Aditama A. P. R, Asiyah S, Hariyani T, Nuraisya W, Mustofa L. A, Setyowati A. Enhancement of Osteoblastogenesis within the hFOB 1.19 Cell Line by the Induction of the n-Butanol Fraction Derived from Marsilea crenata C Presl. Leaves. Biomed Pharmacol J 2024;17(3).
Manuscript accepted on :21-05-2024
Revealed on-line on: 17-07-2024
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Agnis Pondineka Ria Aditama1, Siti Asiyah2*
, Tintin Hariyani2, Wahyu Nuraisya2
, Linda Andri Mustofa2
and Anis Setyowati2
1Well being Polytechnic of Jember, Jember, Indonesia.
2Division of Midwifery, STIKES Karya Husada Kediri, Kediri, Indonesia.
Corresponding Creator E-mail:aninkamila@gmail.com
Summary
Osteoporosis is a illness characterised by bone loss, which might be handled by rising the bone formation course of. A marker of elevated bone formation processes is the exercise of the transcription issue Osx which stimulates the formation of Ocn. M. crenata leaves incorporates phytoestrogens that are thought to extend bone formation. The aim of this analysis was to show that the n-butanol fraction from the leaves of M. crenata can enhance bone formation in hFOB 1.19 osteoblast cells by rising the exercise of the transcription elements Osx and Ocn. hFOB 1.19 cells that had reached confluence got n-butanol fraction at a dose of 62.5, 125, and 250 µg/L, and Genistein 2.5 µg/ml as a optimistic management for phytoestrogens. Elevated bone formation was recognized by means of the expression of Osx and Ocn utilizing the immunocytochemical technique with CLSM. The findings point out that the applying of assorted dosages of the n-butanol fraction derived from the leaves of M. crenata has a optimistic affect on bone formation in hFOB 1.19 cells. The very best dose was decided to be 250 µg/L, with a statistically vital distinction of p<0.005. The n-butanol fraction obtained from M. crenata leaves was discovered to extend the expression of Osx and Ocn in hFOB 1.19 cells, indicating its potential to reinforce bone manufacturing.
Key phrases
hFOB 1.19; Marsilea crenata C Presl.; n-butanol fraction; Osteri; Osteocalcin
Copy the next to quote this text: Aditama A. P. R, Asiyah S, Hariyani T, Nuraisya W, Mustofa L. A, Setyowati A. Enhancement of Osteoblastogenesis within the hFOB 1.19 Cell Line by the Induction of the n-Butanol Fraction Derived from Marsilea crenata C Presl. Leaves. Biomed Pharmacol J 2024;17(3).
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Copy the next to quote this URL: Aditama A. P. R, Asiyah S, Hariyani T, Nuraisya W, Mustofa L. A, Setyowati A. Enhancement of Osteoblastogenesis within the hFOB 1.19 Cell Line by the Induction of the n-Butanol Fraction Derived from Marsilea crenata C Presl. Leaves. Biomed Pharmacol J 2024;17(3). Obtainable from: https://bit.ly/3LouPf0
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Introduction
Osteoporosis is a bone illness in people
characterised by decreased bone power and density, resulting in the next danger of
fractures. Bone density typically decreases with age, displaying variations based mostly on
the gender and race of people. Key irreversible elements contributing to
osteoporosis growth are age, menopause, and feminine gender1. Estrogen
deficiency in ladies over 40 results in decreased bone mass, elevating the probability
of osteoporosis2,3.
The method of bone reworking is characterised by
its dynamic nature, whereby osteoclasts have interaction within the resorption of aged bone
tissue and osteoblasts are answerable for the technology of recent bone tissue.
It’s answerable for therapeutic injured bones4,5. The formation stage
includes the proliferation and differentiation of osteoblast precursors,
adopted by the mineralization of the brand new bone matrix4.
The estrogen-receptor (E-ER) complicated within the nucleus
or cell membran might be influenced by estrogen to have an effect on the proliferation,
differentiation, and maturation of osteoblast in several species6,7.
One transcription issue that’s vital for osteoblast growth is named
Osterix (Osx). By upregulating the expression of proteins corresponding to osteocalcin
(Ocn), osteopontin (OPN), bone sialoprotein (BSP), and collagen kind I (COL-I),
it promotes the manufacturing of recent bone8,9. As an grownup, Ocn is a
main protein in bones. It helps bones mineralize by interacting with
γ-carboxyglutamic acid (Gla) residues and rising the absorption of
hydroxyapatite10.
Using estrogen in ladies with estrogen
deficiency, corresponding to long-term hormone substitute remedy (HRT), carries a
carcinogenic danger to feminine reproductive organs and different unintended effects11.
Due to this fact, analysis on estrogen options prefers pure elements,
particularly phytoestrogen compounds, which give bone protecting results by
inhibiting bone resorption and rising bone formation as a consequence of menopause12,13.
Plant chemical substances referred to as phytoestrogens can act like estrogen by making
osteoblast cells work more durable by attaching to the estrogen receptor (ER) with
low unintended effects14.
The aquatic plant Marsilea crenata C Presl. which is often
consumed as a staple meal in Surabaya, East Java, Indonesia, has been
recognized as a supply of phytoestrogens 15,16,17. Quite a few investigations performed on M. crenata have
revealed the presence of phytoestrogens in varied kinds, together with
triterpenoids, steroids, and isoflavones, inside the ethanol extract, n-hexane,
ethyl acetate, and the n-butanol fraction of M. crenata leaves. Moreover, it has the
potential to stimulate the activation of ER-β and improve the elevation of
alkaline phosphatase (ALP) ranges within the MC3T3-E1 preosteoblast cell line,
therefore selling mobile proliferation and differentiation 18,19. In
vivo utilizing female and male mice exhibits that M.
crenata induces the proliferation of trabecular bone osteoblast cells 20,21.
The aim of this analysis is to show that the n-butanol fraction (BF) of M. crenata leaves can enchance bone formation in human fetal osteoblast (hFOB 1.19) by measuring the Osx and Ocn expressions utilizing a confocal laser scanning microscop (CLSM) instrument. These cells are used to check how human osteoblasts differentiate, how osteoblasts work, and the way cytokines have an effect on osteoblast operate22,23.
Materials and strategies
Supplies
UPT Materia Medika, Batu, East Java, recognized M. crenata leaves that have been gathered from the Benowo area of Surabaya, East Java, Indonesia (code: 1a17b-18a-1)24. The hFOB 1.19 cell line (CRL-11372) was obtained from American Sort Cell Tradition (Virginia, USA). Quercetin, dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), bovine serum albumin (BSA), and phosphate-buffered saline (PBS) have been equipped by Sigma-Aldrich (Missouri, USA). Main antibodies in opposition to mice osteocalcin and anti-rabbit osterix have been acquired from Abcam (Cambridge, Britain). The Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS), and G418 have been supplied by Aretha Laboratory (Bandung, Indonesia). Anti-mouse secondary antibody Rhodamine, anti-rabbit secondary antibody fluorescein isothiocyanate (FITC), paraformaldehyde and Tween-80 have been supplied by the Central Laboratory of Life Science at Brawijaya College (Malang, Indonesia).
Technique
Extraction, Fractionation, and Preparation of BF
A method of extraction was carried out utilizing 1.5 kg of M. crenata leaf powder and a solvent consisting of 96% ethanol, with the help of ultrasonics wave. This process yielded a complete of 70 g of extract17. Liquid-liquid extraction was achieved in a 1:1 ratio, the extract was mixed with 600 mL of water and subsequently separated utilizing n-Hexane. Ethyl acetate and n-butanol have been employed to separate the aqueous part. The isolation and drying of the n-butanol part have been performed utilizing a Heidolph Hei-VAP ML/GG3 rotary evaporator.
A inventory answer with a focus of 5000 ppm was ready by combining a 50 mg BF with 0.5% Tween-80 in 0.5% DMSO (w/v). The answer is formulated with concentrations of 62.5, 125, and 250 µg/L.
Cell Tradition
The hFOB 1.19 cells have been cultured in a tradition flask with a whole media together with DMEM, FBS 10%, G418, and penicillin-streptomycin 1%. The flask was then positioned in an incubator with a CO2 5% and maintained at a temperature of 37°C for a interval of 6 days. Cell proliferation was monitored at 24-hour intervals, and the media was routinely replenished till it reached a focus of 80–90%. This was adopted by the switch of the cells to a microplate.
Osx Measurement
After reaching confluence, hFOB 1.19 cells have been handled for 48 hours with BF at doses of 62.5, 125, and 250 µg/L and genistein 2.5 µg/ml as a optimistic management. After that, cells have been fastened with 4% PFA and cleaned with PBS. Subsequently, Osx main antibodies, BSA, and Triton X-100 have been added, and the combination was incubated for a full day at 4°C. Subsequently, Osx expression was examined by immunocytochemistry utilizing an Olympus Fluoview Ver.4.2a CLSM at 488 nm, and anti-rabbit Osx (Secondary Antibody-FITC) was added.
Ocn Measurement
The hFOB 1.19 cells that had reached confluence got BF at a dose of 62.5, 125, and 250 µg/L, and Genistein 2.5 µg/ml as a optimistic management, then incubated for 48 hours. Subsequent, cells have been washed with PBS and stuck with 4% PFA. After that, Triton Adopted by the addition of anti-mouse Ocn (secondary antibody-Rhodamine) and evaluation of Ocn expression utilizing immunocytochemical strategies utilizing the Olympus Fluoview Ver.4.2a CLSM instrument at a wavelength of 543 nm.
Information evaluation
After acquiring information utilizing CLSM, the subsequent step is to
perform quantification to acquire numerical information, which can then be carried
out utilizing one-way ANOVA and post-hoc LSD evaluation utilizing the statistical
software program Statistical Product and Service Options (SPSS) with a major
distinction of p<0.05.
Outcomes and Dialogue
To get 70 g of extract, 1.6 kg of dry powdered M. crenata leaves
have been extracted utilizing 96% ethanol. The extract was then suspended in 1:1 aqua
destillates. The suspension is then fractionated with the n-butanol solvent
liquid extraction technique utilizing a separating funnel. From this course of, the
weight of the BF was 5.97 grams.
Enchancment of the bone formation course of in vitro utilizing the BF from
M. crenata leaves has been studied in
hFOB 1.19 cells. The immunocytochemistry technique utilizing CLSM was carried out to
measure the expression of Osx and Ocn. Measurements have been carried out at a
wavelength of 488 nm for Osx and 543 nm for Ocn. Osx expression was proven by inexperienced
fluorescence depth, whereas Ocn expression was proven by crimson fluorescence
depth in hFOB 1.19 cells (Figures 1 and Determine 3).
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Determine 1: Immunofluorescence Osx of hFOB 1.19 cell line. (A) Adverse management, (B) dose 62.5 µg/ml, (C) dose 125 µg/ml, (D) dose 250 µg/ml, (E) genistein 2.5 µg/ml.
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Determine 2: Expression of Osx in hFOB 1.19 cells after administration of BF of M. crenata leaves. The “*” signifies vital distinction to the destructive management, whereas ** signifies vital distinction to genistein 2.5 µg/ml.
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Determine 1 exhibits Osx immunofluorescence in hFOB 1.19 cells in all
teams. The strongest depth was proven within the genistein optimistic management
group, adopted by the therapy group in response to focus stage, whereas
the weakest depth was proven within the destructive management group. Determine 2 exhibits
the 250 µg/ml dose group gave the most effective outcomes by rising Osx expression
considerably in comparison with the destructive management (p = 0.000) and never
considerably in comparison with 2.5 µg/ml genistein (p = 0.129). The rise in Osx
expression on the dose of 62.5 µg/ml was considerably completely different from the
destructive management (p = 0.000) and from genistein 2.5 µg/ml (p = 0.000), whereas
the 125 µg/ml dose group was additionally considerably completely different from the management
destructive (p = 0.000) and from genistein 2.5 µg/ml (p = 0.000). This exhibits that
genistein and the therapy group have been in a position to enhance Osx expression in hFOB
1.19 cells.
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Determine 3: Immunofluorescence Ocn of hFOB 1.19 cell line. (A) Adverse management, (B) dose 62.5 µg/ml, (C) dose 125 µg/ml, (D) dose 250 µg/ml, (E) genistein 2.5 µg/ml.
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Determine 4: Ocn expression in hFOB 1.19 cells after administration of BF from M. crenata leaves.
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Determine 3 exhibits Ocn immunofluorescence in hFOB 1.19 cells in all
teams. The strongest depth was proven within the genistein optimistic management
group, adopted by the therapy group in response to focus stage, whereas
the weakest depth was proven within the destructive management group. Determine 4 exhibits
the 250 µg/ml dose group gave the most effective outcomes, with a major enhance in
Ocn expression in comparison with the destructive management (p = 0.000) and in addition
vital in comparison with genistein 2.5 µg/ml (p = 0.000). The rise in Ocn
expression on the dose of 62.5 µg/ml was additionally considerably completely different from the
destructive management (p = 0.000) and from genistein 2.5 µg/ml (p = 0.000), whereas
the 125 µg/ml dose group additionally confirmed a major distinction in comparison with the
destructive management (p = 0.000) and genistein 2.5 µg/ml (p = 0.000). In the meantime,
the 250 ppm therapy group was solely considerably completely different from the destructive
group, which exhibits that the power to extend Osx was just like the optimistic
management group.
The BF from M. crenata
leaves has been recognized as a possible phytoestrogen that may operate as an
different to estrogen by way of the ER-β pathway17. Phytoestrogens are
recognized to have the ability to stimulate ER-β activation, which performs a task in regulating
transcriptional gene expression and triggering osteoblast cell proliferation
and differentiation8,9. Within the context of bone formation, osteoblast
cell differentiation turns into a key issue, which is influenced by the
activation of transcription elements corresponding to Osx and runt-related transcription
issue 2 (Runx-2), in addition to the manufacturing of vital proteins corresponding to ALP,
Ocn, kind I collagen, and BSP, together with the mineralization course of and
elevated bone density8,9,10.
As a result of Osx performs a vital position as a selected transcription issue
for osteoblast cells, it regulates the method of osteoblast cell maturation
and bone formation, together with the expression of key proteins like Ocn25.
However, Ocn was chosen as a marker issue as a result of, as the principle
non-collagen protein produced by mature osteoblast cells, it’s typically utilized in
the characterization of human bone cells. Ocn has a vital position in bone
formation and mineralization processes10,26.
Genistein 2.5 µg/ml was chosen as a optimistic management due to its
potential as a phytoestrogen, which may substitute the position of estrogen in preserving
bone steadiness, and in addition due to its potential to extend the bone formation
course of in each in vivo and in vitro experiments27. Genistein is
recognized to have excessive affinity for ER-β28, inhibit nuclear factor-kappa
B (NF-κB) activation29, and enhance Ocn expression14,30.
Exercise of the BF from M. crenata
leaves. Much like genistein, which was used as a optimistic management on this
examine, it’s doable that it has exercise just like genistein. Dose 250 ppm
of the of the BF from M. crenata
leaves. That is essentially the most optimum dose for rising Osx and Ocn as a result of the
quantity and depth measured are just like these of genistein.
Analysis information exhibits that the BF extracted from the leaves of M. crenata is ready to enhance the
expression of Osx and Ocn. These findings point out the presence of
phytoestrogen compounds within the BF, which have a task in rising the
expression of Osx and Ocn, each of that are vital indicators within the bone
formation course of.
Conclusion
The BF of M. crenata leaves at doses of 62.5, 125, and 250 µg/ml has been proven to enchance the expression of Osx and Ocn in hFOB 1.19 cells, with the optimum dose at 250 ppm. These outcomes point out that the BF from M. crenata has the potential to enhance the bone formation course of by inducing osteoblastogenesis. Nonetheless, additional preclinical and medical analysis must be carried out to verify the osteoblastogenesis impact of BF.
Acknowledgment
None
Battle of Curiosity
The authors declare that there are not any doable conflicts of curiosity with respect to the analysis, authors, and/or publication of this text.
Funding Supply
No funding company within the governmental, industrial, non-public, or not-for-profit sectors supplied a selected grant for this analysis.
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