| Objective: To make explicit the effects of surround needling of scalp point on intracranial micro-circulation and nerve function in rats with cerebral infarction through experimental research,the mechanism of surround needling of scalp point which up-regulates the expression of related factors of angiogenesis by Wnt/β-catenin signal pathway is clarified,and the angiogenesis after cerebral infarction was promoted,so as to provide experimental basis for the treatment of facilitating angiogenesis after cerebral infarction with surround needling of scalp point.Methods: The experimental study is divided into four parts.Part 1: The effects of surround needling of scalp point on nerve function,pathological form of cerebral infarction tissue,cerebral infarction volume and cerebral cortical blood perfusion in rats with middle cerebral artery occlusion(MCAO)were observed.A total of 108 male SD rats were randomly divided into sham operation group(Sham),model group(Model)and surround needling of scalp point group(AC group)according to random number table.In accordance with the time points of 7days,14 days and 21 days after surgery,they were divided into three subgroups,with 10 rats in each,and corresponding disposition or treatment respectively was given.The neural function of rats in each group was observed by mNSS scoring method.HE staining was used to observe the pathomorphological changes of the cerebral infarction tissues of rats in each group.The volume of cerebral infarction was observed by TTC staining.The cortical blood flow(rCBF)in the cerebral ischemia area of rats was observed by laser doppler flowmetry.The effects of surround needling of scalp point on behavioristics and morphology of MCAO rats were ensured.Part 2: The effect of surround needling of scalp point on the expression of Wnt/β-catenin signal pathway related protein in MCAO rats was observed.A total of 108 male SD rats were randomly divided into sham operation group(Sham),model group(Model)and surround needling of scalp point group(ACgroup).According to the time points of 7 days,14 days and 21 days after operation,each group was divided into three subgroups,12 rats in each subgroup,numbered and labeled respectively.Another 120 rats were randomly divided into sham operation group(Sham),model group(Model),surround needling of scalp point group(AC group),inhibitor DKK1 group(DKK1)and surround needling of scalp point+inhibitor DKK1 group(AC+DKK1).According to 7 days and 14 days after operation,each group was divided into 2 subgroups,12 rats in each subgroup were numbered and labeled.Take corresponding measures.The expression of Wnt3 a,β-catenin,TCF4,cyclin D1 and GSK-3β in cerebral infarction organization by immunohistochemistry,Western blot and RT-PCR.so as to clear the effect of surround needling of scalp on Wnt/β-catenin signal pathway key protein and gene expression in MCAO rats.Part 3: The effects of surround needling of scalp point on VEGF and other angiogenesis related factors in rats with cerebral infarction were observed.The number and grouping method of rats were the same as part 2.The expression of VEGF,Ang2,Flk1 and b FGF were detected by immunohistochemistry,Western blot and RT-PCR.It is clear that surround needling of scalp point can promote the expression of angiogenesis related factors after cerebral infarction.From the point of view of Wnt/β-catenin signaling pathway mediates the up regulation of angiogenesis related factors,the mechanism of surround needling of scalp point promoting angiogenesis after cerebral infarction was revealed.Results:Part 1:(1)MNSS evaluation method were used to observe the rats’ nerve function defect degree in each group: compared with the model group,the scores of AC group were significantly lower at 7days,14 days and 21days(P<0.05).In AC group,the time points of 7days,14 days and 21 days decreased gradually,and 21 days decreased significantly compared with 7days(P< 0.05).(2)HE staining was used to observe the infarction tissue pathological and morphological changes: in the model group,there were obvious tissue loose,structure disorder,irregular shape,unclear arrangement and number of nerve cells,obvious deformation of microvascular lumen,even collapse,cytoplasmic light staining and nucleolysis in the cerebral infarction tissues,until 21 days,they were slightly improved but not obvious.Compared with model group,the AC group was significantly improved at three time points,increased number of nerve cells around the infarct.Compared with the model group,the structure and morphology of rats were slightly regular.The arrangement trend was tighter than that of the model group,the deformation of microvascular lumen was less,the number of microvessels was more than that of the model group.At 7 and 14 days,the pathological changes of brain tissue in AC group were significantly improved compared with model group.At 21 days,compared with the model group,the number of peripheral nerve cells increased,the structure and morphology were regular,the arrangement trend was tight,the microvascular lumen had a little deformation,and the number of microvessels increased.(3)Cerebral infarction volume was observed by TTC staining:comparison of three time points,the volume of cerebral infarction in AC group was significantly smaller than that in model group(P<0.05).The volume of cerebral infarction in AC group decreased significantly in 21 days compared with 7 days(P<0.05).The results show that the volume of cerebral infarction can be reduced obviously by surround needling of scalp point.(4)Laser doppler blood flow meter test was used to detect rats’ cortex blood flow(rCBF)at each time point before and after experiment in cerebral infarction: at the time points of 7 days,14 days and 21 days,the rCBF of the model group was significantly lower than that before the experiment(P<0.01,P<0.05).After the experiment,the rCBF of each time point in the model group did not increase significantly with the extension of time.After theexperiment,the rCBF of AC group was close to that before the experiment.After 21 days,the difference in rCBF between the postoperative period and the pre-experiment period was not statistically significant(P>0.05).Part 2:(1)The expression of Wnt3 a,β-catenin,TCF4,Cyclin D1 and GSK-3βwere observed by immunohistochemistry: after acupuncture intervention,the expression of Wnt3 a and β-catenin in AC group was significantly higher than that in model group(P<0.05,P<0.01),and the peak lasted to 14 days,and then tended to decline slightly in 21 days.The expression of TCF4 in the three time points of AC group was significantly higher than that in sham group(P<0.05),and the protein expression peaked on the 7th day,remained at a high level until the 21 st day.In the three time points,the expression of cyclin D1 in AC group was significantly higher than that in sham group(P<0.01),which was significantly different from that in model group(P<0.05),the peak appeared on the 14 th day.The expression of GSK-3β in AC group was significantly lower than that in sham group(P<0.05).(2)Western blot method was used to detect the expressions of Wnt3 a,β-catenin,TCF4,Cyclin D1 and GSK-3β: the protein expression of Wnt3 a andβ-catenin increased at three time points.Compared with sham group,AC group and model group increased significantly(P<0.01,P<0.05).The expression of TCF4 and cyclin D1 in AC group was significantly higher than that in model group(P<0.05).The expression of GSK-3β protein in AC group was significantly lower than that in model group(P<0.05).surround needling of scalp point circumcision can up regulate Wnt 3a,β-Catenin,TCF4,cyclin D1 protein expression and down regulate GSK-3β protein expression,especially Wnt 3a and β-catenin are more sensitive to head acupoint circumcision.After inhibitor intervention,Wnt3 a and β-catenin protein increased significantly in AC+DKK1 group and model group at 7 and 14 days(P<0.01).Compared with model group,DKK1 group decreased significantly(P<0.01).(3)The expression of Wnt 3a,β-catenin,TCF4,Cyclin D1,GSK-3β mRNA was detected by RT-PCR: at three points in time,The expression of Wnt3 a,β-catenin,TCF4,Cyclin D1 mRNA in AC group was significantly higher than that in model group(P<0.01).The expression of GSK-3β decreased gradually compared with the model group,the difference was statistically significant(P<0.05).The expression of Wnt 3a and β-catenin mRNA was more sensitive after acupuncture,especially on the 7th and 14 th day.After adding inhibitor,the expression of Wnt 3a and β-catenin mRNA was significantly different between AC+DKK1 group and model group(P<0.05).Compared with model group,DKK1 group decreased significantly(P< 0.01).Part 3:(1)The expression of VEGF,Ang2,Flk1 and b FGF were observed by immunohistochemistry: the expression of VEGF and Ang2 in AC group was significantly higher than that in sham group and model group(P< 0.01),and the peak period of 7days continued to 14 days.After 21 days,it stabilized and still significantly exceeded Sham group(P<0.01).FLK1 expression was significantly increased in the AC group compared with the sham group and the model group,the difference was statistically significant(P<0.01),reached a peak at 7 days and continued to decline slightly until 21 days,significantly exceeding the model group(P<0.05).The expression of bFGF began to increase at 7 days,the model group was significantly higher than the sham group(P<0.05),and began to gradually decline at 14 days.The AC group was significantly higher than the model group at three time points(P< 0.01).(2)Western blot was used to detect the expressions of VEGF,Ang2,Flk1 and bFGF: at three time points in AC group,VEGF protein was significantly higher than that in model group and sham group,the difference was statistically significant(P< 0.01).The expression of Ang2,FLK1,b FGF in AC group increased to peak at 14 days,which was significantly higher thanthat in model group and sham group,the difference was statistically significant(P< 0.05).The expression of VEGF and Ang2 protein in the acupuncture group increased significantly on the 7th and 14 th days.After the addition of inhibitors,the expression of VEGF and Ang2 protein was significantly increased at 7 and 14 days at two time points,AC+DKK1 group and model group(P< 0.05).Compared with the Model group,the DKK1 group decreased significantly,and the difference was statistically significant(P< 0.01).(3)The mRNA expressions of VEGF,Ang2,Flk1 and bFGF were detected by RT-PCR: the expression of VEGF,Ang2,Flk1,b FGF mRNA was higher in the AC group than in the model group,the difference was statistically significant(P < 0.05).Among them,VEGF and Ang2 mRNA expression reached a peak on the 7th day and continued to 14 days,gradually decreasing.After adding inhibitors,the expression of VEGF and Ang2 mRNA was observed.The expression of AC+DKK1 group was significantly higher than that of model group at two time points of 7 days and 14 days(P< 0.01).Compared with the model group,the DKK1 group decreased significantly(P <0.05).Conclusion:1.The surround needling of scalp point can reduce the degree of neurological deficit,reduce the volume of cerebral infarction,increase the blood flow and perfusion in cerebral infarction area,and restore the neurological function of cerebral infarction rats.2.The surround needling of scalp point can up-regulate the related proteins of Wnt/β-catenin signal pathway and activate the expression of downstream angiogenesis factors.3.The surround needling of scalp point can up-regulate the protein expression of the factors related to angiogenesis,promoting the micro-angiogenesis in the infarction area,and reconstructing the microcirculation in the infarction area.4.The surround needling of scalp point promotes the expression of proteins related to angiogenesis through the regulation of Wnt/β-catenin signal pathway,thereby promoting angiogenesis and reducing the degree of nerve function injury in rats with cerebral infarction. |
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