European Review for Medical and Pharmacological Sciences 2019; 23: 4458-4464 Effect of ulinastatin on myocardial ischemia reperfusion injury through ERK signaling pathway H. CHE, Y.-F. LV, Y.-G. LIU, Y.-X. HOU, L.-Y. ZHAO Department of Anesthesiology, Beijing Anzhen Hospital of Capital Medical University, Beijing, China Abstract. – OBJECTIVE: To study the ef- Introduction fect of ulinastatin (UTI) on myocardial isch- emia-reperfusion injury (MIRI) through the ex- Acute myocardial infarction ranks first in the tracellular signal-regulated kinase (ERK) signal- cause of death in patients in China. The area and ing pathway. severity of myocardial infarction seriously affect MATERIALS AND METHODS: A total of 24 the prognosis of patients. Although early reperfu- Sprague-Dawley rats were randomly divided in- sion therapy is the most direct and effective means to sham group (n=8), I/R group (n=8), and UTI group (n=8), and the rat model of MIRI was to reduce the area of myocardial infarction, the established. The changes in the content of se- dysfunction and structural damage of ischemic rum biochemical indexes, including superoxide myocardium cannot be repaired in the first time dismutase (SOD) and malondialdehyde (MDA), or even become worse after reperfusion, which is were detected using the kits, and the changes in known as ischemia-reperfusion injury1, seriously the expressions of serum inflammatory factors, hindering the greatest efficacy of reperfusion including interleukin-6 (IL-6) and tumor necro- therapy. Therefore, finding new treatment means sis factor-α (TNF-α), were detected using the to protect ischemic myocardium has become a quantitative Reverse Transcription-Polymerase problem urgently to be solved in the reperfusion Chain Reaction (qRT-PCR) and enzyme-linked therapy of acute myocardial infarction2. immunosorbent assay (ELISA) kits. Moreover, the ERK phosphorylation level in myocardial This ischemia-reperfusion injury is a very com- tissues was detected using the immunofluores- mon pathophysiological phenomenon in ischemic cence method, and the ERK phosphorylation heart diseases and during the perioperative period level and cleaved caspase-3 expression were of cardiac surgery, so simulating such reperfusion detected via qRT-PCR and Western blotting. injury in basic research is of great importance in RESULTS: Compared with those in sham directly observing the effect of drugs on cells and group, the serum SOD content significantly de- further exploring the mechanism deeply3. A large clined, while the MDA content was significantly number of studies have demonstrated that extracel- increased in I/R group, and they were significant- lular signal-regulated kinase (ERK) protein is lowly ly improved in UTI group (p<0.01). The results of detection using qRT-PCR and ELISA kits revealed expressed in normal myocardial cells, while it will that the inflammatory factors (IL-6 and TNF-α) in be significantly activated and highly expressed in UTI group were significantly improved (p<0.01). the case of myocardial ischemia-reperfusion injury The immunofluorescence results showed that the (MIRI)4. According to many studies, the ERK sig- ERK phosphorylation level in myocardial tissues nal transduction pathway plays an important role was significantly increased in UTI group. The in MIRI5. The main pharmacological activity of results of qRT-PCR and Western blotting mani- ulinastatin (UTI) is the protease inhibitor, which fested that both ERK phosphorylation level and has a significant inhibitory effect on such serine cleaved caspase-3 expression were significantly proteases as trypsin, plasmin, and chymotrypsin, improved in UTI group (p<0.01). CONCLUSIONS: and the anti-inflammatory and anti-shock biological UTI can play a protective role 6,7 in MIRI through up-regulating the ERK signal- functions of UTI have been confirmed . ing pathway. In the present study, UTI was used to inter- vene in the rat model of MIRI, and the protec- Key Words: tive effect of UTI in injured myocardial tissues Ulinastatin, ERK, Myocardial ischemia-reperfusion injury, Rats. through the ERK signaling pathway and its pos- sible mechanism were preliminarily investigated. 4458 Corresponding Author: Liyun Zhao, MD; e-mail: [email protected] Ulinastatin in myocardial ischemia reperfusion injury Materials and Methods Detection of Changes in Serum IL-6 and TNF-α Content via ELISA Animals The myocardial tissues of rats cryopreserved A total of 30 clean-grade male Sprague-Daw- at -80°C were taken, placed into Eppendorf (EP) ley rats weighing (200±20) g were provided by tubes, added with an appropriate amount of tissue the Capital Medical University Laboratory An- lysis buffer, thoroughly ground with a grinder, imal Center They were fed in strict accordance and placed on ice for 15 min, followed by centrif- with the standards of animal laboratory and ugation at 12000 g and 4°C for 5 min. Then, the had free access to food and water. This study supernatant was taken into another new batch of was approved by the Animal Ethics Committee EP tubes, and the protein concentration was deter- of Capital Medical University Animal Center. mined using the BCA method and quantified. The After the rats were anesthetized with pento- expression levels of inflammatory factors TNF-α barbital sodium (20 mg/kg), the left anterior and IL-6 in myocardial tissues of rats were detect- descending coronary artery was ligated at 0.5 ed according to the instructions of the ELISA kits. mm using the surgical suture to block the blood flow, causing ischemia. Then, it was found that Immunofluorescence Detection the distal myocardial tissues of the left anterior of p-ERK Expression Level descending coronary artery gradually turned in Myocardial Tissues dark purple. The surgical suture was loosened First, the myocardial tissues fixed were embed- to simulate the reperfusion process and, then, ded in paraffin, sliced, and deparaffinized. The the distal myocardial tissues of the left anterior endogenous peroxidase was inactivated with 3% descending coronary artery gradually returned H2O2, followed by microwave antigen retrieval. to be bright red. At 30 min before the opera- The tissues were sealed with serum at an appro- tion, UTI (2500 U/kg) was administered intra- priate concentration, marked with the immunohis- venously in UTI group, while an equal amount tochemical pen, and added with the primary anti- of normal saline was given in sham group and body for incubation in a wet box at 4°C overnight. I/R group. On the next day, the tissues were washed with PBS for 3 times, dropwise added with the fluorescence Reagents and Instruments secondary antibody for incubation for 30 min, UTI (Sigma-Aldrich, St. Louis, MO, USA), and washed again with PBS for 3 times, followed immunohistochemical staining kit SP-9001 by color development using diaminobenzidine (Beijing Zhongshan Goldenbridge Biotechnol- (DAB). Finally, the resin adhesive was dropwise ogy Co., Ltd., Beijing, China), ribonucleic acid added and the sections were covered with the (RNA) extraction kit (Invitrogen Life Technol- cover glass, followed by photography under a mi- ogies, Carlsbad, CA, USA), quantitative Re- croscope (TE2000-U, Nikon, Tokyo, Japan). verse Transcription-Polymerase Chain Reac- tion (qRT-PCR) kit (Tiangen, Beijing, China), Detection of p-ERK and Cleaved ERK, cleaved caspase-3 and glyceraldehyde Caspase-3 mRNA Expression 3-phosphate dehydrogenase (GAPDH; Cell Sig- Levels Via qRT-PCR naling Technology Inc., Shanghai, China), bi- After 50 mg myocardial tissues of rats cryopre- cinchoninic acid (BCA) protein quantification served at -80°C were taken into the EP tube, 200 kit and cell lysis buffer (Hanbio, Shanghai, μL TRIzol (Invitrogen, Carlsbad, CA, USA) was China), interleukin-6 (IL-6) and tumor necro- added into each tube, the tissues were ground us- sis factor-α (TNF-α) enzyme-linked immuno- ing the grinder on ice, and then, TRIzol was added sorbent assay (ELISA) kits (Hanbio, Shanghai, until the total volume was 1 mL. After standing for China). 5 min, chloroform was added, shaken violently for 15 s, and placed for 15 min. Then, the water-phase Detection of Biochemical layer was taken into another new batch of EP tubes, Indexes SOD and MDA added with isopropanol and turned upside down for After the blood was drawn and centrifuged, several times, followed by centrifugation. The white the serum was carefully separated to detect the precipitate at the bottom of the tube was RNA. content of SOD (xanthine oxidase colorimetry) After RNA was washed and dissolved in water, and MDA (thiobarbituric acid method) according its concentration and purity were detected. The to the instructions. qualified RNA (A260/A 280 =1.8-2.0) was reversely 4459 H. Che, Y.-F. Lv, Y.-G. Liu, Y.-X. Hou, L.-Y. Zhao Table I. qRT-PCR primer sequences. Gene Primer Primer sequence Forward primer 5'-CTCATCGCAGATGCCTGGAA-3' ERK Reverse primer 5'-TTCAGGTAATAGGCACCCTTGAAGA-3' Forward primer 5'-ACGCACGACGTCTTCCAGTA-3' Caspase-3 Reverse primer 5'-CCACCTGGTTCAACTCACTCC-3' Forward primer 5'-GCTTCGGCAGCACATATACTAAAAT-3' GAPDH Reverse primer 5'-CGCTTCACGAATTTGCGTGTCAT-3' transcribed into complementary deoxyribose nu- (Bio-Rad Laboratories, Hercules, CA, USA). Fi- cleic acid (cDNA), and the mRNA expression level nally, the gray scale was analyzed and compared, was detected via qRT-PCR. The primer sequences with GAPDH as an internal reference. are shown in Table I. Reaction conditions are as follows: 94°C for 5 min, amplification at 94°C for Statistical Analysis 30 s, 57°C for 30 s, and 72°C for 30 s for a total The data were expressed as mean ± standard of 40 cycles, and 72°C for 5 min. The data were deviation and processed using Statistical Prod- processed using Microsoft Excel, and the relative uct and Service Solutions (SPSS) 17.0 software expression level of the target gene was calculated (International Business Machines Corporation, using the 2-∆∆Ct method according to the following Armonk, NY, USA). The comparison between formula, with GAPDH as a control gene: ∆Ct (target groups was done using the One-way ANOVA test gene) = Ct (target gene) – Ct (control gene), ∆∆Ct = followed by the Post-Hoc Test (Least Significant ∆Ct (target gene) – ∆Ct (standard value).