白及胶/微米三七膏对大鼠糖尿病足溃疡炎症微环境的分子机制研究

Molecular mechanism of hyacinth bletilla/micron pseudo-ginseng cream on inflammatory microenvironment of diabetic foot ulcers in rats

目的探讨白及胶/微米三七膏对大鼠糖尿病足溃疡(DFU)炎症微环境的分子机制。方法制备白及胶/微米三七膏;将SPF级健康SD大鼠32只,随机分成4组,每组8只。空白组(正常喂养),其余3组另分别给予不同药物浓度白及胶/微米三七膏:含药血清低剂量组(5μmol/L)、含药血清中剂量组(10μmol/L)、含药血清高剂量组(20μmol/L)。灌胃7d,第7天重复给药2次,间隔时间1h。并于末次给药后30 min,无菌操作,水合氯醛麻醉,腹主动脉采血制备含药血清;LPS诱导RAW264.7建立细胞损伤炎症微环境模型(LPS刺激组),ELISA法验证该模型;用MTT法分析白及胶/微米三七含药血清对RAW264.7细胞毒性,用ELISA法检测细胞炎症因子TNF-α、IL-6,用Griess法检测NO分泌。结果白及胶/微米三七含药血清中有相关血药浓度,符合作为作用于细胞药物的基本要求;炎症模型较正常培养细胞中TNF-α、IL-6均有显著性增加(P<0.05);不同浓度的含药血清(5~40μmol/L)对RAW264.7细胞无明显毒性(P>0.05);不同浓度含药血清作用的细胞炎症损伤模型中TNF-α、IL-6比较差异有统计学意义(P<0.05),与LPS刺激组相比,不同浓度含药血清对NO分泌显著减少(P<0.05)。结论白及胶/微米三七含药血清对细胞炎症损伤模型中的TNF-α、IL-6和NO有抑制作用,减轻了DFU局部炎症反应,降低血管和神经损伤,提高局部组织免疫力,改善炎症微环境,促进创面愈合。

Objective To study the molecular mechanism of hyacinth bletilla/micron pseudo-ginseng cream on inflammatory microenvironment of diabetic foot ulcers in rats.Methods Hyacinth bletilla/micronpseudo-ginseng cream was prepared.32 SPF grade healthy SD rats were randomly divided into four groups.8 rats in each group.The control group was normally feed.The other three groups were given different concentrations of hyacinth bletilla/micron pseudoginseng cream including low dose group(5μmol/L),middle dose group(10μmol/L)and high dose group(20μmol/L).The administration was repeated twice on the 7 th day with an interval of 1 h.At 30 minutes after the last administration,the serum containing drugs was prepared by aseptic operation,chloral hydrate anesthesia and blood collection from abdominal aorta.RAW264.7 was induced by LPS to establish a cell injury inflammation microenvironment model(LPS stimulation group),which was verified by ELISA.MTT method was used to analyze the cytotoxicity on RAW264.7.TNF a and IL 6 were detected by ELISA,and Griess method was used to detect the cytokines NO secretion was detected.Results The serum containing hyacinth bletilla/micron pseudo-ginseng has the related blood concentration,which meets the basic requirements of acting as a cell drug.The inflammatory model has a significant increase in TNF a and IL 6 compared with the normal cultured cells(P<0.05).The serum containing hyacinth bletilla/micron pseudo-ginseng(5-40 pmol/L)has no significant toxicity to RAW264.7 cells(P>0.05).The serum containing drugs with different concentrations has no significant toxicity to RAW264.7 cells(P>0.05).TNF a and IL 6 in the model of inflammatory cell injury were statistically significant(P<0.05).Compared with LPS stimulation group,no secretion in different concentrations of serum were significantly reduced(P<0.05).Conclusion Hyacinth bletilla/micronpseudo-ginseng containing serum inhibits TNF-α,IL-6 and NO in a model of cellular inflammatory injury,which can reduce local inflammatory response and vascular and nerve damage,improve local tissue immunity,and improve the inflammatory microenvironment and promote the wound healing.