YQHX Formula Alleviate PM2.5 Exposure Aggravated Myocardial Ischemia Reperfusion Injury Via PEAMIR-miR-29b CeRNA Pair

Environmental pollutants are closely related with human health,which has become a hot worldwide topic.PM2.5,the particles with aerodynamic diameter less than 2.5um,has been widely regarded as the most harmful and representative air pollutants to human health.PM2.5 has many characteristics,including small particle size,small volume,relatively large surface area with adsorbed toxic substances,long suspension time in the air,high deposition rate and strong penetration in lung tissue.PM2.5 has clear pulmonary toxicity.Moreover,inhaled PM2.5 is easy to penetrate through alveolar endothelium and infiltrate into blood circulation to invade systemic tissues and organs,causing a series of extrapulmonary injuries such as cardiovascular events,acute kidney injury and hematonosis.There are many researches sites on the cardiotoxicity of PM2.5 exposure,and our study focuses on heart failure(HF)and heart ischemia-reperfusion(IR)injury.This study is mainly divided into two parts:Part one:Meta-analysis of air pollutants and hospitalization and death in patients with heart failureIn this part,we first retrieved the English literatures focus on the relationship between air pollutants(CO,SO2,NO2,O3,PM2.5 and PM10)and hospitalization and death events in HF patients from the Pubmed database.The retrieval period is from the database construction to October 2021.After two-rounds screening,the final included literature was determined and the literature quality was evaluated.Then,the parameters of the enrolled literatures were extracted,and the OR values of different concentration pollutants in different literatures were uniformly corrected by the formula,and the lag effects were also combined.Finally,51 studies were included.Meta-analysis results suggested that for every lppm increase in CO,the pooled OR values of total effect,lag0 and lag1 were 1.0346(95%CI 1.0233-10.46),1.0225(95%CI 1.0083-1.0368)and 1.0227(95%CI 0.9897-1.0587),respectively.For every 10ppb increase of SO2,the pooled OR values of total effect,lag0 and lagl were 1.022(95%CI 1.0106-1.0335),1.0457(95%CI 1.0141-1.0783)and 1.0493(95%CI 1.0273-1.0717),respectively.For each 10ppb increase of NO2,the pooled OR(95%CI)values of total effect,lag0 and lag1 were 1.0207(95%CI 1.0138-1.0277),1.0152(95%CI 1.013-1.0174)and 1.0165(95%CI 1.001-1.0323),respectively.For each increase of O3 by 10ppb,the pooled OR values of total effect,lag0 and lagl were 1.0095(95%CI 1.0025-1.0166),1.002(95%CI 0.9905-1.0136)and 1.0045(95%CI 1.0016-1.0074),respectively.For every 10μg/m3 increase in PM2.5,the pooled OR values of total effect,lag0,lag1,lag2 and lag3 were 1.0129(95%CI 1.0093-1.0165),1.01(95%CI 1.0067-1.0132),1.0067(95%CI 1.0017-1.0117),1.0028(95%CI 1.0013-1.0042)and 1.0027(95%CI 0.9996-1.0058),respectively.For PM10 increased by 10μg/m3,the pooled OR values of total effect,lag0,lag1,lag2 and lag3 were 1.013(95%CI 1.0102-1.0157),1.0128(95%CI 1.0059-1.0196),1.0026(95%CI 0.9962-1.009),0.9973(95%CI 0.9921-1.0025)and 1.0006(95%CI 0.9983-1.0029),respectively.Subgroup analysis was conducted based on age,study type and outcome,and the pooled OR values of each pollutant subgroup did not change direction.In terms of publication bias,there was no publication bias by Begg’s method(P>0.05).Egger’s test showed that there was publication bias in studies related to CO(T=4.06,P=0.001),NO2(T=3.62,P=0.002)and PM2.5(t=4.66,P<0.001)exposure and hospitalization or death in HF patients.These results suggest that exposure to gaseous pollutants(CO,SO2,NO2 and O3)and particulate pollutants(PM2.5 and PM 10)are associated with adverse events(hospitalization and death)in HF patients.Part Ⅱ:YQHX formula alleviate PM2.5 exposure aggravated myocardial ischemia reperfusion injury via PEAMIR-miR-29b ceRNA pairIn the second part,PM2.5 aqueous solution was prepared,and the rat model of PM2.5 exposure to IR injury and H9c2 cell model of PM2.5 exposure to HR injury were built respectively.In vivo,TTC staining was used to measure the area of myocardial infarction,cardiac function and structure were evaluated by echocardiography,and serum myocardial enzyme level was detected by ELISA.These tests were used to evaluate the degree of cardiac injury.The levels of inflammatory factors(IL-1β and TNF-α)were detected by RT-PCR and ELISA to evaluate the degree of inflammatory response.TUNEL was used to detect cell apoptosis,immunohistochemical method was used to detect the expression of Bcl-2 and Bax in myocardial tissue,and WB was used to detect the expression of cleaved caspase-3 and total-Caspase-3 to evaluate the degree of apoptosis.In vitro experiments,cell proliferation level was tested by CCK-8 to evaluate the degree of cell damage.RT-PCR and ELISA were used to detect the levels of inflammatory factors to evaluate the degree of inflammatory injury.Apoptosis was detected by Annexin V/PI double staining and caspase-3 expression was detected by WB to evaluate apoptosis level.Then,the key lncRNA was identified by high-throughput Illumina sequencing+RT-PCR,and the lncRNA-miRNA-mRNA network was initially constructed based on bioinformatics software combined with literature review.The lncRNA-miRNA-mRNA interaction was confirmed by double luciferase reporter gene analysis.Adenovirus were used to up-regulate or down-regulate the expression of key lncRNA in H9c2 cells.The expression of lncRNA-miRNA-mRNA was analyzed by RT-PCR,and the activation level of signal pathway was confirmed by Western Blot.Cell viability was evaluated by CCK8 and apoptosis was detected by Annexin V-FITC/PI double labeled flow cytometry.These tests were used to confirm lncRNA-miRNA-mRNA network.To evaluate the prevention and treatment effects of YQHX formula on IR injury after PM2.5 exposure and screened ceRNA pair.We prepared the serum containing medicine of YQHX formula and treated in H9c2 cell HR model of PM2.5 exposure.Cell damage was evaluated from the perspectives of cell proliferation,inflammation and apoptosis.Our results suggested that infarct size,CK-MB(4656.5±886.3 U/L vs.3253.2±876.7 U/L,t=2.757,P=0.0202)and LDH(2025.3±353.2 U/L vs.1353.2±265.3 U/L,t=3.727,P=0.0039)in PM+IR group were significantly higher than those in IR group.The myocardial apoptosis index in PM+IR group was significantly higher than that in IR group(25.63±3.26%vs.20.11±2.65%,t=3.218,P=0.0092),and the bcl-2/Bax level in PM+IR group was significantly lower than that in IR group(0.35±0.04%vs.0.46±0.04%,t=4.763,P=0.0008).LVEF level in PM+IR group was significantly lower than that in IR group(62.5±6.30%vs.70.66±6.80%,t=2.328,P=0.0422).These results suggest that PM2.5 exposure aggravates cardiac injury in IR rats.Based on high-throughput Illumina sequencing,top 10 differential expressed lncRNA were selected for further RT-PCR verification,and NONRATT003473.2 was selected as the key lncRNA,which we named it as PM2.5 exposure aggravated myocardial IR injury(PEAMIR).Up-regulation of PEAMIR could significantly improve cell viability,apoptosis and inflammatory damage in H9c2 cell HR model of PM2.5 exposure,while down-regulation of PEAMIR presented adverse effects.By miRanda database and bioinformatics analysis,it was predicted that there was a binding site between miR-29b-3p and PEAMIR,and PEAMIR might inhibit the biological function of miR-29b-3p by spongy adsorption.RT-PCR showed that level of miR-29b-3p in PM+IR group was significantly higher than that in IR group(P<0.05),and level of miR-29b-3p in PM+HR group was significantly higher than that in HR group(P<0.05).Up-regulation or down-regulation of PEAMIR could significantly inhibit or increase miR-29b-3p level,respectively.Dual luciferase reporter assay confirmed that PEAMIR could not bind to mut-miR-29b-3p.We confirmed that PEAMIR-miR-29b involved in the inflammation and apoptosis in H9c2 cell HR model of PM2.5 exposure.Using target-SCAN database and bioinformatics analysis,it predicted the existence of binding sites between miR-29b-3p and PI3K(p85a).Based on the our previous studies,PI3K/Akt/GSK3b/p53 pathway was selected as the potential downstream target pathway of PEAMIR-miR-29b.In H9c2 cell,PM2.5 exposure could significantly enhance the effect of HR on the PI3K/Akt/GSK3b/p53 pathway,and LV-PEAMIR could resist the amplification of PM2.5 on PI3K/Akt/GSK3b/p53 pathway(P<0.05).MiR-29b-3p mimic could block this effect of PEAMIR(P<0.05).YQHX could improve the LVEF level of IR rats exposed to PM2.5(P<0.05).The serum containing medicine of YQHX formula could significantly improve the cell viability,reduce apoptotic cell mediate PEAMIR-miR-29b ceRNA pair and decrease inflammatory factors in H9c2 cell HR model of PM2.5 exposure(P<0.05).In conclusion,our results indicate that PM2.5 exposure can significantly increase IR injury in vivo and vitro.PEAMIR-miR-29b,as a specific ceRNA pair,participates in the pathological process of IR injury aggravated by PM2.5 exposure via PI3K/Akt/GSK3b/p53 pathway.YQHX can improve cardiac function of IR rats after PM2.5 exposure.The serum containing medicine of YQHX formula could significantly reduce damages in H9c2 cell HR model of PM2.5 exposure.