Ethanol extracts of Rhaponticum uniflorum (L.) DC inflorescence ameliorate LPS-mediated acute lung injury by alleviating inflammatory responses via the Nrf2/HO-1 signaling pathway.

Rhaponticum uniflorum (L.) DC is a member of the Compositae family. Loulu flowers (LLF) is the inflorescence of this plant, which is a commonly used Mongolian medicine for the treatment of inflammatory diseases due to its heat-clearing and detoxifying properties. It is used caused by. However, its anti-inflammatory mechanisms are not clear. We investigated whether ethanol extracts of LLF can alleviate LPS-induced acute lung injury and explored the mechanism involved. BALB/C mice were intragastrically administered with sodium carboxymethyl cellulose (0.5%, 1 mL/100 g) or ethanol extracts of LLF at a dose of 100, 200, and 400 mg/kg, once daily, for 3 days. Subsequently, mice models of acute lung injury were established by LPS and used for the determination of anti-inflammatory effects of LLF. After 6 h of treatment, mice were sacrificed to collect lung tissues and bronchoalveolar lavage fluid (BALF). H&E staining assay was performed on the tissues for pathological analysis. The ELISA test was conducted to measure NO, IL-6, TNF-α, MPO, SOD, CAT, MDA and GSH-PX levels. The expression level of proteins associated with the Nrf2/HO-1 and MAPK/NF-κB signaling pathways were determined using Western blot analysis. Levels of F4/80 and Nrf2 in lungs were quantified using immunohistochemistry. Oral administration of LLF extracts alleviated LPS-induced pathological alterations, reduced lung W/D weight ratio, decreased levels of TP, pro-inflammatory factors (TNF-α and IL-6), and NO in BALF. Pretreatment with LLF extract downregulated F4/80 expression in lung tissue and suppressed LPS-induced elevations in BALF and lung tissue levels of MPO. Moreover, treatment with LLF extract reduced the expression level of proteins associated with the MAPK signaling pathway (p-p38, p-JNK, p-ERK) and TLR4/NF-κB signaling pathways (TLR4, Myd88, p-IκB, p-p65). Moreover, LLF extract upregulated Nrf2, HO-1 and NQO1 protein levels, downregulated Keap1 protein level. Immunohistochemical analysis revealed that LLF reduced the LPS-induced increase in Nfr2 expression in lung tissues. Ethanol extracts of LLF ameliorated LPS-induced acute lung injury by suppressing inflammatory response and enhancing antioxidation capacity, which correlated with the MAPK/NF-κB and Nfr2/HO-1 signaling pathways.

Zhen D ，Liu C ，Huang T ，Fu D ，Bai X ，Ma Q ，Jiang M ，Gong G ... -  《-》

The effect of ethanol extracts of loulu flower on LPS-induced acute lung injury in mice.

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. To reveal the effects of LLF on mice with lipopolysaccharide (LPS)-stimulated acute lung injury (ALI) and elucidate the underlying mechanisms. 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. 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). 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.

Wurentuya ，Han S ，Mei S ，Lai M ，Sirigunqiqige ，Luoricuo ，Yang M ，Feng Y ，Zhong G ，Zhu J ，Li M ... -  《-》

Bakuchiol regulates TLR4/MyD88/NF-κB and Keap1/Nrf2/HO-1 pathways to protect against LPS-induced acute lung injury in vitro and in vivo.

Bakuchiol (Bak) possesses a protective effect in acute lung injury (ALI). Nonetheless, the molecular processes that regulate the protective activity of Bak in ALI remain elusive. Lipopolysaccharide (LPS)-treated rats and RLE-6TN cells were used as the ALI models in vivo and in vitro to investigate the function and mechanism of Bak. Rats were divided into four groups: control, LPS, LPS + Bak (30 mg/kg), and LPS + Bak (60 mg/kg). RLE-6TN cells were assigned into four groups: control, LPS, LPS + Bak (10 µM), and LPS + Bak (20 µM). Myeloperoxidase (MPO) and 4-hydroxy-2-nonenal (4-HNE) levels were detected by immunohistochemistry (IHC). The levels of TNF-α, IL-6, and IL-1β were quantified by ELISA. Apoptosis was analyzed by TdT-mediated dUTP nick-end labeling (TUNEL) staining and flow cytometry. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and reactive oxygen species (ROS) were assayed to evaluate oxidative stress. In LPS-induced rats, Bak attenuated pathological injury, lung wet/dry weight ratio, MPO expression, and protein concentration and cell number in bronchial alveolar lavage fluid (BALF). Bak decreased the secretion of TNF-α, IL-6, and IL-1β in BALF. Bak reduced MDA content and 4-HNE expression, and increased SOD and GSH-Px activities in lung tissues. Bak also repressed pulmonary apoptosis by decreasing Bax expression and enhancing Bcl-2 expression. In LPS-treated RLE-6TN cells, Bak downregulated the mRNA levels of TNF-α, IL-6, and IL-1β and inhibited the protein expression of iNOS and COX2. Bak decreased MDA level and ROS production and increased SOD and GSH-Px activities. Bak also suppressed cell apoptosis, reduced Bax expression, and increased Bcl-2 expression. Moreover, Bak decreased the expression of TLR4, MyD88, p-IκBα, and p-p65. Additionally, Bak inhibited Keap1 expression and increased Nrf2 and HO-1 levels. Bak protects against LPS-induced inflammation, oxidative stress, and apoptosis in ALI by regulating TLR4/MyD88/NF-κB and Keap1/Nrf2/HO-1 pathways.

Zhao L ，Zhang Z ，Li P ，Gao Y ，Shi Y ... -  《-》

Botanical formulation, TADIOS, alleviates lipopolysaccharide (LPS)-Induced acute lung injury in mice via modulation of the Nrf2-HO-1 signaling pathway.

TADIOS is an herbal formulation prepared from a mixture of Taraxacum officinale (L.) Weber ex F.H.Wigg, Dioscorea batatas Decaisne and Schizonepeta tenuifolia (Benth.) Briquet. These plants have traditionally been used in Asia to treat a variety of respiratory diseases. A bulk of literature on traditional Korean medicine describe their activities and functions for respiratory problems. Therefore, we hypothesized that the combination of these plants might be effective in alleviating respiratory symptoms. In this study, we investigated whether TADIOS ameliorates LPS-induced acute lung injury via regulation of the Nrf2-HO-1 signaling pathway. The LPS-induced acute lung injury mouse model was used to determine the anti-inflammatory and anti-oxidative stress effects of TADIOS. The amount of marker compounds contained in TADIOS was quantified using high-performance liquid chromatography (HPLC) analysis. The protein level of pro-inflammatory cytokines in culture supernatant was measured by ELISA. Changes in the RNA level of pro-inflammatory cytokines in mice lungs and RAW264.7 cells were measured by quantitative RT-PCR. The relative amounts of reactive oxygen species (ROS) were measured by DCF-DA assay. Western blot analysis was used to evaluate expression of cellular proteins. Effects of TADIOS on antioxidant responsive elements (AREs) were determined by luciferase assay. The severity of acute lung injury was evaluated by Hematoxylin & Eosin (H&E) staining. To test the effects of TADIOS on LPS-induced oxidative stress, myeloperoxidase (MPO) activity and the total antioxidant capacity were measured. TADIOS was prepared by extraction of a blend of these three plants by ethanol, and quality control was performed through quantification of marker compounds by HPLC and measurement of bioactivities using cell-based bioassays. In the murine macrophage cell line RAW264.7, TADIOS effectively suppressed the production of pro-inflammatory cytokines such as IL-6 and IL-1β, and also ROS induced by LPS. When RAW264.7 cells were transfected with a luciferase reporter plasmid containing nucleotide sequences for AREs, TADIOS treatment increased the level of relative luciferase units in a dose-dependent manner. In the LPS-induced acute lung injury mouse model, orally administered TADIOS alleviated lung damage and neutrophil infiltration induced by LPS. Consistent with the in vitro data, treatment with TADIOS inhibited the LPS-mediated expression of pro-inflammatory cytokines and oxidative stress, and activated the Nrf2-HO-1 axis. Our data suggest the potential for TADIOS to be developed as a safe and effective therapeutics for the treatment of acute respiratory distress syndrome.

Lee W ，Lee CH ，Lee J ，Jeong Y ，Park JH ，Nam IJ ，Lee DS ，Lee HM ，Lee J ，Yun N ，Song J ，Choi S ，Kim S ... -  《-》

Ethanol extracts of Rhaponticum uniflorum (L.) DC flowers attenuate doxorubicin-induced cardiotoxicity via alleviating apoptosis and regulating mitochondrial dynamics in H9c2 cells.

Loulu flowers (LLF) is the inflorescence of Rhaponticum uniflorum (L.) DC. (R. uniflorum), a member of the Compositae family. This plant possesses heat-clearing properties, detoxification effects, and is therefore frequently used for the treatment of cardiovascular diseases. This study aimed to investigate the cardioprotective effects of ethanol extracts of LLF against doxorubicin (DOX)-induced cardiotoxicity and explore the associated mechanisms. Ethanol extracts of LLF were prepared and analyzed by LC-ESI-MS/MS. DOX-treated H9c2 cells and DOX-treated zebrafish models were used to explore the cardioprotective effect of ethanol extracts on myocardial function. The effects of LLF on DOX-induced cytotoxicity in H9c2 cells were investigated by MTT assay. Reactive Oxygen Species (ROS) levels, mitochondrial membrane potential (MMP), and nuclear translocation of NF-κB p65 were examined using fluorescent probes. The expression level of Bax, Bcl-2, PARP, caspase-3, cleaved-caspase3, caspase9, IκBα, p-IκBα, IKK, p-IKK, p65, p-p65, OPA1, Mfn1, MFF and Fis 1 and GAPDH was determined by western blotting. Twenty-five compounds were detected in ethanol extracts of LLF, include Nicotinamide, Coumarin, Parthenolide, and Ligustilide. Pre-treatment with LLF attenuated the DOX-induced decrease in viability and ROS production in H9c2 cells. Moreover, LLF treatment maintained the mitochondrial membrane integrity and suppressed apoptosis by upregulating expression level of Bcl-2 and downregulating the expression level of Bax, cleaved-caspase-3, cleaved-caspase-9 and cleaved-PARP. In addition, LLF significantly inhibited the DOX-induced activation of NF-κB signaling. Cells treated with DOX showed aberrant expression of mitochondrial dynamics related proteins, and these effects were alleviated by LLF pre-treatment. In conclusion, these results show that LLF can alleviate DOX-induced cardiotoxicity by blocking NF-κB signaling and re-balancing mitochondrial dynamics. Ethanol extracts of LLF is a potential treatment option to against DOX-induced cardiotoxicity.

Hu B ，Zhen D ，Bai M ，Xuan T ，Wang Y ，Liu M ，Yu L ，Bai D ，Fu D ，Wei C ... -  《-》