The Lingzhi Mushroom (Ganoderma lucidum) Extract Reduce the

Number of Lymphocyte in Diabetics Rats with Periodontitis: In Vivo

Experimental on Sprague dawley Rats

Pratitis Widi Seno

, Amilia Ramadhani

, Saryono

, Haris Budi Widodo

and Christiana C. Prihastuti

School of Dentistry, Medical Faculty of Jenderal Soedirman University, Purwokerto, Indonesia

Departement of Biomedicine, School of Dentistry, Medical Faculty of Jenderal Soedirman University, Purwokerto,

Indonesia

Departement of Biochemistry, Nursing of Jenderal Soedirman University, Purwokerto, Indonesia

Departement of Oral Biology, School of Dentistry, Medical Faculty of Jenderal Soedirman University, Purwokerto,

Indonesia

Keywords: Garnoderma lucidum, lymphocyte, periodontitis, diabetes mellitus.

Abstract: Periodontitis is a chronic inflammation on the periodontal tissue that is characterized by attachment loss and

bone loss. Diabetes mellitus deteriorated the condition of periodontitis. Ganoderma lucidum has the potential

as anti-inflammation and anti-oxidant agents that can hasten the convalescence of periodontitis disease. This

research aims to understand the effect of oral administration of Ganoderma lucidum extract on the number of

lymphocytes in Sprague dawley rats induced with diabetes mellitus and periodontitis. The results showed that

the estimated number of lymphocytes in groups that given the extract decreased as the doses increased. The

group with 20 mg/kgBW dose has the lowest lymphocyte count and approached the healthy control group.

The results of the Kruskal-Wallis test showed that there are significant differences among the five treatment

groups for the estimation of lymphocyte counts (p <0.05). The Mann-Whitney test showed a significant

difference between groups in estimating lymphocyte counts (p <0.05). This research concludes that there is

an effect of G. lucidum extract to the amount of lymphocyte in Sprague dawley rats designed with diabetes

mellitus and periodontitis.

1 INTRODUCTION

Diabetes mellitus is a chronic disease that disrupts the

body's metabolism so the body can’t regulate blood

glucose levels. Diabetes mellitus has several

complications in the oral cavity, such as periodontitis,

dry mouth, gingivitis, calculus, and alveolar bone

resorption. Periodontitis occurs in patients with

diabetes mellitus, with a prevalence of 75%.

According to research conducted by Sari et al. (2017),

88.24% of people with diabetes in Sardjito Regional

Hospital suffer from periodontitis.

https://orcid.org/0000- 0002-9957-501X

https://orcid.org/0000- 0001-8902-5164

https://orcid.org/0000- 0002-3012-5328

https://orcid.org/0000- 0003-2154-902X

https://orcid.org/0000- 0002-0611-7651

Periodontitis is a chronic inflammatory disease

caused by specific microorganism or groups of

specific microorganisms. This infection was resulting

in progressive destruction of the periodontal ligament

and alveolar bone with increased probing depth

formation, recession, or both (Newman et al, 2015).

Lymphocytes, plasma cells and macrophages mediate

the body's response during chronic inflammation

(Stryer et al., 2012). Lymphocytes are specific

immune defences to remove antigens, release

antibodies, and provide cytokines for other immune

cells (Kumar et al., 2015).

254

Seno, P., Ramadhani, A., Saryono, ., Widodo, H. and Prihastuti, C.

The Lingzhi Mushroom (Ganoderma lucidum) Extract Reduce the Number of Lymphocyte in Diabetics Rats with Periodontitis: In Vivo Experimental on Sprague dawley Rats.

DOI: 10.5220/0010490902540259

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 254-259

ISBN: 978-989-758-499-2

The hyperglycemia condition in diabetes mellitus

can worsen the periodontal disease. High blood

glucose level induces high secretion of pro-

inflammatory cytokines and decreases the activity of

chemotaxis, phagocytosis, and intracellular activity

of lymphocyte. This alteration of the immune system

stimulates delayed wound healing (Daniel et al.,

2012; Mealey et al., 2006)

Diabetes mellitus and periodontitis have a two-

way relationship. Diabetes mellitus produces

Advanced Glycation End Products (AGEs), which

can increase local expression of pro-inflammatory

cytokines and enhanced the destruction of periodontal

tissue (Preshaw et al. 2012). Otherwise, high levels of

pro-inflammatory cytokines dysregulated blood

glycemic levels and also increased risk of diabetic

nephropathy, retinopathy, and heart disease. Diabetes

mellitus and periodontitis conditions will increase the

expression of IL-6. The high level of IL-6 will also

increase the number of lymphocytes in the gingival

tissue and worsen the periodontal inflammation (Wu

et al., 2015).

Scaling, root planning, curettage of gingiva, and

Host Modulating Therapy (HMT) were the golden

standard of periodontitis therapy (Andriani 2012;

Notoharjo & Sihombing 2015). Non-steroid anti-

inflammation medicines such as ibuprofen and

flurbiprofen can be used as host modulating therapies.

In the contrary, the long-term use of anti-COX2 may

lead to stomach ulcers and haemorrhage (Newman et

al., 2015). The Ganoderma lucidum (G. lucidum) has

a potent anti-inflammation effect and has a minimum

long-term side effect (Li et al., 2011).

Ganoderma lucidum (Lingzhi mushroom or

Reishi mushroom) contains triterpenoid, ganoderic

acid, polysaccharide, protein, and unsaturated fat

(Huang et al. 2018). These active substances have a

potential effect of antioxidant, anti-inflammation, and

anti-hyperglycemic (Ma et al. 2015; Ratnaningtyas et

al., 2018; Huang et al., 2018). The previous study

confirmed that ganoderic acid could inhibit the

production of COX-2, and prevent the inflammation

process (Ratnaningtyas et al., 2018).

Huang et al (2018) has proved that the provision

of 10 mg/kg BB doses of G. lucidum extract in 7 days

can decrease the amount of polymorphonuclear cells

(PMN) and in 10 days, it can lead the bone forming.

Polymorphonuclear cells are innate immune system

of non specific body defense system while a specific

immune system takes part in periodontitis (Berglundh

& Donati 2005). One of the cells that takes part in the

specific immune system is lymphocyte (Newman et

al., 2015). The aim of this study was to examine the

effect of oral administration of G. lucidum extract at

a dose of 5 mg/kg BW, 10 mg/kg BW, and 20 mg/kg

BW for 7 days on the total number of lymphocytes in

Sprague dawley rats induced with diabetes mellitus

and periodontitis.

2 MATERIAL AND METHOD

This research was an experimental laboratory with

posttest-only control group design. The study was

conducted for 3 months, started from March to May

2019. This research used 30 rats with inclusion

criteria of male Sprague dawley rats weighing about

200-300 grams, age of 2-3 months, and induced with

periodontitis and diabetes mellitus with blood glucose

levels >126 mg/dL. The rats were divided into 5

groups which include healthy control group (K1),

negative control group (K2), 5 mg/kg BW dose group

(P1), 10 mg/kg BW dose group (P2) and 20 mg/kg

BW dose group (P3).

This study was approved by the Ethics

Commission of Faculty of Medicine, Jenderal

Soedirman University under Ref: 1394 / KEPK / III /

2019. The rats were adapted and placed in a room

with sufficient air flow and light for one week. The

extract was produced by dissolving 300 grams of G.

lucidum mushroom simplicia using 96% ethanol in a

ratio of 1: 5, then it was soaked for 3x24 hours. The

mushroom extract filtrate was evaporated using a

rotary evaporator until a thick extract was obtained.

The extract was then dissolved with the dose of 5 mg

/ kg BW, 10 mg/kg B, and 20 mg / kgBB.

All the K2, P1, P2, P3 group samples were

induced with diabetes mellitus by injecting

streptozotocin (STZ) intraperitoneally at a dose of 40

mg / kg in 0.1 M citrate buffer pH 4.5 (Furman et al.,

2015; Zulkarnain et al., 2013). Measurement of blood

glucose was carried out on the third day after STZ

induction (day 10). The diabetics rats were rats with

glucose blood level >126 mg/dL (Su et al., 2006).

On day 11, K2, P1, P2, and P3 groups were

induced with periodontitis by inoculating P.

gingivalis bacteria into the labial maxillary incisors at

a dose of 1 McFarland according to the standard

bacterial test which is equivalent to a density of 108

i.e. 200 microliters or 0.2 mL everyday within 4 days.

On day 15, clinical examination showed that the rats’

gingiva were reddish and suppurating. One rat of each

groups were euthanized to examine the alveolar bone

resorption radiographically (Figure 1). The oral

administration of G. lucidum extracts and distilled

water were given every 24 hours for 7 consecutive

days using gastric tube. On day 25, the rats’ blood

samples were taken from the tail to measure post-

The Lingzhi Mushroom (Ganoderma lucidum) Extract Reduce the Number of Lymphocyte in Diabetics Rats with Periodontitis: In Vivo

Experimental on Sprague dawley Rats

255

treatment blood sugar levels. The rats were

euthanized using ether and the anterior maxillary

gingival tissues were carefully extracted and stored in

10% formalin solution.

Figure 1: Radiograph image of rats alveolar bone (A)

healthy rats ; (B) periodontitis rats; Yellow arrow showed

bone resorption.

The dehydration, clearing, and embedding phases

were performed in a tissue processor (Tissue Tex®,

USA). The embedding process was carried out by the

embedding center tool (Leica®, Germany). Pre-

dehydration and post-clearing images of gingival

tissue were documented for the calculation of the

volume shrinkage. A grid composed of regularly

spaced array of points made up using Image J®

program was placed over the gingival tissue pre-

dehydration and post-clearing images. The distance

between points were 1.5 mm. The number of points

falling on the images of gingival tissues were counted

and used to calculate the volume shrinkage using the

following formula (Nyengaard, 1999):

(1)

Paraffin blocks were sectioned at nominal

thickness of 5µm using a Systematic Uniform

Random Sampling (SURS) technique (Altunkaynak

et al., 2012; Tschanz et al., 2014). A number between

1 and 20 was randomly chosen and this number

pointed to the number of the section to be sampled

together with its adjacent section. The following 18

sections were discarded and the subsequent pairs of

section were sampled. This procedure was continued

until the whole gingival tissue was exhaustively

sectioned. The average number of pairs of section was

6 pairs. All sample sections were mounted on to glass

slides and stained with Hematoxylin-eosin.

The gingival tissue volume was estimated using

the Cavalieri principle. Images of the gingival tissue

of one section from each pair of section of each rat

were viewed under Olympus CX21FS1 (Olympus

Singapore PTE, LTD) light microscope at 40X

magnification and captured using Optilab CX-21

camera (PT Minocos, Indonesia). These images were

combined in order to make a complete picture of

gingival tissue using Adobe photoshop® CS6

software (Adobe System Incorporated, United

States). The complete image was then viewed using

ImageJ

software (NIH Image; National Institutes of

Health, Bethesda, MD) and superimposed with

regularly spaced array of test points at a distance of

3.4 mm between points. The areas represented by

each point (a/p) were 11.56 mm

. All points (P) which

hit the gingival tissue were counted. The volume of

gingival tissue was estimated using the following

formula (Pulungan et al., 2018) :

(2)

where “T” is the distance between sections (mm),

“a/p” is the area per point (mm

), and ∑P is the

total number of points.

The numerical densities of gingival tissue’s

lymphocytes were determined using a physical

dissector probe. The gingival tissue were viewed and

captured at 400x magnification. The counting frame

of 60 x 60 mm

was used for counting the profile of

gingival tissue’s lymphocytes (Figure 2). The

numerical density (Nv) of the lymphocytes was

calculated using the following formula (Pulungan et

al., 2018).

(3)

where “ΣQ-”is the sum of nucleoli profiles; “a” is the

area of counting frames (μm

); “h” is the height of the

disectors which is equal to the section thickness (5

μm); and “ΣP” is the total number of counting frames.

The total number of gingival tissue’s lymphocytes

were estimated by multiplying the volume with the

numerical density.

Figure 2 : Example of paires of adjacent section; (A) Look

up section; (B) Reference section. Dashed line of counting

frame represent inclusion line, while full drawn line

represent exclusion line. Yellow arrow showed counted

lymphocyte.

The differences of total lymphocytes’ number of

in all groups were analyzed using Kruskal-Wallis test

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

256

with confidence level of 95% (p<0,05), since the data

were not normally distributed. The Mann Whitney

test were carry out with a confidence level of 95% (p

<0.05) to observe the differences of total

lymphocytes’ number between groups.

3 RESULT

The table 1 showed the rats’ blood glucose level pre-

treatment and post-treatment. The Levene test

showed that the hyperglycemic state of each rat was

different at the beginning of the study (p<0.05).

Tabel 1: Blood glucose level (mg/dL) ± SD

No. Group Pre treatment Post treatment

1. P1 214,2±43,2 240,2±52,9

2. P2 220,8±73,0 188,0±50,9

3. P3 439,4±97,6 352±76,9

4. K1 79,8±24,0 -

5. K2 363,2±151,76 312,75±107,2

P1: Groups of treatment with a dose of 5 mg/KgBB, P2:

Groups of treatment with a dose of 10 mg/KgBB, P3:

Groups of treatment with a dose of 20 mg/KgBB), K1:

Healthy control, K2: Negative control

The total number of lymphocytes in each groups were

shown in table 2. The total number of lymphocytes in

the treatment group decreased as the doses of G.

lucidum extract increased.

Oral administration of 20

mg/KgBW G.lucidum extract decreased the total

number lymphocytes related to healthy control.

Table 2: Total number of lymphocytes each groups

No. Groups Mean of estimated

hoc

te ± SD

1. P1 992,64 ± 262,43 0,04

2. P2 848,70± 358,06

3. P3 322,85± 226,73

4. K1 325,74 ± 57,8

5. K2 1.006,49 ± 488,08

P1: Groups of treatment with a dose of 5 mg/kgBW, P2:

Groups of treatment with a dose of 10 mg/kgBW, P3:

Groups of treatment with a dose of 20 mg/kgBW), K1:

Healthy control, K2: Negative control; p: Result of

Kruskal-Wallis test

The Kruskal-Wallis test results showed that there

were significant differences of the total number of

lymphocytes between all groups (p <0.05). Mann-

Whitney Test results show that there is no significant

differences of total number of lymphocytes between

the 5 mg / kgBW dose group (P1), 10 mg / kgBW

dose group (P2), and the negative control group (K2).

Otherwise, the total number of lymphocytes of the 20

mg / kgBB dose group (P3) was significant difference

to the P1, P2, and K2 groups. This result showed that

the total number of lymphocytes in group P3 similar

to the healthy control group, since there were no

significant difference with the healthy control group.

4 DISCUSSION

Inflammatory responses of periodontitis in

conjunction with DM were different than

periodontitis alone. DM condition accompanied by

periodontitis reduces the function of neutrophils,

monocytes, and macrophages in chemotaxis and

phagocytosis (Mealey, 2006). According to Otton et

al. (2002), the DM conditions affect lymphocyte

metabolism. Glucose and glutamine metabolism of

lymphocytes were altered so it requires more glucose

but cannot oxidize their metabolites efficiently and

caused decreased function of lymphocyte in immune

system.

Hyperglycemic conditions caused some

disruption in healing process such as a longer

inflammatory phase. Mirza et al. (2014) stated that in

the condition of diabetes mellitus, the inflammatory

phase will reach the peak on the 5th day and start to

decrease on the 10th day. Meanwhile, in the non-

diabetes mellitus condition, the proliferation phase

has begun on the 6th day. Changes in the period of

inflammation will affect the healing process. In

hyperglycemic conditions, reparative cells in the

periodontium and fibroblast cannot work optimally so

that the newly formed-collagen fibers were easily

destroyed by MMP (Mealey, 2006). The DM

condition modify the microvascular integrity, that is

the formation of AGEs (Mirza et al., 2014).

Inflammatory cells have AGEs receptor on the cell

surface. When AGEs bound to inflammatory cells

such as macrophages, monocytes, and lymphocytes,

the production of proinflammatory cytokines will

increase and resulted in the hyper-responsiveness of

the immune system (Mealey, 2006).

The oral administration of 20 mg/kgBW G.

lucidum extract showed a significant decrease of total

number of lymphocytes compared to negative control

group. Dudhgaonkar et al. (2009) argued that

ganoderic acid and polysaccharides from G. lucidum

extract decreased the proinflammatory cytokines

such as TNF-α, IL-6, and PGE2 from LPS induced

macrophages. As in vivo study showed that G.

The Lingzhi Mushroom (Ganoderma lucidum) Extract Reduce the Number of Lymphocyte in Diabetics Rats with Periodontitis: In Vivo

Experimental on Sprague dawley Rats

257

lucidum extract act as anti-inflammatory and

antiproliferative agents via inhibition of the NF-κB

and AP-1 signaling cycles in macrophages. Izzaty et

al., (2014) study showed that hyperglycemic

conditions cause a longer inflammatory phase,

regarding to high number of lymphocytes observed

on the 7th day of healing process. Our recent study

showed that oral administration of G. lucidum extract

for 7 consecutive days reduce the number of

lymphocytes, so that the healing process accelerated.

Triterpenoids as antioxidants play a role to bind

reactive ROS compounds such as superoxide anions

(O2-), so that the inflammatory response will

decrease (Chen et al., 2016). The decrease in ROS

will result in acceleration of proliferation phase by

decreasing the production of IL-7, so the migration of

leukocyte cells such as neutrophils and macrophages

will decrease (Pushparani, 2015; Sari et al., 2017).

Polysaccharides optimize the macrophages,

lymphocytes, and plasma cells to modulate the

inflammatory process (Ratnaningtyas et al., 2018).

Polysaccharide extract of G. lucidum has a

hypoglycemic effect by reducing glucose levels in

plasma in which it enhanced the activity and

metabolism of lymphocytes. The treatment groups of

the 5 mg/kgBW and 10 mg/kgBW G. lucidum extract

statistically showed no significant difference with the

negative control group. This shows that both doses

have not reached the maximum effect in reducing the

number of lymphocytes.

The oral administration of 20 mg/kg BW G.

lucidum extract was the most optimum dose to reduce

the number of lymphocytes in rats with periodontitis

and diabetes mellitus condition. This result different

from Huang et al. (2018) study that showed decreased

number of neutrophils of periodontitis rats on day 7

after administration of 10 mg/kgBW G. lucidum

extract. The persistent inflammatory response of

neutrophils triggered lymphocytes to form a chronic

inflammatory response. In this study, the

hyperglycemic condition is responsible for the

increase in the severity of periodontitis and the

delayed of healing response.

The oral administration of 5 mg/kgBW, 10

mg/kgBW and 20mg/kgBW G. lucidum extract did

not affect the hyperglycemic conditions of the rats.

The post treatment glucose levels were decreased, but

still remained in hyperglycemic conditions. Previous

study confirmed antidiabetic effect of G. lucidum

extract in higher dose. The administration of 50

mg/kg BW and 100 mg/kgBW G. lucidum extract

prove to provide antidiabetic effects as measured by

blood glucose levels and insulin levels of rats

(Sirisidthi et al., 2016; Zhang & Lin, 2004).

This recent study showed that the oral

administration of 20 mg/kgBW G. lucidum extract

reduced the total number of gingival tissues’

lymphocytes pronounced to the healthy control

group. As the decreased number of lymphocytes

indicate the reduced in periodontal inflammation

process (Taubman & Kawai, 2001). The higher dose

of G. lucidum extract will comprised the higher

concentration of triterpenoids and polysaccharides

and escalated the anti-inflammatory effect.

5 CONCLUSION

In conclusion, the G. lucidum extract intensify the

inflammatory phase of healing process of

periodontitis with diabetes mellitus. The total number

of lymphocytes were decline resembling the healthy

groups after oral administration of 20 mg/kg BW G.

lucidum extract. Further study is required to examine

the potential of G. lucidum extract to the next phases

of healing process of periodontitis with diabetes

mellitus.

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The Lingzhi Mushroom (Ganoderma lucidum) Extract Reduce the Number of Lymphocyte in Diabetics Rats with Periodontitis: In Vivo

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