Chinese Journal of Physiology

: 2020  |  Volume : 63  |  Issue : 2  |  Page : 68--76

Repetitively hypoxic preconditioning attenuates ischemia/reperfusion-induced liver dysfunction through upregulation of hypoxia-induced factor-1 alpha-dependent mitochondrial Bcl-xl in rat

Pei-Lei Chou1, Kuo-Hsin Chen2, Tzu-Ching Chang1, Chiang-Ting Chien1,  
1 School of Life Science, National Taiwan Normal University, Taipei, Taiwan
2 Department of Surgery, Division of General Surgery, Far-Eastern Memorial Hospital; Department of Electrical Engineering, Yuan Ze University, Taoyuan City, Taiwan

Correspondence Address:
Prof. Chiang-Ting Chien
No. 88, Sec. 4, Tingzhou Road, School of Life Science, National Taiwan Normal University, Taipei 11677
Prof. Tzu-Ching Chang
No. 88, Sec. 4, Tingzhou Road, School of Life Science, National Taiwan Normal University, Taipei 11677


Repetitive hypoxic preconditioning (HP) enforces protective effects to subsequently severe hypoxic/ischemic stress. We hypothesized that HP may provide protection against ischemia/reperfusion (I/R) injury in rat livers via hypoxia-induced factor-1 alpha (HIF-1α)/reactive oxygen species (ROS)-dependent defensive mechanisms. Female Wistar rats were exposed to hypoxia (15 h/day) in a hypobaric hypoxic chamber (5500 m) for HP induction, whereas the others were kept in sea level. These rats were subjected to 45 min of hepatic ischemia by portal vein occlusion followed by 6 h of reperfusion. We evaluated HIF-1α in nuclear extracts, MnSOD, CuZnSOD, catalase, Bad/Bcl-xL/caspase 3/poly-(ADP-ribose)-polymerase (PARP), mitochondrial Bcl-xL, and cytosolic cytochrome C expression with Western blot and nitroblue tetrazolium/3-nitrotyrosine stain. Kupffer cell infiltration and terminal deoxynucleotidyl transferase-mediated nick-end labeling method apoptosis were determined by immunocytochemistry. The ROS value from liver surface and bile was detected by an ultrasensitive chemiluminescence–amplification method. Hepatic function was assessed with plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. HP increased nuclear translocation of HIF-1α and enhanced Bcl-xL, MnSOD, CuZnSOD, and catalase protein expression in a time-dependent manner. The response of HP enhanced hepatic HIF-1α, and Bcl-xL expression was abrogated by a HIF-1α inhibitor YC-1. Hepatic I/R increased ROS levels, myeloperoxidase activity, Kupffer cell infiltration, ALT and AST levels associated with the enhancement of cytosolic Bad translocation to mitochondria, release of cytochrome C to cytosol, and activation of caspase 3/PARP-mediated apoptosis. HP significantly ameliorated hepatic I/R-enhanced oxidative stress, apoptosis, and mitochondrial and hepatic dysfunction. In summary, HP enhances HIF-1α/ROS-dependent cascades to upregulate mitochondrial Bcl-xL protein expression and to confer protection against I/R injury in the livers.

How to cite this article:
Chou PL, Chen KH, Chang TC, Chien CT. Repetitively hypoxic preconditioning attenuates ischemia/reperfusion-induced liver dysfunction through upregulation of hypoxia-induced factor-1 alpha-dependent mitochondrial Bcl-xl in rat.Chin J Physiol 2020;63:68-76

How to cite this URL:
Chou PL, Chen KH, Chang TC, Chien CT. Repetitively hypoxic preconditioning attenuates ischemia/reperfusion-induced liver dysfunction through upregulation of hypoxia-induced factor-1 alpha-dependent mitochondrial Bcl-xl in rat. Chin J Physiol [serial online] 2020 [cited 2020 Jun 1 ];63:68-76
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Full Text


Under hypoxic/ischemic conditions, severe hypoxia/ischemia leads to cell and tissue impairment. However, the tolerance resistant to hypoxia/ischemia can be achieved by hypoxic preconditioning (HP),[1] ischemic preconditioning,[2],[3] and hyperthermic preconditioning.[4] The mechanisms of these preconditioning methods possibly involved the release of adenosine, prostacyclin, reactive oxygen species (ROS), and Akt signaling transmitters.[5],[6],[7],[8] The single hypoxic or ischemic preconditioning technique exerts mitochondrial-independent and mitochondrial-dependent protective pathways to the following ischemic damage.[8],[9],[10],[11],[12] Furthermore, repetitive HP can afford efficiently and significantly longer protection than single preconditioning.[6],[13]

The mitochondrion is an important cellular component responsible for regulating energy, oxidative metabolism, and adaption to hypoxic/ischemic condition. Mitochondrial dysfunction following ischemia/reperfusion (I/R) injury can induce apoptotic cell death, since the mitochondrial apoptogenic factor, cytochrome C, is released into the cytoplasm to activate caspase-3-mediated apoptosis.[14] Cytochrome C leakage to cytosol can be triggered by pro-apoptogenic Bax/Bad[14],[15] and inhibited by Bcl-2/Bcl-xL[16] and heat shock protein 70 (HSP70).[17] On the other hand, hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1β and HIF-1α subunits,[18] enhances protective genes, including Bcl-2,[7] Bcl-xL,[19] erythropoietin, HSP70,[13] and vascular endothelial growth factor (VEGF)[1],[20] and protects against oxidative injury. HIF-1α activation could also downregulate the proapoptotic Bax/Bid/Bad expression.[19],[21] Under hypoxia, HIF-1α is upregulated to high levels at 3–6 h and decreased thereafter.[22] Our previous study confirmed that HP reduced renal I/R injury through HIF-1-mediated Bcl-2[23] or HSP70[13] protective pathways. HP can promote the survival of cardiac progenitor cells because HP time dependently enhances the Pim-1 kinase to inhibit proapoptotic factors and restores mitochondria Bcl-2/Bcl-xL to decrease mitochondrial injury.[19] In addition, long-term cycles of hypoxia/normoxia conditioning decreased oxidative stress and liver dysfunction but increased the antioxidant superoxide dismutase levels and improved mitochondrial respiratory function in the rat liver.[24] We hypothesized that HP might induce mitochondrial protection to reduce hepatic I/R injury-induced oxidative injury via HIF-1α-dependent pathway. In clinical trials, the expression of the cytoprotective protein heme oxygenase-1 is significantly upregulated in the liver within minutes of intermittent clamping of the porta hepatis, which is commonly performed during liver surgery to reduce blood loss and has been reported to precondition livers, resulting in improved outcome after liver surgery in humans.[25] Large population studies have displayed that living at higher altitudes with lower oxygen exposure is associated with the reduced cardiovascular disease mortality by the role of HIF-1α in increasing anti-inflammatory IL-10 transactivation.[26]

In this study, we address the possible role of HIF-1α and several antioxidant and antiapoptotic proteins in HP-induced protective mechanism in rat livers.

 Materials and Methods

Repetitive hypoxic preconditioning

Female Wistar rats (200–250 g) were purchased from BioLASCO Taiwan Co. Ltd. (Taipei) and housed at the Experimental Animal Center of National Taiwan University with constant temperature and consistent light cycle (light from 07:00 to 18:00). The animal care and experimental protocols were approved by the National Taiwan University of Public Health Institutional Animal Care and Use Committee (IACUC ethic code No: 20040321) and were in accordance with the guidelines of the Ministry of Science and Technology of the Republic of China. The HP induction was performed as described previously.[27] In brief, the animals were placed in a simulated high altitude chamber (HP) 15 h/day, whereas age-matched control animals were maintained at sea level (normoxic group, SL). We selected the 380 torr (5500 m) for hypoxic induction, because, at this altitude, these rats can well adapt. The body weight of the animals was determined every week. Food and water were provided ad libitum.

YC-1 on hypoxic preconditioning-induced hypoxia-induced factor-1 alpha and Bcl-xL

YC-1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole, was provided by Prof. C. M. Teng, Department of Pharmacology, College of Medicine, National Taiwan University. YC-1 was developed for inhibiting HIF-1 activity in vitro and in vivo.[13] We examined the effects of YC-1 on HP-enhanced Bcl-xL protein levels. We pretreated the HIF-1α inhibitor YC-1 (3.2 μM) intraperitoneally (i.p) before HP induction.

Induction of liver ischemia/reperfusion injury

We followed a surgical procedure as described previously.[14] The rats were anesthetized with urethane (50 mg/kg, i.p.) and were tracheotomized. We catheterized one PE50 tubing to the femoral arterial for arterial blood pressure recording and blood sampling. One PE50 tubing was introduced to induce ischemia in the liver; the portal vein was clamped for 45 min with a small vascular clamp. Sham-operated animals underwent similar operative procedures, without occlusion of the portal vein. Reperfusion was done by removing the clamp for 6 h. After experiment, these rats were sacrificed with intravenous KCl and the blood was collected. The livers were removed and washed with cold saline. One part was stored in 10% formalin and the other was frozen in liquid nitrogen and stored at −70°C.

Determination of myeloperoxidase activity, plasma alanine aminotransferase, and aspartate aminotransferase

The liver samples were used to assay the myeloperoxidase (MPO) activity by the use of H2O2 and o-dianisidine as described previously.[28] The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were evaluated using commercial kits (Sigma, St. Louis, MO, USA).

Western blot

Mitochondrial Bcl-xL protein downregulation and mitochondrial leakage of cytochrome C to cytosol are important indexes for triggering apoptotic pathway.[29] Total homogenized proteins from livers were subjected to differential centrifugation to obtain the mitochondrial and cytosolic fractions. Protein concentration was determined by a BioRad protein assay (BioRad Laboratories). Ten micrograms of protein was electrophoresed as described below. The primary antibody of polyclonal rabbit antihuman cytochrome C (Santa Cruz), mouse antihuman Bcl-xL (Santa Cruz), goat polyclonal heat shock protein 60 (HSP60, Santa Cruz), and β-actin (Sigma) was used.

Nuclear extracts from rat livers were prepared as described previously.[30] The antibodies raised against HIF-1α (BD Biosciences), Bad (Chemicon), and Bcl-xL and the activation fragments (32 kD of proenzyme and 17 kD of cleaved product) of caspase 3 (Upstate Biotechnology), poly-(ADP-ribose)-polymerase (PARP) (Promega), MnSOD and CuZnSOD (Enzo Life Sciences), catalase (Calbiochem), and β-actin (Sigma) were used. The density of the band with the appropriate molecular mass was determined semiquantitatively by densitometry using an image analyzing system (Alpha Innotech).

In vivo hepatic reactive oxygen species and in vitro bile reactive oxygen species determination

The ROS generation in response to hepatic I/R injury was determined from the liver surface in vivo and bile in vitro by our developed method, as described in detail previously.[28] The bile was collected from one PE-50 tubing in the bile duct. The measurement of bile ROS was also detected by a lucigenin-amplified method, as previously described.[28] The ROS signal was monitored continuously for an additional 300 s. The total amount of lucigenin ROS chemiluminescence was calculated by integrating of the area under the curve and subtracting it from the background level.

In situ demonstration of nitroblue tetrazolium, 3-nitrotyrosine, ED-1, and apoptosis

A nitroblue tetrazolium (NBT) perfusion method was used for localizing de novo ROS generation in the liver.[28] The value of blue NBT deposits/total section area was counted by Adobe Photoshop 7.0.1 image software analysis.

We suggest that I/R injury might increase hepatic oxidative stress, monocyte/macrophage (Kupffer cells) infiltration, and apoptotic cellular death in the liver. We evaluated the level of oxidative stress by 3-nitrotyrosine (3-NT) accumulation,[31],[32] Kupffer cell infiltration, and apoptosis formation[28] in the paraffin-embedded sections of hepatic tissues with immunocytochemistry. The hepatic sections were deparaffinized, rehydrated, and stained immunohistochemically for the presence of 3-NT (Alpha Diagnostic International) diluted at 1:50. The percentage of 3-NT was calculated as 3-NT-stained area/total area ×100% and analyzed by Adobe Photoshop 7.0.1 image software analysis.

The method for the terminal deoxynucleotidyl transferase-mediated nick-end labeling method (TUNEL) was performed as described previously.[31]

For hepatic monocyte/macrophage (Kupffer cells, ED-1) staining, the tissue sections were incubated overnight at 4°C with a mouse antirat antibody to ED-1 (CD68, Serotec). A biotinylated secondary antibody (Dako) was then applied followed by streptavidin conjugated to HRP (Dako). Twenty high-power (×200) fields were randomly selected for each liver section, and the value of Kupffer cells determined by ED-1-positive cells was counted.

DNA laddering

The analysis of DNA laddering was performed as previously described.[31]

Statistical analysis

All these values were expressed as mean ± standard error of the mean. Differences within groups were evaluated by paired t-test. One-way analysis of variance was applied to compare differences among groups. Intergroup comparisons were analyzed with Duncan's multiple-range test. A P< 0.05 was considered statistically significant.

Ethics approval statement

All studies in this article have received the institutional review board and ethics approval.


Hypoxic preconditioning evokes an increase in nuclear hypoxia-inducible factor-1 alpha protein expression in livers

We determined whether HP enhanced HIF-1α expression in the livers by exploring nuclear HIF-1α protein in the rat livers under different time schedules of HP (10%). Our results demonstrated that after 1 week of our developed HP model, HIF-1α expression was significantly upregulated and maintained at the high level during 1–3 weeks of HP in rat livers [[Figure 1]A, [Figure 1]A-1].{Figure 1}

Hypoxic preconditioning evokes an increase in antiapoptotic and antioxidant protein expression in livers

We further examined the antiapoptotic protein expression of Bcl-xL and antioxidant proteins, MnSOD, CuZnSOD, and catalase in the livers from SL and HP kidneys by Western blot. [Figure 1]B and B1 to B4 demonstrate that the expression of hepatic Bcl-xL [Figure 1]B, [[Figure 1]B-1], MnSOD [Figure 1]B, [[Figure 1]B-2], CuZnSOD [Figure 1]B, [[Figure 1]B-3], and catalase [Figure 1]B, [[Figure 1]B-4] was significantly enhanced and well maintained during 1–3 weeks of HP.

Hypoxic preconditioning enhances hypoxia-inducible factor-1 alpha-dependent Bcl-xL expression in the rat livers

As shown in [Figure 2], we determined the effect of HIF-1α inhibitor YC-1 on nuclear HIF-1α and total proteins of hepatic Bcl-xL expression in the rat livers. [Figure 2] shows that YC-1 treatment significantly depressed HIF-1α and Bcl-xL expression in response to different time schedule of HP in the livers.{Figure 2}

Hypoxic preconditioning decreases ischemia/reperfusion injury by attenuating liver and bile reactive oxygen species

The basal level of hepatic ROS was similar between SL and HA rats. In response to hepatic I/R injury, the level of liver ROS [Figure 3]a and [Figure 3]b and bile ROS [Figure 3]c was significantly increased after 6 h of reperfusion as compared to SL or HP control. HP treatment significantly depressed I/R-enhanced parameters.{Figure 3}

Hypoxic preconditioning decreases ischemia/reperfusion injury by enhancing liver myeloperoxidase, alanine aminotransferase, and aspartate aminotransferase levels

In response to hepatic I/R injury, the level of liver MPO activity [Figure 4]a, plasma ALT concentration [Figure 4]b, and plasma AST concentration [Figure 4]c was significantly increased after 6 h of reperfusion as compared to SL or HP control. HP treatment significantly depressed I/R-enhanced parameters.{Figure 4}

Hypoxic preconditioning efficiently inhibited ischemia/reperfusion injury-induced mitochondrial dysfunction

In [Figure 5], HP significantly increased the basal level of Bcl-xL in the total proteins and mitochondrial proteins. I/R injury significantly upregulated Bad [Figure 5]a, CPP32 [Figure 5]c, and PARP [Figure 5]d expression but decreased Bcl-xL [Figure 5]b expression in the SL rat livers. HP significantly upregulated baseline Bcl-xL expression and thus inhibited the increased level of Bad, CPP32, and PARP in the HP livers. We further determined the HP effect on mitochondrial function. HP inhibited Bad translocation by I/R injury [Figure 5]a and caused restoration of mitochondrial Bcl-xL [Figure 5]e and reduction of cytosolic cytochrome C levels [Figure 5]f.{Figure 5}

Hypoxic preconditioning efficiently inhibited ischemia/reperfusion-induced oxidative stress, inflammation, and apoptosis

As shown in [Figure 6], I/R increased de novo production of oxidative stress in blue NBT deposits, brown 3-NT stain [Figure 6]a,[Figure 6]b,[Figure 6]c,[Figure 6]d, Kupffer cells infiltration [Figure 6]e,[Figure 6]f,[Figure 6]g,[Figure 6]h, and apoptosis [Figure 6]i, [Figure 6]j, [Figure 6]k, [Figure 6]l formation in the in the SL rat livers. However, these I/R-enhanced oxidative stress [Figure 6]m, Kupffer cell number [Figure 6]n, and TUNEL-positive cells [Figure 6]o were significantly depressed by HP in the HP rat livers. I/R markedly induced DNA laddering in SL rats, whereas HP efficiently inhibited I/R-induced DNA laddering [Figure 6]p.{Figure 6}


HP increases survival of cardiac progenitor cells and survival and engraftment in the heart through inhibition of proapoptotic elements (cytochrome C and cleaved caspase-3), preservation of the mitochondria Bcl-2 and Bcl-xL, subsequently leading to attenuate mitochondrial damages.[19] Several lines of evidence indicated that HP treatment increases tolerance to I/R injury insults by upregulation of Bcl-2 in the kidney[33] and in the bladder,[27] HSP70 in the kidney,[13] and Bcl-xL protein expression in the brain,[34] subsequently resulting in the reduction of apoptosis and oxidative injury. Our present data further indicated that HP significantly enhanced hepatic Bcl-xL, MnSOD, Cu/ZnSOD, and catalase expression. Our data further evidenced that by the use of YC-1, a HIF-1α inhibitor Bcl-xL enhancement, was dependent on HIF-1α activation. These data could confirm previous finding that hypoxia through HIF-1α induced Bcl-xL expression, leading to antiapoptosis.[35] Furthermore, the upregulated Bcl-xL expression associated with the downregulated Bax expression. All these data implicated that HP evokes a systemically and wide range protection to reduce oxidative stress in several organs/tissues.

It is important to evaluate the possible application potential for future. Our previous study indicated that 4 weeks of simulated 18,000-feet (5500 m) hypoxia led to polycythemia, which causes right ventricular hypertrophy and pulmonary hypertension by an increased pulmonary vascular resistance.[36] According to our data [Figure 1], 1 week of HP can induce a protective capability like 4 weeks in the induction of HIF-1α, Bcl-xL, MnSOD, Cu/ZnSOD, and catalase. We suggest that 1 week of HP induction is enough to induce HIF-1α-related defensive mechanism in the liver and can avoid the possible adverse effect on pulmonary vasoconstriction and right ventricular hypertrophy. Cumulated evidence from basic researches also demonstrated that the HP technique can afford protective potential against oxidative injury to the kidney,[13],[23] urinary bladder,[27] brain,[34] and cardiac progenitor cells.[19] It is also possible that the application of selective HIF-1α inducer may be available to induce several antioxidant and antiapoptotic proteins in the target organ against I/R injury during surgery.

Augmented mitochondrial Bcl-2[35] or Bcl-xL[32] protein expression by intrarenal adenoviral bcl-2 or bcl-xL gene transfer protects the kidney cells from I/R injury via the antioxidant and antiapoptotic actions. In the present study, we found that HP through a hypobaric hypoxia enhanced hepatic HIF-1α protein expression, which subsequently increased hepatic and mitochondrial Bcl-xL expression in the livers. Our data further informed that the upregulation of mitochondrial Bcl-xL inhibited cytosolic Bad translocation to mitochondria and mitochondrial cytochrome C release to cytosol in response to I/R injury. Upregulation in the HIF-1α/Bcl-xL by HP decreases cytosolic Bad translocation and mitochondrial cytochrome C release and attenuates ROS production in I/R injury liver.

HIF-1 plays a central physiological role in oxygen and energy homeostasis and is activated during hypoxia by stabilization of the subunit HIF-1α. The PI3K/Akt pathway was required for hypoxia-induced expression of HIF-1α and VEGF, whereas the MEK/ERK pathway was required only for VEGF in laser-induced rat choroidal neovascularization.[33] Our previous study reported that HP would enhance HIF-1α-dependent HSP70 in the renal tubular cells and kidney and this upregulated HSP70 protected the kidney against renal I/R injury.[13] Hypoxia induces a group of physiologically important genes that include erythropoietin and VEGF. Osada et al.[37] found that a flavoprotein, NADPH-P450 reductase located at the plasma membrane, could induce erythropoietin mRNA under hypoxic conditions. Cycling hypoxia induced Bcl-xL expression via the ROS-mediated HIF-1α mechanism, leading to antiapoptosis and chemoresistance, and the use of Bcl-xL knockdown and Tempol treatment inhibited hypoxia-induced chemoresistance.[35] Two clinical trials with 68 subjects reported ischemic preconditioning conferred protection after hepatic I/R injury based on the higher level of HIF-1α in ischemic preconditioning group compared with control group.[38] Ischemic preconditioning in clinical trials also significantly improved biochemical markers of liver function and HIF-1α and reduced the need for reoperation in the postoperative period in the donor liver transplantation.[39] As far we know, the technique of HP has not yet been done in the clinical trials. Luks et al.[40] reported that to improve the quality of healthcare, people with chronic medical conditions are experiencing better quality of life and increasingly participating in a wider capacity for activities, including travel to high altitude or hypoxic exposure. In their review, the liver transplant (liver I/R) patients may tolerate high altitude (hypoxia) exposure without difficulty. It should be determined carefully the duration, the frequency, and the degree of hypoxia in future.

In recent studies, it is indicated that there are two subtypes of macrophages in liver, Kupffer cells, and monocyte-derived macrophages (MDMs). Kupffer cells belong to yolk-sac derived resident macrophage, and MDMs differentiate from bone marrow-derived or circulating monocytes. Under homeostatic conditions and liver injury, Kupffer cells are refilled by local proliferation of mature Kupffer cells. However, while Kupffer cells depletion, MDMs are recruited to the Kupffer cell niche and differentiate into Kupffer cells.[41],[42],[43] It has been reported that Kupffer cells and MDMs express distinct transcriptomes.[44],[45] Moreover, it reported recently that Kupffer cells and MDMs possess differential sensitivity to bacterial LPS.[46] While response to LPS, MDMs express more TNFα, Cxcl1, and Cxcl2 than Kupffer cells do.[46] It indicates that MDMs have more proinflammatory phenotypes. The markers in Kupffer cells are F4/80high CD11blow Cx3crllow whereas F4/80low CD11bhigh Cx3crlhigh in MDMS.[46] In this study, we detected Kupffer cell infiltration with monoclonal antibody, ED-1, to against CD68. CD68 is a pan marker for the various macrophage lineages, including monocytes, Kupffer cells, and osteoclasts. We found that I/R injury increased CD68-positive cells [Figure 6]g and [Figure 6]n while it significantly depressed by HP [Figure 6]h and 6n] in livers. We considered these CD68-positive cells as resident Kupffer cells; however, the proper subtypes of these CD68-positive cells need to be further elucidated.


In summary, I/R injury is frequently occurred during hepatic surgical procedures. Our results show that HP via the upregulation of HIF-1α-dependent Bcl-xL protein protects the liver against subsequent I/R injury by inhibition of oxidative stress, inflammation, and apoptosis. HP through pAkt/pERK-mediated pathways upregulates HIF-1α-dependent Bcl-xL protein expression in the livers. The upregulation of mitochondrial Bcl-xL could inhibit I/R-induced mitochondrial Bad and cytosolic cytochrome C translocation and subsequently reduce oxidative stress, inflammation, and apoptosis in the I/R livers.

Financial support and sponsorship

This work was supported in part by the National Science Council of the Republic of China (NSC96-2320-B-002-007 and NSC96-2221-E-002-256-MY3).

Conflicts of interest

There are no conflicts of interest.


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