Expression of Hypoxia-inducible Factor-1α (HIF-1α) Related to Oxidative Stress in Liver of Rat-induced by Systemic Chronic Normobaric Hypoxia

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1 ORIGINAL ARTICLE Expression of Hypoxia-inducible Factor-1α (HIF-1α) Related to Oxidative Stress in Liver of Rat-induced by Systemic Chronic Normobaric Hypoxia Sri Widia A. Jusman*, Abdul Halim S.**, Septelia I. Wanandi*, Mohamad Sadikin* * Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Indonesia - Cipto Mangunkusumo Hospital. Jl. Diponegoro no. 71, Jakarta Pusat 10430, Indonesia. ** Biomedical Program, Faculty of Medicine, University of Indonesia, Jakarta Pusat, Indonesia. Correspondence mail to: sriwidia@fk.ui.ac.id ABSTRACT Aim: to observe the expression of -inducible factor-1a (HIF-1a) and its relation with oxidative stress in liver of rats induced by systemic chronic normobaric. Methods: twenty five male, 6-8 weeks old rats were induced by systemic. Rats were divided randomly into 5 groups (n = 5 per group). The control group was exposed to normal environment while the hypoxic groups were kept in hypoxic chamber (10 % O 2 ) for 1, 3, 7, and 14 days. Animals were sacrificed, the liver isolated and homogenized. Total RNA was extracted and isolated and expression of HIF-1a mrna was measured by real-time RT PCR using Pffafl method. Malondialdehyde (MDA), product of lipid peroxidation was measured by tbars assay. Glutathione (GSH), an abundant endogenous antioxidant in the liver tissue was measured using Ellman method. Results: study showed that expression of HIF-1a mrna was increased in group treated for 1 day of hypoxic condition, and then decreased in group treated for 3, 7 and 14 days of hypoxic condition related with duration of hypoxic condition. The MDA level in liver tissue increased, but not significant in all groups of hypoxic condition and persisted along duration time of hypoxic condition. The GSH level was decreased significantly (p<0.005) in all groups of hypoxic condition. Conclusion: expression of HIF-1a mrna was higher at the early phase of and decreased as continued. Systemic induction caused increased ROS formation during, and depleted the GSH concentration in the liver. Oxidative stress present in liver of rat was induced by systemic. Key words: systemic chronic normobaric, HIF-1α mrna, tbars, GSH, oxidative stress, liver tissue. INTRODUCTION Hypoxia is a condition, characterizes by insufficiency of oxygen supply to meet cellular demand. Another form of environmental is normobaric, in which the total atmosphere pressure is normal, but the partial oxygen pressure is lower than it should be, which is found only in experimental condition. 1 Hypoxia inducible factor-1 (HIF-1) is ubiquitous intracellular protein, whose content increases in any hypoxic cell. HIF-1 is a heterodimer protein, consists of 2 subunits, α and β. The stabilized HIF-1α joins HIF-1β and the whole protein translocates into nucleus and binds the HIF-1 response element (HRE), found in various target genes. 2-5 Thanks to these processes, many genes can be regulated in order to optimize the utilization of the available oxygen Hypoxia enhances also the generation and the release of mitochondrial reactive oxygen species (ROS), which in turn regulates the cellular response to the low oxygen tension. 2, 10, 12 Inhibition of electron transport with rotenone, proximal to complex III, decreases the production of ROS by complex III. 13,14 There is a controversy about HIF-1α activation, induced by ROS in and HIF-1α deactivation induced by ROS in normoxia. 10, Glutathione can counter-balance the 10, 19, 21, 22 ROS, catalyzed by glutathione peroxidase. The aim of this study is to improve understanding of the mechanism of oxidative stress in liver tissue by observing the correlation of HIF-1α gene expression with oxidative stress in rat s liver, induced by systemic chronic normobaric. 17

2 Sri Widia A. Jusman Acta Med Indones-Indones J Intern Med METHODS All chemicals were reagent grade and purchased from Sigma. Twenty five male Sprague-Dawley rats, 6-8 weeks old, weighing grams kept in the animal house of Department of Biochemistry and Molecular Biology, Faculty of Medicine University of Indonesia. To induce the hypoxic condition, animals were placed in hypoxic chamber (10%O 2 : 90%N 2 ). Rats were provided with food and water ad libitum. Animals were randomly divided into 5 groups (n = 5 per group). The control group was exposed to normal environment while the hypoxic groups were kept in hypoxic chamber for 1, 3, 7, and 14 days. All procedures were approved by Ethical Committee of Center Research and Health Development, Ministry of Health Republic of Indonesia (BALITBANGKES RI) No LB /KE/4783/08. Animals were sacrificed by ether anesthesia. Liver tissues were taken out, weighed, divided into aliquots, and immediately frozen - 86 C. Total RNA was extracted from liver tissues by using TriPure Reagent Isolation Reagent (Roche). RNA concentration was determined using UV spectrophotometer. Five hundred nanograms of RNA were reversetranscribed and amplified to cdna using real time RT- PCR with iscript One-Step RT-PCR Kit with SYBR Green (BioRad, USA). b-actin gene was used as internal control. Primers used for HIF-1a and b-actin were designed with Primer3 based on GeneBank (NM for HIF-1α; NM for b-actin). HIF-1α primers: Forward 5 -CGA AGA ACT CTC AGC CAC AG-3 and Reverse 5 - AGC TCG TGT CCT CAG ATT CC-3 ; b-actin primers: Forward 5 - CAC TGG CAT TGT GAT GGA CT-3 and reverse 5 - CTC TCA GCT GTG GTG GTG AA-3. Real time RT PCR product for each primer pair was 174 bp for HIF-1α and 178 bp for b-actin. Real-time RT PCR reaction mix consists of 25 ml 2x SYBR Green RT-PCR reaction Mix; 1.5 ml of each primer, 19 ml nuclease-free water; 2 ml RNA template and 1 ml iscript Reverse Transcriptase. Realtime RT PCR conditions were: synthesis cdna at 50 C for 10 minutes; inactivation of iscript Reverse Trancriptase at 95 C for 5 minutes; 39 cycles at 95 C for 10 seconds, 60 C for 30 seconds and 72 C for 30 seconds. A melting curve was performed to verify the presence of a single amplicon. Non template control (NTC) was used as a negative control. Real-time RT PCR data were calculated according to Pffafl method. 23 Expression ratio = DCt target (calibrator target) (Efficiency target) DCt ref (calibrator ref) (Efficiency reference) To measure the MDA concentration, mg of liver tissue in 1 ml PBS, ph 7.0 was homogenized with micropestle in microtube. One ml 20 % TCA was added to 200 ml liver homogenate to precipitate the protein, and centrifuged. Supernatants were collected and 2 ml thiobarbituric acid (TBA) solution was added to the supernatants. After boiling for 10 minutes in water bath, the absorbance was measured at l,530 nm. Concentration of MDA in supernatants of liver homogenate was calculated using the standard curve of MDA standard solution (0;0.625;1.25;2.5;5.0 nmol/ml). MDA concentration was expressed as mg MDA/g liver tissue. GSH concentration 24 was measured from 250 ml of liver homogenate in 8.9 ml phosphate buffer ph 8.0 and then 5% TCA was added, to precipitate liver protein. After centrifugation, 50 ml dithiobisnitrobenzoate (DTNB) solution was added to the supernatants of liver homogenate, and incubated for 1 hour. The absorbance was measured at l 412 nm. Concentration of GSH in liver tissue was calculated using the standard curve of GSH standard solution (0; 10;20;40;50;100 mg/ml). GSH concentration was calculated as mg GSH/g of liver protein. The liver protein concentration was calculated by using standard curve of bovine serum albumin (BSA) solution. Data of HIF-1α gene expression, MDA and GSH concentration in liver tissue between normoxic and hypoxic groups were reported as the mean + SEM and analyzed with ANOVA followed by LSD test. RESULTS Body Weight of Rats The body weight of all treatment groups were decreased significantly during induction. The differences of body weight after and before treatment for the day-1; day-3; day-7 and day-14 were 4.609; 7.473; and g respectively. Liver Tissue Weight The weight of liver tissue from the control group; day-1; day-3; day-7 and day-14 were 4.924; 5.672; 6.108; and g respectively, showing differences between the control group compared to the treatment groups, but showing no differences between all the treatment groups. Ratio of Liver Tissue Weight/Body Weight The ratio between the liver tissue weight /body weight from the control group; day-1; day-3; day-7 and day-14 were 0.026; 0.031; 0.028; and 0.31 respectively, showing no differences among the groups. 18

3 Vol 42 Number 1 January 2010 Expression of Hypoxia-inducible Factor 1α (HIF 1α) Related to Oxidative Stress Total RNA The purity index of RNA isolation from majority samples is >1.75 (Mean ). Real Time RT-PCR Product of real time RT-PCR was detected as fluorescence absorbance of SYBR Green. Threshold of fluorescence curve was set-up to achieve the optimum efficiency of expression. The threshold values for all fluorescence curves were standardized so all data can be comparable and analyzed (Figure 1). actin HIF-1a mrna relative expression level was elevated on the first day of (1.4-fold elevated), and then was gradually lowered during the time of of day- 3 (0.69-fold lowered) and day-7 (0.29-fold lowered), and slightly elevated on the 14 days of (0.41-fold elevated) compared to normoxic control group (Figure 3) Relative expression of mrna HIF-1 HIF threshold Ct NTC -actin 0.00 Series 1 Control day-1 day-3 day-7 day Figure 3. Relative expression of HIF-1α mrna in liver of rat-induced by systemic chronic normobaric Figure 1. Cycle threshold (Ct) of HIF-1α mrna, β-actin and nontemplate control (NTC) Analysis of melting curve, using HIF-1α and b-actin primers showed 1 peak for each primer pairs (Figure 2), 82 C for HIF-1α and 85.5 o C for β-actin. Electrophoresis on 2% agarose (inzet) showed only one band each for β-actin and HIF-1α equal to 174 bp and 178 bp respectively. It is proved that there was no primer dimer present. MDA Content in Liver Tissue Amount of malondialdehyde (MDA) reflects the lipid peroxide quantities, which is formed as results of ROS attack on the lipid. As can be seen on Table 1, statistical analysis reveals the increase of liver MDA content during the experiment. However, the differences are significant (p <0.05) between control, day-1, day-3, day- 7 and day-14. The MDA content still increased in the day-7 and day-14, but the differences between them and day-3 as well as among themselves are not significant any longer (p >0.01). fluorescence temperature Figure 2. Melting curve of HIF-1α and β-actin Expression of HIF-1α mrna Relative expression of HIF-1α mrna of treatment group was normalized to control group (expression level of calibrator = 1) Liver MDA content (nmol/mg liver tissue) Liver MDA content control day-1 day-3 day-7 day Figure 4. MDA content in liver tissue of rat induced by systemic chronic normobaric 19

4 Sri Widia A. Jusman Acta Med Indones-Indones J Intern Med Figure 4 shows that MDA concentration is elevated progressively from the day-1 of (1.90 fold elevated), during the period of the observation time of on day-3 (2.85 fold elevated), and day-7 (3.02 fold elevated), and 3.42 fold elevated on the day-14 compared to normoxic control group. The changes have been statistically significant since the day-1 (p < 0.05) (Table 1). Table 1. MDA content in liver tissue of rat-induced by systemic chronic normobaric (nmol/g liver tissue) No GSH Content in Liver Tissue Reduced glutathione (GSH) is the most abundant thiol in the cell and is generally found in the millimolar range. Statistical analysis, as shown in Figure 5 and Table 2, indicates that GSH decreases progressively. Figure 5 shows that GSH concentration is decreased progressively during the period of observation from the day-1 of (0.76 fold), on day-3 (0.60 fold), and day-7 (0.49 fold), and 0.40 fold on the day-14 compared to normoxic control group. The differences between control group and day-1, day-3, day-7 and day-14, and almost among all experimental groups, except between day-7 and day-14, are highly significant (p< 0.05) (Table 2). g/mg protein Control 1-day 3-day Liver GSH content 7 day control day-1 day-3 day-7 day day Mean ± SD * ± * ± *^ ± *^ ± *^ ± *Significant (p < 0.05, ANOVA followed by LSD test) between control, day-1, day-3, day-7 and day-14). ^ Not significant (p > 0.05, ANOVA followed by LSD test) between day-3, day-7, day-14 Figure 5. GSH content in liver tissue of rat induced by systemic chronic normobaric Table 2. GSH content in liver tissue of rat-induced by systemic chronic normobaric (µg/mg liver protein) Correlation of HIF-1α mrna Expression and MDA Content in Liver Tissue The correlation between HIF-1α mrna and MDA content in rat liver tissue underwent the total body during 2 weeks is pictured in Figure 6. Statistical analysis (Pearson) indicated that there is a strong-moderate negative correlation (R = , p < 0.05) between both parameters. MDA No Control 1-day 3-day Correlation mrna HIF-1 & MDA mrna 7 day 14-day Mean ± SD 0.054* ± * ± * ± * ± *Significant (p < 0.05, ANOVA followed by LSD test) between control, day-1, day-3, day-7 and day-14) * ± 0.00 Figure 6. Correlation of HIF-1a mrna expression and MDA content in liver tissue of rat induced by systemic chronic normobaric Correlation of HIF-1α mrna Expression and GSH Content in Liver Tissue Figure 7 pictured the correlation of HIF-1α mrna and GSH content in rat liver. It can be seen at a glance that the HIF-1α expression varied in parallel with the GSH content. Statistical analysis revealed that the correlation is positive strong to moderate (Pearson, R = 0.737, p<0.05). Correlation of Liver GSH and MDA Content The relation of liver GSH and MDA content is presented in Figure 8. As it can be seen immediately, the relationship of liver GSH and MDA is inversed. The decrease of GSH is accompanied by the increase of MDA. Statistical analysis (Pearson, R = 0.993, p<0.05) 20

5 Vol 42 Number 1 January 2010 Expression of Hypoxia-inducible Factor 1α (HIF 1α) Related to Oxidative Stress GSH Figure 7. Correlation of HIF-1a mrna expression and GSH content in liver tissue of rat- induced by chronic systemic normobaric MDA Correlation mrna HIF-1 & GSH mrna HIUF-1 Correlation GSH & MDA GSH Figure 8. Correlation of GSH and MDA content in liver tissue of rat-induced by systemic revealed that there is a strong negative correlation between both parameters. DISCUSSION The body weight of rats during induction was decreased significantly, despite the fact that there are no differences of liver tissue weight and ratio of liver tissue weight to body weight from all the groups. These observations showed that the liver tissue has a very large capacity to maintain its vital functions. Systemic chronic seems to have a tendency to cause decrease of body weight (hypoxic cachexia). This condition could be caused by muscle protein degradation, to supply the tissue with substrate for anaerobic metabolism. Recent studies showed that HIF-1α is a sensitive marker of. Hypoxia and various other stimuli induce HIF-1α signaling cascade, and then transcriptionally activate multiple genes. Under a continuous hypoxic stimulus, HIF-1α was induced in the liver within 1 hour of exposure and peaked after 2 hours. 14 ROS formation during can induced HIF-1α signaling cascade. 10 Decreased expression of HIF-1α mrna on the day-3 and day-7 may be due to stabilization of HIF- 1α protein which is not degraded by ubiquitin-proteasome system. 2, 25 Stabilization of HIF-1α protein is also carried 10, 12, 25, 26 by increased production of ROS during. It seems that kinetic of HIF-1α expression is organspecific and are differed from one organ to the other organ. In brain, expression of HIF-1α mrna level was not significantly increased in hypoxic condition, may be caused by high activity of MnSOD in brain tissue. 28, 29 In contrast, heart tissue is very responsive to hypoxic condition, showed by increased expression of HIF-1α 29, 30 mrna and reached peak at 21 days of. The elevated MDA concentration is due to increased ROS formation in mitochondria as a consequence of. The increased level of MDA in liver tissue also proved by Jun et al, who found that oxidative stress caused by exposure to intermittent for 1 week resulted in a trend to an increase in MDA level in liver tissue, while in the aorta and heart, intermittent did not affect the MDA level. 31 Increased ROS formation from mitochondria will trigger the redox signaling cascade. ROS activate tyrosine kinase receptor or inhibit tyrosine posphatase protein which activate the mitogen-activated protein kinase (MAPK), phospholipase Cg (PLCg), protein kinase C (PKC) pathways and in turn activate transcription factor and expression of target gene. 10 HIF-1α is one of many target genes which is activated by ROS. Increased level of ROS during systemic in this experiment is counter-balanced by antioxidant system in liver tissue. The glutathione level is decreased from the early phase of and continued until the end of observation of on the day-14. This condition is caused by decreased antioxidant capacity of the liver. Antioxidant capacity of the liver is maintained by glutathione in reduced form (GSH), enzymatic antioxidant such as glutathione peroxidase and glutathione reductase, which is supported by the HMP shunt pathway producing the NADPH. 21, 22 Although liver was containing much of antioxidant, but since GSH were used to counter-balance the increased formation of ROS formation during time of hypoxic condition, the GSH level was more decreased at the day-14 of. Glutathione is one of endogenous antioxidant which protects tissues from oxidative stress. Glutathione is a substrate for glutathione peroxidase, the enzyme that reduces hydroperoxides and organic peroxides, including lipid peroxide. 22 Depletion of GSH due to oxidative stress was also proved by Samarsinghe et al, who found that oxidative stress due to followed by reoxygenation showed the fall of cellular GSH level to 37% compared to the normoxic control group (p<0.001)

6 Sri Widia A. Jusman Acta Med Indones-Indones J Intern Med It seemed that increased formation of ROS at the early phase of is accompanied by increased expression of HIF-1α mrna and significant depletion of GSH. As continued, it was accompanied by more increase of ROS formation (Figure 6, 7, 8). The increased level of ROS was not accompanied by increased expression of HIF-1α due to stabilization of HIF-1α protein which was not degraded by ubiquitin-proteasome system. Hence, there is no need to increase the expression of HIF-1α gene. But, from this experiment it was not proved that HIF-1α protein becomes stabile. Increased expression of HIF-1α present in the early phase of, showed that liver tissue has different pattern from other tissue such as heart and brain. Increased formation of MDA concentration and depletion of GSH concentration showed that oxidative stress present in liver tissue of rats, induced by chronic systemic normobaric. The effect of systemic normobaric on liver tissue might be found in condition such as obstructive lung disease, obstructive sleep apnoea (OSA), chronic anemia and congestive heart failure accompanied with failure of blood perfusion to the liver tissue. CONCLUSION Expression of HIF-1α gene was increased in liver tissue of rat at the early phase of systemic normobaric and decreased as continued. Systemic normobaric induction caused increased formation of MDA in liver tissue of rat due to increased ROS formation during. Systemic normobaric induction caused depletion of the GSH concentration in the liver tissue of rat. It is suggested that oxidative stress present in liver of rat is induced by systemic normobaric. Systemic normobaric could lead to insufficient blood perfusion to the liver tissue which might be found in conditions such as obstructive lung disease, obstructive sleep apnea, chronic anemia or congestive heart failure. ACKNOWLEDGEMENT This research was funded by Grant Research for Post-Graduate Student, DRPMUI, Academic Year 2007, No. 243B/DRPM-UI/N1.4/2008. Thanks to Dr. Frans Ferdinal, MS for hypoxic chamber apparatus. REFERENCES 1. Vaupel P, et al. Tumor : Causative factors, compensatory mechanism and cellular response. Oncologist. 2004;9(Suppl 5): Giaccia AJ, Simon MC, Johnson R. The biology of : The role of oxygen sensing in development, normal function, and disease. Genes and Development. 2004;18: Schuler B, Lundby C, Gassmann M. HIF-1 and the adaptation of man to high altitude. Sportmedizin und Sporttraumatologie. 2005;53(2); Lee JW, et al. Hypoxia-inducible factor (HIF-1a): Its protein stability and biological functions. Exp Mol Med. 2004;369(1); Wenger R. Cellular adaptation to : O 2 -sensing protein hydroxylases, -inducible transcription factors, and O 2 - regulate gene expression. FASEB. 2002;16: Zagorska A, Dulak J. HIF-1: The knowns and unknowns of sensing. Acta Biochimica Polonica. 2004;51(3): Semenza GL. HIF-1 and human disease. Gene & Development. 2000;14: Semenza GL. Hydroxylation of HIF-1a: Oxygen sensing at the Molecular Level. Physiology. 2004;19: Semenza GL. HIF-1a: Mediator of physiological and pathophysiological responses to. J Appl Physiol. 2000;88: Hermes-Lima M. Oxygen in biology and biochemistry: Role of free radicals. In: Storey KB, ed. Functional metabolism: Regulation and adaptation. Hoboken, New Jersey: Wiley Liss, Inc; p Weidemann A, Johnson RS. Biology of HIF-1a. Cell Death and Differentiation. 2008;15; Schumacker PT, Guzzy RD. Oxygen sensing by mitochondria at complex III: The paradox of increased ROS during. Experimental Physiology. 2006;91: Chen Q, Vasquez EJ, Moghadas S, Hoppel CL, Lesnefsky EJ. Production of reactive oxygen species by mitochondria. Central role of complex III. J Biol Chem. 2003; 278(38): Stroka D, Candinas D. Hypoxia-inducible factor-1 signaling system. In: Dufour JF, Clavien PA, Trautwein C, Graf R, eds. Signaling pathways in liver disease part II. Berlin: Heidelberg Springer; 2005; 26. p Michiels C. Physiological and pathological responses to. Am J Pathol. 2004;164: Hervouet E, et al. HIF and reactive oxygen species regulate oxidative phosphorylation in cancer. Carcinogenesis. 2008;29: Giordano FJ. Oxygen, oxidative stress, and heart failure. J Clin Invest. 2005;115: Halliwell B, Gutteridge JMC. Oxygen is a toxic gas. An introduction to oxygen toxicity and reactive species. Free radicals in biology and medicine. 4 th edition. London: Oxford University Press; p Haddad JJ. Oxygen sensing mechanism and the regulation of redox-responsive transcription factors in development and pathophysiology. Respir Res. 2002;3: Wills ED. Evaluation of lipid peroxidation in lipids and biological membranes. In: Snell K, Mullock B, eds. Biochemical toxicology: A practical approach. London: Oxford;

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