| Objective:Cerebral infarction can lead to the destruction of blood brain barrier(BBB)permeability,and the protection of BBB is considered to be the core link in the treatment of cerebral infarction,and the regulation of matrix metalloproteinase network is the key to protect BBB.Xingnao Kaiqiao acupuncture is one of the most effective methods in the treatment of cerebral infarction,but the protective mechanism of this method on BBB is still unclear.Therefore,this study aims to observe the protective effect of Xingnao Kaiqiao acupuncture on cerebral infarction BBB,and elucidate its mechanism from the regulatory pathway of matrix metalloproteinase network.Methods:Experiment 1:the protective effect of Xingnao Kaiqiao acupuncture on BBB in cerebral infarction model rats:the healthy adult male SPF SD rats were randomly divided into sham group,model group,Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group.The model of middle cerebral artery occlusion(MCAO)was made by modified Longa thread occlusion method as the research object of cerebral infarction.The acupoints of Xingnao Kaiqiao acupuncture group were Neiguan(bilateral),Shuigou and Sanyinjiao(affected side),while those of Yangming meridian acupuncture group were Quchi,Hegu,Huantiao and Zusanli(all on the affected side).The two groups received acupuncture intervention at 2h,24h,48h and 72h after successful modeling,4 times in total,and the needles were kept for 30min each time.Sham operation group and model group only received the same fixation at the same time without acupuncture intervention.The brain tissues of the Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group were taken immediately after the completion of acupuncture intervention for 72 hours after modeling,while the brain tissues of the sham operation group and the model group were taken at the same time points.Eighteen rats were successfully enrolled in each group after neurobehavioral scoring(six rats were used to measure cerebral infarction volume,six rats were used to measure BBB permeability,and six rats were used to measure cerebral water content).Cerebral infarction volume was determined by TTC staining,BBB permeability was determined by Evans blue(EB)osmosis,and brain moisture was determined by dry-wet weight method.Experiment 2:regulation mechanism of matrix metalloproteinase network on the protective effect of Xingnao Kaiqiao acupuncture on BBB in cerebral infarction model rats:the animals were grouped and sampled at the same time as experiment 1.Twelve rats were successfully enrolled in each group after neurobehavioral scoring(six rats were used for Western Blot,Real-time PCR and ELISA,six rats were used for immunofluorescence(IF)detection and transmission electron microscopy detection);the indicators detected by Western Blot were TNF-α,IL-1β,ZO-1 and Occludin,the indicators detected by Real-time PCR were TNF-α and IL-1β,and the indicators detected by ELISA were serum S100β protein,IF detected MMP2 and MMP9,and transmission electron microscopy observed the ultrastructure of BBB.Results:Experiment 1:the protective effect of Xingnao Kaiqiao acupuncture on BBB in cerebral infarction model rats1.Comparison of neurobehavioral scores of rats in each group(1)Score after 24h intervention:compared with model group,the neurobehavioral score of Xingnao Kaiqiao acupuncture group was lower(P<0.01),there was no significant difference in neurobehavioral score in Yangming meridian acupuncture group(P>0.05).Compared with Yangming meridian acupuncture group,the neurobehavioral score of Xingnao Kaiqiao acupuncture group was lower(P<0.01).(2)Score after 48h intervention:compared with model group,the neurobehavioral scores in the Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group were lower(all P<0.01).Compared with Yangming meridian acupuncture group,the neurobehavioral score of Xingnao Kaiqiao acupuncture group was lower(P<0.01).(3)Score after 72h intervention:compared with model group,the neurobehavioral scores in the Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group were lower(all P<0.01).Compared with Yangming meridian acupuncture group,the neurobehavioral score of Xingnao Kaiqiao acupuncture group was lower(P<0.05).2.Comparison of cerebral infarction volume of rats in each groupThe cerebral infarction volume in the Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group was smaller than that in model group(all P<0.01).Compared with the Yangming meridian acupuncture group,the cerebral infarction volume was smaller than that in the Xingnao Kaiqiao acupuncture group(P<0.05).3.Comparison of BBB permeability of rats in each groupThe content of EB in the Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group was smaller than that in model group(all P<0.01).Compared with the Yangming meridian acupuncture group,the content of EB was smaller than that in the Xingnao Kaiqiao acupuncture group(P<0.05).4.Comparison of brain water content of rats in each groupThe brain water content in the Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group was smaller than that in model group(all P<0.01).Compared with the Yangming meridian acupuncture group,the brain water content was smaller than that in the Xingnao Kaiqiao acupuncture group(P<0.01).Experiment 2:regulation mechanism of matrix metalloproteinase network on the protective effect of Xingnao Kaiqiao acupuncture on BBB in cerebral infarction model rats1.Comparison of TNF-α,IL-1β mRNA and protein expression of rats in each groupReal-time PCR results showed that compared with model group,the mRNA expressions of TNF-α and IL-1β in Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group were decreased(both P<0.01).Compared with Yangming meridian acupuncture group,the expression of TNF-α mRNA in Xingnao Kaiqiao acupuncture group was decreased(P<0.05),there was no significant difference in IL-1β mRNA expression(P>0.05).Western Blot results showed that compared with model group,the protein expression of TNF-α and IL-1β in Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group were decreased(both P<0.01).Compared with Yangming meridian acupuncture group,the protein expression of TNF-α in Xingnao Kaiqiao acupuncture group was decreased(P<0.05),there was no significant difference in IL-1β protein expression(P>0.05).2.Comparison of MMP2 and MMP9 expression of rats in each groupThe results of IF showed that compared with model group,the expression levels of MMP2 and MMP9 in Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group decreased(all P<0.01).Compared with Yangming meridian acupuncture group,the expression level of MMP2 in Xingnao Kaiqiao acupuncture group decreased(P<0.05),MMP9 expression level decreased(P<0.01).3.Comparison of the expression of ZO-1 and Occludin of rats in each groupThe results of Western Blot showed that compared with model group,the expression of ZO-1 and Occludin in Xingnao Kaiqiao acupuncture group increased(all P<0.01);the expression of ZO-1 protein in Yangming meridian acupuncture group increased(P<0.01),Occludin protein expression increased(P<0.05).Compared with Yangming meridian acupuncture group,the expression of ZO-1 protein in Xingnao Kaiqiao acupuncture group increased(P<0.01),Occludin protein expression increased(P<0.05).4.Ultrastructural changes of BBB in each groupTransmission electron microscopy showed that the ultrastructure of BBB in model group was damaged,which showed obvious swelling of endothelial cells and basement membrane,deformation and irregularity,and loose tight connection.The ultrastructural damage of BBB in Xingnao Kaiqiao acupuncture group was significantly reduced,which showed that the swelling of endothelial cells and basement membrane was significantly ’reduced,the morphology was regular,and the damage of tight connection was significantly reduced.The ultrastructural damage of BBB in Yangming meridian acupuncture group was reduced,which showed that the swelling of endothelial cells and basement membrane was reduced,the morphology was regular,and the damage of tight connection was reduced.5.Comparison of serum S100β protein content in each groupThe results of ELISA showed that compared with model group,the content of serum S100β protein in Xingnao Kaiqiao acupuncture group and Yangming meridian acupuncture group decreased(all P<0.01).Compared with Yangming meridian acupuncture group,the content of serum S100β protein in Xingnao Kaiqiao acupuncture group was lower(P<0.01).Conclusion:1.Both Xingnao Kaiqiao acupuncture and Yangming meridian acupuncture could improve the neurological function,reduce the volume of cerebellar infarction,reduce the permeability of BBB and reduce the water content of brain in rats with cerebral infarction model.Among them,Xingnao Kaiqiao acupuncture had a better protective effect on BBB and had a faster effect on improving the neurological function.2.Both Xingnao Kaiqiao acupuncture and Yangming meridian acupuncture could inhibit the expression of inflammatory indicators(TNF-α and IL-1β),then down-regulate the expression of matrix metalloproteinase core indicators(MMP2 and MMP9),and up-regulate the expression of tight junction(ZO-1 and Occludin)to protect BBB.These results indicate that acupuncture could achieve this effect by regulating the matrix metalloproteinase network pathway.In general,Xingnao Kaiqiao acupuncture had better protective effect on BBB than Yangming meridian acupuncture.This effect was further verified by electron microscopy at the subcellular level of BBB ultrastructure and serum level of S100 β protein content.3.Xingnao Kaiqiao acupuncture is a more effective acupuncture method for cerebral infarction,which provides a deep theoretical basis for clinical treatment. |
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