The Mechanism Research Of Apigenin Up-regulating Mir-503-5p To Inhibit Airway Inflammation In Chronic Asthma

Background:Asthma is a chronic airway inflammation disease involving many inflammatory cells and cellular components.It is involved in all ages.The incidence rate of chronic respiratory diseases,especially asthma,is increasing year by year due to the serious air pollution and the increase of fine particles in industrialized countries.At present,there are about 300 million people suffering from asthma in the world.The prevalence of asthma in adults is 1.2%to 25.5%,and that in children is 3.3%to 29.0%.It has become a serious public health problem.Up to now,extensive studies on the pathogenesis of asthma have shown that chronic airway inflammation,airway hyperresponsiveness and airway remodeling are important characteristics of asthma.Therefore,it is of great significance to study the occurrence,development,pathological and molecular characteristics of these three important characteristics in the pathogenesis of asthma for revealing the etiology,treatment and looking for effective drugs or methods for the treatment of asthma.Flavonoids are the secondary metabolites of plants,which are abundant in common foods in daily life and widely exist in fruits,vegetables,spices,herbs,nuts,vegetables and other crops.Apigenin(API)is a common flavonoid in daily food.It is named for its highest content in celery.In addition,it is rich in warm tropical vegetables and fruits,such as Verbenaceae,Daphne odorifera and Selaginellaceae.A large number of previous studies have shown that Apigenin plays an important role in antioxidation and anti-inflammatory.In RAW264.7 cells,Apigenin-7-o-glucoside(Ag)can inhibit the production of reactive oxygen species(ROS)induced by H202,thus reducing the level of oxidative stress,In LPS treated RAW264.7 cells,Apigenin derivative Ag significantly inhibited NF-?B/NLRP3/caspase-1 signaling pathway and decreased the secretion of inflammatory factors,thereby reducing the inflammatory response.In nonalcoholic steatohepatitis(NASH),Apigenin significantly inhibits liver fat deposition and cell proliferation,and effectively inhibits inflammatory response in Nash model by reducing the synthesis and secretion of IL-8,TNF-?,GLUT-4 and IR.Apigenin plays an important role in anti-inflammatory response.Many researchers have conducted in-depth studies on the molecular mechanism of apigenin.In the model of ischemia-reperfusion injury,inflammatory cells in ischemic lesions are activated,infiltrate ischemic tissues and secrete a variety of inflammatory factors,including interleukin-1?,IL-6,TNF-?,nitric oxide(NO)and prostaglandin E2(PGE2).Apigenin treatment can significantly reduce the contents of iNOS,COX-2,no and PGE2 in vivo,which proves that apigenin plays an anti-inflammatory role in the inflammatory injury induced by ischemia-reperfusion.In ACN induced germ cell inflammation and apoptosis,apigenin up-regulated the content of lactate dehydrogenase(LDH)and sorbitol dehydrogenase(SDH),down regulated the expression of IL-1?,TNF-?,IL-6 and NF-?B,thus alleviating ACN induced inflammation.In addition,in leukemia,breast cancer,prostate cancer and melanoma,apigenin has antitumor effect by blocking cell cycle.MicroRNA,or miRNA,is a single strand RNA molecule with a length of 22 NT,which is widely involved in posttranscriptional regulation in plants and animals.Previous literature reported that apigenin plays a variety of biological functions in the presence of miRNA.In the mouse peritoneal mesothelial cells(MMCs)induced by Decoction,apigenin treatment down regulated the expression of miR-34a,promoted cell proliferation and apoptosis,thus inhibited the fibrosis of mouse peritoneal mesothelial cells.According to Wang and other researchers,apigenin treatment of glioma cell line U87 can significantly up regulate the expression level of mir16,and induce tumor cell apoptosis by inhibiting BCL2 protein and NF ?B/MMP9 signaling pathway.In the model of chronic asthma,the current research mainly focuses on inflammatory response,airway hyperresponsiveness and airway remodeling,but there is little research on signal transduction pathway,especially miRNA/mRNA signaling pathway.This study aims to explore the effect of Apigenin in the treatment of ova chronic asthma mouse model and its molecular mechanism through miRNA/mRNA.Methods:SPF BALB/C female mice aged 6-8 weeks were randomly divided into normal control group,ova chronic asthma model group,15 mg/kg Apigenin treatment group and 30 mg/kg apigenin treatment group.ASMCs were cultured in vitro and intervened by PDGF-BB.After the establishment of ova chronic asthmatic mouse model,the lung lobes were taken,and the pathological characteristics and inflammatory cell infiltration of lung tissue were observed by HE staining;the airway mucus secretion was observed by PAS staining;the contents of inflammatory factors INF-?,IL-2 and IL-12 and oxidative stress proteins ROS,SOD and MDA in ASMCs induced by bronchoalveolar lavage fluid(BALF)and PDGF-BB were detected by ELISA The protein expression levels of p-Akt,NF-?B,IRF-3 and IRF-7 were detected by RT-PCR,and the proliferation of ASMCs induced by PDGF-BB was analyzed by CCK8 assay.miRNA microarray was used to screen differentially expressed miRNAs in ASMCs induced by PDGF-BB;according to the obtained miRNAs,relevant literature and reports were consulted to screen mir-503-5p which is most closely related to asthma or cell proliferation;RT-qPCR was used to detect the expression level of mir-503-5p gene after apigenin treatment;the expression of mir-503-5p gene was detected by RT-qPCR microRNA.org.The target gene STAT3 of mir-503-5p was screened,and the effect of mir-503-5p on STAT3 was analyzed by Luc fluorescence reporting system STAT3 3'UTR binding and regulation.The expression of mir-503-5p was inhibited in ASMCs induced by PDGF-BB.RT qPCR and Western blot were used to detect STAT3 after apigenin treatment In ASMCs induced by PDGF-BB,the effect of apigenin on the proliferation of ASMCs was detected by CCK8.Results:HE staining showed that airway inflammation infiltration was obvious and mucus secretion was high in the lung tissue of model mice.ELISA results showed that the secretion of INF-y,IL-2 and IL-12 inflammatory cytokines in BALF of asthmatic model mice increased;the content of ROS and MDA increased,while the content of SOD decreased;western blot analysis showed that the expression of TNF-y,IL-2 and IL-12 in BALF of asthmatic model mice increased The expression levels of p-Akt,NF-?B,IRF-3 and IRF-7 were significantly increased,which indicated that the levels of inflammatory response and oxidative stress in the lung tissue of ova mice with chronic asthma were significantly increased,and the ova mice with chronic asthma were successfully established.In order to study the role and value of Apigenin in asthma,the asthmatic mice model was established successfully.15 mg/kg and 30 mg/kg Apigenin treatment can effectively alleviate the asthma symptoms,pulmonary mucosal edema,airway obstruction,inflammatory cell infiltration,reduce the expression levels of inflammatory factors INF-y,IL-2 and IL-12,p-Akt,NF-?B,IRF-3 and IRF-7,ROS and MDA,and increase the expression level of SOD,which is a negative regulator of oxidative stress signaling pathway.In PDGF-BB-induced ASMCs,the expression levels of inflammatory factors INF-?,IL-2 and IL-12,inflammatory response related proteins p-Akt,NF-?B,IRF-3 and IRF-7,and oxidative stress proteins ROS and MDA were increased,while the level of SOD,a negative regulator of oxidative stress pathway,was decreased.Similarly,apigenin treatment significantly inhibited PDGF-BB-induced inflammatory response and oxidative stress in ASMCs.The expression levels of inflammatory cytokines inf-?,IL-2 and IL-12,inflammatory response related proteins p-Akt,NF-?B,IRF-3 and IRF-7,and pro-oxidative stress response proteins ROS and MDA were decreased,while the level of SOD protein in oxidative stress signaling pathway was increased.These results suggest that Apigenin treatment or treatment can effectively reduce the level of inflammatory response and oxidative stress,and play a role in the model of chronic asthma.In addition,the phenomenon of airway remodeling in ova chronic asthma model mice is serious.CCK8 experiment results show that Apigenin treatment can significantly inhibit the proliferation of ASMCs,and Apigenin may also alleviate asthma symptoms by inhibiting airway remodeling in mice.A total of 20 differentially-expressed miRNAs were screened from PDGF-BB-induced cells and normal control cells by miRNA microarray analysis,including 10 up-regulated miRNAs,namely mir-196a-5p,mir-107,mir-4640,mir-6838,mir-497,miR-424,mir-374b,mir-367b,mir-203-5p and mir-228,and 10 down regulated miRNAs,namely mir-199-3p,mir-1285,mir-536-3p,miR-141 and MI MiR-9,mir-20b,mir-19a,mir-251,mir-503-5p and mir-288.According to literature reports,mir-503-5p plays an important role in the carcinogenesis of colon cancer by down regulating the expression of VEGF-A and inhibiting the occurrence,angiogenesis and lymphangiogenesis of colon cancer.Moreover,in the further analysis,the expression of mir-503-5p in the lung tissue of ASMCs and ova chronic asthma model mice treated with PDGF-BB was significantly up-regulated,while apigenin treatment inhibited the expression of mir-503-5p.Therefore,it is speculated that mir-503-5p may play a role in the process of chronic asthma and be further studied.RT qPCR showed that the expression of mir-503-5p decreased in ASMCs induced by PDGF-BB.Luc fluorescence assay showed that mir-503-5p could specifically bind to the 3'UTR region of STAT3 and inhibit its expression.When the miRNA binding site of STAT33'UTR region was mutated,the inhibitory effect disappeared.In PDGF-BB-induced ASMCs,the level of STAT3 protein was increased.Apigenin treatment could down regulate this effect,and inhibition of mir-503-5p expression could increase the level of STAT3 protein.In addition,PDGF-BB increased the contents of inflammatory cytokines INF-y,IL-2 and IL-12 in ASMCs.Apigenin treatment could down regulate the increase induced by PDGF-BB.Inhibition of mir-503-5p or overexpression of STAT3 could inhibit the inhibitory effect of apigenin on the expressions of INF-?,IL-2 and IL-12.In addition,CCK8 results showed that overexpression of STAT3 or inhibition of mir-503-5p in ASMCs could reverse the inhibitory effect of Apigenin on the proliferation of ASMCs.Conclusion:Apigenin can effectively alleviate asthma symptoms and airway inflammation in OVA mice with chronic asthma.The mechanism may be that apigenin further inhibits airway inflammation,oxidative stress and airway remodeling through mir-503-5p/STAT3 pathway.