PI3K/Akt Signaling Involved with Osteoinductive Effects of Achatina fulica Mucus

Kantawong, Fahsai; Jearasakwattana, Thananat; Nira, Alisa; Chewae, Jumaila; Sajjamongkol, Phusanisa; Phothong, Pajaree; E-kobon, Teerasak; Chumnanpuen, Pramote

doi:10.1590/1678-4324-2020180501

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Article - Biological and Applied Sciences • Braz. arch. biol. technol. 63 • 2020 • https://doi.org/10.1590/1678-4324-2020180501 linkcopy

PI3K/Akt Signaling Involved with Osteoinductive Effects of Achatina fulica Mucus

Authorship SCIMAGO INSTITUTIONS RANKINGS

Mesenchymal stem cells and osteoblasts play important roles in bone formation. Achatina fulica mucus presented the property of osteoinduction. This study aimed to examine the effects of A. fulica mucus on human mesenchymal stem cell (hMSC) and human fetal osteoblastic cell line (HFOB) differentiation. The integrated effects of A. fulica mucus and polycaprolactone (PCL) on the differentiation of hMSCs were tested. The cell viability of hMSCs treated with A. fulica mucus was investigated by the MTT assay. The cell mineralization was observed by Alizarin Red S staining, the gene expression was investigated using RT-PCR, and the PI3K activation was studied using flow cytometry. The results indicated that A. fulica mucus induced osteogenic differentiation in hMSCs and HFOBs by upregulation of the osteogenic markers; osteopontin (OPN) and osteocalcin (OCN). The results of the Alizarin Red S staining indicated that A. fulica mucus supported mineralization in both hMSCs and HFOBs. The hMSCs cultured on PCL supplemented with A. fulica mucus showed significantly increased RUNX2 and OPN expressions. A. fulica mucus was observed to increase PI3K activation in hMSCs. The findings of this study suggested that A. fulica mucus and biomaterials could be applied together for use in bone regeneration in the future.

Keywords:
Achatina fulica; polycaprolactone; PI3K/Akt; glycosaminoglycan

Authorship

person Fahsai Kantawong *

schoolChiang Mai University, Faculty of Associated Medical Sciences, Department of Medical Technology, Chiang Mai, Thailand; Chiang Mai UniversityThailandChiang Mai, ThailandChiang Mai University, Faculty of Associated Medical Sciences, Department of Medical Technology, Chiang Mai, Thailand;

0000-0003-0323-3021

person Thananat Jearasakwattana

0000-0001-9253-8542

person Alisa Nira

0000-0002-6401-612X

person Jumaila Chewae

0000-0001-8799-798X

person Phusanisa Sajjamongkol

0000-0003-1863-6464

person Pajaree Phothong

0000-0003-4673-6948

person Teerasak E-kobon

schoolKasetsart University, Faculty of Science, Department of Genetics, Bangkok, Thailand; Kasetsart UniversityThailandBangkok, ThailandKasetsart University, Faculty of Science, Department of Genetics, Bangkok, Thailand; schoolKasetsart University, Computational Biomodelling Laboratory for Agricultural Science and Technology, Bangkok, ThailandKasetsart UniversityThailandBangkok, ThailandKasetsart University, Computational Biomodelling Laboratory for Agricultural Science and Technology, Bangkok, Thailand

0000-0002-3919-9841

person Pramote Chumnanpuen

schoolKasetsart University, Faculty of Science, Department of Zoology, Bangkok, Thailand; Kasetsart UniversityThailandBangkok, ThailandKasetsart University, Faculty of Science, Department of Zoology, Bangkok, Thailand; schoolKasetsart University, Computational Biomodelling Laboratory for Agricultural Science and Technology, Bangkok, ThailandKasetsart UniversityThailandBangkok, ThailandKasetsart University, Computational Biomodelling Laboratory for Agricultural Science and Technology, Bangkok, Thailand

0000-0003-3072-1733

*Correspondence:
emailFahsai.k@cmu.ac.th; Tel.: +66-53-935085 (F.K.)

Conflicts of Interest:

The authors declare no conflicting interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

SCIMAGO INSTITUTIONS RANKINGS

Chiang Mai University, Faculty of Associated Medical Sciences, Department of Medical Technology, Chiang Mai, Thailand; Chiang Mai UniversityThailandChiang Mai, ThailandChiang Mai University, Faculty of Associated Medical Sciences, Department of Medical Technology, Chiang Mai, Thailand;

Kasetsart University, Faculty of Science, Department of Genetics, Bangkok, Thailand; Kasetsart UniversityThailandBangkok, ThailandKasetsart University, Faculty of Science, Department of Genetics, Bangkok, Thailand;

Kasetsart University, Faculty of Science, Department of Zoology, Bangkok, Thailand; Kasetsart UniversityThailandBangkok, ThailandKasetsart University, Faculty of Science, Department of Zoology, Bangkok, Thailand;

Kasetsart University, Computational Biomodelling Laboratory for Agricultural Science and Technology, Bangkok, ThailandKasetsart UniversityThailandBangkok, ThailandKasetsart University, Computational Biomodelling Laboratory for Agricultural Science and Technology, Bangkok, Thailand

Figures

Figures (5)

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Figure 1: The MTT assay indicated that A. fulica mucus at the concentrations of 75 µg/mL and 150 µg/mL had an effect on hMSC viability at 48 h (* P < 0.05 and ** P < 0.01, respectively).

Thumbnail

Figure 2: (A) Morphology of hMSCs cultured in complete DMEM for 18 days. (B) Morphological changes of hMSCs cultured in complete DMEM supplemented with 150 µg/mL of A. fulica mucus for 18 days. (C) The Alizarin Red S staining of hMSCs cultured in complete DMEM for 18 days. (D) The Alizarin Red S staining of hMSCs cultured in complete DMEM supplemented with 150 µg/mL of A. fulica mucus for 18 days.

Thumbnail

Figure 3: (A) The Alizarin Red S staining of hFOBs cultured in complete DMEM for 8 days. (B) The Alizarin Red S staining of hFOBs cultured in complete DMEM containing 50 µg/mL of A. fulica mucus for 8 days. (C) The hFOBs supplemented with 50 µg/mL of A. fulica mucus showed a significantly higher number of mineralization nodules compared to the control group (P < 0.01).

Thumbnail

Figure 4: (A) OCN and OPN expressions of hMSCs cultured in a medium supplemented with 150 µg/mL of A. fulica mucus were higher than control group at 18 days culture. The significance levels of the OCN and the OPN expressions were determined at P = 0.002 and P = 0.03, respectively. (B) The OPN expressions of HFOBs cultured in a medium supplemented with 50 µg/mL of A. fulica mucus was higher than control at 8 days culture (* P = 0.012). No significance of OCN expression was observed. (C) When hMSCs were cultured on PCL sheets, increased expressions of the RUNX2 gene (* P < 0.001) was observed in hMSCs supplemented with 50 µg/mL A. fulica mucus at 5 days culture. Increased expression of OPN gene (* P < 0.0001) was observed in the hMSCs supplemented with 50 µg/mL A. fulica mucus at 14 days. hMSCs supplemented with 25 µg/mL A. fulica mucus did not show significant OPN expression at 14 days.

Thumbnail

Figure 5: (A) PI3K activation in control group = 1.90%. (B) PI3K activation in the hMSCs cultured in mucus 35 µg/mL for 5 h = 4.40%. (C) PI3K activation in the hMSCs cultured in complete DMEM supplemented with snail mucus for 12 h was higher than the time-correlated control groups. (D) PI3K activation in the hMSCs cultured in complete DMEM supplemented with snail mucus for 48 h was higher than the time-correlated control groups.

imageFigure 1: The MTT assay indicated that A. fulica mucus at the concentrations of 75 µg/mL and 150 µg/mL had an effect on hMSC viability at 48 h (* P < 0.05 and ** P < 0.01, respectively). open_in_new

imageFigure 2: (A) Morphology of hMSCs cultured in complete DMEM for 18 days. (B) Morphological changes of hMSCs cultured in complete DMEM supplemented with 150 µg/mL of A. fulica mucus for 18 days. (C) The Alizarin Red S staining of hMSCs cultured in complete DMEM for 18 days. (D) The Alizarin Red S staining of hMSCs cultured in complete DMEM supplemented with 150 µg/mL of A. fulica mucus for 18 days. open_in_new

imageFigure 3: (A) The Alizarin Red S staining of hFOBs cultured in complete DMEM for 8 days. (B) The Alizarin Red S staining of hFOBs cultured in complete DMEM containing 50 µg/mL of A. fulica mucus for 8 days. (C) The hFOBs supplemented with 50 µg/mL of A. fulica mucus showed a significantly higher number of mineralization nodules compared to the control group (P < 0.01). open_in_new

imageFigure 4: (A) OCN and OPN expressions of hMSCs cultured in a medium supplemented with 150 µg/mL of A. fulica mucus were higher than control group at 18 days culture. The significance levels of the OCN and the OPN expressions were determined at P = 0.002 and P = 0.03, respectively. (B) The OPN expressions of HFOBs cultured in a medium supplemented with 50 µg/mL of A. fulica mucus was higher than control at 8 days culture (* P = 0.012). No significance of OCN expression was observed. (C) When hMSCs were cultured on PCL sheets, increased expressions of the RUNX2 gene (* P < 0.001) was observed in hMSCs supplemented with 50 µg/mL A. fulica mucus at 5 days culture. Increased expression of OPN gene (* P < 0.0001) was observed in the hMSCs supplemented with 50 µg/mL A. fulica mucus at 14 days. hMSCs supplemented with 25 µg/mL A. fulica mucus did not show significant OPN expression at 14 days. open_in_new

imageFigure 5: (A) PI3K activation in control group = 1.90%. (B) PI3K activation in the hMSCs cultured in mucus 35 µg/mL for 5 h = 4.40%. (C) PI3K activation in the hMSCs cultured in complete DMEM supplemented with snail mucus for 12 h was higher than the time-correlated control groups. (D) PI3K activation in the hMSCs cultured in complete DMEM supplemented with snail mucus for 48 h was higher than the time-correlated control groups. open_in_new

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