Based On The Inflammatory Resolution To Compare The Cardioprotective Effect Of EA Per-conditioning And Post-conditioning On Myocardial Ischemia-reperfusion Injury And Its Mechanism

Background and PurposePrompt and effective myocardial reperfusion is the first choice for patients with myocardial infarction(MI)to reduce acute myocardial injury and limit the infarct size.However,the reperfusion process itself can induce myocardial cell death,i.e.,myocardial ischemia-reperfusion injury(MIRI),and there is still no effective treatment.Based on the classical theory of "Neiguan(PC 6)" in traditional Chinese medicine,this study compared the cardioprotective effects of Neiguan EA per-conditioning and post-conditioning against MIRI and explored the mechanisms of the two therapies to relieve MIRI from the perspective of pro-inflammatory regression,to provide a theoretical basis for the clinical application of EA of MIRI.MethodsThis experiment was divided into three parts.The first part focused on comparing the cardioprotective effects of EA per-conditioning and post-conditioning against MIRI.Male SD rats were randomly divided into control group(Con),model group(M),EA per-conditioning group(Per-EA)and EA post-conditioning group(Post-EA),with an additional EA per-conditioning group after 5 min of ischemia(Per5min-EA)according to the experimental results.In the Con group,no modeling and no EA were performed,and all other groups were subjected to 30 min ischemia and 24 h reperfusion(I/R)treatment.EA was applied at the onset of ischemia in the Per-EA group,at the onset of reperfusion in the Post-EA group,after 5 min of ischemia in the Per5min-EA group.After 24 h I/R,the cardioprotection of each group against MIRI was compared by echocardiography,infarct size,mortality,myocardial zymogram,and H&E staining of myocardial tissue.The second part is mainly based on SDF-1α to explore the mechanism of Per5min-EA alleviating MIRI by promoting inflammation resolution.This part is based on the Con group,M group and Per5min-EA group in the first part,adding Per5min-EA+SDF-1α(border zone)group,SDF-1α(infraction)group and AMD3100 group.1)After 24 h I/R,MPO+Tunel+DAPI immunofluorescence staining was used to observe the number and apoptotic rate of neutrophils in the infarcted myocardium.qRT-PCR was used to detect the gene expression of MPO,IL-1β and TNF-α in infarcted myocardium to preliminarily explore the pro-inflammatory effect of Per5min-EA.2)After 24 h I/R,Western Blot was used to detect the protein expression of SDF-1α in the infarct area to explore whether the cardioprotective effect of Per5min-EA was related to the expression of SDF-1α.qRT-PCR and ELISA were used to detect the expression of SDF-1α in infarcted myocardium and serum,respectively,to determine whether it was synthesized in the myocardium.Western Blot was used to detect and compare the expression of SDF-1α protein in the infarct area and peri-infarct area,to explore whether there was a concentration gradient of SDF-1α.Western Blot was used to detect and compare the expression of CXCR7 protein in the infarct area and peri-infarct area,and to investigate whether the concentration gradient of SDF-1α was related to the scavenger effect of CXCR7.3)In the Per5min-EA+SDF-1α(border zone)group,the active SDF-1α was injected to the edge of the LAD dominant zone before ischemia to destroy its concentration gradient,and the rest of the treatments were consistent with the Per5min-EA group;In the SDF-1α(infraction)group,the active SDF-1α was injected into the center of the LAD dominated area before ischemia to construct its concentration gradient,and the rest of the treatments were the same as those in the M group.The effect of anti-MIRI in each group was compared by echocardiography,infarct size and mortality after 24 h reperfusion to explore whether the central distribution of SDF-1α in the infarct area is the key to the cardioprotective of Per5min-EA.4)After 24 h I/R,Western Blot was used to detect the protein expression of CXCR4 in the infarcted myocardium to explore whether the cardioprotective effect of Per5min-EA was related to CXCR4.Western Blot was used to detect the protein expression of CXCR4 in the infarcted myocardium of the SDF-1α(infraction)group,to explore whether SDF-1α was a signaling factor for Per5min-EA regulating CXCR4.Western Blot was used to compare the expression of ERK/NF-κB signaling pathway in the infarcted myocardium of Per5min-EA and SDF-1α(infraction)groups to further explore the functional activation of the CXCR4 receptor.MPO+Tunel+DAPI immunofluorescence staining was used to compare the infiltration and apoptotic rate of neutrophils in the Per5min-EA and SDF-1α(infraction)groups.The protein expressions of IL-1β in the infarcted myocardium were detected by Western Blot.qRT-PCR detection of iNOS,CD206 and Arg 1 mRNA expression in infarcted myocardium to explore whether SDF-1α is a signaling factor through which Per5min-EA promotes inflammation resolution.5)Based on the first part of the study,this part continues to add AMD3100 group to explore whether the inhibition of CXCR4 is the key to the cardioprotective effect of Per5min-EA.In the AMD3100 group,AMD3100 was intraperitoneally injected 5 min before ischemia,and the rest of the treatments were the same as in the M group.The cardioprotective effects of anti-MIRI in each group were compared by echocardiography,myocardial infarction size,and mortality after 24 h reperfusion.6)After 15 min I/R,the protein expressions of SDF-1α and CXCR4 in the infarcted myocardium were detected by Western Blot,and the ERK protein expressions at two time points,I/R 15 min and 24 h,were compared to explore the regulatory ability of Per5min-EA on CXCR4 receptor,and then to determine the versatility of SDF-1α and CXCR4.The third part mainly compares the difference in cardioprotective mechanism between EA-Per and EA-Post and explores the mechanism of Post-EA alleviating MIRI based on the inhibition of vascular permeability by CXCR7-A2b.This part is based on the Con group,M group and Per5min-EA group in the first part,adding Post-EA+MRS 1754 group.In the Post-EA+MRS 1754 group,MRS 1754 was intraperitoneally injected 5 min before LAD ligation,and the rest of the treatments were the same as in the Post-EA group.1)Western Blot,qRT-PCR,and ELISA were used to detect SDF-1α in infarcted myocardium and serum,respectively.Western Blot and qRT-PCR were used to detect CXCR7 in infarcted myocardium to explore whether the cardioprotective effect of Post-EA was also related to SDF-1α concentrated expression.2)Western Blot was used to detect the expression of CXCR4 in infarcted myocardium to explore whether the cardioprotective effect of Post-EA was also related to CXCR4.3)Western Blot and qRT-PCR were used to detect A2b receptors in infarcted myocardium to compare the mechanism differences of the cardioprotective effects of Post-EA and Per5min-EA.MRS 1754 was used to inhibit A2b receptor,and the effect of anti-MIRI was compared in each group by echocardiography,mortality and myocardial enzymes after 24 h reperfusion to explore whether A2b receptor was a key factor in the cardioprotective effect of Post-EA.4)Western blot was used to detect the phosphorylation of CREB and NF-κ p65 downstream of A2b receptor in the infarcted myocardium of each group to explore the causal relationship between Post-EA and A2b receptor.5)CD34+Fibrinogen+DAPI immunofluorescence staining was used to observe the vascular permeability in the infarcted myocardium of each group,and Western Blot was used to detect the expressions of occludin(OCLDN),tight junction protein 1(ZO-1)and VEGFa in the infarcted myocardium level to explore the effect of Post-EA on vascular permeability.The expressions of TNF-α and IL-1β were detected by Western Blot,to evaluate the effect of Post-EA on vascular permeability from the perspective of inflammation resolution.ResultsPart Ⅰ:Different effects of Neiguan EA per-conditioning and post-conditioning on alleviating myocardial ischemia-reperfusion injuryBoth Per-EA and Post-EA could significantly improve the cardiac function of rats after 24 h I/R(P<0.01,P<0.001),and the difference between the two groups was not statistically significant(P>0.05).However,we found that Per-EA was prone to sudden death within 5 minutes of ischemia,and the mortality rate within 24 hours(37.5%)was higher than that of the M group(33.3%).Therefore,we adjusted the timing of EA intervention and performed EA after 5 min of ischemia.The results showed that Per5min-EA could also obtain cardiac function similar to Per-EA(P>0.05),and the mortality rate(6.7%)was lower than that of Per-EA and Post-EA(16.7%).Further research showed that both Per5min-EA and Post-EA could significantly reduce the infarct size after 24 h I/R(both P<0.01),as well as the expression levels of cTnI and CK-MB in serum(both P<0.001).The difference between the two groups was not statistically significant(P>0.05).At the same time,H&E results showed that both Per5min-EA and Post-EA could significantly improve the destruction of myocardial fiber structure and the infiltration of inflammatory cells after 24 h I/R.Part Ⅱ:Per5min-EA promotes inflammation resolution and mitigates myocardial ischemia-reperfusion injury based on the time-dependent effects of SDF-1α/CXCR4.1)After 24 h I/R,Per5min-EA significantly increased neutrophil apoptosis(P<0.0001),which in turn decreased neutrophil infiltration within the infarcted myocardium(P<0.01),as well as gene expression of the pro-inflammatory cytokines MPO,IL-1β and TNF-α(P<0.05,P<0.05,P<0.001).2)After 24 h I/R,Per5min-EA significantly increased the protein expression of SDF-1α in infarcted myocardium(P<0.0001),but failed to affect the expression of SDF-la mRNA(P>0.05).EA significantly increased the protein expression of SDF-1α in serum(P<0.0001).Further research showed that the protein expression of SDF-1α in the infarcted myocardium was significantly higher than that in the peri-infarct area(P<0.001),while the protein expression of CXCR7 in the infarcted myocardium was lower than that in the peri-infarct area(P<0.001).3)After 24 h I/R,SDF-1α(infarction)significantly improved cardiac function LVEF and LVFS(both P<0.001),decreased infarct size(P<0.001)and mortality,and the effect was similar to that of Per5min-EA group(P>0.05);Per5min-EA+SDF-1α(border zone)could not improve cardiac function LVEF and LVFS(P>0.05),nor could it reduce infarct size(P>0.05)and mortality.4)After 24 h I/R,Per5min-EA significantly decreased the protein expression of CXCR4 in infarcted myocardium(P<0.05),and SDF-1α(infarction)also decreased the expression level of CXCR4(P<0.05).Both SDF-1α(infarction)and Per5min-EA could significantly inhibit the phosphorylation of downstream ERK(both P<0.001)and NF-κB p65(P<0.05,P<0.01).Further research found that SDF-1α(infarction)can also increase the infiltration and apoptotic rate of neutrophils(P<0.0001)in the infarcted myocardium,which is similar to the effect of Per5min-EA group(P>0.05).Both SDF-1α(infarction)and Per5min-EA could reduce the protein levels of IL-1β(both P<0.05),and iNOS mRNA(both P<0.01),and increased the mRNA levels of CD206(both P<0.01)and Arg 1(P<0.001,P<0.0001)in infarcted myocardium.5)After 24 h I/R,AMD3100 significantly improved the cardiac function(P<0.05),and the effect was similar to that of the Persmin-EA group(P>0.05);AMD3100 also reduced the infarct size(P<0.05).0.01),but bigger than that in the Per5min-EA group(P<0.01).6)After 15 min I/R,Per5min-EA increased the protein expression of CXCR4/ERK in infarcted myocardium(P<0.001,P<0.05),which was opposite to the trend of I/R 24 h(P<0.05).At both time points,the expression of SDF-1α in the infarcted myocardium was increased(P<0.05).Part Ⅲ:Post-EA inhibits vascular permeability through CXCR7-A2b receptor,promotes inflammation resolution,and relieves myocardial ischemia-reperfusion injury.1)After 24 h I/R,Post-EA also increased the protein expression of SDF-1α in infarcted myocardium and serum(P<0.01,P<0.0001),but did not affect the expression of SDF-1αmRNA(P>0.05).Post-EA also inhibited the protein expression of CXCR7 in infarcted myocardium(P<0.01)but failed to affect the expression of the CXCR4 receptor(P>0.05).2)After 24 h I/R,Post-EA increased the protein expression of A2b in infarcted myocardium(P<0.01),while Per5min-EA had no such effect(P>0.05).A2b receptor inhibition with MRS 1754 decreased cardiac function LVEF(both P<0.01)and LVFS(P<0.01,P<0.05),increased serum CK-MB and cTnI levels(both P<0.0001),and mortality.3)After 24 h I/R,Post-EA promoted the activation of CREB and NF-κB p65 downstream of A2b(all P<0.0001),and inhibited the release levels of TNF-α and IL-1β(P<0.001,P<0.05),while MRS 1754 abolished the above effects.4)After 24 h I/R,Post-EA stabilized the expression of Tight Junction Protein OCLDN and ZO-1(both P<0.0001),decreased the level of VEGFa(P<0.05),and decreased vascular permeability(both P<0.05).<0.001),while MRS 1754 abolished the above effects.Conclusion1)Both Per5min-EA and Post-EA are effective in improving myocardial function after 24 h I/R,reducing infarct size and serum myocardial zymogram,and improving myocardial fibrous structure destruction and inflammatory cell infiltration in the infarcted area.It is important to note that the timing of the intervention with Per-EA needs to be avoided within 5 min ischemia,which significantly reduces mortality.2)Both Per5min-EA and Post-EA increase the expression of SDF-1α in infarcted myocardium.However,SDF-1α is not synthesized in the myocardium.On the one hand,it is derived from increased expression of SDF-1α in serum.On the other hand,CXCR7 expression is reduced in infarcted myocardium,inhibiting the internalized clearance of SDF-1α.A concentration gradient of SDF-1α from the center of infarcted area towards the per-infarcted area was constructed,favoring precise migration of myeloid cells.3)The concentrated distribution of SDF-1α is a key factor in the cardioprotective effect of Per5min-EA.SDF-1αexhibits versatility through CXCR4 receptors at different time points:after15 min reperfusion,SDF-1α activates the CXCR4/ERK pathway,which prevents massive apoptosis at the onset of reperfusion;after 24 h reperfusion,the continuously expressed SDF-1αsaturation signal degrades the CXCR4/ERK/NF-κB pathway,promoting inflammation resolution and alleviating myocardial ischemia-reperfusion injury.4)Post-EA down-regulates the A2b/CREB/NF-κB pathway by inhibiting the CXCR7 receptor,stabilizes vascular tight junction protein,inhibits vascular permeability,and prevents a large number of inflammatory cells in the recanalized blood from excessively infiltrating the myocardium,thereby leading to inflammation resolution and relieve myocardial ischemia-reperfusion injury.