## 4 Discussion  
JAUN has been continuously improved and used since the Edo period in Japan, with a history of use exceeding 400 years. Although many recipes for producing JAUN have been developed, _Lithospermrum erythhorhizon_ and _Angelica gigas_ are indispensable. The ingredients in multiple herbal blends often have complementary effects, working together to enhance the therapeutic or other properties of the mixture [21]. This study examined whether JAUN reduces UVB-induced photoaging by modulating the Nrf/ARE and TGF-/Smad signaling pathways by examining the effects of JAUN therapy on UVB-irradiated HaCaT cells. ROS are byproducts of the mitochondrial aerobic metabolism electron transfer chain and are regarded as the primary source of intrinsic aging apart from genetic factors [22]. UVB irradiation leads to the overproduction of ROS, accelerating the endogenous aging process, so to confirm the occurrence of anti-photoaging effects, it is necessary to verify changes in ROS. Here, we showed that treatment with 100 \(\upmu\)g/mL JAUN effectively reduced the 35.46% increase in ROS induced by UVB irradiation (Figure 4). Nrf/ARE is a key player in the protection of the skin against photoaging, and the activation of Nrf/ARE can coordinate cellular defense mechanisms, including DNA repair, phase II detoxification, antioxidant response, and inflammatory signals [23]. Research by [PERSON] et al. showed that Nrf/ARE combats ROS by transcriptionally upregulating the expression of detoxifying enzymes and antioxidants, showing an anti-photoaging effect [24]. Therefore, we examined the levels of Nrf signaling pathways in UVB-irradiated HaCaT cells to identify Nrf-related mechanisms controlled by JAUN. Interestingly, the outcomes revealed that the degree of Nrf upregulation by 100 \(\upmu\)g/mL JAUN was 1.6 times higher than that of the commonly used anti-photoaging cosmetic additive ascorbic acid (Figure 9). Reactive oxygen species generation is known to trigger UVB-irradiated induction of MMPs. The zinc-dependent endopeptidase's MMPs represent a large class of regulatory proteases in the ECM that is secreted by dermal keratinocytes and are responsible for the degradation of connective tissue and ultimately skin aging [25]. Therefore, inhibiting the secretion of MMPs to alleviate ECM degradation is an effective way of countering photoaging. Recently, Carica papaya leaf has been shown to achieve anti-photoaging effects by downregulating the secretion of MMPs [26]. Our results presented in Figures 6 and 7 demonstrate that treatment with JAUN decreases the release of MMP-1 and MMP-3. UVB-induced ROS production activates the MAPK signaling pathway and then stimulates MMP gene transcription by activating the AP-1 transcriptional factor. Therefore, MAPK/AP-1 signaling activation has been thought to play a dominant role in inducing the release of MMP-1. Research by [PERSON] et al. revealed that the MAPK/AP-1 signaling pathway is capable of controlling MMP secretion [27]. The AP-1 complex protein is immediately phosphorylated upon activation of the MAPK signaling pathway, and the AP-1 transcription factor is primarily in charge of controlling MMP production and activity. From the results shown in Figure 8, JAUN also exhibited an excellent effect of downregulating MAPK/AP-1 phosphorylation, which may explain why JAUN downregulates MMPs. The TGF-\(\beta\)/Smad signaling pathway mainly promotes the expression of fibrotic proteins in the ECM, including collagen [28]. By the way, UV irradiation has been suggested to trigger ROS, cause the loss of TGF-\(\beta\) receptor expression, and increase Smad 7 expression, which in turn blocks the TGF-\(\beta\)/Smad signaling axis. Recently, a study has shown that UVB irradiation can downregulate TGF-\(\beta\) expression, but unsaponifiable matter from Perilla seed meal reverses this effect [29]. Therefore, we hypothesized that the mechanism of action of JAUN in combating photoaging also includes the TGF-\(\beta\)/Smad signaling pathway.