{"id":59877,"date":"2024-09-30T10:48:34","date_gmt":"2024-09-30T10:48:34","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=59877"},"modified":"2024-10-09T18:32:06","modified_gmt":"2024-10-09T18:32:06","slug":"enhancement-of-osteoblastogenesis-in-the-hfob-1-19-cell-line-by-the-induction-of-the-n-butanol-fraction-derived-from-marsilea-crenata-c-presl-leaves","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/enhancement-of-osteoblastogenesis-in-the-hfob-1-19-cell-line-by-the-induction-of-the-n-butanol-fraction-derived-from-marsilea-crenata-c-presl-leaves\/","title":{"rendered":"Enhancement of Osteoblastogenesis in the hFOB 1.19 Cell Line by the Induction of the n-Butanol Fraction Derived from Marsilea crenata C Presl. Leaves"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Osteoporosis is a bone disease in humans\ncharacterized by reduced bone strength and density, leading to a higher risk of\nfractures. Bone density often decreases with age, showing variations based on\nthe gender and race of individuals. Key irreversible factors contributing to\nosteoporosis development are age, menopause, and female gender1<\/sup>. Estrogen\ndeficiency in women over 40 leads to reduced bone mass, raising the likelihood\nof osteoporosis2,3<\/sup>.<\/p>\n\n\n\n

The process of bone remodeling is characterized by\nits dynamic nature, wherein osteoclasts engage in the resorption of aged bone\ntissue and osteoblasts are responsible for the generation of new bone tissue.\nIt is responsible for healing injured bones4,5<\/sup>. The formation stage\ninvolves the proliferation and differentiation of osteoblast precursors,\nfollowed by the mineralization of the new bone matrix4<\/sup>. <\/p>\n\n\n\n

The estrogen-receptor (E-ER) complex in the nucleus\nor cell membran can be influenced by estrogen to affect the proliferation,\ndifferentiation, and maturation of osteoblast in different species6,7<\/sup>.\nOne transcription factor that is important for osteoblast development is called\nOsterix (Osx). By upregulating the expression of proteins such as osteocalcin\n(Ocn), osteopontin (OPN), bone sialoprotein (BSP), and collagen type I (COL-I),\nit promotes the production of new bone8,9<\/sup>. As an adult, Ocn is a\nmajor protein in bones. It helps bones mineralize by interacting with\n\u03b3-carboxyglutamic acid (Gla) residues and increasing the absorption of\nhydroxyapatite10<\/sup>.<\/p>\n\n\n\n

The use of estrogen in women with estrogen\ndeficiency, such as long-term hormone replacement therapy (HRT), carries a\ncarcinogenic risk to female reproductive organs and other side effects11<\/sup>.\nTherefore, research on estrogen alternatives prefers natural ingredients,\nespecially phytoestrogen compounds, which provide bone protective effects by\ninhibiting bone resorption and increasing bone formation due to menopause12,13<\/sup>.\nPlant chemicals called phytoestrogens can act like estrogen by making\nosteoblast cells work harder by attaching to the estrogen receptor (ER) with\nlow side effects14<\/sup>.<\/p>\n\n\n\n

The aquatic plant Marsilea crenata<\/em> C Presl. which is commonly\nconsumed as a staple meal in Surabaya, East Java, Indonesia, has been\nidentified as a source of phytoestrogens <\/em>15,16,17<\/sup>. Numerous investigations conducted on M. crenata<\/em> have\nrevealed the presence of phytoestrogens in various forms, including\ntriterpenoids, steroids, and isoflavones, within the ethanol extract, n-hexane,\nethyl acetate, and the n-butanol fraction of M. crenata<\/em> leaves. Additionally, it has the\npotential to stimulate the activation of ER-\u03b2 and enhance the elevation of\nalkaline phosphatase (ALP) levels in the MC3T3-E1 preosteoblast cell line,\nhence promoting cellular proliferation and differentiation 18,19<\/sup>. In\nvivo using male and female mice shows that M.\ncrenata<\/em> induces the proliferation of trabecular bone osteoblast cells 20,21<\/sup>.<\/p>\n\n\n\n

The purpose of this research is to prove that the n-butanol fraction (BF) of M. crenata<\/em> leaves can enchance bone formation in human fetal osteoblast (hFOB 1.19) by measuring the Osx and Ocn expressions using a confocal laser scanning microscop (CLSM) instrument. These cells are used to study how human osteoblasts differentiate, how osteoblasts work, and how cytokines affect osteoblast function22,23<\/sup>.<\/p>\n\n\n\n

Material and methods<\/strong><\/p>\n\n\n\n

Materials<\/strong><\/p>\n\n\n\n

UPT Materia Medika, Batu, East Java, identified M. crenata<\/em> leaves that were gathered from the Benowo region of Surabaya, East Java, Indonesia (code: 1a17b-18a-1)24<\/sup>. The hFOB 1.19 cell line (CRL-11372) was obtained from American Type Cell Culture (Virginia, USA). Quercetin, dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), bovine serum albumin (BSA), and phosphate-buffered saline (PBS) were supplied by Sigma-Aldrich (Missouri, USA). Primary antibodies against mice osteocalcin and anti-rabbit osterix were acquired from Abcam (Cambridge, Britain). The Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS), and G418 were provided by Aretha Laboratory (Bandung, Indonesia). Anti-mouse secondary antibody Rhodamine, anti-rabbit secondary antibody fluorescein isothiocyanate (FITC), paraformaldehyde and Tween-80 were provided by the Central Laboratory of Life Science at Brawijaya University (Malang, Indonesia).<\/p>\n\n\n\n

Method<\/strong><\/p>\n\n\n\n

Extraction, Fractionation, and Preparation of BF<\/strong><\/p>\n\n\n\n

A technique of extraction was performed using 1.5 kg of M. crenata<\/em> leaf powder and a solvent consisting of 96% ethanol, with the assistance of ultrasonics wave. This procedure yielded a total of 70 g of extract17<\/sup>. Liquid-liquid extraction was done in a 1:1 ratio, the extract was combined with 600 mL of water and subsequently separated using n-Hexane. Ethyl acetate and n-butanol were employed to separate the aqueous phase. The isolation and drying of the n-butanol phase were conducted using a Heidolph Hei-VAP ML\/GG3 rotary evaporator.<\/p>\n\n\n\n

A stock solution with a concentration of 5000 ppm was prepared by combining a 50 mg BF with 0.5% Tween-80 in 0.5% DMSO (w\/v). The solution is formulated with concentrations of 62.5, 125, and 250 \u00b5g\/L.<\/p>\n\n\n\n

Cell Culture<\/strong><\/p>\n\n\n\n

The hFOB 1.19 cells were cultured in a culture flask with a complete media including DMEM, FBS 10%, G418, and penicillin-streptomycin 1%. The flask was then placed in an incubator with a CO2<\/sub> 5% and maintained at a temperature of 37\u00b0C for a period of 6 days. Cell proliferation was monitored at 24-hour intervals, and the media was routinely replenished until it reached a concentration of 80\u201390%. This was followed by the transfer of the cells to a microplate.<\/p>\n\n\n\n

Osx Measurement<\/strong><\/p>\n\n\n\n

After reaching confluence, hFOB 1.19 cells were treated for 48 hours with BF at doses of 62.5, 125, and 250 \u00b5g\/L and genistein 2.5 \u00b5g\/ml as a positive control. After that, cells were fixed with 4% PFA and cleaned with PBS. Subsequently, Osx primary antibodies, BSA, and Triton X-100 were added, and the mixture was incubated for a full day at 4\u00b0C. Subsequently, Osx expression was examined by immunocytochemistry using an Olympus Fluoview Ver.4.2a CLSM at 488 nm, and anti-rabbit Osx (Secondary Antibody-FITC) was added.<\/p>\n\n\n\n

Ocn Measurement<\/strong><\/p>\n\n\n\n

The hFOB 1.19 cells that had reached confluence were given BF at a dose of 62.5, 125, and 250 \u00b5g\/L, and Genistein 2.5 \u00b5g\/ml as a positive control, then incubated for 48 hours. Next, cells were washed with PBS and fixed with 4% PFA. After that, Triton Followed by the addition of anti-mouse Ocn (secondary antibody-Rhodamine) and analysis of Ocn expression using immunocytochemical techniques using the Olympus Fluoview Ver.4.2a CLSM instrument at a wavelength of 543 nm.<\/p>\n\n\n\n

Data analysis<\/strong><\/p>\n\n\n\n

After obtaining data using CLSM, the next step is to\ncarry out quantification to obtain numerical data, which will then be carried\nout using one-way ANOVA and post-hoc LSD analysis using the statistical\nsoftware Statistical Product and Service Solutions (SPSS) with a significant\ndifference of p<0.05.<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

To get 70 g of extract, 1.6 kg of dry powdered M. crenata leaves\nwere extracted using 96% ethanol. The extract was then suspended in 1:1 aqua\ndestillates. The suspension is then fractionated with the n-butanol solvent\nliquid extraction method using a separating funnel. From this process, the\nweight of the BF was 5.97 grams.<\/p>\n\n\n\n

Improvement of the bone formation process in vitro using the BF from\nM. crenata<\/em> leaves has been studied in\nhFOB 1.19 cells. The immunocytochemistry method using CLSM was carried out to\nmeasure the expression of Osx and Ocn. Measurements were carried out at a\nwavelength of 488 nm for Osx and 543 nm for Ocn. Osx expression was shown by green\nfluorescence intensity, while Ocn expression was shown by red fluorescence\nintensity in hFOB 1.19 cells (Figures 1 and Figure 3).<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Immunofluorescence Osx of hFOB 1.19 cell line. (A) Negative control, (B) dose 62.5 \u00b5g\/ml, (C) dose 125 \u00b5g\/ml, (D) dose 250 \u00b5g\/ml, (E) genistein 2.5 \u00b5g\/ml.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Expression of Osx in hFOB 1.19 cells after administration of BF of M. crenata<\/em> leaves. The \u201c*\u201d indicates significant difference to the negative control, while ** indicates significant difference to genistein 2.5 \u00b5g\/ml.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Figure 1 shows Osx immunofluorescence in hFOB 1.19 cells in all\ngroups. The strongest intensity was shown in the genistein positive control\ngroup, followed by the treatment group according to concentration level, while\nthe weakest intensity was shown in the negative control group. Figure 2 shows\nthe 250 \u00b5g\/ml dose group gave the best results by increasing Osx expression\nsignificantly compared to the negative control (p = 0.000) and not\nsignificantly compared to 2.5 \u00b5g\/ml genistein (p = 0.129). The increase in Osx\nexpression at the dose of 62.5 \u00b5g\/ml was significantly different from the\nnegative control (p = 0.000) and from genistein 2.5 \u00b5g\/ml (p = 0.000), while\nthe 125 \u00b5g\/ml dose group was also significantly different from the control\nnegative (p = 0.000) and from genistein 2.5 \u00b5g\/ml (p = 0.000). This shows that\ngenistein and the treatment group were able to increase Osx expression in hFOB\n1.19 cells.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Immunofluorescence Ocn of hFOB 1.19 cell line. (A) Negative control, <\/strong>(B) dose 62.5 \u00b5g\/ml, (C) dose 125 \u00b5g\/ml, (D) dose 250 \u00b5g\/ml, (E) genistein 2.5 \u00b5g\/ml.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 4: Ocn expression in hFOB 1.19 cells after administration of BF from M. crenata <\/em>leaves. <\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Figure 3 shows Ocn immunofluorescence in hFOB 1.19 cells in all\ngroups. The strongest intensity was shown in the genistein positive control\ngroup, followed by the treatment group according to concentration level, while\nthe weakest intensity was shown in the negative control group. Figure 4 shows\nthe 250 \u00b5g\/ml dose group gave the best results, with a significant increase in\nOcn expression compared to the negative control (p = 0.000) and also\nsignificant compared to genistein 2.5 \u00b5g\/ml (p = 0.000). The increase in Ocn\nexpression at the dose of 62.5 \u00b5g\/ml was also significantly different from the\nnegative control (p = 0.000) and from genistein 2.5 \u00b5g\/ml (p = 0.000), while\nthe 125 \u00b5g\/ml dose group also showed a significant difference compared to the\nnegative control (p = 0.000) and genistein 2.5 \u00b5g\/ml (p = 0.000). Meanwhile,\nthe 250 ppm treatment group was only significantly different from the negative\ngroup, which shows that the ability to increase Osx was similar to the positive\ncontrol group.<\/p>\n\n\n\n

The BF from M. crenata<\/em>\nleaves has been identified as a potential phytoestrogen that can function as an\nalternative to estrogen via the ER-\u03b2 pathway17<\/sup>. Phytoestrogens are\nknown to be able to stimulate ER-\u03b2 activation, which plays a role in regulating\ntranscriptional gene expression and triggering osteoblast cell proliferation\nand differentiation8,9<\/sup>. In the context of bone formation, osteoblast\ncell differentiation becomes a key factor, which is influenced by the\nactivation of transcription factors such as Osx and runt-related transcription\nfactor 2 (Runx-2), as well as the production of important proteins such as ALP,\nOcn, type I collagen, and BSP, along with the mineralization process and\nincreased bone density8,9,10<\/sup>.<\/p>\n\n\n\n

Because Osx plays a crucial role as a specific transcription factor\nfor osteoblast cells, it regulates the process of osteoblast cell maturation\nand bone formation, including the expression of key proteins like Ocn25<\/sup>.\nOn the other hand, Ocn was chosen as a marker factor because, as the main\nnon-collagen protein produced by mature osteoblast cells, it is often used in\nthe characterization of human bone cells. Ocn has a crucial role in bone\nformation and mineralization processes10,26<\/sup>.<\/p>\n\n\n\n

Genistein 2.5 \u00b5g\/ml was chosen as a positive control because of its\nability as a phytoestrogen, which can substitute the role of estrogen in preserving\nbone balance, and also because of its ability to increase the bone formation\nprocess in both in vivo and in vitro experiments27<\/sup>. Genistein is\nknown to have high affinity for ER-\u03b228<\/sup>, inhibit nuclear factor-kappa\nB (NF-\u03baB) activation29<\/sup>, and increase Ocn expression14,30<\/sup>.\nActivity of the BF from M. crenata<\/em>\nleaves. Similar to genistein, which was used as a positive control in this\nstudy, it is possible that it has activity similar to genistein. Dose 250 ppm\nof the of the BF from M. crenata<\/em>\nleaves. This is the most optimal dose for increasing Osx and Ocn because the\namount and intensity measured are similar to those of genistein.<\/p>\n\n\n\n

Research data shows that the BF extracted from the leaves of M. crenata<\/em> is able to increase the\nexpression of Osx and Ocn. These findings indicate the presence of\nphytoestrogen compounds in the BF, which have a role in increasing the\nexpression of Osx and Ocn, both of which are important indicators in the bone\nformation process.<\/p>\n\n\n\n

Conclusion<\/strong><\/strong><\/p>\n\n\n\n

The BF of M. crenata<\/em> leaves at doses of 62.5, 125, and 250 \u00b5g\/ml has been shown to enchance the expression of Osx and Ocn in hFOB 1.19 cells, with the optimal dose at 250 ppm. These results indicate that the BF from M. crenata<\/em> has the potential to improve the bone formation process by inducing osteoblastogenesis. However, further preclinical and clinical research needs to be carried out to confirm the osteoblastogenesis effect of BF.<\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

None <\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors declare that there are no possible conflicts of interest with respect to the research, authors, and\/or publication of this article.<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

No funding agency in the governmental, commercial, private, or not-for-profit sectors provided a particular grant for this research.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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