Osteoarthritis (OA), a degenerative disorder, is considered to be one of the most common forms of arthritis. Limonin (Lim) is extracted from lemons and other citrus fruits. Limonin has been reported to have anti-inflammatory effects, while inflammation is a major cause of OA; thus, we propose that limonin may have a therapeutic effect on OA. In this study, the therapeutic effect of limonin on OA was assessed in chondrocytes in vitro in IL-1β induced OA and in the destabilization of the medial meniscus (DMM) mice in vivo. The Nrf2/HO-1/NF-κB signaling pathway was evaluated to illustrate the working mechanism of limonin on OA in chondrocytes. In this study, it was found that limonin can reduce the level of IL-1β induced proinflammatory cytokines such as INOS, COX-2, PGE2, NO, TNF-α, and IL-6. Limonin can also diminish the biosynthesis of IL-1β-stimulated chondrogenic catabolic enzymes such as MMP13 and ADAMTS5 in chondrocytes. The research on the mechanism study demonstrated that limonin exerts its protective effect on OA through the Nrf2/HO-1/NF-κB signaling pathway. Taken together, the present study shows that limonin may activate the Nrf2/HO-1/NF-κB pathway to alleviate OA, making it a candidate therapeutic agent for OA.

Random-pattern skin flap is a vital technique frequently applied in reconstruction surgeries for its convenience and effectiveness in solving skin defects. However, ischemic necrosis, especially in the distal areas of the flap, still needs extra attention after surgery. Earlier evidence has suggested that paeoniflorin (PF) could stimulate angiogenesis and suppress ischemic cardiovascular disease. However, few studies have focused on the role of PF in flap survival. In this study, we have demonstrated that the human umbilical vein endothelial cells (HUVECs) treated with PF can alleviate tert-butyl hydroperoxide (TBHP)-stimulated cellular dysfunction and apoptosis. To better evaluate, HUVECs’ physiology, cell tube formation, migration, and adhesion were assessed. Mechanistically, PF protects HUVECs against apoptosis via stimulating the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway. PF also downregulates mitochondrial ROS production to reduce excessive intracellular ROS production induced by TBHP and restore TBHP-induced mitochondrial depolarization. As a result, silencing Nrf2 partially abolishes the protective effect of PF exposure on HUVECs. In in vivo experiments, the oral administration of PF was shown to have enhanced the vascularization of regenerated tissues and promote flap survival. However, the PF-mediated protection was partially lost after co-treatment with ML385, a selective Nrf2 inhibitor, suggesting that PF is a crucial modulator regulating the Nrf2/HO-1 signaling pathway. In summary, our data have provided a new insight into PF as a potential therapy for enhancing random-pattern flap viability.

Increasing evidence indicates that pyroptosis, a new type of programmed cell death, may participate in random flap necrosis and play an important role. ROS-induced lysosome malfunction is an important inducement of pyroptosis. Transcription factor E3 (TFE3) exerts a decisive effect in oxidative metabolism and lysosomal homeostasis. We explored the effect of pyroptosis in random flap necrosis and discussed the effect of TFE3 in modulating pyroptosis. Histological analysis via hematoxylin-eosin staining, immunohistochemistry, general evaluation of flaps, evaluation of tissue edema, and laser Doppler blood flow were employed to determine the survival of the skin flaps. Western blotting, immunofluorescence, and enzyme-linked immunosorbent assays were used to calculate the expressions of pyroptosis, oxidative stress, lysosome function, and the AMPK-MCOLN1 signaling pathway. In cell experiments, HUVEC cells were utilized to ensure the relationship between TFE3, reactive oxygen species (ROS)-induced lysosome malfunction and cell pyroptosis. Our results indicate that pyroptosis exists in the random skin flap model and oxygen and glucose deprivation/reperfusion cell model. In addition, NLRP3-mediated pyroptosis leads to necrosis of the flaps. Moreover, we also found that ischemic flaps can augment the accumulation of ROS, thereby inducing lysosomal malfunction and finally initiating pyroptosis. Meanwhile, we observed that TFE3 levels are interrelated with ROS levels, and overexpression and low expression of TFE3 levels can, respectively, inhibit and promote ROS-induced lysosomal dysfunction and pyroptosis during in vivo and in vitro experiments. In conclusion, we found the activation of TFE3 in random flaps is partially regulated by the AMPK-MCOLN1 signal pathway. Taken together, TFE3 is a key regulator of ROS-induced pyroptosis in random skin flaps, and TFE3 may be a promising therapeutic target for improving random flap survival.