肺纤方对小鼠肺纤维化模型及NIH3T3成纤维细胞作用机制的研究  

Effect of Feixian Fang on mouse pulmonary fibrosis model and NIH3T3 fibroblasts

作　　者：谢纬[1] 唐明文[1] 郭竹英 陈生[1] 黄俊浩[2] 曾梓苑 XIE Wei;TANG Mingwen;GUO Zhuying;CHEN Sheng;HUANG Junhao;ZENG Ziyuan(Shenzhen Hospital of Traditional Chinese Medicine,Shenzhen 518033,China;不详)

机构地区：[1]深圳市中医院,广东深圳518033 [2]广州中医药大学第四临床医学院,广东深圳518033

出　　处：《现代医院》2021年第5期792-797,共6页Modern Hospitals

基　　金：广东省首批省名中医师承项目(粤中医函[2015]20号);深圳市知识创新计划基础研究项目基金(JCYJ20170307154848369);深圳市“医疗卫生三名工程”项目(SZSM201812063)。

摘　　要：目的在前期二次造模实验基础上,建立小鼠肺纤维化模型,观察肺纤方对肺纤维化小鼠模型的干预作用,并探讨不同浓度的肺纤方煎剂对体外培养NIH3T3成纤维细胞细胞增殖的影响及可能的作用机制。方法将60只雄性昆明小鼠,随机取出其中50只,气管内雾化吸入博莱霉素(8 mg/kg)造模,造模第10天后再次给予吸入博来霉素(8 mg/kg)二次造模,第25天将其随机分为5组:模型组、甲泼尼龙组、肺纤方高、中、低剂量组,每组10只。其余10只小鼠气管内滴入及灌胃等容生理盐水作为空白对照组。处死后取肺脏标本进行肺组织肺泡炎、纤维化评价方法和肺组织羟脯氨酸(Hyp)测定。培养小鼠NIH3T3成纤维细胞,分为对照组、肺纤方低、中、高剂量组,分别给予生理盐水、低、中、高浓度剂量肺纤方煎剂处理,一段时间后进行平板克隆形成实验和Western blot法测定细胞增殖和CyclinD1、MMP2、TIMP1蛋白的表达水平。结果采用肺纤方各组及甲泼尼龙组小鼠肺泡炎、纤维化评分和Hyp含量均明显低于模型组(P<0.05),且肺纤方各组上述指标值随着肺纤方剂量的增加而降低,但高剂量组与甲泼尼龙组比较,无显著性差异(P>0.05);肺纤方各组具有抑制NIH3T3细胞的增殖能力,且呈时间依赖,但无浓度依赖性;肺纤方各组对CyclinD1和MMP2蛋白的表达均有下调,且一定范围内随药物浓度增加,CyclinD1蛋白表达下调越明显;同时肺纤方各组TIMP1蛋白的表达有所增加,但是差异不显著;而肺纤方各组GAPDH条带灰度比值无明显差异。结论肺纤方能够有效抑制博来霉素致小鼠肺纤维化,同时能抑制NIH3T3细胞的增殖,可能机制为其抑制CyclinD1蛋白表达和改善MMPs/TIMPs系统平衡,但上述效应与药剂浓度无正相关性。Objective On the basis of the previous secondary modeling experiment, a mouse model of pulmonary fibrosis was established to observe the intervention effect of Feixian Fang on the mouse model of pulmonary fibrosis, and to explore the effect of different concentrations of Feiqian Fang Decoction on the proliferation of NIH3 T3 fibroblasts in vitro and the possible mechanism of action. Methods Fifty male Kunming mice were randomly taken out from sixty male Kunming and inhaled bleomycin(8 mg/kg) by intratracheal atomization for modeling, then inhaled bleomycin(8 mg/kg) was given again for modeling on the 10 th day. On the 25 th day, they were randomly divided into 5 groups: model group, methylprednisolone group, Feixianfang high-dose, medium-dose and low-dose groups, with 10 mice in each group. The other 10 mice were inhaled and fed constant volume normal saline as blank control group. After death, lung specimens were collected for the evaluation of alveolitis and fibrosis in lung tissue and the determination of Hyp in lung tissue. The cultured NIH3 T3 fibroblasts were divided into control group, Feixianfang low-dose, medium-dose and high-dose groups, and treated with normal saline, low-dose and high-dose Feixianfang decoction respectively. After a period of time, the cell proliferation and the protein expression levels of CyclinD1, MMP2 and TIMP1 were determined by plate clone formation experiment and Western blot. Results The alveolitis score, fibrosis score and HYP content in mice treated with Feixianfang and methylprednisolone group were significantly lower than those in model group(P<0.05), and the above indexes decreased with the increase of the dosage of Feixian prescription, but there was no significant difference between the high-dose group and methylprednisolone group(P>0.05). Feixianfang could inhibit the proliferation of NIH3 T3 cells in a time-dependent manner but not in a concentration-dependent manner. The expression of CyclinD1 and MMP2 proteins were down-regulated in all groups of Feixianfang, and th

关 键 词：肺纤方 肺纤维化 NIH3T3成纤维细胞 作用机制 

分 类 号：R289.51[医药卫生—方剂学] R-332[医药卫生—中药学]