Luteolin Pretreatment Ameliorates Myocardial Ischemia/Reperfusion Injury by lncRNA‐JPX/miR‐146b Axis
Tongda Xu, Yuanyuan Zhang, Gege Liao, Haochen Xuan, Jie Yin, Jieli Bao, Yang Liu, Dongye Li
Abstract
Background . In the present study, we aimed to find out whether luteolin (Lut) pretreatment could ameliorate myocardial ischemia/reperfusion (I/R) injury by regulating the lncRNA just proximal to XIST (JPX)/microRNA‐146b (miR‐146b) axis. Methods . We established the models in vitro (HL‐1 cells) and in vivo (C57BL/6J mice) to certify the protection mechanism of Lut pretreatment on myocardial I/R injury. Dual luciferase reporter gene assay was utilized for validating that JPX could bind to miR‐146b. JPX and miR‐146b expression levels were determined by RT‐qPCR. Western blot was utilized to examine apoptosis‐related protein expression levels, including cleaved caspase‐9, caspase‐9, cleaved caspase‐3, caspase‐3, Bcl‐2, Bax, and BAG‐1. Apoptosis was analyzed by Annexin V‐APC/7‐AAD dualstaining, Hoechst 33342 staining, as well as flow cytometry. Animal echocardiography was used to measure cardiac function (ejection fraction (EF) and fractional shortening (FS) indicators). Results . miR‐146b was demonstrated to bind and recognize the JPX sequence site by dual luciferase reporter gene assay. The expression level of miR‐146b was corroborated to be enhanced by H/R using RT‐qPCR ( P < 0.001 vs. Con). Moreover, JPX could reduce the expression of miR‐146b, whereas inhibiting JPX could reverse the alteration ( P < 0.001 vs. H/R, respectively). Western blot analysis demonstrated that Lut pretreatment increased BAG‐1 expression level and Bcl‐2/Bax ratio, but diminished the ratio of cleaved caspase 9/caspase 9 and cleaved caspase 3/caspase 3 ( P < 0.001 vs. H/R, respectively). Moreover, the cell apoptosis change trend, measured by Annexin V‐APC/7‐AAD dualstaining, Hoechst 33342 staining, along with flow cytometry, was consistent with that of apoptosis‐related proteins. Furthermore, pretreatment with Lut improved cardiac function (EF and FS) ( P < 0.001 vs. I/R, respectively), as indicated in animal echocardiography. Conclusion . Our results demonstrated that in vitro and in vivo , Lut pretreatment inhibited apoptosis via the JPX/miR‐146b axis, ultimately improving myocardial I/R injury.