Phenylbutyrate

4-Phenylbutyrate Benefits Traumatic Hemorrhagic Shock in Rats by Attenuating Oxidative Stress, Not by Attenuating Endoplasmic Reticulum Stress

Objective: Vascular dysfunction such as vascular hyporeactivity following severe trauma and shock is a major cause of death in injured patients. Oxidative stress and endoplasmic reticulum stress play an important role in vascular dysfunction. The objective of the present study was to determine whether or not 4-phenylbutyrate can improve vascular dysfunction and elicit antishock effects by inhibiting oxidative and endoplasmic reticulum stress.

Design: Prospective, randomized, controlled laboratory experi- ment.

Setting: State key laboratory of trauma, burns, and combined injury.

Subjects: Five hundred and fifty-two Sprague-Dawley rats. Interventions: Rats were anesthetized, and a model of traumatic hemorrhagic shock was established by left femur fracture and hemorrhage. The effects of 4-phenylbutyrate (5, 20, 50, 100, 200, and 300 mg/kg) on vascular reactivity, animal survival, hemody- namics, and vital organ function in traumatic hemorrhagic shock rats and cultured vascular smooth muscle cells, and the relation- ship to oxidative stress and endoplasmic reticulum stress was observed.

Measurements and Main Results: Lower doses of 4-phenylbu- tyrate significantly improved the vascular function, stabilized the hemodynamics, and increased the tissue blood flow and vital organ function in traumatic hemorrhagic shock rats, and markedly improved the survival outcomes. Among all dosages observed in the present study, 20 mg/kg of 4-phenylbutyrate had the best effect. Further results indicated that 4-phenylbutyrate significantly inhibited the oxidative stress, decreased shock-induced oxidative stress index such as the production of reactive oxygen species, increased the antioxidant enzyme levels such as superoxide dis- mutase, catalase, and glutathione, and improved the mitochondrial function by inhibiting the opening of the mitochondrial permeabil- ity transition pore in rat artery and vascular smooth muscle cells. In contrast, 4-phenylbutyrate did not affect the changes of endo- plasmic reticulum stress markers following traumatic hemorrhagic shock. Furthermore, 4-phenylbutyrate increased the nuclear levels of nuclear factor-E2–related factor 2, and decreased the nuclear levels of nuclear factor κB in hypoxic vascular smooth muscle cells.

Conclusions: 4-phenylbutyrate has beneficial effects for trau- matic hemorrhagic shock including improving animal survival and protecting organ function. These beneficial effects of 4-phenyl- butyrate in traumatic hemorrhagic shock result from its vascular function protection via attenuation of the oxidative stress and mitochondrial permeability transition pore opening. Nuclear factor-E2–related factor 2 and nuclear factor-κB may be involved in 4-phenylbutyrate-mediated inhibition of oxidative stress. (Crit Care Med 2016; 44:e477–e491) approaches for patients with vascular hyporesponsiveness remain the major research priorities.

Key Words: 4-phenylbutyrate; endoplasmic reticulum stress; mitochondria; oxidative stress; traumatic hemorrhagic shock; vascular hyporeactivity

Vascular hyporesponsiveness is a severe vascular dysfunction that is manifested in association with multiple critical illnesses, such as severe trauma, shock, and sepsis, which is character- ized by a reduced vascular reactivity to vasoconstrictors and vasodilators. Previous studies in our laboratory and others have demonstrated that improving vascular hyporesponsive- ness can be beneficial in shock patients (5–8). However, the mechanisms responsible for vascular hyporesponsiveness and effective prevention and treatment measures are in need of fur- ther investigation.

4-phenylbutyrate (PBA) is a low-molecular weight fatty acid that has been approved for the treatment of urea cycle dis- orders (UCDs) as an ammonia scavenger (9). More recently, PBA has been shown to provide protection against various noxious stimuli, such as ischemic injury, diabetes, cancer, and neurodegenerative diseases (10–12). The multiple actions of PBA are mainly attributed to its multiple biological activities, including ammonia scavenging, endoplasmic reticulum (ER) stress inhibition, histone deacetylase (HDAC) inhibition, and antioxidative effect. Among them, the antioxidative and anti- ER stress activities are generally accepted as the main mecha- nisms of the protective effect of PBA (10, 13).

Oxidative stress is defined as oxidant/antioxidant imbal- ance. It is known that hemorrhage and resuscitation may cause oxidative stress, which plays a key role in the development of organ injuries (e.g., liver, kidney, lung, brain, and heart) and death of injured patients (14–16). ER stress is another novel and fundamental mechanism for various cardiovascular dis- eases (17). Various factors, including ischemia, hypoxia, oxida- tive stress, and infections, may trigger ER stress. Several studies have reported that ER stress plays a fundamental role in the development and progression of multiple diseases, including ischemic heart disease, atherosclerosis, cancer, and diabetes (17, 18). Furthermore, accumulating evidence has suggested a close link between oxidative stress and ER stress, in which there is a direct link between the production of reactive oxygen spe- cies (ROS) and ER dysfunction. Oxidative stress and ROS may initiate and be a major contributor to ER stress, and ER stress can also induce oxidative stress, depending on the experimen- tal model and pathologic process (19, 20). Some studies have shown that PBA exerts a protective effect against oxidative or ER stress injuries in models of ischemia, cancer, or neurode- generative diseases (21, 22). It is not known, however, whether or not PBA exerts protective effects on vascular function and traumatic hemorrhagic shock via antioxidative and ER stress.

Mitochondria are the key organelles that play a criti- cal role in regulating cell function and determining cell fate. Recent studies have shown that there is a tight link between mitochondria, oxidative stress, and ER stress. It is known that mitochondria are the main source of ROS production, and increased ROS damages mitochondrial biogenesis and causes mitochondrial dysfunction (23). Some studies indicate that ER and mitochondria form a direct contact between their surfaces, and these ER-mitochondria interactions have pivotal roles in numerous cellular functions (24). It is suggested that mito- chondria may be the common target of oxidative stress and ER stress. The mitochondrial permeability transition pore (MPTP) is a high-conductance pore located in the inner mitochondrial membrane, which will open due to ischemia or other stresses and cause disruption of mitochondrial function and structure, thus resulting in cell dysfunction or cell death (25). Miki et al (26). reported that increased ER stress in the diabetic myocar- dium impairs some protective signal-mediated inhibition of MPTP opening and facilitates myocardial necrosis and apop- tosis after ischemia/reperfusion. Our recent data showed that cyclosporine A (an inhibitor of MPTP) may restore the vascu- lar hyporeactivity in septic shock (unpublished observations). Therefore, we hypothesize that PBA may improve shock- induced vascular dysfunction and have an antishock effect through oxidative stress and/or ER stress and the associated mitochondrial pathway. To test this hypothesis, we determined whether or not PBA is beneficial for traumatic hemorrhagic shock by improving vascular and other organ function and investigated the relationship with oxidative stress and ER stress, and the underlying mechanism using traumatic hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs). Meanwhile, to know the clinical application poten- tial of PBA, we further compared the effects of norepineph- rine (a first-line recommended vasoactive agent for refractory shock in clinic) versus norepinephrine + PBA, and observed the effects of different administration time in the treatment of traumatic hemorrhagic shock rats.

MATERIALS AND METHODS

The current study conformed to the principles of the “Guide for the Care and Use of Laboratory Animals” (eighth edi- tion, 2011, National Academies Press, Washington, DC) and was approved by the Research Council and Animal Care and Use Committee of the Research Institute of Surgery (Daping Hospital, Third Military Medical University, Chongqing, P. R. China).

Animal Preparation

Five hundred and fifty-two male and female Sprague-Dawley rats (SD) (180–220 g) were used in the current study. The rats were anesthetized with sodium pentobarbital (initial dosage, 30 mg/kg intraperitoneal) and Jingsongling (xylidinothiazole; initial dosage, 0.1 mg/kg IM), then the two drugs were added until the rats had no response to a needle stimulus. The right femoral artery and vein were catheterized with a polyethylene catheter filled with heparinized saline for monitoring the mean arterial pressure (MAP)/bleeding and drug administration, respectively. The right carotid artery and vein were catheter- ized for monitoring hemodynamics or cardiac output (CO).

A traumatic hemorrhagic shock model was prepared as previously described (27). Briefly, the left femur fracture was made, and hemorrhage was induced via the right femoral artery catheter until the MAP decreased to 40 mm Hg. When this pressure was maintained for 3 hours, traumatic hemor- rhagic shock was established for subsequent experiments. At the end of all experiments, the animals were euthanatized with a pentobarbital-based euthanasia solution (Sleepaway, 2 mL IV; Fort Dodge Laboratories, Fort Dodge, IA) via femoral vein.

Cell Culture and Hypoxia Treatment

VSMCs were obtained from the superior mesenteric arteries (SMAs) of 40 SD rats using an explant technique, as previ- ously described (27). VSMCs were cultured in Dulbecco-mod- ified Eagle medium-F12 supplemented with 20% fetal bovine serum (Hyclone, Logan, UT) and 1% antibiotics. The third-to- fifth passage cells were used in the present study. For hypoxic experiments, VSMCs were transferred into a hypoxia culture compartment (MIC-101, Billups-Rothenberg, Del Mar, CA) equilibrated with 95% N2 and 5% CO2, in which the estimated
oxygen concentration was less than 0.2% (28). Under hypoxic conditions, VSMCs were incubated for 3 hours, and then used for subsequent experiments.

Experimental Protocol

The in vivo experiments consists of three phases. Phase I was the traumatic hemorrhagic period (shock model stage), in which the femur was fractured and the MAP was main- tained at 40 mm Hg for 3 hours. Phase II was the period of PBA (Sigma-Aldrich, St. Louis, MO) administration. When the shock model was established (at the end of Phase I), rats received a continuous infusion of PBA (5, 20, 50, 100, 200, or 300 mg/kg) with two volumes of blood loss of lactated Ring- er’s (LR) solution within 30 minutes. Phase III was the obser- vation period, in which animal survival, hemodynamics, CO, blood gases, and vital organ and mitochondrial function were observed (Fig. 1).
All experiments were performed in five parts: part 1, effects of PBA on survival in traumatic hemorrhagic shock rats; part 2, systemic effects of PBA; part 3, the organ effects of PBA; part 4, the mechanisms of PBA in the protection of vascular and other vital organ function (molecular and organelle effects of PBA); and part 5, the clinical application potential evaluation of PBA. (detailed outline in supplemental material, Supplemental Digital Content 1, http://links.lww.com/CCM/B503).

Part 1: Effects of PBA on Survival in Traumatic Hemorrhagic Shock Rats. The first part was aimed to investigate whether PBA is beneficial for traumatic hemorrhagic shock. Ninety-six SD rats were randomly divided into six groups (n = 16 per group), as follows: normal control (sham-operated), shock control,shock + LR, and shock + LR + PBA (5, 20, and 50 mg/kg). At the end of shock, the animals in the PBA groups received an infusion of PBA (5, 20, or 50 mg/kg) in two volumes of LR. The LR group received an equal volume of LR. The animals in the shock control group did not receive any treatment after shock. Sham-operated animals underwent the same operation, but not hemorrhaged. After the treatment period, the cathe- ters were removed, the incisions were closed, and the animals received analgesia with xylidinothiazole (0.2 mg/kg intramus- cular every 6 hr). The animal survival time and 24-hour sur- vival rate were recorded.

Figure 1. Timeline of the experimental phases. Phase I: the traumatic hemorrhagic shock model stage. Phase II: the treatment period with lactated Ringer or 4-phenylbutyrate (PBA). Phase III: the observation period, to 24 hr (animal survival) or 2 hr after resuscitation (hemodynamic parameters, etc).

Part 2: Systemic Effects of PBA on Hemodynamics, Cardiac Function, and Blood Gases. The second part was aimed to investigate the systemic effects of PBA, including effects on hemodynamics, cardiac function, and blood gases (acid-base balance in vivo). Forty-eight SD rats were randomly divided into six groups (n = 8 per group) treated identically as part 1: sham-operated, shock control, shock + LR, and shock
+ LR + PBA (5, 20, and 50 mg/kg). At baseline, at the end of phase I (shock model stage), and at 1 and 2 hours in phase III (observation period), the hemodynamics and CO were recorded, and blood sample was withdrawn for the measure- ment of blood gases. Hemodynamic parameters including left intraventricular systolic pressure (LVSP) and the maximal change rate of left intraventricular pressure (± dp/dtmax) were
determined with a polygraph physiologic recorder (SP844; AD Instruments, Castle Hill, Australia). CO was assessed using a Cardiomax-III Thermodilution Cardiac Output System (Columbus Instruments, Columbus, OH) (3). Blood gases were determined using a blood gas analyzer (Phox plus L; Nova Biomedical, Waltham, MA).

Part 3: Organ Effects of PBA. The third part was aimed to investigate the organ protection of PBA including the vascular function (vascular reactivity) and liver and kidney function. Forty-eight SD rats were randomly divided into six groups (n = 8 per group): sham-operated, shock, LR, PBA 5, 20, and 50 mg/kg. The animal management and treatment were the same as part 1. At 2 hours after resuscitation, the rats under- went a laparotomy, and the blood flow in the liver and kidney was measured by a laser Doppler blood flowmeter (Feriflux system 5000; Perimed, Stockholm, Sweden). Blood sample was withdrawn for determination of liver and kidney function, including aspartate aminotransferase (AST), alanine amino- transferase (ALT), blood urea nitrogen (BUN), and serum creatinine (SCr) by a biochemical analyzer (Beckman, Fuller- ton, CA). Thereafter, animals were euthanatized by a venous injection of a pentobarbital-based euthanasia solution as described above, and liver and kidney tissues were removed to measure mitochondrial function with a mitochondrial func- tion analyzer (MT 200; Strathkelvin, Lanarkshire, Scotland). Mitochondrial function was represented by the respiration control rate (the ratio of oxygen consumption in the presence or absence of substrate and adenosine diphosphate), as previ- ously described (29).

Fifty-six SD rats were randomly divided into seven groups (n = 8 per group): sham-operated, shock, LR, PBA (5, 20, and 50 mg/kg); and apocynin (an antioxidant; 10 mg/kg; Sigma). At 2 hours after resuscitation, the SMAs (SMAs) were obtained for the measurement of vascular contraction and dilation function to norepinephrine (GrandPharma, Wuhan, China) and acetylcholine (Sigma) using an isolated organ perfusion system (Scientific Instruments, Barcelona, Spain), as described previously (27). The cumulative concentration-response curves of SMAs to norepinephrine or acetylcholine were then constructed. The vasoconstriction reactivity of SMAs was reflected by a percentage of the increased vascular contractile tension induced by norepinephrine to the 124 mmol/L K+- induced reference contraction, and the vasodilator reactivity was expressed as the percentage of the decreased tension by acetylcholine to the increased tension by norepinephrine.
Part 4: The Mechanism of PBA in Protection of Vascular and Other Organ Function: Molecular and Organelle Effects of PBA. The fourth part was aimed to investigate the protec- tive mechanisms of PBA on vascular function and other vital organ function including the relationship to oxidative stress, ER stress, and MPTP opening.

Part 4.1. Effects of PBA on oxidative stress biomarkers in vivo and in vitro experiments: the blood samples and SMA tis- sues from rats in experiment part 3 were collected at 2 hours after resuscitation. The levels of oxidative stress biomarkers including malondialdehyde, nitric oxide (NO), superoxide dis- mutase (SOD), catalase, and glutathione were detected with commercial assay kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. To further determine the role of PBA on oxidative stress, the ROS levels in SMAs from shock rats of experiment part 3 and hypoxia/PBA-treated VSMCs were measured. In vitro experi- ments, cultured VSMCs were divided into five groups: normal control, positive control (ROSup, the compound mixture that inducing ROS production, 50 μg/mL, 30 min; Beyotime Institute of Biotechnology, Haimen, China); 3-hour hypoxia; 3-hour hypoxia + PBA (10 mmol/L, 1 hr); and 3-hour hypoxia + apocynin (100 μmol/L, 1 hr). Intracellular ROS levels in rat arteries and VSMCs were measured using the 2′,7′-dichloro- fluorescin diacetate (DCF-DA) method (30). Briefly, SMAs or VSMCs were incubated with 10 μmol/L of DCF-DA (Sigma) for 20 minutes at 37°C. DCF fluorescence was detected at 488- nm excitation and 525-nm emission by a Leica TCS SP5 con- focal system (Leica Microsystems, Wetzlar, Germany). Images were collected, and the mean intensities of DCF fluorescence were calculated using Leica TCS software.

Part 4.2. Effects of PBA on ER stress in vivo and in vitro experiments: to determine the role of PBA on ER stress, the protein expression of ER stress biomarkers including 78-kDa glucose-regulated protein (GRP78), inositol-requiring enzyme (IRE)-1α, pancreatic ER kinase (PERK), and activating tran- scription factor (ATF)-6 in SMAs from shock rats and hypoxia/ PBA-treated VSMCs were determined by western blot (27). Forty-eight SD rats were randomly divided into six groups (n = 8 per group): sham-operated, shock, LR, PBA 5, 20, and 50 mg/ kg. The animal management and treatment were the same as part 1, and the SMAs were collected at 2 hours after resuscitation.

In vitro experiments, VSMCs were divided into three groups: normal control, 3-hour hypoxia, and 3-hour hypoxia + PBA (10 mmol/L, 1 hr). After treated with hypoxia and/or PBA, VSMCs were collected for the measurement of the expression of ER stress markers. The following antibodies were used in the measurement: GRP78 (1:1,000; Cell Signaling, Danvers, MA, USA), IRE-1α (1:200; Santa Cruz Biotechnology, Santa Cruz, CA); PERK (1:1,000; Cell Signaling), ATF-6 (1:1,000; Abcam, Cambridge, MA); and β-actin (1:5,000; Sigma).

Part 4.3. Effects of PBA on MPTP opening and intracellular calcium concentration in VSMCs: to investigate the relation- ship between the MPTP opening and the beneficial effects of PBA on vascular function, the effects of PBA and atractylo- side (ATR, a MPTP opener; Sigma) on MAP and the pressor response of norepinephrine (reflecting the vascular reactivity in vivo) in traumatic hemorrhagic shock rats, and the effects of PBA on MPTP opening in hypoxia-treated VSMCs were observed. For in vivo experiment, 24 SD rats were randomly divided into three groups (n = 8 per group): shock + LR + PBA (20 mg/kg), shock + LR + ATR (5 mg/kg), and shock + LR + ATR + PBA. At the end of shock, rats in shock + LR + ATR + PBA group received an infusion of ATR with one volume of blood loss of LR, and then PBA was infused along with another one volume of LR. Rats in shock + LR + PBA and shock + LR + ATR groups were given PBA or ATR in two volumes of blood loss of LR. MAP and the pressor response of norepinephrine were observed at baseline, at the end of phase I, and at 1 and 2 hours in phase III. In vivo, the pressor response was presented as the increase of MAP after administration of norepineph- rine (3 μg/kg bolus IV injection) (31). For in vitro experi- ments, VSMCs were divided into three groups: normal control, 3-hour hypoxia; and 3-hour hypoxia + PBA (10 mmol/L, 1 hr).
After treatment with hypoxia and/or PBA, MPTP opening in VSMCs was measured by monitoring calcein fluorescence in the absence and presence of CoCl2 (32). In normal condition, CoCl2 can quench calcein fluorescence in the cytoplasm, but does not affect calcein fluorescence in mitochondria. However, if the MPTP is over opened, the calcein fluorescence in the mitochondrial matrix can be quenched by CoCl2, which showed decreased intensity of calcein fluorescence. Briefly, the treated cells were loaded with 2 μmol/L of calcein-AM (Sigma) and 100 nmol/L of MitoTracker deep red (Sigma) for 30 minutes.

Cells were washed twice with PBS, then exposed to 2 mmol/L of CoCl2 for 15 minutes. The fluorescence was detected with an excitation of 488 nm (calcein) or 633 nm (MitoTracker) using a Leica confocal microscope. Given the important role of calcium in the contraction of VSMC, the effect of PBA on the calcium concentration ([Ca2+] ) in VSMCs was observed. VSMCs were also divided into three groups: normal control, 3-hour hypoxia, and 3-hour hypoxia + PBA (10 mmol/L, 1 hr). The calcium concentration ([Ca2+] ) in VSMCs was measured using a Confocal micro- scope after incubation with Fura-2/AM (2 µmol/L; Sigma) at 37°C for 20minutes (7).Part 4.4. Mechanisms responsible for the effect of PBA on oxidative stress: to investigate the mechanisms by which PBA regulates oxidative stress in traumatic hemorrhagic shock, fol- lowing experiments were performed: 1) effects of PBA on the cytoplasmic and nuclear expression of NF-E2–related factor 2 (Nrf2), Sirtuin 1 (Sirt1), nuclear factor κB (NF-κB), and acti- vator of transcription 1 (STAT1) (the four important regula- tory molecules in oxidative stress pathway) in hypoxia-treated VSMCs were observed. VSMCs were divided into three groups: normal control, 3-hour hypoxia, and 3-hour hypoxia + PBA (10 mmol/L, 1 hr). The cytoplasmic and nuclear extracts from VSMCs were obtained with the norepinephrine-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL). Western blot analysis was performed using antibodies against Nrf2 (1:200; Santa Cruz), Sirt1 (1:1,000; Cell Signaling), NF-κB p65 (1:1,000; Cell Signaling), and STAT1α (1:1,000; Cell Signaling), β-actin (1:5,000; Sigma), and Lamin B1(1:1,000; Cell Signaling). Bands were detected with fluorescent secondary antibodies and quantified with Odyssey CLx Infrared Imaging System (LI-COR, Lincoln, NE). The cytoplasmic and nuclear protein expressions were normalized to β-actin and Lamin B1, respectively. 2) Effects of alteration of these molecules on PBA- regulating oxidative stress in VSMCs were observed. VSMCs were divided into six groups: normal control, 3-hour hypoxia, 3-hour hypoxia + PBA, 3-hour hypoxia + PBA + brusatol (5 μmol/L for 30 min prior to PBA, the Nrf2 inhibitor from JONLN, Shanghai, China), 3-hour hypoxia + PBA + Ex527 (10 μmol/L for 30 min prior to PBA, the Sirt1 inhibitor from Sigma), and 3-hour hypoxia + PBA + lipopolysaccharide (LPS; 10 μg/mL for 30 min prior to PBA, used as NF-κB and STAT1 activating agent; Sigma). The levels of NO, inducible NO synthase (iNOS), SOD, catalase, and glutathione in VSMCs and in culture medium were detected using assay kits as described above.

Part 5: Evaluation of Clinical Application Potential of PBA. To evaluate the therapeutic application of PBA in hemor- rhagic shock, following experiments were performed: 1) effects of higher doses of PBA (100, 200, and 300 mg/kg) on traumatic shock; 2) the potentiation effect of PBA on norepinephrine; 3) effects of PBA without LR resuscitation; and 4) effects of early application of PBA on traumatic shock.Part 5.1. Effects of higher doses of PBA on traumatic hem- orrhagic shock and the potential side effects: 72 SD rats ran- domly divided into three groups: PBA 100, 200, and 300 mg/kg groups (24 rats per group). At the end of shock, rats received an infusion of PBA (100, 200, or 300 mg/kg) with two volumes of blood loss of LR as described above. Among them, 48 rats were used for observation of animal survival (n = 16 per group). Other 24 rats were used for the measurement of MAP, vascular reactivity, and liver and kidney function parameters (n = 8 per group) as described above in experiment parts 1 and 3.

Part 5.2. The potentiation effect of PBA on norepinephrine in traumatic hemorrhagic shock rats: 48 SD rats randomly divided into two groups: norepinephrine (1 μg/kg) and nor- epinephrine + PBA (20 mg/kg) groups (24 rats per group); among them, 32 rats were used for observation of animal survival (n = 16 per group), and the others were used for the measurement of hemodynamics and the pressor response of norepinephrine (n = 8 per group). At the end of shock, the rats in norepinephrine group received one volume of blood loss of LR infusion, and then norepinephrine (1 μg/kg) was infused along with another one volume of LR. The rats in norepineph- rine + PBA group also received one volume of blood loss of LR infusion first, and then norepinephrine (1 μg/kg) + PBA (20 mg/kg) were infused along with another one volume of LR. The hemodynamic parameters and the pressor response were observed at baseline, at the end of phase I, and at 1 and 2 hours in phase III as described above. After measurement, the animal survival was observed.

Part 5.3. Effects of PBA with or without LR resuscitation in traumatic hemorrhagic shock rats: 48 SD rats were used and divided two groups: PBA (20 mg/kg) with and without LR infusion. Thirty-two rats were used for observation of animal survival (n = 16 per group), and the others were used for the measurement of hemodynamics and the pressor response (n = 8 per group). A bolus injection of PBA (20 mg/kg) in 1-mL LR/kg body weight was given at the end of shock in no LR infu- sion group. Two volumes of blood loss of LR were infused in PBA with LR infusion group. The hemodynamic parameters and the pressor response were observed at baseline, at the end of phase I, and at 1 and 2 hours in phase III. After measure- ment, the animal survival was observed.

Part 5.4. Effects of early application of PBA in traumatic hemorrhagic shock rats: 24 SD rats were used to investigate the effects of early application of PBA in traumatic hemorrhagic shock. Similarly, 16 rats were used for observation of animal survival, and eight for the measurement of hemodynamics and the pressor response. A bolus injection of PBA (20 mg/kg) was given in the initial stage of shock (at the beginning of phase I), and two volumes of blood loss of LR were infusion at the end of phase I. The animal survival, hemodynamics, and the pres- sor response were observed. For comparison, the late appli- cation of PBA (20 mg/kg, at the end of phase I) was used as control (from experiment part 5.3).

Statistical Analysis

Data are expressed as the mean ± se. The animal survival was analyzed using Kaplan-Meier survival curve, and the differ- ences among groups were analyzed by chi-square test. In the other experiments, the differences among groups were ana- lyzed by repeated measures analysis of variance, followed by post hoc Tukey tests. The analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL). Kaplan-Meier survival curves were drawn using GraphPad Prism 5.0 (GraphPad Soft- ware, La Jolla, CA), and other graphs were drawn using Origin 5.0 (Origin Lab, Northampton, MA). A p value of less than 0.05 was considered statistically significant.

RESULTS

Part 1: Effects of PBA on the Survival Time and 24-Hour Survival Rate of Traumatic Hemorrhagic Shock Rats PBA 20 mg/kg significantly prolonged the survival time and 24-hour survival rate of traumatic hemorrhagic shock rats when compared with the LR-alone group. The mean survival time and 24-hour survival rate in the PBA 20 mg/kg group were 19.9 hours and 75% (12/16), respectively, which were sig- nificantly higher than in the shock control (3.2 hr; 0/16) and LR-alone groups (10.8 hr; 3/16; p < 0.01). The other two doses of PBA (5 and 50 mg/kg) also increased the survival time and 24-hour survival rate of shock rats (16.2 hr; 9/16 and 13.7 hr; 7/16 in the PBA 5 and 50 mg/kg groups, respectively), but there was no significant difference when compared with the LR group (Fig. 2, A and B). Figure 2. Effects of 4-phenylbutyrate (PBA) on the survival rate and survival time after traumatic hemorrhagic shock (n = 16 per group). A, Kaplan- Meier survival curve; (B) survival time. *p < 0.05, **p < 0.01 compared with the sham-operated group; #p < 0.05, ##p < 0.01 compared with the lactated Ringer (LR) group. Sham = sham-operated, S = shock. Part 2: Systemic Effects of PBA in Traumatic Hemorrhagic Shock Hemodynamics. All hemodynamic parameters, including MAP, LVSP, and ± dp/dtmax, were significantly decreased after shock. Two volumes of LR infusion slightly increased these parameters. The administration of PBA at 20 mg/kg signifi- cantly improved all of these parameters in shock rats when compared with the LR group (p < 0.05 or p < 0.01). The LVSP and the ± dp/dtmax in the PBA 20 mg/kg group were nearly restored to normal levels at 2 hours after resuscitation. PBA 5 and 50 mg/kg slightly increased parameters, but there were no significant differences when compared with the LR group (Fig. 3, A–D).CO. CO was significantly reduced at the end of the shock period. Two volumes of LR infusion significantly restored the decreased CO of the shock animals. The CO in the LR group was recovered to 97.6% and 82.8% of the baseline level at 1 and 2 hours after resuscitation, respectively. PBA did not further increase the CO; there were no significant differences among the three PBA groups and the LR group at all time- points (Fig. 3E). Blood Gases. At the end of phase 1 (shock period), the level of blood lactic acid was significantly increased; it was approxi- mately seven-fold higher than the normal control group. Two volumes of LR slightly decreased the level of blood lactic acid. PBA significantly decreased the level of lactic acid in arterial blood. One hour after resuscitation, the lactic acid level in the PBA groups (5, 20, or 50 mg/kg) was significantly lower than the LR group (p < 0.05 or p < 0.01), and 2 hr after resuscitation the lactic acid level in the PBA 20 mg/kg group was significantly lower than the LR group (p < 0.01; Fig. 3F). Blood HCO – and base excess (BE) levels were significantly reduced after shock. The PBA 20-mg/kg infusion increased the HCO – and BE levels, which were significantly higher than the LR group (p < 0.05). There were no significant differences in the HCO – and BE levels among the PBA 5- and 50-mg/kg groups and the LR-only group. LR and the three doses of PBA all restored the decreased pH to a normal level (Fig. 3, G–I). Part 3: Protective Effects of PBA on Organ Function in Traumatic Hemorrhagic Shock Blood Flow of the Liver and Kidney. At the end of the shock period, blood flow in the liver and kidney was signifi- cantly reduced when compared with the baseline level. Two volumes of LR slightly increased the blood flow. Infusion of PBA 20 mg/kg significantly increased blood flow in the liver and kidney, which were significantly higher than the LR group (p < 0.01). Infusion of PBA 5 and 50 mg/kg significantly increased renal blood flow, but not hepatic blood flow, when compared with the LR group (p < 0.01) (Fig. 4, A and B). Liver and Kidney Function. The parameters of liver and kidney function, including AST, ALT, BUN, and SCr, were sig- nificantly increased after shock. Infusion of PBA 20 mg/kg sig- nificantly decreased the levels of ALT and SCr when compared with the LR group (p < 0.05 or 0.01), whereas AST and BUN were not affected by LR or PBA (Fig. 4, C–F). Mitochondrial Function. At the end of the shock period, the respiratory control rate of mitochondria in the liver and kidney was significantly reduced. Two volumes of LR did not increase the mitochondrial function. PBA at 20 mg/kg significantly increased the mitochondrial function both in liver and in kidney when compared with LR resuscitation (p < 0.01). PBA at 5 and 50 mg/kg only increased the mitochon- drial function of kidney, but not increased the mitochondrial function of liver (Fig. 4G). Figure 3. Effects of 4-phenylbutyrate (PBA) on hemodynamic parameters, cardiac output (CO), and blood gases after traumatic hemorrhagic shock (n = 8 per group). A, Mean arterial pressure (MAP); (B) left intraventricular systolic pressure (LVSP); (C and D) maximal change rate in left intraventricular pressure (± dp/dtmax); (E) CO; (F) lactic acid (Lac); (G) bicarbonate (HCO –); (H) base excess (BE); (I) blood pH. *p < 0.05, **p < 0.01 compared with the sham-operated group; #p < 0.05, ##p < 0.01 compared with the LR group. Sham = sham-operated; LR = lactated Ringer’s solution, B = baseline, S = shock. of 20 mg/kg of PBA signifi- cantly decreased the levels of malondialdehyde and NO when compared with the LR group (p < 0.01). Application of apocynin (10 mg/kg) also reduced the MDA level (Fig. 5, A and B). There appeared a protective increase in the key antioxidant enzymes including SOD, catalase, and glutathione in vessel tissues and blood after traumatic hemorrhagic shock; LR did not enhance these anti- oxidant enzymes. Infusion of PBA 20 mg/kg significantly increased the levels of SOD, catalase, and glutathione in blood vessels and blood when compared with the LR group (p < 0.01). Apocynin also increased the SOD and catalase levels (Fig. 5, C–E). Intracellular ROS Levels in Mesenteric Arteries and VSMCs. At the end of shock, the intracellular ROS levels in mesenteric arteries were sig- nificantly increased compared with normal rat mesenteric arteries. PBA and apocynin Vascular Reactivity. The vascular constriction reactivity of SMAs to norepinephrine was significantly reduced after shock, as stated in our previous report. LR slightly increased constriction reactivity. PBA (5, 20, and 50 mg/kg) markedly restored the decreased constriction of SMAs, which was sig- nificantly higher than the LR group (p < 0.05 or 0.01). Apoc- ynin, an antioxidant, also increased the constriction reactivity of SMAs when compared with the LR group (p < 0.01). The vasodilator reactivity of SMAs was also significantly attenuated after shock. PBA (5, 20, and 50 mg/kg) and apocynin signifi- cantly restored the decreased vasodilator reactivity of SMAs. PBA 20 mg/kg showed the best improvement in vascular reac- tivity (Fig. 4H). Part 4: The Mechanism of PBA Protecting the Vascular and Other Vital Organ Function: The Relationship to Oxidative Stress, ER Stress, and MPTP Opening Part 4.1: Effects of PBA on Oxidative Stress Levels of Malondialdehyde, NO, SOD, Catalase, and Glutathi- one in Blood Samples and Mesenteric Artery Tissues. The levels of malondialdehyde and NO were significantly increased after traumatic hemorrhagic shock in vascular tissues and blood. Infusion of LR alone further increased the levels of malondial- dehyde and NO in the blood vessels and blood. Administration markedly reduced shock-increased intracellular ROS levels in mesenteric arteries (Fig. 5, F and G). Studies involving VSMCs showed a similar change in trend. Three-hour hypoxia caused a significant increase in ROS levels in VSMCs from SD rats, and similar changes were observed in the positive control (ROSup) group. Treatment with PBA and apocynin reduced ROS levels in hypoxic VSMCs (Fig. 5, H and I). Part 4.2: Effects of PBA on ER Stress. After 3 hours of shock, the expression of the key ER stress markers (GRP78, IRE-1α, PERK, and ATF-6) did not show significant changes, and infu- sion of LR or PBA did not alter the levels of these markers in the SMAs of rats. There were no significant differences among the groups (Fig. 6, A and B). Similar results were also observed in hypoxia-treated VSMCs from the SMAs of rats. The expres- sion of GRP78, IRE-1α, PERK, and ATF-6 protein was not altered after 3-hour hypoxia or treatment with PBA in VSMCs (Fig. 6, C and D). Part 4.3: Effects of PBA on MPTP Opening and Intracellular Calcium Concentration in VSMCs MPTP Opening. In vivo experiment, the MPTP opener ATR abolished the protective effect of PBA. MAP and the pressor response of norepinephrine in ATR + PBA group were signifi- cantly lower than that in PBA-alone group (p < 0.01) (Fig. 7, A and B). In vitro experiment, 3-hour hypoxia induced MPTP opening in VSMCs, and the mitochondrial calcein fluorescence which suggested that hypoxia caused the oxidative injury and the activation of protective anti- oxidant system in vitro. PBA significantly decreased the levels of NO and iNOS and further increased the levels of SOD, cat- alase, and glutathione. Brusatol, the Nrf2 inhibitor, antagonized the PBA-induced increase in the levels of catalase and glutathi- one in VSMCs and the culture medium (Fig. 8, I and J). Inter- estingly, Brusatol also blocked PBA-induced down-regulation of NO and iNOS (Fig. 8, F and G). LPS pretreatment, used as NF-κB and STAT1 activat- ing agent, abolished the PBA- induced decrease of the levels of NO and iNOS (Fig. 8, F and G). Ex527, the Sirt1 inhibitor, had no significant influence on the action of PBA on oxidative stress markers. In addition, the three chemical reagents had no effect on the up-regulation of SOD induced by PBA (Fig. 8H). Figure 4. Effects of 4-phenylbutyrate (PBA) on blood flow in the liver and kidney and their function, and vascular reactivity of superior mesenteric arteries (SMAs) after traumatic hemorrhagic shock (n = 8 per group). A, Hepatic blood flow (HBF), (B) renal blood flow (RBF), (C) aspartate aminotransferase (AST); (D) alanine aminotransferase (ALT); (E) blood urea nitrogen (BUN); (F) serum creatinine (SCr); (G) mitochondrial function (respiration control rate [RCR]) in the liver and kidney; (H) vascular reactivity of SMA to norepinephrine (NE) and acetylcholine (ACh). *p < 0.05, **p <0.01 compared with the sham-operated group; #p < 0.05, ##p < 0.01 compared with the lactated Ringer’s (LR) group. Sham = sham-operated, S = shock, Apo = apocynin. Part 5: Evaluation of the Clinical Application values in the hypoxia group were significantly lower than the normal control group (p < 0.01). PBA incubation inhibited hypoxia-induced MPTP opening, represented by an increase in calcein fluorescence (Fig. 7, C and D). Intracellular Calcium Concentration. After exposure to hypoxic conditions (3-hr hypoxia), the changes of [Ca2+] in VSMCs were not noticeable. PBA had no significant influence on the [Ca2+] in hypoxic VSMCs (Fig. 7, E and F). Part 4.4: The Mechanisms for the Effect of PBA on Oxidative Stress. The Expression of Nrf2, Sirt1, NF-κB, and STAT1α in Cytoplasm and Cell Nuclei in VSMCs. Immunoblot analysis showed that 3-hour hypoxia induced an increased expression of nuclear Nrf2 and NF-κB in VSMCs. PBA treatment further promoted the expression of nuclear Nrf2, with an accompanying decrease of cytoplasmic Nrf2 (Fig. 8, A and B). In addition, PBA effec- tively prevented the increase of nuclear NF-κB levels in hypoxic VSMCs, and reduced the cytoplasmic NF-κB levels (Fig. 8, A and D). The expression of Sirt1 and STAT1α did not show sig- nificant changes in nuclear and cytoplasmic fractions of VSMCs after PBA and/or hypoxia treatment (Fig. 8, A, C, and E). Effects of Alteration of Nrf2, Sirt1, NF-κB, and STAT1 on PBA- Regulating Oxidative Stress. Similar to in vivo findings, 3-hour hypoxia caused a significant increase in the levels of NO, iNOS, SOD, catalase, and glutathione in VSMCs and the culture medium,Potential of PBA in Traumatic Hemorrhagic Shock Part 5.1: Effects of Higher Doses of PBA in Traumatic Hemor- rhagic Shock. Survival. Similar to effects of PBA at 50 mg/kg on animal survival, PBA 100, 200, and 300 mg/kg also slightly prolonged the animal survival. The mean survival time and 24-hour survival rate in the PBA 100, 200, and 300 mg/kg groups were 12.5 hours, 6 of 16; 15.4 hours, 8 of 16; 14.4 hours, 7 of 16, respectively (Supplemental Fig. S1A and S1B, Supplemental Digital Content 2, http://links.lww.com/CCM/B504). MAP, Vascular Reactivity, and Liver and Kidney Function. Compared with PBA 20 mg/kg, the improvement of higher doses of PBA (100, 200, and 300 mg/kg) in MAP and vascu- lar reactivity was slightly reduced (Supplemental Fig. S1C and S1D, Supplemental Digital Content 2, http://links.lww.com/ CCM/B504). The higher doses of PBA induced a dose-dependent increase in the levels of ALT and AST (the index of liver func- tion, which also reflect the degree of liver damage), but did not increase the levels of BUN and SCr (the index of kidney func- tion) (Supplemental Fig. S1E–H, Supplemental Digital Con- tent 2, http://links.lww.com/CCM/B504). Part 5.2: The Potentiation Effect of PBA on Norepinephrine in Traumatic Hemorrhagic Shock. Survival. When compared with norepinephrine group, PBA (20 mg/kg) in combination with norepinephrine Figure 5. Effects of 4-phenylbutyrate (PBA) on oxidative stress markers and reactive oxygen species (ROS) levels in vivo and in vitro. A and B, The levels of malondialdehyde (MDA) and nitric oxide (NO) in blood samples and mesenteric vascular tissues (n = 8 per group); (C–E) the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in blood samples and mesenteric vascular tissues (n = 8 per group); (F) images acquired by confocal microscopy showing intracellular ROS levels in superior mesenteric arteries (SMAs); (G) quantitative data of the mean intensity of 2′,7′-dichlorofluorescin diacetate (DCF, a probe of ROS) fluorescence, experiments were repeated thrice; (H) images showing intracellular ROS levels in vascular smooth muscle cells (VSMCs); (I) quantitative data of the mean intensity of DCF fluorescence (n = 30 cells per group). *p < 0.05, **p < 0.01 compared with the sham-operated/normal control group; #p < 0.05, ##p < 0.01 compared with the lactated Ringer’s (LR) group; ^p < 0.05, ^^p < 0.01 compared with the hypoxia group. Sham = sham-operated, S = shock, Apo = apocynin. Hemodynamics and Pressor Response. PBA+ norepinephrine significantly improved the hemodynamics of traumatic shock rats when compared with norepinephrine group. All hemody- namic parameters including the MAP, LVSP, and ± dp/dtmax in norepinephrine + PBA group were higher than those in norepi- nephrine group at 1 and 2 hours after resuscitation (p < 0.05) (Supplemental Fig. S2B-E, Supplemental Digital Content 3, http://links.lww.com/CCM/B505). The pressor effect of nor- epinephrine (reflecting the vascular reactivity in vivo) was sig- nificantly reduced after shock, as stated in our previous report. Norepinephrine infusion did not restore the decreased pressor response after shock. Infusion of norepinephrine + PBA signifi- cantly increased the pressor response of traumatic shock rats when compared with that of norepinephrine group (p < 0.01). At 2 hours after resuscitation, the pressor response was restored to 95.3% of the baseline level in norepinephrine + PBA group, whereas in norepinephrine group, the pressor response was only 64.2% of the baseline level (Supplemental Fig. S2F, Supplemen- tal Digital Content 3, http://links.lww.com/CCM/B505). Part 5.3: Effects of PBA With or Without LR Resuscitation. Survival. PBA (20 mg/kg) without LR infusion did not show a beneficial effect on traumatic hemorrhagic shock rats. The survival time and 24-hour survival rate were 6.9 hours and 6.3% (1/16), respectively. They were significantly lower than PBA with LR infusion (p < 0.01) (Supplemental Fig. S3A, Supplemental Digital Content 4, http://links.lww. com/CCM/B506). Hemodynamics and Pressor Response. When compared with PBA with LR resuscitation, PBA (20 mg/kg) without LR infu- sion did not improve the hemodynamics of shock rats including the MAP, LVSP, and ± dp/dtmax (Supplemental Fig. S3B–E, Supplemental Digital Content 4, http://links.lww.com/CCM/ B506). Also, PBA without LR did not restore the decreased pressor response after shock (Supplemental Fig. S3F, Supple- mental Digital Content 4, http://links.lww.com/CCM/B506). Part 5.4: Effects of Early Application of PBA in Traumatic Hemorrhagic Shock. Survival. Early application of PBA (20 mg/kg, at the initial stage of shock) significantly improved the survival outcomes in traumatic hemorrhagic shock rats. The survival time and 24-hour survival rate were 21.1 hours and 81.3% (13/16), respectively. They were a little higher than the late application of PBA (20 mg/kg) (Supplemental Fig. S4A, Supplemental Digital Content 5, http://links.lww.com/CCM/B507). Hemodynamics and Pressor Response. Early application of PBA (20 mg/kg) significantly improved the hemodynam- ics and restored the pressor response of traumatic shock rats. When compared with PBA administration at the end of shock, early application of PBA showed a little better beneficial effect on traumatic shock (Supplemental Fig. S4B-F, Supplemental Digital Content 5, http://links. lww.com/CCM/B507). Figure 6. The expression of endoplasmic reticulum (ER) stress markers (78-kDa glucose-regulated protein [GRP78], inositol-requiring enzyme [IRE]-1α, pancreatic ER kinase [PERK], and activating transcription factor (ATF)-6) in vivo and in vitro. A and C, The expression of GRP78, IRE-1α, PERK, and ATF-6 in superior mesenteric arteries and in vascular smooth muscle cells (VSMCs), immunoblot analyses were repeated thrice; (B and D) the ratio of the optical density for proteins/β-actin. *p < 0.05, **p < 0.01 compared with the sham- operated group; #p < 0.05, ##p < 0.01 compared with the lactated Ringer’s (LR) group. N = sham-operated/ normal, S = shock, Hy = hypoxia. DISCUSSION The present study showed that early or late application of the proper dosage of PBA has a beneficial effect on trau- matic shock by improving the decreased vascular reactivity and other organ function. It has been shown that the protective effects of PBA are primarily attributed to its two important functions (inhibiting oxida- tive stress and inhibiting ER stress) (10, 13). It is known that oxidative stress is involved in the pathogenesis of numer- ous cardiovascular diseases, including myocardial infarc- tion, atherosclerosis, stroke, and diabetic vascular lesion. Thus, there has been increasing interest in antioxidant therapy for cardiovascular diseases. PBA has shown promise in the treatment of ischemia and neurodegenerative diseases by attenuating oxidative stress (21, 33). The current study showed that PBA can protect vascular function against oxidative stress injury after traumatic hemorrhagic shock. Application of PBA can significantly decrease the levels of oxidative stress mark- ers such as malondialdehyde and NO, and further increase the levels of the key antioxidant enzymes such as SOD, catalase, and glutathione in the vessel tissues and blood after traumatic hemorrhagic shock. The results further showed that shock and hypoxia cause a significant increase in ROS levels in vessel tis- sues and VSMCs from SD rats, which was prevented by PBA. Figure 7. Effects of 4-phenylbutyrate (PBA) on mitochondrial permeability transition pore opening and intracellular calcium concentration in vascular smooth muscle cells (VSMCs) after hypoxia (Hy). A and B, Mean arterial pressure (MAP) and the pressor effect of norepinephrine (n = 8); (C) images acquired by confocal microscopy showing VSMCs loaded from calcein-AM + CoCl2 and stained with MitoTracker deep red. Left column, Calcein fluorescence inside mitochondria; middle column: MitoTracker fluorescence, MitoTracker is a mitochondrion-selective fluorescent dye; and right column: merged image. Experiments were repeated thrice; (D) quantitative data of the mean intensity of calcein fluorescence (n = 30 cells per group); (E) images acquired by confocal microscopy showing intracellular calcium concentration in VSMCs. F, Quantitative data of the mean intensity of calcium fluorescence (n = 30 cells per group). *p < 0.05, **p < 0.01 compared with the normal group; #p < 0.05, ##p < 0.01 compared with the Hy group; ^p < 0.05, ^^p < 0.01 compared with the shock (S) + PBA group. ATR = atractyloside, B = baseline. To explore the mechanisms by which PBA reduces oxida- tive stress after shock, we observed the effect of PBA on the main regulatory factors of oxidative stress pathway, includ- ing Nrf2, Sirt1, NF-κB, and STAT1 (34–37). The results showed that PBA treatment could further increase the levels of nuclear Nrf2 and prevent the increase of NF-κB levels in cell nuclei in hypoxic VSMCs. Brusatol, an Nrf2 inhibitor, antagonized PBA-induced increase in the levels of cata- lase and glutathione in hypoxia-treated VSMCs. LPS, as the activation agent of NF-κB and STAT1, abolished PBA- induced decrease of the levels of NO and iNOS. Ex527, the Sirt1 inhibitor, had no significant influence on the action of PBA on oxidative stress. These results suggest that the mechanisms by which PBA reduces the oxidative stress may be associated with increasing the antioxidants (catalase and glutathione) levels via Nrf2 pathway, as well as inhibiting NO and iNOS expression via NF-κB pathway. Interestingly, PBA- induced down-regulation of NO and iNOS was also inhib- ited by Brusatol, whereas up-regulation of SOD induced by PBA was not affected by the three chemical reagents, which suggests that there may be other mechanisms involved in. Of course, further investigations are needed to elucidate the precise mechanism. Research showed another principal function of PBA is inhibiting ER stress as a chemical chaperone (10, 11). ER stress plays a vital role in the synthesis and folding of pro- teins, calcium homeostasis, and intracellular redox potential (12). It is not known, however, whether or not ER stress is involved in the protective effects of PBA on traumatic hemor- rhagic shock. The current study showed that the levels of ER stress markers, including GRP78, IRE-1α, PERK, and ATF-6,did not significantly change after shock/resuscitation. The in vitro studies with cultured rat VSMCs exposed to acute physi- ologic hypoxia also showed that these ER stress markers did not increase after hypoxia. Taken together, with the changes in oxidative stress markers, it is suggested that traumatic hemor- rhagic shock results in increased oxidative stress, but not ER stress. PBA elicits the antishock effects and vascular function protection mainly through inhibition of oxidative stress. Figure 8. Mechanisms underlying the effect of 4-phenylbutyrate (PBA) on oxidative stress. A, The cytoplasmic and nuclear expression of NF-E2– related factor 2 (Nrf2), Sirtuin 1 (Sirt1), nuclear factor κB (NF-κB), and activator of transcription (STAT)-1α in vascular smooth muscle cells (VSMCs), immunoblot analyses were repeated thrice; (B–D) the ratio of the optical density for nuclear proteins/Lamin B1 or cytoplasmic proteins/β-actin; (F–J) the levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in VSMCs and the culture medium (n = 8); (K) schematic diagram showing the possible signaling mechanisms of PBA’s effects on oxidative stress. *p < 0.05, **p < 0.01 compared with the normal (N) group; #p < 0.05, ##p < 0.01 compared with hypoxia (Hy) group; ^p < 0.05, ^^p < 0.01 compared with PBA group.LPS, lipopolysaccharide, Ex = Ex527, Bru = brusatol. Mitochondria are the key cellular organelles and partici- pate in the regulation of multiple cell functions. Accumulating evidence has revealed that the interactions between oxidative stress and mitochondria are very important in cell life and death, and oxidative stress might affect the function of mito- chondria (23, 38). The MPTP, which is located in the inner mitochondrial membrane, plays an essential role in mitochon- drial function and structure (25). Our recent studies showed that the MPTP takes part in the regulation of vascular reac- tivity after shock (unpublished observations). In the present study, PBA incubation inhibited the opening of the MPTP in VSMCs after hypoxia, and the MPTP opener ATR abolished the protective effect of PBA on traumatic hemorrhagic shock. It is suggested that the protection of vascular function induced by PBA may be attributed to the decreased opening of the MPTP via inhibition of oxidative stress. The precise mecha- nism, however, needs further investigation. In the current study, we used six doses of PBA (5, 20, 50, 100, 200, and 300 mg/kg), 20 mg/kg of PBA was shown to have the best beneficial effects, whereas PBA greater than 50 mg/ kg resulted in less improvement, including in the 24-hour survival rate, tissue blood flow, and vital organ function and caused liver function damage. PBA is a Food and Drug Administration approved drug to treat UCDs, and the cur- rent data from a series of clinical trials showed that PBA was well tolerated and had few side effects. The reported adverse events in patients included abdominal pain upper, nausea, emesis, decreased appetite, cardiac murmur, dermatitis con- tact, Kussmaul respiration, metabolic acidosis, headache, fatigue, lethargy, etc (9, 39). This difference of dose-effect response in shock animals was difficult to explain with these reported efficacy and side effects of PBA in patients with UCDs or other disease. Our data showed that higher doses of PBA (50, 100, 200, and 300 mg/kg) were associated with a dose-dependent increase in the level of enzymatic biomark- ers including ALT and AST, but did not cause a significant increase in the index of kidney function. In addition, higher doses of PBA (> 50 mg/kg) had a slightly reduced improve- ment in many parameters such as vascular reactivity. It is sug- gested that over some dosages, PBA may cause organ function impairment.

Another unexpected result in this study was that the lev- els of ER stress markers in SMAs of traumatic shock rats, and hypoxia-treated VSMCs did not increase. ER stress has been reported to contribute to ischemia/reperfusion injury in the brain and other organs, and PBA can exhibit protective effects against ER stress injury (21, 40). The conflicting findings may be related to the severity and stage of shock. In the pres- ent study, what we observed was the acute effect of PBA on traumatic shock. However, ER stress–induced cell damage and apoptosis during ischemia-reperfusion was generally found in the late stage of ischemia. Whether the chaperone activity of PBA on ER stress is involved in the late stage of hemorrhagic shock is unclear, which needs further investigation.

Antioxidative therapeutic strategies hold great prom- ise for numerous diseases. However, most of clinical stud- ies showed disappointing results. Only a few therapeutic agents are clinically available for the prevention of oxidative stress injury, such as edaravone (41). The conflicting results between experimental and clinical trials may be due to the dosage of antioxidants, limitation of single antioxidant, other medication, the stage and severity of the disease, and concomitant disease (42, 43). Thus, there has been increasing interest in exploring new combinatory antioxidant strategies (such as an antioxidant cocktail) and development of new drugs with multiple functions in the regulation of oxidative stress. It is known that PBA has multiple biological activi- ties (10–13, 44). Our present data suggested that the anti- oxidant activities of PBA may be involved in multiple targets and mechanisms, so it may prove highly useful in the regula- tion of oxidative stress, which may partially explain why PBA exerts protective effects in traumatic hemorrhagic shock via antioxidative stress. In addition, there may be other mecha- nisms that PBA play protective effects in traumatic hemor- rhagic shock. For example, the HDAC inhibitory function of PBA has been reported to provide protection toward isch- emic damage (45).

In addition, our results also found that PBA could poten- tiate the beneficial effect of norepinephrine (the first-line vasoactive for shock) (46) in the resuscitation of hemorrhagic shock, and found early or late application of PBA all had ben- eficial effect in traumatic hemorrhagic shock. These findings suggest that PBA may be a promising therapeutic drug with a wide therapeutic time window for the treatment of severe trauma/shock.

In addition, the current study had some limitations. First, all of the experiments were performed in small animals; whether or not the antishock effect of PBA can be extrapolated to larger animals and humans needs further investigation. Second, only a severe model of hemorrhagic shock was used, so the effec- tiveness of PBA on moderate or less serve hemorrhage model is needed. Third, this study showed different effects of PBA on liver, kidney, heart, and the vascular system, but the basis for the differences was not determined. Fourth, we only observed a short-term effect of PBA and its relationship to oxidative and ER stress in the present study; the long-term effect needs fur- ther investigation.

CONCLUSIONS

Our results indicated that PBA has good beneficial effect for traumatic hemorrhagic shock including improving animal survival and protecting organ function. These beneficial effects of PBA result from its vascular function protection and hemo- dynamic stabilization via attenuation of the oxidative stress and the associated MPTP opening. Nrf2 and NF-κB may be involved in PBA-mediated inhibition of oxidative stress. PBA may be a promising measure for the treatment of severe trauma and shock, especially for refractory shock.