观察卡奴卡叶提取物对人真皮成纤维细胞(HDFs )活性、增殖及迁移能力、胶原合成功能的影响及对大鼠创面愈合的影响。

使用不同浓度卡奴卡叶提取物刺激HDFs后，经CCK-8法及Calcein/PI染色筛选药物最优浓度。采用划痕实验检测细胞迁移能力，细胞免疫荧光及蛋白免疫印迹法定性和定量检测功能性目的蛋白(Ki67蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、α-平滑肌肌动蛋白)，观察卡奴卡叶提取物对细胞增殖能力以及胶原合成功能的影响。建立大鼠背部皮肤缺损自身对照模型，分别使用卡奴卡叶提取物及蒸馏水处理创面，比较创面愈合时间。通过HE染色、Masson染色及组织免疫荧光染色观察创面愈合后组织形态及胶原沉积情况。

CCK-8实验及Calcein/PI染色结果显示，1%浓度的卡奴卡叶提取物可显著提高HDFs细胞活性，并明显增加细胞Ki67表达量[(0.71±0.16) vs (0.18±0.06)，t＝7.156，P<0.001]，促进HDFs增殖。划痕实验证实，卡奴卡叶提取物可增强HDFs迁移能力。细胞免疫荧光、蛋白免疫印迹法结果显示，卡奴卡叶提取物可增加HDFs的Ⅰ型胶原蛋白、Ⅲ型胶原蛋白及α-平滑肌肌动蛋白表达量[(43.22±7.58)vs(16.71±1.68)，t＝7.635, P<0.001；(46.62±5.90) vs (27.44±1.60)，t＝7.016，P<0.01；(1.32±0.14) vs (0.46±0.06)，t＝9.947，P<0.01]，提高细胞的胶原合成能力。动物实验结果显示，卡奴卡叶提取物处理侧创面各时间点愈合率均高于对照侧，且创面愈合时间明显短于对照侧[(13.6±1.5)d vs (16.7±1.6)d，t＝6.270，P<0.001]。HE染色和Masson染色结果显示，卡奴卡叶提取物处理侧创面愈合后皮肤厚度及组织形态更加接近正常皮肤，组织内Ⅰ型胶原蛋白含量明显高于对照侧[(84.36±7.82) vs (67.72±5.24)，t＝5.595，P<0.001]。

卡奴卡叶提取物可促进HDFs的增殖、迁移以及胶原合成，且可能通过增强真皮成纤维细胞的生物学功能进而促进大鼠创面修复。

To investigate the effects of kunzea ericoides leaf extract on human dermal fibroblasts (HDFs) concerning the migration, proliferation, and collagen synthesis ability and to observe the impact of kunzea ericoides leaf extract on wound healing in rats.

After stimulation of kunzea ericoides leaf extract in different concentrations, assays of CCK-8 and Calcein/PI staining were applied to screen the optimal concentration of kunzea ericoides leaf extract on HDFs. The expression of Ki67 was detected to observe the proliferation ability of HDFs. Wound-healing assay was applied to detect the migration ability of HDFs after incubation with kunzea ericoides leaf extract. The expressions of typeⅠcollagen, type Ⅲ collagen, and α-smooth muscle actin were detected using the immunofluorescence and western blot assay, representing the collagen synthesis ability change of HDFs. The models of rat skin defect were established and treated with or without kunzea ericoides leaf extract, and the healing time was compared. Furthermore, the wound area tissue's morphology and collagen composition were observed using HE and Masson staining assays after complete healing.

The optimal concentration of kunzea ericoides leaf extract for HDFs was determined as 1% based on CCK-8 assay and Calcein/PI staining. In addition, the Ki67 expression was increased significantly [(0.71±0.16) vs (0.18±0.06), t=7.156, P<0.001], kunzea ericoides leaf extract enhanced the cell proliferation. The wound-healing assay confirmed that kunzea ericoides leaf extract improved the migration ability of HDFs. The results of the immunofluorescence and western blot assay showed that kunzea ericoides leaf extract increased the expressions of typeⅠcollagen, type Ⅲ collagen, and α-smooth muscle actin in HDFs [(43.22±7.58) vs (16.71±1.68), t=7.635, P<0.001; (46.62±5.90) vs (27.44±1.60), t=7.016, P<0.01; (1.32±0.14) vs (0.46±0.06), t=9.947, P<0.01], which meant kunzea ericoides leaf extract improved the collagen synthesis ability of HDFs. The healing time in the kunzea ericoides leaf extract group was (13.6±1.5)d, significantly shorter than (16.7±1.6)d of the control group (t=6.270, P<0.001). The results of HE and Masson staining suggested that after applying kunzea ericoides leaf extract, the skin tissue from the wound area resembled normal skin morphology and demonstrated more collagen deposition [(84.36±7.82) vs (67.72±5.24), t=5.595, P<0.001].

Kunzea ericoides leaf extract can promote the proliferation, migration, and collagen synthesis ability of HDFs, and might promote wound healing in rats by enhancing the function of dermal fibroblasts.