| Background:Acute lung injury (ALI) is a common clinical critical illness that occurs when the body is in severe infection,shock or trauma,with an acute onset and high mortality rate.In Traditional Chinese Medicine (TCM),it belongs to the category of ’asthma’,’dehiscence’ and ’knotted chest’,and is mainly located in the lung,and the pathogenesis is mostly due to heat pressing blood stasis,stagnation of blood stasis and heat,congestion of the qi mechanism,and failure to promote and descend.As the disease develops,it may involve the large intestine,kidney,heart,spleen and liver.The current treatment for ALI is mainly supportive therapy and pharmacological interventions,including mechanical ventilation,antioxidants,protease inhibitors,complement inhibitors and corticosteroids,but no satisfactory results have been achieved.TCM,based on the principle of evidence-based treatment,has given full play to the characteristics of individualized and precise treatment,and has exerted better clinical efficacy in the prevention and treatment of ALI,but still lacks uniform standards of medication and accessibility to patients.Therefore,screening natural active ingredients based on TCM with anti-inflammatory effects could provide an important source of new drug development for the treatment of ALI.Strictosamide (STR) is a natural indole alkaloid derived from the Chinese herbal medicine Nauclea officinalis,which has various pharmacological effects such as antiinflammatory,antipyretic,analgesic and antiviral.Chinese patent medicines using STR as the main active ingredient are widely used in clinical practice to treat respiratory tract infections such as acute tonsillitis,paediatric viral colds and lower respiratory tract infections,which have the potential to treat ALI.Therefore,exploring the anti-inflammatory mechanism of action of STR against ALI can provide a scientific basis for the development of natural drugs for ALI.Objective:The aim of this study is to investigate the pharmacodynamic evaluation of STR against ALI using a lipopolysaccharide (LPS) induced ALI mouse model,combined with histopathological and molecular biology experiments.The binding ability of the two was verified by protein expression purification techniques,surface plasmon resonance (SPR),cellular thermal shift assay (CETSA),biotin-labeled microarray experiments and molecular docking to obtain key target proteins of STR;finally,we conducted in vitro and in vivo experiments to validate the anti-inflammatory mechanism of the target protein in STR for ALI treatment through LPS-induced ALI mouse model and BEAS-2B cell inflammation model.Methods:1.The LPS intratracheal drip method was used to construct a mouse model of ALI.LPSinduced lung inj ury in mice was assessed by hematoxylin-eosin (HE) staining,lung tissue injury score,lung tissue wet weight/dry weight (W/D) ratio,myeloperoxidase (MPO) assay,and mRNA expression levels of lung tissue inflammatory factors IL-6,TNF-α and IL-10,as well as the therapeutic effect after STR intervention.2.The STR-biotin compound was synthesized by chemical synthesis labeling technology and screened by human proteome microarray to obtain the potential target proteins of STR.Bioinformatics analysis using IPA software was used to obtain key target proteins and related signalling pathways of STR for the treatment of ALI.In vitro expression of STR target proteins was performed with the help of protein expression purification technology,and SPR assays were carried out to verify the binding ability of the two in vitro,CETSA assays to verify the binding ability of the two in vivo,and biotin labeling microarray assays and molecular docking to predict the binding mode of the two.3.In vivo and in vitro experiments were carried out by LPS-induced ALI mouse model and BEAS-2B cell inflammation model.HE staining,lung tissue damage score,lung tissue W/D ratio,MPO assay,mRNA expression levels of lung tissue inflammatory factors IL-6,TNFα and IL-10 were used to clarify the therapeutic effect of STR on ALI,and immunohistochemical experiments,and immunofluorescence assay to verify the staining of target proteins in lung tissues.We also performed immunohistochemical assays and immunofluorescence assays to verify the anti-inflammatory effect of the target proteins in the treatment of ALI by transfecting BEAS-2B cells with the target proteins and the mRNA expression levels of the key proteins of the corresponding signalling pathways.Results:1.After the construction of ALI mouse model by LPS intratracheal drip method,the lung tissues of mice were infiltrated with a large number of neutrophils,macrophages and other inflammatory cells in the interstitium and alveolar space,the integrity of alveoli was destroyed,the alveolar cavity was heavily collapsed,the alveolar wall was thickened and accompanied by alveolar cavity congestion,the pathological score of ALI,the W/D ratio of lung tissues and MPO activity were significantly higher than those of Control group (P <0.01),and inflammatory factors IL-6 and TNF-α were significantly up-regulated (P <0.01) and IL-10 was significantly down-regulated (P <0.01),reflecting the severe damage and inflammatory response in the lung tissue of ALI mice.After intervention with STR 20 mg/kg,40 mg/kg and dexamethasone (DEX) 30 mg/kg,compared with the LPS group,the pathological manifestations of lung tissue injury in mice were significantly improved,and the ALI pathological score,lung tissue W/D ratio and MPO activity were significantly decreased (P <0.01),and the inflammatory factors IL-6,TNF-α were significantly down-regulated (P <0.01)and IL-10 were significantly up-regulated (P <0.01),indicating that STR has good antiinflammatory effects in the treatment of ALI.2.The STR biotin probe was chemically synthesised to obtain strictosamide 4-oxo-4-((2(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamido)ethyl)amino)butanoate,which was characterised by high resolution mass spectrometry and nuclear magnetic resonance hydrogen spectroscopy.The STR-biotin compound has the ability to bind to streptavidin and can be linked to fluorescent moieties for protein microarray experiments.276 STR potential target proteins were screened.The top 5 diseases or functions associated with the 276 potential STR target proteins in the Diseases and disorders analysis module were Cancer,Organismal injury and Abnormalities,Endocrine System Disorders,Infectious Diseases,Gastrointestinal Diseases,and Gastrointestinal Diseases.Infectious Diseases,and Gastrointestinal Diseases.46 STR potential target proteins were included in the infectious diseases most associated with ALI.Among the 46 potential target proteins,the most critical were Extracellular regulated protein kinases (ERK2) (17 links to other proteins),STAT3 (14 links to other proteins),HRAS (13 links to other proteins) and BCL2 (11 links to other proteins).Analysis of the interactions between the 276 target proteins of STR and the 7944 target genes network of ALI showed that they together contain 76 genes,and ERK2 is also at the core of the key 76 target proteins of STR for ALI,with 18 linkages to other proteins.The analysis of signalling pathways based on the 76 target proteins showed that inflammation-related signalling pathways such as NF-κB Signaling,HIF-1α Signaling,TREM1 Signaling,BAG2 Signaling Pathway and IL-23 Signaling Pathway play an important role in STR for ALI.The role of inflammation-related signaling pathways in the treatment of ALI with STR is important.3.The ERK2 plasmid was constructed,and ERK2 protein was expressed and purified based on the E.coli system.The accuracy of the final expression vector was confirmed by enzymatic digestion;the amount of ERK2 protein was measured by SDS-PAGE based on protein gel staining solution and by peptide-based protein mass spectrometry.The results showed that the ERK2 protein expression and purification were as expected and could be used for subsequent STR-ERK2 binding validation experiments.The results of the biotin-labelled microarray showed that ERK2 was significantly more able to bind to STR-biotin compared to biotin,and that ERK2 bound to STR-biotin was degraded but not completely inactivated after heating at 90℃ for 3 min,indicating that there is a spatial structure requirement for ERK2 to bind to STR and that its thermal stability can be enhanced.Molecular docking experiments showed that the affinity between STR and ERK2 was-9.0 kcal/mol and that STR was able to interact with GLY-32 and LYS-52 amino acid residues forming hydrogen bonds,resulting in a stable binding between the two.The results of the CETSA assay showed that the protein degradation of ERK2 gradually increased with increasing temperature in BEAS-2B cells supplemented with STR,but the ERK2 protein was more stable and less degraded at the same temperature compared to the Control group without STR,demonstrating that STR can bind to ERK2 and thus increase the thermal stability of ERK2 protein.In BEAS-2B cells intervened with different STR concentrations,ERK2 was in a stable state at 37℃ with changes in STR concentration,and there was no significant difference in protein content;while at 55℃,the degradation of ERK2 protein increased with decreasing STR concentration,demonstrating that STR concentration also affects the thermal stability of ERK2 protein.The thermal melting curve of ERK2 shifted to the right as the temperature increased,also demonstrating that STR was able to bind to ERK2 in vivo,improving the thermal stability of ERK2 protein.4.In vivo results of the LPS-induced ALI mouse model showed that the Control group had an intact alveolar structure with clear alveolar cavity boundaries and no significant inflammatory cell infiltration in the interstitial spaces of the lung tissues.After the intervention with STR 20 mg/kg, 40 mg/kg,DEX 30 mg/kg and Mitogen-activated protein (MEK) inhibitor U0126 30 mg/kg, the pathological manifestations of lung tissue damage in mice were significantly improved,and the ALI pathological score,lung tissue W/D ratio,and MPO activity were significantly reduced (P <0.01).The pathological manifestations of lung tissue injury in mice with ALI were significantly improved by the intervention of U0126 30 mg/kg,with a significant decrease in ALI pathological score,lung tissue W/D ratio and MPO activity (P <0.05).Immunohistochemical results showed that there was no difference in the positive results of ERK immunolabeling among the six experimental subgroups and the semi-quantitative results were not statistically significant (P> 0.05),while the expression of P-ERK was elevated in the LPS group compared with the Control group in the form of brownish-yellow-colored granules and the semi-quantitative results were significantly different (P <0.01).P-ERK expression was down-regulated in the lung tissues of ALI mice after intervention with STR 20 mg/kg,STR 40 mg/kg, DEX 30 mg/kg and U0126 30 mg/kg (P <0.05),and the immunofluorescence results showed that there was no significant difference in the immunofluorescence labeling results of ERK2 (Red light) and nucleus (Blue light) in the six experimental subgroups;while compared with the Control group,the LPS P-ERK expression was significantly higher in the LPS group compared to the Control group (Green light);P-ERK expression was downregulated in the lung tissues of ALI mice after intervention with STR 20 mg/kg and STR 40 mg/kg;while the downregulation of P-ERK expression in the lung tissues of ALI mice after intervention with DEX 30 mg/kg and U0126 30 mg/kg was better than the effect of the two drug concentrations of STR,suggesting that The results suggest that STR can be used to treat ALI by affecting the phosphorylation level of ERK after targeted binding.5.The results of in vivo experiments on NF-κB signaling pathway protein expression in STR-treated ALI showed that ERK,IκB kinase α (Inhibitor kappa B kinase α,IKKα),IκBα and P65 protein expression were not significantly altered in the six experimental subgroups;compared with the Control group,P-ERK,P-IKKα,P-IκBα and P-P65 protein expression in the LPS group were P-ERK,P-IKKα,P-IκBα and P-P65 protein expression were significantly increased in the LPS group compared to the Control group;after intervention with STR 20 mg/kg,STR 40 mg/kg,DEX 30 mg/kg and U0126 30 mg/kg,P-ERK,P-IKKa,P-IκBα and PP65 protein expression were decreased.semi-quantitative WB analysis showed that compared to the Control group,the expression ratios of P-ERK/ERK,P-IKKα/IKKα,P-IκBα/IκBα and PP65/P65 were up-regulated in the LPS group with significant differences (P <0.01);after STR intervention with a drug concentration of 20 mg/kg,the expression ratios of P-ERK/ERK and P-P65/P65 was down-regulated (P <0.05) and the expression ratios of P-IKKα/IKKα and PIκBα/IκBα were significantly down-regulated (P <0.01);after intervention with STR at a drug concentration of 40 mg/kg,DEX 30 mg/kg and U0126 30 mg/kg,the expression ratios of PERK/ERK,P-P65/P65,P-IKKα/IKKα and P-IκBα/IκBα expression ratios were significantly down-regulated (P <0.01).RT-PCR results of mouse lung tissues showed that compared with Control group,lung tissues of ALI mice showed significant up-regulation of IL-1β,IL-6,IL-8,IL-15,IL-18,IL-33 and TNF-α after LPS tracheal drip,while IL-10 was significantly downregulated,and all were significantly different (P <0.01),indicating that lung tissues of ALI mice showed more severe inflammatory imbalance.After pretreatment with STR at a concentration of 20 mg/kg,the expression of IL-1β,IL-6,IL-8,IL-15,IL-18,IL-33 and TNFa in the lung tissues of ALI mice was reduced and the expression of IL-10 was increased,whereas after pretreatment with STR at a concentration of 40 mg/kg,DEX at 30 mg/kg and U0126 at 30 mg/kg,the expression of IL-10 in the lung tissues of ALI mice was reduced.After pretreatment,the expression of IL-1β,IL-6,IL-8,IL-15,IL-18,IL-33 and TNF-α in lung tissue of ALI mice was significantly down-regulated and IL-10 expression was significantly upregulated (P <0.01),and the effect of DEX was more significant.The experimental results suggest that STR can reduce the inflammatory response in the lung tissue of ALI mice by regulating the inflammatory imbalance.6.The results of STR intervention in the LPS-induced inflammation model of BEAS-2B cells in vitro showed that IKKα,IκBα and P65 protein expression were not significantly altered in any of the eight experimental subgroups,but ERK2 protein expression levels were decreased in all si-ERK plasmid-transfected groups.Compared with the Control group,P-ERK,P-IKKα,P-IκBα and P-P65 protein expressions were significantly increased in the LPS group;after intervention with STR 25 μM and STR 50μM,P-ERK,P-IKKα,P-IκBα and P-P65 protein expressions were all decreased.While P-ERK,P-IKKα,P-IκBα and P-P65 protein expression were significantly higher in the si-LPS group compared to the si-Control group;after intervention with STR 25 μM,STR 50 μM, P-IKKα,P-IκBα and P-P65 protein expression decreased,but due to the decrease in ERK protein expression,P-ERK decreased to a lesser extent.WB semi-quantitative analysis showed that the expression ratios of P-ERK/β-actin, PIKKα/IKKα, P-IκBα/IκBα and P-P65/P65 were up-regulated in the LPS group compared to the Control group with significant differences (P <0.01);at drug concentrations of 25 μM,50 μM STR after intervention,the expression ratios of P-ERK/β-actin,P-IKKα/IKKα,P-IκBα/IκBαand P-P65/P65 were significantly down-regulated (P <0.01).Compared with the si-Control group,the expression ratios of P-ERK/β-actin,P-IKKα/IKKα, P-IκBα/lκBα and P-P65/P65 were upregulated in the si-LPS group with significant differences (P <0.01);after STR intervention with drug concentrations of 25 μM,50 μM,the expression ratios of P-ERK/βactin,P-IKKα/IKKα,P-IκBα/IκBα and P-P65/P65 expression ratios were all down-regulated after STR interventions at 25 μM and 50 pM,but the overall down-regulation effect was weaker than that without si-ERK intervention.RT-PCR results showed that compared with the Control group,the LPS group had significantly higher expression ratios of IL-1β,IL-6,IL-8,IL-15,IL18,IL-33,and TNF-α were significantly upregulated,whereas IL-10 was significantly downregulated,and all were significantly different (P <0.01),indicating a more severe inflammatory imbalance in BEAS-2B cells.After pretreatment with STR at concentrations of 25 μM and 50 μM, the expression of IL-1β,IL-6,IL-8,IL-15,IL-18,IL-33 and TNF-α begin to be reduced and that of IL-10 was elevated,and the effect of 50 μM STR was more pronounced.Compared with the si-Control group,the si-LPS group also showed significant upregulation of IL-1β,IL-6,IL-8,IL-15,IL-18,IL-33 and TNF-α,while IL-10 was significantly down-regulated,and all of them were significantly different (P <0.01).After pretreatment with STR at concentrations of 25 μM and 50 μM,IL-1β,IL-6,IL-8,IL-15,IL-18,IL-33,TNF-αexpression,although all decreased and IL-10 expression increased,were weakly compared with the effect of no si-ERK intervention.The experimental results suggest that ERK2 has a very important role in STR targeting to mediate the NF-κB signaling pathway to play a role in regulating the inflammatory response.STR plays a role in the treatment of ALI explicitly by binding to ERK2,thereby down-regulating ERK2 phosphorylation levels and inhibiting PIKKα,P-IκBα and P-P65,and adjusting the balance of anti-inflammatory factor/inhibitory factor expression.Conclusion:1.In this study,276 potential target proteins of STR were screened by biotin chemical modification and protein microarray technology,and extracellular signal-regulated kinase 2 was confirmed to be a direct target protein of STR by surface plasmon resonance and cellular thermal shift assay.2.By targeting ERK2 to reduce its phosphorylation level and thereby inhibiting the phosphorylation levels of IKKα,IκBα and P65 in the NF-κB signalling pathway,STR restores the balance of anti-inflammatory factor/inhibitory factor expression,thereby reducing the LPSinduced inflammatory response in C57BL/6 mice and BEAS-2B cells,and is a potential natural drug precursor for the treatment of acute lung injury,providing a new option for drug development. |
