H. Changes in the amount of melanocytes stimulating cytokines secreted by fibroblasts
IV. DISCUSSION
1 2
The results of this study show that FB was affected by PDT indirectly through cytokines 3
from KC. The PDT also has inhibitory effect of melanogenesis in the human MC. This 4
inhibitory effect of melanogenesis thought to be regulated indirectly by cytokines from KC 5
and FB as well as directly by PDT (Fig. 10).
6
In the previous report, the investigators demonstrated direct effect of PDT on FB which 7
is result of photorejuvenation effect such as increased collagen fiber. They suggested that the 8
PDT induced increase of MMP-3 and collagen type Iα through prolonged activation of ERK 9
(Jang et al., 2013). Increased level of MMP-3 may induce destruction of old damaged 10
collagen and then induce new collagen synthesis. In the present study, the indirect effect of 11
PDT on FB mediated by cytokines from KC was evaluated. After stimulation of FB with 12
KC-conditioned medium taken after PDT, The mRNA level of MMP-1 was decreased and 13
the mRNA level of collagen type Iα was increased in FB. However, mRNA level of MMP-3 14
and TGF-β were not changed significantly. According to these data, PDT significantly 15
affected FB indirectly through KC-conditioned medium. Among the FB stimulating 16
cytokines, IL-1α, IL-6, and TNF-α were elevated in the KC-conditioned medium after PDT.
17
Although it was not significant, TGF- β was also increased at 48 h in the KC-conditioned 18
medium after PDT. IL-1α was increased at 24 h and rapidly decreased at 48 h after PDT. In 19
photoaging process, increased secretion of inflammatory cytokines induces expression of 20
MMPs which results in degradation of collagen fibers in the dermis and makes wrinkles 21
(Shin et al., 2005). In the present results, although IL-1α, IL-6, and TNF-α were increased in 22
- 28 -
KC-conditioned medium after PDT, mRNA of MMP-1 was decreased and mRNA of 1
collagen Iα in FB was increased after stimulation with KC-conditioned medium taken after 2
PDT. Similar context with previous reports (Jang et al., 2013), there may be possible 3
explanation for these inconsistent results. At initial phase of PDT, secretion of inflammatory 4
cytokines may be increased to induce MMPs and degradation the old collagen fiber.
5
Breakage of old damaged collagen may allow induction of new collagen synthesis. At later 6
phase of PDT, inflammatory cytokines and MMPs may be decreased than initial phase which 7
allow accelerated synthesis of new collagen.
8
In the result of this study, although mRNA level of collagen Iα in FB was significantly 9
increased at 12 h after stimulation with KC-conditioned medium taken after PDT, it became 10
almost equal at 24 h in PDT group and control group (Fig 3D). The similar finding was also 11
detected in the result of TGF- β (Fig 3C). Although it was not significant, the mRNA level of 12
TGF-β in PDT group was rather become lower than the control group. It may be due to the 13
reduced effect of PDT after 12 h. In previous result, the authors also presented that rapid and 14
induced new collagen synthesis may be mediated not only by direct effect of PDT on FB but 19
also by indirect effect of PDT on FB through cytokines from KC.
20
As we mentioned above, PDT has been known to improve photoaged skin by induction 21
of new collagen synthesis in human FB (Park et al., 2010; Jang et al., 2013). However, 22
- 29 -
inhibition of melanogenesis of human MC by PDT has rarely been reported. According to 1
this study, it was clearly demonstrated that PDT inhibited melanogenesis not only by direct 2
effect to MC but also by indirect effect of PDT through cytokines from KC and FB.
3
In the present study, melanogenesis was reduced in the MC after PDT without 4
influencing cell viability. According to the figure 5A, the melanin content was increased at 5
48h compared with 24h in both control and PDT group. It may be due to stabilization of the 6
MC at 48h than 24h. It may also caused by the increase of culture time and the consequent 7
increase in cell number.
8
Melanogenesis was regulated by various cytokines through interactions between KC-9
MC and FB-MC. SCF, ET-1 and GM-CSF are representative cytokines secreted by KC and 10
stimulated MC (Imokawa, 2004). According to this study result, among above cytokines, 11
SCF was reduced in the KC-conditioned medium after PDT. Therefore, SCF is thought to be 12
a major cytokine that induces inhibitory effect of melanogenesis in the MC after PDT. There 13
are several signaling pathways regulating melanogenesis of human MC. SCF/c-kit pathway 14
is one of the major melanogenesis pathway in human MC. SCF lead to activation of 15
mitogen-activated protein kinase (MAPK) cascade by binding to its corresponding receptor 16
c-kit. MAPK activates microphthalmia-associated tramscription factor (MITF) and result in 17
transcription of the melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1 18
(TRP-1) and TRP-2 (Imokawa et al., 2000). Therefore, decreased secretion of SCF by KC 19
after PDT may lead to inhibition of melanogenesis through this SCF/c-kit pathway in MC.
20
Future studies should be focused on the downstream cascade of SCF/c-kit pathway in MC 21
after stimulating with KC-conditioned medium after PDT.
22
- 30 -
In the present result, SCF was detected at lower level in KC-conditioned medium 1
compared to the previous report (Fig 7A) (Okazaki et al., 2005). However, in seven 2
independent experiments, the data showed similar result that the SCF level was significantly 3
decreased after stimulation with KC-conditioned medium taken after PDT. If the experiment 4
meaningful experiment to investigate restoring effect of PDT in photo-damaged cell.
8
induced inhibitory effect of melanogenesis in the MC after PDT. HGF have been known as a 12
mitogens for human MC in vitro (Imokawa, 2004). In the previous study, stimulation of MC 13
with 10 nM of HGF induced proliferation of cultured human MC (Matsumoto et al., 1991).
14
However, in the present study, there was no significant proliferation of MC after PDT 15
compared to the control. In dermatofibroma, a benign dermal tumor characterized by 16
proliferation of FB-like cells, hyperpigmentation in the overlying epidermis is supposed to 17
be mediated by high expression of HGF of dermal FB (Shishido et al., 2001). The high 18
expression of HGF in the dermis of dermatofibroma was detected at both the gene and the 19
protein levels (Imokawa, 2004). To date, precise mechanism of HGF in enhancement of 20
melanogenesis in human MC is not fully demonstrated. However, much evidence support a 21
distinct role of HGF in stimulating epidermal MC. Taken together with these data, decreased 22
- 31 -
secretion of HGF from FB after PDT thought to induce inhibition of melanogenesis in the 1
MC in our study.
2
According to the results showing reduced melanin content and dopa oxidase activity in 3
MC after stimulation with KC-conditioned medium taken after PDT, the melanin content and 4
dopa oxidase activity also reduced in MC stimulated with KC-conditioned medium without 5
PDT compared to the MC stimulated with KC medium itself (Fig 6). In addition, the melanin 6
content and dopa oxidase activity also reduced in MC stimulated with FB-conditioned 7
medium without PDT compared to the MC stimulated with 1:1 mixture of FB medium and 8
MC medium (Fig 8). It may be caused by growth factors in supplements of KC and FB 9
medium. The various kinds of growth factors, such as human epidermal growth factor, 10
bovine insulin, hydrocortisone, bovine pituitary extract, bovine transferrin and FBS, exist in 11
the supplement of KC and FB medium. These growth factors in media, which is able to 12
stimulate MC to synthesis of melanin, may be used up during culture of KC or FB. Therefore, 13
decreased level of growth factors in KC-conditioned medium and FB-conditioned medium 14
may result in reduced melanin content and dopa oxidase activity in MC stimulated with KC-15
conditioned medium without PDT or FB-conditioned medium without PDT.
16
Contrast to our study, Karrer et al. suggested that KC stimulated by PDT produced 17
proinflammatory cytokines such as IL-1a and TNF-a, which influenced FB to produce 18
increased amounts of MMP-1 and MMP-3. They suggested that this paracrine induction of 19
collagen-degrading MMPs results in the reduction of collagen volume after PDT (Karrer et 20
al., 2004). However, they used higher light energy in PDT procedure compare to this study;
21
100 µM/L of ALA and 24 J /cm2 of red light. We used “low-level PDT” (0.1 mM /L of ALA 22
- 32 -
and 3 J /cm2 of red light) to induce activation of FB without cytotoxic effects. Therefore, 1
“low-level PDT” used in this study, can be an explanation about different result compare 2
with previous studies (Karrer et al., 2003, 2004).
3
In the similar context, the different results of other studies examined PDT effect in 4
melanogenesis can be explained. There are a few studies that demonstrated PDT induced 5
hyperpigmentation in vivo (Monfrecola et al., 2002; Juzeniene et al., 2008). To begin with, 6
in vivo study might differ from in vitro study. Also, “low-level PDT” may induce different 7
result compare to previous studies which used conventional parameter of PDT. In future 8
study, if we apply “low-level PDT” to the human skin in vivo, we may be able to observe 9
inhibition of melanogenesis.
10 11
- 33 - 1
Fig. 10. A schematic diagram of low level photodynamic therapy induced 2
photorejuvenation. PDT induced new collagen synthesis may be mediated by indirect effect 3
of PDT on FB through cytokine from KC such as IL-1α, IL-6, and TNF-α as well as directly 4
by PDT. PDT induced inhibitory effect of melanogenesis thought to be regulated indirectly 5
by KC and FB as well as directly by PDT. Decreased secretion of SCF from KC and HGF 6
from FB may have crucial role which induce inhibition of melanogenesis of MC after PDT.
7
Black dotted arrow=direct effect of PDT, black solid arrow=indirect effect of PDT.
8
- 34 -
V. CONCLUSION
1 2
Based on above results, PDT induced new collagen synthesis may be mediated by 3
indirect effect of PDT on FB through cytokine from KC such as IL-1α, IL-6, and TNF-α. In 4
addition, PDT has inhibitory effect of melanogenesis in the human MC. This inhibitory 5
effect of melanogenesis thought to be regulated indirectly by KC and FB as well as directly 6
by PDT. Decreased secretion of SCF from KC and HGF from FB may have crucial role 7
which induce inhibition of melanogenesis of MC after PDT.
8 9 10
- 35 -
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4 5
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로 생각된다. 본 저자는 또한 광역동치료가 멜라닌세포에서 멜라닌 형성을 억제 1
한다고 제안한다. 이 멜라닌 형성 억제 효과는 광역동치료가 멜라닌세포에 미치 2
는 직접적인 영향뿐 아니라 각질세포와 섬유아세포에 의한 광역동치료의 멜라닌 3
세포에 대한 간접적인 영향에 의해서 발생하는 것으로 생각된다. 각질세포에서 4
분비되는 SCF와 섬유아세포에서 분비되는 HGF가 광역동치료 후 멜라닌세포의 5
멜라닌 형성 억제를 유도하는 주요 싸이토카인으로 보인다.
6 7
핵심어: 광역동 치료, 광회춘, 각질세포, 섬유아세포, 멜라닌세포 8
9