The Prevention Of Chlorogenic Acid And Its Cyclodextrin Inclusion Complexes On Lung Injury

Lung injury is a disease caused by a variety of direct and indirect traumatic factors.When lung injury occurs,the alveolar capillary endothelium-epithelial barrier is impaired,inflammatory cell infiltration,uncontrolled inflammatory response,and oxidative stress damage cause a large number of neutrophils accumulated in the lung to be activated.Oxidative stress caused by a large number of oxygen free radicals and reactive oxygen species causes diffuse pulmonary interstitial and alveolar edema and hypoxic respiratory insufficiency.Acute respiratory distress syndrome?ARDS?can be induced in severe cases,and its morbidity and mortality have been high,which has a great impact on public health.Inducing factors of lung injury include pulmonary infection,inhalation injury?including toxic gas,acid reflux,drowning,etc.?,mechanical injury,iatrogenic lung damage caused by radiotherapy and chemotherapy,severe pancreatitis,sepsis or severe non-Chest trauma and so on.Chlorogenic acid is an important biologically active substance with anti-inflammatory,anti-bacterial,anti-viral,anti-tumor,blood pressure lowering,blood lipid lowering,free radical scavenging and excitatory central nervous system.It is a clinical hotspot in the field of natural products research.However,since it has a plurality of alkylhydroxy groups and a plurality of phenolic hydroxyl groups,it is unstable,and is easily decomposed by heat or light.At present,there are few studies on the anti-oxidation effect of chlorogenic acid in vivo.This paper is divided into two parts:1 Based on the nature of chlorogenic acid and the characteristics of pulmonary inhalation,chlorogenic acid was prepared into a chlorogenic acid inclusion complexs,which can allows the drug to directly act on lung tissue by the intubation administration.The chlorogenic acid inclusion complexs were used to treat acute lung injury?ALI?induced by hydrogen peroxide?H₂O₂?and evaluated its efficacy;2 Established a model of radiation-induced lung injury in mice,and explored whether chlorogenic acid can prevent radiation-induced lung injury?RILI?,and initially explored its underlying mechanism.1 Preparation and properties of chlorogenic acid inclusion complexThe chlorogenic acid inclusion complexs were prepared by a grinding method according to chlorogenic acid:?-cyclodextrin?mol:mol?=1:1,and chlorogenic acid inclusion complex white powders was obtained by lyophilization.The chlorogenic acid inclusion loading amount was 21.9%which was obtained by HPLC method.The angle of repose was 28.98±0.072°by the funnel method,and the fluidity was good.The particle diameter D₅₀ of the chlorogenic acid inclusion complex was 11.57±0.11?m.The bulk density was determined by the cylinder method to be 0.2040±0.0024 g/cm³,and the aerodynamic particle diameter?D_a?was calculated to be 5.35±0.046?m.Scanning electron microscopy showed that the CGA-?-CD powder was granular and relatively uniform.The deposition rate of chlorogenic acid inclusion complex powder in the lungs in vitro is 23.9%,which could effectively enter the deep lungs.2 Cytotoxicity of chlorogenic acidThe cytotoxicity of chlorogenic acid on human bronchial epithelial BEAS-2B cells was examined by MTT colorimetry of tetramethyl azozolium salt.We evaluated the toxicity of chlorogenic acid to BEAS-2B by MTT assay.The results showed that chlorogenic acid at a concentration of 3.91?M to 500?M showed no significant toxicity in BEAS-2B cells and could be used in the next experiment.3 Protective effects of chlorogenic acid and chlorogenic acid inclusion complexs on rats with acute lung injuryAfter the rat model of acute lung injury?ALI?was established by intratracheal injection of H₂O₂,the animals were divided into 5 groups:normal control group;model group;CGA-?-CD lung delivery group;CGA lung delivery group;dexamethasone lung delivery group.HE staining was used to observe the pathological changes of lung tissue.The content of TNF-?and IL-1?in alveolar lavage fluids was detected by ELISA.The content of reduced glutathione?GSH?and malondialdehyde?MDA?were detected in the lung tissues.Histopathology showed that CGA-?-CD and CGA had significant therapeutic effects on H₂O₂-induced ALI.Compared with the model group,the content of TNF-?and IL-1?in the alveolar lavage fluid were significantly decreased in the CGA-?-CD group.In the lung tissue,the level of MDA was significantly reduced,and the level of GSH was significantly increased in the CGA-?-CD group compared to the model group.CGA and CGA-?-CD can alleviate ALI in rats by inhibiting inflammatory factors and alleviating oxidative damage.4 Establishment of a model of radiation-induced lung injuryC57BL/6J mice were irradiated with 14,16 and 18 Gy in the whole lung.The general condition of the mice and the histopathological changes of 4w and 8w were observed.HE staining,Masson staining and the content of IL-6,TNF-?and IL-1?in the alveolar lavage fluid of mice were used to evaluate the model of radiation-induced lung injury.The content of IL-6,TNF-?and IL-1?in the alveolar lavage fluid were determined by ELISA test.Histopathology showed that the alveolar and capillary structures of the normal control group were intact and clear,and there was no manifestation of oozing and exudation.The lung histopathology of the model group had interstitial congestion and edema at 4w and 8w,a large number of inflammatory cell infiltration,and extensive deposition of collagen fibers and extracellular matrix.The content of TNF-?,IL-6 and IL-1?were significantly increased in the alveolar lavage fluid of the model group?P<0.01?.5 Protective effect of chlorogenic acid on mice with radiation-induced lung injuryC57BL/6J mice were randomly divided into normal control group,irradiation control group,administration group?irradiation+treatment?,and positive control group W2721group.The irradiated control group,the drug-treated group and the positive control group WR2721 mice were subjected to a single X-ray chest irradiation with a self-designed fixation and shielding device,and the total dose was 18 Gy.The mice were sacrificed at 4w and 8w after irradiation,respectively.The general condition of the mice was observed after irradiation.The right lung tissues of the 4w and 8w mice were stained with hematoxylin and eosin staining?HE?and Masson to observe the pathological changes of lung tissue in each group.Immunohistochemistry?ELISA?was used to detect the levels of TNF-?,IL-6 and IL-1?in alveolar lavage fluid collected from mice at 4w and 8w;immunohistochemical staining?IHC?was used to detect each the expression of TNF-?,IL-?1 and?-SMA protein in the lung tissues of the 4w and 8w mice in the group.Histopathology showed that CGA had significant therapeutic effects on RILI;compared with the model group,The content of TNF-?,IL-6 and IL-1?in the alveolar lavage fluid of the CGA group were significantly decreased at 4w and 8w in the treated group.CGA can alleviate early lung injury in mouse RILI by inhibiting inflammatory factors and the like.6 Protective effect of chlorogenic acid on radiation-damaged Beas-2B cells and discussion on its mechanism its mechanismFlow cytometry was used to detect the effect of chlorogenic acid on the apoptosis of Beas-2B cells in radiation injury.The protective effect of CGA on DNA damage of Beas-2B cells was detected by?H2AX staining.The fluorescence of ROS in cells was detected after irradiation.The intensity indicated that the ROS in Beas-2B cells treated with CGA for 24 h was significantly reduced,thereby protecting lung epithelial cells.The expression of inflammatory protein TGF-?1 and COX-2 protein was detected by Western Blot.It can be seen that the 250?M CGA pretreatment group canreduce the expression of inflammatory protein.