Effects of Ellagic Acid and Berberine on Hind Limb Ischemia Reperfusion Injury: Pathways of Apoptosis, Pyroptosis, and Oxidative Stress

<i>Background and Objectives</i>: Hind limb ischemia-reperfusion (I/R) injury is a serious clinical condition that requires urgent treatment and develops as a result of a sudden decrease in blood flow in the extremity. Antioxidant combinations are frequently used in diseases today. This study aimed to investigate and compare the effectiveness of ellagic acid (EA) and berberine (BER), which are important antioxidants, and the combination on hind limb I/R injury to evaluate their therapeutic power. <i>Materials and Methods</i>: Thirty-five male Sprague Dawley rats were randomly divided into five groups: sham, I/R, EA+I/R, BER+I/R, and EA/BER+I/R. In the I/R procedure, the infrarenal abdominal aorta was clamped and reperfused for 2 h. EA (100 mg/kg, ip) and BER (200 mg/kg, ip) were administered in the 75th minute of ischemia. Oxidative stress markers (MDA, GSH, SOD, and CAT) and TNF-α were measured. Apoptosis (Bax, Bcl-2, and Cleaved caspase-3) and pyroptosis (Nrf2, NLRP3, and Gasdermin D) pathways were evaluated via Western blot. Muscle tissue was examined histopathologically by hematoxylin eosin staining. One-way ANOVA and post hoc LSD tests were applied for statistical analyses (<i>p</i> < 0.05). <i>Results</i>: Bax levels increased in the ischemia group and decreased with EA and BER (<i>p</i> < 0.05). Bcl-2 levels decreased in the ischemia group but increased with EA and BER (<i>p</i> < 0.05). The highest level of the Bax/Bcl-2 ratio was in the I/R group (<i>p</i> < 0.05). Cleaved caspase 3 was higher in the other groups compared to the sham group (<i>p</i> < 0.05). While Nrf2 decreased in the I/R group, NLRP3 and Gasdermin D increased; EA and BER normalized these levels (<i>p</i> < 0.05). In the histopathological analysis, a combination of EA and BER reduced damage (<i>p</i> < 0.05). TNF-α levels were similar between groups (<i>p</i> > 0.05). MDA levels were reduced by EA and BER, but GSH, SOD, and CAT levels were increased (<i>p</i> < 0.05). <i>Conclusions</i>: It was concluded that TNF-α levels depend on the degree and duration of inflammation and that no difference was found in relation to duration in this study. As a result, EA, BER, and their combination could be potential treatment agents on hind limb I/R injury with these positive effects.