Abstract：
BACKGROUND: In Mongolian medicine, Loulu flower (LLF), the dried inflorescence of Rhaponticum uniflorum (L.) DC. from the Compositae family, has been used to clear heat and relieve toxicity for millennia, particularly in the treatment of pneumonia.
OBJECTIVE: To reveal the effects of LLF on mice with lipopolysaccharide (LPS)-stimulated acute lung injury (ALI) and elucidate the underlying mechanisms.
METHODS: ALI was established in BALB/c mice via nasal drops administration of LPS (5 mg/kg). The mice were then orally administrated with various doses of LLF extracts and the positive drug dexamethasone (DEX, 5 mg/kg), once daily for seven consecutive days. Last day, after being stimulated with LPS for 6h, the mice were closed dislocation of cervical vertebra, the serum, bronchus alveolar lavage fluid (BALF) and lung tissue were put into the EP tube and stored at -80 °C for further analysis. The changes of histopathology were tested by hematoxylin and eosin stain (H&E), the levels of, IL-1β, IL-18, TNF-α and IL-4 in BALF and serum were measured by ELISA. The pathways related to the treatment of ALI were predicted by network pharmacology. The expression levels of TLR4/NF-κB and NLRP3 signaling pathway-associated proteins, COX-2 and ERK were tested by western blotting. The levels of P65 and NLRP3 in lung tissues were determined by immunofluorescence analysis.
RESULTS: LLF total extract and the extract parts could alleviate the inflammatory cell infiltration, thicken the alveolar walls in lung tissues, reduce the levels of IL-18, IL-1β in BALF, the TNF-α in both BALF and serum, meantime enhance the level of IL-4 in BALF and serum in mice with LPS-induced ALI. Our network pharmacology and comprehensive gene ontology analyses revealed the active constituents of LLF and the pathways, including TLR4/NF-κB, NLRP3 and MAPK signaling pathways, which play significant roles in ALI. Furthermore, both the total extract and its extraction portions suppressed the expressions of proteins related with the COX-2, p-ERK and TLR4/NF-κB signaling pathway (TLR4, p-IκB, p-p65), as well as the NLRP3 signaling pathway (NLRP3, cleaved caspase-1, caspase-1, IL-1β).
CONCLUSIONS: LLF could improve the pathological changes and reducing inflammatory reactions in mice induced by LPS. The mechanism may be related to the modulation of the TLR4/NLRP3 signaling pathways.
OBJECTIVE: To reveal the effects of LLF on mice with lipopolysaccharide (LPS)-stimulated acute lung injury (ALI) and elucidate the underlying mechanisms.
METHODS: ALI was established in BALB/c mice via nasal drops administration of LPS (5 mg/kg). The mice were then orally administrated with various doses of LLF extracts and the positive drug dexamethasone (DEX, 5 mg/kg), once daily for seven consecutive days. Last day, after being stimulated with LPS for 6h, the mice were closed dislocation of cervical vertebra, the serum, bronchus alveolar lavage fluid (BALF) and lung tissue were put into the EP tube and stored at -80 °C for further analysis. The changes of histopathology were tested by hematoxylin and eosin stain (H&E), the levels of, IL-1β, IL-18, TNF-α and IL-4 in BALF and serum were measured by ELISA. The pathways related to the treatment of ALI were predicted by network pharmacology. The expression levels of TLR4/NF-κB and NLRP3 signaling pathway-associated proteins, COX-2 and ERK were tested by western blotting. The levels of P65 and NLRP3 in lung tissues were determined by immunofluorescence analysis.
RESULTS: LLF total extract and the extract parts could alleviate the inflammatory cell infiltration, thicken the alveolar walls in lung tissues, reduce the levels of IL-18, IL-1β in BALF, the TNF-α in both BALF and serum, meantime enhance the level of IL-4 in BALF and serum in mice with LPS-induced ALI. Our network pharmacology and comprehensive gene ontology analyses revealed the active constituents of LLF and the pathways, including TLR4/NF-κB, NLRP3 and MAPK signaling pathways, which play significant roles in ALI. Furthermore, both the total extract and its extraction portions suppressed the expressions of proteins related with the COX-2, p-ERK and TLR4/NF-κB signaling pathway (TLR4, p-IκB, p-p65), as well as the NLRP3 signaling pathway (NLRP3, cleaved caspase-1, caspase-1, IL-1β).
CONCLUSIONS: LLF could improve the pathological changes and reducing inflammatory reactions in mice induced by LPS. The mechanism may be related to the modulation of the TLR4/NLRP3 signaling pathways.
摘要：
背景：在蒙古医学中，Loulu花(LLF),单花Rhaponticumunflorum的干燥花序（L.)DC。来自菊科，几千年来一直被用来清热和缓解毒性，特别是在治疗肺炎方面。
目的：揭示LLF对脂多糖（LPS）刺激的小鼠急性肺损伤（ALI）的作用及其机制。
方法：通过LPS(5mg/kg)滴鼻在BALB/c小鼠中建立ALI。然后对小鼠口服各种剂量的LLF提取物和阳性药物地塞米松（DEX，5mg/kg），每天一次，连续七天。最后一天,用LPS刺激6h后，小鼠颈椎闭合脱位，血清，将支气管肺泡灌洗液（BALF）和肺组织放入EP管中，并在-80°C下储存以进行进一步分析。采用苏木精和曙红染色(H&E)检测组织病理学改变,的水平，IL-1β,ELISA法测定BALF和血清中IL-18、TNF-α和IL-4的含量。通过网络药理学预测与ALI治疗相关的通路。TLR4/NF-κB和NLRP3信号通路相关蛋白的表达水平,通过蛋白质印迹测试COX-2和ERK。通过免疫荧光分析测定肺组织中P65和NLRP3的水平。
结果：LLF总提取物及提取物部位能减轻炎症细胞浸润，增厚肺组织的肺泡壁，降低BALF中IL-18，IL-1β的水平，BALF和血清中的TNF-α，同时提高LPS诱导的ALI小鼠BALF和血清中IL-4的水平。我们的网络药理学和全面的基因本体论分析揭示了LLF的活性成分和途径，包括TLR4/NF-κB，NLRP3和MAPK信号通路,在ALI中起着重要作用。此外，总提取物及其提取部分均抑制了与COX-2，p-ERK和TLR4/NF-κB信号通路相关的蛋白质的表达（TLR4，p-IκB，p-p65),以及NLRP3信号通路（NLRP3，裂解的caspase-1，caspase-1，IL-1β）。
结论：LLF能改善LPS诱导的小鼠病理变化，减轻炎症反应。其机制可能与TLR4/NLRP3信号通路的调节有关。
目的：揭示LLF对脂多糖（LPS）刺激的小鼠急性肺损伤（ALI）的作用及其机制。
方法：通过LPS(5mg/kg)滴鼻在BALB/c小鼠中建立ALI。然后对小鼠口服各种剂量的LLF提取物和阳性药物地塞米松（DEX，5mg/kg），每天一次，连续七天。最后一天,用LPS刺激6h后，小鼠颈椎闭合脱位，血清，将支气管肺泡灌洗液（BALF）和肺组织放入EP管中，并在-80°C下储存以进行进一步分析。采用苏木精和曙红染色(H&E)检测组织病理学改变,的水平，IL-1β,ELISA法测定BALF和血清中IL-18、TNF-α和IL-4的含量。通过网络药理学预测与ALI治疗相关的通路。TLR4/NF-κB和NLRP3信号通路相关蛋白的表达水平,通过蛋白质印迹测试COX-2和ERK。通过免疫荧光分析测定肺组织中P65和NLRP3的水平。
结果：LLF总提取物及提取物部位能减轻炎症细胞浸润，增厚肺组织的肺泡壁，降低BALF中IL-18，IL-1β的水平，BALF和血清中的TNF-α，同时提高LPS诱导的ALI小鼠BALF和血清中IL-4的水平。我们的网络药理学和全面的基因本体论分析揭示了LLF的活性成分和途径，包括TLR4/NF-κB，NLRP3和MAPK信号通路,在ALI中起着重要作用。此外，总提取物及其提取部分均抑制了与COX-2，p-ERK和TLR4/NF-κB信号通路相关的蛋白质的表达（TLR4，p-IκB，p-p65),以及NLRP3信号通路（NLRP3，裂解的caspase-1，caspase-1，IL-1β）。
结论：LLF能改善LPS诱导的小鼠病理变化，减轻炎症反应。其机制可能与TLR4/NLRP3信号通路的调节有关。
