Effect of Artemisia capillaris flower extract on the microRNA-regulated HYBID expression in human dermal fibroblasts
364
Presented by: Kazal Boron Biswas
Introduction: Depolymerization of hyaluronic acid (HA) has been reported to be mediated by a novel protein called HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization) which binds and degrades high molecular size (>1000 kDa) HA into intermediate size fragments with molecular weights ranging from 10 kDa to 100 kDa. Although the multifaceted role of high molecular size HA is well-understood, the function of its intermediate size fragments in dermal fibroblasts is yet to be determined. Moreover, the regulation of HYBID expression by the presence of extrinsic aging factors such as UV or inflammation is well-documented both in vitro and in vivo, but its expression regulated by intrinsic aging factors is still absent. Therefore, this study aims to evaluate the function of intermediate size fragments of HA, to investigate the microRNA (miRNA)-based regulation of HYBID expression in newborn and adult fibroblasts, and to search for the effective material which can inhibit the expression of HYBID, thereby, can reduce not only intrinsic aging but also extrinsic aging of skin.
Methods: Normal human dermal fibroblasts (NHDF) from newborn (NB) and adult (AD) were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum. Different sizes of HA such as high molecular size HA (H2: 1,200-1,600 kDa), medium size HA (M2: 600-1,120 kDa), small size HA (S2: 40-80 kDa), and ultra-small size HA (U2: 5-10 kDa) were used in this study. The effect of these different sizes of HA on the cellular function was investigated in NHDF-NB by measuring the mRNA expression of IL-1β, IL-6, and MMP-1 in the presence or absence of a potent inflammatory agent, TNF-α. Histamine, a well-known factor released from Mast cells upon UV irradiation, was considered as a model of extrinsic skin aging and, hence, was used to induce HYBID expression in NHDF-NB. Histamine, a well-known factor released from Mast cells upon UV irradiation, was considered as a model of extrinsic skin aging and, hence, was used to induce HYBID expression in NHDF-NB. In search for intrinsic aging factor, miRNA which directly targets and inhibits HYBID protein expression was investigated in both NHDF-NB and NHDF-AD. About 400 plant extracts were screened and the best one was finally selected based on its efficacy of inhibiting HYBID expression induced by histamine in NHDF-NB.
Results: In the absence of TNF-α, none of the different sizes of HA (H2, M2, S2, and U2) showed any effect on the expression of IL-1β, IL-6, and MMP-1. On the other hand, when mimicking cells under inflammatory state by the addition of just 1 ng/ml of TNF-α to the pre-treated fibroblasts with different sizes of HA, expression of IL-1β, IL-6, and MMP-1 was accelerated HA size-dependently and the U2 size showed the highest effect. Several miRNAs, which have already been confirmed as validated targets of HYBID in different cell-types, were primarily screened based on their expression patterns in NHDF. It was found that the expression level of a selected miRNA was decreased in NHDF-AD compared to NHDF-NB. Artemisia capillaris flower extract was found to not only inhibit the histamine-induced mRNA expression of HYBID in NHDF-NB concentration-dependently, but also increase the expression of miRNA in NHDF-AD. Finally, the plant extract is expected to improve the condition of sagging and wrinkle in human clinical study.
Discussion and conclusion: The above data suggest that although HA with its intact size provides strong structural and functional support against aging, it may somehow get involved in inducing wrinkle when degraded into different intermediate size fragments. We have revealed this characterization of intermediate size fragments in dermal fibroblasts for the first time. We have also confirmed the miRNA-regulated expression of HYBID in skin fibroblast. The decreased level of miRNA in NHDF-AD compared to NHDF-NB suggest that it may be associated, at least partially, in the cause of intrinsic aging of skin. We have proposed a specific plant ingredient, Artemisia capillaris flower extract, with interesting properties for skin aging care. This plant extract may be able to take care of both extrinsic and intrinsic skin aging by downregulating the stress-induced expression of HYBID and by upregulating the chronologically decreased expression of miRNA, respectively.
Methods: Normal human dermal fibroblasts (NHDF) from newborn (NB) and adult (AD) were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum. Different sizes of HA such as high molecular size HA (H2: 1,200-1,600 kDa), medium size HA (M2: 600-1,120 kDa), small size HA (S2: 40-80 kDa), and ultra-small size HA (U2: 5-10 kDa) were used in this study. The effect of these different sizes of HA on the cellular function was investigated in NHDF-NB by measuring the mRNA expression of IL-1β, IL-6, and MMP-1 in the presence or absence of a potent inflammatory agent, TNF-α. Histamine, a well-known factor released from Mast cells upon UV irradiation, was considered as a model of extrinsic skin aging and, hence, was used to induce HYBID expression in NHDF-NB. Histamine, a well-known factor released from Mast cells upon UV irradiation, was considered as a model of extrinsic skin aging and, hence, was used to induce HYBID expression in NHDF-NB. In search for intrinsic aging factor, miRNA which directly targets and inhibits HYBID protein expression was investigated in both NHDF-NB and NHDF-AD. About 400 plant extracts were screened and the best one was finally selected based on its efficacy of inhibiting HYBID expression induced by histamine in NHDF-NB.
Results: In the absence of TNF-α, none of the different sizes of HA (H2, M2, S2, and U2) showed any effect on the expression of IL-1β, IL-6, and MMP-1. On the other hand, when mimicking cells under inflammatory state by the addition of just 1 ng/ml of TNF-α to the pre-treated fibroblasts with different sizes of HA, expression of IL-1β, IL-6, and MMP-1 was accelerated HA size-dependently and the U2 size showed the highest effect. Several miRNAs, which have already been confirmed as validated targets of HYBID in different cell-types, were primarily screened based on their expression patterns in NHDF. It was found that the expression level of a selected miRNA was decreased in NHDF-AD compared to NHDF-NB. Artemisia capillaris flower extract was found to not only inhibit the histamine-induced mRNA expression of HYBID in NHDF-NB concentration-dependently, but also increase the expression of miRNA in NHDF-AD. Finally, the plant extract is expected to improve the condition of sagging and wrinkle in human clinical study.
Discussion and conclusion: The above data suggest that although HA with its intact size provides strong structural and functional support against aging, it may somehow get involved in inducing wrinkle when degraded into different intermediate size fragments. We have revealed this characterization of intermediate size fragments in dermal fibroblasts for the first time. We have also confirmed the miRNA-regulated expression of HYBID in skin fibroblast. The decreased level of miRNA in NHDF-AD compared to NHDF-NB suggest that it may be associated, at least partially, in the cause of intrinsic aging of skin. We have proposed a specific plant ingredient, Artemisia capillaris flower extract, with interesting properties for skin aging care. This plant extract may be able to take care of both extrinsic and intrinsic skin aging by downregulating the stress-induced expression of HYBID and by upregulating the chronologically decreased expression of miRNA, respectively.