2 Modeling and Administration

The model group, western medicine group and saikosaponin A group were fed in single cage respectively, and the chronic unpredictable stress stimulation (CUMS) for 21d was carried out with reference to Katz method [7]. The stimulation methods were as follows: tail clamping (90s), electric shock on the plantar (current intensity 1mA, voltage 45mV, 10s/ time, 30 times in total), high-speed horizontal vibration (60s), fasting (24h), water Prohibition (24h), and heat stress (45 ℃) 5 min), swimming in ice water (45 ℃ 5 min), binding (8 h), day and night reversed (24 h). One stimulus mode was randomly arranged every day, and each stimulus mode was used no more than three times in the experimental cycle. The control group rats were not given any stimulation. At the same time of modeling, rats in the western medicine group were given 1.2mg/kg/d fluoxetine (Sino US joint venture Sinochem Pharmaceutical Industry Co., Ltd.) by gavage, saikosaponin a group was given 25mg / kg / D saikosaponin a by gavage, the control group and the model group were given the same volume of normal saline for 21 days.

2.3 Behavioral evaluation of rats

Open field test: after the completion of modeling and administration, prepare a 25 square box without cover, which is 40cm high, 80cm long at the bottom, black on four walls of the box, white at the bottom, and 16cm × 16cm with black lines. Put each rat into the box separately, and record the number of bottom grids and the number of upright in 3min. Scoring criteria: place the rats in the center of the floor, and the rats are counted as 1 point when they pass through one grid (horizontal movement) or when their feet leave the bottom (vertical movement).

Determination of sugar water preference: before the experiment, on the 7th, 14th and 21th day after the experiment, the amount of drinking 1% sucrose water and pure water was measured, and the sugar water preference was calculated. Sugar water preference = 1% sucrose water consumption / (1% sucrose water consumption + pure water consumption) × 100%.

2.4 Detection of Th17 and Treg cell subsets

The peripheral blood of tail vein was collected and anticoagulated with heparin. The peripheral blood mononuclear cells (PBMC) of rats were extracted from 100 UL peripheral blood with Ficoll. The concentration of PBMC was adjusted to 1.5 × 106 / ml in RPMI-1640 medium containing 10% FBS, and 25 ng / L of phorbol ester, 1 ug / ml of ionomycin, 3 UG / ml of sigma were added to the cell suspension. 1 ml of suspension was inoculated into 24 well plate and cultured for 5 hours at 37 ℃. The cells were resuspended in a flow tube, washed twice with PBS, centrifuged for 3 min at 800 g, and the supernatant was discarded. Add 5ul anti rat CD4 FITC antibody (Treg detection tube needs to add 5ul anti rat CD25 PE antibody at the same time), and incubate at room temperature in dark for 25min. Wash PBS twice, centrifugation, discard supernatant. Add 200 μ l Th17 and Foxp3 special fixative and incubate at room temperature in dark for 25min. Wash PBS twice, centrifugate the supernatant, add 200 μ l Th17 and Treg cell specific membrane breaker, and incubate at room temperature in dark for 25min. PBS was washed twice. 5ul anti PE anti IgG1 homotype control antibody was added to the control tubes of each group of experiments. 5ul anti rat IL-17 and Foxp3 APC antibodies were added to the experimental tubes. They were incubated at room temperature in dark for 25min. After 3 times of PBS washing, cells were resuspended in 500ul PBS and detected by flow cytometry. Use the cellquest software to analyze the test results.

2.5 Detection of cytokine level

The peripheral blood of tail vein was collected, centrifuged at 5000rpm for 10min, and the upper serum was taken and stored at - 80 ℃. The expression levels of IL-17, IL-23, TNF -α, IL-10 and TGF - β in peripheral blood of rats were detected by ELISA Kit (biyuntian biotechnology company). The specific method shall be operated according to the instructions of testing kits of relevant companies.

2.6 Statistical methods

Spss19.0 statistical software was used for statistical analysis, and mean ± standard deviation was used for measurement data(x ± s) indicates that the sample comparison between groups adopts t test; the count data adopts rate (%), and the comparison between groups adopts χ2test. P ＜ 0.05 means the difference is statistically significant.

3. Results

3.1 Behavioral comparison of rats

Compared with the control group, the scores of horizontal movement, vertical movement and sugar preference in the model group decreased significantly (P ＜ 0.05); compared with the model group, the scores of horizontal movement, vertical movement and sugar preference in the western medicine group and saikosaponin a group increased significantly (P ＜ 0.05). The results are shown in Tables 1, 2 and 3.

Note: compared with the control group, AP ＜ 0.05; compared with the model group, BP ＜ 0.05 Group: 7d, 14d, 21d before the experiment

Note: compared with the control group, AP ＜ 0.05; compared with the model group, BP ＜ 0.05Group: 7d, 14d, 21d before the experiment

Group Before the experiment Part 7d Part 14d Part 21d control group 75.44±8.41 75.93±6.88 79.00±6.03 73.25±6.97 Model group 76.79±7.96 59.74±6.61a 49.53±5.72a 32.51±4.26a Western medicine group 78.06±7.77 66.60±7.02 57.14±5.68b 43.42±5.20b Saikosaponin a group 77.38±8.03 68.59±5.83b 62.34±6.53b 50.11±5.87b

3.2 Comparison of Th17 and Treg cell subsets in rats

The results of flow cytometry showed that compared with the control group, the ratio of Th17 cells in the model group increased, the ratio of Treg cells decreased, the ratio of Th17 / Treg cells increased significantly (P ＜ 0.05); compared with the model group, the ratio of Th17 cells in the western medicine group and saikosaponin a group decreased, the ratio of Treg cells increased, the ratio of Th17 / Treg cells decreased, the difference was statistically significant (P ＜ 0.05) Significance (P ＜ 0.05), see Table 4.

Table 4 statistical analysis of Th17 and Treg cell subsets in peripheral blood of mice (±s)

Table 4 statistical analysis of Th17 and Treg cell subsets in peripheral blood of mice (±s)

Note: compared with the control group, AP ＜ 0.05; compared with the model group, BP ＜ 0.05

Group Th17(%) Treg(%) Th17/Treg control group 2.56±0.29 7.80±0.61 0.31±0.04 Model group 6.44±0.50a 3.79±0.40a 1.68±0.12a Western medicine group 3.02±0.33b 7.03±0.58b 0.44±0.05b Saikosaponin a group 3.45±0.39b 6.76±0.62b 0.53±0.06b

3.3 Comparison of serum cytokine levels in rats

Compared with the control group, the serum levels of IL-17, IL-23 and TNF -αin the model group increased, while the serum levels of IL-10 and TGF - β in the model group decreased (P ＜ 0.05); compared with the model group, the serum levels of IL-17, IL-23 and TNF -αin the western medicine group and saikosaponin a group decreased, and the serum levels of IL-10 and TGF - β increased, the difference was statistically significant (P ＜ 0.05), as shown in Table 5.

4. Discussion

Depression belongs to the category of "depression syndrome" in traditional Chinese medicine. Traditional Chinese medicine believes that depression is the stagnation of liver Qi caused by a variety of factors. The main efficacy of Chaihu is to soothe the liver and relieve the depression, relieve the external heat and reduce the fever. It is the most common traditional Chinese medicine compound used in the clinical treatment of depression, such as Chaihu Shugan powder, Xiaoyao Powder, Xiaochaihu Decoction and Chaihu longoyster Decoction [8]. Pharmacological studies have shown that saikosaponin can significantly improve the depressive behavior of depressed rats, mainly involving monoamine neurotransmitters in the brain center, BDNF signaling pathway, hippocampal neuron activity, hypothalamus pituitary adrenal axis and immune system and many other targets [9-10]. The mechanism of action is complex anduncertain.

Table 5 serum concentrations of IL-17, IL-6, IL-23, TNF -α, IL-10 and TGF - β in mice (±s, pg / ml)

Table 5 serum concentrations of IL-17, IL-6, IL-23, TNF -α, IL-10 and TGF - β in mice (±s, pg / ml)

Note: compared with the control group, AP ＜ 0.05; compared with the model group, BP ＜ 0.05

Group IL-17 IL-23 TNF-αα IL-10 TGF-β control group 31.55±4.66 56.61±6.29 45.64±5.16 29.66±3.15 72.45±6.86 Model group 112.13±15.02a 193.16±15.33a 152.06±12.73a 5.44±0.52a 23.62±3.01a Western medicine group 43.57±5.81b 70.04±8.84b 61.15±7.42b 24.71±3.05b 68.51±6.96b Saikosaponin a group 53.87±6.24b 86.56±9.43b 72.10±6.68b 18.33±2.12b 59.09±5.83b

Cytokine hypothesis is one of the research focuses of depression pathogenesis in recent years [11]. The study shows that with the occurrence of neurological disorders in patients with depression, the expression of proinflammatory cytokines such as IL-1, IL-6, TNF -αin serum is significantly up-regulated, while the expression of anti-inflammatory cytokines such as IL-10, IL-4, TGF - β, IL-13 is significantly down regulated, suggesting that the systems of neuritis, neuroendocrine and neurotrophic factors play an important role in the development of depression Function [12]. Some studies have shown that Bupleurum and its traditional Chinese medicine compound can alleviate depression like symptoms by regulating the expression of brain-derived neurotransmitter and nerve growth factor in the nervous system. The purpose of this study was to investigate the effect of saikosaponin a on Th17 / Treg immune balance in depressed rats.

Th17 and Treg cell subsets are the main members of effector T cells, which play an important role in autoimmune diseases, infectious diseases and tumor immunity. Th17 cells mainly secrete IL-6, IL-17, IL-22, TNF -α and other cytokines to play the role of immune regulation and promotion of inflammation, and then participate in the occurrence and development of immune diseases, inflammatory diseases and tumors [13]. Treg cells mainly secrete IL-4, IL-10, TGF - β and other cytokines to inhibit the immune response of the body, so as to maintain the immune tolerance of the body [14]. Under normal circumstances, Th17 / Treg maintains a state of dynamic balance in the body, and the imbalance of Th17 / Treg cells will cause abnormal immune response [15].

Our results showed that Th17 cells increased, Treg cells decreased and Th17 / Treg cells ratio increased in the serum of depression rats, while Th17 cells decreased, Treg cells increased and Th17 / Treg cells ratio decreased in the serum of depression rats treated with western medicine or saikosaponin a, suggesting that the balance of Th17 / Treg cells in depression rats was unbalanced; in addition, IL-17, IL-23, TNF -αin depression rats The serum level of IL-10 and TGF - β decreased with the increase of serum level; the serum level of IL-17, IL-23 and TNF -αdecreased with the increase of IL-10 and TGF - β in the western medicine or saikosaponin a group, which indicated that the pathogenesis of depression was closely related to the imbalance of Th17 / Treg cell subsets and the abnormal expression of inflammatory cytokines. Chen et al. Also showed that saikosaponin a can restore the normal of neuroinflammation, neuroendocrine and neurotrophic factor system by down regulating the expression level of IL-1 β, IL-6 and TNF -αin the depression model of chronic female rats, so as to play an antidepressant role [16]. However, the relationship between depression and immune balance of Th17 / Treg cells was not described in the above studies.

In conclusion, saikosaponin a can improve the immune imbalance of Th17 / Treg cells in depressed rats, which is expected to provide an important theoretical basis for clinical medication and new drug development of depression.