RECORD OF A RESEARCH

INTRODUCTION:
Adenosine monophosphate-activated protein kinase (AMPK) activation is suggested to relax smooth muscle by endothelial nitric oxide synthase (eNOS) phosphorylation.

AIM:
To assess the mechanism and effect of a novel AMPK activator, beta-lapachone, upon cavernosal smooth muscle relaxation and the therapeutic potential for erectile dysfunction.

METHODS:
Human umbilical vein endothelial cells (HUVECs) were treated with beta-lapachone. The lysates were blotted with specific antibodies for phosphorylated AMPK (p-AMPK) or phosphorylated eNOS (p-eNOS). The membranes were re-blotted for total AMP total eNOS, or beta-actin. The eNOS activity was measured by the conversion of L-14C-arginine to L-14C-citrulline in HUVECs lysates. In a separated experiment, cavernosal strips from New Zealand white rabbits were harvested for organ bath study and the relaxation effect of beta-lapachone on phenylephrine-induced contracted strips was evaluated and compared with sodium nitroprusside, zaprinast, metformin, and aminoimidazole carboxamide ribonucleotide (AICAR). Methylene blue and L-NAME were used to assess the inhibition of cyclic guanosine monophosphate/nitric oxide pathway. Zinc-protoporphyrin-IX (ZnPP) was also used to investigate the contribution of mevalonate pathway.

MAIN OUTCOME MEASURES:
The expression of p-AMPK, p-eNOS, AMPK and eNOS induced by beta-lapachone in HUVECs study and the percent relaxation of cavernosal tissue in organ bath study.

RESULTS:
Beta-lapachone clearly induced AMPK phosphorylation and, as a consequence, eNOS phosphorylation in HUVECs. Beta-lapachone-induced upregulation of eNOS activity was also observed in HUVECs and steadily increased up to 1 hour. In organ bath study, beta-lapachone significantly relaxed the phenylephrine pretreated strips in a dose-dependent manner. This relaxation effect was not totally blocked by methylene blue or L-NAME. After removing endothelium, the relaxation was totally blocked by ZnPP.

CONCLUSIONS:
A novel AMPK activator, beta-lapachone has a strong relaxation effect on precontracted cavernosal smooth muscle strips in the rabbit. And phosphorylation of AMPK and eNOS strongly related to the action of beta-lapachone. Mevalonate pathway also might be considered as a suggestive mechanism

In this study, we demonstrate that activation of AMP-activated protein kinase (AMPK) with glabridin alleviates adiposity and hyperlipidemia in obesity. In several obese rodent models, glabridin decreased body weight and adiposity with a concomitant reduction in fat cell size. Further, glabridin ameliorated fatty liver and plasma levels of triglyceride and cholesterol. In accordance with these findings, glabridin suppressed the expression of lipogenic genes such as sterol regulatory element binding transcription factor (SREBP)-1c, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD)-1 in white adipose tissues and liver, whereas it elevated the expression of fatty acid oxidation genes such as carnitine palmitoyl transferase (CPT)1, acyl-CoA oxidase (ACO), and peroxisome proliferator-activated receptor (PPAR)α in muscle. Moreover, glabridin enhanced phosphorylation of AMPK in muscle and liver and promoted fatty acid oxidation by modulating mitochondrial activity. Together, these data suggest that glabridin is a novel AMPK activator that would exert therapeutic effects in obesity-related metabolic disorders.

FIG. 1.

Glabridin decreases body weight and food intake in obese mice. A: Structure of glabridin. Glabridin is one of the major flavonoids in the hydrophobic fraction of G. glabra extract. B–E: HFD-fed obese mice (n = 7–8 per group) were orally administered with vehicle or glabridin (150 mg/kg) or used for pair-feeding, for four weeks. B: Gross images of whole body (top) and abdominal fat (bottom) of mice in each group. C, D: Body weight and food consumption were measured every day throughout the experimental period. E: Body temperature during 4 h of cold exposure (n = 6 per group) was measured at indicated time points. Data represent mean ± SE. *P < 0.05 versus vehicle; **P < 0.01 versus vehicle; #P < 0.05 versus pair-feeding (Student t-test). Each experiment was independently performed at least twice.

FIG. 2.

Glabridin ameliorates adiposity and fat tissue inflammation in obese mice. A, B: Appearance of epididymal (upper panel) and retroperitoneal (lower panel) white adipose tissue (WAT) and coronal (upper panel) and transverse (lower panel) MRI sections of whole body from vehicle- or glabridin-treated obese mice. C, D: Histological analysis of epididymal WAT from vehicle- or glabridin-treated obese mice and adipocyte size was quantified. E: Total RNA was isolated from WAT of obese mice. Relative mRNA levels of inflammatory genes, such as mcp-1 and inos, from WAT were determined using qRT-PCR and normalized with cyclophilin mRNA. Data represent mean ± SE of triplicates. *P < 0.05 versus vehicle (Student t-test). Epi. AT, epididymal adipose tissue; Ret. AT, retroperitoneal adipose tissue.

FIG. 3.

Glabridin alleviates fatty liver and hyperlipidemia. A, B: Gross appearance and histological (H and E staining) analysis of liver from vehicle- or glabridin-treated obese mice. C: Plasma levels of triglyceride, total cholesterol, and LDL cholesterol (n = 4–5 per group) were determined as described in Materials and Methods. D: Hepatotoxicity was assessed by measuring plasma levels of ALT and AST (n = 4–5 per group). Each bar represents mean ± SE. *P < 0.05 versus vehicle, **P < 0.01 versus vehicle (Student t-test).

FIG. 4.

Glabridin improves glucose tolerance in obese mice. Plasma levels of glucose (A), insulin (B), and adiponectin (C) were measured (n = 4–5 per group). Each bar represents mean ± SE. *P < 0.05 versus vehicle (Student t-test). D: IPGTT. Mice (n = 4–6 per group) were fasted and injected with glucose as described in Materials and Methods. Blood glucose levels were measured at indicated time points. Data represent mean ± SE. *P < 0.05 versus vehicle (Student t-test).

FIG. 5.

Glabridin regulates expression of genes involved in lipid metabolism. Total RNA was isolated from epididymal WAT, liver, and muscle of vehicle- or glabridin-treated obese mice. Relative mRNA levels of metabolic genes: (A) WAT and (B) liver lipogenic genes; (C) muscle fatty acid oxidation and mitochondrial biogenesis genes; and (D) liver gluconeogenic genes were determined using qRT-PCR. The gene expression levels were normalized by the level of cyclophilin mRNA. Experiments were independently performed at least three times. Data represent mean ± SE. *P < 0.05 versus vehicle; **P < 0.01 versus vehicle (Student t test).

FIG. 6.

FIG. 7.

for 3 h in prior to glabridin (20 μM) treatment for 30 min (C, E), and differentiated C2C12 cells were infected with Ad-GFP or Ad-DN-AMPKα2 (D, F) as described in Materials and Methods. Then cells were treated with vehicle or glabridin (20 μM) for 30 min. Total cell lysates were subjected to Western blot analysis (C, D). Fatty acid oxidation assays (E, F) were conducted as described in Materials and Methods. Each experiment was independently performed at least four times. Each bar represents mean ± SE. *P < 0.05 versus vehicle; #P < 0.05 versus glabridin.

Glabridin stimulates AMPK in vivo and in vitro. A–C: Total lysates from epididymal WAT (A), liver (B), or muscle (C) from vehicle- or glabridin-treated obese mice were subjected to Western blot analysis using specific antibodies (pAMPK, total AMPK, pACC, and total ACC). The pAMPK/AMPK and pACC/ACC ratios were quantified. More than three independent experiments were performed. D, E: Differentiated adipocytes (3T3-L1), hepatoma (FAO) cells, and differentiated myotube (C2C12) cells were treated with various concentrations (0, 0.2, 2, 20, and 50 μM) of glabridin for 30 min (D) or were incubated with glabridin (20 μM) for different time points as indicated (E). Cells were lysed and total cell lysates were subjected to Western blot analysis. Each experiment was independently performed more than twice.

FIG. 8.

Glabridin affects mitochondrial functions and cellular AMP/ATP ratio. A: AMPK activity was determined with SAMS peptide. FAO cells were treated with 2 or 20 μM glabridine or 10 μM berberine (BBR) for 1 h. Then AMPK activity was determined by measurement of phosphorylation of SAMS peptides as described in Materials and Methods. Two independent experiments were performed. Each bar represents mean ± SE. * P < 0.05 versus vehicle; ** P < 0.01 versus vehicle. B–E: ATP production rates (B, C) and activities of mitochondrial complexes I and II (D, E) in FAO cells. Mitochondria were isolated from FAO cells treated with glabridin or berberine and then challenged with 5 mM glutamate/malate (B, D) or 5 mM succinate (C, E). Each experiment was independently performed more than twice. Each bar represents mean ± SE. **P < 0.01 versus vehicle. F, G: Cellular AMP contents and AMP/ATP ratio were determined in (F) FAO cells treated with 20 μM glabridine or 10 μM berberine for 30 min or 12 h and (G) liver from vehicle- or glabridin-treated obese mice. Each bar represents mean ± SE. *P < 0.01 versus vehicle; **P < 0.01 versus vehicle.

Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARgamma (peroxisome proliferator-activated receptor gamma) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARgamma gene is a novel target gene of LXR, since the PPARgamma promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRalpha. Moreover, activated LXRalpha exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARgamma, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRalpha by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.

FIG. 1.

Expression of LXRα mRNA in adipocytes and mouse tissues. (A) Relative amounts of mRNA expression of adipogenic genes as determined by DNA microarray analysis. Total RNA was isolated from confluent preadipocytes and fully differentiated 3T3-F442A adipocytes and used for DNA microarray analysis. Relative amounts of mRNA expression of 11 genes are shown. Apo CI, apolipoprotein 1; Apo C2, apolipoprotein 2. (B) LXRα mRNA expression in C57BL/6 mouse tissues. Northern blot analysis was performed by using 20 μg of total RNA and cDNA probes for LXRα, ADD1/SREBP1c, PPARγ, aP2, and 36B4. B, brain; F, white adipose tissue (epididymal fat); K, kidney; M, muscle; Lu, lung; Li, liver; S, spleen; H, heart.

FIG. 2.

Adipogenic effect of LXR activation in preadipocytes. 3T3-L1 cells (A and B) and HSVCs (C) were differentiated into adipocytes in the presence or absence of the LXR agonist T0901317, 22(R)-hydroxycholesterol [22-(R) HC], or the PPARγ agonist TZD. (A and C) Microscopic pictures were taken 10 days after differentiation. (B and C) Differentiated adipocytes were stained with Oil Red O and photographed. EtOH, ethyl alcohol.

FIG. 3.

Stimulation of adipogenic marker gene expression following LXR activation during adipocyte differentiation (differ.). (A and B) 3T3-L1 cells were differentiated into adipocytes in the absence (A) or presence (B) of T0901317 (3 μM), and cells were harvested at the indicated time points. Northern blots (20 μg of total RNA) were hybridized with FAS, ADD1/SREBP1c, PPARγ, LXRα, aP2, and 36B4 cDNA probes. (C) Data from panels A and B were quantified and normalized relative to the loading control to show relative mRNA expression. Experiments were independently repeated three times.

FIG. 4.

Induction of lipogenic and adipogenic genes following acute LXR activation in differentiated adipocytes. (A) Differentiated 3T3-L1 adipocytes were treated with 0, 1, 3, or 10 μM T0901317 for 24 h. Northern blot analysis results were quantified and normalized relative to 28S rRNA levels. (B) 3T3-L1 adipocytes were treated for 0, 24, and 48 h with 3 μM T0901317, after which cells were harvested for Northern blot analysis. The results obtained were quantified and normalized relative to 28S rRNA levels. Northern blots (20 μg of total RNA) were hybridized with FAS, ADD1/SREBP1c, PPARγ, LXRα, and aP2 cDNA probes. Each experiment was independently repeated two times.

FIG. 5.

ChIP assay of mouse ADD1/SREBP1c promoter. Differentiated 3T3-L1 adipocytes were incubated in the absence (−) or presence (+) of 10 μM T0901317 for 24 h. Cells were cross-linked and immunoprecipitated with rabbit polyclonal antibodies against LXRα (LXRα Ab) (A) or polyclonal antibodies against acetylated histone H3 (H3-Ac Ab) (B). Immunoprecipitated DNA fragments were amplified by PCR (see Materials and Methods). Lane 1 shows the amplified mouse ADD1/SREBP1c promoter and GAPDH from 1% of the input DNA. GAPDH fragments were also amplified for the normalization of input DNA. PCR-amplified products of the mouse ADD1/SREBP1c promoter normalized with the amplified GAPDH fragments were quantitated. The putative sterol regulatory element (SRE; ellipse), LXRE (white square), and E-box (black triangle) are indicated. Data from a representative of three independent experiments are shown. IP, immunoprecipitant.

FIG. 6.

In vivo effects of LXR activation on adipogenic gene expression in white adipose tissue and liver. C57BL/6 mice were treated with T0901317 (50 mg/kg) or vehicle for 0, 1, 3, or 5 days. Epididymal fat and liver tissues were collected, and total RNA was isolated for Northern blotting (A and C) or RT-PCR analysis (E) to examine the mRNA expression of several genes, including the FAS, LXRα, ADD1/SREBP1c, PPARγ, and aP2 genes. (A) Expression in epididymal fat. (B) Data in panel A were quantified and normalized relative to 28S rRNA levels. (C and E) Expression in liver. (D) Data in panel C were quantified and normalized relative to 28S rRNA levels. Data from a representative of two independent experiments are shown.

FIG. 7.

Direct binding of LXRα to the mouse PPARγ promoter. (A) Sequence comparison of putative LXREs (DR4) in mouse and human PPARγ promoters. (B) In vitro-translated LXRα and RXRα proteins were used for EMSA with 32P-labeled PPARγ LXRE oligonucleotide. Sequence-specific competition assays were performed with the addition of a 100-fold molar excess of unlabeled sterol regulatory element (SRE) (lane 4), Cyp7A1 LXRE (lane 5), and PPARγ LXRE (lane 6) oligonucleotides. (C) ChIP assays of the mouse PPARγ promoter. Differentiated 3T3-L1 adipocytes were incubated with (+) or without (−) LXR agonist T0901317 (10 μM) for 0, 2, or 12 h. Cells were cross-linked and immunoprecipitated with rabbit polyclonal antibodies against LXRα or polyclonal antibodies against acetylated histone H3. Immunoprecipitated DNA fragments were amplified by PCR (see Materials and Methods). Lane 1 shows the amplified mouse PPARγ promoter and GAPDH from 1% of the input DNA. GAPDH fragments were also amplified for the normalization of input DNA. IP, immunoprecipitant. (D) h293 cells were cotransfected with the pADD1/SREBP1c −600-Luc reporter DNA (100 ng/well) and expression vectors for LXRα and RXRα (lanes 3 and 4). pADD1/SREBP1c −600-Luc is a luciferase reporter containing the region comprising bp −600 to +89 of the mouse ADD1/SREBP1c promoter. In parallel, h293 cells were cotransfected with the mouse pPPARγ −1,022-LXRE-Luc reporter (wild-type) DNA (100 ng/well) or the mutant pPPARγ −1,022-mLXRE-Luc reporter DNA (100 ng/well) and expression vectors for LXRα and RXRα (lanes 2 and 3). The pPPARγ −1,022-LXRE-Luc reporter is a luciferase reporter containing bp −1022 to +26 of the mouse PPARγ promoter. The pPPARγ −1,022-mLXRE-Luc reporter is a luciferase reporter containing the mutation in the LXRE motif of the mouse PPARγ promoter. After transfection, cells were treated with (+) or without (−) LXR agonist T0901317 (10 μM) for 24 h.

FIG. 8.

Effect of LXRα knockdown by siRNA during adipogenesis. 3T3-L1 cells were infected and selected with pSUPER retroviruses including mock, siLXRαSR933, and siLXRαSR1246 (see Materials and Methods). Those infected cells were differentiated into adipocytes and were harvested for total RNA preparation at the indicated time point. (A) The cells were stained with Oil Red O and photographed. (B) Northern blots (20 μg of total RNA) were hybridized with FAS, ADD1/SREBP1c, PPARγ, LXRα, LXRβ, and aP2 cDNA probes. Mock and siLXRαSR1246 were used as negative controls. (C) Effects of PPARγ ligand on 3T3-L1-LXRα siRNA stable cells. Mock and 3T3-L1-LXRα siRNA stable cell lines were differentiated into adipocytes under normal differentiation conditions (see Materials and Methods) in the absence (DMSO) or presence of T0901317 (1 μM) or rosiglitazone (0.1 μM).

FIG. 9.

Effects of LXR or PPARγ ligand on DN-ADD1/SREBP1c-expressing cells or PPARγ-deficient MEF cells. (A) 3T3-L1 cells were infected and selected with pBabe retroviruses including mock and DN-ADD1/SREBP1c. (B) MEF cells deficient in the PPARγ or the PPARγ heterozygote mutant or both of them were differentiated into adipocytes in the absence (DMSO) or presence of T0901317 (1 μM) or rosiglitazone (0.1 μM).

Fas/APO-1/CD95, a member of the tumor necrosis factor (TNF) receptor superfamily, is a potential anti-cancer factor as it can induce apoptosis in tumor cells. However, despite the fact that many cancer cells express Fas on the membrane, some tumors such as prostate cancer display resistance to Fas-induced apoptosis. In these cases, combination therapy using chemotherapeutic agents and Fas may be more suitable than therapy using Fas alone. In the present study, we demonstrate that the apoptosis inhibitory protein, Bcl-2, was highly expressed in response to Fas in DU145 prostate cancer cells, thereby conferring resistance to apoptosis. We have screened a number of naturally occurring products that may overcome this resistance. Here we report that cryptotanshinone, the major tanshinone isolated from Salvia miltiorrhiza Bunge, can suppress Bcl-2 expression and augment Fas sensitivity in DU145 cells. We further show that JNK and p38 MAPK act upstream of Bcl-2 expression in Fas-treated DU145 cells, and that cryptotanshinone significantly blocked activation of these kinases. Moreover, cryptotanshinone sensitized several tumor cells to a broad range of anti-cancer agents. Collectively, our data suggest that cryptotanshinone has therapeutic potential in the treatment of human prostate cancer.

Metabolic disorders, including type 2 diabetes and obesity, represent major health risks in industrialized countries. AMP-activated protein kinase (AMPK) has become the focus of a great deal of attention as a novel therapeutic target for the treatment of metabolic syndromes, because AMPK has been demonstrated to mediate, at least in part, the effects of a number of physiological and pharmacological factors that exert beneficial effects on these disorders. Thus, the identification of a compound that activates the AMPK pathway would contribute significantly to the treatment and management of such syndromes. In service of this goal, we have screened a variety of naturally occurring compounds and have identified one compound, cryptotanshinone, as a novel AMPK pathway activator. Cryptotanshinone was originally isolated from the dried roots of Salvia militorrhiza, an herb that is used extensively in Asian medicine and that is known to exert beneficial effects on the circulatory system. For the first time, in the present study, we have described the potent antidiabetic and antiobesity effects of cryptotanshinone, both in vitro and in vivo. Our findings suggest that the activation of the AMPK pathway might contribute to the development of novel therapeutic approaches for the treatment of metabolic disorders such as type 2 diabetes and obesity.

IBACKGROUND/OBJECTIVES:
Colitis is a serious health problem, and chronic obesity is associated with the progression of colitis. The aim of this study was to determine the effects of natural raw meal (NRM) on high-fat diet (HFD, 45%) and dextran sulfate sodium (DSS, 2% w/v)-induced colitis in C57BL/6J mice.

MATERIALS/METHODS:
Body weight, colon length, and colon weight-to-length ratio, were measured directly. Serum levels of obesity-related biomarkers, triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), insulin, leptin, and adiponectin were determined using commercial kits. Serum levels of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were detected using a commercial ELISA kit. Histological study was performed using a hematoxylin and eosin (H&E) staining assay. Colonic mRNA expressions of TNF-α, IL-1β, IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were determined by RT-PCR assay.

RESULTS:
Body weight and obesity-related biomarkers (TG, TC, LDL, HDL, insulin, leptin, and adiponectin) were regulated and obesity was prevented in NRM treated mice. NRM significantly suppressed colon shortening and reduced colon weight-to-length ratio in HFD+DSS induced colitis in C57BL/6J mice (P < 0.05). Histological observations suggested that NRM reduced edema, mucosal damage, and the loss of crypts induced by HFD and DSS. In addition, NRM decreased the serum levels of pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6 and inhibited the mRNA expressions of these cytokines, and iNOS and COX-2 in colon mucosa (P < 0.05).

CONCLUSION:
The results suggest that NRM has an anti-inflammatory effect against HFD and DSS-induced colitis in mice, and that these effects are due to the amelioration of HFD and/or DSS-induced inflammatory reactions.

FIG. 1.

Effects of NRM on body weights of mice treated with HFD (45%) and/or DSS.

Norm.: normal control mice; HFD: mice tre Fig. 2

Effects of NRM on colon length and colon weight-to-length ratio in HFD (45%) and/or DSS-treated mice.

Norm.: normal control mice; HFD: mice treated with a high-fat diet (HFD, 45%); DSS: mice treated with DSS (2%) and a normal diet; HFD+DSS: mice treated with HFD and DSS; HFD+DSS+NL: mice treated with HFD and DSS with 30% NRM in diet; HFD+DSS+NH: mice treated with HFD and DSS with 70% NRM in diet. Superscript with different letters on the bars are significantly different (P < 0.05) by Duncan's multiple range test.

ated with a high-fat diet (HFD, 45%); DSS: mice treated with DSS (2%) and a normal diet; HFD+DSS: mice treated with HFD and DSS; HFD+DSS+NL: mice treated with HFD and DSS with 30% NRM in diet; HFD+DSS+NH: mice treated with HFD and DSS with 70% NRM in diet. Results are presented as mean ± SD. Superscript with different letters on 8th week are significantly different (P < 0.05) based on Duncan's multiple range test.

FIG. 3.

Histological observations of colon tissue damage in mice treated with NRM, HFD (45%), and/or DSS The arrow (→) indicates specific points of inflammation.

Norm.: normal control mice; HFD: treated with a high-fat diet (HFD, 45%); DSS: mice treated with DSS (2%) and a normal diet; HFD+DSS: mice treated with HFD and DSS; HFD+DSS+NL: mice treated with HFD and DSS with 30% NRM in diet; HFD+DSS+NH: mice treated with HFD and DSS with 70% NRM in diet.

FIG. 4.

Effect of NRM on the serum levels of TNF-α, IL-1β, and IL-6 in HFD (45%) and/or DSS-treated mice.

Norm.: normal control mice; HFD: mice treated with a high-fat diet (HFD, 45%); DSS: mice treated with DSS (2%) and a normal diet; HFD+DSS: mice treated with HFD and DSS; HFD+DSS+NL: mice treated with HFD and DSS with 30% NRM in diet; HFD+DSS+NH: mice treated with HFD and DSS with 70% NRM in diet. Superscript with different letters on the bars are significantly different (P < 0.05) by Duncan's multiple range test.

FIG. 5.

Effect of NRM on the mRNA levels of TNF-α, IL-1β, and IL-6 of HFD (45%) and/or DSS-treated mice.

Norm.: normal control mice; HFD: mice treated with a high-fat diet (HFD, 45%); DSS: mice treated with DSS (2%) and a normal diet; HFD+DSS: mice treated with HFD and DSS; HFD+DSS+NL: mice treated with HFD and DSS with 30% NRM in diet; HFD+DSS+NH: mice treated with HFD and DSS with 70% NRM in diet. Band intensities were measured using a densitometer and are expressed as folds of the control (HFD+DSS treated groups). Fold ratio: Gene expression / β-actin × control numerical value (Control fold ratio = 1). Superscript with different letters on the bars are significantly different (P < 0.05) by Duncan's multiple range test.

FIG. 6.

Effect of NRM on the mRNA levels of iNOS and COX-2 in the colon tissues of HFD (45%) and/or DSS-treated mice.

Norm.: normal control mice; HFD: mice treated with a high-fat diet (HFD, 45%); DSS: mice treated with DSS (2%) and a normal diet; HFD+DSS: mice treated with HFD and DSS; HFD+DSS+NL: mice treated with HFD and DSS with 30% NRM in diet; HFD+DSS+NH: mice treated with HFD and DSS with 70% NRM in diet. Band intensities were measured using a densitometer and are expressed as folds of control (HFD+DSS treated groups). Fold ratio: Gene expression / β-actin × control numerical value (Control fold ratio = 1). Superscript with different letters on the bars are significantly different (P < 0.05) by Duncan's multiple range test.

We investigated the combined moisturizing effect of liposomal serine and a cosmeceutical base selected in this study. Serine is a major amino acid consisting of natural moisturizing factors and keratin, and the hydroxyl group of serine can actively interact with water molecules. Therefore, we hypothesized that serine efficiently delivered to the stratum corneum (SC) of the skin would enhance the moisturizing capability of the skin. We prepared four different cosmeceutical bases (hydrogel, oil-in-water (O/W) essence, O/W cream, and water-in-oil (W/O) cream); their moisturizing abilities were then assessed using a Corneometer®. The hydrogel was selected as the optimum base for skin moisturization based on the area under the moisture content change-time curves (AUMCC) values used as a parameter for the water hold capacity of the skin. Liposomal serine prepared by a reverse-phase evaporation method was then incorporated in the hydrogel. The liposomal serine-incorporated hydrogel (serine level=1%) showed an approximately 1.62~1.77 times greater moisturizing effect on the skin than those of hydrogel, hydrogel with serine (1%), and hydrogel with blank liposome. However, the AUMCC values were not dependent on the level of serine in liposomal serine-loaded hydrogels. Together, the delivery of serine to the SC of the skin is a promising strategy for moisturizing the skin. This study is expected to be an important step in developing highly effective moisturizing cosmeceutical products.

FIG. 1.

Effect of formulation type on moisture content of the skin (A). Changes in moisture content of the skin as a function of time (B) are plotted to assess the moisture hold capacity of the skin by calculating the area under the moisture content change-time curve. Mean±SD (n=40~50).

FIG. 2.

Effect of serine, blank liposomes, and hydrogel loaded with liposomal serine on the moisture content of the skin. The profiles of changes in the skin moisture contents were produced after monitoring the moisture content values of the skin measured at 0, 0.5, 1, 1.5, 2, 2.5 and 3 h of the experiments. Mean±SD (n=40~50).

FIG. 3.

Effect of serine levels in the liposomes incorporated in hydrogel bases on moisture content of the skin. The profiles of changes in skin moisture contents were produced after monitoring the moisture content values of the skin measured at 0, 0.5, 1, 1.5, 2, 2.5 and 3 h of the experiments. Mean±SD (n=40~50).