| Hypertrophic scars (HS), resulting from pathological processes of cutaneous wound healing, are characterized by continuous proliferation of dermal tissue with a fibroblastic hyperproliferation and excessive deposition of fibroblast-derived extracellular matrix proteins, especially collagen. HS associated with high incidence often destroy patients'cosmetics or result in dysfunction of organs, who suffer from psychological and somatic pain, and remain a major challenge for modern medicine presently. Zhangshi-Bahen-Zhiyang-Ruanhua cream (ZSC) is composed of five Chinese herbs including Melaphis chinensis, Clematis chinensis, Lycopus lucidus, Paeonia suffruticosa and Ligusticum chuanxiong. Since the 1960s, It has been used in the clinical treatment of HS and showed significant effects on clinical application. However, the active ingredient of ZSC and its best compatibility are still not confirmed.Hypertrophic fibroblasts (FB) were cultured from HS tissue. Melaphis chinensis, Clematis chinensis, Lycopus lucidus, Paeonia suffruticosa and Ligusticum chuanxiong were mixed according to the ratio of 100:8:20:10:50 (w/w) and refluxed by 90% ethanol, which was removed by evaporation under reduced pressure, then extracted with petroleum ether (PE) to get PE sub-fraction (PEF). The remaining solution was fractioned using macroporous adsorption resin (D101) column which was eluted with distilled water, 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol in a sequence to obtain five sub-fractions, i.e., F00, F30, F50, F70 and F90. PEF, F70 and F90 were found to be more active than others in the inhibition of the proliferlation of FB. These three fractions were analyzed to comprise volatile oil, gallic acid, oleanolic acid and paeonol. MTT cell viability assay and uniform design were performed to get their best compatibility. The complex prescription was composed of essential oils of chuanxiong and chinese gall, gallic acid and oleanolic acid in accordance with the concentration ratio of 2:2:1:1. In order to observe the effects of complex prescription on apoptotsis of FB, cultured FB were exposed to various concentrations (0, 25, 50, 100 and 200μg/mL) of the complex prescription for different time (12, 24 and 48 h).In this study, we further investigated the mechanism of FB apoptosis by Flow Cytometer (FCM) assay. The apoptotsis rate was detected by FCM at 24h with annexin V-FITC/propidium iodide (PI) double staining. DCFH-DA was used for ROS detection and the level of ROS was measured after 12h exposure. On the other hand, rhodamine 123 was used in order to observe changes of mitochondrial membrane potential (?Ψm) and the caspase-3 activity was determined by using the caspase-3 colorimetric assay kit after 24h of treatment. In conclusion, the complex prescription can promote apoptosis and suppress proliferation of FB via ROS generation, capsase-3 activation and disruption of ?Ψm. Mitochondrial membrane potential depolarization might play a major role in apoptosis.HS in the rabbit ear model were established to verify the efficacy of the complex prescription. In this study, The rabbit ears were randomly divided into six groups viz. normal group, model group, low-dose (5%, v/v) group, middle-dose (10%, v/v) group, high-dose (20%, v/v) group, and positive control group. The scars were administered with drugs once a day on the fifth day after wounding. After 14, 28 days of treatment, the thickness was measured by vernier digital caliper and the expressions of collagenⅠ, collagenⅢ, TGF-β1 and MMP-1 were investigated by enzyme-linked immunoadsorption assay (ELISA). Hypertrophic scar tissue was histologically evaluated with hematoxylin-eosin (HE) staining. The results suggest that the scar cream could significantly reduce hypertrophic scar formation through the inhibition of collagenⅠ, collagenⅢ, TGF-β1 expression and the improvment of the MMP-1 activity. |
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