Page 48 - GPD-2-2

Gene & Protein in Disease Dunaliella salina & myocardial ischemia-reperfusion injury

(ECG) were continuously sampled using the BL-420S synthesized by reverse transcription using a high-capacity
biological and functional experimental system. complementary reverse transcription kit (Applied
Biosystems): denatured at 70°C for 10 min, amplified at
2.3. Creatine kinase (CK) and lactate dehydrogenase 37°C for 60 min, and inactivated at 94°C for 10 min. The
(LDH) activity measurement SYBR™ Green PCR Master Mix kit (CWBio) was used
The CK and LDH activity of the perfusate were for qPCR amplification. The messenger RNA (mRNA)
measured using a commercial test kit (Nanjing Jiancheng levels of NQO1, HO1, IL-1β, and IL-6 were detected.
Biotechnology, China). qPCR was performed in the StepOnePlus qPCR system
(Applied Biosystems), that is, predenaturation at 94°C for
2.4. Triphenyltetrazolium chloride (TTC) staining 20 s, then 94°C for 15 s and 60°C for 1 min for 40 cycles.
Myocardial infarction area was detected by TTC staining. Genes were quantified by the 2 -ΔΔCq method, and the
Each heart was removed and frozen at −20°C for 20 min. internal reference gene was glyceraldehyde-3-phosphate
The whole heart was cut into 5–6 pieces of 2 mm tissue dehydrogenase (GAPDH) gene. The primer sequences are
slides, which were placed in 1% 2,3,5-TTC (lot number: shown in Table S1.
abs47011070, Absin) to stain evenly. After washing in 2.8. Western blot analysis
phosphate-buffered saline (PBS), the heart pieces were
[19]
fixed in 10% formalin for 24 h and then photographed. Total proteins were extracted from the left ventricle with
All the non-infarct areas (red) and infarct areas (white) radioimmunoprecipitation assay lysis buffer at 4°C. Equal
were measured and calculated from each sample in the amounts of protein (30 µg) were subjected to 10% sodium
corresponding group by the image analysis software dodecyl sulfate-polyacrylamide gel electrophoresis
Image-Pro. The percentage of infarct area was calculated analysis and transferred to polyvinylidene difluoride
according to the following formula: membranes (EMD Millipore). The membranes were sealed
with 5% bovine serum albumin containing 0.05% Tween-
Myocardial infarct area (%) = Myocardial infarct area/
total myocardial area × 100%. 20 (TBST) for 2 h at room temperature and then incubated
overnight with the following primary antibody at 4°C:
2.5. Hematoxylin and eosin (H&E) staining NRF2 (1:500 Proteintech 163961AP), KEAP1 (1:500
Proteintech 105032AP), JAK2 (1:1000 CST 3230S), phos-
At the end of reperfusion, part of the left ventricle tissue JAK2 (1:1000 CST 3774S), STAT3 (1:1000 CST 12640S),
was immediately placed in 4% paraformaldehyde for phos-STAT3 (1:1000 CST 9145S), and GAPDH (1:1000
more than 24 h. After embedding the myocardial tissues BOSTER. BM3896). After the primary antibody incubation
in paraffin, the tissues were sliced, dewaxed, stained with overnight, the membranes were incubated with secondary
hematoxylin, re-stained with eosin, dehydrated, sealed, antibody for 1 h at room temperature. Protein bands were
and then observed under a microscope and photographed.
developed with excellent chemiluminescent substrate
2.6. Superoxide dismutase (SOD) activity and (ECL; catalog no. 180–501, Tanon). GAPDH was used as
maleic dialdehyde (MDA) content in myocardial the internal reference of total protein, and ImageJ 1.80 was
homogenate used for band quantification.

At the end of reperfusion, the hearts were taken off the 2.9. Statistical analysis
Langendorff apparatus and placed in an icy plate. The left Data were expressed as mean ± standard error of the mean.
ventricles were sliced to about 50 mg and homogenized at Statistical analysis was conducted with GraphPad Prism
4°C in saline (1:9). The homogenates were centrifuged at 8.0. One-way analysis of variance was used for comparing
3000 rpm for 15 min, and then, the supernatant was taken out multiple groups with one factor. P < 0.05 was considered
for copper zinc (Cu/Zn) SOD activity and MDA content assay. statistically different.
The activity levels of Cu/Zn SOD and the content of MDA
were determined by enzyme-linked immunosorbent assay 3. Results
(ELISA) using the assay kit (Nanjing Jiancheng Biotechnology,
China) and following the manufacturer’s instructions. 3.1. D. salina pre-treatment improved cardiac
function after myocardial ischemia reperfusion in
2.7. RNA extraction and quantitative polymerase mice
chain reaction (qPCR) The drug treatment and building steps of isolated MIRI are
RNA was extracted from the myocardium using TRIzol shown in Figure 1A. The cardiac function was monitored
®
reagent (Invitrogen), and copy DNA (cDNA) was during myocardial ischemia reperfusion. D. salina pre-

Volume 2 Issue 2 (2023) 3 https://doi.org/10.36922/gpd.387