[關(guān)鍵詞]
[摘要]
目的 探究補肺活血膠囊對小鼠肺纖維化的影響及作用機制。方法 90只C57BL/6J小鼠隨機分為對照組、模型組、NLRP3抑制劑(MCC950,10 mg/kg)組、補肺活血膠囊(0.32、0.63、1.62 g/kg、)組,每組15只小鼠。通過對小鼠氣管內(nèi)單次注射2.5 mg/kg的博來霉素來誘導(dǎo)肺纖維化,對照組小鼠接受單次氣管注射等體積的磷酸緩沖鹽溶液(PBS)。造模成功后第1天開始給藥,持續(xù)給藥21 d。統(tǒng)計小鼠每天的存活率和體質(zhì)量,采集小鼠肺泡灌洗液(BALF)并測定總細胞與總蛋白,測定小鼠肺組織濕干質(zhì)量比(W/D)與羥脯氨酸(HYP);采用蘇木精–伊紅(HE)染色和Masson染色觀察肺組織病理學(xué)變化,采用qRT-PCR檢測上皮間質(zhì)轉(zhuǎn)化(EMT)相關(guān)蛋白[E-鈣黏蛋白(E-cad)、波形蛋白(Vim)、α-平滑肌肌動蛋白(α-SMA)、轉(zhuǎn)化生長因子β1(TGF-β1)]的mRNA表達;采用Western blotting檢測EMT相關(guān)蛋白以及Nod樣受體蛋白3(NLRP3)通路相關(guān)蛋白[NLRP3、凋亡相關(guān)斑點樣蛋白(ASC)、mature-半胱氨酸天冬氨酸蛋白酶(Caspase-1)、mature-白細胞介素(IL)-1β、mature-IL-18]表達。結(jié)果 相較于模型組,MCC950與補肺活血膠囊干預(yù)后肺纖維化小鼠存活率明顯提高,體質(zhì)量明顯增加,肺組織W/D、BALF總細胞與總蛋白、HYP含量均明顯降低(P<0.05、0.01);肺組織病理形態(tài)明顯改善;肺組織上皮細胞標(biāo)志物E-cad mRNA及蛋白的表達增加,間質(zhì)細胞標(biāo)志物Vim、α-SMA、TGF-β1 mRNA和蛋白的表達下降,NLRP3信號通路中相關(guān)蛋白表達水平降低(P<0.05、0.01)。結(jié)論 補肺活血膠囊能通過抑制NLRP3通路介導(dǎo)的EMT改善博來霉素誘導(dǎo)的肺纖維化程度。
[Key word]
[Abstract]
Objective To investigate the effect and mechanism of Bufei Huoxue Capsules on pulmonary fibrosis in mice. Methods A total of 90 C57BL/6J mice was randomly divided into control group, model group, NLRP3 inhibitor (MCC950, 10 mg/kg), and Bufei Huoxue Capsules (0.32, 0.63, and 1.62 g/kg) group, each group had 15 mice. Mice were given a single intratracheal injection of 2.5 mg/kg bleomycin to induce pulmonary fibrosis, mice in the control group received a single intratracheal injection of an equal volume of PBS. The drug was administered first day after successful modeling, and the whole administration process lasted for 21 d. The survival rate and body weight of mice were counted. Bronchoalveolar lavage fluid (BALF) was collected and the total cell count and total protein were determined. The wet/dry weight ratio (W/D) and hydroxyproline (HYP) of lung tissue were measured. The pathological changes of lung tissue were observed by hematoxylin and eosin (HE) staining and Masson trichrome staining. qRT-PCR was used to detect the mRNA expression of EMT-related proteins (E-cad, VIM, α-SMA, and TGF-β1). Western blotting was used to detect the expression of EMT-related proteins and NLRP3 pathway-related proteins (NLRP3, ASC, mature-Caspase-1, mature-IL-1β, mature-IL-18). Results Compared with the model group, the survival rate of pulmonary fibrosis mice after MCC950 and Bufei Huoxue Capsules intervention was significantly improved, the body weight was significantly increased, the W/D of lung tissue, the total cell count and total protein and HYP content of BALF were significantly decreased (P< 0.05, 0.01), the pathological morphology of lung tissue was significantly improved, the expression of epithelial cell marker E-cad mRNA and protein in lung tissue was increased, the expression of mesenchymal cell marker Vim, α-SMA, TGF-β1 mRNA and protein was decreased, and the expression of related proteins in NLRP3 signaling pathway was decreased (P < 0.05, 0.01). Conclusion Bufei Huoxue Capsules can improve bleomycin-induced pulmonary fibrosis in mice by inhibiting NLRP3 pathway-mediated EMT.
[中圖分類號]
R965;R974
[基金項目]
山東省中醫(yī)藥科技項目(2020Q074);青島市中醫(yī)藥科研項目(2019-WJZD053)