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.