Gamma-enolase predicts lung damage in severe acute pancreatitisinduced acute lung injury

Lawrence Owusu1,2,5 · Caiming Xu1, · Hailong Chen 1,2 · Geliang Liu3 · Guixin Zhang1 · Jinwen Zhang1 · Zhankai Tang1 · Zhongwei Sun1 · Xin Yi

Abstract

Severe acute pancreatitis (SAP) associated acute lung injury (ALI) accounts for about 70% mortality of SAP patients. However, there are no precise biomarkers for the disease currently. Herein, we evaluated the potential of gamma-enolase (ENO2), against its universal isoform alpha-enolase (ENO1), as a marker of SAP–ALI in a rat model. Firstly, 16 male Sprague–Dawley rats were randomly divided into two groups, Sham (n = 8) and SAP–ALI (n = 8), for pancreatitis induction. Ultra-structure examination by electron microscopy and HE staining were used for lung injury assessment. Lung tissue expressions of alpha-enolase and gamma-enolase were evaluated by qRT-PCR and immunohistochemistry. In a prospective validation experiment, 28 rats were used: sham (n = 8), SAP–ALI at 3 h (3 h, n = 10), and SAP–ALI at 24 h (24 h, n = 10). Lung tissue damage, tissue expression and circulating alpha-enolase and gamma-enolase levels were evaluated. Elevated serum levels of α-amylase and TNF-α were observed in SAP rats but not in sham-operated rats. Histological examination of pancreatic and lung tissues indicated marked damage in SAP rats. While alpha-enolase was universally expressed, gamma-enolase was expressed only in damaged lung tissues. Gamma-enolase was detected in lung tissues, BALF, and serum as early as 3 h post-surgery when physical pathological damage was not apparent. Unlike alpha-enolase, secreted and/or circulating gamma-enolase level progressively increased, especially in serum, as lung damage progressed. Thus, gamma-enolase may signal and correlate lung tissue damage well before obvious physical pathological tissue damage and might be a candidate diagnostic and/or prognostic marker.

Keywords Severe acute pancreatitis-induced acute lung injury · Alpha-enolase · Gamma-enolase · Sprague–Dawley rats

Background

Acute lung injury (ALI) or mild acute respiratory distress syndrome (MARDS) is the commonest distant organ complication with high rates of morbidity and mortality in severe acute pancreatitis (SAP) (Renzulli et al. 2005). It is associated with 60–70% deaths of patients within the first week (Guice et al. 1988). To help unravel the pathogenesis of the syndrome, several types of animal models of the disease have been used over the years (Guice et al. 1988; Samuel et al. 2010). Generally, systemic inflammation is accepted as prerequisite for SAP-induced ALI pathogenesis (SAP–ALI), and it is characterized by immune cells infiltration of the lungs, local and systemic release of several and abundant proinflammatory mediators, and activation of multiple inflammatory pathways and genes (Abraham et al. 2000; Xu et al. 2010; Konrad and Reutershan 2012). Although great strides have been made in understanding the disease, there are yet several unknown molecular factors to permit the comprehensive molecular understanding, the diagnosis and/ or prognosis of the disease, and thus no known specific treatment for the disease yet.
Gamma enolase (ENO2), also known as neuron-specific enolase (NSE), is an isoenzyme of the ubiquitous glycolytic enzyme alpha enolase (ENO1). ENO2 is predominantly expressed in neurons but has also been indicated as marker for neuroendocrine and paraneuronal cells, and found in lower quantities in non-neuronal and non-neuroendocrine tissues or cells, including platelets, erythrocytes, prostate, lung, uterus and breast (Schmechel et al. 1978; Haimoto et al. 1985; Soh et al. 2011). Appreciable gamma-enolase levels have also been observed in cells with high and quick energy demand or turnover, such as myoepithelial cells and spermatogonia, for their normal function (Haimoto et al. 1985). During glucose metabolism, enolases catalyze the interconversion of 2-phospho-d-glycerate to phosphoenolpyruvate by processes of dehydration and hydration in the catabolic and anabolic (gluconeogenesis) directions, respectively. Although the homologous genes that encode enolases are present in almost all cells, they are not considered housekeeping due to variations in expression that occurs under various physiological and pathological conditions.
The expressions of both ENO1 and ENO2 are up-regulated in several cancer cells where enhanced glycolysis is essential to compensate for impaired ATP generation from oxidative phosphorylation (Niklinski and Furman 1995; Lamerz 1998; Takashima et al. 2005; Kondoh et al. 2007; Yan et al. 2011). Increased ENO2 level in extracellular fluids has been associated with cancer progression and are typical for metastatic cancers in advance stages, especially pulmonary tumors (Allen et al. 1995; Lamerz 1998; Hao et al. 2004; Kasprzak et al. 2007). Alpha-enolase, in addition to its significance in cancer, however, has been proposed as a biomarker for the early diagnosis of acute myocardial infarction (Mair 1997; Takashima et al. 2005). In contrast to alpha-enolase, gamma-enolase has no C-terminal lysine but rather a PDZ-binding motif for possible interactions with PDZ-domain containing proteins. These characteristics and differences have led to the revelation that the enolases may engage in other cellular functions and signaling pathways events besides glycolysis (Diaz-Ramos et al. 2012; Vizin and Kos 2015).
The current study uses an established rat model of SAP–ALI to explore the potential of ENO1 and ENO2 as biomarkers for the disease.

Experimental procedures

Animals and SAP model

All animal studies were carried out in strict accordance with the recommendations in the EU animal management practices (1986). The model protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Dalian Medical University, Dalian, China (# SCXK (Liao) 2013-0003). Sixteen clean-grade healthy male Sprague–Dawley rats (8 weeks old, weight: 200–220 g) were purchased from the specific pathogen free (SPF) Animal Center of Dalian Medical University. The animals were randomly divided into two groups of sham operated (healthy, n = 8) and SAP–ALI group (n = 8). The SAP model was established as we have previously described (Zhang et al. 2015). Briefly, animals were fasted for 12 h, but with free access to drinking water, and anesthetized with 10% chloral hydrate by i.p injection (3 ml/kg) prior to operation. Under sterile conditions, 1.5% sodium deoxycholic acid (Baier Di Biotechnology, Beijing, China, 1 ml/kg dose, speed of 0.1 ml/min) was injected through the bile pancreatic duct into the pancreas. This procedure effectively induced severe acute pancreatitis and eventually SAP-induced acute lung injured rats. The acute nature of the model and short time (24 h) for ALI development could not permit the use of the solvent which was used to dissolve the salt to be injected (vehicle control) into the bile pancreatic duct of control animals. Such approach may induce mild acute pancreatitis which because of the limited model window, may not sufficiently resolve and hence compromise the principal clinical differences between SAP and control rats. Control animals were thus operated upon but only had their pancreas marginally rotated (Sham operated). At 24 h post-surgery, animals were anesthetized with 10% chloral hydrate by i.p. injection (3 ml/kg) prior to tissues harvesting. For validation of the candidate biomarkers, 28 additional animals were used for the model and sacrificed at 3 h (early phase SAP–ALI, n = 10) or 24 h post-surgery (late phase SAP–ALI, n = 10), or sham operated (24 h, n = 8). Abdominal aortic blood was collected for serum harvesting within 1 h of blood sampling, and stored at − 80 °C until used. Broncheoalveolar lavage (BAL) was performed by instilling 3 ml sterile iced PBS three times via a blunted 18-gauge needle into the trachea of the lung by alternating ligation of the left and right primary bronchi to sample BAL fluid (BALF) randomly from both the left and right lungs of rats within a group. Recovered BALF was centrifuged at 10,000×g for 10 min and the supernatant stored at − 80 °C until used. Tissues from unlavaged lung were quickly harvested and portions fixed in neutral phosphate formaldehyde or glutaraldehyde, and others frozen (− 80 °C).

Enzyme‑linked immunosorbent assays (ELISA)

The levels of serum amylase (α-AMY), a clinical marker for pancreatitis, tumor necrosis factor alpha (TNF-α; in serum and BALF), alpha enolase (ENO1; in serum and BALF) and gamma enolase (ENO2; in serum and BALF) were determined using commercially available ELISA kits per the manufacturer’s instructions. The ELISA kits for rat TNFα, α-AMY and ENO1 quantification were purchased from Lengton Company, Shanghai, China, and the ELISA kit for ENO2 quantification was purchased from Proteintech Inc., IL, USA. Briefly, 50 µl standards or samples (serum diluted 1:3 for ENO1 and ENO2; 1:4 for TNF-α and α-AMY; undiluted BALF) were carefully added to specific enzyme (TNFα, α-AMY, ENO1 or ENO2) pre-coated wells of ELISA strip plates. Blank or control wells were appropriately included in the setup. The wells were sealed with closure membranes that were included in the kits and the plates were incubated at 37 °C for 30 min. Wash buffer was diluted from 30× to 1× with ddH2O and used to wash the wells following incubation. The washing procedure was repeated five times with 30 s buffer holding time before draining. After the final wash, 50 µl HRP-conjugated reagent was added to each well, except blank wells. The plates were sealed with closure membrane and incubated for 30 min at 37 °C. Washing of wells were repeated as indicated above. Fifty microliters each of chromogen A and B solutions were added to each well in the dark and incubated for 10 min at 37 °C. Following incubation, stop solution (50 µl) was added to each well. Absorbance was read at 450 nm wavelength within 15 min of adding the stop solution using a Wallac Victor 3 plate reader (PerkinElmer, Turku, Finland). Using a specialized quantitation software associated with the plate reader, the amount of the analytes in the samples were estimated from a 4-parameter logistic standard curve.

Pathological examination by optical microscopy and transmission electron microscope (TEM)

Lung tissues previously fixed in neutral phosphate formaldehyde were paraffin embedded, sectioned (2 microns serial sectioning), and transferred onto glass slides for routine hematoxylin and eosin (HE) staining according to the protocol by Fischer et al. 2008. Briefly, glass slides bearing tissue sections were deparaffinated in three changes of xylene for 2 min each. The sections were rehydrated by transferring the slides through graduated ethanol of 100% (three times, 2 min each time), 95% for 2 min, 70% for 2 min, under running tap water for 2 min, and then immersed in water for 30 s with agitation by hand. The slides were dipped in Mayer’s hematoxylin solution with gentle agitation for 2 min and then rinsed in water for 3 min. The slides were transferred into 1% eosin Y solution for 1 min with gentle agitation. The sections were then dehydrated using the rehydration steps as outlined above in reverse order. A drop or two of mounting medium was used to affix coverslips over the tissue sections on the slides. Pathological examinations were made with an optical microscope (Olympus BX63, Tokyo, Japan).
For TEM investigation, lung samples (1 mm3 in size) were fixed twice with 2.5% glutaraldehyde. The tissues were rinsed three times (15 min/time, 4 °C) in PBS (0.1 mol/l). Before being embedded in araldite, the samples were prefixed with 2% osmic acid for 2 h and rinsed three times with PBS and then dehydrated through graded series of alcohol (50, 70, 80, 90, and 100% × 2). Ultrathin sections were cut using ultramicrotome and stained with uranyl acetate and lead citrate. The stained ultrathin sections were washed in distilled water thrice and air dried. The sections were observed under a JEM-100SX transmission electron microscope (JEOL USA Inc.)

Quantitative real‑time polymerase chain reaction (qRT‑PCR)

qRT-PCRs were used to quantify cDNAs of genes of interest using EvaGreen 2X qPCR MasterMix in an ABI ViiA7 Dx real-time PCR system. Total RNA was extracted from lung tissues with TRIzol reagent (Invitrogen Life Technologies, CA, USA) for reverse transcription using 5X All-InOne RT MasterMix (Applied biological materials Inc., BC, Canada) in a T100 Thermal Cycler (Bio-Rad Technologies Inc., CA, USA). qRT-PCRs were prepared in 20 µl reaction volumes by mixing 10 µl 2X EvaGreen qPCR MasterMix, 5 µl cDNA (500 ng), 0.6 µl each of forward (10 µM) and reverse (10 µM) primers, and 4.8 µl RNase-free water. The reaction conditions were: One cycle of 95 °C for 10 min for enzyme activation, 40 cycles of denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 30 s. The forward and reverse primer sequences for ENO1 were 5′-AGG TCA TCA GCA AGG TCG TG-3′ and 5′-TGC GGT GTA GAG ATC CAC CT-3′, and 5′-TCT TTC TTG CTG TCC CGA CC-3′ and 5′-TAG AAG GGA AAG TGG GCA GA-3′ for ENO2. The endogenous reference gene used was GAPDH: forward primer sequence as 5′-AGT GCC AGC CTC GTC TCA TA-3′ and reverse sequence as 5′-GAC TGT GCC GTT GAA CTT GC-3′. All primers were obtained from TaKaRa Biotechnology (Takara Bio, Dalian, China).

Immunohistochemistry (IHC)

Lung tissue sections on glass slides were deparaffinated and rehydrated by passing through xylene and graded series of alcohol, and then rinsed under running cold tap water using the same protocol for rehydration of tissues for HE staining as indicated above. Antigen retrieval was done with Tris/ EDTA buffer (10 mM/1 mM, pH 9) in a scientific microwave at 750 W for 20 min and allowed to cool at room temperature for 10 min under running tap water. Slides were rinsed twice in PBST (PBS with 0.025% Triton X-100) for 5 min/rinse. Tissues were blocked in 10% goat serum (Zsbio, Beijing, China) for 1 h at room temperature. Primary monoclonal antibodies (rabbit anti ENO1, and ENO2; Proteintech, IL, USA) were diluted in 1% BSA in PBST and applied to the tissues, and incubated overnight at 4 °C. Slides were washed thrice in PBST, incubated in 3% H 2O2 for 30 min to quench endogenous peroxidase activity, and then washed twice in PBST. Secondary biotinylated antibody and streptavidin solution (Kit #9000, Zsbio, Beijing, China) were applied per manufacturer’s instructions. Slides were rinsed thrice for 5 min each and then developed with diaminobenzidine (DAB, Zsbio, Beijing, China). Color development was stopped by rinsing slides in running cold tap water for 1 min and counterstained in Mayer’s hematoxylin for 1 min. Slides were rinsed in water bath five times and then washed under running tap water for 10 min. Tissues were dehydrated by reversing the rehydration protocol and then air dried for 30 min. Sections were mounted and observed under an optical microscope (Olympus BX63, Japan).

Statistical analysis

All data are mean ± SD. Student’s t test or ANOVA with Benferroni multiple comparison test was used for statistical differentiation between groups, where appropriate. p < 0.05 was considered to indicate statistically significant difference. GraphPad Prism 5 (GraphPad software, San Diego California USA) was used for all statistical computations.

Results

Severe acute pancreatitis‑induced acute lung injury (SAP–ALI) model

To evaluate the success of the model, two clinically relevant markers were used. Alpha amylase is a diagnostic marker of pancreatitis. Significantly higher serum α-amylase level was measured in the serum of SAP rats as compared with sham (Fig. 1a), thus, indicative of successful pancreatitis in model animals. Tissue pathology examination showed marked destruction of pancreas lobular structure, necrosis, and large hemorrhage of many pancreatic acinar cells (Fig. 1c, upper role) to confirm pancreatitis (Dervenis et al. 1999). Tumor necrosis factor alpha (TNF-α), a potent proinflammatory cytokine and a marker for systemic inflammation, indicated significantly higher inflammatory status in all SAP rats (Fig. 1b). Twenty-four hours post SAP induction, lung damage was obvious, characterized by alveoli septum thickening, large number of inflammatory cell infiltration, and large hemorrhage areas, in SAP rats (Fig. 1c, middle role). Ultrastructural examinations also revealed increased number of lamellar bodies, extensive disappearance of microvilli around alveolar type II epithelial cells, damaged basement membrane, and irregularly shaped or damaged nuclei in lung tissues of SAP rats as compared with sham-operated rats (Fig. 1c, bottom role). Thus, the injection of 1.5% bile salt into pancreatic duct successfully induced severe acute pancreatitis which progressed to cause acute lung injury (SAP–ALI) in the experimental animals.

Tissue expression of ENO1 and ENO2 in SAP–ALI lung tissue

Specific antibodies directed at ENO1 and ENO2 were used to examine the tissue expression of the enolase variants by immunohistochemistry. Alpha-enolase was highly expressed in lung tissues irrespective of the pathological state of the tissue. However, gamma-enolase was markedly expressed only in damaged lung tissues as compared with lung tissues from sham-operated rats (Fig. 2). This suggested a possibility of gamma-enolase overexpression being associated with injury of the lung.

Validation of ENO2 as marker of lung tissue damage

To validate the association of differential enolase expression with SAP-induced lung damage, a time-point SAP–ALI model was used. Alpha enolase (ENO1) and gamma enolase (ENO2) specific expressions were determined in lung tissues by qRT-PCR and immunohistochemistry. The expressions of the enolase isoforms were investigated at early (3 h) and late (24 h) phases of SAP–ALI development. Figure 3a, b show consistent increase in serum levels of α-amylase and TNF-α (BALF as well). Thus, indicating the progressive development of the syndrome. However, albeit both biomarkers were significantly elevated in the blood and BALF of SAP rats at 3 h, no obvious corresponding lung tissue damage was seen at same time point (Fig. 3c). Interestingly, the expression of ENO2 at both the gene (3.34 ± 1.9 fold change at 3 h) and protein level markedly increased at this stage, as compared with expression in normal lung tissues (Fig. 3e, f), and persisted to the late phase (3.17 ± 0.26 at 24 h) when tissue damage was obvious (Fig. 3c, e, f). ENO1 mRNA expression was fairly unchanged at 3 h (0.89 ± 0.19 fold change) as compared to expression in sham tissues but significantly decreased in expression at 24 h (0.43 ± 0.02; p = 0.0099) (Fig. 3d). The protein expression pattern of ENO1 in lung tissues was generally consistent with its mRNA expression (Fig. 3d, f), except that the ENO1 protein expression at 24 h was not as drastically reduced as its mRNA expression.

Evaluation of secreted ENO2 as a marker for early detection of SAP–ALI

To explore ENO2 as a potential clinical marker for early detection of SAP–ALI, serum and bronchoalveolar lavage fluid (BALF) time-point samples were analyzed by ELISA. Secreted levels of ENO1 remained fairly unchanged in both the BALF and serum (Fig. 4a, c, respectively) but ENO2 levels varied significantly between sham-operated, 3 and 24 h SAP rats (Fig. 4b, d). The relative change in enzyme isoform expression as the disease progressed was also in favor of ENO2 (Fig. 4e), and this was more noticeable in serum than in BALF (Fig. 4e). Additionally, a strong and significant positive correlation was observed between serum ENO2 and TNF-α, a clinically validated biochemical marker for acute lung injury (Fremont et al. 2010; Blondonnet et al. 2016), as shown in Table 1.

Discussion

ALI remains a clinical challenge for the lack of comprehensive molecular understanding of its pathobiology, and hence the lack of specific therapy to date. Recently, several clinical and experimental studies have critically attempted to propose reliable markers that may be important in the diagnosis and/prognosis of ALI (Fremont et al. 2010; Blondonnet et al. 2016). In this current experimental study, we demonstrated the potential of gamma-enolase as a biomarker that could indicate lung injury in a rat model of SAP–ALI essentially before pathological lung tissue damage.
Infusion of 1.5% sodium deoxycholate (a component of bile salt) into the pancreatic duct of rats in this model mimicked human gallstone-induced acute pancreatitis with ensuing development of SAP–ALI. The potency of bile salt to cause pancreatitis is well documented (Flexner 1906), as well as gallstone-induced pancreatitis in humans (Acosta and Ledesma 1974). Significantly higher serum α-amylase level, a diagnostic maker of pancreatitis, in SAP rats (Fig. 1a), was indicative of successful pancreatitis in model subjects, as confirmed by tissue pathology examination (Fig. 1c) which showed destruction of pancreas lobular structure, necrosis, and large hemorrhage of many pancreatic acinar cells (Flexner 1906). Elevated tumor necrosis factoralpha (TNF-α), a potent proinflammatory cytokine and a marker for systemic inflammation, was indicative of the spread of a local inflammation (here, induced pancreatitis) into a systemic inflammatory burst. The lung is very susceptible to inflammatory assaults and also the most common extrapancreatic target of complications due to pancreatic injury (Rubenfeld et al. 2005). The inflammation promotes immune cells’ trafficking to the lungs, surge in secretion of proinflammatory factors, and activation of multiple inflammatory pathways coupled with the release of tissue degrading enzymes to collectively orchestrate lung tissue damage (Abraham et al. 2000; Xu et al. 2010; Konrad and Reutershan 2012). Thus, injection of 1.5% bile salt into pancreatic duct successfully induced severe acute pancreatitis which progressed to cause acute lung injury (SAP–ALI) in the experimental animals. These observations agreed with previous reports from our lab and by others (Luan et al. 2013; Liu et al. 2014; Tang et al. 2014).
One of the major challenges to the provision of timely medical intervention prior to acute lung injury in severe acute pancreatitis is the lack of early diagnostic markers. In a recent study by Bhargava et al. (2014), using isobaric tags for absolute and relative quantification (iTRAQ) coupled with LC MS/MS technique, to identify differentially expressed proteins in early phase and late phase survivors of acute respiratory distress syndrome (ARDS) from BALF, carbohydrate catabolism was identified as one of the significant ontologies. Alpha-enolase was among the glycolytic enzymes identified in their study. However, assessment of enolase expression in our study revealed that alpha-enolase was ubiquitously expressed and secreted irrespective of disease state (Figs. 2, 3). Of note was the reduced expression of ENO1 gene in damaged lungs, although such drastic decrease in protein expression was not observed. This could be due to increased degradation and/ or decrease in transcription of the gene but without a corresponding lineal, but marginal, effect on protein translation as insignificant decreases were observed in ENO1 tissue expression and secretion into BALF and serum (Figs. 3f, 4a, c). Gamma-enolase, on the other hand, was differentially expressed in lung tissue, and secreted in BALF and serum to correspond to acute lung injury (Figs. 3, 4). Aside neural cells, gamma-enolase localizes largely in neuroendocrine cells, particularly those of the amine precursor uptake and decarboxylation (APUD) lineage, such as the intestine, thyroid and pituitary gland, lung and pancreas (Tiainen et al. 2003; Suresh 2005). In SAP–ALI, two (at least) of such APUD lineage cells (and thus, organs) are directly affected in the disease process. This makes it plausible for the cells to express and/or secrete gamma-enolase, as one of the many factors, due to stress or damage. Increased gamma-enolase expression and secretion has largely been implicated in cancers and obesity where altered glucose metabolism and inflammation are hallmarks of disease progression (Nakatsuka et al. 2002; Leiherer et al. 2016). In some specific cases such as neuroblastoma, intracerebral hemorrhage, brain damage, seizures, post comatose resuscitation, ischemic stroke, and in small cell lung cancer, it has proved reliable as a diagnostic and/or prognostic marker (Isgrò et al. 2015). Thus, in the absence of any of the above-mentioned disease conditions or complications, gamma-enolase might be of diagnostic and/or prognostic significance in SAP–ALI, and possibly a therapeutic target.
In a study involving 135 patients with benign pulmonary diseases, elevated serum level of gamma-enolase was observed in 11.1% of the subjects (Collazos et al. 1994). The highest gamma-enolase levels were found in subjects with lung infiltrates related to local hypoxia or direct damage to the lung epithelium which lead to the conclusion that alveolar, interstitial, or distal airway damage or perhaps local hypoxia could contribute to the release of gamma-enolase (Collazos et al. 1994). In a smaller study involving 13 resuscitated patients after circulatory arrest due to cardiopulmonary etiologies, one of the seven surviving patients with elevated serum gamma-enolase (76 µg/l; normal range: ≤ 15 µg/l) but normal cortical potential developed acute respiratory distress syndrome on the fourth day post-resuscitation (Stelzl et al. 1995). Our data corroborate these clinical observations and suggest that gamma-enolase may predict the lung damage in severe acute pancreatitis conditions before pathological lung tissue damage becomes obvious (Figs. 3, 4). The serum levels of gamma-enolase, but not BALF, positively correlated with the levels of TNF-α which is one of the markers proposed for the diagnosis of acute lung injury (Fremont et al. 2010; Blondonnet et al. 2016). Thus, serum as the source of measurement of gamma-enolase may be reliable for SAP–ALI diagnostic and/or prognostic purposes. Notwithstanding, the absence of direct clinical data to confirm our experimental observation, and the lack of data to indicate whether gamma-enolase is an etiologic or salvage factor in the pathogenesis of the disease call for more studies.
In summary, gamma-enolase may be relevant in SAP–ALI and that as an early phase damage-predictive marker, it could be useful for clinical applications and possibly target for therapy.

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