Hui Liang ,Huimin Yang ,Xiao Wang ,Yimin Gong ,Bo Zhou ,Wenxian Jia ,Yanjun Tan,,Shengfeng Zheng,Suixia Chen,Jiayi Liu,6,Fuli Qin,Zhouquan Li,Bo Zhang,Wenpu Zuo,Yang Jiao*

Abstract Objective:To investigate the protective effects and the potential targets of Nervilifordin F (NF) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats.Methods:All rats were given an intratracheal injection of LPS to induce ALI,and then high,medium,and low doses (10.0,5.0,2.5 mg/kg) of NF and 3 mg/kg dexamethasone (DEX)were used for intervention.Hematoxylin-eosin (HE) staining was used to observe the pathological damage of lung tissue.Enzyme-linked immunosorbent assay(ELISA)was used to detect inflammatory factors in peripheral blood and lung tissue.Real-time quantitative polymerase chain reaction (RT-qPCR)was used to detect the mRNA expression of autophagy pathway-related genes in tissues.Western blotting and immunohistochemistry (IHC) were used to detect the expression of autophagy pathway-related proteins in tissues.Molecular docking was used to predict and simulate the combinations of the small drug molecule and large protein molecules.Results:NF significantly attenuated the pathological damage of lung tissue,and reduced inflammatory response as evidenced by the significant decrease in the levels of tumour necrosis factor alpha(TNF-α),interleukin 6 (IL-6) and interleukin-1beta (IL-1β) in serum and lung tissue (P＜0.05).Moreover,NF treatment markedly enhanced the expressions of autophagy-associated gene 5 (ATG5) and Beclin 1 (ATG6),and the ratio of microtubule associated protein 1 light chain 3 beta II (LC3B II)/LC3B I as well (P＜0.05).Conclusion:NF can alleviate LPS-induced ALI by promoting autophagy,which may be developed as a potential agent for the treatment of ALI.

Keywords Nervilifordin F;lipopolysaccharide;acute lung injury;autophagy

Introduction

Acute lung injury (ALI)is a common clinical disease with high incidence caused by an uncontrolled inflammatory response[1].So far,it’s still a momentous source of mortality in the critically ill people[2].Recently,a large number of studies have shown that its pathogenesis involves a variety of inflammatory mediators,effector cells,and is a cascade-amplified inflammation mediated by multiple pathogenesis [3-4].The causes for acute lung injury can be divided into direct pulmonary causes and indirect extrapulmonary causes[5].Direct causes include inhalation,physical lung injury and severe pneumonia,etc.,while indirect causes include other severe shocks,infection and trauma and so on.The“two-hit”holds that traumatic shock,bacterial displacement,ischemia-reperfusion and other first-hit can activate immune cells in vivo,whichlead to an uncontrolled inflammatory response if the second-hit occurs [6].And ALI may further deteriorate into the acute respiratory distress syndrome (ADRS)that has complex and multifactorial pathobiology,ultimately leading to multiple organ dysfunction syndromes(MODS)and even death[7].

Nervilia fordii(Hance) Schltr,a common herb used in Asia,has been extensively used for the treatment of pulmonary inflammatory diseasesbecause of its antiinflammatory and anti-oxidant effects [8].According to the record of Quality Standard of Yao Medicinal Material in Guangxi Zhuang Autonomous Region(volume Ⅱ),Nervilia fordii(Hance) Schltr has been usually used in improving lung cough.In previous studies,we found that Nervilifordin F (NF),an active ingredient inNervilia fordii(Hance) Schltr,inhibited the release of inflammatory cytokines in LPS-induced macrophage line RAW264.7 [9].Considering the development and progression of LPS-induced ALI hinges on a set of inflammatory responses,we assumed that NF may alleviate ALI by inhibiting inflammation.However,up to now,there are still few reports on NF,and its molecular mechanism for treating ALI remains unclear.

Autophagy is an intracellular digestion process that removes damaged macromolecules and organelles to perform self-protectionin vivo[10].Autophagy plays critical roles in the activation of NOD-like receptor family pyrin domain-containing 3(NLRP3)inflammasome,the clearance of bacterial and viral infections and what is more,the function of adaptive immune cells [11].The outbreak of ALI can be exacerbated by inhibition of autophagy in the cell [12],and to induce autophagy may be a new therapeutic strategy for ALI.To investigate the anti-inflammatory effects of NF on both direct (LPS) and indirect (intestinal ischemia/reperfusion,II/R) induced ALI,our team established two rat models of ALI.We found NF significantly alleviates ALI indirectly induced by II/R in rats [13].In this study,we applied LPS-a direct pulmonary inflammatory factor-to induce ALI,explored the antiinflammatory effects of NF and discussed the autophagy pathways in which NF might be involved.

Materials and methods

Drug and reagents

LPS was purchased from Sigma-Aldrich (batch number:320V031,America).NF (its structure was shown in Figure 1) was provided by Zhipu Biology Technology Co.Ltd.(＞99.5%,Shanghai,China).Dexamethasone (DEX) was purchased from Hubei Qian Jiang Pharmaceutical Co.Ltd.(batch number:20180301,Hubei,China).Kit for H&E staining was obtained from Shanghai Beyotime Biotechnology Co.Ltd.(Shanghai,China).ELISA kits were provided by Hangzhou Lianke Biotechnology Co.Ltd.(Hangzhou,China).The reverse transcription kit and SYBR Green Mix reagent for RT-qPCR were acquired from Takara(Dalian,China)and Roche(Germany),respectively.Primary antibodies:anti-Beclin 1(ab207612)andanti-ATG5(ab108327)were purchased from Abcam,US.Anti-β-actin (cst4970) and anti-LC3B (cst43566)were purchased from Cell Signaling Technology,Inc.The secondary antibody:Goat Anti-Rabbit IgG H&L(HRP) (ab205718) was purchased from Abcam,US.IHC kit was obtained from Beijing Zhongshan Jinqiao Biotechnology Co.Ltd.(Beijing,China).

Figure 1.NF structure.Its chemical name is methyl rhamnosin-3-O-3-glucopyranosyl-4'-O-4'-glycosylmethylacetyl-glucoside,which is C31H36O18.

Animals

Adult male SPF Sprague-Dawley Rats (SD rats),weighing(200±20)g,were purchased from the Experimental Animal Center of Guangxi Medical University (Laboratory animal license number:SYXK Gui 20140003).The experiment was carried out after 3 days of adaptive feeding.Before invasive surgery,the rats were fasted for more than 12 h,while they had free access to the drinking water.

Animal model and administration procedures

Adult male SD rats were randomly divided into 6 groups:control (normal saline) group,LPS group(model),DEX group(positive control),and LPS+NFhigh (H),medium (M) and low (L) dose groups.All rats were narcotized byintraperitoneal injection of pentobarbital sodium and given an intratracheal injection of 10 mg/kg LPS to induce ALI,while the normal control group was fed the same amount of saline[14].After induced by LPS,NF intervention groups were given NF 10.0,5.0,2.5 mg/kg for 5 days,and other groups were raised the equal amount of saline while DEX group was injected intraperitoneally with DEX 3 mg/kg on the 5th day.All animals were finally anesthetized for collectingabdominal aortic blood and lung tissues.

Histopathological analysis

About 1 cm3of anterior lobe of the right lung waschopped and fixed in 4% paraformaldehyde,embedded in paraffin and cut into 3 μm thickness sections to make paraffin sections.According to the routine histopathological examination,HE stainingwas performed.The final stained sections sealed with neutral gum were photographed under a light microscope at 200×magnification.

Lung injury score

The lung injury scoring system was used to score the H&E staining results,which assessed the severity of lung injury according to alveolar septum,alveolar hemorrhage,intra-alveolar fibrin and intra-alveolar infiltrate[15].Each slide was evaluated by two separate investigators in a blinded manner.All the points for each category were added and weighted,based on their relative importance.The injury score was calculated in the light of the following formula:injury score=[(alveolar hemorrhage points/no.of fields) +2×(alveolar infiltrate points/no.of fields)+3×(fibrin points/no.of fields)+(alveolar septal congestion/no.of fields)]/total number of alveoli counted.

In addition,some pathological changes of lung tissue were assessed to score the severity of lung injury,on account of the conditions of pulmonary congestion and hemorrhage,alveolar exudation,PMN infiltration of the alveolar and vascular wall,thickening of alveolar wall and formation of hyaline membrane.The score was divided into five levels:0-5 means no damage,minor damage,moderate damage,severe damage and extremely severe damage.Besides,the degree of lung injury was indicated based on the total score in 10 randomly selected fields in sections of each group using high power fields(×200).

ELISA analysis

To assess the changes in the levels of some inflammatory factorsin vivoafter treatment of NF,the inflammatory factors TNF-α,IL-6 and IL-1β levels in serum and lung tissue were measured using ELISA kits according to the manufacturer's instructions.

Western blotting

Total proteins were extracted with RIPA lysis buffer and quantified using the BCA kit.Proteins were separated using a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF)membrane.After blocking with 5% (w/v) fat-free milk for 1 h,the membranes were incubated with primary antibodies overnight at 4oC and secondary antibodies for 1 h at room temperature.All antibodies were diluted as follows:anti-β-actin (1:1,000),anti-Beclin 1 (1:2,000),anti-ATG5 (1:4,000),anti-LC3B(1:1,000) and the secondary antibodiy was diluted 1:3,000.Proteins were visualized using an electrochemiluminescence (ECL) kit,developed on a membrane sweeper and quantified using Image J software.

RT-qPCR

Total RNA was extracted from the lung tissues in each group using Trizol reagent according to the manufacturer’s introduction.The cDNA obtained by reverse transcription of 1 μg total RNA was used to detect the mRNA expression using the SYBR Green Mix and ABIPRISM STEPONE rapid real-time PCR system according to the manufacturer's protocol,and each sample was repeated three times.The 2-ΔΔCtmethod was taken to calculate the expression level of these geneswith actin as reference.The qPCR primers were designed using Premier 5.0 softwareand the primer sequences used were as follows:ATG5 forward,5’-ACCTCGGTTTGGCTTGGTT-3’and reverse,5’-AAACCACACGTCTCGAAGCA-3’;Beclin 1 forward,5’-TCTCGTCAAGGCGTCACTTC-3’and reverse,5’-CCTTAGACCCCTCCATTCCTCA-3’;LC3B forward,5’-GACAGCACTGGCTGTGTAAG-3’and reverse,5’-TGCCTACGTTCTGATCTGTGG-3’;.β-actin was utilized for the standardization,and its primer sequences were:forward,5’-AGATCAAGATCATTGCTCCTCCT-3’and reverse,5’-ACGCAGC-TCAGTAACAGTCC-3’.

IHC analysis

IHC was performed with specific antibodies against LC3B protein and the samples were then counterstained with Hematoxylin.The immunoreactivity score (IRS) system was used to analyze the results and the staining intensity was divided into 0-3 points,which means unstained,weak staining,medium staining,and strong staining,respectively;while the cell staining ratio was divided into 0-4 points,which were negative,1%-10%,11%-50%,51%-80%,and ＞80%severally.IRS is the product of the intensity score multiplied by the staining ratio,and the total score was greater than 3 was deemed to be positively expressed.

Docking simulations

In order to clarify the binding mode of the compounds and provide straightforward information for further structural optimization,primary molecular docking was performed with the Surflex-Dock module of Sybyl-X 2.0 program.The crystal structure of protein was downloaded from the Protein Data Bank(http://www.rcsb.org/).Protein was prepared using protein structure preparation module of the SYBYL X-2.0 software.All the water molecules and ligands were eliminated,and hydrogen atoms were added to the crystal structure with Gasteiger–Huckel charges.Besides,the Powell energy gradient method was used for molecule optimization,with the maximum number of optimizations set as 10,000.The energy convergence was limited to 0.005 kJ/mol and other parameters were set to default values.The results were visualized with PyMOL.

Statistical analysis

ANOVA was employed for analysis when the data meets its requirements.Normality test and homogeneity test of variance were performed for analyzing the difference between the measurement data sets by oneway ANOVA.At the same time,the count data were analyzed by theχ2test.P＜0.05 andP＜0.01 were considered statistically significant.

Results

NF alleviated lung tissue pathological damage and reduced the levels of inflammatory factors in se⁃rum and lung tissue in LPS-induced ALI rats

As shown in Figure 2A-B,the results of HE staining showed that LPS induced several obvious inflammatory histological changes in lung tissues,such as pulmonary congestion,thickening of the alveolar wall,and inflammatory infiltration.After NF intervention,the classical pathological changes of ALI were improved in the lung tissue of LPS-induced rats,and the lung injury scores were reduced.Moreover,the protective degree of NF was increased in a dose-dependent manner.Then,the effects of NF on LPS-induced intrapulmonary inflammatory responses in rats were further investigated by ELISA.It found that the rising trend in the levels of the inflammatory factors (TNF-α,IL-6 and IL-1β) in serum and lung tissues in LPS-induced rats were significantly reversed by NF intervention(P＜0.01)(Figure 2C-D).

NF stimulated the expression of autophagy-associ⁃ated genes in LPS-induced ALI rats

The mRNA (Figure 3A-C) and protein (Figure 3DG) expression levels of ATG5,Beclin 1 and LC3B in the LPS group were evidently reduced compared with the control group (P＜0.01),but significantly increased after different doses of NF intervention (P＜0.01).Compared to the LPS group,the mRNA expression of LC3B was obviously increased after NF intervention,and the ratios of protein expression of LC3B II/LC3B I in the LPS+NF-L and LPS+NF-H groups were still markedly higher,though dropped in LPS+NF-M group(P＜0.01).

Immunohistochemistry results showed (Figure 3H-I)LC3B protein was mainly expressed in the cytoplasm,and its positive cells were seen in the control group.There was no statistical difference in LC3B protein expression between the LPS group and the control group (P＞0.05).After NF intervention,the expression of LC3B had a significant increase in LPS+NFL,LPS+NF-M,and LPS+NF-H groups compared with the LPS group(P＜0.01).

Figure 2.NF alleviated ALI and suppressed the production of TNF-α,IL-6 and IL-1β.A:HE staining of pathological damage in lung tissue under a microscope (×200,the scale bar represents 100 µm).B:Quantitative histopathology score of lung injury.C-D:ELISA results of inflammatory factors in serum and lung tissues of rats.n=10,*P＜0.01 vs.Control group;#P＜0.01 vs.LPS group.

Figure 3.NF stimulates autophagy in LPS-induced ALI rats.A-C:RT-qPCR results.D-G:Western blotting results and relative protein expression.n=10,*P＜0.01 vs.Control group;#P＜0.01 vs.LPS group.H-I:IHC staining results and immunoreactivity score of LC3B(scale bar:100µm).

Docking

To explore the possibility that NF affects the gene expression of autophagy signal pathway,we further used Sybyl X-2.0 to evaluate the potential intermolecular interactions between NF and autophagy-related proteins.The docking results between NF and ATG5 were the best (total score 5.7695),followed by LC3B(total score4.788) and Beclin 1 (total score 1.1696)(Table 1).

Table 1.Molecular docking results

As depicted in Figure 4,NF bound to ATG5 protein by forming nine hydrogen bonds with Lys 51,Asp 50,Leu 38,Arg 15 and Asp 60 sites (Figure 4A,D).NF also formed four hydrogen bonds with Glu 36,Lys 8 and Gly 85 sites of LC3B protein (Figure 4B,E),and five hydrogen bonds with Phe 419,Asn 420,Arg 358 and Gly 356 sites of Beclin 1 protein (Figure 4C,F).And in particular,it was observed that the main structure of NF,methylrhamnosin-3-O-β-6''' acetylglucosyl,formed a cavity with Asp 50 and Lys 51,which may be beneficial to the formation of stable drug molecule and protein macromolecule complexes,making the docking score of NF and ATG5 higher.

Figure 4.The results of molecular docking between NF and protein ATG5 (A,D),LC3B (B,E),Beclin 1 (C,F).A-C:Hydrogenbond interaction of compound NF at the active sites.The hydrogen bonds are shown as yellow dotted lines with the distance unit of Å.D-F:Docked view of NF at the active sites of these 3 proteins.

Total score represents binding affinities,which was expressed in log (Kd) units.Consensus score integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active sites of a receptor and reports the output of the total score.Crash is the degree of inappropriate penetration of ligands into proteins and the degree of mutual penetration between ligand atoms separated by rotatable bonds.Crash scores close to 0 are favorable,revealing the inappropriate penetration into the binding site.Polar score is the contribution of the polar non-hydrogen bonding interactions to the total score,which may be useful for excluding docking results that make no hydrogen bonds.Total score≥5 and Consensus score≥4 was considered to be good.

Discussion

The essence of ALI is lung inflammation,which works through the mediation of a variety of inflammatory cells and inflammatory mediators and the release of cytokines[17].LPS is the primary component of the G-bacterial cell wall,currently causing uncontrolled inflammatory responses and organ failure,even death [18].At this point,we built a model of ALI through intratracheal instillation of LPS,and then the validity of models was verified by examining the lung tissue pathology and the changes about infiltrating inflammatory factors and chemokines as well.Early studies have found that total flavonoids ofNervilia fordiicould alleviate the alveolar wall capillary congestion and thickening,also observably reduce PMN exudation of LPS-induced ALI in mice and have a good anti-inflammatory activity.Among them,the NF has particularly prominent anti-inflammatory activity and was found to be substantially not cytotoxic [9].In this study,based on the successful establishment of reliable ALI model,our observations show an anti-inflammatory effect of NF on the inflammatory factors TNF-α,IL-6 and IL-1β in LPS-induced ALI rats.

Considering another our research on II/R-induced ALI [13],we found that the lung injury was significantly alleviated and the histopathology of lung improved after the treatment of NF.What’s more,the levels of inflammatory factors TNF-α,IL-6,and IL-1β in serum and lung tissue of LPS-induced ALI(pulmonary) and II/R-induced ALI (extrapulmonary) rats both were decreased after NF intervention.Interestingly,we found that the degree of lung injury on LPSinduced ALI rats decreased as the concentration of NF increased,however,the II/R-induced ALI rats were decreased more significant after middle dose(5.0 mg/kg) of NF intervention.Their potential mechanisms are still unclear,and we will supplement samples and replicate other inflammatory-related models to complete the mechanism network of the protective effect of NF on ALI induced by pulmonary and extrapulmonary causes.

Several studies have found that autophagy is closely related to the occurrence of pulmonary inflammatory diseases [19].A variety of ATGs are involved in the formation of autophagic vacuoles,such as ATG5 [20]and ATG6 (also known as Beclin1) [16,21],which were offered a large part in the formation of autophagosomes.LC3B is a homologous protein of ATG8,and a central protein in the autophagy pathway involved in the selection of autophagy substrates and the initiation of autophagosomes [22],which was present both inside and outside of autophagosomes,making this protein an autophagosomal marker [23].After NF intervention,the expression levels of ATG5,Beclin 1 and LC3B were significantly increased in the lung tissue of LPS-induced ALI rats.ATGs operate in multiple processes of autophagosome formation,especially the early phase of bona fideautophagic responses (e.g.,initiation,nucleation,and elongation)[24-26],and ATGs could be stimulated to induce the synthesis of autophagy-related proteins (Beclin 1,Rab7,etc.),reducing pro-inflammatory responses and thereby reducing lung damage when ALI occursin vivo[12].Direct interaction between NF and ATG5/LC3B was predicted by molecular docking,which suggested that NF might interact with ATG5 and LC3B directly to participate in the autophagy signal pathway and reduce ALI in rats.However,we have not yet understood the mechanisms through which NF affects the autophagy pathway,whether NF inhibits inflammatory-related pathways by other upstream mechanism or not,and which genes are the key to the anti-inflammatory effect of NF.These questions will be focused on in our further study.

Taken together,NF has a protective effect on ALI due to its anti-inflammatory effect.Our results support the hypothesis that NF stimulates the autophagy signal pathway in LPS-induced ALI rats.These results suggested that NF might be a candidate for effective treatment of ALI as an anti-inflammatory drug.

AcknowledgmentsThis work was supported by the Natural Science Foundations of Guangxi Province(No.2018GXNSFAA138021).