马勃孢子粉醇提物对小鼠全层皮肤缺损性创面的治疗作用考察

Effect of Alcohol Extract of Spore Powder of Lasiosphaera Calvatia on Wound Healing of Full-thickness Skin Defect in Mice

目的:比较马勃孢子粉和子实体的不同溶剂提取物对成纤维细胞和小鼠全层皮肤缺损性创面的药效差异,筛选马勃治疗创面的最佳用药部位。方法:将马勃孢子粉和子实体的水提物、醇提物对体外培养的小鼠皮肤成纤维细胞(MSF)进行干预,通过细胞增殖与活性检测(CCK-8)法比较细胞增殖、划痕实验比较细胞迁移、流式细胞技术探究细胞周期、酶联免疫吸附测定法(ELISA)测定Ⅰ型和Ⅲ型胶原蛋白生成量,同时建立小鼠背部全层皮肤缺损性创面模型,考察马勃孢子粉不同溶剂提取物的治疗效果,并采用紫外-可见分光光度法测定其不同溶剂提取物中指标成分的含量。结果:马勃水提物具有一定细胞增殖抑制作用,醇提物具有促进MSF增殖、迁移、分泌Ⅰ型和Ⅲ型胶原蛋白的作用,且孢子粉功效显著优于子实体。在质量浓度为10 mg·L^(-1)时,马勃孢子粉醇提物处理的细胞增殖率最高可达(159.22±15.95)%,并促进MSF从G_(0)/G_(1)期的静止状态进入S期和G_(2)/M期的增殖状态,增殖指数增大,增高成纤维细胞迁移率及Ⅰ型、Ⅲ型胶原蛋白的生成量。马勃孢子粉醇提物能有效促进创面愈合、促进创面组织上皮化完整、减少炎性细胞浸润、生成新生胶原纤维。成分含量测定结果显示,与子实体醇提物比较,孢子粉醇提物中多酚及黄酮类成分含量更高。结论:马勃孢子粉醇提物是马勃促进创面愈合的主要活性部位,具有较好的开发前景,其药效成分可能为多酚和黄酮类。

Objective:To compare the effect of different solvent extracts of spore powder and fruiting body of Lasiosphaera Calvatia on fibroblasts and wound healing of full-thickness skin defect,in order to screen the optimal medication part of Lasiosphaera Calvatia.Method:The effect of water extract and alcohol extract of spore powder and fruiting body on cell proliferation and cell migration of mouse skin fibroblasts(MSF)were examined in vitro.Cell proliferation and activity test(CCK-8)method was used for cell proliferation,scratch assay was used for cell migration,flow cytometry was conducted to explore cell cycle,enzyme-linked immunosorbent assay(ELISA)was used to determine the production of collagenⅠandⅢ.At the same time,a full-thickness skin defect wound model was established to investigate the therapeutic effect of different solvent extracts of spore powder.Ultraviolet-visible spectrophotometry was used to measure the contents of index components in different solvent extracts.Result:The water extract of spore powder and fruiting body had certain cytotoxicity,while the alcohol extract could promote proliferation,migration and production of collagenⅠandⅢof MSF,and the effect of spore powder was significantly higher than that of fruiting body.When the concentration was 10 mg·L^(-1),the cell proliferation rate of alcohol extract of spore powder was as high as(159.22±15.95)%,and could promote MSF from the G_(0)/G_(1)phase to S phase and G_(2)/M phase with an increased proliferation index.The alcohol extract also promoted the migration of fibroblasts,secreted collagenⅠandⅢ.On in vivo model,the alcohol extract of spore powder significantly accelerated wound healing on mice,effectively promoted the complete epithelialization of wound tissue,and generated new collagen fiber.The results of determination showed that the contents of polyphenols and flavonoids in the alcohol extract were higher than the alcohol extract of fruiting body.Conclusion:The alcohol extract of spore powder in Lasiosphaera Calvatia has active components in the treatment of wounds with good development prospect,and the medicinal components may be polyphenols and flavonoids.