Fengjuan Yuan1, Rui Liu2, Mengying Hu3, Xiaojuan Rong3, Liping Bai2, Lei Xu3, Yan Mao3, Hamulati Hasimu3, Yuhua Sun4, Jinhua He5. 1. Xinjiang Institute of Materia Medica, Urumqi 830004, China; Xinjiang Uygur Autonomous Region for Food and Drug Control, Urumqi 830002, China. 2. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050,China. 3. Xinjiang Institute of Materia Medica, Urumqi 830004, China. 4. Xinjiang Institute of Materia Medica, Urumqi 830004, China. Electronic address: 1641636482@qq.com. 5. Xinjiang Institute of Materia Medica, Urumqi 830004, China. Electronic address: hejh1216@163.com.
Abstract
BACKGROUND: Hyssopus cuspidatus Boriss has been used to treat bronchial asthma for many years in Uighur medicine. JAX2, an ethanol extract from this plant, has effectiveness against bronchial asthma. However, the molecular basis for the anti-inflammatory effects of JAX2 remains unclear. PURPOSE: This study aimed to evaluate the mechanism of JAX2 against bronchial asthma. METHODS: We established an asthma model in rats using ovalbumin (OVA), and an inflammatory model in RAW264.7 cells using lipopolysaccharide (LPS) stimulation. To evaluate the anti-inflammatory effects of JAX2, the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-6, IL-17, eotaxin and immunoglobulin (Ig)E were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was investigated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay. Further, nitric oxide (NO) and reactive oxygen species (ROS) were determined using Griess reagent and 2,7-dichlorofluorescein diacetate. The phosphorylation of p-extracellular signal-regulated kinase (ERK), p-Jun-NH2-terminal kinase (JNK), p38 kinases (p38) and p-inhibitor of nuclear factor kappa-B kinase (IKK), and nuclear translocation of p-p65 kinases (p-p65) were determined by immunofluorescence to uncover the effects of JAX2 on the Mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways. RESULTS: After JAX2 administration to rats, Interferon (IFN)-γ concentrations in BALF increased significantly. Further, the concentrations of TNF-α, IL-4, IL-6, IL-17 and eotaxin in BALF, and IgE in serum decreased. JAX2 decreased TNF-α, IL-6 and NO in cell supernatant, and reduced ROS intracellularly. Concurrently, IFN-γ concentrations increased in cell supernatant significantly. In LPS-induced RAW264.7 cells, JAX2 inhibited phosphorylation of p-ERK, p-JNK and p-38 MAPK. The subsequent phosphorylation of p-IKK and nuclear translocation of the p-p65 subunit of NF-κB were also suppressed. CONCLUSION: Based on these findings, we believe that JAX2 has both preventive and treatment effects in bronchial asthma. Furthermore, in the RAW264.7 cell inflammatory model, JAX2 also inhibited NF-κB and MAPK signaling pathways.
BACKGROUND:Hyssopus cuspidatus Boriss has been used to treat bronchial asthma for many years in Uighur medicine. JAX2, an ethanol extract from this plant, has effectiveness against bronchial asthma. However, the molecular basis for the anti-inflammatory effects of JAX2 remains unclear. PURPOSE: This study aimed to evaluate the mechanism of JAX2 against bronchial asthma. METHODS: We established an asthma model in rats using ovalbumin (OVA), and an inflammatory model in RAW264.7 cells using lipopolysaccharide (LPS) stimulation. To evaluate the anti-inflammatory effects of JAX2, the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-6, IL-17, eotaxin and immunoglobulin (Ig)E were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was investigated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay. Further, nitric oxide (NO) and reactive oxygen species (ROS) were determined using Griess reagent and 2,7-dichlorofluorescein diacetate. The phosphorylation of p-extracellular signal-regulated kinase (ERK), p-Jun-NH2-terminal kinase (JNK), p38 kinases (p38) and p-inhibitor of nuclear factor kappa-B kinase (IKK), and nuclear translocation of p-p65 kinases (p-p65) were determined by immunofluorescence to uncover the effects of JAX2 on the Mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways. RESULTS: After JAX2 administration to rats, Interferon (IFN)-γ concentrations in BALF increased significantly. Further, the concentrations of TNF-α, IL-4, IL-6, IL-17 and eotaxin in BALF, and IgE in serum decreased. JAX2 decreased TNF-α, IL-6 and NO in cell supernatant, and reduced ROS intracellularly. Concurrently, IFN-γ concentrations increased in cell supernatant significantly. In LPS-induced RAW264.7 cells, JAX2 inhibited phosphorylation of p-ERK, p-JNK and p-38 MAPK. The subsequent phosphorylation of p-IKK and nuclear translocation of the p-p65 subunit of NF-κB were also suppressed. CONCLUSION: Based on these findings, we believe that JAX2 has both preventive and treatment effects in bronchial asthma. Furthermore, in the RAW264.7 cell inflammatory model, JAX2 also inhibited NF-κB and MAPK signaling pathways.