| Acute lung injury(ALI)is a life-threatening medical disease with high morbidity and mortality.ALI refers to the damage of alveolar epithelial cells and pulmonary capillary endothelial cells caused by non-cardiogenic factors.The clinical manifestations are respiratory distress and refractory hypoxemia,which can further develop into acute respiratory distress syndrome.ALI can cause excessive inflammation,trigger a large amount of neutrophils to penetrate the lung tissue,and induce the release of inflammatory cytokines and the damage of lung endothelial cells and epithelial cells.Lipopolysaccharide(LPS),also known as endotoxin,is the main component of the gram-negative outer membrane.It has obvious pathogenicity and is one of the important causes of ALI.Most of the existing drugs for the treatment of ALI are corticosteroid drugs,but these drugs usually cause serious side effects.Therefore,the development of potentially safe and effective drugs without side effects is of great significance for the treatment of ALI.Due to its special characteristics,such as low toxicity or non-toxicity,small size,and high solubility,the new nano-components gradually developed in the field of biological applications have opened a broad prospect for a new generation of nano-medicine for the treatment of various diseases.In addition,a large amount of evidence shows that nanocomponents derived from different precursors or preparation conditions exhibit different properties,mainly in terms of size,charge,and chemical groups.These differences in properties have been shown to be closely related to the biological activities of nano-components,which further explains the different activities of nano-components found so far,including antibleeding,anti-inflammatory and antioxidant effects.On this basis,the potential of nanocomponents in the control or treatment of other diseases such as ALI has not yet been discovered and is worth studying has become the focus of current research.In this study,we used nanoscience technology to find that a new substance,the nano-component of Armeniacae Semen Amarum carbonisata(ASAC-NCS),was produced in the process of burning Armeniacae Semen Amarum into charcoal from a new perspective,and further studied the protective effect of this nano-component on LPS-induced ALI in rats and its possible mechanism.Objective:(1)The preparation process parameters and structural characteristics were combined to preliminarily screen the optimal preparation conditions of ASAC-NCs.(2)Pharmacodynamic experiments were further utilized to optimize the best preparation process of ASAC-NCs.(3)Characterization information such as microstructure and optical characteristics of ASAC-NCs isolated from ASAC was fully obtained,and the fluorescence quantum yield was calculated.(4)CCK-8 cytotoxicity test was used to preliminarily study the safety of ASAC-NCs.(5)The protective effect of ASAC-NCs on LPS-induced ALI in rats was studied,and its possible mechanism was initially explored,so as to clarify the new material basis of ASAC.Methods:(1)The preparation conditions of ASAC-NCs were optimized by using temperature and time as the standard and combining with the principle of "charcoal storage".Transmission electron microscope(TEM)and high resolution transmission electron microscope(HRTRM)were utilized to observe and compare the microstructure characteristics of ASAC-NCs prepared under different conditions,while using ultraviolet-visible spectrophotometer(UV-vis)and fluorescence spectroscopy(FL),Fourier Infrared Spectroscopy(FT-IR)and other methods to compare the optical characteristic information of the ASAC-NCs prepared under different conditions,and preliminarily optimize the preparation conditions.(2)Pharmacodynamic experiments such as the xylene-induced ear swelling experiment in mice and the effect of the ASAC-NCs on the four parameters of blood coagulation in rats were utilized to further screen the optimal preparation process conditions of ASAC-NCs.(3)High performance liquid chromatography(HPLC)was utilized to eliminate the interference of small molecules.The analysis method of TEM,HRTRM and X-ray diffraction technology(XRD)were utilized to obtain the microstructure of ASAC-NCs under the optimal preparation process conditions,such as morphology,size,distribution characteristics,lattice spacing,etc.information.UV-Vis and FL methods were used to analyze the optical characteristics of the ASAC-NCs.FTIR and X-rays Photoelectron spectroscopy(XPS)were utilized to analyze the surface functional group distribution and element composition of the ASAC-NCs.(4)The CCK-8 cytotoxicity test was utilized to preliminarily evaluate the safety of ASACNCs.(5)Histological study was utilized to observe the protective effect of ASAC-NCs on LPSinduced ALI in rats.IL-1β,IL-6 and TNF-α in serum and lung tissue myeloperoxidase(MPO),malondialdehyde(MDA),superoxide dismutase(SOD),glutathione(GSH)and other indicators were determined to preliminarily explore their possible mechanisms.Results:(1)Comparing the AS AC prepared under different temperature and time conditions,it was found that 300℃0.5h,300℃lh and 350℃0.5h are relatively reasonable for ASAC by using the principle of "charcoal storage" as the standard.After processing,the characters of ASAC remained relatively intact,and the carbonization yield was relatively stable between 80%-87%.In addition,TEM and HRTEM were used to observe that the ASAC-NCs prepared under the conditions of 300℃0.5h,300℃lh and 350℃0.5h had better particle size distribution in this experiment.At the same time,spectrometers were further used to compare the ASAC-NCs prepared under various temperature and time conditions in this experiment,and results showed that the ASAC-NCs prepared under different temperature and time have certain commonalities in optical characteristics,such as having certain fluorescence characteristics,and the surface contains functional groups such as hydroxyl,carboxyl and amino groups.At the same time,the results also showed certain differences,such as the position and strength of functional groups.(2)The anti-inflammatory effects of ASAC-NCs prepared under different conditions were evaluated through the xylene-induced ear swelling experiment.The experimental results showed that ASAC-NCs prepared in the 300℃0.5h group and 300℃lh group are more effective and stable in the treatment of xylene-induced ear swelling in mice.Further experiments were used to detect the effects of ASAC-NCs on rat blood coagulation and platelets showed that the preparation of ASAC-NCs at 300℃ for 1h had the best pharmacological effect.Finally,the optimal preparation conditions for ASAC-NCs were screened at 300℃ for 1h.(3)The ASAC-NCs extracted and separated at 300℃ for 1h was characterized in detail by using modern instruments such as TEM,HRTRM,UV-Vis,FL,FTIR,XRD,XPS,etc.,The results showed that ASAC-NCs,which were mainly composed of three elements of C,O,and N,were a kind of spherical particles with a diameter of 1.5-5.5 nm in microstructure,with a lattice spacing of about 0.215 nm.The optical characteristics showed that the maximum excitation wavelength was 353 nm,the maximum emission wavelength was 436 nm,the fluorescence quantum yield was 3.17%,and the surface had hydrophilic groups such as hydroxyl,carboxyl,and amino groups.(4)In the LPS-induced rat ALI model,the ASAC-NCs with abundant groups on the surface had a significant anti-inflammatory effect,which was preliminarily proved to reduce serum inflammatory cytokines(including IL-6,IL-1β is related to TNF-α)and reduce lung inflammation to a certain extent.On the one hand,ASAC-NCs could reduce the content of MPO and MDA in lung tissues,indicating that ASAC-NCs could reduce the damage to lung inflammation by enhancing the antioxidant effect.On the other hand,ASAC-NCs could increase the activity of SOD and the content of GSH in lung tissue to a certain extent,which also implies the antioxidant activity of ASAC-NCs.ASAC-NCs had a certain protective effect on LPS-induced ALI,and its preliminary mechanism was possibly related to its antioxidant activity and inhibiting inflammation.Conclusion:In this study,the optimal preparation process conditions of ASAC-NCs at 300℃ for 1h were selected by taking temperature and time as the conditions,processing appearance and structural characteristics as indicators,and combining with pharmacological and pharmacodynamic experiments.A novel type of nano-components was successfully isolated from the ASAC.In the LPS-induced ALI model in rats,it could be seen from the pathological slices of lung tissues that the ASAC-NCs with abundant groups on the surface exerted an obvious protective effect,and the mechanism of its effect was initially proved to be related to the reduction of the levels of inflammatory cytokines such as IL-6,IL-1β and TNF-α.It could also reduce lung inflammation to a certain extent and enhance the body’s antioxidant activity.This study provides an important basis for the application of ASAC-NCs in clinical antipneumonia and lays an experimental foundation for the research and development of ASACNCs. |
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