## 3 Discussion  
In the current study, (2'S)-columnianetin, a bioactive coumarin isolated from _C. heterocarpa_, was studied for its anti-photoaging properties in UVA irradiated dermal fibroblasts. The (2'S)-columnianetin was shown to possess important bioactivities which include protective effects against UVB-induced damages in keratinocytes, cells forming the upper epidermis layer of skin, via antioxidant and MMP-1 inhibitory activities [22]. In order to confirm its photoprotective effects against UVA irradiation in dermal fibroblasts, the present study investigated the effects of (2'S)-columnianetin on UVA-irradiated changes in MMP-1 and collagen production using human dermal fibroblast cell line. UVA rays can penetrate deeper than UVB until the bottom layers of the dermis [5]. Subtoxic UVA exposure induced several detrimental effects in the dermis such as premature skin aging, also known as photoaging [7]. Cells exposed to UVA expressed increasing amounts of MMPs. MMPs are key enzymes in ECM building and maintenance due to their ability to cleave main ECM components such as collagen. MMP-1 is a member of the collagenase subfamily of MMPs and responsible for the breakdown of ECM during regular cellular processes such as tissue remodeling and recovery as well as diseases such as metastasis by initially cleaving the type I, II and III collagen [24; 25]. However, UVA irradiation upregulates the MMP-1 production and subsequently reduces the collagen synthesis in the dermal fibroblasts. The main outcome of these changes is loss of skin elasticity and strength [7; 8]. It was shown in Figure 3 that UVA exposure increased the MMP-1 production and decreased the type I\(\alpha\)1 pro-collagen. Treating dermal fibroblasts with 10 \(\upmu\)M (2'S)-columnianetin notably ameliorated the effects of UVA irradiation by increasing the type I\(\alpha\)1 pro-collagen production which is the precursor of type I collagen and decreasing the MMP-1 release (Figure 3). This was further evidenced by analyzing its effects on mRNA and protein expression levels of MMP-1 and type I pro-collagen. The (2'S)-columnianetin reversed the effects of UVA on mRNA and protein levels: increasing pro-collagen expression while suppressing MMP-1 (Figure 4). It was reported that UVA irradiation upregulated the production of MMP-1 through activation of MAPKs via cell surface cytokine and stress receptors due to elevated oxidative stress [26]. UVA-induced activation of ERK, JNK and p38 MAPKs further activates the AP-1 transcription factor which stimulates the transcriptional expression of MMP genes [26; 27]. The overexpression of MMPs in fibroblasts causes ECM to lose its integrity. As seen in Figure 5A, UVA irradiation significantly increased the phosphorylation of p38, ERK and JNK in HDFs. The (2'S)-columnianetin (10 \(\upmu\)M) treatment slightly decreased the ERK activation while significantly suppressing the activation of JNK. On the other hand, it did not affect p38 phosphorylation. Results indicated that (2'S)-columnianetin downregulated the UVA-induced MMP-1 production through a JNK-mediated signaling which speculatively resulted in decreased AP-1 activity. The collagen synthesis in dermal fibroblasts is partly regulated by TGF\(\beta\) and Smad cascades [28]. Stimulated TGF\(\beta\) receptor facilitates the phosphorylation of Smad2 and Smad3 which together form an active complex with Smad4. The Smad2/3/4 complex then initiates the type I pro-collagen expression after its nuclear translocation. This cascade is negatively regulated by Smad7 protein which inhibits the formation of active Smad2/3/4 complex [28; 29]. UVA exposure caused a decrease in the both production of TGF\(\beta\) cytokine and phosphorylation of Smad2/3 complex (Figure 5B). As expected, this was accompanied by an increase in Smad7 level. Presence of 10 \(\upmu\)M (2'S)-columnianetin relieved the suppression on p-Smad2/3 levels. Likewise, Smad7 levels were decreased by **(2'S)-columnianetin** treatment.