摘要
目的:观察牛珀至宝微丸(NPZB)对快速肺纤维化小鼠胶原沉积的影响并探索其潜在的细胞机制。方法:将40只C57BL/6小鼠随机分为正常组、模型组、地塞米松组(1.0 mg·kg^(-1))、NPZB低剂量组(0.75 g·kg^(-1))、NPZB高剂量组(1.5 g·kg^(-1))。基于文献研究和前期结果,采用“脂多糖(LPS)三次打击”方案建立快速肺纤维化小鼠模型,造模后给予NPZB灌胃或地塞米松腹腔注射治疗,每天1次,第5天取材。苏木素-伊红(HE)染色观察肺组织病理改变,马松三色(Masson)染色评价胶原沉积情况;蛋白免疫印迹法(Western blot)检测肺组织中Ⅰ型胶原蛋白(ColⅠ)、骨桥蛋白(OPN)、转化生长因子-β_(1)(TGF-β_(1))的表达水平;免疫组化法(IHC)对肺组织中的CD163阳性巨噬细胞进行定位;免疫荧光检测CD163阳性巨噬细胞和OPN的共定位情况;流式细胞术检测肺组织CD163阳性巨噬细胞的数量。制备快速肺纤维化小鼠模型,使用磁性分选从肺组织中分离CD163阳性巨噬细胞;细胞增殖与活性检测法(CCK-8)明确NPZB对CD163阳性巨噬细胞的无毒性剂量;酶联免疫吸附测定法(ELISA)检测NPZB对CD163阳性巨噬细胞OPN分泌水平的影响。将24只CD163敲除(CD163^(-/-))小鼠随机分为正常组、模型组、NPZB高剂量组(1.5 g·kg^(-1)),以野生型小鼠作为对照。采用流式细胞术检测CD163的敲除情况;采用Western blot检测敲除CD163后肺组织ColⅠ、OPN的表达水平。苏木素-伊红(HE)染色和马松(Masson)染色观察肺组织病理改变和胶原沉积情况。结果:与正常组比较,模型组小鼠胶原沉积显著增加(P<0.01),肺组织中ColⅠ、OPN和TGF-β_(1)蛋白的表达水平明显升高(P<0.05,P<0.01),CD163阳性巨噬细胞数量显著增多(P<0.01);与模型组比较,NPZB低、高剂量组胶原沉积明显减少(P<0.05,P<0.01),肺组织ColⅠ、OPN和TGF-β_(1)表达明显降低(P<0.05,P<0.01),CD163阳性巨噬细胞数量显著减少(P<0.01)。与正常组比较,NPZB显著降低CD163阳性巨噬细胞上清的OPN水平(P<0.01)。与野生型小鼠模型组比较,CD163^(-/-)模型组和NPZB组小鼠肺组织胶原沉积范围显著减小(P<0.01),肺组织ColⅠ和OPN表达水平降低(P<0.01)。结论:NPZB通过降低CD163阳性巨噬细胞的数量及其OPN分泌水平,抑制快速肺纤维化小鼠的胶原沉积。
Objective:To observe the effect of Niupo Zhibao Pellet(NPZB)on collagen deposition in a mouse model of rapid pulmonary fibrosis(RPF)and explore the underlying cellular mechanisms involved.Methods:The 40 C57BL/6 mice were randomly divided into a control group,a model group,a dexamethasone group(1.0 mg·kg^(-1)),a low-dose NPZB group(0.75 g·kg^(-1)),and a high-dose NPZB group(1.5 g·kg^(-1)).Based on the previous studies and results,the"lipopolysaccharide(LPS)three-hit"protocol was employed to establish a mouse model of RPF.Following model establishment,NPZB was administered via gavage,and dexamethasone was delivered through intraperitoneal injection once a day.On the 5th day,the lung tissue was collected.Hematoxylin-eosin(HE)staining was employed to observe pathological alterations in the lung tissue,while Masson's trichrome staining was used to assess collagen deposition.Western blot was employed to detect the expression level of ColⅠ,osteopontin(OPN),and transforming growth factor(TGF)-β_(1) in the lung tissue.Immunohistochemical(IHC)staining was conducted to localize CD163-positive macrophages(CD163+Mφ),and immunofluorescence(IF)staining was performed to detect the colocalization of CD163+Mφand OPN.Flow cytometry was utilized to quantify the number of CD163+Mφin the lung tissue.The mouse model of RPF was prepared,and CD163+Mφwere isolated from the lung tissue by magnetic-activated cell separation.The non-toxic dosages of NPZB for these cells were determined by the cell counting kit-8(CCK-8).Subsequently,an enzymelinked immunosorbent assay(ELISA)was used to detect the effect of NPZB on the OPN secretion level of CD163+Mφ.The 24 CD163 knockout(CD163-/-)mice were randomly assigned to a control group,a model group,and a high-dose NPZB group(1.5 g·kg^(-1)),with wild-type mice serving as controls.Flow cytometry was utilized to confirm the knockout of CD163,and Western blot was conducted to assess the expression level of OPN and ColⅠin the lung tissue of CD163-/-mice.HE staining was used to observe pathological changes,and Masson staining was employed to assess collagen deposition.Results:In comparison to the control group,the model group exhibited a significant increase in collagen deposition(P<0.01),the expression level of ColⅠ,OPN(P<0.01),and TGF-β_(1)(P<0.01),as well as the number of CD163+Mφ(P<0.01)in the lung tissue.Compared to the model group,both the low-dose and high-dose groups of NPZB demonstrated a reduction in collagen deposition(P<0.05,P<0.01),the expression of ColⅠ(P<0.05,P<0.01),OPN(P<0.01),and TGF-β_(1)(P<0.05,P<0.01)in lung tissue,and the number of CD163+Mφ(P<0.01).Furthermore,compared with the control group,NPZB significantly decreased the OPN level in the supernatant of CD163+Mφ(P<0.01).Compared to the model group in wild-type mice,CD163-/-mice exhibited a significant reduction in collagen deposition(P<0.01)and a decrease in the expression level of ColⅠand OPN(P<0.01).Conclusion:NPZB inhibits collagen deposition in mice with RPF by reducing the number of CD163+Mφand their OPN secretion levels.
作者
邓穗晖
丁文俊
陈贤杰
叶森
李春
黎晖
DENG Suihui;DING Wenjun;CHEN Xianjie;YE Sen;LI Chun;LI Hui(School of Basic Medical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;State Key Laboratory of Traditional Chinese Medicine Syndrome,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;School of Nursing,Guangzhou University of Chinese Medicine,Guangzhou 510006,China)
出处
《中国实验方剂学杂志》
北大核心
2025年第23期51-62,共12页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(82274256,82405080)。
