INTRODUCTION:

Probiotics can be defined that live microorganisms have many beneficial effects to human body, they can be isolated from many sources like food, so they are considered as dietary supplements that improve the health by balance the intestinal microbe. Probiotics include Lactic Acid Bacteria (LAB) group and Bifidobacterium¹.

LAB group has special properties are gram positive bacteria ,not spore forming, not produce any toxin, safe and ferment the sugar to lactic acid².

Pediococcus is one of LAB group that being effective against most Gram-positive and negative pathogens ³. Recent studies revealed that Pediococcus activate the immune system, resulting in the prevention of immune disease⁴. Immunomodulation capacity of Pediococcus strains is considered a criterion for probiotic assessment⁵. In other side, Macrophages play key roles in innate and adaptive immunity because Macrophages present invasion of pathogens by releasing inflammatory and cytotoxic molecules⁶. Probiotics control the inflammatory cytokine responses of macrophage⁷. Other studies revealed that probiotic induced IL-12 production by macrophages when stimulated by probiotic components⁸.

In our previous studies, the effect of many genus of LAB group on pathogenic bacteria was studied^9,10,11, that isolated from different sources: cosmetic, paper currency, urinary tract infection and vagina^12-15.

In this study Pediococcus acidilactici strain with the highest ability to inhibit test. Pathogenic bacteria was selected to assess its ability to immune regulate by macrophages and model of immunosuppression because of nave specific cytotoxic effects against normal, high doses of cyclophosphamide lead to decrease the body weight, also spleen and thymus may be decrease in weights relatively, in addition to that decrease of phagocyte index. Cyclophosphamide is alkyl that used extensively as alkylation agent, in addition to that cyclophosphamide; it considered the main component of associated regimens with chemotherapy¹⁶. Therefore, this study tried to find an immunomodulator with safe properties able to improving the immunity and reduce the effects of cyclophosphamide.

MATERIAL AND METHODS:

Pediococcus acidilactici:

P. acidilactici strain tested in this study was isolated from Donkey milk, it was collected from (AL-Fdhailia), but in sterilized tube and brought to the laboratory cultured in Man-Rogosa-Sharpe (MRS) broth medium activate the bacteria incubated in 37ºCfor 24 hr in anaerobic condition, after that serial dilution was made to 10¹⁰to gain single colony, and cultured from the last one on MRS Agar in the same conditions, examined microscopically and diagnosed by Vitek 2 compact system device.

Experimental Animal:

20 Mice BALB/C mice male with 6-8 weeks of age were obtained from Iraqi Center for Cancer and Medical genetics research (Baghdad/Iraq), 20-25gram were mice body weight that housed in groups, four mice per cage at 20-25ºC for 12 hr light/dark cycle and provided with feed that contain carbohydrates, protein, vitamin and mineral source.

Cyclophosphamide material:

(CPM) was obtained from Scbt, regard to Scbt this material was used for immunosuppression of mice.

Oral suspension Preparation:

To prepare a suspension of P.acidilactici fresh growth for 24 hr Of P.acidilactici in MRS broth was centrifuged (1000RPM/10min) then washed twice with phosphate buffer saline (PBS), after that suspension of P.acidilactici was concentrated to 10⁸ and 10⁹CFU/ml, this concentration was diluted in sterile PBS to prepare the doses in oral administration.

Design of experiment:

Five groups of mice were found to complete the experiment depending on the type of dose as following:

Group 1	Normal control (injected with normal saline), this is considered positive control
Group 2	Only (CPM) 80mg/kg/d (no probiotic), this is considered negative control
Group 3	(CPM) with probiotic 6×10⁸ CFU/ml (first treated)
Group 4	(CPM) with probiotic 6×10⁹ CFU/ml (second treated)
Group 5	Only probiotic 6×10⁹ CFU/ml

Intraperitoneally (IP) injection with sterile saline was used to inject the three groups (2,3,4) 80mg/kg of body weight, the injection was used to induce immunosuppression effect, and then probiotic was given every day to mice groups for 20 days. One of the effects of immunosuppression is the body weight because it is considered as measure of immunosuppression, after that, blood and spleen was taken to detect the immune level (macrophages).

All treatments were submitted to oral administration once daily with 20ųl/kg body weight, this oral administration continued for twenty days, whereas (G2) group was injected with normal saline and considered as immunosuppression group¹⁷.

Body weight analysis:

The weight of mice bodies were monitored in 1^st and after 20 days from the experiment.

Immune organ index analysis:

Immune organ indexes mg/g =organ weight

The treated groups were weighted 20 days before they scarified after start the oral administration. Both of thymus and spleen organs were surgically replaced and weighted immediately .The following equation was used for calculating the thymus and spleen index^18,19:

Prepare the Candida albicans:

Candida albicans was grown on Sabouraud dextrose (SD) broth (Difco) at 30°C for activate the cells of yeast, orbital shaking at 150 RPM was continued with grown for 16 hr. To maintain the yeast cells alive and it was kept on SD agar. For preparing the yeast to binding and phagocytosis test: C. albicans cells were centrifuged 10 000rpm/ 10 min, washed with phosphate buffer (3) times in 0.01M killed by heat at 100°C, stored in PBS that contain sodium azide (0.05%) at 4°C until use, the heat killed (HK) cells were suspended to 4 × 10⁶/ml and examined viability test.

Viability test:

Fifty ml from cell suspension were placed in tube, 50μl from trypan blue was added then mixed by pipetting, and the mixture was incubated less than 3 min (room temperature) cell death leads to inaccurate the viability.

Isolation of peritoneal macrophage from mice²⁰

1. Injected mice with 0.5ml of glycogen solution after 4 days injected mice with (5ml) of Phosphate buffer Saline (PBS) supplemented with heparin in peritoneal fluid.

2. Peritoneal cavity was performed by a gentle massage, killed mice by cervical dislocation.

3. Abdomen of each mouse was washed with 70% ethanol. A lateral incision was performed along the bottom using a scissor. Abdominal skin was pulled back by using a forceps to expose the transparent peritoneal skin.

4. The fluid was aspirated carefully with puncturing any organ, dispensed into 10ml siliconized tubes.

5. Centrifugation 2000rpm/10 min, discard supernatant, washed twice with PBS buffer and cell pellet was re-suspended in RPMI-1640 medium.

6. Cells were counted by a hemocytometer, and then adjusted to cell density 1×10⁶/ml.

Determination of phagocytic index of peritoneal macrophages:

The peritoneal cells of scarified mice were harvested with medium (4ml of RPMI-1640) that supplemented with 10% heat inactivated FBS. Harvesting cells were done by peritoneal lavage and last collected in silicon tube, and then each three milliliter of the cell-rich lavage fluid was aspirated and by 2000rpm/10 min was centrifuged. The pellet was seeded in class slide after it was re-suspended at 1×10⁶ cells/ml in medium that called RPMI-1640²¹.

Washed three times, so, the attached cells (adherent cells) were used as peritoneal macrophages where as the non-adherent cells were removed by aspiration²².

After that, the cells were washed to remove the excess dye, washing was done by PBS buffer, then. They were blotted dry. The expression of the percentage the phagocytic index was expressed by calculates the percentage of phagocyte macrophages in 200 cells of the total cells by the optical microscope the count was performed.

Added killed Candida albicans was concentrated (4 × 10⁶) incubated at 37°C for 1hr. under 5% CO₂, after was discarded and was washed thrice with PBS buffer, left at room temperature to dried and fixed with absolute methanol, stained by Giemsia stain for 20 min. After that, the cells were washed to remove the excess dye , washing was done by PBS buffer, then, they were blotted dry. The expression of the percentage the phagocytic index expressed by calculate the percentage of phagocyte macrophages in 200 cells of the total cells, by the optical microscope the count was performed²³.

Statistical analysis:

The data was analyzed with SPSS 20.0 software (SPSS Inc., Chicago, IL, USA), expression the results was by using mean ± standard deviation (SD) in three replicates and determining the significance of data and comparison among them in statistical analysis was done by one-way analysis of variance. Duncan’s multiple range was followed the tests last, p < 0.05 value was considered to be significant statically.

RESULTS:

The weight of mice body:

The five groups of mice were monitored at zero, 20 times during the experiment. The study revealed the big differences in body weights initially in day (0), and after 20 days during acclimatization in the animal house subsequently, compared with the first time, whereas the immunosuppressed group(G2) showed decreasing in body weight after injection the mice with CPM (p˂0.05), compared with the zero time (Table1).

Table 2 show were big differences body weights in G2 (immunosuppressed group) compare with control group while in other three mice groups, there were no significant differences when compared with the NC group, the five group (G5) (administrated only probiotic) increased significant differences vs. NC group (25.00±0.00gm) (28.50±1.73gm) respectively, G4: cyclophosphamide plus P.acidilactici 6×109 CFU/ml, G5: P.acidilactici 6×109 CFU/ ml revealed the increase in body weight during the experiment, while in other three mice groups, there were no significant differences when compared with the NC.

The index of Immune organ

The thymus indexesin G5 and G4 groups compared with the CPM mice, were significantly (p<0.05), but P.acidilactici with low doses 6×10⁸ CFU/ml had not significant difference (p<0.05), also, there were increasing significantly (p<0.05) in spleen indexes, for both treated P.acidilactici 6×10⁸ and G5 groups compared with NC group. In current study P.acidilactici 6×10⁸(G5) effect was the strongest in the thymus index compared with mice were treated with CPM plus P.acidilactici 6×10⁸at 5×10⁹CFU/ml (0.2ml) with statistically significant (p< 0.05) (table 3).

Table 3: Pediococcus acdilactici effect on mice thymus and spleen indices

Mean	Groups
Mean	G1 (gm)	G2 (gm)	G3 (gm)	G4 (gm)	G5 (gm)
Spleen index mg/g	1.78 ± 0.7	0.323 ± 0.34	0.772 ± 0.38	0.856 ± 0.35	1.4 ± 0.7
P-value compare to G1	----	0.050*	0.05*	0.05*	0.05*
Thymocyte index mg/g	0.097 ± 0.23	0.039 ±0.022	0.102 ± 0.14	0.147 ±0.030	0.1973 ±0.005
P-value compare to G1	----	0.05*	0.660	0.05*	0.05*

*significant differences

Effect of pediococcus acdilactici on phagocytic activity of peritoneal macrophage:

Macrophages were isolated in day 20 from mice treated with P.acidilactici and CPM by oral administration, and then macrophages were examined for phagocytosis and pinocytosis activity. So, this activity was measured by engulf of candida albicans method, (figure1). Whereas phagocytosis activity in mice that treated with CPM just (G2) group was reduced of the macrophage, mice treated with P.acidilactici 6 × 10⁸Cfu/ml or P.acidilactici 6 × 10⁹Cfu/ml were restored it with significantly (p<0.05). P.acidilactici 6×10⁸Cfu/ml and P.acidilactici 6×10⁹Cfu/ ml effects were comparable to that of CPM group. However, the high dose of P.acidilactici 6×10⁹Cfu/ ml revealed a stronger effect of macrophages in phagocytic activity than P.acidilactici 6×10⁸Cfu/ ml in treated mice groups with significant differences (p˂0.0001).

Figure 1: P.acidilactici effect on macrophage activity in mice macrophages.

Table 4: P.acidilactici effect on macrophages of mice

Mean	Groups
Mean	G1 (gm)	G2 (gm)	G3(gm)	G4 (gm)	G5 (gm)
Phagocytic index	65.125 ± .39	35.375 ± 0.10	76.625 ± .108	72.125 ±1.65	87.750 ± 1.32
P-value compare to G1	----	0.000000 P < 0.001	0.0001**	0.003**	0.000003**

**significant differences

DISCUSSION:

CPM has significant available effect, it is alkylation agent, so it used extensively clinically and chemotherapy, it has numerous technique to destroy cells like; damage the structure of DNA, or interfere with the proliferation and differentiation of B and T cells, also CPM can immune cells. The CPM effect on T and B cells by reducing the of normal T and B cells, so by this way it can reduce the levels of inflammatory cytokines (25,26), this is leading to suppressing the organism immune responses by suppress the cellular and humeral responses²⁴.

In this study treated mice with CPM were considered as animal model of the immunosuppression, in the same time this study showed enhancement of immunity by P. acidilactici. CPM could reduce mice body weight notably when used (80mg/kg, i.p). In the current study, also, there were obvious effect on immune organ index, it could inhibit both of proliferation of spleen cells and activity of the phagocytosis of macrophages, the study results are accompanied with many reports^25,26,27,28. In addition to that, results revealed immune BALB/c mice functions were suppressed by CPM as the results that noticed above expressly, this suppression was obvious significantly, so, this model of mice was successes to establish the immunosuppressive model. P. acidilactici had been indicated to its activity on immunity, because it is (like other lactic acid bacteria) has more than components enable to influence on humeral, cellular immunity^29,30.

In one of reports P. acidilactici was improved the ability of enhancing plasma cells to produce IgA , this result agreed with current study that found P. acidilactici was capable of reinstate mice immunosuppression that induced by CPM. The thymus and spleen have the importance according to the body organs because of they are considered the places of immunological cells, either growth or proliferation the cells or both of them, Thymus is considered immune organ because of T lymphocytes developed, proliferation, differentiation and finally mature there. In the same time spleen organ contain T and B cells, the effect of P. acidilactici on thymus and spleen organ. Indices were determined, the index of each immune organ is determined to reflect the growth of this organ and evaluate the probiotic effect as immunoregulatory^31,32.

In one research, Li et al.,³³ indicated that some genus of lactobacilli revealed indexed in immune organ and significantly, also, in the current study and at the end of experiment, results improved that three treated groups were reported greater index in thymus and spleen obviously than CPM (G2) group.

The results concluded that P. acidilactici indicated to keep the influence of immune organs development during its influence of immunosuppression. So, we can summarized the study that P. acidilactici is very effective agent as immunomodulatory because it could improved the immunity, it act as promoter to immune organ and will cause the development of macrophages phagocytosis activity. Finally, P. acidilactici may be used with human chemotherapy in immune function

ACKNOWLEDGMENTS:

The authors would like to thank Mustansiriyah University, Iraq (www.uom ustansiriyah.edu.iq) for its support of the current work.

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Received on 03.03.2021 Modified on 02.05.2021

Research J. Pharm.and Tech 2022; 15(2):605-610.

DOI: 10.52711/0974-360X.2022.00099

Mean	Groups
Mean	G1 (gm)	G2 (gm)	G3 (gm)	G4 (gm)	G5 (gm)
Mean of weight ± S.D. In zero days	25.00 ± 0.00	27.50 ± 2.88	24.25 ± 1.50	25.00 ± 0.00	24.50 ± 1.00
Mean of weight ± S.D. After 20 days	25.00 ± 0.00	19.00 ± 1.15	25.50 ± 2.64	28.25 ± 0.95	28.50 ± 1.73
P-value	0.0 N.S.	0.002**	0.443	0.0005**	0.007**

Mean	Groups
Mean	G1(gm)	G2(gm)	G3(gm)	G4(gm)	G5(gm)
Mean of weight ± S.D. After 20 days	25.00 ± 0.00	19.00 ± 1.15	25.50 ± 2.64	28.25 ± 0.95	28.50 ± 1.73
P-value compare to G1	0.0 N.S.	0.002**	0.443	0.0005**	0.007**