The study investigates the cardioprotective effects of Munziq on myocardial perfusion injury in rats, particularly those with abnormal body fluid. We aim to determine if Munziq pretreatment can mitigate myocardial ischemia-reperfusion injury, induce pathological changes, protect cardiac function, and explore its mechanisms, especially via the NF-kappa B signaling pathway. Our research group has been focusing on studying myocardial ischemia-reperfusion injury.
Our finding suggests that inhibiting the NF-kappa B pathway can alleviate myocardial ischemia-reperfusion injury, which may mitigate myocardial ischemia-reperfusion injury by inhibiting the NF-kappa B pathway, affecting mitochondrial function and myocardial metabolism. Our research identifies Munziq as a potential myocardial ischemia-reperfusion injury therapeutic, advancing the field by exploring a natural medicine approach. This discovery is significant as it targets NF-kappa B pathway, a novel mechanism for myocardial ischemia-reperfusion injury treatment.
Our findings paves the way for further clinical application of traditional medicine in cardiovascular disease management. To begin, house the rats in the abnormal body fluid, or ABF group, in a controlled environment within climate boxes and provide ordinary feed. Provide the rats with ordinary feed mixed with dry cold food, namely barley and coriander seeds, for 21 days to establish the ABF model.
Using an intragastric administration technique, administer five grams per kilogram of Munziq to the Munziq group rats for 21 days prior to myocardial ischemia-reperfusion injury, or MIRI surgery. To establish the MIRI model, after 21 days of pretreatment, place the anesthetized rat on a sterile operating table. Perform a tracheostomy to enable ventilator-assisted breathing in the rat.
Before opening the chest, wash the surgical site with soapy water, shave the area, and disinfect it with an iodine solution. Next, using sterile scissors, forceps, and retractors, open the chest wall to expose the heart and identify the left anterior descending artery, which lies on the heart's surface. Using a 6-0 suture, ligate the artery to induce regional ischemia for 30 minutes.
Confirm effective occlusion by observing a pale color in the myocardium. After 30 minutes, release the ligature and allow reperfusion for 120 minutes. Confirm reperfusion of the myocardium when it returns to a bright red color.
Following reperfusion, use a vacuum blood collection tube to collect one to two milliliters of blood from the abdominal aorta. After euthanizing the rat, use sterile forceps and scissors to collect myocardium tissue from the infarct area in the left ventricle. Place the collected tissue in a sterile container for further analysis.
After collecting myocardium infarcted heart from the MIRI established rat, use sterile scissors and blade to transect the heart horizontally into two halves along the midpoint of the left ventricular long axis perpendicular to the heart's direction. Divide one half of the apical part into two portions. Preserve one portion in 4%paraformaldehyde at room temperature for two to 24 hours for morphological examination.
Place the other portion in glutaraldehyde at four degrees Celsius for one to four hours for electron microscopy. Next, divide the base part of the heart, including both ischemic and non-ischemic areas, into two portions. Place one portion into a cryovial and rapidly freeze it in liquid nitrogen.
Transfer the frozen tissue to a minus 80 degrees Celsius freezer for molecular biology testing. Centrifuge the blood collected from the inferior vena cava at 1, 000 G for 10 minutes. Store the separated serum in a minus 80 degrees Celsius freezer.
Place the formaldehyde-fixed tissue sections in a 65 degrees Celsius incubator to bake for 1.5 to two hours. Immerse the baked tissue sections in xylene for 10 minutes. Next, sequentially immerse the tissue sections in anhydrous alcohol one and two for five minutes each, followed by 95%90%80%and 70%alcohol, and finally in distilled water for five minutes each.
Stain the tissue sections with hematoxylin for three minutes. Perform acidic differentiation by briefly immersing the tissue sections in a hydrochloric acid alcohol solution for one to two seconds. Terminate differentiation by rinsing in tap water for five minutes.
Next, immerse the tissue sections in distilled water, then in 70%80%90%and 95%alcohol for three minutes each. After immersing the section in anhydrous alcohol one and two, stain the sections with 0.5%eosin in ethanol for one minute. Now, rinse the sections in 95%ethanol to remove excess red color.
Then, immerse the sections in anhydrous ethanol for five minutes, followed by immersion in xylene one and two for five minutes each. Mount the stained sections with neutral balsam. Observe pathological changes in the tissue under a microscope.
Myocardial tissue in the sham group exhibited granular and vacuolar degeneration, limited red blood cell and lymphocyte infiltration, and occasional vascular dilation and congestion. In the MIRI group, there was severe myocardial tissue damage with extensive granular and vacuolar degeneration. Myocardial damage was notably severe in the ABF MIRI group compared to the control MIRI group, showing amplified signs of tissue degeneration and infiltration.
Myocardial cells treated with Munziq in both control and ABF MIRI groups displayed reduced granular and vacuolar degeneration with fewer red blood cells, lymphocyte infiltration, and congestion. Myocardial cells in the sham group maintained intact myofibril structure, uniform sarcomere length, and numerous mitochondria. In the MIRI group, myocardial cells displayed swelling, varied sarcomere length, and disrupted myofilaments, with extensive mitochondrial damage.
Munziq treatment reduced myocardial cell swelling and preserved myofibril, sarcomere, and mitochondrial structure, similar to sham conditions. Serum cTn-T, CK-MB, and ICAM-1 levels were significantly higher in the ABF MIRI group than in the control MIRI group. Munziq pre-treatment notably reduced levels of cTn-T, CK-MB, and ICAM-1 in both ABF and control MIRI groups.
The ABF MIRI group showed increased malondialdehyde levels and decreased nitric oxide levels in myocardial tissue compared to the control MIRI group. Munziq pretreatment significantly lowered lactate dehydrogenase and malondialdehyde content while increasing nitric oxide levels in ischemic myocardium. The ABF MIRI group exhibited elevated interleukin one beta, interleukin six, and TNF alpha levels, particularly at protein levels.
Munziq pretreatment reduced serum and mRNA levels of interleukin one beta, interleukin six, and TNF alpha, particularly at protein levels in the myocardium. NF-kappa B pathway markers were upregulated in MIRI tissue, indicating inflammation. Munziq treatment decreased the expression of pathway markers, indicating reduced NF-kappa B pathway activation.
