Mangosteen Extract Reduces Apoptosis via Inhibiting Oxidative Process

in Rat Model of Traumatic Brain injury

Aldy S. Rambea*, Suzy Indhartyb, Michael Lumintangb, Wibi Riawanc

a
Department of Neurology, Faculty of Medicine, University of Sumatera Utara/Haji Adam Malik General
Hospital, Medan 20155, Indonesia
b
Department of Neurosurgery, Faculty of Medicine, University of Sumatera Utara/Haji Adam Malik General
Hospital, Medan 20155, Indonesia
c
Department of Biochemistry, Faculty of Medicine, University of Brawijaya, Malang 65145, Indonesia

Correspondence

Aldy S. Rambe

Email aldysr02@yahoo.com
Abstract

Backround. Traumatic brain injury is one of primary causes of mortality, morbidity, and

economic burden, especially in young population. Following initial primary injury, there is

secondary insult, resulting in neuroinflammatory response and free radical generation.

Oxidative stress is a powerful precursor of apoptosis. Mangosteen is a powerful natural

antioxidant that also has neuroprotective property.

Method. Thirty Sprague dawley rats were randomized into three treatments group, i.e sham-

operated controls, closed head injury (CHI), and CHI with mangosteen extract (treatment

group) In treatment group, we gave mangosteen extract once daily every day after CHI for 7

days. As oxidative process marker, we investigated MDA expression. For apoptosis marker,

we investigated AIF, caspase 8, and caspase 9.

Result. MDA, AIF, caspase 8, and caspase 9 were up-regulated in CHI group compared to

sham-operated group (p<0.05). MDA, AIF, caspase 8, and caspase 9 were down-regulated

significantly in treatment group compared to sham-operated group and CHI group (p<0.05).

Conclusion. Mangosteen extract decreased neuronal apoptosis in traumatic brain injury by

down-regulated MDA, AIF, caspase 8, and caspase 9.

Introduction

Traumatic brain injury (TBI) is a primary cause of mortality, morbidity, and economic

burden.1 Head trauma causes two kinds of damage in the brain tissue. One is the primary

injury, which refers to the initial physical forces applied to the brain at the moment of impact.

The other is secondary injury, which occurs over hours or days after the initial trauma,

resulting in a neuroinflammatory response and free radical generation.2

Reactive oxygen species (ROS) are generated after TBI, and they are involved in the

secondary brain injury.3–6 Under normal conditions, ROS are maintained by the action of pro-

oxidant and antioxidant in cerebral tissue, and they are continuously formed in small amounts

as products of oxidative metabolism. TBI results in an increase of ROS and leads to lipid

peroxidation, among other effects. Malondialdehyde (MDA) is an end-product formed during

this lipid peroxidation, because of degeneration of cellular membrane phospholipids.7,8 MDA

is released into extracellular space and finally into the blood; it has been used as an effective

biomarker of lipid oxidation.7,8

Oxidative stress is a powerful precursor of apoptosis.9 Following oxidative stress, the

mitochondria release apoptotic proteins such as cytochrome c and apoptosis inducible factor

(AIF) into the cytosol.10 The release of cytochrome c (cyt c) and other pro-apoptotic proteins

can induce caspase activation and apoptosis. Once freed into the cytosol from the

mitochondrial intermembrane space, cyt c binds with apoptotic protein-activating factor-1

(Apaf-1) and procaspase-9 to form an “apoptosome”, which actived caspase-9 and caspase-3

respectively. Activated caspase-3 cause a wide range of homeostatic disturbance, reparative

and cytoskeletal proteins disruption and leads to neuron cell death.11

α-Mangostin, the first xanthone extracted from mangosteen fruit,12 is a yellow coloured

matter that can be optained from bark and dried sap of Garcinia mangostana, a tropical fruit

from the family of Guttiferae. G. Mangostana is cultivated in tropical rainforest of Indonesia,

Malaysia, Sri Lanka, The Philippines and Thailand. For many years, people in these countries

have used the pericarp (peel, rind, hull or ripe) of G. Mangostana as a traditional herbs for

indigestion, infected wounds, and chronic wound.13-15

Study showed that alpha-mangostin can inhibit the oxidation of low-density lipoprotein

(LDL) in vitro16 and scavenge singlet oxygen, superoxide anion and peroxynitrite anion. 17

This antioxidant as a free radical scavenger ameliorates the neuronal death induced by 3-

nitropropionic acid (3-NP).18 Treatment of alpha-mangostin provided a neuroprotective

effect on cerebellar granule neurons (CGNs) which was associated with the induction of

heme oxygenase-1 (HO-1).19

In considering the variety of antioxidant and protective properties described for α mangostin,

in this study we evaluated whether this agent may decrease the expression of MDA and

apoptosis.

Material and Method

Mangosteen extract (ME) preparation

Mangosteen were collected from Malang regency in East Java, Indonesia. The fruit bodies of

mangosteen were cleaned to remove any residual compost. The pericarp were separated and

then dried. All dried pericarp was placed in 70% ethanol (100 gr in 500 cc), shake with speed

50 rpm in 24 hours. The macerate filtrate was evaporated using rotary evaporator in 2 hours.

From 100 gr dried mangosteen pericarp, 50 cc ME was obtained.

Mouse model of closed head injury

Unilateral focal brain injury was induced on the right frontal area using rat model of closed

head injury (CHI), performed with the modified Feeney’s weight-drop protocol.20 This study

was approved by local ethical committe. Thirty-three Sprague dawley rats weighing 250-400

gr were randomized into three treatments group, i.e sham-operated controls, CHI only

(Positive control group), and CHI with mangosteen extract (treatment group). All animals

were given ketamine HCl (Intramuscular dosage 100 mg/kg) and xylazine base’

concentration 20mg/ml (Intramuscular dosage 0.15 ml/kg). The scalp was cleaned with

povidone iodine and aseptic techniques were used throughout surgery. The scalp was opened

on the right frontal. Then, the rats were placed securely in stereotactic apparatus. We gave 40

mg metal mass from 1.5 m height (figure 1A). Mangosteen extract (100mg/Kg per oral) was

given once daily until seventh day via nasogastric tube. Afterward, animals were sacrificed

through cervical dislocation after giving ketamine HCl (100 mg/kg, intramuscular). In

positive control group as well as treatment group, subjects were divided into three groups

based on sacrificial timing, i.e. first, third, and seventh day respectively. The brains were

removed (figure 1B) and fixed in 10% buffered formalin. The specimens were then processed

for paraffin-embedded for immunohistochemistry staining preparation. Sham operated rat

underwent anesthesia and surgery, without trauma and treatment.

Immunohistochemistry staining

As oxidative process marker, we investigated MDA expression. For apoptosis marker, we

investigated AIF, caspase 8, and caspase 9. The expression of all markers was investigated

on paraffin-embedded sections using the avidin-biotin-peroxidase complex method. Five-

millimeter-thick paraffin sections were dewaxed, rehydrated, and microwave for 10 minutes.

The endogenous peroxidase activity of the investigated specimens was blocked with 3
percent H2O2 for 10 minutes, followed by 25 minutes washing with phosphate-buffered saline

(PBS). The tissue sections were incubated with normal rabbit serum for 10 minutes, and then

the slides were incubated at room temperature with monoclonal mouse MDA, SOD, AIF,

caspase 8, and caspase 9 (Santa Cruz). Sections were washed with PBS and incubated with a

secondary antibody for 30 minutes. Sections were washed twice with PBS, developed with

0.05% 3,3 diamino-benzinetetrahydrochloride for five minutes, and slightly counterstained.

Apoptotic cells were detected by TUNEL (terminal transferase-mediated dUTP nick end

labeling) staining in (para)formaldehyde-fixed paraffin-embedded (FFPE) using the In Situ

Cell Death Detection Kit from Roche’s protocols.

All samples were evaluated by first author (not blinded to specimen). Positive signal for

MDA, SOD, AIF, caspase 8, and caspase 9 and the number of apoptosis cells in brain tissue

was quantitatively estimated on the basis of distribution of positive stained cells in cortical

brain. Cell counts were carried out using light binocular microscope with 1000 times

magnification in 20 high power fields.

Statistical Analysis

The total stained cells were reported in mean and standard deviation. When comparisons

were made between groups, significance in between-group variability was analyzed using the

one way Anova test with Tukey as post hoc test. Differences were considered significant at

the P <0.05.
RESULT

Thirty-three rats were included in this research, divided into three groups, i.e sham-operated

controls, CHI, and CHI + mangosteen extract were given once daily. During the follow up,

three rats died directly after trauma procedure. The brain was removed after craniocervical

dislocation under anesthesia, using ketamine HCl and xylazine base (Figure 2).

α-mangostin inhibit the MDA expression

In our study, MDA expression was devided in 3 groups, which is positive control group,

negative control group and treatment group. We found that the lowest MDA expression is

found in treatment group with the significant correlation of MDA expression within 3 groups

(Table 1, fig. 3, p= 0.0001).

In the positive control group, MDA expression is shown to decrease in day 3 and day 7

compare to the first day with the value of of 8,67 ± 1.53 in day 7 compare to 13.67 ± 1.53 in

day 1. It is shown to be a significant correlation within MDA groups in positive control group

(Table 2, fig. 3, p= 0.041).

In the treatment group, the lowest expression of MDA is shown in the third day and a slight

increase is shown in day 7 (5 ± 1.58 and 5.2 ± 1.92 respectively). We noted that there are no

correlation in MDA expression in treatment group (Table 3, fig. 3).

α-mangostin inhibit the caspase 8, caspase 9 and AIF expression

α-mangostin was shown to have significant correlation in the expression of apoptotic

pathway such as caspase 8, caspase 9 and AIF expression in treatment group compare to

positive and negative control group (p= 0.0001 in all apoptotic pathway) in which was shown

that the expression was lowest in the treatment group (Table 1, fig. 4-6).

The p value of all apoptotic pathway in positive control group was below 0.05, which means

that there is a significant correlation of inhibition of apoptotic pathway by α-mangostin

(Table 2, fig 4-6).

In the treatment group, the apoptotic pathway expression is shown to be the lowest in day 7.

It is also supported by the p value of 0.031, 0.008 and 0.005 in caspase 8, caspase 9 and AIF

respectively (Table 3, fig. 4-6).

α-mangostin lowers the apoptosis profile

Apoptosis profile is shown to have a significant correlation in the 3 groups (p value of

0.0001). This means that α-mangostin lowers the apoptosis profile in treatment group

compare to control group (Table 1, fig 7).

In positive control group, apoptosis profile tends to be higher in day 7 compare to first day

(18.67 ± 1.15 vs 10.33 ± 1.53). there is a significant correlation of apoptosis profile in the

positive control group p value= 0.002 (Table 2, fig 7).

In treatment group, apoptosis profile is shown to decrease in day 7 with the value of 6 ± 2.45

compare to first day (9.4 ± 1.14). There is also a significant correlation in the treatment group

with p value of 0.0034 (Table 3, fig 7).

DISCUSSION

Since TBI is a multi-pathway process, medicinal plants have been evaluated as resource of

agents for alternative treatments. Medicinal plants usually have multiple compounds and

works via multipathways. Mangosteen has been used as traditional medicine for years. Many

reports have been published regarding the pharmacological activities of  and  mangostin,

the active substance in mangosteen pericarp.21 It has strong effect in reducing the expression

of inflammatory mediators, such as tumor necrosis factor  (TNF ) and interleukin 6 (IL-

6).22,23 Xanthone from mangosteen, especially  mangostin, inhibits the NMDA and

glutamate receptors.24

In this study, we investigated the effect of mangosteen extract on expression of NMDA as the

marker of lipid peroxidation and apoptosis. We observed through immunohistochemistry that

mangosteen extract inhibits oxidative process and apoptosis after traumatic brain injury.

Based on these data, we investigated the molecular mechanism of mangosteen extract on the

suppression of apoptosis. The result showed that mangosteen extract reduced the expression

of caspase 8, caspase 9, and apoptosis inducing factor. This result was interesting, since in

many research in oncology,  mangostin induces apoptosis. It proposed that  mangostin

triggered the loss of mitochondrial membrane permeability and activation of caspase

family.25,26
Caspases are a family of genes important for maintaining homeostasis through regulating cell

death and inflammation. Caspases involved in apoptosis have been subclassified by their

mechanism of action and are either initiator caspases (caspase-8 and -9) or executioner

caspases (caspase-3, -6, and -7).27 Pathologically, insults such as trauma, at the appropriate

severity that occur below the threshold required for necrosis, may result in cellular apoptosis.

Apoptosis may also assume an important role in the pathogenesis of many illnesses. “Too

little” apoptosis may cause uncontrolled cell growth, as found in cancer. Conversely, “too

much” apoptosis may contribute to several neurodegenerative processes, like Alzheimer’s

and Huntington’s disease.28 In TBI, apoptosis is one of long term consequences.29 Inhibiting

apoptosis may be one alternative modality in traumatic brain injury.

In summary, we had demonstrated the down regulation of oxidative stress and apoptosis in

traumatic brain injury after administration of mangosteen extract. To the best of our

knowledge, this is the first study that demonstrate the effect of mangosteen extract in

apoptotic pathway after traumatic brain injury. The main limitation of this study was the

nature of injury. We only demostrated focal injury in this research. Meanwhile, diffuse injury

is commonly found after traumatic brain injury and correlates significantly with secondary

injury.

Inhibiting apoptosis after traumatic brain injury remains controversial because apoptosis is

vital mechanism for biological system to eliminate abnormal cell. Therefore, the study about

effect of antiapoptosis should not be limited to the expression of molecular status only, but

also to clinical in functional outcome.

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TABLES
Table 1. Profile of apoptosis and apoptotic pathway (Mean±SD)

MDA Caspase 8 Caspase 9 AIF Apoptosis

Group 1 5.78 ± 1.99 5.67 ± 1.87 4.33 ± 2.12 6.56 ± 1.81 5.67 ± 1.66
Group 2 11.67 ± 2.83 15 ± 2.74 12.67 ± 2.12 12.67 ± 2.12 14.67 ± 3.87
Group 3 6 ± 2.27 6 ± 3.32 6.27 ± 3.51 5.87 ± 3.14 7.67 ± 2.16
p 0.0001* 0.0001* 0.0001* 0.0001* 0.0001*
*
Significant if p<0.05. Group 1: negative control, group 2: CHI, group 3: treatment. Post-hoc
MDA: 1 vs 2 p= 0.0001, 1 vs 3 p= 0.0001; caspase 8: 1 vs 2 p= 0.0001, 1 vs 3 p= 0.0001;
caspase 9: 1 vs 2 p= 0.0001, 1 vs 3 p= 0.0001; AIF: 1 vs 2 p= 0.0001, 1 vs 3 p= 0.0001;
apoptosis: 1 vs 2 p= 0.0001, 1 vs 3 p= 0.0001. CHI: closed head injury; MDA:
malondialdehyde; AIF: apoptosis inducing factor.

Table2. Temporal profile of apoptosis and apoptotic pathway in CHI group (Mean ± SD)
MDA Caspase 8 Caspase 9 AIF Apoptosis
Day 1 13.67 ± 1.53 17 ± 1 13.67 ± 1.53 13.33 ± 2.08 10.33 ± 1.53
Day 3 12.67 ± 2.52 16 ± 2 14 ± 1 13 ± 1 15 ± 2
Day 7 8.67 ± 1.53 12 ± 2 18.33 ± 1.53 18.67 ± 2.08 18.67 ± 1.15
p 0.041* 0.027* 0.031* 0.014* 0.002*
*
Significant if p<0.05. Post-hoc MDA: 1 vs 7 p= 0.043; Caspase 8: 1 vs 7 p= 0.028; caspase
9: 3 vs 7 p= 0.039; AIF: 1 vs 7 p= 0.025, 3 vs 7 p= 0.019; apoptosis: 1 vs 7 p= 0.002, 1 vs 3
p= 0.027. CHI: closed head injury; MDA: malondialdehyde; AIF: apoptosis inducing factor.

Table 3. Temporal profile of apoptosis and apoptotic pathway in treatment group (Mean ±
SD)
MDA Caspase 8 Caspase 9 AIF Apoptosis
Group 3 Day 1 7.8 ± 2.39 9 ± 3.54 9.4 ± 3.85 8.4 ± 2.89 9.4 ± 1.14
Day 3 5 ± 1.58 4.6 ± 2.7 6.2 ± 1.64 6.4 ± 2.07 14.7.6 ±
1.52
Day 7 5.2 ± 1.92 4.4 ± 1.34 3.2 ± 1.3 2.8 ± 1.3 6 ± 2.45
p 0.083 0.031* 0.008* 0.005* 0.0034*
*
Significant if P<0.05. Post-hoc caspase 8: 1 vs 7 p= 0.046; caspase 9: 1 vs 7 p= 0.006; AIF:
1 vs 7 p= 0.004; Apoptosis 1 vs 7 p= 0.027. MDA: malondialdehyde; AIF: apoptosis
inducing factor.
FIGURES

Figure 1. Animal model. A. Modified Feeney’s weight-drop model after CHI ; B. cranial
defect after CHI.

Figure 2. Mice brain after removal. A. Sham-operated group ; B. CHI group ; C. Treatment
group
Figure 3. MDA expression in sham control, CHI, and treatment group.

Figure 4. Caspase 8 expression in sham control, CHI, and treatment group.

Figure 5. Caspase 9 expression in sham control, CHI, and treatment group.

Figure 6. AIF expression in sham control, CHI, and treatment group.

Fig 7. Apoptosis in sham control, CHI, and treatment group