We observed that ALA-PDT had a direct effect on sebaceous gland in acne vulgaris in clinical. To verify this, we detected if ALA accumulated in targeting cells and tissues. As shown in Fig. 1A, red fluorescence of PpIX in sebocytes induced by ALA was observed by fluorescence microscope. As the fluorescence intensity of PpIX was related to the effect of ALA-PDT, we investigated the optimum parameters of ALA. Our results showed that fluorescence intensity of PpIX was strongest in sebocytes when incubated with 0.5 mM ALA for 6 h (Fig. 1B, 1C). But if incubated for 6 h, the proliferation of sebocytes would be decreased significantly. No significant differences were detected when incubated for 4 h (Fig. 1D). So the optimum parameter of ALA was 0.5 mM for 4 h on sebocytes.
To further confirm that ALA was accumulated in sebaceous gland directly, 8% ALA was applied on the right sebaceous gland on back of golden hamster for 3 h. As shown in Fig. 1E, red fluorescence of PpIX was observed around both right and left sebaceous gland. Freezing section of the tissue also showed that PpIX was accumulated in follicle sebaceous gland units directly (Fig. 1F). Taken-together, ALA could specifically accumulate in sebaceous gland.
2. ALA-PDT suppresses proliferation and lipid secretion of sebocytes
To substantiate the inhibitory effect we evaluated the proliferation, apoptosis and lipid secretion of sebocytes after ALA-PDT. After 0.5 mM ALA-PDT (10 J/cm2), ROS accumulates in sebocytes (Fig. 2A). According to the previous study, the accumulation of ROS was related to the effect of ALA-PDT.
Next we used CCK-8 assay to evaluate the cell viability. As the energy intensity of 0.5 mM ALA-PDT increased from 0 to 40 J/cm2, cell viability of sebocytes was reduced from 100 ± 8.99% to 20.18 ± 8.09% and IC50 was at 10 J/cm2 (Fig. 2B). The apoptotic population in sebocytes was evaluated by flow cytometry. As expected, 0.5 mM ALA-PDT (10 J/cm2) significantly decreased the relative number of live sebocytes and concurrently increased the relative number of apoptotic sebocytes (Fig. 2C, 2D). Fluorescence microscope was used to detect the secretion of lipids in sebocytes treated by ALA, Red light and ALA-PDT respectively. (Fig. 2E). Lipid in sebocytes was found to be significantly reduced 24 h after ALA-PDT. The above results indicated that ALA-PDT inhibited the ability of lipid secretion and proliferation of sebocytes.
3. ALA-PDT induces up-regulation of CXCL8 in sebocytes and sebaceous gland.
Clinically, intense inflammatory reaction occurs in the early phase after ALA-PDT. Therefore, we tested whether inflammatory cytokines changed after ALA-PDT. Sebocytes treated by ALA, Red light and ALA-PDT were collected 24 h later. The results showed that the expression of CXCL8, IL-6, TNF-α and IL-1β was significantly increased after ALA-PDT (Fig. 3A). In view of the effect of the recruitment of CXCL8 on immune cells, our research focused on the regulatory effect of ALA-PDT on CXCL8.
Further experiment substantiated that the expression of CXCL8 was elevated by ALA-PDT in sebaceous gland of golden hamster. After treated by ALA-PDT, tissues were taken from treated sites at 1 h, 3 h, 6 h, 12 h, 24 h after ALA-PDT and untreated tissues were used for comparisons. We found that positive staining for CXCL8 started to increase at 3 h after ALA-PDT around sebaceous gland. These findings illustrated ALA-PDT upregulated the expression of CXCL8 on sebocytes and sebaceous gland.
4. CXCL8 amplifies intense inflammatory response after ALA-PDT in sebaceous gland.
CXCL8 is a kind of chemokines, recruiting inflammatory cells. To further investigate the role of CXCL8, we detected the expression of CD3, CD11b, CD19 and CD68 in sebaceous gland after ALA-PDT by immunohistochemistry. After treated by ALA-PDT, tissues were taken from treated sites at 1 h, 3 h, 6 h, 12 h, 24 h after ALA-PDT and untreated tissues were used for comparisons. In HE staining, inflammatory cells started to collect towards sebaceous gland at 1 h after ALA-PDT. The results showed that positive staining for CD3(+) T cells and CD11b (+) neutrophils started to increase gradually at 1 h after ALA-PDT around sebaceous gland. Expression of CD19(+) B cells and CD68(+) macrophages slightly increased from 3 h after ALA-PDT (Fig. 4). Collectively, the results implied that CXCL8 could recruited several inflammatory cytokines, which were mainly T cells with a few neutrophils, B cells and macrophages.
5. ALA-PDT regulates the expression of CXCL8 via p38 signaling pathway.
The activation of CXCL8 gene promoter, trans-activation by JNK pathways and stabilization by p38 pathway are involved in the up-regulation of CXCL8 expression. To further study the mechanism of ALA-PDT’s regulation of CXCL8 in sebocytes, we analyzed the expression of p38, Erk1/2, and JNK pathways by western blot. Sebocytes were collected for western blotting after ALA-PDT immediately. The results showed that ALA-PDT upregulated p-p38 and decreased expression of p-Erk1/2 and p-JNK significantly (Fig. 5A, 5B).
Next, to verify whether suppression of p38 pathway could block the effect of ALA-PDT in sebocytes, p38 inhibitor SB203580 was employed. Cells were collected for further assays at 24 h post-treated with ALA-PDT and SB203580 (10 µM). The results showed that pretreated with SB203580 attenuated the upregulated CXCL8 mRNA expression by ALA-PDT in sebocytes (Fig. 5C). The results indicated that ALA-PDT induce CXCL8 production through p38 pathway.