Discussion
As a stress-activated serine/threonine kinase, protein kinase D1 is crucial in various fundamental physiological and biological functions, including cell growth, apoptosis, adhesion, motility, and angiogenesis16, 20, 21. Because of the low axial blood flow and IR, distal skin flap necrosis is a frequent postoperative complication6, 22. As a result, the axial vascular distribution must be carefully considered. The perfusion pressure of the flap blood and the nascent capillaries is critical for flap survival23. As a result, PKD1 may be able to increase the random flaps survival rate. Our findings indicate that PKD1 can enhance random skin flap survival through inhibiting oxidative stress and apoptosis and increasing distal flap neovascularization.
According to some studies, angiogenesis is crucial for random skin flaps survival7, 24. Angiogenesis entails a number of complex processes, including preexisting cell connections destruction, endothelial cell sprouting, mitosis, and maturation of new capillaries25. According to some studies, VEGF is required for endothelial cell mitosis and can increase microvascular permeability26, 27. By integrating VEGF-A signaling and interacting with the extracellular matrix, PKD1-regulated alphavbeta3 transport promotes angiogenesis28. Increasing evidence indicates that the PKD1-mediated signaling pathway is vital for endothelial cells, particularly in VEGF-induced angiogenesis regulation29-31. It is a critical signal for angiogenesis32. MMP9 is involved in the angiogenic switch and can aid in VEGF release33. Cadherin 5 prevents the formation of disorganized endothelial aggregates in new blood vessels and forms intercellular junctions34. According to IHC and western blotting analysis, PKD1 treatment increased Cadherin 5 and VEGF expression in stromal cells and blood vessels in the dermis. The present study demonstrated that PKD1 stimulates new blood vessels formation and increases microvessels number in ischemic skin flaps, thereby increasing the regenerated blood vessels number and quality. Thus, we hypothesize that PKD1 promotes neovascularization in the flap’s dermis by increasing VEGF, cadherin 5, and MMP9 levels.
When the flap’s blood supply is restored, the tissue may experience I/R damage and produce an abnormal amount of reactive oxygen species (ROS)8, finally resulting in necrosis in the flap’s distal region35. PKD1 protects cells from oxidative stress and minimizes hypoxia-induced damage36. Additionally, PKD1 regulates the detoxification of reactive oxygen and nitrogen species in the mitochondria, leading to cells protection against oxidative stress37, 38. Cell survival and cell death in dopaminergic neuronal cells are regulated by the PKD1-mediated protective mechanism38. SOD is a well-known antioxidant enzyme that converts superoxide radicals to hydrogen peroxide39. The PKD1-induced protective signaling pathways are hypothesized to be involved in the manipulation of mROS effects and age-related diseases associated with mitochondrial dysfunction40, 41. HO1 and eNOS are both involved in the process of oxidative stress and have antioxidant properties42. As a result, we predicted that PKD1 could help improve flap survival by inhibiting oxidative stress. IHC and WB analysis revealed that PKD1 enhanced eNOS, SOD1, and HO1 expression, all of which are considered to reduce oxidative stresses. Thus, our findings indicated that PKD1 inhibited oxidative stress and subsequently alleviated IRI in random skin flaps.
During flap I/R injury, the primary mode of cell death is apoptosis43. PKD1 acts as a critical anti-apoptotic kinase, protecting neuronal cells from oxidative stress throughout the early phases. Dopaminergic neuronal cells subjected to H2O2 or 6-OHDA caused phosphorylation of the PKD1 activation loop (PKD1S744E/748E) long before neuronal cell death was induced38. Numerous cellular stressors stimulate the apoptotic pathway (mitochondria-mediated), causing a mitochondrial release of CYC, which in turn leads to activation of the caspase cascade and ultimately CASP344. Bax considerably increases the outer mitochondrial membrane permeability during cell death, resulting in mitochondrial swelling45. We investigated CASP3, CYC, and Bax expression levels in response to various treatments to assess apoptosis extent. The PKD1 group suppressed the expression of CASP3, Bax, and CYC, according to western blotting analysis. A significantly lower CASP3 expression was noted in the PKD1 group than the control group, showing that PKD1 attenuated apoptosis levels in ischemic skin flaps.
The worst results were reported in the CID755673 group, which may indicate that PKD1 improves the prognosis and survival rate of skin flaps.