Immunomodulating effect of Polysaccharide Krestin from Cariolus versicolor grown in Indonesia against Rheumatoid arthritis in Rat

 

Diah Purwaningsari^1,3, Jusak Nugraha³, Sri Puji Astuti Wahyuningsih², Suhailah Hayaza², Raden Joko Kuncoroningrat Susilo², Hunsa Punnapayak^2,4, Ruey-An Doong^2,5, Win Darmanto²*

¹Faculty of Medicine, University of Hang Tuah, Surabaya, Indonesia.

²Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya 60115, Indonesia.

³Department of Medicine, Faculty of Medicine, Universitas Airlangga, Surabaya 60115, Indonesia.

⁴Plant Biomass Utilization Research Unit, Department of Botany, Faculty of Science,

Chulalongkorn University, Bangkok 10330, Thailand.

⁵Institute of Analytical and Environmental Sciences, National Tsing Hua University,

Sec. 2 Kuang Fu Road, Hsinchu 30013, Taiwan

 

ABSTRACT:

Rheumatoid arthritis (RA) is a chronic inflammatory disease that can lead to joint destruction, deformity, and loss of function. The aim of this study is to investigate the therapeutic effect of polysaccharides krestin from Cariolus versicolor grown in Indonesia against complete Freund's adjuvant arthritis rat. Sixty-six male Wistar white rats (Rattus norvegicus) were induced by adjuvant arthritis. Samples were divided into 6 groups, 3 control (KP1, KP2, KP3) and 3 treatment groups (KP4, KP5, KP6). There were 3 different times of data collection: 1 week, 2 weeks, and 3 weeks after booster administration. Intragastric injection of polysaccharides krestin was given at a dose of 50 mg/kg body weight per day in all treatment groups. Flowcytometry blood tests were performed to examine the TGF-β1 level and T regulator percentage. The joint scale was observed using Elisa and histopathological analysis. The analysis of various arthritic assessment parameters used in this study revealed that polysaccharides krestin from Cariolus versicolor has a considerable effect in preventing the inflammation and severe damage in adjuvant arthritis rat’s joint. The correlation between polysaccharides krestin-TGF-β1, levels-CD4⁺CD25⁺FoxP3⁺Treg cells expression, and histopathological joint scale during the second weeks showed a strong correlation (p ≤0.05). These findings suggest that the administration of PSK from C. versicolor appears to be promising as immunomodulator and medication in chronic arthritis, therefore preclinical and clinical studies in human are highly recommended.

 

 

 

INTRODUCTION:

Rheumatoid arthritis (RA) is one of the most common chronic autoimmune inflammatory disease, which can cause persistent synovitis, systemic inflammation, and autoantibodies¹. It is a common cause of disability, which usually appears early on the onset of this disease. The overall prevalence of clinically diagnosed RA was 0.5–2% of the population with a higher prevalence in developed countries². Rheumatoid arthritis patients have a 50% greater risk of heart attack and have a two times higher risk of heart failure^3,4.

 

This fact shows that RA disease, although the incidence is small, but needs to get considerable attention.

 

One of the factors causing RA disease is the imbalance of Th17 (as a proinflammatory factor) and T-regulator (as an anti-inflammatory factor), along with Th1 and Th2. Transforming Growth Factor-β1 (TGF-β1) acts as an anti-inflammatory cytokine in inducing the formation of CD4⁺CD25⁺FoxP3⁺ T-regulator cells in rats and humans. It can suppress T-cells effector in autoimmune diseases via direct or indirect pathways.

 

Rheumatoid arthritis patient needs lifelong treatment. Therefore, more patients prefer using natural product remedies. Coriolus versicolor is a common medicinal mushroom used by locals in Asian countries. It is known as Yunzhi in Chinese and belongs to the subclasses of Homobasidiomycetes and the family of Polyporaceae. Polysaccharides have long been recognized as the anticancer agent with low toxicity and slight of side effects⁵. Both polysaccharides krestin (PSK) and polysaccharides peptide (PSP) from C. versicolor are active substances that could modify immune responses. Polysaccharides peptide has demonstrated immunomodulatory effect while combined with cyclosporin on treating human lymphocyte homeostasis⁶. Pramudya and Wahyuningsih have reported that crude polysaccharides from C. versicolor has the ability to modulate the immune response in mice infected with Neisseria gonorrhoeae⁷. Powdered polysaccharide krestin of C. versicolor contains 91-93% active compound β-glucan in its carbohydrate. It has also been previously reported that the presence of β-glucan component is able to induce B-cell activation and enhance cytokine production⁸.

 

However, data regarding the effect of PSK as immunomodulators on RA disease is still lacking. Therefore, the present study was undertaken to examine the therapeutic effect of C. versicolor PSK in adjuvant arthritis (AA) rat models, including TGF-β1 levels, CD4⁺CD25⁺FoxP3⁺Treg cells, and joint scale of AA rat, as to see the improvement the inflammatory conditions.

 

MATERIAL AND METHODS:

Ethical approval:

All procedures involving animal care in a laboratory were based on the letter of ethics no. 541-KE from Animal Care and Use Committee of Veterinary Faculty, Universitas Airlangga, Surabaya, Indonesia.

 

Experimental animal:

Sixty-six male Wistar white rats (Rattus norvegicus), aged 16 weeks, weighing 200-250g, were obtained from Faculty of Pharmacy, Universitas Airlangga (Surabaya, Indonesia). The animals were maintained in cages made of plastic at 20^oC, with 12-hour light/12-hour dark cycle, fed and watered by ad libitum.

 

Experimental design:

The research method used was an experimental laboratory using a randomized post-test only group design. The chemicals used in this study were from Sigma-Aldrich (USA) and Merck (Germany). Sixty-six rats were divided into 6 groups: 3 treatment groups and 3 control groups. Arthritis was induced by injecting 0,1ml Complete Freund’s Adjuvant (P Code 1001821390, Sigma-Aldrich, St. Louis, MO, USA) intradermally in rats’ tail and injecting 0.05ml CFA, as a booster, in both rat’s feet two weeks after the first injection². The boosting injection of CFA was considered as day-0. The study used six groups divided into the arthritis control group for 1 week (K1), the arthritis control group for 2 weeks (K2), the arthritis control group for 3 weeks (K3), the PSK treatment group at the dose rate 50mg/kg BW for 1 week (K4), the PSK treatment group with dose rat of 50mg/kg BW for 2 weeks (K5), the PSK treatment group with dose rate of 50mg/kg BW for 3 weeks (K6). Flowcytometry blood tests were performed to examine the levels of TGF-β1 and T regulator. Elisa was performed to observe joint tissue retrieval for histopathology and immunohistochemistry in order to measure the joint scale.

 

Preparation of PSK from C. versicolor:

Coriolus versicolor was collected from Surabaya, East Java, Indonesia. The taxonomic identification of the C. versicolor was carried out by Dr. Ni’matuzahroh from the Department of Biology, Faculty of Science and Technology, Airlangga University, Surabaya, Indonesia. Polysaccharides krestin was prepared in the laboratory with a method based on Cui & Christy⁹. A crude powder of 200grams C. vesicolor was added with 3L of distilled water and heated at a temperature of 80 - 98⁰C for 2-3 hours. Then, it was separated between the undissolved part (pulp) and the dissolved part. The pulp was added with 2L of distilled water and heated at a temperature of 80 - 98⁰C for 2-3 hours. Furthermore, the dissolved part was filtered with Whatman paper to obtain the extract solution and dried by lyophilization. The dry mushroom extract was precipitated with 90% ammonium sulfate (65.7g ammonium sulfate + 100ml distilled water + 2g dried mushroom extract). The extract was then dissolved in 30mL phosphate-buffered saline and dialyzed through nitrocellulose membrane for 24 h in PBS at 4^oC. The aqueous solution was freeze-dried and crude PSK of C. vesicolor was obtained.

 

Sample preparation:

Rats were anesthetized and sacrificed. Surgery was performed in the thoracic region. Blood was collected from the ascending aorta using a 3cc syringe, with the maximum amount possible, divided into 2 tubes. One tube was mixed with EDTA for flow cytometry examination, while the other one was without EDTA for ELISA examination. After collecting the blood, surgery on the ankle joint (tibio-tarsal) was carried out. The joint was immediately put into fixative solutions and labeled. Samples were later used for a histopathology examination.

 

Measurement of TGF-β1 using ELISA:

Blood cells were added in the 96 well plates coated with antibody. Then, 100μL of Biotin-Conjugated anti-rat TGF beta 1 antibody (Abcam Kit ab119558) was added to each well, incubated at room temperature for 1 hour. Cells were then being washed and added 100μL of Streptavidin-HRP Conjugate to all wells, incubated for 30 min at room temperature. Then, the plate was washed and added 100μL of TMB Substrate Solution to each well, incubated for 30 minutes. Lastly, 100μL of stop solution was added to each well. The absorbance was read using ELISA reader at 450nm wavelength.

 

Measurement of CD4⁺ CD25⁺ FoxP3⁺ T-regulator cells with flowcytometry

Blood cells were added in the 96 well plates and washed the cells once by resuspending the pellet in 200μl Facs buffer. Cells were centrifuged (1600 rpm, 5 min, 4°C), the supernatant was discarded very carefully. 2x10⁵ cells were resuspended in 25μl Facs buffer containing 10% mouse serum and incubated for 5 min (4°C). Then, the antibodies were added and incubated for 20 min. The antibodies used were monoclonal antibody of FITC Anti-Rat CD4, PE Anti-Rat CD25, and Alexa Fluor 488 Anti-Rat FoxP3 from Biolegend. Lastly, the cells were fixed by adding 50μl Fixation buffer per well and transferring the samples to the Nunc Micronic tubes containing 150μl Facs buffer.

 

Histopathology scale examination of rat’s joint:

Samples were observed under a light microscope with 200x magnification. Histological samples had been softened by decalcification and stained with Hematoxylin and Eosin. Histopathology of the joints was analyzed using scoring from modification of several references. Joint histology features were assessed with the following histopathological scale: 1=There are no abnormalities in the joint (normal); 2=normal synovium with 1-2 inflammatory cells per field of view, the cartilage surface is not slippery; 3=Two or more synovial cell layers with infiltration of 3-5 inflammatory cells per field of view, cartilage surface has mild erosion; 4=Synovium hyperplasia with infiltration of more than 5 inflammatory cells per field of view, the cartilage surface is experiencing moderate erosion; 5 = Synovitis, pannus formation and severe erosion of cartilage and subchondral bone erosion begins^10,11.

 

Statistical analysis:

The normality test of all variables was analyzed using the Saphiro-Wilk test, with P=0.05. To find out whether there were significant differences from variables, TGF-β levels, the number of CD4⁺CD25⁺FoxP3⁺Treg cells, and Diameter of rats’ feet, a statistical test of Multivariate Analysis of Variance (MANOVA) was done, followed by the test of between-subjects’ effects, p=0.05. All of these analytic tests were performed using SPSS Statistic 18 Software (IBM Corporation, USA).

 

RESULT:

Level of TGF- β1

Data of TGF-β1 levels from all groups are shown in Table 1. The Shapiro-Wilk normality test showed p-value > 0.05, which means that the data was normally distributed. ANOVA test results showed p-value <0.001, which means that there were at least significant differences in TGF-β1 levels in 2 groups. Multiple comparison LSD showed a significant difference between KP1 and KP4 (p = 0.17). A KP2 and KP5 were also significantly different as well as KP3 and KP6. This shows that the administration of PSK from C. vesicolor was able to increase TGF-β1 levels in the blood of AA' models compared to the control group. The result also shows that the longer the administration of PSK, the more increase TGF-β1 levels in rat blood.

 

Table 1. Data of TGF-β1 levels from all groups. Polysaccharides krestin was given at dose rate 50 mg/kg BW. Same superscript letter indicates no significant difference (p-value ≤0.05).

Group	n	TGF-b1 (pg/ml)		P
		Mean	Std. Dev.
KP1	11	847,409a	101,585	<0,001*
KP2	11	912,345ab	62,857
KP3	11	962,136b	98,497
KP4	11	982,818b	142,341
KP5	11	1150,236c	160,908
KP6	11	1314,682d	176,270

*p-value=0,05. Same superscript letter indicates no significant difference.

 

Percentage of CD4⁺CD25⁺FoxP3⁺Treg cells:

The results of the comparative test showed that there was a significant difference between K1 and K4 (p= 0.008, see Table 2). There was also a significant difference between KP2 and KP5, as well as KP3 and KP6. This shows that the administration of PSK could increase the number of CD4⁺CD25⁺FoxP3⁺Treg cells in the blood of AA models compared to the control group that was sacrificed at the same time.

 

Data analysis of the number of CD4⁺CD25⁺FoxP3⁺Treg cells between treatment groups showed that KP4 and KP5 have a significant difference. KP 5 and KP6 also has a significant difference, as well as KP4 and KP6. This shows that the longer the administration of PSK, the more increase CD4⁺CD25⁺FoxP3⁺Treg cells in rat blood.

 

Table 2. Data of CD4+CD25+FoxP3+Treg cells from all groups. Polysaccharides krestin was given at dose rate 50 mg/kg BW. Same superscript letter indicates no significant difference (p-value ≤0.05.)

Group	n	% T-reg cells		P
		Mean	Std. Dev.
KP1	11	6,64a	1,03	<0,001*
KP2	11	7,09a	1,30
KP3	11	8,91b	2,51
KP4	11	9,18b	3,19
KP5	11	14,27c	4,15
KP6	11	21.73d	9.54

*p-value=0,05. Same superscript letter indicates no significant difference.

 

Joint histopathology scale analysis:

There were significant differences between the treatment group and the control group that was sacrificed at the same time (Table 3). But, among the treatment groups, there was no significant difference between KP4 and KP5. On the other hand, KP5 and KP6 showed significant differences, as well as KP4 and KP6. This result shows that giving PSK for 1, 2, and 3 weeks have caused a decrease in joint scale (Fig.1). The picture of joint tissue in Fig.2 shows thickening in all control groups (KP1, KP2, KP3) due to an inflammatory cell (yellow arrow). The cartilage surface of all control groups also experienced heavy erosion (red arrow). On the other hand, treatment groups (KP4, KP5, KP6) shows a minimum inflammation. The cartilage surface also looks smooth (no erosion).

 

Table 3. Data of joint histopathology scale from all groups. Polysaccharides krestin was given at dose rate 50 mg/kg BW. Same superscript letter indicates no significant difference (p-value ≤0.05).

Group	n	Histopathology Scale		P
		Median	Interquartile Deviation
KP1	11	4,00a	0,50	<0,001*
KP2	11	4,00a	0,00
KP3	11	4,00a	0,50
KP4	11	2,00b	0,00
KP5	11	2,00b	0,00
KP6	11	1,00c	0,50

*p-value=0,05. Same superscript letter indicates no significant difference.

 

 

Figure 1: Joint histopathology scale of each groups. Control group was given 1 week, 2 week, and 3 weeks of water. Treatment group consists of 1 week, 2 weeks, and 3 weeks of Polysaccharides krestin administration at dose rate 50 mg/kg BW.

 

 

Figure 2: Histopathology of foot joints of RA rat with HE staining, 200x magnification. Yellow arrow indicate cell inflammatory; red arrow shows the cartilage surface; blue arrow indicated cartilage cell inflammatory. KP1, KP2, KP3 are control group for 1, 2, and 3 weeks respectively. KP4, KP5, KP6 are treatment groups given PSK (50 mg/kg BW) for 1, 2, and 3 weeks respectively.

DISCUSSION:

Transforming growth factor-β (TGF-β) is a homodimeric protein with pleiotropic action in embryonic development, immune response, inflammation, and amelioration. Excessive TGF-β1 signaling is associated with pathological scarring and fibrotic disease. TGF-β activation and signaling have many regulatory points that alter their functional immunoregulatory effects¹².

 

TGF-β is a cytokine with pleiotropic function in hematopoiesis, angiogenesis, cell proliferation, differentiation, migration, and apoptosis. Although its role in rheumatoid arthritis is not well defined, TGF-β activation leads to functional immunomodulatory effects according to environmental conditions. TGF-β function in the development of arthritis in murine models has been extensively studied with controversial results. Studies of TGF-β in T-cell responses have shown results in its role as inflammatory inhibitors¹². In our study, there was an increase in TGF-β1 levels in experimental animals after the administration of PSK, contained an active β-Glucan. This product can balance the immune system affected by arthritis.

 

In this study, the administration of PSK which contained the active ingredient of β-Glucan has increased the TGF-β1 levels in experimental animals. This material was balancing the immune system affected by arthritis. The statistical test results of TGF-β1 level indicate that there were significant differences between the treatment groups and the control group (Table 1). Without the administration of PSK, TGF-β1 levels did not perform much improvement within 3 weeks. On the other hand, all three treatment groups showed significant differences to one another. This result proved that the duration of therapy was directly proportional to the increased level of TGF-β1 in rats.

 

Regulatory T cells (Treg cells) are cells that mediate suppression, play a role in the mechanisms of immune-mediated inflammation, including in autoimmune and autoinflammatory disorders, allergies, acute and chronic infections, cancer and metabolic inflammation¹³. Treg cells maintain immune balance by preventing autoimmune, reducing inflammation and minimizing tissue damage. Different subsets of Treg cells can have different functions or roles in maintaining immune homeostasis and therefore, it can be targeted differently in autoimmune or cancerous diseases^14,15.

 

This shows that the provision of PSK material can increase the number of CD4⁺CD25⁺FoxP3⁺Treg cells in rats compared to the control group. The longer the administration of PSK, the more increase of T-reg cells in mice blood. Moreover, Treg cell number also has a strong correlation with TGF-β1 level (p <0.001) in all treatment groups. The higher the TGF-β1 levels, the more the Treg cells number. The presence of CD4⁺CD25⁺FoxP3⁺Treg cells will suppress the function of T-effector cells and inhibit further inflammation. The number of Treg cell increased during arthritis, especially in spleen and lymph node, which indicated selective tropism to the disease site. The inhibition of inflammation was also proved by the histopathological analysis of rat's joint in all treatment groups that shows sign of tissue amelioration (Fig 2).

 

β-Glucan is a biologically active polysaccharide group that has proven pleiotropic (dual) immunomodulating effects¹⁶. It could restore the imbalances among different T-lymphocyte subpopulations. Immunomodulatory activities of polysaccharides may be due to direct or indirect interaction with immune system components¹⁷. The component of crude polysaccharides in G. lucidum was able to decreased the levels of ALT, AST, and MDA and increased the levels of SOD and CAT in CCl4-treated mice¹⁸. Study by Wang et al., explain that other types of polysaccharide from Ephedra sinica could treat arthritis and inhibit cytokine expression, although its molecular mechanism is not yet completely clear, assuming the role of ESP-B4 which decreases cytokine production arising from stimulation LPS, then suspected ESP-B4 reduces the stimulation of LPS cell core translocation-from p65 NF-kB subunits¹⁹. Lee et al. proved that PSP from C. versicolor showed cyclosporin-like activity in controlling aberrant lymphocyte activation⁶. In the present study, the decreasing tendency of cellular infiltration during the initial phase of arthritis supports the assumption that HA provides a temporary protective barrier on the cartilage and thus protects the bone against CFA.

 

CONCLUSION:

In summary, the provision of PSK as immunomodulator at dose 50 mg/ kg body weight/ day orally potentially improved the rheumatoid arthritis in the rat (Rattus norvegicus) by increasing TGF-β1 levels, CD4⁺CD25⁺FoxP3⁺T-regulatory cells, and ameliorating joint scale. These conditions were mediated by activation of the glycan from PSK.

 

ACKNOWLEDGEMENT:

This work was financially supported by Mandat Grant of Institute of Innovation and Research in Universitas Airlangga and Ministry of Research, Technology, and Higher Education of the Republic of Indonesia.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 07.02.2020           Modified on 16.04.2020

Accepted on 10.05.2020         © RJPT All right reserved