Protective effect of the total alkaloid extract from Bulbus Fritillariae Pallidiflorae on cigarette smoke-induced Beas-2B cell injury model and transcriptomic analysis.

Background: Bulbus Fritillariae Pallidiflorae (BFP) is a traditional Chinese medicine that has long been used to treat lung diseases, but the active components and mechanism are still unclear. Objective: This study aimed to investigate the effect and mechanism of the total alkaloid extract from BFP (BFP-TA) on cigarette smoke extract (CSE)-induced Beas-2B cells injury. Design: The Beas-2B cells injury model was induced by 2% CSE, then the effect of BFP-TA on the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA) was detected according to the instructions of the T-AOC assay kit, the SOD detection kit and the MDA detection kit, and the production of ROS was detected by fluorescence microscopy. The effect of BFP-TA on Beas-2B cells apoptosis was detected by flow cytometry, and the effect of BFP-TA on related protein expression was detected by western blot. Subsequently, the effect of BFP-TA on differentially expressed genes (DEGs) in CSE-induced Beas-2B cells was studied by transcriptomic sequencing, and the expression of DEGs was verified by quantitative real-time polymerase chain reaction (qPCR). Results: The results showed that BFP-TA could attenuate CSE-induced oxidative damage in Beas-2B cells by elevating T-AOC and SOD levels while inhibiting ROS and MDA levels, and the mechanism was potentially related to the SIRT1/Nrf2/Keap1 signaling pathway. Furthermore, BFP-TA could inhibit CSE-induced apoptosis by inhibiting the protein expression of Bax, MST1 and FOXO3a, and exert anti-inflammatory effect by inhibiting the activation of MAPK signaling pathway. Subsequently, transcriptome analysis and qPCR validation showed that BFP-TA could alleviate inflammation, oxidative stress, apoptosis and lipid metabolism disorders by regulating the expression of DEGs in PPAR and PI3K-Akt signaling pathways, thereby exerting a protective effect against CSE-induced Beas-2B cell injury. Conclusion: This study is the first to demonstrate that BFP-TA could exert a protective effect on CSE-induced Beas-2B cell injury by exerting anti-inflammatory, antioxidant, anti-apoptotic and regulate lipid metabolism disorders.

Protective Effect of the Total Alkaloid Extract from Bulbus Fritillariae pallidiflorae in a Mouse Model of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

Purpose: In recent years, the incidence of chronic obstructive pulmonary disease (COPD) has been increasing year by year, but therapeutic drugs has no breakthrough. The total alkaloid extract from Bulbus Fritillariae pallidiflorae (BFP-TA) is widely used in treating lung diseases. Therefore, this study aimed to investigate the protective effect and mechanism of BFP-TA in COPD mice. Methods: BFP-TA was prepared by macroporous adsorbent resin, and the material basis of BFP-TA was analyzed by HPLC-ELSD and UHPLC-MS/MS. Then, the COPD mouse model was induced by cigarette smoke (CS) for 12 weeks, administered at weeks 9-12. Subsequently, the body weight, lung-body ratio, pulmonary function, histopathology, and the levels of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and oxidative stress markers in the serum of mice were determined. The expressions of related protein of EMT and MAPK signaling pathways in the lung tissues of mice were detected by Western blot. Results: The alkaloid relative content of BFP-TA is 64.28%, and nine alkaloids in BFP-TA were identified and quantified by UHPLC-MS/MS. Subsequently, the animal experiment showed that BFP-TA could improve pulmonary function, and alleviate inflammatory cell infiltration, pulmonary emphysema, and collagen fiber deposition in the lung of COPD mice. Furthermore, BFP-TA could decrease the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), MMPs (MMP-9 and MMP-12) and MDA, while increase the levels of TIMP-1 and SOD. Moreover, BFP-TA could decrease the protein expressions of collagen I, vimentin, α-SMA, MMP-9, MMP-9/TIMP-1, Bax, p-JNK/JNK, p-P38/P38, and p-ERK/ERK, while increase the level of E-cadherin. Conclusion: This study is the first to demonstrate the protective effect of BFP-TA in CS-induced COPD mouse model. Furthermore, BFP-TA may improve airway remodeling by inhibiting the EMT process and potentially exert anti-inflammatory effect by inhibiting the MAPK signaling pathway.

A distinction between Fritillaria Cirrhosa Bulbus and Fritillaria Pallidiflora Bulbus via LC-MS/MS in conjunction with principal component analysis and hierarchical cluster analysis.

Fritillaria Cirrhosa Bulbus (known as chuanbeimu in Chinese, FCB) is one of the most used Chinese medicines for lung disease. However, a variety of substitutes have entered the market, with Fritillaria Pallidiflora Bulbus (FPB) being the most common. Due to their similarity in appearance, morphology, and chemical composition but a large price difference, the FCB has frequently been adulterated with the FPB, posing a serious challenge to the distinction and quality of the FCB. Therefore, we aimed to distinguish FCB and FPB based on their main nine isosteroidal alkaloid contents and test the potential of chemometrics as a discrimination approach for evaluating quality. The nine major isosteroidal alkaloids were measured using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach in 41 batches of FCB and 17 batches of FPB. Additionally, they were categorized and distinguished using the methods of hierarchical cluster analysis (HCA) and principal component analysis (PCA). Quantitative analysis revealed that the nine alkaloids were present in different amounts in the two types of Fritillariae bulbus. In FCB, the highest amount was peimisine (17.92-123.53 µg/g) and the lowest was delavine (0.42-29.18 µg/g), while in FPB, imperialine was higher (78.05-344.09 µg/g), but verticinone and verticine were less than the other seven alkaloids. The FCB and FPB were successfully classified and distinguished by the HCA and PCA. Taken together, the method has a good linear relationship (R2 > 0.9975). The LOD and LOQ of the nine alkaloids were in the range of 0.0651-0.6510 and 0.1953-1.9531 ng/mL, respectively. The intra- and inter-day precision were shown to be excellent, with relative standard deviations (RSDs) below 1.63% and 2.39%, respectively. The LC-MS/MS method in conjunction with HCA and PCA can effectively differentiate FCB and FPB. It may be a promising strategy for quality evaluation and control at the FCB.