马勃子实体和发酵物促进创面组织修复的药效物质基础及作用机理研究

Lasiosphaera seu Calvatia (LsC) posesses the effect of cleaning lung, anastalsis and promoting granulation and used to acure sorts of ulcer such as bedsores, burns, burns, diabetic foot ulcers in clinical, but the wild resources are scarce. The group screened out a kind of high-yield strains which was Scleroderma polyrhizum Pers. applying liquid fermentation. Results of antiblastic experiments in vitro as well as animal model of antiphlogistic, antalgic, hemostatic and healing of wounded tissue showed that the efficacy of liquid leavening was better than that of the wild fruiting bodies. In this program, to pick out the bioactive parts and compounds of promoting restoring wounded tissue and clarify the pharmacodynamic material basis of it, different parts of Scleroderma polyrhizum Pers．and its liquid leavening were separated by applying chemical method, and monomeric compounds were seperated and purified by sereval sorts of chromatographic technique. In order to discuss the mechanism of promoting restoring wounded tissue, immunohistochemistry and molecular biology were utilized to study the effect of the bioactive parts and compounds on the microcirculation of wounded surface, expression of Smad3,TGF-β1, collogenⅠand collogen Ⅲ, and the content of EGFR as well as FN. This paper provided scientific foundation for liquid fermentation instead of wild resources of LsC.

中药马勃具有清肺、利咽、止血、生肌之功效。临床治疗外伤出血、各种溃疡（褥疮、烧伤、烫伤、糖尿病足溃疡）有奇效，但野生资源匮乏。课题组前期筛选出一株能够利用液体发酵生产的高产菌种- - 星裂硬皮马勃 Scleroderma polyrhizum Pers．菌。通过体外抑菌试验和抗炎、镇痛、止血、创面愈合动物试验证明，液体发酵物药效好于野生子实体。本项目以星裂硬皮马勃野生子实体和液体发酵物为研究对象，通过化学手段分离不同部位，利用各种柱色谱技术分离纯化单体化合物，筛选促进创面组织修复活性部位和活性化合物，阐明其药效物质基础。利用免疫组织化学和分子生物学方法与技术，研究活性部位及单体化合物对创面微循环（毛细血管数、血红蛋白及羟脯氨酸、VEGF含量）、Smad3,TGF-β1表达、Ⅰ型和Ⅲ型胶原表达、EGFR及FN含量的影响，探讨其促进创面组织修复的作用机理。为马勃液体发酵生产代替野生资源提供科学依据

中药马勃具有清肺、利咽、止血、生肌之功效。课题组共采集马勃菌13种，通过菌种的分离纯化获得纯菌株种。对13个菌株进行发酵工艺研究，优选出星裂硬皮马勃菌株，其发酵工艺简单稳定，生物量高。对星裂硬皮马勃发酵物做了以下各方面的研究。. 抑菌实验：发酵液乙酸乙酯萃取物对大肠杆菌、绿脓杆菌、金黄色葡萄球菌抑菌作用最强。抗炎、镇痛、创伤组织修复实验：发酵液好于子实体和发酵菌丝。小鼠RAW 264.7巨噬细胞炎症反应：显著降低巨噬细胞产生的TNF-α, IL-6及IL-1β水平，机制是通过抑制MAPK通路, NF-kB 和 COX-2的激活而减弱炎症反应。急性肺损伤小鼠模型抗炎作用：显著降低肺组织中中性粒细胞，降低支气管肺泡灌洗液中TNF-α，IL-1和IL-6水平，机制是通过影响急性肺损伤小鼠体内NF-кB的活性发挥抗炎作用。乙醇型胃溃疡的保护：通过提高胃溃疡大鼠血清SOD活性，降低MDA含量，升高胃液NO含量对胃溃疡发挥保护作用。止血作用：不同部位均有缩短大鼠TT、PT、APTT时间，能较好的提高凝血过程中纤维蛋白原的利用度，通过内外源凝血酶的共同途径达到止血效果。促进伤口愈合作用：能提高大鼠皮肤创伤愈合时间，提高肉芽组织中EGFR、VEGF-A、Fn、羟脯氨酸含量，说明马勃不同部位均可以改善创面微循环促进创面愈合，作用机制为促进血管新生以及羟脯氨酸的生成从而达到促进伤口愈合的作用。分离纯化得到5个单体化合物，甘露醇，麦角甾醇，麦角甾酮、麦角甾醇葡萄糖苷，吲哚类生物碱。麦角甾醇对急性肺损伤炎症反应的影响：使脂多糖诱导的炎症细胞浸润减弱，肺泡灌洗液中炎症细胞因子（TNF-α，IL-1β，IL-6）降低，提高肺功能。抑制NF-κB、COX-2和 iNOS通路。麦角甾醇对糖尿病小鼠肾损伤的影响：可回调链脲霉素刺激导致的小鼠血糖上调和血清胰岛素含量下降；可控制尿酸和肌酐水平急剧升高；降低血清甘油三酯和总胆固醇在糖尿病小鼠异常高表达。麦角甾醇可下调糖尿病小鼠磷酸化的PI3K和 NF-κB。说明通过PI3K / Akt/ NF-κB信号通路缓解慢性炎症，从而降低血糖浓度。