| Purpose:The purpose of this Thesis was to investigate the effect and mechanism of acupuncture on early brain injury after subarachnoid hemorrhage.In this study,an animal model of subarachnoid hemorrhage was prepared by thread puncture method.CARD9 protein and its downstream pathway were used as the entry point.Through acupoint transformation of experimental animals,neurological function score,blood-brain barrier permeability detection,brain water content determination,protein quantitative analysis,immunohistochemistry,immunofluorescence,qPCR and other experimental methods were used.To observe the effects of acupuncture at Shuigou,Baihui,Neiguan,Hegu,Zusanli and other acupoints on nerve function,histopathology,early brain injury-related inflammatory factors and blood-brain barrier permeability in mice with subarachnoid hemorrhage,so as to explore the mechanism of acupuncture alleviating early brain injury and protecti ng blood-brain barrier after subarachnoid hemorrhage.To provide experimental basis for treating early brain injury after subarachnoid hemorrhage with acupuncture.Material and method:In this Thesis,a total of 412 adult C5 7B6J mice weighing 22-30g were reared in 12h light/12h no light cycle environment.62 mice were selected in Thesis 1 and randomly divided into Sham group,SAH group and SAH+AC group.The subarachnoid hemorrhage mouse model was prepared by thread threading in SAH group,and acupuncture intervention was given in SAH+AC group.In Thesis 2,150 mice were used,and the randomized group and intervention methods were the same as in paper 1.In Thesis 3,200 mice were randomly divided into Sham group,SAH+NS group,SAH+AC+NS group,S AH+AC+C ARD9 group,and SAH+AC+CARD9 siRNA group.Solvent,CARD9 recombinant protein and CARD9 small interfering RNA were administered by lateral ventricle injection.The Ethics committee approved all experimental procedures and protocols.Laboratory procedures follow the National Institutes of Health Guidelines for the Care and Use of Laboratory animals(NIH Publication No.85-23,revised 1996).Thesis 1:CARD9 expression peak time and changes after acupuncture in mice with subarachnoid hemorrhage1.The difference of CARD9 protein expression in time course and cell location after Subarachnoid hemorrhage was evaluated.The 42 mice were randomly divided into 7 groups(n=6):Sham group,SAH 3 h group,SAH 6 h group,SAH 12 h group,SAH 24 h group,SAH 48 h group and SAH 72 h group.CARD9 expression in the left hemisphere cortex was detected by Western blotting(WB).2.Five acupuncture points were selected:Baihui,Hegu,Shuigou,Neiguan and Zusanli.The corresponding locations of the acupoints of Baihui,Hegu,Shuigou,Neiguan and Zusanli in mice were determined through the transformation of animal degrees.Then,after modeling,the mice were fixed on the fixator every day for 0,12,24,3 6,and 48 hours and stimulated with acupuncture needles(20min).Baihui:The middle of the skull,stabbed back 1 mm,The positions of selected acupoints after transformation are as follows:Baihui:Center of skull,stab back 1mm;Zusanli:Below the knee joint,under the fibula small head 0.3 mm muscle groove directly stabbed 3mm,can be acupuncture;Hegu:Direct acupuncture of 1 mm between the first and second metacarpals of the forelimbs can be used for acupuncture;Ditch:the middle of the nose tip,straight or upward oblique stab 1mm;Neiguan:The inside of the forelimb,0.3 mm above the wrist between the radial and ulna,1-2mm straight stab.3.Thirty C5 7B6J adult mice were randomly divided into three groups:Sham group,SAH group and SAH+AC group CARD9 protein expression was detected by Western Blot after each group was given the above stimulus and time point.Thesis 2:Effect of acupuncture on regulating inflammatory response to early brain injury after subarachnoid hemorrhage in mice.Part 1:Study on the effect of acupuncture on early brain injury and nerve function after SAH in mice.1.Neurological function score:Thirty C5 7B6J adult mice were selected and randomly divided into 3 groups(Sham group,SAH group SAH+AC group),with 6 mice in each group successfully(total 18 mice).Neurological function score was performed after 0,12,24,3 6,and 4 8 hours of minor stimulation,respectively.Neural function in mice was assessed using the modified Garcia score and the balance beam test(n=6)(48 h).2.Thirty mice were selected and divided into 3 groups(Sham group,SAH group SAH+AC group).A total of 1 8 mice were used to evaluate blood-brain barrier injury by Evans blue(n=6).3..Thirty mice were selected and divided into 3 groups,SAH group and SAH+AC group with 6 successful mice.After the previous intervention,the dry weight of each part of the samples was measured and the brain water content was recorded after drying at 55℃ for 48h in the back of the brain.4.HE staining(n=6)was used to observe the damage of nucleus and tissue cells in the brain tissue of mice after acupuncture,and TUNEL was used to detect the apoptosis of brain tissue cells after acupuncture,and WB was used to detect whether the expression of Claudin 5 and ZO-1 was increased after acupuncture.5.collagen iv and lectin were detected by double-labeling immunofluorescence to determine the injury of blood-brain barrier,to check whether the degree of fracture of vascular endothelium was reduced,and to determine whether vascular integrity and blood-brain barrier were improved after acupuncture treatment.Part 2:Study on the inhibitory effect of acupuncture on early brain injury after subarachnoid hemorrhage in mice.The protein contents of IL1β,TNF-α and IL6 were quantitatively determined by ELISA kit.NF-κB was detected by WB to observe whether acupuncture could inhibit the expression of NF-κB after SAH in mice,which might inhibit the expression of inflammatory response.Thesis 3:Study on the effect and signaling pathway of acupuncture regulating CARD9 expression and inhibiting inflammation on early Blood-brain barrier injury after subarachnoid hemorrhage in mice.1.Effective doses of CARD9 recombinant protein and CARD9 siRNA were injected into the lateral ventricle.A total of 13 5 adult C57B6J mice were randomly divided into 5 groups:Sham(n=12),SAH+NS(2μLDMSO mixed with normal saline at 1:9 ratio),(n=12),SAH+AC+NS(acupuncture time was the same as in paper 1,n=12),SAH+AC+CARD9 recombinant protein(n=12),SAH+AC+CARD9 siRNA(n=12).In addition,the blood-brain barrier injury was analyzed by Evans blue permeability assessment at 48 h.At the same time,the continuity of cortical endothelial cells was detected by immunostaining 4 8 h after SAH acupuncture(n=3).2.Western Blot and PCR were used to detect CARD9 expression on genes and proteins.In order to explore the mechanism of BBB destruction,immunofluorescence was used to detect the expression levels of NLRP3 and CARD9 after the administration of CARD9 recombinant protein and CARD9 siRNA.3.Western Blot was used to detect NLRP3,GSDMD-G,GSDMD-N,pro-caspase-1,caspase-1 and IL-1 β in order to explore the signaling pathway of CARD9 inhibiting SAH inflammatory response and repairing BBB damage.IL-1 β,IL-18,TNF-α and IL6 were detected by ELISA.4..Blood-brain barrier permeability test,50 mice were selected and divided into Sham,SAH+NS,SAH+AC+NS,SAH+AC+CARD9,SAH+AC+CARD9 siRNA groups.After injecting Evans blue into mice,The measured values were substituted into the formula to calculate the sample Evans blue content to evaluate the blood-brain barrier permeability.5.collagen iv and lectin were co-labeled by immunofluorescence after CARD9 inhibitor and recombinant protein were administered after acupuncture.To evaluate the damage of blood-brain barrier,check whether the fracture degree of vascular endothelium is reduced,and explore the effect of regulating CARD9 expression on early blood-brain barrier injury after SAH in mice.Results:Thesis 1:CARD9 expression peak time and changes after acupuncture in mice with subarachnoid hemorrhage1.CARD9 protein expression was significantly increased in 3h,6h,12h,24h,4 8h and 72h groups.The expression peaked about 48 hours after simultaneous subarachnoid hemorrhage.2.CARD9 protein expression was significantly increased 4 8 h after subarachnoid hemorrhage,and was significantly decreased after 2 0 min of acupuncture stimulation at the five acupoints of Baihui,Hegu,Shuigou,Neiguan and Zusanli at 0,12,24,36,and 48h after model building(P<0.05).Thesis 2:Effect of acupuncture on regulating inflammatory response to early brain injury after subarachnoid hemorrhage in micePart I:Study on the effect of acupuncture on early brain injury and nerve function after SAH in mice1.The modified Garcia score and balance beam test(n=6)(48 h)were used to evaluate the neurological function of mice.The Garcia score in SAH group was significantly lower than that in Sham group(P<0.0 5),while the score in SAH+AC group was higher than that in SAH group(P<0.05).In the balance beam experiment,the score of SAH group was significantly higher than that of Sham group(P<0.0 5),and the score of SAH+AC group was lower than that of SAH group(P<0.05).2.The blood-brain barrier permeability in SAH group was significantly higher than that in Sham group(P<0.05),and the blood-brain barrier permeability in SAH+AC group was lower than that in SAH group(P<0.05).3.The content of cerebral water in SAH group was significantly higher than that in Sham group(P<0.05),and the content of cerebral water in SAH+AC group was lower than that in SAH group(P<0.05).4.HE staining results showed that the brain cells in the Sham group showed normal morphology and tissue structure.In SAH group,the nerve nuclei showed obvious shrinkage while the white matter became loose,the tissue structure was obviously disordered,the color was white,and the space between the nucleus and the white matter became large and irregular.After acupuncture intervention,the shrinkage degree of brain cell nucleus and the disorder degree of brain tissue were significantly reduced.5.TUNEL test indicated the apoptosis of brain tissue cells after acupuncture.It was obvious from the results that the apoptosis of SAH group was significantly increased,while the apoptosis of SAH group was significantly decreased after intervention.There was statistical significance among all groups by fluorescence analysis(P<0.05).6.CARD9 protein expression was significantly increased 4 8 h after subarachnoid hemorrhage,and was significantly decreased after 2 0min at 0,12,24,3 6 and 48h after acupuncture acupuncture at the five acupoints of Baihui,Hegu,Shuigou,Neiguan and Zusanli(P<0.05).The protein concentrations of Claudin 5 and ZO-1 in SAH group were lower than those in Sham group(P<0.05).The content of NF-κB protein in the acupuncture group was significantly higher than that in the SAH group(P<0.05).The content of NF-κB protein in the SAH group was higher than that in the SHAM group and the SAH+AC was lower than that in the SAH group(P<0.05).7.Collagen iv and lectin were co-labeled by immunofluorescence.In the Sham group,the vascular endothelium was intact,and the degree of vascular endothelium fracture increased significantly after subarachnoid hemorrhage.After acupuncture intervention,the continuity of endothelium was enhanced,and the degree and number of fracture were significantly reduced.These results indicate that acupuncture treatment can significantly reduce the permeability of blood-brain barrier after subarachnoid hemorrhage.Part 2:Study on the inhibitory effect of acupuncture on early brain injury after subarachnoid hemorrhage in mice1.The protein contents of IL1β,TNF-α and IL6 were determined quantitatively by ELISA.The IL1β protein in SAH group was higher than that in Sham group(P<0.05),and the SAH+AC in SAH group was lower than that in SAH group(P<0.05).TNF-a protein in SAH group was higher than that in Sham group(P<0.0 5),and SAH+AC was lower than that in SAH group(P<0.0 5).IL6 protein in SAH group was higher than that in Sham group(P<0.0 5),and SAH+AC was lower than that in SAH group(P<0.05).2.The content of NF-κB protein in SAH group was higher than that in Sham group and SAH+AC was lower than that in SAH group by WB detection(P<0.05).Thesis 3:Study on the effect and signaling pathway of acupuncture regulating CARD9 expression and inhibiting inflammation on early Blood-brain barrier injury after subarachnoid hemorrhage in mice1.The results of WB and PCR showed that CARD9 siRNA could effectively inhibit the expression of CARD9 after SAH,and injection of CARD9 5 0 ng/mpa recombinant protein could improve the expression of CARD9.2.NLRP3 and CARD9 were detected by immunofluorescence,indicating that the expression of NLRP3 increased significantly after the increase of CARD9 expression,and decreased when the expression of CARD9 decreased.At the same time,NLRP3 expression decreased significantly after acupuncture treatment,and the difference was statistically significant after fluorescence analysis:The expression of CARD9 in SAH+NS group was higher than that in Sham group(P<0.05),that in SAH+AC+CARD9 group was higher than that in SAH+AC+NS group(P<0.0 5),and that in SAH+AC+CARD9 siRNA group was lower than that in SAH+AC+NS group(P<0.05).Meanwhile,the expression of NLRP3 in SAH+NS group was higher than that in Sham group(P<0.05),that in SAH+AC+CARD9 group was higher than that in SAH+AC+NS group(P<0.0 5),and that in SAH+AC+CARDS siRNA group was lower than that in SAH+AC+NS group(P<0.05).3.WB was used to detect the expression of NLRP3,GSDMD-G,GSDMD-N,pro-caspase-1,caspase-1,IL-1 β and NF-κB proteins under various intervention factors:There was no difference in the expression of pro-caspase-1.The expression of NLRP3 protein in SAH+NS group was higher than that in Sham group(P<0.05),and that in SAH+AC+CARD9 group was higher than that in SAH+AC+NS group(P<0.05).SAH+AC+CARD9 siRNA group was lower than that of SAH+AC+NS group(P<0.05).The expression of GSDMD-G protein in SAH+NS group was higher than that in Sham group(P<0.0 5),that in SAH+AC+CARD9 group was higher than that in SAH+AC+NS group(P<0.05),and that in SAH+AC+CARD9 siRNA group was lower than that in SAH+AC+NS group(P<0.0 5).The expression of GSDMD-N protein in SAH+NS group was higher than that in Sham group(P<0.05),that in SAH+AC+CARD9 group was higher than that in SAH+AC+NS group(P<0.05),and that in SAH+AC+CARD9 siRNA group was lower than that in SAH+AC+NS group(P<0.05).The expression of Caspase-1 protein in SAH+NS group was higher than that in Sham group(P<0.05),that in SAH+AC+CARD9 group was higher than that in SAH+AC+NS group(P<0.05),and that in SAH+AC+CARD9 siRNA group was lower than that in SAH+AC+NS group(P<0.0 5).The expression of IL-1β in SAH+NS group was higher than that in Sham group(P<0.0 5),that in SAH+AC+CARD9 group was higher than that in SAH+AC+NS group(P<0.05),and that in SAH+AC+CARD9 siRNA group was lower than that in SAH+AC+NS group(P<0.05).The expression of NF-κB protein in SAH+NS group was higher than that in Sham group(P<0.05),that in SAH+AC+CARD9 group was higher than that in SAH+AC+NS group(P<0.05),and that in SAH+AC+CARD9 siRNA group was lower than that in SAH+AC+NS group(P<0.05).4.The protein contents of IL-1 β,IL-18,TNF-α and IL6 were determined quantitatively by ELISA.The expression of SAH+NS group was higher than that of Sham group(P<0.0 5),and that of SAH+AC+CARD9 group was higher than that of SAH+AC+NS group(P<0.05).The SAH+AC+CARD9 siRNA group was lower than the SAH+AC+NS group(P<0.05).The SAH+NS group was higher than the Sham group,and the SAH+AC+NS group was lower than the SAH+NS group,and the expression of SAH+AC+CARD9 group was increased while SAH+AC+CARD9 group siRNA group decreased expression(P<0.05).5.The blood-brain barrier permeability in SAH+NS group was significantly higher than that in Sham group(P<0.0 5).Permeability of SAH+AC+NS group was lower than that of SAH+NS group(P<0.05),permeability of SAH+AC+CARD9 group was higher than that of AH+AC+NS group(P<0.05),and permeability of SAH+AC+CARD9 siRNA group was lower than that of AH+AC+NS group(P<0.0 5).6.Collagen iv and lectin were co-labeled by immunofluorescence.In the Sham group,the vascular endothelium was intact,and the integrity of endothelial cells and tight-junction protein was damaged by labeling and staining after subarachnoid hemorrhage.After acupuncture intervention,the continuity of endothelium was enhanced,and the degree and number of fractures were significantly reduced.After the administration of CARD9 small interfering RNA,the damage degree of blood vessel was reduced and the continuity of endothelium was enhanced.After the administration of CARD9 recombinant protein,the degree of vascular breakage was significantly increased,and the vascular breakage was more inflammatory than that after subarachnoid hemorrhage alone.Conclusion:1.CARD9 protein expression peaks at 4 8 h in mouse brain tissue after SAH.2.Acupuncture of Baihui,Hegu,Shuigou,Neiguan and Zusanli can reduce the expression of CARD9 protein in the brain of mice after SAH,thus inhibiting the inflammation in early brain injury after SAH.3.Acupuncture can relieve brain tissue edema,reduce brain cell apoptosis and vascular endothelial injury in SAH mice,and reduce the permeability of the blood-brain barrier in mice after SAH,thus improving nerve function.4.CARD9 can cause blood-brain barrier damage by enhancing the expression of NF-κB and NLRP3,while activating downstream inflammatory factors such as caspase-1 and IL-1 β.5.Acupuncture may be an effective method to reduce early brain injury after SAH,and CARD9 may also be a potential target to protect the blood-brain barrier after SAH. |
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