| Acute lung injury(ALI)is a progressive respiratory disease caused by trauma,infection and other factors,which is an over-dramatic and uncontrolled acute inflammatory response in essence.It is characterized by the disruption of endothelial and epithelial integrity,neutrophils infiltration,pulmonary permeability edema,uncontrolled oxidative stress and inflammatory response.The pathogenesis of acute lung injury is complicated.It mainly focus on symptomatic supportive treatment in clinic,but the mortality remains high for many years.Thus,it is urgent to develop effective medications and innovative therapies.Garlic oil has been considered as the main active ingredient of garlic which possesses various pharmacological properties,including anti-bacteria,anti-cancer,anti-inflammation,anti-oxidation and immune regulation activities.Although the current research on the formulation and pharmacological effects of garlic oil has been continuously developing,there are no reports on related aerosol products or prevention effect on ALI.L-Menthol is an anthraquinone with antioxidant,antibacterial,anti-inflammatory and other activities.The previous results of the research group show that L-Menthol is compatible with garlic sulfide,which has the functions of cooling and reducing the pungent stimulating effect of sulfide,and does not affect the pharmacological activity and metabolic kinetics of sulfide.Therefore,the development of a compound aerosol of garlic oil and L-Menthol has broad application prospects.In summary,the project aims to develop a garlic oil compound aerosol for the treatment of ALI.The main contents include:(1)Pre-prescription study of garlic oil compound aerosol;(2)The anti-inflammatory effect study of garlic oil compound preparation in vitro;(3)The prevention effect and mechanism of garlic oil compound preparation on lipopolysaccharide(LPS)-induced ALI rats.The main contents of the pre-prescription study of garlic oil compound aerosol included the screening of propellants and excipients,the establishment of pharmaceutical analysis methodology and the preliminary quality evaluation.The prescription screening of garlic oil compound aerosol included the selection of appropriate propellants,determination of solubility of drug in propellants,screening of appropriate surfactant,thus the formulation of the garlic oil compound aerosol was initially determined to be garlic oil,L-Menthol,0.86%Span-85 and an appropriate amount of hydrofluoroalkane propellant.In order to make a preliminary evaluation of garlic oil compound preparation,a GC-MS method for the simultaneous detection of L-Menthol and organic sulphides in garlic oil was established and verified.Then,the preliminary stability study was carried out by using the accelerated test and the long-term test.The methodological experiments results showed that the established GC-MS method was accurate and reliable,and was suitable for the analysis of various components in garlic oil compound preparation.The preliminary preparation evaluation results showed that the garlic oil compound preparation should be stored in dark place at low temperature.The study on anti-inflammatory effect of garlic oil compound preparation in vitro consisted of the establishment of acute inflammation model in NR8383 cells and the evaluation of anti-inflammatory effect and mechanism.Firstly,we used LPS-induced NR8383 cells as an acute inflammation model,and then the expressions of NO,TNF-α,IL-6 and NF-κB were determined after administration.The levels of NO in the supernatant of the cells were detected using a NO assay kit.The mRNA expression of TNF-α and IL-6 were measured by RT-PCR.The expressions of iNOS,NF-κB p65 and IκBaα were determined by western blot.The results revealed that the garlic oil compound preparation could significantly reduce the amount of NO by inhibiting the expression of iNOS,while weaken the expressions of TNF-α and IL-6 at the gene level in a dose-dependent manner.The anti-inflammatory effect of the garlic oil compound preparation was achieved by inhibiting the activation of NF-κB signal pathway in vitro.Studies on the protective effect and mechanism of garlic oil compound preparation on ALI rats included the establishment of ALI model,the evaluation of anti-inflammatory and antioxidant effects and the exploration of signal pathways.The ALI rat model was established by intratracheal instillation of LPS.A CHT inhalation exposure system was used to achieve the inhalation administration.The protective effect of garlic oil compound preparation on ALI was evaluated by counting inflammatory cells,calculating the lung wet-to-dry weight ratio,measuring inflammatory and oxidation cytokines in bronchoalveolar lavage fluid and lung homogenate,and obversing the morphological changes of lung tissue.In addition,western blot and immunohistochemistry were used to investigate the molecular biological mechanism.The results showed that the garlic oil compound preparation could reduce the total number of inflammatory cells and neutrophils in lung tissue of ALI rats,ameliorated histopathological changes and attenuate pulmonary edema,which revealed a protective effect on ALI rats.Beyond that,the garlic oil compound preparation decreased TNF-α,IL-1β and IL-6 secretions,increased IL-10 secretion,down-regulated MPO and MDA levels,as well as up-regulated the SOD activity,which reversed the imbalance of inflammation and oxidative stress caused by LPS.The western blot and immunohistochemistry results suggested that the mechanism of garlic oil compound medicine against ALI in vivo might be associated with the inhibition of NF-κB signal pathway and the activation of Nrf2 signal pathway.In conclusion,the project first carried out the preliminary pre-prescription study of garlic oil compound aerosol,and then evaluated the protective effect and mechanism of the new preparation on the ALI in vitro and in vivo.The results showed that the newly developed garlic oil compound preparation could effectively inhibit both inflammation and oxidative stress induced by ALI through regulating NF-κB and Nrf2 signaling pathways simultaneously.Therefore,it is expected to be developed into a specific medicine with multiple targets for the treatment of ALI through further research in the future. |
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