Ixora parviflora Protects against UVB-Induced Photoaging by Inhibiting the Expression of MMPs, MAP Kinases, and COX-2 and by Promoting Type I Procollagen Synthesis

Ixora parviflora with high polyphenol content exhibited antioxidant activity and reducing UVB-induced intracellular reactive oxygen species production. In this study, results of the photoaging screening experiments revealed that IPE at 1000 μg/mL reduced the activity of bacterial collagenase by 92.7 ± 4.2% and reduced the activity of elastase by 32.6 ± 1.4%. Therefore, we investigated the mechanisms by which IPE exerts its anti-photoaging activity. IPE at 1 μg/mL led to an increase in type I procollagen expression and increased total collagen synthesis in fibroblasts at 5 μg/mL. We found that IPE inhibited MMP-1, MMP-3, and MMP-9 expression at doses of 1, 5, and 10 μg/mL, respectively, in fibroblasts exposed to UV irradiation (40 mJ/cm2). Gelatin zymography assay showed that IPE at 50 μg/mL inhibited MMP-9 secretion/activity in cultured fibroblasts after UVB exposure. In addition, IPE inhibited the phosphorylation of p38, ERK, and JNK induced by UVB. Furthermore, IPE inhibited the UVB-induced expression of Smad7. In addition, IPE at 1 μg/mL inhibited NO production and COX-2 expression in UV-exposed fibroblasts. These findings show that IPE exhibits anti-inflammatory and anti-photoaging activities, indicating that IPE could be a potential anti-aging agent.


Introduction
Skin is directly exposed to ultraviolet irradiation, which is a prooxidant agent. Skin aging can be divided into two basic processes, intrinsic and extrinsic aging. UV irradiation is the major cause of extrinsic aging, namely, sun exposure or photoaging, which is characterized by severe wrinkling, sagging, and hyperpigmentation [1]. Disorganization, fragmentation, and dispersion of collagen bundles are prominent features of photodamaged human skin. The role of UVB in skin tumor induction has been accepted for decades. UVB irradiation can cause skin damage by indirectly inducing oxidative stress or directly forming pyrimidine dimers and C-T mutation in DNA, which can lead to photoaging and cancer development [2][3][4].
The most abundant structural protein in skin connective tissue is type I collagen (90% of extracellular matrix in dermis), which is responsible for conferring strength and resiliency [5,6]. Type I collagen is synthesized primarily by fibroblasts residing within skin connective tissue (dermis). It is synthesized as a soluble precursor, type I procollagen, which is secreted from fibroblasts and proteolytically processed to form insoluble collagen fibers. Loss of the structural integrity of the collagenous extracellular matrix is believed to be primarily responsible for the wrinkled appearance of photodamaged skin. UV irradiation induces the synthesis of matrix metalloproteinases (MMPs), which degrade collagen in the skin. UVB has been shown to induce the overexpression of MMP-1, -3, and -9 in normal human epidermis in vivo [7,8]. Studies have shown that MMP-mediated collagen destruction accounts, in large part, for the connective tissue damage that occurs in photoaging [9]. Under normal physiological conditions, MMPs are regulated by endogenous inhibitors known as tissue inhibitors of metalloproteinases (TIMP), especially TIMP-1 [10][11][12]; however, UVB disrupts the balance between MMPs and TIMPs [13]. UV stimulates 2 Evidence-Based Complementary and Alternative Medicine inhibitory Smad (I Smad; Smad 6 and Smad 7) leading to receptor-activated Smad protein (R Smad; Smad 2 and Smad 3) phosphorylation. It will block R Smad complex translocating into the nucleus [14], thereby reducing expression of type I procollagen and elastin [15].
Besides regulating of MMPs and collagen, excessive UV irradiation can cause acute skin inflammation and lead to the development of skin cancer. Prostaglandins (PGs) and nitric oxide (NO) play important roles in the inflammatory process [16]. Prostaglandins are the products of the cyclooxygenase (COX) pathway of arachidonic acid metabolism. UVB induces cyclooxygenase-2 (COX-2) production, which is the rate-limiting enzyme in PGs generation [17]. UV-induced lipid peroxides are also involved in PGs generation, such as PGE2, an important factor in skin inflammation [18,19]. UV irradiation also upregulates the expression of inducible nitric oxide synthase (iNOS) to produce NO at sufficiently high levels to react with superoxide, leading to the production of peroxynitrite and other reactive oxygen species (ROS) [20,21]. ROS drive MAP kinase activation (including ERK, JNK, and p38) which recruits c-Fos and c-Jun to the nucleus and NF-κB activation, and the gene related to proinflammatory would be upregulated subsequently [22].
Polyphenols are abundant in fruits, vegetables, green tea, and wine. Polyphenols such as epigallocatechin-3-gallate (EGCG) found in tea have been shown to have photoprotective properties by hampering collagen destruction and collagenase activation [23,24]. Ixora parviflora, a member of the Rubiaceae family of flowering plants, is rich in polyphenols and used as a folk medicine in India [25][26][27]. In our previous study, Ixora parviflora extract (IPE) exhibited ROS scavenging activity and, therefore, may protect skin from photodamage by diminishing UV-induced ROS production [27]. The aim of this study was to investigate the potential mechanisms by which IPE counteracts UVB-induced overexpression of COX and MMPs, the secretion of MMPs, and the reduction in type I procollagen levels.

Gelatin Digestion Assay.
The assay was carried out as previously described [8]. Briefly, agarose solution was prepared in a collagenase buffer with porcine gelatin. Various concentrations of IPE (125, 250, 500, and 1000 μg/mL; 10 μL) and IPH1-IPH4 (1000 μg/mL; 10 μL) dissolved in 50% propylene glycol were incubated with bacterial collagenase-1 in collagenase buffer for 1 h at room temperature. Doxycycline hyclate (100 μg/mL) was used as the positive control, and 50% propylene glycol served as the negative control. The samples were loaded onto paper disks, placed on gelatinagarose gel, and then incubated. The degree of gelatin digestion in agarose gel was visualized by the Coomassie Blue staining. Following destaining, the area of the light translucent zone over the blue background was determined densitometrically to estimate gelatinase activity.
The inhibition rate of collagenase was calculated by the following equation: where A indicates the absorbance without enzyme and sample, B indicates the absorbance with enzyme but without sample, and C indicates the absorbance with enzyme and sample.

MMP Activity Assays by Fluorescent Gelatin.
The assay was performed as previously described [8].
where A control is the absorbance of collagenase and substrate without IPE and A sample is that with IPE.

Measurement of Elastase
Activity. The ability of IPE to inhibit elastase activity was investigated using elastase from porcine pancreases as previously reported with minor modifications [28]. Elastase inhibitor I (250 μM) was used as the positive control, and 50% propylene glycol served as the negative control. Elastase (500 U) was dissolved in 5 mL of 10 mM tris buffer solution (pH 6.0), and 5 mg of elastase substrate IV was dissolved in 5 mL of 100 mM tris buffer solution (pH 8.0 (3)

Cell Culture.
Human foreskin fibroblasts (Hs68) were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 • C in 5% CO 2 humidified air. The cells were subcultured following trypsinization, and cells were used in the 20th to 35th passages.

Cell Viability
Test. The fibroblasts were plated at a density of 4×10 4 cells/well, HaCaT cells were plated at a density of 1.5 × 10 4 cells/well, and B16-F0 cells were plated at a density of 8 × 10 4 cells/well in 24-well plates per 1 mL medium. The cells were allowed to attach overnight and were treated with 1 mL of various concentrations of IPE dissolved in DMEM and DMSO (<0.1%) for 24 h. The cytotoxicity of IPE was evaluated in cells that had been cultured for 4 h using the MTT assay as previously described [8].
2.6. UVB Irradiation. Cells were washed twice with PBS covered with PBS, and then exposed to UV irradiation (302 nm, CL-1000 M, UVP, USA). In our preliminary study, a dose of 40 mJ/cm 2 UVB irradiation was determined to induce MMP and MAP kinase expression without being cytotoxic (data not shown). After UVB irradiation, PBS was replaced with a serum-free medium with or without sample and then incubated for 24 h for assay of total collagen, MMPs, MAP kinases, and Smad activity.

Western Blot Analysis.
Cells were harvested and homogenized with lysis buffer (10 mM Na 3 VO 4 , 10 mg/mL PMSF, 10 mg/mL leupeptin and RIPA buffer, pH 7.4). The lysates were then centrifuged, and protein content was determined using the Bradford method. Cell lysates containing equal amounts of total protein (30 μg) were separated by electrophoresis on SDS-polyacrylamide gel and then transferred to a PVDF membrane (Hybond ECL, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Blots were blocked with 5% (w/v) skim milk in TBS buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.05% Tween 20 (TBST). The membrane was incubated overnight at 4 • C with goat polyclonal antibodies against MMP-1 (1 : 500) and type I procollagen (1 : 500) and mouse polyclonal antibodies Immunoreactive proteins were detected with the ECL western blotting detection system (Fujifilm, LAS-4000). Signal strengths were quantified using a densitometric program (Multi Gauge V2.2).

Gelatin Zymography for MMP-9.
Cell-free medium (30 μL) after UVB exposure was mixed with tris-glycine SDS sample buffer without reducing agent and electrophoresed. After electrophoresis, the gels were incubated with washing buffer for 30 min at room temperature. The MMP-9 reaction buffer (1 M Tris-HCl, 1 M CaCl 2 , 1% NaN 3 and 1% Brij-35, pH 7.4) was then added to the gel and incubated at 37 • C for 24 h. The gels were stained with 0.05% Coomassie Blue G-250 until proteinase bands were clearly visible in a blue background. The area of light translucent zones over the blue background was determined by a densitometric program (Multi Gauge V2.2) to estimate gelatinase activity.

Measurement of Total Collagen.
Total collagen synthesis in fibroblasts after UVB exposure was measured by the Sircol-soluble collagen assay kit (Biocolor Ltd., UK) according to the manufacturer's protocol. Briefly, cell culture medium was mixed with Sircol dye reagent and incubated at room temperature for 30 min. After centrifugation, ice-cold acidsalt washing reagent was added to the precipitate and then centrifuged. The precipitate was dissolved with the Alka reagent, and the absorption was determined at 555 nm (Tecan, Austria).
2.11. Statistical Analysis. Differences between groups were analyzed by ANOVA followed by the Scheffe test. A P value <0.05 was considered to indicate statistical significance.

Inhibitory Effect of IPE and IPH on Bacterial Collagenase-1 Assessed by Gelatin Digestion Assay.
For visual investigation and screening of the inhibitory effect of IPE and IPHs on MMP expression, an indirect assay was developed using bacterial collagenase-1 as previously described [8]. As shown in Figure 1(a), the control group treated with reaction products of bacterial collagenase-1 and propylene glycol exhibited the highest gelatinolytic activity in the discrete zone, representing no enzyme inhibition. The inhibition of doxycycline (100 μg/mL, as positive control) was 100.4 ± 0.3%. Following incubation of bacterial collagenase-1 with various concentrations of IPE and IPHs, the inhibition of enzyme activity was compared with enzyme activity in the control group. Gelatinolytic activity decreased in a dose-dependent manner following treatment with IPE. The inhibitory effect of IPE on gelatin digestion by collagenase is shown in Figure  1(b). The rates of collagenase-1 inhibition were 100.2 ± 3.9% for IPH1, 92.4 ± 5.1% for IPH2, 100.3 ± 2.8% for IPH3, and 92.7 ± 4.2% for IPH4. The concentration of IPH in each assay was 1000 μg/mL.

Fluorometric Analysis of the Inhibitory Effect of IPE and IPH on Bacterial
Collagenase-1. Fluorescence-conjugated gelatin was used to measure the inhibitory effect of IPE and IPH on bacterial collagenase-1 protein expression. Fluorescence-conjugated substrate was incubated with bacterial collagenase-1 for 20 h in the presence of different concentrations of IPE, IPH, or doxycycline hyclate (positive control) at 37 • C. IPE and IPH exhibited significant dose-dependent inhibitory effects on bacterial collagenase-1. As shown in Figure 1(c), treatment with 500 μg/mL of IPE decreased the activity of bacterial collagenase-1 by 97%. The hydrolysates in high concentrations showed similar inhibitory action against collagenase. The effect of IPE and IPH1, 3, and 4 was similar to that of the positive control at 500 μg/mL.
According to the results of gelatin digestion assay and fluorometric analysis on the inhibition of collagenase, the activity of collagenase of IPE was superior to IPHs; therefore, IPE was used in the following study for the effect and mechanism on antiphotoaging.

The Effect of IPE on Elastase
Activity. This assay measured the synthesis and activity of elastase in cells exposed to IPE. As shown in Figure 2, IPE significantly suppressed elastase activity in a dose-dependent manner. The effect of IPE on reduction of elastase activity was similar to that of the positive control (250 μM elastase inhibitor I) at 500 μg/mL.

Effect of IPE on Cell
Viability. Hs68, HaCaT, and B16-F0 cells were treated with various concentrations of, IPE and cell viability was measured using the MTT assay. As shown in Figure 3, the survival curve indicates that IPE (5-500 μg/mL) did not exhibit cytotoxic effects on the proliferation of the tested cell lines.

Effect of IPE on Type I Procollagen Expression.
According to our preliminary study, cell viability was greater than 95% relative to that of the control after exposure to 20-80 mJ/cm 2 UVB irradiation (data not shown). We also found that UV irradiation at a dose of 40 mJ/cm 2 led to an increase in MMP secretion levels and a decrease in type I procollagen synthesis in UV-exposed fibroblasts (data not shown). This dose is equivalent to about 30 seconds of exposure at noon in the month of July in Central Taiwan as measured by a UV meter (UVP, USA) [28]. Therefore, 40 mJ/cm 2 was chosen as the exposure dose in the following experiment. Fibroblasts were pretreated with IPE (1-50 μg/mL) for 1 h, exposed to UVB, and then treated with IPE for 48 h. The expression of type I procollagen is shown in Figure 4(a). UVB exposure resulted in 0.4-fold of type I procollagen level relative to the level in control cells; however, after IPE treatment (1-50 μg/mL), type I procollagen levels reached as high as 1.2fold of those in the control group.

Effect of IPE on Total Collagen Synthesis.
Fibroblasts were pretreated with IPE (1-50 μg/mL) for 1 h, exposed to UVB, and then treated with IPE for 24 h. As shown in Figure  4(b), UVB exposure led to a 52.3% reduction in total collagen synthesis. IPE treatment (1, 5, 10, and 50 μg/mL), however, resulted in a dose-dependent restoration of collagen (Figure 4(b)). Figure 5, Smad 7 expression was 2-fold higher than that in the control group after UVB irradiation. Smad 7 expression in fibroblasts pretreated with IPE, however, was 1.8-fold higher in cells treated with 1 μg/mL IPE, 1.7-fold higher at a concentration of 5 μg/mL, 1.2-fold higher in cells treated with 10 μg/mL IPE, and 1.2-fold higher in cells exposed to 50 μg/mL IPE relative to the control group.

Effect of IPE on MAP Kinase Expression. As shown in
in phosphorylation of the above-mentioned MAP kinases expression.

The Anti-Inflammatory Effect of IPE
3.7.1. Nitric Oxide Scavenging. The content of NO in fibroblasts increased to 2-fold after exposure to UVB; however, IPE treatment (1, 5, 10 and 50 μg/mL) significantly reduced the levels of NO (Figure 9(a)).

IPE on COX-2 Expression.
The levels of COX-2 were 1.6-fold higher in fibroblasts exposed to UVB (40 mJ/cm 2 ) than that in EGCG-exposed control cells (Figure 9(b)). In addition, IPE exhibited a dose-dependent reduction in

Discussion
In our previous study, the pH value of IPE in water, methanol, and propylene glycol ranged from 4.9 to 6.7, which is similar to that of skin [27]. Therefore, IPE preparations do not seem to irritate skin.
UV irradiation enhances collagenase activity and contributes to wrinkle formation through degradation of collagen in the dermal extracellular matrix [32,33]. Collagenase inhibitors have been identified as potential therapeutic agents that can protect against photoaging and wrinkle formation [34]. In the screening experiments, our results indicate that IPE and IPHs inhibit collagenase activity, although the effect of IPE was superior to that of IPHs. The inhibitory activity of IPE at 1000 μg/mL on elastase expression was similar to that of elastase inhibitor I. These findings indicate that IPE may be a potential topical agent to protect against UV-induced skin damage or aging-related disorders. Thus, the IPE was used in following experiments for effects and mechanisms study in Hs68.
UV irradiation upregulated the phosphorylation of p38, ERK, and JNK, and IPE treatment inhibited phosphorylation of those MAP kinases. Polypodium leucotomos extract has been shown to inhibit the activities of MMP-1, -2, -3, and -9 preventing UV damage [44]. In addition, Moon et al. showed that erythrodiol-3-acetate isolated from Styrax japonica inhibited UV-induced MMP-1 expression [45]. MAP kinase activation is not only one of the photoaging pathways but is also a factor in MMP production in fibroblasts [22,46,47]. In this study, we found that IPE was not only a potent MMP inhibitor but that it also inhibited the MMP-1-initiated degradation of type I collagens. In our previous study, IPE was a potent suppressor of UVB-induced ROS generation [27] and, therefore, might be a promising agent to protect against UVB-induced photodamage. Ho et al. reported that UVBinduced ROS generation promoted downstream signal transduction in human skin fibroblasts [48]. Thus, inhibition of ROS production would help protect skin from photoaging. We speculate that the inhibitory effect of IPE against collagen degradation is most likely due to its antioxidant activity, since the injury to skin caused by UVB may be due to ROS generation.
UV stimulates inhibitory Smad leading to receptoractivated Smad protein phosphorylation causing inhibition of R Smad complex translocating into the nucleus, thereby reducing expression of type I procollagen and elastin [14,15]. In this study, after UV exposure, IPE treatment resulted in increasing type I procollagen and reducing Smad 7. We expected that the effect on inhibiting of Smad 7 of IPE leading to enhance Smad 2 and Smad 3 phosphorylation may inhibit type I procollagen synthesis. UV irradiation induced the expression of TIMP-1 expression in fibroblasts, and the levels were 1.4-fold higher than control levels in cells exposed to IPE treatment.
UV irradiation leads to direct or indirect DNA damage and the formation of ROS, induces an inflammatory response (induced COX-2 and iNOS), and damages the integrity of the extracellular matrix [49]. Natural products with anti-inflammatory activities can protect DNA from ROSinduced damage. Our studies have shown that IPE possesses antioxidant and anti-inflammatory activities. EGCG has been shown to suppress the UV-induced inflammatory response by inhibiting the activation of NFκB [50]. UV irradiation has been shown to upregulate the expression of inducible nitric oxide synthase (iNOS) to produce NO at sufficiently high levels to react with superoxide, leading to the production of peroxynitrite and other reactive oxygen species. These ROS then induce MAP kinases to produce COX-2 [22]. In this study, we found that the concentration of NO was significantly increased in human fibroblasts that had been exposed to UVB (40 mJ/cm 2 ); however, IPE at 1 μg/mL led to a significant reduction in NO, and IPE at 50 μg/mL resulted in NO levels similar to those in control cells. We also found that IPE attenuated the UV-induced overexpression of COX-2. Studies have shown that 1α,25dihydroxyvitamin D3 inhibits UV-induced DNA damage and leads to a reduction in NO levels by inhibiting NOS in keratinocytes [51]. Furthermore, Porphyra dentate extract containing catechol, rutin, and hesperidin has been shown to decrease NO production by inhibiting UV-induced NFκB and iNOS expression [52]. In our previous study, IPE exhibited potent ROS scavenging and metal-chelating activities [27]. The scavenging of ROS by IPE may lead to a blockade of the MAP kinase pathway, which would in turn inhibit the activation of NFκB and AP-1 and, hence, inhibit the expression of MMP-1, -3, -9 and COX-2. Inhibition of the MAP kinase pathway, therefore, would prevent the phosphorylation of ERK and the expression of Smad 7, thereby enhancing the expression of type I procollagen.
In summary, our results show that IPE attenuated UVBinduced photodamage and inflammation by modulating the expression of MMPs, MAP kinases, and COX-2. IPE, therefore, appears to be a potent antiphotoaging agent. Further studies on the effect IPE has on other biomarkers in photoaging as well as the identification of the major active components in IPE are warranted.