Correlation between endoplasmic reticulum stress and macrophage polarization in myocardial ische-mia-reperfusion injury
Objective To investigate the correlation between endoplasmic reticulum stress(ERS)and macrophage polarization in myocardial ischemia-reperfusion injury(MIRI).Methods C57BL/6 mice,20-40 g,20(half male and half female),provided by the Experimental Animal Center of Guangxi Medical University,were divided into four groups of 5 mice each using the random number table method,the sham operation group(Sham group),the myocardial ischemia-reperfusion group(I/R group),the clin-damycin+I/R group(TM+I/R group),and the 4-phenyl-butyric acid+I/R group(4-PBA+I/R group).In the Sham group,the coronary artery was padded after simple chest opening;in the I/R group,the ante-rior descending branch of the left coronary artery was ligated for 30 min and reperfused for 2.5 h.In TM+I/R and 4-PBA+I/R groups,TM and 4-PBA were injected intraperitoneally,respectively,and the anteri-or descending branch of the left anterior descending branch was ligated for 30 min and then reperfused for 2.5 h.The number of macrophages recruited was detected by flow cytometry;the myocardial tissues were stained by hematoxylin and eosin(HE)staining and immunohistochemistry Myocardial cell morphology was observed;protein expression of myocardial ERS marker protein glucose regulated protein 78(GRP78),macrophage polarization marker protein inducible nitric oxide synthase(iNOS)and nuclear factor-KB(NF-κB)pathway marker protein p65 were detected by Western blotting.comparisons between groups were made using a t-test for two-sample comparisons in a completely randomized design.Results The results of flow cytometry,the number of macrophages recruited was higher in the 4-PBA+I/R group(0.192±0.019),the 1/R group(0.308±0.043)and the TM+I/R group(13.112±0.462)than in the Sham group(0.116±0.03,t=8.835,9.928,62.941,P<0.05),and the 4-PBA+I/R group was lower than the I/R group(t=-13.485,P<0.05),while the TM+I/R group was higher than the I/R group(t=62.011,P<0.05).Immunohistochemistry of myocardial ERS marker protein GRP78 and macrophage po-larization marker protein iNOS showed weak positive cytoplasmic staining in myocardial tissue staining in the Sham group[(9.3±3.2)%,(7.6±3.9)%,P<0.05;and(18.4±2.4)%,(22.3±2.1)%,P<0.05]in the 4-PBA+I/R group.Weak positive cytoplasmic staining;positive cytoplasmic staining ap-peared in the I/R group[(46.0±1.3)%,(33.6±4.9)%,P<0.05],and strong positive cytoplasmic staining appeared in the TM+I/R group[(76.2±9.3)%,(79.3±5.2)%,P<0.05].The results of Western blotting showed that the 4-PBA+I/R group,I/R and TM+1/R groups had higher GRP78,iNOS,and p65 protein expression than the Sham group(t=1.298,3.934,and 6.128,P<0.01),and the GRP78 protein expression in the 4 groups was 0.716±0.305,0.308±0.043,1.589±0.349,0.518±0.141,4 groups of iNOS protein expression were 0.416±0.207,0.748±0.274,1.323±0.298,0.223±0.132,and 4 groups of p65 protein expression were 0.687±0.214,1.101±0.496,1.697±0.959,1.697±0.959.4-PBA+I/R group protein expression(0.716±0.305,0.416±0.207,0.687±0.214)was lower than that of the I/R group(0.308±0.043,0.748±0.274,1.101±0.496,t=-13.485,P<0.01),TM+I/R group protein expression(1.589±0.349,1.323±0.298,1.697±0.959)were higher than that of the I/R group(0.308±0.043,0.748±0.274,1.101±0.496,t=62.011,P<0.01);and GRP78 had a positive correlation with iNOS protein expression(correlation coeffi-cient(r1)=0.998,P<0.01);p65,GRP78 had positive correlation with iNOS protein expression[corre-lation coefficient(r2)=0.989,(r3)=0.978,P<0.01].Conclusion In MIRI,ERS,and M1-type macrophages,which have a positive correlation,ERS is an important regulator of macrophage polarization;myocardial ERS may regulate macrophage polarization through NF-κB p65.
Myocardial ischemia-reperfusion injuryEndoplasmic reticulum stressMacro-phage Ml polarizationGlucose regulated protein 78Nuclear factor-KB