Protective Effect And Mechanism Of Echinacea Polyphenols On LPS Induced Acute Lung Injury

Acute lung injury（ALI）and acute respiratory distress syndrome（ARDS）are common life-threatening clinical syndromes characterized by diffuse alveolar damage,pulmonary edema,and uncontrolled inflammatory response.In recent years,ALI has been reclassified as moderate to mild ARDS.The clinical manifestations of ALI/ARDS are bilateral infiltration,severe hypoxemia,non-cardiogenic pulmonary edema,and eventually respiratory failure.At present,the clinical efficacy of mechanical ventilation and glucocorticoids is limited,and the mortality rate is still as high as 40%.Therefore,it is urgent to find new therapeutic agents and therapeutic targets for ALI/ARDS.Natural drugs are abundant in resources.Screening exogenous compounds for prevention and control of ALI is an important way to solve the current clinical problems of ALI.Echinacea,a perennial plant of the Asteraceae family native to North America,has been documented in several pharmacopeias and other monoids since 1999 due to its high nutritional and medicinal value.In this paper,we systematically investigated whether echinacea polyphenol（EPP）has a protective effect on LPS-induced ALI,and further studied the protective mechanism of EPP against ALI.The main research contents and results are summarized as follows:1.Acute toxicity test and subchronic toxicity test of EPPKunming SPF mice were given EPP by gavage,and four dose groups of EPP（30.8 g/kg,15.4 g/kg,7.7 g/kg,3.85 g/kg）and normal saline were set for acute toxicity test.No mice in each group died,and the mental activity state was not abnormal.Ten specific pathogen-free Kunming mice were divided into 30.8 g/kg EPP group and normal saline group by gavage（0.8 m L/20 g）twice within 24 hours.The results showed that no death occurred in the mice within 7 days,the mental activity was normal,and no abnormal lesions were found in any organs.Therefore,the LD₅₀of EPP was greater than 30.8 g/kg without obvious acute toxic effects.The rats were given three doses of EPP（30.8 g/kg,15.4 g/kg,and 7.7 g/kg）by gavage once a day for 28 days.The results showed that EPP had no significant effect on the body weight,blood routine,serum biochemical indexes,and organ coefficient,and there was no subchronic toxic effect.2.Protective effect and mechanism of EPP on LPS-induced ALI in miceThe model of ALI was established by direct tracheal infusion of LPS solution into the glottis cleft in mice,and the LPS solution flowed into the lung with spontaneous respiration.In the formal test,mice were given EPP solution once a day for seven consecutive days before LPS solution was injected.After LPS administration,the clinical symptoms of the mice were observed.12 h later,the blood of the mice was collected through the retroorbital venous plexus.After anesthesia,the lungs of the mice were dissected,observed and collected.HE histopathologic sections were used to observe the lung tissue injury in each group.Lung wet-dry ratio and lung coefficient were calculated to evaluate the degree of pulmonary edema.Serum concentrations of inflammatory factors（TNF-α,IL-18,IL-1β）and MPO activity were determined by ELISA and myeloperoxidase（MPO）assay kit,respectively.The protein expression level in mouse lung tissue were detected by Western blot.Autopsy and HE showed that EPP could significantly inhibit the swelling,hardening and bleeding of lung tissue,inflammatory cells and red blood cell infiltration,alveolar septum thickening,and alveolar structure disappearance caused by LPS.The results of ELISA showed that EPP could significantly reduce the content of inflammatory factors in serum of model group mice.Western Blot showed that EPP inhibited the expression levels of NLRP3 inflammasome,pyroptosis,apoptosis and necroptosis-related proteins in the lung tissue of ALI mice.3.Protective effect and mechanism of EPP on LPS treated A549 cellsThe appropriate concentration and time of LPS and EPP were determined by CCK-8 test.NLRP3 expression in A549 cells was detected by immunofluorescence assay.The ratio of apoptosis and necroptosis cells was determined by flow cytometry and YO-PRO-1/PI detection kit.Western Blot was used to investigate the protein expression in A549 cells.In the research of this chapter,EPP concentrations of 40μg/m L and 80μg/m L were used for 2 h,followed by 10μg/m L LPS for 22 h.Immunofluorescence assay showed that EPP inhibited NLRP3 overexpression in A549 cells induced by LPS.The results of flow cytometry and YO-PRO-1/PI kit showed that EPP inhibited the apoptosis and necroptosis of A549 cells induced by LPS.Western Blot showed that EPP had a protective effect on LPS-treated A549 cells by reducing the expression levels of NLRP3 inflammasome,pyroptosis,apoptosis and necroptosis-related proteins.The i NOS inhibitor SMT was used to pretreat A549 cells,and the effect was compared with EPP to explore the role of NO in PANoptosis signaling pathway in ALI and the mechanism of EPP to inhibit PANoptosis in ALI.The results showed that 80μg/m L EPP and 0.1 mg/m L SMT inhibited the increase of i NOS expression and NO production in LPS-induced cells.Both 0.1 mg/m L SMT pretreatment and 80μg/m L EPP pretreatment reduced the expression levels of NLRP3 inflammasome,pyroptosis,apoptosis and necroptosis-related proteins,and inhibited LPS-induced apoptosis and necroptosis in each group.The results indicated that EPP may inhibit PANoptosis by inhibiting the expression of i NOS and the production of NO in LPS-induced ALI.