| Objective: BML-111 is a synthetic,more stable lipoxygen A4 analog and lipoxygen A4 receptor agonist.Lipoxin A4,as an endogenous anti-inflammatory mediator,can exert a wide-ranging anti-inflammatory and regressive effect by binding to LXA4 receptor.Our previous research also confirmed that LXA4 can improve cerebral ischemic injury by exerting anti-inflammatory effects.Further research found that BML-111 has a neuroprotective effect on cerebral ischemic injury,but its mechanism is still unclear.This study observed that the effect of BML-111 on the expression of TNF-?,IL-1? and the percentage of M1 microglia/macrophages,as well as P2X7 in rats with permanent cerebral ischemia to further explore its potential neuroprotective mechanism.Methods: Sixty healthy male Sprague-Dawley rats(8-10 weeks old,weight280-320 g)were divided into four groups randomly: Sham group,cerebral ischemia(CI)group,BML-111 group,BML-111+BZATP(P2X7 receptor agonist)group,15 rats in each group.Permanent middle cerebral artery occlusion(pMCAO)model was established by modified Longa suture.At 30 min before pMCAO,the rats in BML-111+BZATP group were given BZATP(5?g/5?L)through left lateral ventricle injection and the remaining groups were injected with equal volume of normal saline.BML-111 group and BML-111+BZATP group were given BML-111(1mg/kg)by left intraperitoneal injection immediately after pMCAO and continued to be administered for 7 days.The sham group and CI group were injected with equal volume of normal saline at the same time.At 7 days after pMCAO,rats were evaluated for neurological deficits.The volume of cerebral infarction was measured by 2,3,5-triphenyl tetrazolium chloride(TTC)staining.The histological damage was observed by hematoxylin-eosin(HE)staining.The expression of TNF-? and IL-1? were detectedby enzyme linked immunosorbent assay(ELISA)and Real time PCR(RTPCR).The percentage of M1 microglia/macrophages were detected by Flow cytometry.In addition,the expression of P2X7 was detected by Immunohistochemistry.Results: 1.Neurological deficit score: The Sham group had no symptoms of neurological deficit,and the remaining groups had varying degrees of neurological deficit.Compared with the CI group,the neurological deficit score was significantly improved in BML-111 group(P<0.05);compared with the BML-111 group,the neurological deficit score in BML-111+BZATP group was significantly increased(P<0.05).2.Cerebral infarction volume: no infarction was seen in the sham operation group,and other groups had white infarctions of different sizes.Compared with the CI group,the infarct volume of the BML-111 group was significantly reduced(P <0.05);compared with the BML-111 group,the infarct volume of BML-111+BZATP group was significantly increased(P <0.05).3.Histological damage: the Sham group showed the rat brain tissue structure was clear and complete,the nerve cells were densely arranged,the neurons were rich in cytoplasm,the nucleoli was clear,and the space around the cells was dense without edema.The remaining groups had varying degrees of nerve cell number reduction,disordered arrangement,nuclear shrinkage,cytoplasmic vacuole,interstitial looseness and other tissue and cell damage.Compared with CI group,the damage of brain tissue cells in BML-111 group was reduced;compared with BML-111,the damage of brain tissue cells in BML-111+ BZATP group was more severe.4.The expression of IL-1?: Compared with sham operation group,the expression of IL-1? in CI group was up-regulated(P<0.05);compared with CI group,the expression of IL-1? was down-regulated in BML-111 group(P<0.05);compared with BML-111 group,the expression of IL-1? was up-regulated in BML-111+BZATP group(P<0.05).5.The expression of TNF-?: Compared with Sham group,the expression of TNF-? inCI group was up-regulated(P<0.05);compared with CI group,the expression of TNF-? was down-regulated in BML-111 group(P<0.05);compared with BML-111 group,the expression of TNF-? was up-regulated in BML-111+BZATP group(P<0.05).6.The expression of P2X7: Compared with the Sham group,the expression of P2X7 in the CI group increased(P<0.05);compared with the CI group,the expression of P2X7 decreased in the BML-111 group(P<0.05);compared with the BML-111 group,the expression of P2X7 increased in BML-111+BZATP group(P <0.05).7.The percentage of M1 microglia/macrophages: Compared with the Sham group,the percentage of M1 microglia/macrophages in the CI group increased(P<0.05);compared with the CI group,the percentage of M1 microglia/macrophages in the BML-111 group decreased(P<0.05);compared with the BML-111 group,the percentage of M1 microglia/macrophages in the BML-111+BZATP group increased(P<0.05).Conclusions: 1.BML-111 has a protective effect on permanent cerebral ischemic injury in rats.2.It may be one of the important mechanisms of neuroprotective effect of BML-111 on permanent cerebral ischemia by inhibiting P2X7 activation,decreasing M1 microglia/macrophage phenotypic polarization,down regulating the expression of TNF-? and IL-1?,then inhibiting excessive inflammatory response... |
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