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Scutellarin Improves Apoptosis and Accelerates Neurite Outgrowth via Regulating Neurotrophins-3 in Hypoxia Ischemia Injured Neonatal Rats


Abstract
Objective: To explore the role of scutellarin and its relative molecular mechanism in recovery of
pathogenesis after hypoxic ischemic encephalopathy.
Methods: Neonatal Sprague–Dawley (SD) rats (seven-day old) were randomly divided into
4 groups: normal group, sham group, hypoxia ischemia group and NT3-si group. Above all,
assessing by quantify reverse transcription polymerase chain reaction (qRT-PCR), the expression
of TNFα was detected in lung, hippocampus and cortex. After that, the expression of NT3 in
cortex-right, and TRKC in cortex-right and heart was examined after interfering TNFα. Then,
immunofluorescence staining was used to determine the localization of TNFα and NT3 in cortex
left, cortex-right, hippocampus-left and hippocampus-right. The different relative expression of
NT3 after administrating scutellarin of 15μm and 50 μm was also determined by using qRT-PCR.
Moreover, the Effective siRNA Fragment of NT3 were screened out and validated successfully
by Cy3 staining and qRT-PCR. Furthermore, the neurite growth and cell apoptosis was observed
by TUJ1 and TUNEL staining. Last but not least, the expression of relative molecular mechanism
was detected by qRT-PCR.
Results: Observing from the results of qRT-PCR, the relative expression of TNFα was detected
visibly up-regulated after HI injury. After that, using qRT-PCR, the result illustrated that the
relative expression of NT3 in TNFα-shRNA group was significantly up-regulated in relation
to NC group in cortex-right. Beyond that, the protein expression of TRKC both in cortex-right
and heart was found visibly increased after the interference of TNFα. Besides, co-localization
of TNFα and NT3 in cortex and hippocampus was observed by immunofluorescence staining.
Moreover, the expression of NT3 was determined after administration of scutellarin for 15 μm
and 50 μm, respectively. However, it was observed that different from the up-regulation of NT3
after administration of scutellarin for 15 μm, the NT3 was found visibly down-regulated after
administration of scutellarin for 50 μm. Furthermore, the segment of F3 was screened out to be the
most effect fragment of siRNA, and it was transfected in neurons. After administrating scutellarin,
it could be indicated that administration of scutellarin could inhibit cell apoptosis and accelerate
the growth of neurite, which was associated with NT3. Finally, the expression of Bcl-2, BDNF
and NGF was down-regulated after interfering NT3. Thus, the potentially mechanism might be
tightly connected with Bcl-2, BDNF and NGF.
Conclusion: For one thing, the results showed that administration of scutellarin could protect cells
from apoptosis and accelerate the growth of neurite. For another, the scutellarin might come into
play through up-regulating NT3 associated with down-regulating the expression of Bcl-2, BDNF
and NGF.
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