Achatina fulica mucous improves cell viability and increases collagen deposition in UVB-irradiated human fibroblast culture

Abstract

Introduction: Ultraviolet radiation induces skin photoaging by increasing matrix metalloproteinase-1 (MMP-1). MMP-1 degrades type I and III collagen that comprise the dermal connective tissue. Achatina fulica mucous (AFM) is a natural remedy that has protective effects on fibroblasts and collagen. Objective: To investigate the effects of AFM on cell viability and collagen deposition in UVB-irradiated human fibroblast culture. Methods: The mucous was extracted from 50 Achatina fulica snails that were stimulated by a 5-10 Volt electricity shock for 30-60 seconds and converted into powder by the freeze-drying process. The human dermal fibroblast culture was divided into six groups: group 1 were normal fibroblasts without UVB irradiation as normal control, groups 2-5 consisted of 100 mJ/cm² UVB-irradiated fibroblasts. Group 2 had no treatment as negative control, group 3 was treated by PRP 10% as positive control group and groups 4-6 were treated by various concentrations of AFM (3.9; 15.625 and 62.5 μg/mL). At the end of the experiment, the proliferation was assessed with MTT assay, furthermore collagen deposition was measured by Sirius red assay. Real Time-PCR (RT-PCR) was performed to quantify Coll I, Coll III and MMP-1 mRNA expression, then to measured COL 1/COL III ratio. Results: UVB induced significant lower viability, upregulated MMP-1 and downregulated COL I and COL III mRNA expressions. Meanwhile AFM treated groups demonstrated higher cell viability with downregulation of MMP-1 and upregulation of COL I and COL III mRNA expressions. The ratio of COL I/ III expression was significantly (p<0.05) lower in the AFM treated groups compared to the UVB group. Among AFM treated groups, administration of 62.5 μg/mL AFM represented the best result. Conclusion: AFM may ameliorate viability of UVB-irradiated human fibroblast culture which associates with downregulating MMP-1, upregulating COL I and Col III, and reducing COL I/III ratio.

Keywords: Achatina fulica; Collagen; MMP-1; UVB-irradiated human fibroblast culture; Viability.

Figure 1.. Fibroblast viability test of AFM in NDHFs. NDHFs were cultured in DMEM until 80-90% confluence and exposed to 100 mJ/cm2 ultraviolet B (UVB). Cells were added with or without serial concentrations of AFM for 72 h, and afterward the MTT assay was conducted. All data are counted as the average ± SEM of triplicate experiments. Normal control (NC) was NDHFs not exposed to UVB irradiation and received no treatment, UVB was NDHFs exposed to UVB irradiation and received no treatment, UVB+PRP was NDHFs exposed to UVB irradiation and received treatment with PRP 10%, and UVB+AF was NDHFs exposed to UVB irradiation and received treatment with various concentrations (μg/mL) of AFM.

Figure 2.. Quantitative PCR (qPCR) examination of MMP-1 mRNA expression. NHDFs were induced with UVB 100mJ/cm2 and administered with various concentrations of AFM (3.9 μg/mL; 15.625 μg/mL and 62.5 μg/mL). The value is provided as the mean ± SEM. *p < 0.05 indicates the significant versus the normal control. #p< 0.05 indicates the significant versus a UVB. $ p<0.05 indicates the significant versus PRP. Normal control (NC) was NDHFs not exposed to UVB irradiation and received no treatment, UVB was NDHFs exposed to UVB irradiation and received no treatment, UVB+PRP was NDHFs exposed to UVB irradiation and received treatment with PRP 10%, and UVB+AF was NDHFs exposed to UVB irradiation and received treatment with various concentrations (3.9 μg/mL; 15.625 μg/mL and 62.5 μg/mL) of AFM.

Figure 3.. A. Representative pictures of Sirius red staining for collagen deposition. Positive staining was reddish shown. B. Quantitative PCR (qPCR) interpretation of COL I, COL III, and COL I/III ratio mRNA expressions. UVB+AF62 group demonstrated significantly higher COL I and COL III mRNA expressions compared with the NC group. UVB group demonstrated significantly lower expressions of both COL I and COL III mRNA compared with the NC group. *p < 0.05 indicates the significant versus the normal control. #p< 0.05 indicates the significant versus a UVB. $ p<0.05 indicates the significant versus PRP.