Type of Manuscript:

Research

PEG-4000 ameliorates morphine-induced constipation in mice through

inhibition of AQP-3 mRNA expression

Mahardian Rahmadi1*, Zuhaela Iqbal2, Ikbar Nanda Pratama1, Rifky Anindita Karunia1, Arina
Derry Puspitasari1, Khoirotin Nisak1, Aniek Setiya Budiatin1
1
Department of Pharmacy Practice, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60115, Indonesia
2
Post-graduate student, Master of Pharmacy, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60115,
Indonesia

*Corresponding Author:
Mahardian Rahmadi
Department of Pharmacy Practice, Faculty of Pharmacy, Universitas Airlangga, Surabaya,
Indonesia
Nanizar Zaman Joenoes Building, Mulyorejo, Surabaya, East Java 60115, Indonesia.
Email: mahardianr@ff.unair.ac.id

1
ABSTRACT:

Morphine is μ-opioid receptor (MOR) agonist that is used clinically for patients suffering from moderate to
severe pain. Morphine can cause constipation due to activation of μ-opioid receptors in the central nervous
system and the nervous system in the gastrointestinal tract. It is caused by increasing the expression of AQP-3 in
the instestine through increased of serotonin by enterochromaffin cells. PEG 4000 is one of the osmotic
laxatives used to treat Opioid Induced Constipation (OIC). PEG 4000 affects the upregulation of serotonin re-
uptake which can lead to expression enhancement of AQP-3. The purpose of this study was to analyze the effect
of PEG 4000 on expression changes of aquaporin-3 in mice colon induced acute constipation with
morphine.Constipation conditions and the effectiveness of laxative therapy are indicated by constipation
parameters in the form of fecal water content and stool weight. This study used 36 male mice of the Balb/c line
genus which were divided into 3 groups, normal saline, morphine, and morphine + PEG 4000. Each group was
further divided into 2 subgroups based on the time of observation, namely the first hour and fifth hour after
morphine induced. The expression of AQP-3 was observed using Polymerase Chain Reaction (PCR) method.
The result of this study showed that the administration of PEG 4000 to mice induced constipation with morfine
could decrease the expression of AQP-3 from (146,413 ± 1,736) to (118,411 ± 3,476) with p value <0,05,
increase the percentage of fecal water content from (0,000 ± 0,000 %) to (28,903 ± 12,930 %) with p value
<0,05 and increase the stool weight from (0,000 ± 0,000 g) to (0,928 ± 0,178 g) with p value <0,01 From these
results, it can be concluded that the administration of PEG 4000 in morphine induced constipation to mice was
effective in reducing AQP-3 expression as well as increasing the percentage of fecal water content and stool
weight.

KEYWORDS: Aquaporin-3, PEG 4000, Constipation, Morphine.

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INTRODUCTION :

Constipation is a condition in which a person experiences bowel movements within a week or defecates with
hard, dry and small stools that are painful or difficult to pass. Constipation can be active which means time and
lasts a short time, which lasts a long time, even years 1. Constipation can arise as a result of pathophysiological
interactions that have lifestyle factors and factors from drugs. Common factors or disorders that can cause
dietary constipation include low fiber, lack of physical activity, use of drugs, complaints to defecate, nervous
and metabolic disorders, and problems and disorders of the gastrointestinal tract1.

Opioid analgesics constitute one of the most important drug groups of pharmacotherapy agents in clinical
practice settings. These drugs play a very important role in the management of pain therapy, especially in
moderate to severe cancer and visceral pain in various health care settings. The key to optimal pain management
is the ability to treat the patient effectively to ensure optimal pain relief, while also creating or managing side
effects resulting from the patient's pain medication3.

Morphine is the most important opioid analgesic drug in pain management. Morphine is derived from the
extraction of an alkaloid plant, namely the wild opium poppy, Papaver somniferum 3. Morphine is a -opioid
receptor (MOR) agonist that is used clinically for patients with moderate to severe pain 4. Morphine has become
the “gold standard” in pain therapy because of its strong analgesic effect and its availability in various dosage
forms. The analgesic effect of morphine occurs through -opioid receptors in the central nervous system.
However, the opioid receptors themselves can be expressed through both the central and peripheral nervous
systems5.

Although morphine and MOR agonists exhibit strong analgesic effects, they can cause side effects such as
constipation, nausea and drowsiness, often making it difficult for the user to take them continuously. Among
these side effects, opioid-induced constipation is estimated to occur in approximately 40-64% of patients given
opioids for pain management6. Constipation is the most serious effect in clinical practice. The prevalence of
cancer patients with morphine as therapeutic management and experiencing side effects of constipation is
around 23% to more than 90%, which has an impact on the quality of life of cancer patients with opioid
therapy7. From the results of a survey study of 322 patients in the United States and Europe who were treated
with opioid analgesics orally, it showed that as many as 81% of patients experienced constipation. Patients
receiving morphine may experience side effects of constipation via opioid receptors in the peripheral nerves.
Morphine-induced constipation may develop at analgesic doses or less than analgesic doses. Therefore, almost
every patient taking morphine is constipated5.

To date, many pharmacological studies have been conducted on the mechanism of constipation induced by
MOR agonists. MOR is thought to exert a direct effect on inhibition of gastrointestinal transit 9. MOR expression
has been detected in many central and peripheral tissues. In addition, MOR is known to be highly expressed in
the gastrointestinal tract in the myenteric plexus and to a lesser extent in the submucosal plexus 10. The myenteric
plexus is responsible for controlling the contraction and relaxation of intestinal motility, while the submucosal
plexus is responsible for osmotically releasing enzymes and hormones in the intestinal lumen. Opioids acting on
MOR in the central nervous system in gut neurons can indirectly cause constipation by altering gastrointestinal
motility9.

Constipation caused by administration of opioids (such as: morphine) can be called Opioid Induced
Constipation (OIC). Opioid Induced Constipation (OIC) is systemically caused by activation of -opioid
receptors in the gastrointestinal tract and central nervous system. This causes a decrease in contraction of the
intestinal muscles so that the transit time of feces in the intestine becomes longer. The long transit time of feces
in the intestine is caused by the absorption of water in the digestive tract which has increased so that the water
content in the feces becomes less. Induction of morphine can also trigger the release of serotonin 5-HT which is
secreted by enterochromaffin cells in the colon. Serotonin 5-HT is secreted, will undergo reuptake by SERT into
the intracellular and activate PPARγ. PPARγ activation can cause an increase in AQP3 expression, which can
cause a constipating effect 10. The intestinal tract is a major source of endogenous serotonin (5-HT), which has an
important role in the regulation of motility and fluid and electrolyte transport. The availability of serotonin 5-HT
is regulated by the serotonin transporter (SERT) 11. The serotonin transporter SERT is localized to the apical
membrane of intestinal epithelial cells and has a role for serotonin 5-HT re-uptake. Increased levels of high 5-

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HT can cause gastrointestinal motility disorders that can trigger constipation. A study showed that the process of
increasing serotonin 5-HT re-uptake is also influenced by Epidermal Growth Factor (EGF) while also increasing
SERT transcription in human intestinal epithelial cells. EGF can influence this by binding to the EFG receptor
(EGFR) on human intestinal epithelial cells12.

The use of morphine with laxatives can be used to treat constipation experienced by patients. According to the
World Health Organization in Palliative Care: Symptom Management and End of Life Care, there are two types
of therapy to prevent or treat constipation, namely pharmacological therapy and non-pharmacological therapy.
Pharmacological therapy can be given a laxative in the form of bisacodyl 5-15 mg once a day one tablet at night
or senna 7.5 mg with an initial administration of two tablets twice a day every four hours depending on the
response. As for non-pharmacological therapy, it can be done by drinking water frequently, eating fruits,
vegetables and foods that have a high fiber content 13. Laxatives are divided into 5 classifications based on the
mechanism of providing laxative effects, namely bulk laxatives, stool softeners, osmotic laxatives, stimulant
laxatives, and prokinetic agents14. For now, laxative types such as stimulant laxatives and osmotic laxatives have
been used empirically as symptomatic therapy in patients with constipation due to the use of morphine 5.
However, the use of stimulant laxatives can have side effects of stomach cramps, so its use should be avoided in
patients who use opioid pain therapy regularly15.

One example of an osmotic laxative is Polyethylene glycol (PEG). PEG is a water-soluble biological polymer
that is minimally absorbed from the gastrointestinal tract. PEG acts as an osmotic laxative interacting with water
molecules by forming hydrogen bonds in a ratio of 100 water molecules per one PEG molecule leading to an
increase in the water content of the colon. In many countries PEG is the first choice of laxative therapy for the
treatment of constipation16. PEG 4000 is one type of PEG. PEG 4000 is a non-toxic, water-soluble, high
molecular weight polymer, which is not absorbed from the gastrointestinal tract. PEG 4000 acts as an osmotic
agent by increasing the water content of feces. In various studies, PEG 4000 has been shown to be effective in
the treatment of constipation in adults and children 17. The use of PEG is also recommended as an effective
choice of laxative because it can increase cost effectiveness compared to other prescribed laxatives such as
lactulose18. Based on clinical trials, the use of PEG 4000 compared to PEG 3350 in general has the same
effectiveness but differs in terms of comfort, namely taste. PEG 4000 is more widely accepted by patients
because it tastes better than PEG 3350. Another clinical trial comparing the effectiveness of using PEG 4000
with lactulose, PEG 4000 has an effectiveness of 97.6% compared to lactulose in relieving constipation
conditions19.

The effectiveness of PEG's osmotic activity depends on its ability to absorb water into the intestinal lumen 18. In
a study showed that PEG can reduce EGFR in the colon of mice 20. Where EGFR is a receptor that affects EGF
which plays a role in the regulation of serotonin 5-HT re-uptake in intestinal epithelial cells. Increased levels of
EGF can trigger an increase in serotonin 5-HT re-uptake in intestinal epithelial cells, so that it can increase
AQP3 expression which can cause constipation12,14. The osmotic effect works by decreasing the transport of
water from the luminal to the vascular side due to a decrease in AQP3 expression in colonic mucosal epithelial
cells21. Aquaporin (AQP) is a membrane water transport channel in the human body and plays an important role
in the regulation of water homeostasis. There are 13 types of AQP in humans, namely AQP0 to AQP12 which
are expressed in various tissues. The types of AQP that are most commonly expressed in the intestinal tract are
AQP1, AQP2, AQP3, AQP4, and AQP 821. The expression of AQP3 and AQP4 is dominant in both human and
mouse colonic mucosal epithelial cells22.

Administration of morphine can cause constipation by increasing AQP3 expression in rats 6. However, in another
study on the induction of opioid agonists, it caused a decrease in AQP3 expression in mice which were then
administered with naringenin laxative and resulted in an increase in AQP39 expression. These differences in
results resulted in the need for research related to AQP expression. The research showed that there was an
increase in AQP expression in the first hour after morphine induction and the highest changes in AQP3
expression occurred at five hours after morphine induction. Several studies have also suggested that AQP could
be one of the new treatment targets for the treatment of constipation23.

Administration of morphine can cause constipation indicated by low water content in feces. The water content in
feces decreased gradually as AQP3 expression increased and the lowest level was observed at five minutes after
morphine induction12.

Based on this background, a study is needed to determine the effect of PEG 4000 osmotic laxative on changes in
the AQP3 transporter in morphine-induced acute constipation with parameters of stool weight and fecal water
content in the colon of mice.

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MATERIALS AND METHODS:

Materials

The materials used for the research were PEG 4000, morphine hydrochloride, aqua, normal saline, distilled
water, mouse food in the form of complete feed for beef cattle Fat A Pellet produced by PT. Japfa Comfeed
Indonesia Tbk, drinking water, shaved ice, Paclimeda® 6 mg/mL (Oncotec, Pharma Production GmbH Am
Pharmapark, 06861 DessauRoβlau, Germany), PureLink™ RNA Mini Kit (Life Technologies™, USA), GoTaq
Master Mixes (Clever Scientific Ltd), GoScript™ Reverse Transcription System (Promega), QuantiFluor®
RNA Sampel Kit (Promega), Agarose LE, Analytical Grade (Promega), TAE Buffer, 10X, Molecular Biology
Grade (Promega), etanol 99%, UltraPure™ Distilled Water DNAse, RNAse free (Life Technologies™, USA),
RNAse Away® Reagent (Life Technologies™, Mexico), Aqua bidest, Sodium Chloride 0,9% (PT Widatra
Bhakti, Pandaan, Pasuruan, East Java), and primary sequence24.

Laxative Dosage Calculation

In a previous study, the dose of PEG given to experimental mice was 1.5 g/kg BW, 3 g/kg BW, and 6 g/kg BW
then dissolved in 10 mL/kg. Based on experiments conducted by Anggraeni (2016), the effective dose of PEG in
experimental animals is 3 g/kg BW25.

Morphine Injection and Normal Saline

The experimental mice that desired constipation were the positive group and the morphine-PEG 4000 group.
Morphine was injected intraperitoneally at 10 mg/kg BW. While the negative control group was only injected
with normal saline 10 L/g BW intraperitoneally. Morphine and normal saline injections were performed 30
minutes after administration of PEG 4000 or aqua26.

Measurement of Weight and Water Content of Stool

The observed parameter was the water content of the mice's faeces which was accumulated according to the
specified time. Stool levels are calculated in the following20:

Measurement of Aquaporin-3 (AQP3) mRNA Expression

a) Sample preparation

Mice from all groups that had been sacrificing, each mouse was taken from the colon and stored in liquid
nitrogen and then transferred to a freezer at -80°C. After the colon was obtained, total RNA was extracted using
an RNA mini kit22. The RT-PCR used was PCR Electrophoresis (MSMIDI Duo, USA). Complementary DNA
(cDNA) was synthesized 1 g of total RNA at 42°C for 30 minutes. Internal control of RT-PCR using -actin. The
primary sequence of AQP-3 and -actin is as follows23:

mRNA Sequence (5’-3’) PCR Product (bp)

AQP-3 F 5′-GGGCTGTACTACGATGCAATC-3′
420
AQP-3 R 5′-ACACGAAGACACCAGCGATGG-3′
β-actin F 5’-TGTTACCAACTGGGACGACA-3’
573
β-actin R 5’-AAGGAAGGCTGGAAAAGAGC-3’

b) Colonic Tissue Retrieval

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The experimental mice were sacrifice and then sampling was carried out on the colon. The sample that has been
obtained is put into a 1.5 ml tube, then frozen with liquid nitrogen at a temperature of -20°C, which is then put
into a freezer at a temperature of -80°C21.

c) Purification of RNA from Colonic Tissue

1) Lysis and homogenization
1. Add 300 L of Lysis Buffer to the tube (on ice).
2. Smooth the tissue using an RNase-free pestle in an up/down motion and rotate it in the tube until
the tissue is completely crushed.
3. The process was carried out at room temperature: then the lysate was transferred to an RNase-free
tube and centrifuged at 12000 x g for 2 minutes18.
2) Binding, washing and elution
1. Add one volume (equivalent to the previous addition of Lysis Buffer) 70% ethanol on the
homogenized tissue
2. Mix everything by shaking or vortexing to mix any precipitates that may form after the addition of
ethanol.
3. Transfer the 700 L sample into the Spin Cartridge.
4. Centrifuge at 12000 x g for 15 seconds at room temperature. Discard any passing liquid, and
reinstall the Spin Cartridge in the same Collecting Tube.
5. Add 700 L Wash Buffer I to the Spin Cartridge. Centrifuge at 12000 x g for 15 seconds at room
temperature. Discard any passing liquid along with the Collecting Tube. Place the Spin Cartridge in
the new Collecting Tube.
6. Add 500 L Wash Buffer II which has been added with ethanol to the Spin Cartridge.
7. Centrifuge with a rotation of 12000 x g for 15 seconds at room temperature. Discard any passing
liquid, and reinstall the Spin Cartridge in the same Collecting Tube.
8. Repeat steps 6-7 once.
9. Centrifuge Spin Cartridge with a rotation of 12000 x g for 1 minute at room temperature to dry the
membrane (which has captured the RNA). Discard the Collecting Tube and install the Spin
Cartridge in the Recovery Tube.
10. Add 100 L of RNase-Free Water to the center of the Spin Cartridge.
11. Incubate at room temperature for 1 minute.
12. Centrifuge for 2 minutes at 12000 x g at room temperature.
13. Continued analysis of RNA yield and quality23.

14. Measurement of sample concentration (Promega)

1. Prepare 1X TAE Buffer from stock solution of 20X TAE Buffer.
2. Prepare working solution (high standard calibration): dissolve QuantiFluor® RNA Dye 1:400 into
1X TAE Buffer to make QuantiFluor® RNA Dye working solution (high) by adding 9 L
QuantiFluor® RNA Dye into 3591 L 1X TAE Buffer . Protect the working solution from light
using foil or place in a dark place.
3. Prepare a blank sample: add 200 L of QuantiFluor® RNA Dye working solution to an empty 0.5
mL PCR tube. Protect the tube from light.
4. Prepare RNA standard sample: prepare 500 mg standard by adding 5 L of supplied RNA standard
(100 ng/μL) to 200 L QuantiFluor® RNA Dye working solution into 0.5 mL empty PCR tube. Mix,
and protect the tube from light.
5. Prepare the unknown sample: add 5 L of the unknown sample to 200 L of QuantiFluor® RNA Dye
working solution into an empty 0.5 mL PCR tube. Vortex, and protect the tube from light.
6. Incubate the prepared sample at room temperature for 5 minutes, protected from light.
7. Select the RNA protocol on the QuantusTM Fluorometer. Based on the standard calibration that
has been prepared, select high or low.
8. If needed, calibrate the QuantusTM Fluorometer by reading the blank sample (prepared in the third
stage) and standard sample (prepared in the fourth stage) on the calibration screen, then select save.
9. Enter the unknown sample volume (5 L) and the desired concentration unit (ng/μL).

6
 10. Measure fluorescence in unknown samples using the QuantusTM Fluorometer. The number that
appears shows18.

d) First Strand cDNA synthesis

1. Campur dan sentrifuge dengan singkat setiap komponen sebelum digunakan. Gabungkan komponen
dengan kompoisis sebagai berikut:
2. Tutup rapat masing-masing tube RNA. Inkubasi tube pada 70 °C selama 5 menit. Spin down setiap tube
selama 10 detik pada microcentrifuge untuk mengumpulkan kondensat dan memperoleh volume utuh.
Pastikan tube dalam keadaan tertutup dan berada dalam kondisi dingin hingga Reverse Transcription
reaction mix ditambahkan.
3. Siapkan Reverse Transcription reaction mix dengan menggabungkan komponen GoScriptTM Reverse

Transcription System sebagai berikut di dalam tube steril dalam kondisi dingin:

Component 1x reaction 8x reaction

Nuclease-Free Water 6,5 µL 52 µL
GoScript™ 5X Reaction Buffer 4 µL 32 µL
MgCl2 2 µL 16 µL
PCR Nucleotide Mix 1 µL 8 µL
Recombinant RNasin®
0,5 µL 4 µL
Ribonuclease Inhibitor
GoScript™ Reverse Transcriptase 1 µL 8 µL
120 µL
Volume akhir 15 L
4. Tambahkan 15 μL aliquots pada Reverse Transcription reaction mix ke tiap tube dalam kondisi dingin
ke dalam masing-masing 5 μL campuran RNA primer untuk memperoleh volume akhir reaksi sebanyak
20 μL tiap tube.
5. Anneal: inkubasi pada suhu 25°C selama 5 menit.
6. Extend: inkubasi pada suhu 42°C selama 60 menit.
7. Simpan sampel hasil reaksi pada suhu 4°C jika tidak langsung dilanjutkan amplifikasi sampel 10.
e) Amplifikasi Sampel (Cleaver Scientific)
1. Untuk setiap 50 μL reaksi, campur komponen berikut dalam 0,2 mL PCR tu be di atas es.
Komponen amplifikasi sampel sebagi berikut:

Component Volume
GoTaq Master Mixes 12,5 µL
Forward primer, 5-10 μM 2 µL
Reverse primer, 5-10 μM 2 µL
DNA template 2 µL
RNAse Free Water 6,5 µL
2. Campur dengan hati-hati, jika perlu centrifuge singkat, tutup tube dan tempatkan pada thermal cycler.
3. Proses dalam thermal cycler selama 40 siklus
4. Setelah reaksi PCR, dilanjutkan elektroforesis DNA untuk mendeteksi produk PCR8.

f) Elektroforesis Produk PCR

1. Membuat agarosa konsentrasi 1,7% dengan mencampurkan 1,53g agarosa serbuk ke dalam 90 mL 1X
TAE Buffer. Aduk campuran menggunakan magnetik stirer dengan kecepatan 700-1000 rpm di atas hot
plate dengan suhu 200 °C. Pastikan semua serbuk agarosa larut. Tunggu hingga gel mengeras dan siap
untuk digunakan.
2. Siapkan gel agarosa konsentrasi 1,7% ke dalam electrophoresis chamber, lalu isi chamber dengan 1X
TAE Buffer sampai menutupi permukaan gel.
3. Masukkan Ladder 3 μL ke dalam well pertama.

7
 4. Dilanjutkan 1 μL 6X Loading Dye dan 5 μL sampel yang sudah dicampur ke dalam well, diulang
hingga semua sampel masuk ke dalam well.
5. Elektroforesis gel dilakukan pada tegangan 100 V selama 60 menit
6. Setelah elektroforesis, gel direndam dalam larutan 100 mL 1X TAE Buffer yang sudah ditambah
dengan 5 μL Ethidium Bromide selama 25 menit dan lindungi dari cahaya sambil digoyang-goyang
beberapa kali.
7. Bilas gel dengan aqua bidest sebanyak 3X, kemudian rendam kembali gel di dalam aqua bidest selama
5 menit dan lindungi dari cahaya sambil digoyang-goyang beberapa kali.
8. Gunakan UV atau blue-light transilluminator atau UV epi-illuminator untuk memotret gel4.

Analisis Data
Data yang diperoleh dari pengamatan terhadap berat, dan kadar air feses tersebut dianalisis secara satistik
menggunakan two-way ANOVA dengan post hoc Tukey. Untuk data PCR dianalisis menggunakan one-way
ANOVA dengan post hoc Tukey dibantu software ImageJ dan GraphPad Prism.

RESULT:
Pengukuran Ekspresi mRNA AQP3
Pengukuran ekspresi mRNA AQP3 dilakukan dengan metode PCR (Polymerase Chain Reaction). Pengukuran
ekspresi mRNA AQP-3 diambil pada sampel jaringan kolon mencit. Dari metode tersebut didapatkan hasil
ekspresi dari sampel berupa band yang kemudian dianalisis dengan software Image J dan setelah itu didapatkan
nilai mean tiap sampel dari masing-masing kelompok hewan coba. Kemudian setelah itu dilakukan pengolahan
data dengan membandingkan nilai mean dari band mRNA AQP-3 dengan nilai mean dari band beta actin
sehingga diperoleh nilai rasio. Nilai rasio yang diperoleh akan dinormalisasi menjadi 100% yang digunakan
sebagai nilai ekspresi relatif dan kemudian untuk membuat grafik yang menunjukkan ekspresi relatif tiap sampel
kolon dari masing-masing kelompok.

Tabel 1. Data ekspresi relatif mRNA AQP-3

Relative Expression mRNA AQP-3

Group
Hour-1 Hour-5
Normal saline 10 μl/g BB 100,00 ± 5,60 100,00 ± 7,96
Morphine 10 mg/kg BB 146,41 ± 1,74 * 110,96 ± 0,02
Morphine + PEG 4000 3 g/kg
118,41 ± 3,48 # 95,30 ± 0,58
BB
Keterangan:
*p<0,01 terhadap kelompok normal salin 10 µl/g BB jam ke-1. # p<0.05 terhadap kelompok morfin 10 mg/kg BB jam ke-1.

Gambar penampang band

Perlakuan jam
ke-1

AQP-3
(420 bp)
Perlakuan jam
ke-5

b-actin
(573 bp) Perlakuan jam
ke-1

8
 Perlakuan jam
ke-5

Figure 1. Ekspresi relatif mRNA AQP-3 pada sampel kolon mencit (± SEM) diukur pada jam ke-5 setelah induksi morfin (n=2).

Dari hasil analisis data juga terdapat perbedaan yang signifikan antara kelompok morfin 10 mg/kg BB jam ke-1
dengan kelompok morfin + PEG 4000 3g/kg BB jam ke-1 dengan nilai p<0,05. Kadar ekspresi relatif mRNA
AQP-3 mengalami penurunan dari kelompok morfin 10 mg/kg BB sebesar 146,413 ± 1,736 menjadi 118,411 ±
3,476 setelah diberi PEG 4000 pada kelompok morfin + PEG 4000 3g/kg BB. Hal tersebut menunjukkan bahwa
PEG 4000 yang digunakan sebagai terapi laksatif mampu memperbaiki kondisi konstipasi melalui mekanisme
AQP-3 ditandai dengan menurunnya kadar ekspresi mRNA AQP-3 pada kelompok morfin + PEG 4000 3 g/kg
BB. PEG mempunyai mula aksi kerja selama 1-2 jam dan waktu paruh eliminisi sekitar 4-6 jam setelah
pemberian secara oral.

Pengukuran Persentase Kadar Air Feses

Pengukuran persentase kadar air feses dilakukan dengan cara mengukur jumlah kandungan air yang terkandung
dalam feses. Pengamatan dilakukan pada jam ke-1 dan jam ke-5 setelah induksi morfin. Feses awal yang didapat
kemudian langsung ditimbang dan dinyatakan sebagai berat basah. Setelah itu, feses basah dioven selama ± 15
menit pada suhu 100 °C kemudian ditimbang sampai mendapatkan berat yang konstan dan dinyatakan sebagai
berat kering. Pengamatan dilakukan setiap 1 jam sekali selama 5 jam untuk meminimalisir adanya pengotor
pada feses mencit. Data yang diperoleh kemudian dianalisis statistik menggunakan two-way ANOVA dengan
post hoc Tukey. Data pengukuran persentase kadar air feses ditunjukkan pada Tabel 2 dan Gambar 2.

Table 2. Rata-rata Kadar Air Feses (%)

Rata-rata Kadar Air Feses (%)
Kelompok
Jam ke-1 Jam ke-5
Normal Saline 10 μl/g BB 63,09 ± 3,26 64,46 ± 2,32
Morfin 10 mg/g BB 0,00 ± 0,00 * 56,34 ± 1,12 ## Δ
Morfin + PEG 4000 3 g/kg BB 28,90 ± 12,93 ** # 60,02 ± 1,91 ΔΔ
Keterangan:
Pada kelompok morfin 10 mg/kg BB jam ke-1 tidak terdapat feses sama sekali, sehingga nilai dianggap 0. *p<0,0001; **p<0,01 terhadap
kelompok normal salin 10 µl/g jam ke-1. #p<0,05; ##p<0,0001 terhadap kelompok morfin 10 mg/kg BB jam ke-1. Δp<0,05; ΔΔp<0,01
terhadap kelompok morfin + PEG 4000 3 g/kg BB jam ke-1.

Figure 2. Persentase kadar air feses (± SEM) diukur pada jam ke-1 dan jam ke-5 setelah induksi morfin (n=6).

9
Dari hasil analisis data persentase kadar air feses terdapat perbedaan signifikan antara kelompok morfin + PEG
4000 3 g/kg BB jam ke-1 dengan morfin 10 mg/kg BB jam ke-1. Sedangkan pada perlakuan jam ke-5 setelah
induksi morfin diketahui bahwa persentase kadar air feses cenderung sama pada tiap kelompok. Hal tersebut
berkaitan dengan durasi kerja dari morfin yaitu selama 3-4 jam dan percobaan pada mencit konstipasi
mempunyai waktu paruh 25,5-32,3 menit. Selain itu, hal tersebut juga menujukkan bahwa terapi PEG 4000
sebagai laksatif mampu memperbaiki kondisi konstipasi. PEG mempunyai mula aksi kerja selama 1-2 jam
setelah pemberian secara oral.

Pengukuran Berat Feses

Pengukuran berat feses dilakukan dengan mengumpulkan feses mencit selama waktu yang ditentukan yaitu pada
jam ke-1 dan jam ke-5. Feses mencit yang dikumpulkan kemudian ditimbang beratnya. Pengumpulan dan
pengamatan feses dilakukan setiap 1 jam sekali untuk meminimalisir adanya pengotor pada feses yang dapat
mempengaruhi berat feses mencit. Data yang diperoleh kemudian dianalisis secara statistik menggunakan two-
way ANOVA dengan post hoc Tukey. Data pengukuran berat feses ditunjukkan pada Tabel 3 dan Gambar 3.

Table 3. Data rata-rata berat feses

Rata-rata berat feses (g)

Group
Jam ke-1 Jam ke-5
Normal Saline 10 μl/g BB 0,22 ± 0,07 1,42 ± 0,37
Morfin 10 mg/g BB 0,00 ± 0,00 0,53 ± 0,09
Morfin + PEG 4000 3 g/kg BB 0,03 ± 0,02 0,93 ± 0,18 *
Keterangan:
*p<0,01 terhadap kelompok morfin 10 mg/kg BB jam ke-1

Figure 3. Rata-rata berat beses (± SEM) diukur pada pengamatan jam ke-1 dan jam ke-5 setelah induksi morfin (n=6).

Terdapat perbedaan bermakna antara kelompok morfin 10 mg/kg BB jam ke-1 dengan kelompok morfin + PEG
3 g/kg BB jam ke-5 dengan nilai p<0,01. Dari hasil pengamatan, terjadi peningkatan berat feses yang semula
0,000 ± 0,000 g pada kelompok morfin 10 mg/kg BB jam ke-1 menjadi 0,928 ± 0,178 g setelah pemberian PEG
4000 pada kelompok morfin + PEG 4000 3g/kg BB jam ke-5. PEG 4000 bekerja dengan mengikat air molekul
air ke dalam lumen usus sehingga dapat meningkatkan kandungan air dan berat pada feses, dengan demikian hal
tersebut dapat memfasilitasi gerakan usus pada saat defekasi. PEG 4000 mampu menunjukkan pengurangan
kondisi konstipasi sebanyak 94% dengan mengembalikan kondisi pola buang air besar menjadi normal. Kondisi
konstipasi ditandai dengan frekuensi defekasi yang kurang, konsistensi feses yang keras dan sulitnya proses
defekasi26. Dari data hasil analisis, parameter rata-rata berat feses antara perlakuan jam ke-1 dengan jam ke-5
cenderung mengalami peningkatan. Hal tersebut berkaitan dengan pemberian PEG 4000 yang mampu mengatasi
kondisi konstipasi.

DISCUSSION:

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Pada kali ini telah dilakukan penelitian menganilisis efek pemberian PEG 4000 terhadap perubahan ekspresi
AQP-3 pada kolon mencit konstipasi akut yang diinduksi morfin dengan paramater konstipasi berupa persentase
kadar air feses dan berat feses. Penelitian dilakukan pada waktu pengamatan jam ke-1 dan jam ke-5. Perubahan
ekspresi AQP-3 telah diketahui bergantung pada waktu dan perlakuan yang diberikan. Menurut 12 penelitian yang
dilakukan menunjukkan bahwa peningkatan ekspresi AQP-3 terjadi dalam 1 jam setelah pemberian morfin dan
terus mengalami peningkatan hingga 5 jam setelah pemberian. Pada penelitian yang dilakukan sebelumnya,
rentang waktu pemberian obat semakin lama akan menyebabkan tidak adanya perubahan ekspresi dari AQP
karena efek dari obat yang sudah menurun 10. Morfin memiliki waktu paruh eliminasi pada manusia sekitar 1,5-2
jam25. Pada penelitian yang dilakukan oleh16morfin memiliki waktu paruh sekitar 25,5-32,3 menit pada mencit.
Morfin memberikan efek analgesik pada mencit dengan durasi selama 2-3 jam 22. Dari hal tersebut, menjadikan
dasar penelitian menggunakan pemilihan rentang waktu penelitian yaitu pada jam ke-1 dan jam ke-5 setelah
induksi morfin. Pengamatan kadar morfin pada hewan coba diperkirakan masih tinggi pada jam ke-1 dan
mengalami penurunan pada jam ke-5. Pemilihan kedua rentang waktu tersebut diperkirakan juga telah terjadi
perubahan pada parameter kontipasi yang dilakukan dalam penelitian ini.

CONCLUSION:
Berdasarkan hasil penelitian dari pengaruh pemberian PEG 4000 terhadap perubahan ekspresi aquaporin-3 pada
kolon mencit yang diinduksi konstipasi akut dengan morfin, dapat disimpulkan bahwa terjadi peningkatan
ekspresi mRNA AQP-3 pada kolon mencit konstipasi yang diinduksi dengan morfin, pemberian PEG 4000
mampu mengatasi kondisi konstipasi yang diinduksi morfin pada mencit ditunjukkan dengan nilai penurunan
ekspresi mRNA AQP-3 dan pemberian PEG 4000 meningkatkan kadar air feses dan berat feses pada mencit
konstipasi yang diinduksi dengan morfin.

CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
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