Pharmacological effects of Paeoniflorin and Albiflorin on IL-3, GM-CSF, IL-6 and TNF-α in the rats of syndrome of stagnation of liver qi and blood deficiency

Cheng-long Wang, Yu-wang Qin, Shi-xia Pan, Jian-jun Zhang*, Hong-li Teng *

1 Guangxi International Zhuang Medicine Hospital, Nanning, Guangxi, China. 2 Beijing University of Chinese Medicine,Beijing, China.

Background

Baishao (Radix Paeoniae Alba), bitter and acid taste,slightly cold, return to the Liver, Spleen Meridian, not only nourishing the liver blood, but also softening liver[1,2]. It can most conform to the characteristics of liver, is the representative of nourishing blood and supple liver.As a blood tonic, Baishao was mainly used for the treatment of flank rib pain caused by insufficient liver blood and unrelieved liver qi. The Chinese Pharmacopoeia stipulates that paeoniflorin (PF) is a symbolic component of Baishao, modern research also shows that Baishao contains monoterpenes and other glycoside compounds, and PF and Albiflorin (AF) are two components with relatively high contents [3-5].However, few pharmacological studies on AF have been reported. Cytokines are low-molecular-weight soluble proteins produced by stimulating cells with immunogens,mitogens, or other factors. They are bioinformatics molecules, which have the functions of regulating innate and adaptive immune responses, promoting hematopoiesis, and stimulate cell activation, proliferation,and differentiation [6]. In this experiment, the rats model with syndrome of stagnation of liver qi and blood deficiency caused by radiation exposure combined with chronic restraint stress. We monitored the rats body weight and blood cell and investigated the changes in the level of related cytokines and the interference effects of PF and AF on this model to discuss the material base and mechanism of effect of nourishing blood and smoothing the liver of Baishao and the essence of syndrome of stagnation of liver qi and blood deficiency

Materials

Animal

Ninety-six healthy male SD rats were of clean grade and weighing (170-180) g. They were purchased from Speyford (Beijing) Laboratory Animal Science and Technology Co., Ltd. (Beijing China, Approval ID:SCXK 2016-0002). Feeding the clean-class rats feeds that was provided by Beijing Keao Cooperation Feed Co., Ltd.[Beijing, China, Approval ID: （2012）06166]. The rats were housed in the SPF animal room of Beijing University of Traditional Chinese Medicine. During the whole experiment, the rats freely ingested and drank water (except for the behavioral test period), the room temperature was 20-22°C, the relative humidity was 60%-70%, and the lamp lighting cycle was 12h,7:00-19:00 light and 19:00-7:00 dark. The experiment met the relevant ethical requirements.

Drugs

Test drugsPF and AF were self -made (Beijing, China,patent: No. ZL 201110184287.4). Firstly, take the Baishao pieces after smashing and extract to obtain the water extract. Secondly, after the aqueous extract is concentrated, it is passed through a macroporous resin column, and the effluent is discarded. Thirdly, elution,collecting the eluate, obtaining the extract of total glycosides of Baishao. Fourthly, the total glycosides removed impurities from activated carbon. Finally, after the silica gel column, the eluent was washed off. The flow was collected, and the flow of PF and AF was detected with thin layer, and all the solvents were recovered. After repeated purification according to the above steps, PF (content > 98%) and AF (content > 96%)monomer were obtained. (Figure 1)

Figure 1 Chemical structure of PF, AF

Positive drugSiwu granules (Jitaian Pharmaceutical Co.Ltd., Sichuan, China, batch number: 1511133.) and Xiaoyao Pill (concentrated pill) (Jiuzhitang Co., Ltd.,Hunan, China, batch number: 201510006) were used. The content of PF was 32 mg/bag in Siwu granules and 4.19 mg/bag in Xiaoyao Pills, detected by HPLC in the laboratory room, which meet the requirements of the Siwu granules and Xiaoyao Pills in the 2015 edition of Pharmacopoeia of People's Republic of China, and the content of AF is 16.45 mg/g and 2.68 mg/g respectively.The two powders were prepared in our lab and used to prepare the required concentration of the rat drug according to the experimental requirements.

Reagents

Beckman Coulter Ac. T 5diff Rinse (Lot: 05202C2); Ac.T 5diff WBC Lyse (Lot: 20402B); Ac. T 5diff Fix (Lot:21802E); Ac. T 5diff Hgb Lyse (Lot: 06902A2);Interleukin-3 (IL-3) test box (Beijing Huaying Biotechnology Institute, Beijing, China, batch number:201510730); Granulocyte-macrophage Colony-stimulating Factor (GM-CSF) test box (Beijing Huaying Biotechnology Institute Beijing, China, batch number: 201510730); Tumor Necrosis Factor-α (TNF-α)test box (Beijing Huaying Biotechnology Research Institute, Beijing, China, batch number: 201510730);Interleukin-6 (IL-6) test box (Beijing Huaying Institute of Biotechnology, Beijing, China, batch number:201510730); sucrose (Beijing chemical plant, Beijing,China, batch number: 20150303).

Instrument

Rat binding device (self - made), a 20 cm in length and 6 cm in inner diameter tube, can be adjusted by moving the bottom plate. Rats behavior observation open box (self -made); Elevated cross maze (self - made); Electronic balance; Radiation instrument: xh-6020 automatic discharge and exemption counter (xi 'an nuclear instrument factory); Stat Fax 2100 automatic Awareness Technology Inc. USA; Xh-6020 automatic discharge and exemption counter (xi 'an nuclear instrument factory);The radiation source is provided by the radiology institute of the academy of military medical sciences; Beckman Coulter ac. t5 blood cell analyzer (Beckman Coulter Inc.,USA)

Method and Index

Animal grouping and administration method

Ninety-six rats were randomly divided into 8 groups,Twelve for each group, namely blank group, model group,Siwu granule group (2.5 g?kg-1equivalent to 15 g/day of human dosage, 2.5 g Siwu granules Contains 8.225 mg of AF and PF 16 mg), AF group (30 mg?kg-1, 15 mg?kg-1,equivalent to 2 g/kg , 1 g/kg of Baishao pieces ); PF group (30 mg?kg-1, 15 mg?kg-1, equivalent to 1.25 g/kg,0.75 g/kg of Baishao pieces). The first day of the model making was the first day of drug administration, and the dosage was 1ml per 100 g of body weight, once a day.The blank group and the model group were given daily gastric deionized water, and the rest of the groups were given medicine, and total building 21 d.

Syndrome of stagnation of liver qi and blood deficiency model

After 5 days of adaptive feeding in a single cage, the rats were weighed and their sugar and water consumption were measured. Except for the 6 rats in blank group were fed routinely without intervention, and the remaining groups were isolated and bound. Specific operation of binding: put the rat into a stainless-steel binder with a length of 20cm and an inner diameter of 6 cm, which can be adjusted, and bind it for 3 h (8:00-11:00am) every day for 3 consecutive weeks. Binding experiment on the premise of not affect normal circadian rhythm of rats, by the location of the mobile bound within the cylinder bottom, make rats without injury completely fixed to limit rats advance and retreat and turn around, to do not produce oppressive feeling, does not affect the breathing and excretion. On the 8th day of the experiment, except for the blank group, the rest of the groups were irradiated with60Co γ-radiation for 1 time, the irradiation dose was 3.5 Gy, the dose rate was 191.55 Gy·min-1, and the irradiation distance was 4 m. after irradiation, except the blank group, the rest of the group continue to administration in single cage combined with binding for 2 weeks [7].

General observation

Observe the color of the mouth, mouth, eyes, fur, tail and mental state of the rats. The rats in each group were weighed on day 1, 7, 14 and 21 d, and the changes in body mass in each group were recorded and analyzed. In each group, the rats in the 3, 7, 10 and 14 days after irradiation were taken 0.2-0.3 ml blood from the canthus in the eyes of the rats, and the serum was separated. The number of Leucocyte (WBC), Red blood cells (RBC) and Hemoglobin (HGB) in the whole blood was determined by automatic blood cell analysis and counting instrument.

Sample preparation and testing methods

We measured IL-3, IL-6, GM-CSF and TNF-α in serum.After all behavioral experiments are completed, chloral hydrate is applied to anaesthesia, and the abdominal aorta is taken out of the blood and allowed to stand at room temperature, solidification, centrifugal (3500 r/min, 15 min), take supernatant, deposited at -20 °C to be measured.

We also determinate viscera index after all experiment. Beheading of rats after death, quickly open the abdominal cavity and separation on the ice harvesting the spleen and thymus, weighing scales with electronic,then calculated separately with the ratio of weight. The index is calculated by the following formula: spleen (or thymus, adrenal gland) index (g/100g) =100* spleen (or thymus, adrenal gland) weight/body mass.

Statistical analysis

This experiment was statistically analyzed by One-way ANOVA process (every two groups comparison using SNK) or Independent-Sample T Test (SPSS Version 18,USA). Results are expressed as means ± SD. Differences were considered significant when the P value was less than 0.05.

Results

Effects of AF and PF on body weight of rats with syndrome of stagnation of liver qi and blood deficiency

There was no significant difference in the body weight of rats in each administrative group in the 1-day before and 7-day after modeling. From the 14th day of modeling,compared with the blank group, the average body weight of the model group was significantly lower than that of the blank group (P<0.01). At the same time, the body weight of the Siwuf granules group was higher than that of the model group (P < 0.05, P < 0.05). It was found that group of PF 30mg?kg-1and the AF 30mg?kg-1could also increase the mess of the group of rats (P < 0.05, P < 0.05).The rest of the administrative groups showed an increased trend, but there was no statistical difference (P > 0.05).(Table 1)

Effects of AF and PF on the number of WBC in rats with syndrome of stagnation of liver qi and blood deficiency

Compared with the blank group, the number of WBC in the model group was significantly lower than that in the blank group (P < 0.001) from the 3rd day after model establishment, At the same time point, Siwu granule can increase the number of WBC (P < 0.05, P < 0.01)compared with the model group. From the 7th day after modeling, Xiaoyao pill can increase the group WBC count (P < 0.05). From the 10th day of the modeling, AF 30 mg?kg-1can increase the number of WBC in the group(P < 0.05). The number of WBC in the PF 30mg?kg-1group increased on the 14th day of modeling (P < 0.05).(Table 2)

Effect of AF and PF on RBC Number in Rats with syndrome of stagnation of liver qi and blood deficiency

In contrast to the blank group, the model group has a lower number of RBC than the blank group at the 3d of modeling（P < 0.05）, but the rest of the monitoring is slightly lower than the blank group, no statistically significant difference (P > 0.05). In comparison with model group, there are no statistical differences in the number of RBC in each administrative group (P > 0.05).(Table 3)

Table 1 Effects of AF and PF on body weight of rats with syndrome of stagnation of liver qi and blooddeficiency

Table 1 Effects of AF and PF on body weight of rats with syndrome of stagnation of liver qi and blooddeficiency

Compared with the blank group, ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001;Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001

Groups 1d 7d 14d 21d Blank group 204.91 ± 8.10 256.25 ± 9.65 300.91 ± 9.29 327.50 ± 8.15 Model group 204.33 ± 6.86 248.33 ± 9.12 288.83 ± 7.64▲▲ 315.83 ± 7.28▲▲Siwu granules 204.41 ± 7.08 254.58 ± 9.63 297.66 ± 9.75* 324.75 ± 10.01*Xiaoyao pills 204.33 ± 6.51 253.25 ± 8.78 297.58 ± 8.09* 324.58 ± 8.09*PF30mg·kg-1 204.83 ± 7.52 252.58 ± 8.05 296.25 ± 9.34* 323.25 ± 9.34*PF15mg·kg-1 204.58 ± 7.21 250.50 ± 6.59 294.16 ± 7.98 321.16 ± 7.98 AF30mg·kg-1 204.25 ± 8.63 251.33 ± 7.76 296.08 ± 7.50* 323.91 ± 7.56*AF15mg·kg-1 204.33 ± 7.59 249.41 ± 8.30 293.66 ± 9.01 321.50 ± 8.27

Table 2 Effects of AF and PF on the number of WBC in rats with syndrome of stagnation of liver qi and blooddeficiency × 109/L)

Table 2 Effects of AF and PF on the number of WBC in rats with syndrome of stagnation of liver qi and blooddeficiency × 109/L)

Compared with the blank group, ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001;Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001

Groups 1d 7d 14d 21d Blank group 12.50 ± 1.46 12.05 ± 1.21 12.58 ± 1.50 11.28 ± 1.58 Model group 3.28 ± 0.98▲▲▲ 5.86 ± 0.77▲▲▲ 7.47 ± 0.68▲▲▲ 7.53 ± 0.75▲▲▲Siwu granules 4.33 ± 0.95* 6.85 ± 0.95* 8.51 ± 0.81* 8.69 ± 0.82**Xiaoyao pills 4.13 ± 0.86 6.65 ± 0.85* 8.39 ± 0.86* 8.51 ± 1.03*PF30mg·kg-1 3.76 ± 0.89 6.50 ± 0.65 8.24 ± 0.64 8.46 ± 0.64*PF15mg·kg-1 3.44 ± 0.99 5.96 ± 0.78 7.69 ± 0.76 7.88 ± 0.73 AF30mg·kg-1 3.79 ± 0.83 6.62 ± 0.89 8.35 ± 0.89* 8.54 ± 0.85*AF15mg·kg-1 3.50 ± 0.69 6.04 ± 0.70 7.77 ± 0.63 8.25 ± 0.58

Table 3 Effect of AF and PF on RBC Number in Rats with syndrome of stagnation of liver qi and blood deficiency ，n=10，×109/L)

Table 3 Effect of AF and PF on RBC Number in Rats with syndrome of stagnation of liver qi and blood deficiency ，n=10，×109/L)

Compared with the blank group, ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001;Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001

Groups 1d 7d 14d 21d Blank group 9.18 ± 0.71 9.22 ± 0.54 9.20 ± 0.74 8.55 ± 0.90 Model group 8.50 ± 0.82▲ 8.80 ± 0.75 8.87 ± 0.54 7.92 ± 0.78 Siwu granules 8.72 ± 0.58 8.98 ± 0.65 8.96 ± 0.58 8.27 ± 0.79 Xiaoyao pills 8.57 ± 0.70 8.86 ± 0.64 8.83 ± 0.57 8.16 ± 0.64 PF30mg·kg-1 8.55 ± 0.47 8.85 ± 0.48 8.83 ± 0.62 8.15 ± 0.78 PF15mg·kg-1 8.59 ± 0.38 8.88 ± 0.44 8.95 ± 0.60 8.26 ± 0.74 AF30mg·kg-1 8.62 ± 0.55 8.93 ± 0.51 9.00 ± 0.79 8.32 ± 0.91 AF15mg·kg-1 8.42 ± 0.56 8.72 ± 0.57 8.69 ± 0.59 8.02 ± 0.68

Effect of AF and PF on HGB in Rats with syndrome of stagnation of liver qi and blood deficiency

Compared with the blank group, in the 3d after model making, the number of HGB in the model group was lower than that in the blank group (P < 0.05), but there was no statistical difference (P > 0.05) in the other monitoring days that were slightly lower than that in the blank group. Compared with the model group, there was no significant change in RBC number in each administrative group (P > 0.05). (Table 4)

Effects of AF and PF on viscera index of rats with syndrome of stagnation of liver qi and blood deficiency

The index of the spleen and adrenal of the model groups are lower than the blank group（P < 0.01、 P < 0.05）, and the thymus index is lower than the blank group, but the difference is not statistically significant(P>0.05).Compared with the model group, the spleen index of siwu granule group was increased (P < 0.01), while that of AF30 mg?kg-1and PF30 mg?kg-1groups were also elevated (P < 0.05). But No difference was found between the other groups. (Table 5)

Effect of AF and PF on Cytokines content in rats with syndrome of stagnation of liver qi and blood deficiency

It was found that content of IL-3 was significantly reduce(P < 0.01) and TNF-α increase (P < 0.05) in the model group compared with blank group. Compared with the model group, Siwufen granules, AF30mg·kg-1and PF30mg·kg-1can increase the IL-3 content in the group(P < 0.05, P < 0.05) and reduce the TNF-α content (P<0.05, P <0.05), while the other groups had a tendency to reduce the IL-6 content, but the difference was not statistically significant (P > 0.05). (Table 6).

Table 4 Effect of AF and PF on Hemoglobin (HGB) in Rats with syndrome of stagnation of liver qi and blood deficiency n=12，×109/L)

Table 4 Effect of AF and PF on Hemoglobin (HGB) in Rats with syndrome of stagnation of liver qi and blood deficiency n=12，×109/L)

Compared with the blank group, ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001;Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001

Groups 1d 7d 14d 21d Blank group 153.48 ± 7.86 154.50 ± 6.52 155.51 ± 7.82 154.79 ± 6.18 Model group 146.10 ± 7.65▲ 149.29 ± 8.26 151.46 ± 9.82 150.25 ± 9.66 Siwu granules 148.92 ± 5.50 151.27 ± 9.07 153.45 ± 6.07 153.81 ± 6.75 Xiaoyao pills 146.31 ± 9.34 150.33 ± 9.72 152.51 ± 10.62 152.79 ± 8.70 PF30mg·kg-1 147.62 ± 7.22 149.97 ± 8.89 153.81 ± 7.87 152.18 ± 7.50 PF15mg·kg-1 146.92 ± 5.90 149.27 ± 7.53 153.11 ± 6.59 151.48 ± 7.95 AF30mg·kg-1 147.83 ± 7.62 150.18 ± 8.88 154.02 ± 8.15 153.47 ± 6.63 AF15mg·kg-1 146.19 ± 7.73 149.37 ± 8.46 152.38 ± 9.22 152.41 ± 7.17

Table 5 Effects of AF and PF on organ index of rats with syndrome of stagnation of liver qi and blood deficiency ；n=12；g/100g)

Table 5 Effects of AF and PF on organ index of rats with syndrome of stagnation of liver qi and blood deficiency ；n=12；g/100g)

Compared with the blank group, ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001;Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001

Groups Spleen index adrenal index thymus index Blank group 0.69 ± 0.09 0.39 ± 0.06 0.077 ± 0.01 Model group 0.56 ± 0.08▲▲ 0.33 ± 0.07 0.066 ± 0.00▲Siwu granules 0.67 ± 0.10** 0.37 ± 0.07 0.073 ± 0.00 Xiaoyao pills 0.65 ± 0.11* 0.35 ± 0.05 0.075 ± 0.01*PF30mg·kg-1 0.67 ± 0.09* 0.35 ± 0.07 0.072 ± 0.01 PF15mg·kg-1 0.63 ± 0.08 0.34 ± 0.07 0.068 ± 0.00 AF30mg·kg-1 0.67 ± 0.07* 0.36 ± 0.06 0.073 ± 0.00 AF15mg·kg-1 0.64 ± 0.10 0.35 ± 0.07 0.072 ± 0.01

Table 6 Effect of AF and PF on Cytokines Content in Rats with syndrome of stagnation of liver qi and blood deficiency ；n=10；pg/ml)

Table 6 Effect of AF and PF on Cytokines Content in Rats with syndrome of stagnation of liver qi and blood deficiency ；n=10；pg/ml)

Compared with the blank group, ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001;Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001

Groups IL-3 GM-CSF IL-6 TNF-α Blank group 11.50 ± 1.78 95.09 ± 20.92 117.17 ± 13.58 51.50 ± 5.78 Model group 9.00 ± 1.24▲▲ 85.11 ± 27.21 128.55 ± 14.70 59.05 ± 5.61▲Siwu granules 11.16 ± 2.78* 86.54 ± 27.60 118.74 ± 13.91 52.78 ± 5.95*Xiaoyao pills 10.73 ± 1.64* 86.73 ± 25.04 123.22 ± 12.14 53.57 ± 6.78 PF30mg·kg-1 10.70 ± 1.71* 89.29 ± 19.08 118.61 ± 13.63 53.02 ± 4.05*PF15mg·kg-1 9.52 ± 2.15 96.39 ± 20.96 121.63 ± 15.48 54.51 ± 7.98 AF30mg·kg-1 10.82 ± 2.11* 98.06 ± 11.50 119.98 ± 13.73 52.76 ± 7.71*AF15mg·kg-1 9.96 ± 1.35 95.40 ± 27.84 123.45 ± 13.72 54.18 ± 7.53

Discussions

The Syndrome of stagnation of liver qi and blood deficiency is an important part of the syndrome of liver system, which is a common syndrome of traditional Chinese medicine in clinical practice [8]. In this study,multiple stress responses such as radiation irradiation and chronic binding were used to replicate the model of Syndrome of stagnation of liver qi and blood deficiency.The essence of the model was mostly the damage of bone marrow hematopoietic function, which led to changes in the ultrastructure of bone marrow, leading to the reduction of hematopoietic cytokines and blood cell production, suggesting that the pathological changes of blood deficiency and liver depression involve the disorder of bone marrow hematopoietic system [19]. The hematopoietic function of bone marrow is also regulated by multiple hematopoietic growth factors (HGF), of which GM-CSF [20] is a colony-stimulating factor that has extensive proliferation-promoting activity against early hematopoietic progenitor cells; TNF-α is a mononuclear Factor, mainly produced by monocytes and macrophages, is an important pro-inflammatory and immunoregulatory factor [13]; and IL-3 and IL-6 are one of the most potent cytokines to counter radioactivity,which directly participate in the activation process of hematopoietic stem cells and progenitor cells [21,22].Therefore, activation of cytokines can elucidate the mechanism of protecting and repairing of hematopoietic stem/progenitor cells to a certain extent [23,24]. The results of this study show that PF and AF have a clear blood-supplementing effect on the rats with syndrome of stagnation of liver qi and blood deficiency caused by radiation exposure combined with chronic restraint stress.It was further confirmed that PF and AF had similar blood-enriching effects as Siwu granule, both of which could increase the number of WBC in rats with blood deficiency and also improve the spleen index.

Previous studies have shown that the reactions and manifestations of drugs affecting hematopoietic function of bone marrow will eventually be reflected in the blood[9]. The blood cell index used in this experiment can directly reflect the condition of blood cells in the body and indirectly reflect the hematopoietic function of the bone marrow. Li Qiang et al. found that Baishao increased the RBC, HGB and HCT values of blood deficiency in mice caused by blood loss [10]. This effect is also found in the cyclophosphamida-induced blood deficiency model [11]. Gao Yue et al. also revealed that the replenishing blood effect of PF and its mechanism were also systematically studied [12]. In comprehensive bloodletting method to obtain blood deficiency model mice studies, performed by Huang Yinfeng et al., found that the PF and AF both increased the number of WBC in mice with blood deficiency, and the effect of AF on the increase of WBC number was better than PF [13].However, studies on the mouse model of blood deficiency caused by radiation irradiation showed that PF and AF were all effective components of Baishao, and both have similar effects on increasing the number of WBC [14].Zhang Jianjun and Zhu Yingli further revealed [15-17]that AF is a characteristic active ingredient of Baishao and has a clear replenishing blood effect consistent with PF; both of them promote the production of blood cells maintaining the stability of hematopoietic function by regulating of the cytohematopoietic factors, which is the material basis of the effect of Baishao on nourishing blood and astringing yin. In this study, radiation irradiation combined with chronic binding stress was selected to create the model of blood deficiency and liver depression in rats. One of the modeling points was that radiation irradiation was sensitive to the amount of WBC in rats, but it was unstable to RBC and HGB, sometimes it could be decreased and sometimes there was little change [18]. Therefore, under the present experimental conditions, the number of RBC and HGB in the model groups has a decreasing trend but the difference is not obvious. We also analyze that this is related to the short physiological cycle and rapid regeneration of WBC, but long and slow of RBC and HGB. Chinese medicine experts believe the spleen participates in hematopoiesis and blood storage and can regulate the components of peripheral blood. The spleen is also an important immune organ of the human body, and the data of the organ index indicate that the spleen of the model group is significantly decreased compared with the blank group. It also suggests that blood deficiency in the syndrome of stagnation of liver qi and blood deficiency not only destroys the bone marrow hematopoietic system, but also reduces the body mass of the immune organs (spleen,thymus, etc.), suggesting that the immune system suppression exists in rats with blood deficiency and liver depression.

Conclusion

Above all, on the one hand, PF and AF rats have a clear role in enriching blood on rats with syndrome of stagnation of liver qi and blood deficiency, especially the effect on an elevated amount of WBC and similar to Siwu granules positive control drug effect. And the increase of the spleen index indicates that it stimulates the proliferation of the spleen cells in the state of blood deficiency, and regulates the immune function. At the same time, the bone marrow hematopoietic system can be promoted by regulating the action of cytokines, such as IL-3, TNF-α, suggesting that the action mechanism is related to the regulation of cytokines on bone marrow hematopoietic system. On the other hand, in recent years,the "The cytokine hypothesis for depression" has gradually been concerned by some researchers and carried out related mechanism research and experimental research. The theory is that long-term chronic stress activates the peripheral and central immune systems,releases a large number of inflammatory mediators(including cytokines), and the activated immune system influences the progression of depression through interaction with the nervous system and neuroendocrine system [25]. In this experiment, the levels of IL-3 and GM-CSF in the rats of the model groups decreased, while the levels of IL-6 and TNF- increased. Both the AF group and PF group increased the content of IL-3 in the group and reduced TNF-α levels. Therefore, in order to further study the effects of AF and PF, the main effective components of Baishao, on replenishing blood in rats with syndrome of stagnation of liver qi and blood deficiency and related mechanisms, we need to explore the genes and protein levels of hematopoietic cytokines in immune organs (including thymus, bone marrow and other lymphoid tissues).

Acknowledgements

Thanks to Professor Li Wei of the Center for Scientific Research and Experimentation of Beijing University of Traditional Chinese Medicine for their support for this experiment!

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