【作者】 王玉；

【导师】 刘振； 赵万顺；

【作者基本信息】 天津科技大学 ， 制药工程（专业学位）， 2022， 硕士

【摘要】 急性肺损伤是一种急性危重性疾病,是由于各种原因导致的肺部急性弥漫性疾病,急性肺损伤不能及时治疗会导致严重的呼吸窘迫综合征,危及病人的生命。急性肺损伤是炎症和血栓形成的双向关系。因此本研究选用了五味活血化瘀类中药(红花、当归、赤芍、川芎、丹参)预防急性肺损伤的发生,并合理的应用中药配伍达到改善单味中药发挥作用慢,提高药效,减轻毒副作用,增加作用靶点的效果。采用回流提取的方法得到红花、当归、赤芍、川芎、丹参五味中药的总提取物;以脂多糖(LPS)为刺激物刺激小鼠单核巨噬细胞白血病细胞(RAW264.7)建立细胞炎症模型;测定了五味中药总提物的安全作用范围,得到了最佳配伍—红花配伍当归。测定了红花配伍当归对ROS、NO、炎症因子(IL-6、TNF-α、IL-1β)的影响,通过蛋白免疫印迹实验确定相关蛋白表达水平。同时利用LPS腹腔注射建立小鼠急性肺损伤模型,测定小鼠的脏体系数,肺组织的湿/干比,采用尾静脉注射伊文思蓝染料的方法检测肺毛细血管通透性;利用HE染色对小鼠肺组织进行病理学检查;利用蛋白免疫印迹实验确定相关中药配伍抗急性肺损伤的作用机制。本研究首先根据MTT实验结果确定了五味中药总提物的安全作用范围,在安全作用浓度范围内,以NO抑制率>80%和联合指数CI值<1为指标选择红花当归配伍为较优中药配伍。红花与当归配伍使RAW264.7细胞的抗氧化应激能力增加了两倍,抗炎能力增加。研究发现红花与当归配伍使小鼠肺组织的湿/干比降低了50%,治疗组小鼠肺组织中伊文思蓝含量有63μg/100 mg降低到21μg/100 mg。同时本研究发现红花与当归配伍可以改善肺部炎性浸润,肺泡壁增厚。本研究发现红花与当归的抗急性肺损伤作用与MAPK通路及NF-κB通路有关。红花与当归配伍可以显著调节ERK1/2、p38的磷酸化,可以减少IκBα与NF-κB p65/p50的解聚,减少NF-κB p65/p50的核转运,从而发挥抗炎作用;可以抑制PARP、caspase 3的活化,从而改善相关免疫细胞的凋亡,来达到预防肺损伤的作用。本研究发现表明,红花与当归的配伍发挥了由于单味药的作用,二者发挥抗急性肺损伤作用的机制与NF-κB p65的活化与核转运有关,同时可抑制免疫细胞的凋亡。红花与当归配伍可以作为治疗肺损伤的药物组合。更多还原

【Abstract】 Acute lung injury is an acute critical illness,which is an acute diffuse disease of the lungs caused by various reasons,and acute lung injury cannot be treated in time to cause severe respiratory distress syndrome and endanger the patient’s life.Acute lung injury is a two-way relationship between inflammation and thrombosis.Therefore,this study selected five flavors of blood-activating and stasis-reducing chinese medicines(safflower,angelica,red peony,Sichuan root,danshen)to prevent the occurrence of acute lung injury,and rationally applied the compatibility of traditional Chinese medicine to improve the slow function of single-flavor Chinese medicine,improve the efficacy,reduce toxic side effects,and increase the effect of the target.The total extract of safflower,angelica,red peony,Sichuan root and salvia was obtained by reflux extraction method,and the cell inflammation model was established by stimulating mouse mononuclear macrophage leukemia cells(RAW264.7)with lipopolysaccharides(LPS)as the stimulus,and the safety range of the total extract of wuwei Chinese medicine was determined,and the best compatibility was obtainedsafflower compatible angelica was obtained.The effect of safflower compatible angelica on ROS,NO,inflammatory factors(IL-6,TNF-α,IL-1β)was measured,and the relevant protein expression level was determined by western blotting experiments.At the same time,the mouse acute lung injury model was established by LPS intraperitoneal injection,the number of visceral systems and the wet/dry ratio of lung tissue were measured,and the permeability of lung capillaries was detected by tail vein injection of Evans blue dye;the pathological examination of mouse lung tissue was carried out by HE staining;and the mechanism of action of relevant Chinese medicine compatibility anti-acute lung injury was determined by western blotting experiments.In this study,the safety range of the total extract of five Chinese medicine was first determined according to the results of MTT experiments,and within the range of safe action concentration,the no inhibition rate > 80% and the CI value of the joint index <1selected safflower angelica as the better Chinese medicine compatibility.The compatibility of safflower with angelica triples the antioxidant stress capacity of RAW264.7 cells,anti-inflammatory capacity.Its anti-acute lung injury effect at the animal level was further explored.The study found that the compatibility of safflower with angelica reduced the wet/dry ratio of the lung tissue of the mice by 50%,improved pulmonary edema,and reduced the content of Evans blue in the lung tissue of the mice in the treatment group from60 μg/100 mg to 20 μg/100 mg,which further verified the anti-inflammatory ability and antioxidant stress resistance of the drug.At the same time,this study found that the combination of safflower and angelica can improve inflammatory infiltrates in the lungs and thicken the alveolar walls.In this study,it was found that the anti-acute lung injury effects of safflower and angelica were related to the MAPK pathway and the NF-κB pathway.The compatibility of safflower with angelica can significantly regulate the phosphorylation of ERK1/2 and p38,which can reduce the depolymerization of IκBα and NF-κB p65/p50,reduce the nuclear transport of NF-κB p65/p50,thereby exerting anti-inflammatory effects;it can inhibit the activation of PARP and caspase 3,thereby improving the apoptosis of related immune cells to achieve the effect of preventing lung damage.This study shows that the compatibility of safflower with angelica plays a role due to the role of single-flavored drugs,and the mechanism of the two playing a role in seeing lung damage is related to the activation of NF-κB p65 and nuclear transport,and can inhibit the apoptosis of immune cells.Safflower can be combined with angelica as a drug for the treatment of lung injury.更多还原