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Fennel Main Constituent, Trans‑Anethole Treatment Against LPS‑Induced Acute Lung Injury by Regulation of Th17/Treg Function

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Fennel Main Constituent, Trans‑Anethole Treatment Against LPS‑Induced Acute Lung Injury by Regulation of Th17/Treg Function MOLECULAR MEDICINE REPORTS 18: 1369-1376, 2018 Fennel main constituent, trans‑anethole treatment against LPS‑induced acute lung injury by regulation of Th17/Treg function SICHAO ZHANG1*, XI CHEN1*, ICHINKHORLOO DEVSHILT1*, QI YUN2, CONG HUANG1,3, LIJUN AN1, SOSORBURAM DORJBAT1 and XIN HE1 1School of Chinese Materia Medica, Tianjin University Traditional Chinese Medicine, Tianjin 300193; 2Xinjiang Tefeng Pharmaceutical Co., Ltd., Urumqi, Xinjiang 830054; 3School of Basic Medicine, Guiyang College Traditional Chinese Medicine, School of Basic Medicine, Guiyang, Guizhou 550002, P.R. China Received October 30, 2017; Accepted April 25, 2018 DOI: 10.3892/mmr.2018.9149 Abstract. Fennel, commonly used in traditional Chinese IL-10 mRNA expression in isolated lung tissues and resulted medicine, is known as Foeniculum vulgare Mill. And its in a marked elevation in Treg cells and reduction in Th17 cells clinical application has been shown to target many biological in spleen tissues. The results of the present study indicated systems including gastroenterology, endocrinology, gyne- that the main constituent of fennel, trans-anethole may be an cology and respiratory. The main constituent of the fennel anti‑inflammation component, which influenced the regulation essential oil is trans-anethole, which has been described to of Th17/Treg responses. Therefore, this medicinal herb may have anti‑inflammatory and antibacterial activities. The aim support a healing effect on diseases of inflammatory. of the present study was to define the anti-inflammatory influence in acute lung injury (ALI)-bearing mice. For Introduction 3 days, ALI-bearing mice were induced by lipopolysaccha- ride (LPS) suspension in normal saline (24 mg/kg). On the Foeniculum vulgare is from the Umbelliferae family of plants fourth day, the trans-anethole was administrated (36.4, 72.8 and is used as an antiaging agent to promote longevity. It is or 145.6 mg/kg) as well as dexamethasone (5 mg/kg) once per more commonly called fennel and widely used as a medicine day for 7 consecutive days in mice. Following the completion especially in Asia, but also in other places worldwide (1,2). of drug administration mice were sacrificed. Hematoxylin Traditional practices and the scientific assessment of fennel and eosin staining was performed in the lung paraffin section, indicate that it is a widely applied herbal plant in human health for comparisons between monocyte and eosinophil cells in care in many developing and developed countries (3). In fact, bronchoalveolar lavage fluid. The relative gene expression of it has been attested to have a number of therapeutic properties interleukin (IL)-10 and IL-17 was determined by reverse tran- for the treatment of internal organs, especially in the endo- scription-quantitative polymerase chain reaction. These two crine, gastrointestinal tract and respiratory systems (4-6). In cytokines and the proportion of T helper 17 (Th17) cells and addition, fennel is widely used for the treatment of inflamma- T regulatory (Treg) cells were determined by flow cytometry. tory diseases in conventional medicine (7). The main constituent of fennel, trans-anethole, eliminated The volatile chemical composition of wild fennel samples LPS-induced histopathological changes, decreased the number harvested in different geographical areas has been widely of inflammatory cells and resulted in a notable reduction in reported (8). Trans-anethole [1-Methoxy-4-(E)-propenyl-ben IL-17 mRNA expression. In addition, trans-anethole increased zene] is an organic compound isolated from fennel (9). The uses of fennel have been evaluated in a number of biophar- macological studies that suggest that the various constituents of fennel (seeds, leaves and fruits) are rich in molecules with antioxidant activity (10-12). Furthermore, the extracts and Correspondence to: Dr Xin He, School of Chinese Materia components of fennel have been reported to have anti‑inflam- Medica, Tianjin University Traditional Chinese Medicine, matory, anti-bacterial and antiviral effects (13-15). 312 Anshanxi Road, Tianjin 300193, P.R. China E-mail: [email protected] In 1967, acute lung injury (ALI) was defined as mild acute respiratory distress syndrome (ARDS), with a P/F *Contributed equally ratio of <300 mmHg (40 kPa) and positive end-expiratory pressure/continuous positive airway pressure levels of >5 cm Key words: fennel, acute lung injury, trans-anethole, T regulatory H2O, according to the Berlin Definition (16). Classical signs of cells, T helper cell 17 ALI include accumulation of neutrophils, increased alveolar fluid and pro-inflammatory cytokines in the lungs (17). Worldwide, 5% of the population succumb to ALI per year. 1370 ZHANG et al: TRANS-ANTHOLE TREATMENT CONTRIBUTES TO LPS-INDUCED ACUTE LUNG INJURY This syndrome involves acute inflammation of the bronchi in Materials and methods the lungs. The clinical manifestations of ALI are a productive cough, wheezing, shortness of breath, fever and chest disten- Animals and cell culture. According to the guidelines of the tion. Regeneration of the alveolar epithelium after ALI is Tianjin University of Traditional Chinese Medicine (Tianjin, important for maintaining homeostasis, and the latest studies China) and the Animal Ethics Committee of Laboratory demonstrate that the immune system may influence the protec- Animal Care, all animal studies received ethical approval. tion of epithelial surfaces by increasing barrier function and Sixty healthy 5-week-old male C57BL/6 mice (18-22 g) supporting healing (18,19). were ordered from the Animal Research Center of the A recent study demonstrated that CD4+ T lymphocytes, Chinese Academy of Medical Sciences (Beijing, China) and particularly T helper 17 (Th17) cells and regulatory T cells were housed under suitable conditions. Mice were raised on (Tregs) are an essential subject for research into the etiology or standard water and food. pathogenesis of ARDS. Th17 cells, a recently identified active subdivision of CD4+ T cells and the main origin of interleukin Drugs and reagents. LPS, dexamethasone and trans-anethole (IL)-17 (IL-17A), play a major role in protecting the body were purchased from Sigma-Aldrich (Merck KGaA, from microorganisms, such as Staphylococcus aureus (20,21). Darmstadt, Germany). Oligo DNA was purchased from Patients who suffer from ALI/ARDS have increased levels Shanghai Bio-engineering Co. (Shanghai, China). TransScript of T-cell proliferation and activation, especially Th17 First-Strand cDNA Synthesis SuperMix was obtained from cells (22). Tregs act in an anti‑inflammatory role primarily TransGen Biotech (Beijing, China). SuperReal PreMix Plus by contact-dependent destruction of immunocytes or release was obtained from Tiangen (Beijing, China). PE-labeled of cytokines like IL-10 (23). A reduction in the proportion anti-mouse IL-17 and IL-10, fluorescein isothiocyanate of Treg cells or a decrease in Treg cell function is associated (FITC)-labeled anti-mouse CD4, Alexa Fluor® 647-labeled with a number of autoimmune disorders (24). Tregs alter anti-mouse Foxp3, anti-mouse M1/70+ and anti-mouse F4\80+ innate immune reactions during lung injury and may play a were purchased from Santa Cruz Biotechnology, Inc. (Dallas, role in treating ALI as indicated by Guo et al (25). Mature TX, USA). The PE anti-mouse Foxp3 Staining Set was from Tregs can be classified as inducible Tregs (iTregs) or natural eBioscience (Thermo Fisher Scientific, Inc., Waltham, MA, Tregs (nTregs). iTregs are able to perform their inhibitory USA). function by producing immunosuppressive cytokines like IL-10 (26). Tregs differ from Th17 cells and have a powerful Animal model and experimental procedures. These sixty mice pro‑inflammatory action by generating IL‑17. In pre‑clinical were allowed to adapt to normal laboratory conditions during and clinical trials on pulmonary and autoimmune diseases, the experiment. The experimental groups (10 mice/group) were Th17 cells have been reported to play a role (27-30). The as follows: normal control, LPS-induced model, trans-anethole IL-17 from Th17 cells inhibits Treg differentiation (31,32). treatment (low: 36.4 mg/kg; medium: 72.8 mg/kg; and high: Additionally, IL‑17A could be a pro‑inflammatory cytokine 145.6 mg/kg) and dexamethasone treatment (5 mg/kg). As and may play a key role in ALI induced by lipopolysaccharide presented in Fig. 1, the pulmonary delivery model was estab- (LPS), as shown by previous studies (33,34). In addition, lung lished in normal mice. The C57BL/6 mice bronchitis model injury by Bordetella pertussis is promoted by increased levels was induced by intratracheal instillation of LPS suspension in of Th17 cells (35). However, the main mechanism of CD4+ normal saline (24 mg/kg) for 3 days. On the 4th day, the mice T cells in ALI requires further investigation. These studies were administered trans-anethole (36.4, 72.8 or 145.6 mg/kg) have revealed that mice with LPS-induced lung injury may be and dexamethasone (5 mg kg/kg) once a day for 7 consecutive treated by both Cozaar™ and alanylglutamine, which act by days. The mice were sacrificed 24 h after the end of treatment. decreasing the Th17 cell immune responses and affecting the Th17/Treg balance in favor of Treg cells (36). The pathological Histopathology analysis. The lung tissue was stained with mechanism of autoimmune and inflammatory disease involves hematoxylin and eosin and the inflammatory changes and a balance between Th17 and Tregs. A Treg/Th17 imbalance morphological lesions were analyzed using a light microscope is associated with the occurrence of autoimmune diseases. (magnification, x400). Further investigation into these cell populations in airway inflammation may improve the understanding of the immune Inflammatory cell count in BALF. A total of 50 µl/g sterile mechanism of lung disease. saline was lavaged twice, then the BALF was collected and In the present study, we first established a model of centrifuged at 3,000 x g for 15 min. The white blood cells in LPS-induced ALI and then treated the mice with fennel vola- the BALF were divided and counted into two types, containing tile oil.
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