Experimental Study Of Hematoporphyrin Monomethyl Ether-Mediated Photodynamic Therapy For Hypertrophic Scar

Objective:Photodynamic therapy (PDT) is a promising treatment for various kinds of dermatosis. Hypertrophic scar (HS) is a pathological process characterized by fibroblastic hyperproliferation. In this study, we investigated the cellular response to PDT which induced by hematoporphyrin monomerthyl ether (HMME) in human fibroblasts from hypertrophic scar (HSF), and explored the cell signal mechanisms initially. Furthermore, we observed the biological effects of HMME-PDT on hypertrophic scarring in a rabbit ear model.Methods:1. The absorption characteristics of HSF on HMME and the inhibitory effect of HMME-PDT: Fibroblasts were cultured from nontreated burn hypertrophic scars, and the 4-6 passage cells were used in the experiments. HSF was incubated with HMME at different concentrations (0～40μg/ml) and different incubation time (0-120min). The absorption characteristics of HSF on HMME were detected by flow cytometry (FCM). Cell viability after HMME-PDT was detected by MTT assay. The AgNORs expression was determined with the standard silver-staining method and the cell cycle was calculated by FCM.2. TGF-β₁/Smad signal mechanisms in HSF mediated by HMME-PDT: The expression of TGF-β₁ in supernatant of HSF was detected by enzyme linked immunosorbent assay (ELISA). Fluorescence intensity of Smad3 was observed with immunofluorescence staining. The expression of TGF-β₁ Smad3, Phospho-Smad3, and Smad7 was analyzed by Western blot.3. The apoptotic effects of HSF induced by HMME-PDT: The morphological changes in HSF were observed with Hoechst 33258 staining. The rate of apoptotic or necrotic cells was respectively detected by FCM through double staining of Annexin V-FITC and popodium iodide (PI). Caspase-3 activity assay and immunofluorescence staining method were applied to the investigate Caspase-3 expression in HSF by FCM and fluorescence microscope.4. Effect of HMME-PDT on Hypertrophic Scarring in Rabbit Ear Model: After the acute model of dermal hypertrophic scar in the rabbit ear was established, scar wounds randomly received HMME-PDT with different treatment parameters. Hypertrophic index (HI) was measured by slide caliper. Scar histomorphology and thickness were observed with haematoxylin-eosin staining. The microvessel density (MVD) was calculated under microscope. HSF was observed under transmission electron microscope (TEM).Results:1. The absorption of HMME by HSF was increased with increased concentration and incubation time. HMME-PDT inhibited the proliferation of HSF significantly. The growth curve was declined following the increased HMME concentration and light dose. HMME-PDT decreased the expression of AgNORs and led to cell cycle arrest in G₀/G₁ phase.2. HMME-PDT down-regulated the protein level of TGF-β₁ both in supernatant of HSF and in HSF. It prevented the activation of Smad3 while increased the expression of Smad7 protein.3. Marked morphological features of cell apoptosis were viewed under the fluorescent microscope through Hoechst 33258 staining. The analysis of FCM indicated that the apoptotic rate was significantly increased after HMME-PDT, accompanied by the presence of active-caspase-3. The apoptotic rate was still high after used Z-DEVD-FMK which could inactivate caspase-3.4. Compared with the control group, HMME-PDT in the experimental group (photosensitizer dose 10mg/kg, power density 20mw/cm² , energy fluence 5J/cm² ) reduced scar formation, and the HI. Mitochondria, rough ER and Golgi of HSF were abundant in the control group, while decreased and destroyed by PDT. Some fibroblasts underwent marked apoptosis after HMME-PDT.Conclusions:1. Incubation concentration and time are two important factors for the absorption of HMME by HSF. HMME-PDT inhibits the proliferation of HSF which depending on HMME concentration and laser energy density, and also blocks the cell cycle.2. HMME-PDT induces HSF apoptosis, and stimulates caspase-3 activation, but caspase-3 is dispensable for apoptosis in this process.3. HMME-PDT can inhibit the TGF-β₁/Smad signal transduction pathway and prevent HSF's proliferation and collagen's synthesis.4. HMME-PDT can inhibit hypertrophic scarring in rabbit ear. The biological effect is determined by photosensitizer, interval injection of photosensitizer and irradiation, power density and energy fluence. HMME-PDT may have good potent effects on the formation of hypertrophic scar.