Different isoforms of HAS catalyze the synthesis of HA of different molecular weights. Secreted HA with a high molecular weight (2 × 106 Da) is synthesized by HAS1 and HAS2, whereas HA with a low molecular weight (2–3 × 105 Da) is synthesized by HAS3. Similarly, Li et al. reported that the delivery of the HAS2 enzyme into synoviocytes led to the production of endogenous HA with a high molecular weight. In addition, the mRNA expression of PRG4 also increased upon statin treatment. Furthermore, the expression levels of Kir6.2 and ACSIC3, which can modulate HA secretion, were upregulated in FLSs after statin stimulation. By contrast, the ABC transporter MRP5, which modulates HA export, was not influenced by statin. Statin treatment did not modulate SIRT1 expression, although this gene has been reported to inhibit the aggressiveness and inflammatory response of RA FLSs. Therefore, statin may enhance HA production by upregulating HAS1, HAS2, and PRG4 expression and may enhance HA release through the regulation of the cell surface channels Kir6.2 and ACSIC3.
In this study, the Western blot results revealed that statin supplementation increased the production of HAS1 and PRG4 proteins. The mucinous glycoprotein PRG4, a ligand for CD44, controls FLS overgrowth in inflammatory arthropathy, plays a key role in anti-inflammation to modulate cell proliferation, and reduces the expression of catabolic factors in OA FLSs. The activation of HAS1 through the p38-MAPK pathway has been reported, and to be enhanced by statin stimulation. Similarly, Anggiansah et al. reported that the secretion of HA in the synovial joints is regulated by PKC-α in vivo, and our study also indicated that statin increased PKC-α and ERK1/2 expression. Subsequently, the activation of the PKCα–MEK–ERK1/2 cascade may stimulate HA synthesis in FLSs. Collectively, the results indicate that p38-MAPK signaling and PKCα–MEK–ERK1/2 signaling simultaneously modulate HA secretion in FLSs.
Lovastatin supplementation upregulated the expression of HA production-related genes (HAS1, HAS2, and PRG4) and HA secretion-related genes (Kir6.2 and ASIC3). In addition, the Western blot results revealed that lovastatin supplementation enhanced the production of HAS1 and PRG4 proteins and the activation of the PKC-α, ERK1/2, and p38-MAPK signaling pathways. Finally, lovastatin can enhance the intracellular cAMP level to enhance HA secretion.
Where Hyal2 degrades HA fragments beyond 20 kDa, Hyal1 catalyzes the hydrolyzing of 20 kDa HA into LMW HA fragments with pathophysiological effects, as described above. Hyal1 is usually expressed in many different types of cells. The level of expression was found to correlate with tumor stage and grade in bladder cancer and could therefore be used as diagnostic and prognostic biomarker [16]. A number of different inhibitors have been identified. These compounds differ in structure, source and mode of inhibition. Natural compounds and whole plant extracts, as well as synthetically compounds and polymers have been described. Various flavonoids were screened for BTH inhibition; apigenin was investigated several times with differing results (67% inhibition at 250 µM or 57% inhibition at 1 mM). Further well-known BTH inhibitors are L-ascorbic-acid-6-hexadecanoate (Vcpal) (IC50 = 56 µM) and glycyrrhizic acid (IC50 3 - 1300 µM), which are used as reference compounds. It cannot be ignored, at this point, that observed differences in the degree of inhibition were due to the different enzyme activity assays applied.
Two new Hyal1 inhibitors were identified in this study. Testosterone propionate, a steroid derivative, was determined to be moderately active against Hyal1 with an IC50 value of 124 ± 1.1 µM. Regulation of HA metabolism by steroids has been previously described [44,46,59]. Tanyildizi et al. investigated the influence of progesterone and testosterone on hyaluronidase activity in sheep, but came to the conclusion that the compounds were not inhibitorily active. Tranilast, a modified cinnamic acid was identified as a weak hyaluronidase inhibitor in a previous study [60]. Several cinnamides and cinnamic acid derivatives, belonging to the class of phenylpropanoids, were tested here. Out of these compounds, only chicoric acid turned out to be active against Hyal1 with an IC50 value of 171 µM.
By using fluorescent HA and FP, the hyaluronidase inhibitor assay produced reliable measurements with Z′-factor of 0.69. Using this assay, about 3000 compounds from the National Cancer Institute (Approved Oncology Drugs Set, Diversity Set, Natural Products Set and Mechanistic Set) were screened, and Prodelphiniline was selected as a hit compound. After studying the pharmacophores and structure-activity relationship, a much smaller compound, Delphinidin, was identified as a small molecule hyaluronidase inhibitor.
To test the efficacy of delphinidin in increasing HA levels in vivo we treated 3-months-old wild type C57BL/6 mice with vehicle (n = 3) (10% DMSO/90% PBS) or with 50 mg/kg (n = 4) or 100 mg/kg of delphinidin (n = 3). Intraperitoneal (IP) injections of 100 µl of vehicle or delphinidin were administered three times a week for 3 weeks before the mice were sacrificed and skin, muscle, kidney, heart, lung, duodenum, jejunum and colon were collected. Previous studies have shown that IP injections of delphinidin provides reliable systemic distribution. There were no visible adverse effects in the mice, and they maintained weight comparable to the vehicle controls. Slides with paraffin embedded tissues from each of the samples collected from the mice were stained using hyaluronic acid binding protein (HABP) and a fluorescent streptavidin antibody to detect HA in the tissues. For each sample one section was treated overnight with hyaluronidase to degrade any HA before staining. In skin, muscle, heart, kidney and intestines (duodenum, jejunum and colon) fluorescent intensity was significantly increased in animals treated with delphinidin compared to the vehicle control. Indicating that delphinidin inhibits hyaluronidases and increases HA levels in vivo. While we were not able to test the size of the HA in the tissues, the results from our in vitro experiments suggest that HA length is also increased.
We found that supplementation with 100 μM of D3G led to a significant 23.4% increase (p ≤ 0.05) in the mean lifespan of C. elegans.
In conclusion, our study investigated the effects of D3G supplementation on the lifespan and healthspan of C. elegans under various conditions. We found that D3G supplementation significantly increased the mean lifespan and average pharyngeal pumping rate of C. elegans under oxidative stress induced by H2O2. However, supplementation did not significantly affect the nematodes exposed to elevated temperatures or UVA light. Additionally, D3G supplementation did not affect the average number of eggs laid by the nematodes. Our results suggest that D3G has the potential as a protective agent against oxidative stress-induced damage in C. elegans. Further studies are needed to determine the underlying mechanisms of D3G’s protective effects and its potential application as a human dietary supplement.
In this study, we observed that PIC stimulated hyaluronic acid biosynthesis in human skin fibroblasts by upregulating HAS2 expression and downregulating HYAL2 expression. Furthermore, the induction of HAS2 by PIC was impeded by a SIRT inhibitor, suggesting that PIC enhances HAS2 expression via increased SIRT1 expression. Previous clinical trials have demonstrated that PIC intake enhances skin moisture and elasticity [[15], [16], [17]], and our findings suggest that the upregulation of hyaluronic acid biosynthesis by PIC contributes to these improvements in skin function. Notably, PIC has been reported to induce HAS2 mRNA expression in mouse fibroblasts [29], and our study reveals that PIC also suppresses hyaluronic acid degradation, shedding light on the mechanisms underlying its effects on human skin.
PIC has been demonstrated to enhance SIRT1 activity [20] and elevate SIRT1 levels in human monocytic leukemia cell lines [32]. In an in vivo study, mice fed a high-fat diet and orally administered PIC (10 mg/kg/day) for four weeks exhibited increased expression of SIRT1, SIRT3, and SIRT6 in the liver than the high-fat diet group [21]. While there have been no previous reports on the effects of PIC on SIRT1 in skin fibroblasts, our study revealed that PIC also induces SIRT1 in human skin fibroblasts. This induction suggests that PIC may exert beneficial effects on skin through the upregulation of SIRT1.
Additionally, the reduction in HYAL2 expression induced by PIC was not reversed by SIRT1 inhibition, suggesting that SIRT1 may not be directly involved in hyaluronic acid degradation. Recently, the involvement of a long noncoding RNA, HA synthase 2 antisense 1 (HAS2-AS1), has been shown as a new mechanism of action for the regulation of HAS2 gene expression [35].
Piceatannol is a tetrahydric polyphenol and an analogue form of resveratrol. The content of piceatannol in freeze-dried passion fruit seed was 4.8 mg/g when extracted using 70% acetone, which translates into 2.2 mg/g of raw material.
Our results showed that passion fruit contains a very high content of piceatannol in a natural state, which is nearly 50 times larger than that found in grapes (described above). Here, we report for the first time that piceatannol is present in passion fruit seed and that this content of piceatannol is much higher than that known for any other plants. Piceatannol was not detected in the rind or pulp of passion fruit.
Both piceatannol and resveratrol increased soluble collagen in SF-TY cells; piceatannol increased soluble collagen significantly at 5 μM, whereas resveratrol increased soluble collagen significantly at 10 μM.
Piceatannol has been reported to have a wide variety of effects on the skin, including promoting collagen production, inhibiting melanin synthesis, inducing the antioxidant glutathione, and eliminating reactive oxygen species. In this study, a randomized, placebo-controlled, double-blind trial was conducted to clinically evaluate the effects of piceatannol-rich passion fruit seed extract on the skin of healthy Japanese women (age, 35-54 y). Thirty-two women with dry skin received either passion fruit seed extract (5 mg piceatannol) or a placebo (dextrin) for 8 wk. Skin hydration and other parameters on the face were assessed at 0, 4, and 8 wk by using specialized equipment. Furthermore, questionnaire interviews were conducted regarding the physical condition of subjects at 0, 4, and 8 wk. The results showed that consumption of passion fruit seed extract led to significant increases in the moisture content of human skin after 4 and 8 wk compared with that before the trial. The amount of transepidermal water loss decreased over time, although the differences were not significant. Moreover, a stratified analysis of subjects with moisture values of ≤200 μS revealed increased moisture content in the passion fruit seed extract group as compared with the placebo group. Furthermore, the results of questionnaires showed significant reductions in “perspiration” and “fatigue” in the passion fruit seed extract group as compared with the placebo group. These results indicate that oral intake of passion fruit seed extract that is rich in piceatannol could improve the moisture of dry skin and reduce fatigue.
Objectives: The aim of this study was to investigate the effect of a dietary supplement containing piceatannol on skin in healthy subjects with dry skin. Methods: A randomized, double blind, placebo-controlled parallel-group, comparison study was conducted in 36 healthy subjects (30-60 years old) with dry skin. They were randomly divided into two groups and ingested either the test product (125 mL drink containing 10 mg piceatannol derived from passion fruit seed extract) or the control product (placebo drink) three packs per day for 8 weeks. The outcomes about skin condition were evaluated using moisture content of horny layer, Trans-epidermal Water Loss (TEWL) and viscoelasticity of the face. This study was registered in UMIN-CTR (UMIN000030491). Results: The water content and elastic recovery from the pretrial were significantly increased at 8 week in the test product containing piceatannol compared to the placebo group. Conclusions: These result suggested that the daily supplementation of piceatannol derived from passion fruit seed extract is effective for skin moisture.
