A Mechanism Study Of Rhein Involved In Asthmatic Airway Inflammation By Regulating LncRNA Malat1 To Mediate MAPK/NF-kB Pathway

Asthma is a chronic inflammation of the airways with an unknown cause.Due to its refractory nature,it has become a serious public health problem worldwide.The most characteristic asthma features are airway inflammation,airway remodelling,and airway hyperresponsiveness.It is characterised by inflammation of the airways,and is associated with morbidity and mortality in these pathological conditions.Rhein is a common anthraquinone component,and it plays an essential role in an-ti-inflammatory and anti-oxidation.LncRNA malatl,MAPK and NF-κB exhibit significant expressions in various lung diseases,such as inflammatory lung injury,asthma.Rhein exerts various biological functions by interfering with lncRNAs,it inhibits MAPK/NF-κB/NLRP3 signalling pathway and reduces the inflammatory response.In asthma,the mechanism of rhein involved in asthmatic airway in-flammation by regulating lncRNA malat1 to mediate MAPK/NF-kb pathway is seldom reported.Objective:This study aimed to study the action mechanism of rhein that was treating for asthma through network pharmacology,and further study the effect of rhein in the treatment of OVA+LPS asthma model and its molecular mechanism through IncRNA.Methods:1 A detailed step-by-step analysis of the action mechanism of rhein treating for asthma by network pharmacology is given below.The chemical composition of rhein was obtained in PubChem database.The corresponding targets formein were obtained in the TCMSP database.The corresponding gene names for each target protein were obtained in STITCH and Drugbank da-tabase,and the anti-inflammatory targets were obtained in TTD database.Human genes associated with asthma were searched on the NCBI gene database and then mapped to target genes for rhein and asthma and asthma.Interacting proteins of the mapped genes were obtained by using the String Interaction Database and the protein interaction network was constructed.The Enrichr database was used for pathway enrichment of interacting proteins.The MAPK signalling pathway was extracted,and the matching genes were constructed to further excavate the specific mechanism of rhein treating asthma.2 Detect the expression of lncRNA malatl in peripheral blood of children with asthma(1)2ml of venous blood was collected and peripheral blood mononuclear cells were extracted;(2)Total RNA was extracted from samples by Trizol method,and the quality was detected;(3)The expression of lncRNA malat1 in peripheral blood samples of asthmatic children and control(healthy)children was detected by RT-PCR.3 Establish an animal model of asthmatic mice and study on the mechanism of rhein in treating airway inflammation in asthmatic mice.The effect of rhein on lncRNA malat1 mediated MAPK/NF-κB pathway in the treatment of asthma was verified in animal experiments.Here are the steps:(1)Asthma model was induced by OVA+LPS sensitized balb/c mice.Divide all the mice into five groups:model group,normal group,and rhein low-dose group(7.5mg/Kg),rhein middle-dose group(15mg/Kg)and rhein high-dose group(30mg/Kg),each group contains 12 mice.From the beginning of the atomization period,half an hour before each atomization,the administration of rhein was given once a day for 14 days.Observe everyday the general condition of the mice during the period of nebulization;the OVA specific stimulus test scores were recorded on days 31 and 41.(2)After treatment,collect the serum and bronchoalveolar lavage fluid of mice,use ELISA to detect the levels of IgE,IL-4,IL-1β,IL-6,TNF-α.(3)Collect the upper lobe of the left lung;use HE staining and Masson staining to observe the histopathological changes of the lung tissues.(4)The expression levels of lncRNA malat1 and p38MAPK,p65NF-κB,IL-1β,I L-6,TNF-α mRNA were detected by Real-time PCR.(5)The proteins of p38,p-p38 and p65 were detected by Western blot.4 To explore the effect of rhein on inflammation of HBE cells.(1)The inflammatory models of HBE cells were induced by LPS(1μg/ml)+OVA(0.1mg/ml),and control group,model group,rhein groups of different doses(0.1 μM,0.5μM,1.0μM).Set up lncRNA malat1 silenced inflammatory cell models.Use CCK-8 ass ay to evaluate the viability of HBE cells.(2)The expression levels of lncRNA malat1 were detected by Real-time PCR.(3)The proteins of p38,p-p38 and p65 were detected by Western blot after rhein pretreatment in the inflammatory cell model.(4)The expression of lncRNA malat1 was inhibited in the inflammatory cell model,the proteins of p38,p-p38 and p65 were detected by Western blot after rhein pretreatment.Results:1 Network pharmacology analysis results:Obtain 83 human target proteins in all,and 1060 asthma-related human genes in total.The in vivo reaction network of rhein against asthma was constructed through String database.A pathway enrichment analysis of the KEGG database revealed that the top ten pathways of rhein regulating asthma were MAPK signalling path-way,Hepatitis C,Epithelial cell signalling in Helicobacter pylori infection,Proteoglycans in cancer,Bladder cancer,Non-small cell lung cancer,Endometrial cancer,Pancreatic cancer,Central carbon metabolism in cancer,Amyotrophic lateral sclerosis.2 Expression of lncRNA malat1 increased in peripheral blood of children with asthma.3 Animal experiment results:changes in behavior showed that rhein was effective in relieving symptoms in mice(P<0.001).The lung tissue inflammation score showed that rhein prominently reduced inflammatory cell infiltration in the mice(P<0.001).The results of ELISA showed that the levels of IgE,IL-4,IL-1 13,IL-6 and TNF-α in se-rum of asthmatic mice were significantly decreased(P<0.05).The effect of Real-time PCR indicated that the expression of lncRNA malat1,p38MAPK,p65NF-κB,IL-1β,IL-6 and TNF-α in lung tissue of mice was significantly decreased(P<0.05).The results of Western Blot indicated that the expression of p-p38MAPK and p65NF-κB could be markedly reduced by rhein.4 In vitro test results:It was showed that cytotoxicity at the concentration of 0,10,20,and 40μM was not presented obviously by rhein.Therefore,less than 40μM was used for subsequent experiments.LPS+OVA stimulation significantly increased at lncRNA and protein level(P<0.001).Compared to model group(P<0.05),the lncRNA and protein expression was inhibited significantly by Rhein.Conclusions:1 Network pharmacological approaches predict that inflammatory signaling pathways may play an essential role in asthma treatment.2 The expression of lncRNA malat1 increased in patients with asthma.3 Rhein can effectively alleviate asthma symptoms and airway inflammation in OVA+LPS mice with asthma.The mechanism may be that rhein further inhibits airway inflammation through lncRNA malat1/MAPK/NF-κB pathway.