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a

b c
Figure 5: The relative value of Bcl‑2 messenger RNA in all groups 72 h
Figure 4: The results of Western blot, pASK1 relative value and Bcl‑2/Bax following ischemia‑reperfusion. Compared to the ischemia‑reperfusion
ratio in each group 72 h following ischemia‑reperfusion. (a) Representative group, the hyperbaric oxygen, hydrogen‑rich saline, and hyperbaric oxygen
images of Western blots for pASK1, Bcl‑2, and Bax; (b) pASK1 relative + hydrogen‑rich saline groups show higher levels of Bcl‑2 messenger
values in all groups. Compared to the ischemia‑reperfusion group, RNA expression, especially the hyperbaric oxygen + hydrogen‑rich saline
the expression of pASK1 was significantly reduced in the hyperbaric group. IR: ischemia reperfusion, HBO: postoperative hyperbaric oxygen,
oxygen, hydrogen‑rich saline, and hyperbaric oxygen + hydrogen‑rich HRS: hydrogen‑rich saline
saline groups; (c) the ratio between Bcl‑2 and Bax in all groups. Bcl‑2/
Bax ratio reveals the highest level in hyperbaric oxygen + hydrogen‑rich
saline group. IR: ischemia reperfusion, HBO: postoperative hyperbaric the use of an ultrathin silicon sheet (0.1 mm) to avoid
oxygen, HRS: hydrogen‑rich saline revascularization and heparinized saline to avoid
thrombus formation. Analysis of the results showed that
[12]
high concentrations quickly, and excessive hydrogen skin flaps with HBO preconditioning and HRS treatment
can be eliminated from the body via breathing, leaving have the highest rate of survival in an IR model.
no side effects. The protective and therapeutic
[21]
effects of hydrogen in humans have been reported and As a new mechanism of inducing cellular IR injury, a
include such applications as diabetes mellitus type II, TNF‑α‑induced inflammation via mitochondrial ROS (mtROS)
[22]
hemodialysis, inflammatory myopathies, radiotherapy generation has received much attention. mtROS generated
[23]
[24]
for liver cancer, and acute erythematous skin diseases. by TNF‑α can oxidize the reduced thioredoxin‑apoptosis
[25]
[26]
Animal research in the rat skin flap model has also been signal‑regulating kinase 1 complex (Trx [SH] 2‑ASK1), and
performed to test the protective effect of HRS and has then activate ASK1 and its downstream stress signaling
shown that HRS increases the surviving areas of rat targets including JNK [28‑31] to initiate apoptosis. ASK1
abdominal skin flaps while decreasing oxidative stress and could, therefore, be considered to be a bridge during the
inflammation. [12] apoptotic signaling pathway. One pair of proteins plays
a paramount role in regulating apoptosis, specifically the
Based on the above theories, this study focused on the antiapoptotic protein Bcl‑2 and the pro‑apoptotic protein
combined application of HBO preconditioning and HRS Bax. The ratio of Bcl‑2/Bax determines the cellular direction
treatment, demonstrating its synergistic effect in protecting toward apoptosis. Caspase‑3 plays an important role in
[32]
a rat skin flap from IR injury by depressing apoptosis. In increasing the rate of apoptosis. Activated caspase‑3 cuts
this study, an abdominal island skin flap IR model was poly (ADP‑ribose) polymerase and increase the activity
established by ligating the left superficial epigastric artery of Ca /Mg ‑dependant endonuclease to destroy DNA
2+
2+
to investigate cellular and molecular changes following molecules. [33]
HBO, HRS, and combined treatments. This model was
first established by Kuntscher et al. In his study, an In this study, the HBO, HRS and HBO + HRS groups
[27]
extended (6 cm × 10 cm) epigastric adipocutaneous flap showed low levels of apoptotic. HBO preconditioning and
was harvested, and then the flap was sutured back over HRS used cooperatively were more efficient in reducing
a silicone sheet. This flap model has been widely used to cellular apoptosis than HRS or HBO preconditioning
study IR injury for the following reasons: first, the flap used independently. Caspase‑3 could increase the rate
size is large enough for observation of the survival area. of apoptosis. ASK1, as a bridge in the apoptotic process,
In the sham group, the epigastric artery could only sustain determines the integrity of the JNK pathway. The caspase‑3
75.40% ± 10.01% of the blood supply to the flap; second, activity and pASK1 expression were significantly reduced
with a large flap size, the survival area in each group can in the HBO, HRS and HBO + HRS groups. Compared to the
be measured scientifically and easily compared. Second, HBO and HRS groups, the HBO + HRS group showed the
in this model, the flap was supplied by one epigastric lowest level of caspase‑3 activity and pASK1 expression.
artery while other was ligated. A silicon sheet was also Bcl‑2, a mitochondrial anchoring protein, may prevent
[34]
used to prevent revascularization. Thus, the blood supply apoptosis by acting as an antioxidant. The Bcl‑2/Bax
to the model can be easily controlled and manipulated. ratio and Bcl‑2 mRNA expression were increased among
In this study, several modifications were made including the HBO, HRS and HBO + HRS groups. The Bcl‑2/Bax

Plast Aesthet Res || Vol 2 || Issue 6 || Nov 12, 2015 337