| Abstract | Cijeljenje rane je prirodni odgovor na ozljedu tkiva, a u procesu cijeljenja rane
angiogeneza ima ključnu ulogu. Sintetski pentadekapeptid probavnog sustava BPC 157
pospješuje cijeljenje što je dokazano brojnim studijama, a djeluje i na angiogenezu.
Molekularni mehanizam njegovog djelovanja još uvijek je nerazjašnjen.
U ovom istraživanju je analiziran učinak peptida BPC 157 na ranu fazu ekspresije
gena povezanih s angiogenezom na modelu rane izazvane na koži štakora. Analizirani su
uzorci kože i potkožja oko rane uzeti dva, pet, odnosno 10 minuta nakon izazivanja rane i
aplikacije testnih agenasa. Aktivacija ekspresije pojedinih gena istraživana je na razini
analize količine stvorene mRNA. Analiza je napravljena metodom kvantitativne lančane
reakcije polimerazom u stvarnom vremenu (qRT-PCR), a za određivanje relativne
kvantifikacije ekspresije gena korištena je metoda ΔΔCt te su rezultati prikazani kao 2-ΔΔCt
vrijednosti. Istraživani su geni Akt1, Braf, Egfr, Egr1, Grb2, Hdac7, Kras, Mapk1, Mapk3,
Mapk14, Nos3, Pik3cd, Plcg1, Prkcg, Ptk2, Pxn, Src, Srf i Vegfa. Obzirom na distribuciju,
dobiveni su podatci analizirani Studentovim T-testom, a vrijednost p < 0.05 smatrana je
statistički značajnom.
BPC 157 potiče ekspresiju svih navedenih gena. Pojačana ekspresija gena Akt1,
Grb2, Nos3, Pik3cd, Prkcg, Ptk2 i Src javlja se već nakon dvije minute, a vidi se i kroz pet
i deset minuta. Za gene Braf, Egfr, Egr1, Hdac7, Mapk1, Mapk3, Mapk14, Plcg1, Pxn, Srf
i Vegfa, pojačana ekspresija zabilježena je nakon pet i u desetoj minuti, dok je povećana
ekspresija gena Kras detektirana nakon 10 minuta.
Dobiveni podatci ukazuju na to da pojačavanjem ekspresije navedenih gena protein
BPC 157 ima utjecaj i na signalne putove koji su važni za angiogenezu i cijeljenje. Jedan
od njih je prethodno identificiran signalni put PI3K-AKT-eNOS-VEGF koji se aktivira kod
proliferacije, adhezije i migracije stanica. Aktiviraju se i signalni put RAS-RAF-MEKERK/MAPK koji je uključen u regulaciju staničnog ciklusa i migraciju, PTK2(FAK)-SRCPXN, važan za moduliranje stanične adhezije i migracije te signalni put SRC-PLCγ-PKCMEK-ERK uključen u stanični rast i diferencijaciju. Geni čiji su produkti uključeni u
navedene signalne putove imaju različite uloge, a mehanizmi kojima utječu na regulaciju
staničnih procesa su iznimno složeni i često se međusobno isprepliću. Zaključno, BPC 157
ima proangiogeni učinak dokazan u in vivo modelu kožne rane. |
| Abstract (english) | Healing is a complex cascade of events that are not clearly separated, but they
interfere and involve complex interactions of different cell types, mediators, cytokines, and
the vascular system. Briefly, tissue healing is a procedure during which growth factors
cause cell proliferation, leading to the integration of dynamic changes involving soluble
mediators, blood cells, extracellular matrix production, and parenchymal cell proliferation.
We can distinguish several phases of healing: hemostasis, inflammatory phase,
proliferative phase and maturation phase. One of the earliest responses to injury results
from damage to blood vessels in the wound area. Blood coagulation is enabled by various
humoral and cellular ingredients such as fibrinogen and platelets which are also
responsible for signaling that contributes healing. Various cytokines (chemokines,
interleukins, interferons, TNF, etc.) and growth factors (PDGF, VEGF, TGF-β, etc.) are
released. They are signals for regrutation of circulating inflammatory cells to the wound
site, they initiate reepithelialization and connective tissue contraction, stimulate
keratinocyte and fibroblast migration and stimulate the angiogenic response.
Inflammatory cells like neutrophiles and macrophages are attracted to the wound
site where they phagocytise debris, release NO (increase vascular permeability) and
reactive oxygen species – ROS (killing pathogens), secreting proteases (break down
damaged cells), produce and release cytokines, angiogenic, inflammatory and fibrogenic
factors. Epidermis and dermis are regenerated by migration and proliferation of different
cells near the lesion as keranocytes and fibroblasts. For successful wound reparation, it is
necessary to ensure the transport of fluid, nutrients, oxygen and immunocompetent cells to
the newly formed tissue, so the development of new blood vessels is essential.
Angiogenesis is a complex process involving the proliferation, migration and remodeling
of endothelial cells as well as pericyte stabilization. These events are regulated by multiple
cytokines and growth factors. Binding of growth factors to their receptors induce receptor
dimerization which activates receptor kinases, receptor autophosphorylation and
downstream signalling (PI3K-AKT-eNOS, RAS-RAF-MEK-ERK/MAPK, etc.). In the
final stage of healing, it is necessary to establish a normal tissue structure and a scar is
formed with a large number of collagen fibers.
All these processes and events can be modulated by numerous exogenous and
endogenous factors. Some of them will have negative effect (old age, infection, diabetes),
but some of them can enhance wound healing. One of the agens that improves healing is
synthetic pentadecapeptide – BPC 157. It is confirmed by a series of studies: different
types of lesions (skin, muscle, tendon), burns, bone fractures, eye injuries, brain and spinal
cord injuries digestive system impairments etc. In addition to healing, it also has a proven
cellprotective and organoprotective effect. BPC 157 has no proven lethal dose and can be
administrated without a carrier.
The impact of BPC 157 on healing is reflected in rapid development of granulation
tissue, its maturation and degradation, improved epithelialization and increased wound
vascularization. The role of the NO-system is very important in the healing process, and
the effect of BPC 157 on the NO system has been demonstrated in several studies
(activation of VEGFR2-AKT-eNOS signaling pathway). BPC 157 stimulates the
expression of several genes mRNA (Egr1, Nab2, Nos3, Cox2, Vegfa) and phosphorylation
of proteins important for cell adhesion, proliferation and migration (Ptk2, Pxn, Akt1), all
mechanisms important for angiogenesis. Although results of previous studies suggest
promotional effect of BPC 157 on angiogenesis, the molecular level of this mechanism has
not been fully elucidated.
Aim of the study
The aim of this study was to evaluate the effect of the peptide BPC 157 on early
phase expression of the angiogenesis-related genes which was analyzed on the rat skin
wound model. Also, to confirm the effect of BPC 157 on expression of these genes, to
show its angiogenetic effect at the molecular level, to identify affected signaling pathways
and to elucidate the action mechanism of BPC 157.
Material and methods
The study was performed on female Wistar albino rats. The animals were randomly
assigned to two basic groups: control and treated with BPC 157, within which there were
subgroups with different time intervals from wound induction and agens application to
sampling.
Excision wound was performed with a 5 mm diameter biopsy punch in the skin of
the inner part of the hind right limb and immediate intraperitoneal administration of saline
and solution of BPC 157 in saline (2 µg/mL), respectively, was done (10 µg/kg body
weight). After two, five and 10 minutes, respectively, the wound was frozen locally and a
ring-shaped skin and subcutaneous tissue sample was then taken around the wound using a
round 8 mm diameter biopsy knife. Immediately after sampling, the tissue was frozen in
liquid nitrogen and stored until RNA extraction. Prior to extraction of total RNA, tissue
was homogenized. RNA extraction was done using the TRIzol-based method. The
concentration of RNA samples was measured and a reverse transcription reaction was
performed.
Analysis of selected genes expression evaluated on mRNA level was done using
the quantitative real-time polymerase chain reaction method (qRT-PCR). Obtained data
revealed the potential activation of signaling pathways associated with angiogenesis and
the mechanism of peptide BPC 157 activity, respectively. The ΔΔCt method was used to
determine the relative quantification of gene expression, and results were presented as
2
-ΔΔCt values. Akt1, Braf, Egfr, Egr1, Grb2, Hdac7, Kras, Mapk1, Mapk3, Mapk14, Nos3,
Pik3cd, Plcg1, Prkcg, Ptk2, Pxn, Src, Srf and Vegfa genes were analyzed. Endogenous
controls, ie housekeeping genes Gapdh, Actb and Rn18s, were used for results
normalization. Fold change with values < 1 was considered as reduced expression, while
fold change > 1 indicated increased expression.
The data obtained were analyzed, based on the distribution, by the Student's T-test,
and the value of p < 0.05 was considered statistically significant.
Results
BPC 157 increases the expression of all investigated genes. Significant
upregulation of Akt1, Grb2, Nos3, Pik3cd, Prkcg, Ptk2 and Src gene expression was noted
after only two minutes, and can also be detected in five and 10 minutes time periods. As
for the expression of Braf, Egfr, Egr1, Hdac7, Mapk1, Mapk3, Mapk14, Plcg1, Prkcg,
Ptk2, Pxn, Srf and Vegfa genes, significantly increased expression was registred after five
and also after 10 minutes, while increased Kras gene expression was detected 10 minutes
after inducing wound and application of BPC 157.
Discussion and conclusions
This study confirmed most of the data obtained by previous studies. Activation of
Akt1, Nos3, Pik3cd, Ptk2, Pxn, Src and Vegfa gene expression supports the idea that BPC
157 activates the PI3K-AKT-eNOS-VEGF and SRC-CAV-1-eNOS signaling pathways
crucial for cell proliferation, migration and adhesion, and angiogenesis as suggested in
studies by HSIEH et al. in 2017. and 2020., respectively. In the first study they discovered
that, due the BPC 157, expression of VEGFA mRNA and protein was not changed, but
VEGFR2 mRNA and protein indicated an increased expression after six hours with a peak
of expression after 12 hours for mRNA and 9 hours for proteins, respectively (time periods
studied were 3, 6, 12 and 24 hours after treatment). The same study showed, using a
immunofluorescence, that BPC 157, just like VEGF, promotes the internalization of
VEGFR2 on HUVEC cells. Since the internalization of VEGFR2 is followed by activation
of Akt and eNOS, ie initiation of VEGFR2-AKT-eNOS signaling pathway, the expression
of VEGFR2, Akt and eNOS proteins and their phosphorylated (activated) forms was
analyzed in 10, 15 and 30 minutes after treatment. Results showed that BPC 157 causes
phosphorylation of VEGFR2 rapidly, after 10 minutes, as Akt phosphorylation, while
eNOS phosphorylation was noted after 15 minutes. That data suggest that BPC 157 has an
extremely fast effect, as detected in our study with upregulated Akt1 and Nos3 mRNA
expression two minutes and Vegfa five minutes after injury.
In 2020. study HSIEH et al. discovered BPC 157 effect on vasorelaxation induction
by activating eNOS/NO signalling. It also stimulated vascular endothelial cell migration
and activated SRC-CAV1-eNOS signaling pathway. Expression of SRC, CAV1, eNOS
proteins and their phosphorylated forms was analyzed in HUVEC cells using the Western
blot 30 and 60 minutes after treatment. BPC 157 promoted phosphorylation of SRC and
eNOS proteins while the amount of total SRC ans eNOS proteins remained unchanged. A
similar thing happened with the pCAV1 protein, but the amount of total CAV1 protein was
reduced. BPC 157 promotes NO formation (important role in regulation of inflammation),
eNOS phosphorylation and it reduces CAV1-eNOS binding (increased eNOS activity is
associated with decreased CAV1 binding). Data obtained for SRC protein correlates with
previous 2010. in vitro study from CHANG et al. which demonstrated effect of BPC 157
on the increased migration rate of tendon fibroblast cells in a dose-dependent manner. It
was confirmed with Western blot method which showed that the application of BPC 157
had no effect on the increased amount of Fak/Ptk2 protein and paxilin, but enhanced their
phosphorylation. This effect can be associated with the activation of the PTK2(FAK)-SRCPXN signaling pathway which plays an important role in modulating cell adhesion and
migration. Src protein cooperates with many tyrosine kinase receptors and can activate
numerous signaling pathways, as SRC-CAV1-eNOS, PI3K/AKT and PTK2(FAK)-PXN.
In this study, expression of Ptk2, Src and Pxn genes was analyzed, and the obtained data
showed that BPC 157 application increased Ptk2 and Src gene expression after only two
minutes, while the same effect on Pxn was noted after five minutes.
An extensive study by KANG et al. in 2018. on an in vivo model of tumor cachexia
also analyzed molecular mechanisms of BPC 157. RT-PCR analysis was done and
expression of Mfn-2, PGC-1α and Pax mRNA was investigated. Decreased expression of
all these genes was observed in cachexia animals while in BPC 157-treated cachexia
animals the expression level was preserved. BPC 157 corrected both inflammatory and
metabolic changes caused by the tumor. The phosphorylated forms of Foxo, Akt, mTOR i
GSK-3β (transcription factor and regulatory proteins important for muscle proliferation
and myogenesis) were analyzed using the Western blot and their phosphorylation was
reduced in cachexia animals as opposed to those treated with BPC 157.
Some studies investigated BPC 157 effect on PI3K-AKT signalling and eNOS. In
2018. and 2020. VUKOJEVIĆ et al. have shown that BPC 157 has activating effect to
Nos3 expression, while study from CESAREC et al. in 2013. has shown that it reduces
Nos3 expression. The reason for these contradictory data can be found in studies describing
the effect of BPC 157 on the NO system where it interferes with the effects of agonists (Larginine, substrate molecules for NO) and antagonists of the NO system (L-NAME, NOS
inhibitors). It indicates that BPC 157 regulates the amount of NO depending on the
situation in the system. In addition, sampling and analysis were performed at different time
periods: one and 24 hours from wound induction (increased expression of Nos3), at the
beginning of inflammatory phase, and after four days (reduced Nos3 expression), when the
inflammatory phase should enter a downward trajectory.
In vivo studies from VUKOJEVIĆ et al. in 2018. and 2020. also examined the
effect of BPC 157 on the mRNA expression of genes important for cell migration,
proliferation and survival, and for the formation of reactive oxygen species, events
important for overcoming induced injury. Activation of VEGFR2 signaling is very
important for that events. It stimulates phospholipase C gamma (Plcg1) which is
responsible for the activation of protein kinase C (Prkcg), whose downstream signaling
targets are members of the MAPK signaling pathway (Raf, Ras, Mek, Erk2-1/Mapk1-3).
Inferior caval vein ligature samples were analysed one and 24 hours after injury. Results
indicated that BPC 157 increases the expression of the Egr (one hour) and Nos3, Srf, Kras
(one hour and 24 hours) genes and decreases the expression of the Egr (24 hours), Vegfr2
(one hour and 24 hours) and Plcγ (24 hours) genes while the expression the Akt1 gene
remained unchanged. Study done on ischemic/reperfusion injury in rat brain addressing the
effect of BPC 157 discovered its effect on upregulated expression of Egr1, Akt1, Kras, Src,
Foxo, Srf, Vegfr2, Nos3 and Nos1 genes, and downregulated expression of Nos2 and Nfkb
genes, while no effect on Mapk1 gene expression was observed. Data collected in this
study mostly correlates with those, as we showed that BPC 157 has an activating effect on
Prkcg expression after only two minutes, while increased Plcg1 expression was observed
after five minutes. Activation of the MAPK signaling pathway is manifested as increased
expression of Braf, Mapk1 i Mapk3 observed in the fifth minute, and for Kras the same
effect is seen after 10 minutes. BPC 157 also upregulated expression of Mapk14 five
minutes after application. It is also very important in the healing process, especially in the
inflammatory phase, as MAPK p38 (Mapk14) signaling pathway controls cytokine release.
In addition to VEGFR2, activation of EGFR signaling also includes components of
the MAPK signaling pathway and PI3K-AKT signaling pathway, respectively. Egfr
expression analysis showed upregulating effect of BPC 157 noted after five minutes.
Binding of the ligand to Egfr enables activation of Grb2 protein and triggering a MAPK
signaling cascade. Also, it can activate PI3K-AKT, PLCγ-PKC and other signaling
pathways. For this reason, the Grb2 gene was studied and its increased expression was
proven due to the action of BPC 157.
Cellular cytoskeletal proteins play a significant role during angiogenesis as they are
responsible for cell shape, adhesion and motility. One of the main regulators of
cytoskeleton protein expression is Srf – a transcription factor that regulates a number of
genes involved in cell growth, migration, and cytoskeletal organization. Srf is important
for the activation of some genes involved in endothelial proliferation, such as Fos and
Egr1, so in the case of Srf deficiency, VEGF-induced signaling will not be able to activate
the expression of Fos and Egr1. Analysis of the BPC 157 effect on Srf expression in this
study showed an increased expression after five minutes. The same effects on Srf gene
expression were demonstrated in previous studies from VUKOJEVIĆ et al. in 2018. and
2020.
Activation of protein kinase C signaling pathways includes induction of
transcription factors, Nfat and Egr1, respectively, and activation of the angiogenic
response. Egr1 induces cytokines and the production of growth factors as well as the early
formation of collagen, or extracellular matrix, and, like Srf, is a very important component
of the healing process and angiogenesis. An in vitro study from TKALČEVIĆ et al. in
2007. done on Caco-2 cells showed that BPC 157 enhances EGR1 expression. The analysis
was performed by RT-PCR method (effect was detected after 15 minutes) and Western
blot (effect was detected after one hour). That data correlate with the results obtained in
this study where increased expression of the Egr1 gene was demonstrated after only 5
minutes. Studies from 2018. and 2020. performed by VUKOJEVIĆ et al. analysing the
effect of BPC 157 on Egr1 expression also support this data.
Vegfa can cause rapid phosphorylation Hdac7 as early as five minutes, as described
by WANG et al. in 2008. Plcg and Prkc activate Prkd followed by Hdac7 phosphorylation
resulting in endothelial cell proliferation and migration. In this study, therefore, the effect
of BPC 157 on Hdac7 gene expression was investigated and the result indicated increased
expression after five minutes.
The complexity of events in signaling pathways and the large number of
interactions makes it difficult to clearly detect the action mechanism of BPC 157.
However, is is indisputably proven to have a pleiotropic effect, not only on expression of
different genes but also on their protein products triggering complex signaling cascades.
Taking into account the results of this and previous studies, it can be concluded that BPC
157 acts extremely quickly (gene expression effects seen in two, five and ten minutes,
respectively). It can have prolonged impact with modular or regulatory effects on different
events involved in angiogenesis or inflammatory response (regulation of NO system, effect
on expression and activation of certain genes and/or their protein products).
In conclusion, BPC 157 has a proangiogenic effect proven in an in vivo skin wound
model. Studied genes with protein products involved in angiogenic signaling pathways
have different roles and mechanisms they affect as regulation of cellular processes is
extremely complex and often intertwined. That is a puzzle that needs to be solved to
elucidate molecular mechanism of BPC 157 action which can potentially lead to
widespread use in a variety of pathological conditions |
