Immunomodulatory effects of Blaps rynchopetera extract

ABSTRACT Purpose: To explore the potential immunomodulatory effects of total extract and different polar parts from Blaps rynchopetera Fairmaire. Methods: Phagocytic activity was evaluated by neutral red assay, and the effect of the immune function was investigated by normal and immunocompromised mice models. Results: In vitro, total extract, as well as chloroform, ethyl acetate, n-butanol and water fractions could individually enhance the phagocytic ability of mouse peritoneal macrophages; in addition, chloroform and ethyl acetate fractions had an increasing tendency when combined stimulation with lipopolysaccharide (LPS). In vivo, ethyl acetate fraction (EAF) could enhance the immune organ index, increase the serum hemolysin level and peripheral blood immune cells of immunocompromised mice, while for normal mice, the effect was inconspicuous. Conclusions: Blaps rynchopetera extracts had noteworthy immunomodulatory effect, especially for individuals with immune disorders.


Introduction
Immune regulation is one of the primary antitumor mechanisms of the traditional Chinese medicine. Studies have revealed that cancer and autoimmunity are closely related 1,2 , it can escape the immune system and even weaken the immune response of the host 3,4 . By activating the immunity of the body or regulating and balancing immune disorders, it can enhance physical fitness, inhibit tumor growth, improve patient survival time and quality of life, and even eliminate tumors 3,5,6 .
Insects not only have nutritional value but also have medicinal value [7][8][9] , and belong to a group of animal-based drugs widely used in traditional Chinese medicine. Blaps rynchopetera Fairmaire is one of the medicinal insects that belongs to the family Coleoptera Heteromera (Coleoptera) of beetles. It is also known as the smelly fart bug or stink beetle due to its smelly defensive secretion; it is used to treat fever, cough, gastritis, whitlow boils, tumors and other diseases 10,11 . There are reports documenting its medicinal properties such as its hypoglycemic 11,12 , antitumor 13 , antibacterial 14 , and antioxidant characteristics 15 .
A key attribute of the traditional Chinese medicine is the diversity of its effective components and the synergistic effect of multiple targets, which is noticeably different from chemical drugs. If medicine can selectively inhibit the growth of tumor cells and also possess a good immunomodulatory activity, it may have a better therapeutic effect on the tumor. This paper shows that the total extract, chloroform, ethyl acetate, N-butanol, and water fraction of B. rynchopetera could individually increase the phagocytosis index of mouse macrophages to varying degrees. Ethyl acetate fraction (EAF) boasts superior activity and can significantly relieve cyclophosphamide-induced immune deficiency in mice, increase the production levels of the hemolysin antibody, raise the level of immune cells in peripheral blood, and increase the spleen and thymus indexes. Results indicate that this fraction has a positive immune balance regulation effect.

Methods
The experiments follow the rules of the school's animal ethics committee, which approves all experiments for the purpose of controlling and supervising animal experiments (protocol number 2015-0820).

Mouse peritoneal macrophage phagocytosis assay Preparation and purification of mouse peritoneal macrophages
After KM mice were sacrificed with cervical vertebra dislocation, macrophages were collected from the abdominal cavity, and the cell density was adjusted to 2 × 10 6 cells•mL -1 with 1640 medium containing 10% fetal bovine serum. Next, 100 μL per well was inoculated on a 96-well plate and cultured at 5% CO 2 and 37 °C. After 3 h, the unadherent cells were washed away with PBS to obtain purified peritoneal macrophages 16,17 .

Phagocytosis assay
Phagocytic activity was evaluated by neutral red assay 18 . The macrophage of 2 × 10 6 cells•mL -1 was seeded in 96-well plates and set cell control group (cells and culture medium only) and LPS model group (LPS final concentration of 10 μg•mL -1 ). Respectively, sample groups (each with five concentration gradients of 500, 250, 125, 62.5, and 31.25 μg•mL -1 ) with LPS added (first stimulated with LPS for 4 h, then further stimulated with the addition of sample solution) were placed in three wells each. After culturing at 37 °C in 5% CO 2 incubator for 24 h, washed the plate with PBS and added 200 μL neutral red solution to each hole, incubated for 3 h. The supernatant was discarded and the cells were washed with PBS. Then 200 μL cell lysate (containing 50% ethanol, 1% acetic acid, 49% water) was added and the solution was left at 4 °C overnight. Finally, the absorbance was measured at 520 nm. The phagocytic index was calculated as follows (Eq. 1):

Phagocytosis index =
A sample A blank control (1) Immune function assay for normal and immunocompromised KM mice

Preparation of sheep red blood cell (SRBC) suspension
Under aseptic conditions, blood was taken from the external jugular vein of healthy sheep, placed in an Erlenmeyer flask containing glass beads, and shaken for 30 min to remove fibrin. Next, PBS was added and the flask was stored in a refrigerator at 4 °C for later use. Before use, the sample was washed with normal saline and centrifuged for 5 min at 2000 r•min -1 . After the supernatant was discarded, it was diluted with normal saline at a ratio of 3:5 (v/v) to obtain a red blood cell concentration of 2 million cells per microliter.

Preparation of guinea pig serum
Fresh blood was obtained from guinea pigs under aseptic conditions and the serum was separated and stored at -80 °C, then diluted with normal saline at 1:10 (v/v) before use.

Grouping and administration Normal mice model
The immune function assay for normal mice was performed in the same manner as previously described. The mice were randomly divided into six groups: control group, model group, Astragalus polysaccharides (APS) group, and three experimental groups (EAF-H, EAF-M and EAF-L). There were six mice in each group, half male and half female. Each subject in the blank group was given the same quantity of normal saline. The APS group was intraperitoneally injected with APS 160 mg•kg -1 , while the experimental groups were given intragastric administrations of 400, 200, and 100 mg•kg -1 . The medicine was administered once a day for 10 days. The model group was intraperitoneally injected 80 mg•kg -1 of CTX on the 7th day. Each sample group was immunized by an intraperitoneal injection of 0.2 mL 20% sheep red blood cell suspension on the 3rd, 5th, and 7th days of administration 19 .

Immunocompromised mice model
The immune function assay for immunocompromised mice was performed as previously described. The grouping and administration were essentially the same as for the normal mice model. Except for the control group, from the 7th day of administration, each group was injected intraperitoneally with 0.1 mL of 80 mg•kg -1 CTX and injected daily for three days to establish the model of immunosuppression in mice 20 .

Determination of peripheral blood immune cell levels
One hour after the last administration, blood was obtained from the eyeballs of the mice, and 20 μL of whole blood was placed in the prediluted solution. The peripheral blood cells were determined by an animal blood cell analyzer in prediluted mode.

Determination of serum hemolysin levels
The eyeball blood was centrifuged at 2000 r•min -1 , taken the serum and inactivated at 56 °C for 30 min. Diluted and placed in an EP tube, added 0.5 mL of 5% sheep red blood cells and 1.0 mL of diluted guinea pig serum, normal saline as the blank control. All the test tubes were kept at 37 °C for 10 min with water bath and then put into ice bath to terminate the reaction. Centrifuged and took 1.0 mL of the supernatant, added 3.0 mL of Drabkin's reagent; took another test tube and added 0.25 mL of sheep red blood cell suspension and 3.75 mL of Drabkin's reagent 16 . After incubated for 10 min at room temperature, measure the absorbance of each sample at 540 nm and calculate the half hemolysis value (HC 50 ) (Eq. 2) 21 : HC 50 = × Dilution multiple Absorbance value of sample Half absorbance value of red blood cells during half hemolysis (2) Immunomodulatory effects of Blaps rynchopetera extract

Determination of organ index
The mice were sacrificed after blood collection, then the spleen and thymus were removed and accurately weighed to calculate the organ index of the immune organs (Eq. 3).

Organ index =
Organ mass (mg) Mice body weight (g)

Statistical analysis
A one-way analysis of variance was performed with SPSS 19.0 software. After the homogeneity of the variance test, the experimental data with uniform variance were statistically analyzed by the pairwise comparison Fisher's least significant difference method. Besides, the data with uneven variance was analyzed by the rank sum test. Values of p < 0.05 were considered statistically significant.

Effect of B. rynchopetera on phagocytic ability of peritoneal macrophages
Mouse peritoneal macrophages were used as the experimental object and LPS acted as a positive control. The phagocytic ability of macrophages was investigated by single sample stimulation and sample combined with LPS stimulation 22 . The immunological activity of the total extract, chloroform, ethyl acetate, n-butanol, and water fractions of B. rynchopetera were tested.
The results are displayed in Fig. 1 (the left parts). The phagocytosis of mouse peritoneal macrophages was significantly enhanced under the stimulation of LPS, reflected by a phagocytosis index was greater than 1.0 (p < 0.05). Except the water fraction, the total extract and three different polar fractions, i.e., the chloroform, ethyl acetate, and n-butanol fractions, all could enhance the phagocytic ability of macrophages. There were significant differences (p < 0.05) for some fractions at certain concentration, particularly for the chloroform fraction and EAF, while there were no dose-dependent.
When stimulated with sample combined with LPS (the right parts of Fig. 1), only the chloroform fraction showed a trend of enhancing macrophage phagocytosis, the total extract, and other fractions had no synergistic enhancement. On the contrary, all dose groups of n-butanol and water fractions acted a certain inhibitory activity when combined with LPS stimulation. Compared with cell group, *p < 0.05, **p < 0.01; Compared with LPS group, # p < 0.05, ## p < 0.01.

Immune function effect of EAF on normal and immunocompromised KM mice Effect of EAF on immune organs index
In the immune normal model, CTX was used as a model control and APS as a positive control. In the immunocompromised group, except for the control group, all mice were injected intraperitoneally with CTX, and APS was also used as the positive control.
The immune organs index can be used to determine the immune function of animals, while the spleen and thymus indexes can reflect the level of immune response to antigens.
The results in Table 1 and Fig. 2 showed the effect of immune organs index in normal mice. Compared with control group, the liver, spleen and thymus indexes of model group (CTX) showed a slightly decreasing trend, but there was no statistical significance. Compared with the model group, the spleen index of all the experimental groups increased to some extent, but only the APS group and EAF low-dose group displayed statistical differences (p < 0.01), thymus index of all the experimental groups showed an increasing trend, but only the APS group had statistical differences (p < 0.01). As shown in Fig. 2, the changes of spleen in each group were consistent with those in Table 1, but the color was not uniform, which might be caused by different exposures during photography.     Fig. 3 showed the effect of the immune organs index in immunocompromised mice. Compared with the control group, the spleen index and thymus index of model group (CTX) were significantly reduced (p < 0.01). Also, compared with the model group, the spleen index of all the experimental groups were significantly increased (p < 0.01), and even exceeded the control group (p < 0.01). However, there was no statistical difference in the thymus index.

Effect on peripheral blood immune cell levels
Variations in peripheral blood cells can reflect the changes of immune function 23 . The effect on peripheral blood immune cell levels for normal mice were showed in Table 3. Compared with the control group, the levels of white blood cells, lymphocytes, monocytes and granulocytes in the peripheral blood of mice in the model group (CTX) had a decreased tendency, and the decrease of lymphocytes was statistically significant (p < 0.01). The presence of APS can significantly increase the levels of these four immune cells in normal mice (p < 0.01). Additionally, the levels of leukocytes, lymphocytes and monocytes in all dose groups of EAF showed an increasing trend. However, only the levels of lymphocytes and monocytes in the high dose group had a significant difference (p < 0.05), and there was no effect on granulocyte level. Table 3 -Effect of EAF on immune cells in peripheral blood of normal mice ( ± s, n = 6, 1 × 10 9 /L). The effect on immunocompromised mice was very different, as Table 4 illustrates. Compared with the control group, the levels of leukocytes and lymphocytes in CTX group were significantly decreased (p < 0.01). Moreover, levels of the four kinds of immune cells in all the experimental groups were significantly increased (p < 0.01), compared with the model group. Additionally, the levels of white blood cells, monocytes, and granulocytes were higher, exceeding the control group (p < 0.01). Table 4 -Effect of EAF on immune cells in peripheral blood of immunocompromised mice ( ± s, n = 6.1 × 10 9 ·L -1 ).

Effect of EAF on serum hemolysin levels in mice
Serum hemolysin level is one of the commonly-used indicators that evaluates humoral immune response, which reflects the level of antibodies 24 . Table 5 showed the effect of serum hemolysin levels in normal mice. Compared with the control group, the HC 50 of the CTX group was significantly reduced (p < 0.01), while the positive drug APS group was significantly increased (p < 0.01). The EAF exhibited an increase, with the high and medium dose groups presenting a slight rising trend, but only the lowdose group displayed statistical significance (p < 0.05). In Table 6 the effect of serum hemolysin levels in immunocompromised mice can observed. Compared with the CTX group, the HC 50 of all experimental groups were significantly increased (p < 0.01), and the three-dose EAF groups were notably different from APS group (p < 0.01), reaching similar levels to the control group.

Discussion
For the first time, the present study investigated the immunomodulatory effects of Blaps rynchopetera extract. Macrophages are multifunctional immune cells that play a key role in all aspects of the body's immune response 25,26 . Phagocytosis is one of the basic functions of macrophages. Therefore, an experimental model of immune activity of peritoneal macrophages as target cells was constructed to assay the immunomodulatory effects of the extract in vitro 25 .
It has been previously shown that polysaccharides stimulate macrophage proliferation with low cytotoxicity 27,28 . Under the stimulation of LPS, the phagocytosis of macrophages is enhanced, suggesting that the peritoneal macrophages are activated and the innate immune function is enhanced. In this study, macrophages were stimulated with samples alone and in combination with LPS respectively to observe the effects on macrophages, and the phagocytic activity was evaluated by neutral red method. The results of the study indicated that all components can individually enhance the phagocytic ability of mouse peritoneal macrophages. After combined stimulation with LPS, the chloroform fraction and EAF showed that they could further increase the phagocytic ability of macrophages after LPS prestimulation to a certain extent.
In vivo, the effects of EAF on the immune function of normal and immunocompromised mice were further investigated in terms of both cellular and humoral immunity. Astragalus polysaccharide is an extract of Astragalus membranaceus, which is rich in nutrients and has pharmacological effects such as enhancing immunity and improving antioxidant capacity. It has been widely used in clinical medicine 29 . Studies have shown that APS can enhance the cellular and humoral immunity of animals by promoting the development of immune organs and enhancing the functions of T cells, B cells and natural killer cells 30 . In this study, APS was used as a positive control and CTX was used as a chemoinducing agent in an immunosuppressive animal model. After CTX injection, spleen and thymus index decreased, peripheral blood immune cells showed a downward trend, and the secretion level of hemolysin decreased 31 . The spleen is the main organ of humoral immunity, and the thymus is the main organ of cellular immunity. The thymus and spleen indexes can reflect the overall immunity level of the body to a certain extent. The change of the number of peripheral blood cells can reflect the change of immune function 23 . Serum hemolysin is an important index to evaluate humoral immunity 32 .
The results of this study show that APS has a significant effect on both normal and immunocompromised mice. It is noteworthy that EAF could significantly increase the immune organ indexes, the level of peripheral blood immune cells, and the half hemolysis value for immunocompromised mice. Conversely, it has a much smaller influence on normal mice. These results indicate that EAF has a remarkably protective effect on immunocompromised mice induced by CTX, and the regulatory mechanism may be different from that of APS.
The test results of the normal group for both batches of experiments are also quite different, especially for the immune organ index. This reminds the necessity for using animals from the same batch in related subject research in future studies.