Biochemical and Biophysical Research Communications

A small-molecule activator of mitochondrial aldehyde dehydrogenase 2 reduces the severity of cerulein-induced acute pancreatitis

Shengchuan Cao a, b, c, d, Yuan Bian a, b, c, d, ***, Xin Zhou a, b, c, d, Qiuhuan Yuan a, b, c, d, Shujian Wei a, b, c, d, Li Xue a, b, c, d, Feihong Yang a, b, c, d, Qianqian Dong a, b, c, d, Wenjun Wang a, b, c, d, Boyuan Zheng a, b, c, d, Jian Zhang a, b, c, d, Zheng Wang a, b, c, d, Ziqi Han a, b, c, d, Kehui Yang a, b, c, d, Haiying Rui a, b, c, d, Ying Zhang a, b, c, d,
Feng Xu a, b, c, d, **, Yuguo Chen a, b, c, d, *
a Department of Emergency Medicine and Chest Pain Center, Qilu Hospital of Shandong University, Jinan, China
b Clinical Research Center for Emergency and Critical Care Medicine of Shandong Province, Institute of Emergency and Critical Care Medicine of Shandong University, Qilu Hospital of Shandong University, Jinan, China
c Key Laboratory of Emergency and Critical Care Medicine of Shandong Province, Key Laboratory of Cardiopulmonary-Cerebral Resuscitation Research of Shandong Province, Qilu Hospital of Shandong University, Jinan, China
d The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese Ministry of Health and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, Jinan, China

A R T I C L E I N F O

Article history:
Received 7 November 2019
Accepted 19 November 2019 Available online xxx

Keywords:
Acute pancreatitis Aldehyde dehydrogenase 2 Alda-1
Toxic aldehydes
4-Hydroxynonenal

A B S T R A C T

Acute pancreatitis (AP) is one of the leading causes of hospital admission for gastrointestinal disorders. Although lipid peroxides are produced in AP, it is unknown if targeting lipid peroxides prevents AP. This study aimed to investigate the role of mitochondrial aldehyde dehydrogenase 2 (ALDH2), a critical enzyme for lipid peroxide degradation, in AP and the possible underlying mechanisms. Cerulein was used to induce AP in C57BL/6 J male mice and pancreatic acinar cells were used to elucidate underlying mechanisms in vitro. Pancreatic enzymes in the serum, lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and Bcl-2, Bax and cleaved caspase-3 were measured. ALDH2 activation with a small-molecule activator, Alda-1, reduced the levels of the pancreatic enzymes in the serum and the lipid peroxidation products MDA and 4-HNE. In addition, Alda-1 decreased Bax and cleaved caspase-3 expression and increased Bcl-2 expression in vivo and in vitro. In conclusion, ALDH2 activation by Alda-1 has a protective effect in cerulein-induced AP by mitigating apoptosis in pancreatic acinar cells by alleviating lipid peroxidation.
 1. Introduction

Acute pancreatitis (AP) is an inflammatory disorder of the pancreas and is most commonly caused by pancreatic ductal obstruction secondary to gallstones, chronic alcohol use,

Corresponding author. Qilu Hospital of Shandong University, No. 107 Wenhua Xi Road, Jinan, 250012, China.

Corresponding author. Qilu Hospital of Shandong University, No. 107 Wenhua Xi Road, Jinan, 250012, China.
Corresponding author. Qilu Hospital of Shandong University, No. 107 Wenhua Xi Road, Jinan, 250012, China.
E-mail addresses: [email protected] (Y. Bian), [email protected] (F. Xu), [email protected] (Y. Chen).

hyperlipidemia, endoscopic retrograde cholangiopancreatography, etc. [1e3]. The global incidence of AP is approximately 34 per 100,000 people per year, which is constantly increasing year by year [4]. Because of timely and accurate diagnosis and critical care for patients, mortality resulting from AP has decreased over the past decade [5]. However, morbidity and poor long-term prognosis remain substantial and are attributed to the lack of effective pre- ventive and therapeutic agents [6,7]. Therefore, it is necessary to explore new targets for AP.
Toxic aldehydes accumulate in AP, especially 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), which is mainly produced during lipid peroxidation [8]. As a protective mechanism, a series of detoxification enzymes existing in the organisms are reported to remove biogenic aldehydes, such as aldehyde dehydrogenases, aldose reductases, and carbonyl reductases [9e12]. The mito- chondrial aldehyde dehydrogenase (ALDH2) plays a key role in the metabolism of acetaldehyde and other toxic aldehydes [13,14]. Evidence from different laboratories revealed the beneficial role of ALDH2 against acetaldehyde and toxic aldehyde-induced tissue injury, mainly focusing on cardiac or hepatic tissue [13,14]. The ALDH2 rs671 polymorphism, ALDH2*2, an inactive mutant form of the enzyme, is found in up to 30%e50% of East Asian populations [15,16]. ALDH2 activity was also impaired under many pathological conditions, such as myocardial infarction, alcohol-associated liver cancer and nonalcoholic fatty liver disease [16e18]. However, it is unclear whether ALDH2 is involved in the pathogenesis of AP.

Alda-1 is a small-molecule activator of ALDH2 and exerts its
actions by alleviating toxic aldehydes and by regulating autophagy, mitochondrial injuries, endoplasmic reticulum stress, etc. [10,16]. In the present study, we adopted the ALDH2 activator Alda-1 to explore its role in cerulein-induced AP.

2. Materials and methods

2.1. Animals and experimental procedures

C57BL/6 J male mice weighing 25e30 g were randomly assigned to 4 experimental groups (each group contained 12 animals). The mice were housed for one week before the experiments to accli- matize them to new conditions and they were kept in a room with a 12-h light/dark cycles. The experimental procedures were per- formed in accordance with the National Institute of Health guide for the care and use of Laboratory animals and were approved by the Ethics Committee of Qilu Hospital of Shandong University.

2.2. Induction of pancreatitis

AP was induced by cerulein (50 mg/kg, ip, 7 doses at hourly

intervals). In the treatment group, Alda-1 (10 mg/kg, ip) was administered 2 h before the first cerulein injection. The control group received normal saline in the same manner. Twenty-four hours after the first cerulein injection, mice were euthanized, blood samples were collected and pancreases were removed and
snap frozen in liquid nitrogen and then kept at 80 ◦C for later
biochemical measurements. Blood samples were used to determine serum amylase and lipase activities.

2.3. Cell lines, culture conditions and regents

Rat pancreatic acinar AR42J cells were obtained from ProCell (CL-0025, Wuhan, Hubei, China) and cultured in Ham’s Fe12 K medium supplemented with 20% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). AR42J cells were cultured for 12 h and were incubated with cerulein (10 nmol/ L) for another 24 h before the apoptotic assay or western blot was performed.
Alda-1 was purchased from MedChem Express (HY-18936, Monmouth Junction, NJ, USA), and cerulein was purchased from Sigma-Aldrich (C9026, St. Louis, MO, USA). The AKT activator, named SC79, was purchased from Selleck (S7863, Houston, TX, USA), and 4-HNE was purchased from Abcam (ab141502, Cam- bridge, MA, USA). The methyl green staining solution was pur- chased from Leagene Biotechnology (Beijing, China).

2.4. Determination of MDA and 4-HNE

Briefly, after routinely dewaxing and hydration, sections were stained with a primary antibody directed against 4-HNE (Abcam, ab46545, Cambridge, MA, USA) or MDA (Abcam, 6463, Cambridge, MA, USA). Then, sections were incubated in biotinylated secondary antibodies, followed by incubation with DAB as the chromogenic substrate. The stained sections were counterstained with hema- toxylin. Positive immunoreactivity was represented by a brown Fig. 1. Effects of Alda-1 on pancreatic injury in AP mice. A and B. H&E stained pancreatic sections and the histopathological score of the control, Alda-1, AP and Alda-1þAP groups. C. Serum amylase levels in the four groups. D. Serum lipase levels in the four groups. E. Pancreatic weight of the four groups. F. PW/BW (mg/g) of the four groups. AP acute pancreatitis, PW pancreatic weight, BW body weight. *P < 0.05 vs. control group. #P < 0.05 vs. Alda-1 group. All experiments were performed three times (n ¼ 3), and the data were expressed as the mean ± SEM.color under the light microscope and was then quantified using Image-Pro Plus 6.0.

2.5. Histopathological examination and scoring

The mice were decapitated, and a portion of the pancreas was fixed in 4% paraformaldehyde for more than 24 h. Then, the pan- creases were embedded in paraffin to be cut into 5 mm-thick sec- tions and stained with hematoxylin and eosin. Finally, a pathologist blindly observed the tissue sections under the light microscope and scored them (0e4) by evaluating the following criteria: edema, proinflammatory cell infiltration, acinar vacuolization and necrosis (0 is normal and 4 is severe) [19].

2.6. Measurement of apoptosis

Apoptotic cells were determined by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling (TUNEL) assay with an Apoptosis Assay Kit (Roche Applied Science, Penzberg, Germany) or were detected by flow cytometry with PI/Annexin V Apoptosis Detection kits (BD Biosciences). Apoptosis in pancreatic tissue was evaluated by using an ApopTag Plus Peroxidase In Situ Apoptosis Kit (S7101, Merck Millipore, Billerica, MA, USA).

2.7. Western blot analysis

Protein samples extracted from pancreatic tissues by Total

Protein Extraction Kit for Adipose Tissues/Cultured Adipocytes by Invent (AT-022, Minnesota, USA) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA), which were blocked with 5% milk and incubated with anti-ALDH2 (Abcam, ab108306, Cambridge, MA, USA), anti-4-HNE (Abcam, ab46545, Cambridge, MA, USA), anti- malondialdehyde (Abcam, ab243066, Cambridge, MA, USA), anti- Bcl 2 (GeneTex, gtx100064, California, USA), anti-Bax (Pro- teintech, 50599-2-Ig, Wuhan, Hubei, China), anti-caspase-3 (Cell Signaling Technology, 9662, Danvers, MA, USA), anti-AKT (4691, Cell Signaling Technology), anti-phospho-AKT (Ser 473) (4060, Cell Signaling Technology) or anti-b-actin (Proteintech, 60008-1-Ig,
Wuhan, Hubei, China) antibodies overnight at 4 ◦C. The membranes
were incubated with horseradish peroxidase (HRP)-coupled sec- ondary antibodies. The membranes were detected with Amersham Imager 600 (GE, Boston, USA). The intensity of the bands was analyzed densitometrically using ImageJ Software (National In- stitutes of Health, Bethesda, MD, USA). All data were normalized to b-actin internal control data.

2.8. Measurement of mitochondrial membrane potential (DJm)

DJm was determined using a commercial assay kit by incuba- tion with JC-1 (Beyotime, C2006, Shanghai, China) in a serum-free medium for 20 min at 37 ◦C. Then cells were washed with JC-1
staining buffer and imaged under a fluorescence microscope. Normal mitochondria produce red fluorescence, and depolarized or

Fig. 2. Alda-1 reduces the apoptosis and lipid peroxidation products MDA and 4-HNE in vivo. A and B. Histopathological staining of apoptosis in the control, Alda-1, AP, Alda-1þAP groups. C. Electron microscopy showing the structure of the nucleus in the pancreas. Scale bar: 5 mm. D and E. Western blot analysis of the ALDH2 protein and apoptosis-associated markers in the four groups. F and G. 4-HNE expression in pancreatic tissue (immunohistochemical staining 200 × ). H and I. MDA expression in pancreatic tissue (immunohis- tochemical staining 200 × ). *P < 0.05 vs. control group. #P < 0.05 vs. Alda-1 group. All experiments were performed three times (n ¼ 3), and the data are expressed as the mean ± SEM.inactive mitochondria produce green fluorescence. DJm was calculated by the red/green fluorescence ratio.

2.9. Electron microscopy

Sections of pancreatic tissue were fixed in 2.5% glutaraldehyde overnight. After rinsing in 0.1 mmol/L cacodylate buffer with 1% tannic acid, the samples were immersed in 1% osmium tetroxide in
0.1 mmol/L cacodylate buffer for 1 h. After being rinsed again, the samples were dehydrated with alcohol and embedded in Epon 812. Then, the samples were examined using a transmission electron microscope (HT7700, Hitachi, Japan).

2.10. Statistical analysis

All data were expressed as the mean ± SEM. Evaluation was performed using Student’s t-test for two groups. A one-way anal- ysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was used for the statistical significance of multiple treatments as appropriate. Two-sided P < 0.05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Alda-1 administration mitigated the severity of cerulein- induced AP

Alda-1 administration significantly alleviated congestion, edema and inflammatory cell infiltration in the pancreatic tissues of the AP group (Fig. 1A and B). The serum amylase and lipase levels were also evidently decreased by Alda-1 in the AP group (Fig. 1C and D). In addition, the pancreatic weight and the ratio of pancreatic weight to body weight (PW/BW) were lower in the Alda- 1-treated group than in the AP group (Fig. 1E and F).

3.2. Alda-1 administration reduced cerulein-induced pancreatic cell apoptosis and toxic aldehyde accumulation

Then, we determined the effect of Alda-1 on cell apoptosis in the pancreatic tissue induced by cerulein. It was shown that Alda-1 treatment greatly decreased cell apoptosis in the AP group, as examined by the TUNEL assay (Fig. 2A and B), and reduced nuclear consolidation, as shown in the transmission electron microscopy (TEM) images (Fig. 2C). Moreover, Alda-1 administration signifi- cantly reduced the expression levels of Bax and cleaved caspase-3 and elevated the expression level of Bcl-2 in the AP group

Fig. 3. Alda-1 reduces the apoptosis and lipid peroxidation products MDA and 4-HNE in vitro. A and B. Western blot analysis of the ALDH2 protein and toxic aldehyde stimulated by cerulein. C and D. Apoptotic nuclei and total nuclei were respectively identified by TUNEL staining (red) and DAPI staining (blue). Scale bar: 50 mm. E and F. Apoptosis was determined by flow cytometry stained with annexin V and propidium iodide (PI), and the cell apoptosis ratio was expressed as the percentage of annexin Vþ cells. G and H. Western blot analysis of apoptosis-associated markers stimulated by cerulein. I and J. Western blot analysis of apoptosis-associated markers stimulated by 4-HNE. *P < 0.05 vs. control group. #P < 0.05 vs. Alda-1 group. All experiments were performed three times (n ¼ 3), and the data are expressed as the mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

(Fig. 2D and E). We also found that 4-HNE and MDA were decreased in the AP group after Alda-1 administration, as determined by immunohistochemistry (Fig. 2FeI).3.3. Alda-1 reduces cerulein-induced pancreatic cell apoptosis by detoxifying 4-HNE in vitro

We used the AR42J cell line and found that cerulein treatment increased 4-HNE and MDA accumulation, which was mitigated by Alda-1 (Fig. 3A and B). We also found that cerulein induced AR42J cell apoptosis and that Alda-1 reduced apoptosis (Fig. 3CeF). We further discovered that Alda-1 reduced the expression levels of Bax and cleaved caspase-3 and elevated the expression level of Bcl-2 after cerulein treatment (Fig. 3G and H). 4-HNE was reported to induce apoptosis in many cell types [20], and we used 4-HNE to stimulate the AR42J cell line and found that 4-HNE induced obvious pancreatic cell apoptosis accompanied by increased Bax and cleaved caspase-3 and decreased Bcl-2, whereas Alda-1 reversed the effects of 4-HNE (Fig. 3I and J).

3.4. Alda-1 prevents 4-HNE-induced pancreatic cell apoptosis by promoting AKT phosphorylation

We also found that Alda-1 partially reversed cerulein-inhibited

AKT phosphorylation, implying that AKT might be involved in this process (Fig. 4A and B). Then, we used 4-HNE to mimic toxic aldehyde accumulation and found that 4-HNE significantly inhibi- ted AKT phosphorylation, which was improved by Alda-1 (Fig. 4C and D). Finally, we found that 4-HNE-induced pancreatic cell apoptosis was relieved by the AKT activator SC79 (Fig. 4E and F).

4. Discussion

Here, we found that Alda-1 administration decreased the 4-HNE level, reduced cell apoptosis, and attenuated cerulein-induced AP. We also testified that increased 4-HNE production was attributed to cerulein-induced pancreatic cell apoptosis, which can be prevented by ALDH2 activator Alda-1. Mechanically, AKT phosphorylation was involved in 4-HNE-induced pancreatic cell apoptosis.

Toxic aldehydes, such as 4-HNE and MDA, were reported to accumulate in the pancreatic tissue when AP occurred [21e23]; this suggests that targeting aldehydes in the pancreas might be a new way to deal with AP. ALDH2, a key enzyme in alcohol metabolism, is involved in clearing various aldehydes, especially 4-HNE and MDA, under different pathological conditions [10,24,25]. As expected, in this study, we found that Alda-1 significantly reduced toxic alde- hyde accumulation in the pancreas. In addition, we also demon- strated that 4-HNE was responsible for pancreatic cell apoptosis,

Fig. 4. Alda-1 improved pancreatic cell apoptosis mediated by AKT phosphorylation. A and B. Phosphorylation of AKT after stimulation with cerulein. C and D. Phosphorylation of AKT after stimulation with 4-HNE. E and F. Western blot analysis of apoptosis-associated markers induced by 4-HNE and relieved by SC79 (8 mg/ml). *P < 0.05 vs. control group. #P < 0.05 vs. Alda-1 group. & P < 0.05 vs. 4-HNE group. All experiments were performed three times (n ¼ 3), and the data are expressed as the mean ± SEM.which was relieved by Alda-1. Since 30e50% of East Asians carry the ALDH2 mutant phenotype, targeting ALDH2 enzymatic activity might be a new therapy for AP, especially for East Asian populations.

When AP occurred, the pancreatic acinar cells identified their genetic program in a rapid and precise way, with apoptosis as one of the important programs [26]. Many studies have shown that it was protective to reduce apoptosis of pancreatic acinar cells in the process of pancreatitis [27e29], which played a considerable role in affecting the mortality and morbidity of AP. Our in vivo experiments showed that apoptotic cells were increased in wild-type mice treated with cerulein, as identified by TUNEL and cleaved caspase-3 expression analyses. In contrast, the number of pancreatic acinar cells entering the apoptotic program was markedly decreased with the administration of Alda-1. We found similar results in vitro. These results indicated that Alda-1 exerted its protective role by preventing the initiation of apoptosis caused by AP.
The phosphoinositide 3-kinase (PI3K)/AKT pathway has been
shown to play important negative feedback under various patho- logical conditions [30,31], including AP [32e34]. In this study, we found that Alda-1 could improve AKT phosphorylation, which was reduced when AP occurred, and identified AKT as a key regulator of pancreatic cell apoptosis with the application of AKT activator. The 4-HNE- AKT pathway has already been reported to participate in the process of apoptosis in many other diseases [35e37], and me- chanically, 4-HNE can form adducts with AKT to decrease its phosphorylation [37]. Our findings of the 4-HNE-Akt pathway affirmed the role of toxic aldehydes in AP and explained the events in the 4-HNE pathway.
The ALDH2 gene was located in mitochondria [38]; therefore, the improvements in AP by Alda-1 might be due to its effects on mitochondria. In this study, we found that Alda-1 could improve DJm in vitro (Fig. S1). However, we did not further explore the relationship between Caerulein mitochondrial membrane potential and apoptosis with the application of Alda-1.
In summary, we demonstrated that ALDH2 activation played a protective role in AP by reducing the levels of toxic aldehydes and alleviating apoptosis. Our findings will provide a new perspective for new drug development to treat AP.

Declaration of competing interest

No conflict of interest is declared.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (81801942, 81902350, 81570401, 81772036,
81571934, 81671952, 81873950, 81873953), National Key R&D
Program of China (2017YFC0908700, 2017YFC0908703), Natural Science Foundation of Shandong Province (ZR2018BH027, ZR2015HQ020), Taishan Young Scholar Program of Shandong Province (tsqn20161065), Taishan Pandeng Scholar Program of Shandong Province (tspd20181220), Key R&D Program of Shan- dong Province (2018GSF118122, 2016GSF201235, 2016ZDJS07A14,
2018GSF118003, 2019GSF108075) and the Fundamental Research Funds of Shandong University (2014QLKY04, 2018JC011).

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Appendix A. Supplementary data

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