摘要
目的:探讨乌拉草提取物黄酮类成分对大鼠皮肤烫伤后金黄色葡萄球菌(S.aureus)感染的抑菌作用,为新型抗感染中药的研发提供新思路。方法:采用水醇提取法提取乌拉草有效成分并利用紫外分光光度法测定其黄酮含量,采用牛津杯法抑菌试验检测乌拉草有效成分对细菌的抑菌活性,试管稀释法测定其最低抑菌浓度(MIC)。24只SD大鼠采用自制烫伤仪构建Ⅱ度烫伤模型,随机分为生理盐水对照组、青霉素钠(NaBP)对照组和乌拉草提取物CM-70治疗组,每组8只。3组大鼠创面皮内多点注射接种20μL(1.0×10^(8)CFU·mL^(-1))S.aureus,次日3组大鼠分别涂抹无菌生理盐水(0.5 g无菌脱脂棉吸附生理盐水0.5 mL·cm^(-2))、NaBP(0.5 g无菌脱脂棉吸附NaBP,5000 U·kg^(-1)体质量)和10 g·L^(-1)乌拉草CM-70提取物稀释液(0.5 g无菌脱脂棉吸附0.5 mL·cm^(-2)乌拉草CM-70提取液)。观察各组大鼠创面感染情况,采用创面痂下细菌定量实验检测各组大鼠烫伤后第1、2、3、4、5、6和7天创面细菌数(CFU·g^(-1)),酶联免疫吸附试验(ELISA)法检测各组大鼠烫伤前和烫伤后第1、3、7及14天血清白细胞介素1β(IL-1β)、白细胞介素10(IL-10)和肿瘤坏死因子α(TNF-α)水平。结果:体外抑菌活性检测,不同稀释浓度乌拉草CM-70提取物稀释液针对金黄色葡萄球菌形成不同大小抑菌圈,针对大肠埃希菌和白色念珠菌未形成抑菌圈。MIC检测,乌拉草有效成分对S.aureus的MIC值为2.5 g·L^(-1)。各组大鼠烫伤创面感染和愈合情况观察,与生理盐水对照组比较,感染后第3天,乌拉草CM-70提取物治疗组大鼠伤口浆液渗出较少;感染后第7天,伤口浆液渗出减少;红肿减轻,伤口面积缩小;感染后第14天,伤口创面已完全干燥,无浆液渗出,表面形成痂皮,伤口愈合良好,与NaBP对照组相近。烫伤创面痂下细菌定量,与生理盐水对照组比较,同一时间点乌拉草CM-70提取物治疗组大鼠创面痂下细菌数明显减少(P<0.01)。ELISA法检测,与生理盐水对照组比较,乌拉草CM-70提取物治疗组大鼠血清中IL-10水平在烫伤后第1和3天明显升高(P<0.05),在第7和14天明显降低(P<0.05);乌拉草CM-70提取物治疗组大鼠血清中IL-1β和TNF-α水平在烫伤后第1、3、7和14天明显降低(P<0.05)。结论:乌拉草有效成分可明显抑制大鼠皮肤烫伤后S.aureus感染,通过调控血清IL-1β、IL-10和TNF-α水平发挥抗炎和抑菌作用。
Objective:To discuss the becteriostatic effect of flavonoids in the Carex meyeriana Kunth extract on infection of Staphylococcus aureus(S.aureus)of the rats after skin scalding,and to provide the new ideas,for the research on new-style anti-infection traditional Chinese medicine.Methods:The wateralcohol extraction method was used to extract the active ingredients of Carex meyeriana Kunth and the content of flavonoids was determined by UV spectrophotometry,Oxford cup bacteriostatic test was used to detect the bacteriostatic activity of active ingredients of the Carex meyeriana Kunth against bacteria,and its minimal inhibitory concentration(MIC)was determined by tube dilution method.A total of 24 SD rats were used to establishⅡdegree scalding models and randomly divided into normal saline control group,penicillin sodium(NaBP)control group and Carex meyeriana Kunth extract CM-70 treatment group,and there were 8 rats in each group.The rats in three groups were inoculated with 20μL(1.0×10^(8) CFU·mL^(-1))S.aureus by multi-point injection in the wound skin;on the next day,the rats in three groups were smeared with sterile normal saline(0.5 g sterile absorbent cotton adsorbed normal saline 0.5 mL·cm^(-2)),NaBP(0.5 g sterile absorbent cotton adsorbed NaBP,5000 U·kg^(-1) body mass)and 10 g·L^(-1) Carex meyeriana kunth CM-70 extract diluent(0.5 g sterile absorbent cotton adsorbed 0.5 mL·cm^(-2) Carex meyeriana Kunth extract CM-70 extract diluent).The wound infection of the rats in various groups were observed;the bacteria number(CFU·g^(-1))under scalding wound scab of the rats on the 1st,2nd,3rd,4th,5th,6th,and 7th days after scalding was measured by bacteria quantitative test;the serum levels of interleukin-1β(IL-1β),interleukin-10(IL-10),and tumor necrosis factor-α(TNF-α)of the rats in various groups before scalding and on the 1st,3rd,7th,and 14th days after scalding were detected by ELISA method.Results:The in vitro bacteriostatic activity test results showed that different dilution concentrations of Carex meyeriana Kunth extract CM-70 formed different sizes of inhibition zones against S.aureus,and did not formed the inhibition zones against Escherichia and Candida albicans.The MIC detection results showed that the MIC value of active ingredients of the Carex mgyeriana Kunth extract to S.aureus was 2.5 g·L^(-1).The observation results of infection and healing of scalding wounds of the rats in various groups showed that compared with normal saline control group,on the 3rd day after scalding,the exudation of wound serous of the rats in Carex meyeriana Kunth extract CM-70 treatment group was less;on the 7th day after scalding,the wound serous exudation,redness and swelling,and the wound area of the rats were decreased;on the 14th day after scalding,the wounds of the rats were completely dry without serous exudation,the scabs were formed on the surface,and the wounds healed well,which was similar to that in NaBP control group.The number of bacteria under scalding wound scab detection results showed that compared with normal saline control group,the number of bacteria under scalding wound scab of the rats in Carex meyeriana Kunth extract CM-70 treatment group was decreased significantly at the same time(P<0.01).The ELISA results showed that compared with normal saline control group,the level of serum IL-10 of the rats in Carex meyeriana Kunth extract CM-70 treatment group was increased significantly on the 1st day and 3rd day after scalding(P<0.01),and it was decreased significantly on the 7th day and 14th day(P<0.05);the serum levels of IL-1βand TNF-αof the rats in Carex meyeriana Kunth extract CM-70 treatment group were decreased significantly on the 7th day and 14th day(P<0.05).Conclusion:The active ingredients of Carex meyeriana Kunth can significantly inhibit the infection ofS. aureus after skin scalding in the rats and exert the anti-inflammatory and bacteriostatic effects byregulating the levels of serum IL-10,IL-1β, and TNF-α.
作者
孙宏宇
陈子涵
崔佰吉
冯宪敏
刘迪
SUN Hongyu;CHEN Zihan;CUI Baiji;FENG Xianmin;LIU Di(Department of Pathogeny Biology,School of Basic Medical Sciences,Jilin Medical University,Jilin 132013,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2023年第2期377-384,共8页
Journal of Jilin University：Medicine Edition
基金
吉林省卫健委卫生与健康青年科技骨干培养计划项目(2019Q031)
吉林省教育厅科学技术研究项目(JJKH20210499KJ,JJKH20210481KJ)
吉林省中医药管理局中医药科技项目(2019139)。
关键词
乌拉草
皮肤烫伤
感染
金黄色葡萄球菌
炎症因子
抑菌作用
Carex meyeriana Kunth
Skin scalding
Infection
Staphylococcus aureus
Inflammatory factor
Bacteriostasis
