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 years¹. 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 tract^1,2.

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 medication^3,4.

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 somniferum⁵. Morphine is a -opioid receptor (MOR) agonist that is used clinically for patients with moderate to severe pain^6,7. 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 systems^8,9.

Although morphine and MOR agonists exhibit strong analgesic effects, they can cause side effects. Opioid-induced constipation is one of the opioid’s side effect that estimated to occur in approximately 40-64% of patients given opioids for pain management^10,11. 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 therapy¹². 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 constipated^13,14.

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 transit¹⁵. MOR is known to be highly expressed in the gastrointestinal tract in the myenteric plexus and to a lesser extent in the submucosal plexus¹⁰. 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 motility¹⁶.

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^17,18. 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)¹⁹. Increased levels of high 5-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 cells²⁰.

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 5-15mg once a day one tablet at night or senna 7.5mg 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 content²⁰. 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 agents²¹. 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 morphine²². 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 regularly^23,24.

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 constipation²³. 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 children²⁴. 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 lactulose²³. 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 conditions²⁵.

The effectiveness of PEG's osmotic activity depends on its ability to absorb water into the intestinal lumen. In a study showed that PEG can reduce EGFR in the colon of mice²⁶. 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 constipation²⁷. 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 cells²⁵. 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 AQP8²⁸. The expression of AQP3 and AQP4 is dominant in both human and mouse colonic mucosal epithelial cells²⁷.

Administration of morphine can cause constipation by increasing AQP3 expression in rats²⁵. 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 constipation²⁶.

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 induction²⁷.

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 mice²⁸.

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 sequence²⁹.

Laxative Dosage Calculation:

In a previous study, the dose of PEG given to experimental mice was 1.5g/kg BW, 3g/kg BW, and 6 g/kg BW then dissolved in 10mL/kg. The effective dose of PEG in experimental animals is 3g/kg BW³⁰.

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 10mg/kg BW. While the negative control group was only injected with normal saline 10L/g BW intraperitoneally. Morphine and normal saline injections were performed 30 minutes after administration of PEG 4000 or aqua³¹.

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 following³²:

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 kit³³. 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 follows³⁴:

mRNA	Sequence (5’-3’)	PCR Product (bp)
AQP-3 F	5′-GGGCTGTACTACGATGCAATC-3′	420
AQP-3 R	5′-ACACGAAGACACCAGCGATGG-3′	420
β-actin F	5’-TGTTACCAACTGGGACGACA-3’	573
β-actin R	5’-AAGGAAGGCTGGAAAAGAGC-3’	573

b) Colonic Tissue Retrieval:

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°C³⁵.

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 minutes³⁰.

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 500L 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 quality³⁴.

d) First Strand cDNA synthesis:

1. Mix and briefly centrifuge each component before use. Combine the components with the following composition:

2. Close each RNA tube tightly. Incubate the tube at 70 °C for 5 minutes. Spin down each tube for 10 seconds in a microcentrifuge to collect condensate and obtain a complete volume. Make sure the tube is closed and cold until the Reverse Transcription reaction mix is added.

3. Prepare the Reverse Transcription reaction mix by combining the following components of the GoScriptTM Reverse Transcription System in a sterile tube under cold conditions:

Component	Reaction 1 times	Reaction 8 times
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® Ribonuclease Inhibitor	0,5 µL	4 µL
GoScript™ Reverse Transcriptase	1 µL	8 µL
Volume akhir	15 L	120 µL

4. Add 15L aliquots of the Reverse Transcription reaction mix to each tube under cold conditions to 5 L of the primary RNA mixture to obtain a final reaction volume of 20 L per tube.

5. Anneal: incubation at 25°C for 5 minutes.

6. Extend: incubation at 42°C for 60 minutes.Store the reaction product at 4°C if it is not immediately followed by sample amplification^36,37.

e) Sample Amplification (Cleaver Scientific)

1. For every 50L of reaction, mix the following components in a 0.2mL PCR tube on ice. The sample amplification components are as follows:

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. Mix carefully, if necessary a short centrifuge, close the tube and place it on the thermal cycler.

3. Process in thermal cycler for 40 cycles

4. After the PCR reaction, followed by DNA electrophoresis to detect PCR products³⁸.

f) Elektroforesis Produk PCR:

1. Make 1.7% concentration of agarose by mixing 1.53g of powdered agarose into 90mL of 1X TAE Buffer. Stir the mixture using a magnetic stirrer at a speed of 700-1000rpm on a hot plate at 200°C. Make sure all the agarose powder is dissolved. Wait until the gel hardens and is ready to use.

2. Prepare agarose gel with a concentration of 1.7% into the electrophoresis chamber, then fill the chamber with 1X TAE Buffer to cover the surface of the gel.

3. Insert 3 L Ladder into the first well.

4. Followed by 1 L 6X Loading Dye and 5 L of the mixed sample into the well, repeated until all samples enter the well.

5. Gel electrophoresis was carried out at a voltage of 100 V for 60 minutes

6. After electrophoresis, the gel was immersed in a solution of 100mL 1X TAE Buffer which had been added with 5L of Ethidium Bromide for 25 minutes and protected from light while being shaken several times.

7. Rinse the gel with aqua bidest 3 times, then soak the gel again in the aqua bidest for 5 minutes and protect it from light while shaking it a few times.

8. Use UV or blue-light transilluminator or UV epi-illuminator to photograph the gel³⁹.

Data analysis:

The data obtained from the observation of the weight and water content of the feces were analyzed statistically using two-way ANOVA with Tukey's post hoc. The PCR data were analyzed using one-way ANOVA with post hoc Tukey assisted by ImageJ and GraphPad Prism software.

RESULT:

Measurement of AQP3 mRNA expression:

Measurement of AQP3 mRNA expression was carried out using the PCR (Polymerase Chain Reaction) method. Measurement of AQP-3 mRNA expression was taken in the colon tissue samples of mice. From this method, the results of the expression of the sample in the form of bands were then analyzed with Image J software and after that the mean value of each sample from each group of experimental animals was obtained. Then after that, data processing was carried out by comparing the mean value of the AQP-3 mRNA band with the mean value of the beta actin band so that the ratio value was obtained. The ratio value obtained will be normalized to 100% which is used as the relative expression value and then to create a graph that shows the relative expression of each colonic sample from each group⁴⁰.

Table 1. Relative expression data of AQP-3 mRNA

Group	Relative Expression mRNA AQP-3
Group	1^st hour	5^th hour
Normal saline 10 μl/g BW	100,00 ± 5,60	100,00 ± 7,96
Morphine 10 mg/kg BW	146,41 ± 1,74 *	110,96 ± 0,02
Morphine + PEG 4000 3 g/kg BW	118,41 ± 3,48 #	95,30 ± 0,58

*p<0.01 against the normal saline group of 10 l/g BW in the 1st hour. # p<0.05 for the morphine group 10 mg/kg BW in the 1st hour.

Figure 1. The relative expression of AQP-3 mRNA in mouse colon samples (± SEM) was measured at 5 hours after morphine induction (n=2).

Information:

From the results of data analysis, there was also a significant difference between the morphine 10 mg/kg BW group at the 1st hour and the morphine + PEG 4000 3g/kg BW group at the 1st hour with p<0.05. The relative expression level of AQP-3 mRNA decreased from the morphine 10 mg/kg BW group of 146.413 ± 1.736 to 118.411 ± 3,476 after being given PEG 4000 in the morphine + PEG 4000 3g/kg BW group. This shows that PEG 4000 which is used as laxative therapy is able to improve constipation conditions through the AQP-3 mechanism, which is characterized by decreased levels of AQP-3 mRNA expression in the morphine + PEG 4000 group of 3 g/kg BW. PEG has an onset of action of 1-2 hours and an elimination half-life of about 4-6 hours after oral administration.

Measurement of The Percentage of Water Content in Feces

Measurement of the percentage of water content in feces is done by measuring the amount of water content in feces. Observations were made at the 1st and 5th hours after morphine induction. The initial feces obtained were then immediately weighed and expressed as wet weight. After that, the wet feces were baked in the oven for ± 15 minutes at 100 °C and then weighed until it got a constant weight and was expressed as dry weight. Observations were made every 1 hour for 5 hours to minimize the presence of impurities in the feces of mice. The data obtained were then statistically analyzed using a two-way ANOVA with Tukey's post hoc. The data for measuring the percentage of fecal moisture content is shown in Table 2 and Figure 2.

Table 2. Average Water Content of Feces (%)

Group	Average Water Content of Feces (%)
Group	1^st hour	5^th hour
Normal Saline 10 μl/g BW	63,09 ± 3,26	64,46 ± 2,32
Morphine 10 mg/g BW	0,00 ± 0,00 *	56,34 ± 1,12 ## Δ
Morphine + PEG 4000 3 g/kg BW	28,90 ± 12,93 ** #	60,02 ± 1,91 ΔΔ

Information:

In the 10 mg/kg BW morphine group at the 1st hour there was no feces at all, so the value was considered 0. *p<0.0001; **p<0.01 against the normal saline group of 10 l/g at the 1st hour. #p<0.05; ##p<0.0001 against the 10 mg/kg BW morphine group in the 1st hour. p<0.05; p<0.01 against the morphine + PEG 4000 group 3 g/kg BW in the 1st hour.

Figure 2: The percentage of fecal moisture content (± SEM) was measured at the 1st and 5th hours after morphine induction (n=6).

From the results of data analysis on the percentage of fecal moisture content, there was a significant difference between the morphine + PEG 4000 3 g/kg BW group at the 1st hour and morphine 10 mg/kg BW at the 1st hour. Meanwhile, in the 5th hour treatment after morphine induction, it was found that the percentage of fecal moisture content tended to be the same in each group. This is related to the duration of action of morphine which is 3-4 hours and experiments on constipated mice have a half-life of 25.5-32.3 minutes. In addition, it also shows that PEG 4000 therapy as a laxative is able to improve constipation conditions. PEG has an onset of action 1-2 hours after oral administration.

Stool Weight Measurement:

Stool weight measurement was carried out by collecting mouse faeces during the specified time, namely at the 1st hour and the 5th hour. The collected mice feces were then weighed. The collection and observation of feces is carried out every 1 hour to minimize the presence of impurities in the feces that can affect the weight of the mice's feces. The data obtained were then analyzed statistically using a two-way ANOVA with Tukey's post hoc. Stool weight measurement data are shown in Table 3 and Figure 3.

Table 3. Data on average weight of feces

Group	Average weight of feces (g)
Group	1^st hour	5^th hour
Normal Saline 10 μl/g BW	0,22 ± 0,07	1,42 ± 0,37
Morphine 10 mg/g BW	0,00 ± 0,00	0,53 ± 0,09
Morphine + PEG 4000 3 g/kg BW	0,03 ± 0,02	0,93 ± 0,18 *

*p<0,01 against the morphine group 10 mg/kg BW in the 1^st hour

Figure 3. The average weight of the stool (±SEM) was measured at the 1^st and 5^th hour observations after morphine induction (n=6).

Information:

There was a significant difference between the morphine 10mg/kg BW group at the 1st hour and the morphine + PEG 3g/kg BW group at the 5th hour with p<0.01. From the observations, there was an increase in stool weight from 0.000±0.000g in the morphine 10 mg/kg BW group at the 1st hour to 0.928±0.178 g after giving PEG 4000 to the morphine + PEG 40003g/kg BW group at the 5th hour. PEG 4000 works by binding water molecules of water into the intestinal lumen so that it can increase the water content and weight of the feces, thereby facilitating bowel movements during defecation. PEG 4000 was able to show a 94% reduction in constipation by restoring normal bowel patterns. The condition of constipation is characterized by a low frequency of defecation, hard stool consistency and difficulty in the defecation process⁴⁸. From the analysis data, the average faecal weight parameter between the 1st and 5th hours of treatment tends to increase. This is related to the provision of PEG 4000 which is able to overcome constipation conditions⁴⁹.

DISCUSSION:

At this time, a study was conducted to analyze the effect of PEG 4000 on changes in AQP-3 expression in the colon of mice with acute constipation induced by morphine with the parameters of constipation in the form of percentage of fecal water content and stool weight. The study was conducted at the time of observation at the 1st and 5th hours. Changes in AQP-3 expression have been known to depend on the time and the treatment given. According to 12 studies conducted showed that the increase in AQP-3 expression occurred within 1 hour after administration of morphine and continued to increase up to 5 hours after administration. In previous studies, the longer the duration of drug administration will cause no changes in the expression of AQP because the effect of the drug has decreased. Morphine has an elimination half-life in humans of about 1.5-2 hours. In a study conducted by morphine, it has a half-life of about 25.5-32.3 minutes in mice. Morphine provides an analgesic effect in mice with a duration of 2-3 hours. From this, the basis of the study used the selection of the research time span, namely at the 1st hour and the 5th hour after morphine induction. Observation of morphine levels in experimental animals was estimated to be high at the 1st hour and decreased at the 5th hour. The selection of the two time ranges is also estimated to have changed the constipation parameters carried out in this study⁵⁰.

CONCLUSION:

Based on the results of research on the effect of giving PEG 4000 to changes in the expression of aquaporin-3 in the colon of mice induced by acute constipation with morphine, it can be concluded that there is an increase in the expression of AQP-3 mRNA in the colon of mice with constipation induced by morphine. morphine-induced in mice was indicated by a decrease in AQP-3 mRNA expression and administration of PEG 4000 increased fecal water content and stool weight in constipated mice induced by morphine.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 04.11.2021 Modified on 04.08.2022

Research J. Pharm. and Tech 2023; 16(7):3331-3338.

DOI: 10.52711/0974-360X.2023.00550

	*Band* Cross Section
AQP-3 (420 bp)		1^st hour treatment
AQP-3 (420 bp)		5^th hour treatment
b-actin (573 bp)		1^st hour treatment
b-actin (573 bp)		5^th hour treatment