The Effect of Ganoderma Lucidum Powder on some Immune Parameters in Rat

Ali A. Dawood aad@uomosul.edu.iq ABSTRACT The impact of Ganoderma lucidum powder on both chemokines, IgG and C3 complement concentrations, as well as WBC types, was investigated in 60 ester citrine rats split between two groups, one of which was injected with Carbon tetrachloride CcL4 and the other with Ganoderma lucidum powder. They were not given this drug; instead, two concentrations of mushroom powder, 40 mg/ per kilo-gram of body weight and 80 mg/ per kilo gram of body weight, were utilized, along with a control group. The results revealed a clear increase in all immune factors measured, with a clear difference between males and females when compared to control samples, as well as an increase in the numbers of both neutrophils and lymphocytes in males for both concentrations, with a higher increase for the second concentration than the first, while the increase in females for both concentrations was similar. In the groups exposed to CcL4, an increase in the values of chemokines, IgG, and C3 as well as the number of neutrophils appeared in males in varying numbers for both concentrations, whereas the increase in females appeared in the second concentration higher than the first concentration of immune values, and the opposite was true for the number of white cells. The number of monocytes showed slight changes in both males and females under study with clear significant differences between C3, chemokine, white blood cell count, lymphocytes and monocytes.


INTRODUCTION
The third group was given Ganoderma powder manufactured by DXN in Malaysia at a concentration of 40 mg per kg of weight and a dosage of 1 ml per kg, while the fourth group was given Ganoderma powder at an oral dose of 80 mg per kg daily for 30 days.
The fifth group was injected with Ccl4 and the animals were dosed with Ganoderma powder at a concentration of 40 mg/ kg daily for thirty days, the sixth group was injected with Ccl4 twice a week with Canoderma powder at a concentration of 80 mg/kg for 30 days.

Blood sample collection:
After each animal's treatment, blood was drawn from the orb vein through a capillary tube implanted in the eye stone, and blood was allowed to flow through the capillary tube to two types of test tubes, one clean and dry and free of anticoagulant to obtain serum, and the other containing a substance. EDTA anticoagulant for performing blood tests on it, dividing the blood into anticoagulant-free tubes, and storing the serum at -20°C until the analyses are completed (Timm et al., 2021).

Methods
-The ELISA technique was used to determine the values of chemokines, and the principle for this technique was: The test used is a sandwich type Elisa. This test depends on the association of chemokines present in the serum with specific antibodies to it that are fixed at the bottom of the microplate. After adding a specific antibody to chemokines tagged with peroxidase on the microplate. In the appropriate incubation period, the immune complex is formed, and then all the substances not associated with washing are removed. The reducing power of the enzyme is directly proportional to the concentration of interleukin in the sample after incubation with the base substance, which is TMB. Yellow color is formed when the reaction stop solution is added, which is measured at a wavelength of 450 nm and is determined sample interleukin concentration by using the standard curve (Kasha et al., 2020), and the following steps have been followed 1-Dilute capture Ab 1:100 in buffer, and transfer 100 µl of this working solution to each well, incubate 24/ h at room temperature. 2-Remove capture antibody and add 300 µl blocking solution to each well, incubate 1 hour at room temperature. 3-Remove block in solution, transfer 100 µl dilute standard and sample in the wells, incubate 2 hours at room temperature. 4-Wash 5/time with washing buffer. 5-Dilute detection-antibody 1:100-in reagent diluent and transfer 100µl to each well, incubate 2 hours at room temperature. 6-Wash 5/time with washing buffer. 7-Dilute poly-streptavidin 1:1000 in reagent-diluent and transfer 100µl to each well, incubate 20-30 m. at room temperature. 8-Wash 5/time with washing buffer. 9-Add 100 µl of substrate solution to each well and incubate up to 60 Min at room temperature in the dark. 10-Add 50 µl stop solution and read the microplate at wavelength 450 nm and is determined sample interleukin concentration by using the standard curve (Kasha et al., 2020). -While the immunodiffusion technique was used to determine the values of both IgG and C3, and the principle for this technique was: RID test is a type of precipitation reaction that depends on the diffusion of one towards the other (Ag or Ad) on the solid agar medium and the formation of a precipitation ring whose diameter is directly proportional to the concentration of the Ag or Ab in the sample (Parija, 2012). and the following steps have been followed: 1-Place the agar dish and samples at room temperature for 1/2 hour. 2-Added 50µl from serum to the well on the agar.

4-
The diameter of the zoon formed was measured with a graduated magnifying glass. 5-By using table attached to the kit, the concentrations of IgG and C3 were extracted (Parija, 2012) -The CBC Automated Technique was used to determine the numbers of blood cells.
-Statistical analysis was carried out using SSPS Version 25.

RESULTS AND DISCUSSION
White rats of type and ester citrine were used and separated into six groups (control, Ganoderma first concentration G1, Ganoderma second concentration G2, Ccl4, G1+Ccl4, and G2+Ccl4) to determine the effect of Ganoderma lucidum on immunological factors and blood cell counts. As shown in (Table 1).   (1) shows the average values of chemokines and IgG in males and females given Ganoderma powder in both concentrations, as well as those exposed to CcL4 and given Ganoderma powder in both concentrations. In the two doses with the second concentration, the values varied between males and females, while the values for both chemokine and IgG increased among males receiving the two concentrations of Ganoderma powder and CcL4, and the values for C3 increased among males in the second concentration only without the first concentration. The values of both chemokine and IgG decreased in females exposed to Ccl4. and those given Ganoderma powder, and in both concentrations, the values of C3 increased in the higher concentration in females than in the lower concentration, with no significant differences between the concentrations of Ganoderma and the immunological values that were examined P > 0.05 for both sexes.
A study conducted in Singapore in 2003 on the effect LPS of Ganoderma lucidium on the effectiveness of the immune system In which 1800 mg of mushroom powder was used 3 times daily for 12 weeks. The study proved the positive effect of mushroom powder on the effectiveness of the immune system if a significant increase in the production of each of IL-2, IL-6, INF-γ, while non-significant increase in the concentration of each IL-1 and TNF-α .
In China, a study was conducted in 2018 in which Ganoderma lucidium powder was used as a treatment for cancer caused in rats. The study found an active effect of Ganoderma lucidium powder to production of types of cytokines, including IL-2, TNF-α and INF-γ, In addition to increasing the effectiveness of NK cells and T cells (Chunhua et al., 2018).
Chen and his group proved in 2014 demonstration the effect of polysaccharide extract from Ganoderma lucidium on the production of IL-1β in mice (Chan et al., 2014).  Table (2) compares the effects of dosing with Ganoderma powder at concentrations of 40 mg and 80 mg/kg of body weight to control samples on hematological variables such as white blood cell counts, lymphocytes, and monocytes in both sexes (males and females). It was apparent in the white blood cell count in men at a dosage of 40 mg, which was 10.15, and it was much higher at an 80 mg concentration, which was 13.45, compared to the control samples, confirming Ganoderma powder's beneficial effect on raising white blood cell count for both sexes. In the current study, we didn't detect any significant correlation between patients and all parameters, P > 0.05.
In males, the increase was higher in the second concentration, reaching 10.3, and it was lower in the first concentration, reaching 8.45 in males compared to the control samples, and it was close in females at both concentrations, 9.05 and 9.55, respectively, with a clear increase compared to the control samples. Males had a larger rise in monocyte number in the second concentration than in the first, reaching 1.85 when compared to control samples, while females had a higher increase in the first concentration than in the second, reaching 1.8. This effect is due to the presence of polysaccharides in Ganoderma that can boost the immune system in rodents by increasing the production of white blood cells (Cao and Lin, 2006). Many studies indicate that polysaccharides greatly increase the proliferation of T and B lymphocytes.
The proliferative responses of B and T Lymphocyte and the toxic activities of T cells and NK cells were also enhanced with Ganoderma powder without any side effects (Bao et al., 2002). The polysaccharides in Ganoderma also allow the release of TNF-α and IFN-ƴ from resistant T cells in a dose-dependent manner (Zhu et al., 2007). In rats exposed to Ccl4, males had higher numbers of neutrophils in the second concentration than in the first concentration, reaching 12.6 and 11.35, respectively, when compared to rats exposed to the same substance but not dosed with mushroom powder, while females had higher numbers in the concentration. The first concentration was higher than the second concentration, at 14.3 compared to the control samples, as was the number of lymphocytes, with the increase in the second concentration in males being higher than the first concentration, at 10.3, in contrast to females, who had a higher number of lymphocytes in the first concentration. It showed in numbers of 11 with a noticeable rise compared to the control samples in the second concentration, and there was a modest increase in the number of mononuclear cells in the first concentration of the powder than in the second concentration, for both sexes.
This increase is also due to the presence of polysaccharides in Ganoderma that can boost the immune system in rodents by increasing the production of WBC and help remove the effect of toxic substances and oxidizing agents in the body (Bao et al., 2002).
Nk cells can kill a tumor cell and are therefore considered promising tools for treating cancer (Cho and Campana, 2009) and uncontrolled proliferation is a feature of tumors. Research indicates that the polysaccharides in Ganoderma mushroom are not only a normal product that has effective functions in the immune system, but act as an adjuvant therapy that inhibits the proliferation of cell tumor (Cho and Campana, 2009). The increase in the numbers of white blood cells and lymphocytes in rats exposed to Ccl4 is symptomatic. Table (3) shows the statistical analysis of the results obtained, as significant differences appeared between each of the cytokine and complement, as well as between lymphocyte count and white blood cell count at the level of ≥0.01, and significant differences appeared at the level of ≥0.05, There were no clear significant differences between the white blood cell count and the mononcyte count, this indicates that the Canoderma powder does not affect these parameters