| ObjectiveExperiment mainly divided into three parts,the first experiment explores Shenhuo moxibustion of yao medicine effect on nerve protection of ischemia-reperfusion rats after MCAO,observe the effect of nerve function by Shenhuo moxibustion at 6 h,24 h,72 h,7 d and 14 d.From the point of view of cell morphology evaluate the influence of Shenhuo moxibustion of yao medicine on infarction volume and brain tissue cell morphology in rats against ischemia reperfusion,but also estimate the effect on the recovery of neural function defect exists.At the same time,conparing the classic moxibustion therapy,NK-kappa B inhibitors NAC predominate and NAC joint Shenhuo moxibustion.The second part is to observe the effect of Shenhuo moxibustion of yao medical on cell apoptosis,oxidative stress and inflammation related indicators;The third part discusses whether Shenhuo moxibustion of yao medical plays a neuroprotective role and the related mechanism through mediating NF-kappa B pathway.Methods:Animal experiment part:(1)the middle cerebral artery occlusion method(MCAO)was used to establish the animal model of experimental rats with ischemia reperfusion injury,ischemia for 2h and reperfusion for 24h.The behavior of the rats was observed,and the neurological deficit score of the rats was observed by zea-longa scoring method to evaluate whether the model was successful or not.The experimental rats were divided into six groups by random method:sham operation group,model group,Shenhuo moxibustion treatment group,moxibustion treatment group,NAC group,Shenhuo moxibustion+NAC group.In the treatment group,Shenhuo moxibustion and moxibustion therapy were used,and NAC was interfered by ventricular injection.(2)Neurological function scores were observed at different time periods(6h,24h,72h,7d and 14d).(3)Detecting cerebral infarction area by TTC method.(4)Using HE staining to observe morphological changes of brain tissue sections.(5)Nissl staining was used to observe neuron injury.(6)Detecting biochemical indicators related to oxidative stress:peripheral blood of mice was collected at 14d,blood was collected and serum was isolated,MDA and SOD activities in serum at different time periods were observed by ELISA.(7)Peripheral blood of mice was collected at 14d,blood was collected and serum was isolated.The expression of serum inflammatory cytokines(TNF-α,IL-1 and IL-10)was observed by ELISA.(8)Observe TUNNEL apoptosis of each groups.(9)Using immunohistochemical method,western blotting method and qPCR method to measure the proteins related to NF-κB pathway(Cox-2,iNOS,NF-κB/p65,Bax,Bcl-2,Caspase-3)in each group,so as to determine the effect of therapy of Shenhuo moxibustion on related pathway proteins and downstream apoptotic factors.Results1.Effects of Shenhuo moxibustion of yao-medicine on neurologic impairment of ischemia-reperfusion injury in ratsNo abnormal behaviors or limb functions were observed in Sham(Sham surgery group),and the score was O.However,the model group showed contralateral turning or dumping and other neurologic impairments,and the scores of neurologic impairment in the model group were significantly different from those in the sham group.Neurologic impairment scores of each treatment groups was no significant difference at 6h,24h,72h after ischemia reperfusion(P>0.05),comparing with model group,there was no significantly improve neurologic impairment in shenhuo moxibustion treatment and moxibustion treatment group(P>0.05)at 72h after I/R,while at 7d in the treatment,the neurologic impairment in shenhuo moxibustion treatment group had lower compared with model group(P<0.05),the effect last for 14days become obvious gradually(P<0.01),and moxibustion group was no significant difference compared with the model group(P>0.05).However,the NAC group,an antioxidant,was significantly effective at 7d and 14d,significantly improving the neurologic impairment of I/R rats,which was significantly different from the model group(P<0.01),the effect is equivalent to shenhuo moxibustion group.NAC+Shenhuo moxibustion showed a decrease in nerve function score at 7d and 14d(P<0.01).At 14d,the value of neurologic impairment scores decreased in shenhuo moxibustion+NAC group,but the difference was not significant compared with shenhuo moxibustion group.2.Effects of Shenhuo moxibustion of yao medicine on brain injury volume of ischemia-reperfusion ratsThrough image analysis software analysis and calculation,compared with the sham operation group,TTC staining showed obvious cerebral infarction lesions in the coronary tissue sections of rats after MCAO,and the infarct area has no significant difference at 24h after treatment,indicating that no effect was observed on the cerebral infarction volume of I/R rats in each treatment group within 24h.At 14d,there was a statistically significant difference in the cerebral infarction volume in moxibustion group compared with the model group(P<0.05);Compared with the model group,shenhuo moxibustion group、NAC group and NAC+Shenhuo moxibustion treatment group showed a significant decrease in the infarct area of rats,with statistically significant difference(P<0.01),indicating shenhuo moxibustion has obvious neuroprotective effect on cerebral infarction volume,and NAC also has neuroprotective effect.There is no obvious difference in the effect of shenhuo moxibustion and NAC on cerebral infarction volume.The infarct volume decreased in shenhuo moxibustion+NAC group,but the difference is not significant compared with shenhuo moxibustion alone.3.Pathological effects of Shenhuo moxibustion of yao medicine on ischemic reperfusion injury of brain tissue in ratsThe results of HE staining indicated that the morphology of brain tissue cells in the sham group of rats with ischemic brain tissue was basically normal,while the model groups showed necrosis of nerve cells in ischemic center,disorder of tissue structure arrangement,widening of cell gap,swelling of cells,pyknosis of nucleus and serious destruction of structure;However,Shenhuo moxibustion therapy can improve the necrosis process of neuronal cells after ischemia reperfusion,and alleviate ischemia reperfusion injury.Compared with the model group,nerve cell abnormalities are relatively mild,with complete nuclear membrane,uniform chromatin,and less organelle destruction.The morphological changes were consistent with those of the moxibustion group.Under the microscope,nerve swelling in the ischemic brain tissues of rats in the NAC and NAC+Shenhuo moxibustion treatment group was mild,and the number of necrotic cells was significantly reduced compared with that in the model group,indicating that NAC has a clear protective effect as an antioxidant,and the inflammatory reaction was significantly reduced when combined with Shenhuo moxibustion,inflammatory cell infiltration decreased,cell necrosis and nuclear pyknosis decreased significantly.4.Use Nissl staining observing the number of neurons in the ischemic brain tissueNissl staining results showed that Nissl corpuscle number increased in the Shenhuo moxibustion and moxibustion treatment groups compared with the model group,with normal cell morphology and complete structure relatively,suggesting that moxibustion treatment can reduce neuron damage after I/R.However,the number of Nissl corpuscles in the NAC group and NAC+Shenhuo moxibustion treatment group was larger,with most of the cells having normal morphology and complete structure,indicating that NAC and NAC+Shenhuo moxibustion treatment significantly promoted the survival of neurons in the ischemic brain tissue.5.Effects of Shenhuo moxibustion of yao medicine on MDA and SOD expression in ischemia-reperfusion ratsThis experiment showed that after created cerebral ischemia reperfusion model,MDA level in the model group was significantly increased(P<0.01),and SOD activity was significantly reduced(P<0.01)compared with that in the sham operation group,indicating that oxidative stress injury was obvious,SOD activity was significantly inhibited,and antioxidant capacity was decreased,which was consistent with the previous literature reports.When moxibustion was used,MDA level decreased,and there was significant difference compared with the model group(P<0.05),SOD activity increased(P<0.05).After Shenhuo moxibustion,the MDA level significantly decreased(P<0.01),SOD activity increased significant(P<0.01),suggesting Shenhuo moxibustion treatment can improve the ability of SOD activation obviously,enhance the ability of rat brain tissue to resist oxidative stress with I/R injury,reduce oxidative stress related injuries,and through the anti lipid peroxidation ralated reaction to protect I/R injury.While the SOD activity of ischemia-reperfusion rats had significantly increased(P<0.01),MDA content decreased obviously(P<0.01)in lateral ventricle giving NAC injection and NAC joint Shenhuo moxibustion therapy,and the anti-oxidation effect was better than the shenhuo moxibustion alone(P<0.05).It is suggested that NAC can regulate the release of oxygen free radicals,the imbalance of redox stress and alleviate the oxidative stress injury induced by ischemia-reperfusion injury.When combined with Shenhuo moxibustion,the activity of SOD and the reduction of MDA content were significantly better than the single use of shenhuo moxibustion(P<0.01).6.Effect of Shenhuo moxibustion of yao medicine on the expression of inflammatory factors(TNF-α、IL-1、IL-10)The results showed that the levels of TNF-α、IL-1β、IL-10 in the model group increased significantly at 14d compared with that in the Sham group(P<0.01).Compared with the model group,the levels of IL-1β in the moxibustion treatment group decreased significantly(P<0.05),while the levels of TNF-α and IL-10 decreased significantly(P<0.01).After the intervention of Shenhuo moxibustion,the levels of TNF-α、IL-10 and IL-10 were significantly decreased,with statistically significant differences compared with the model group(P<0.01).The levels of TNF-α、IL-1β and IL-10 in the NAC group and NAC+Shenhuo moxibustion treatment group showed the same change trend as that in the Shenhuo moxibustion treatment group,and decreased significantly compared with the model group(P<0.01).However,Compared with the shenhuo moxibustion group,the levels of TNF-α、IL-1βin the NAC+Shenhuo moxibustion group were significantly decreased(P<0.01),and the levels of IL-10 were also decreased(P<0.05),indicating that NAC+shenhuo moxibustion had a more significant effect on the reduction of inflammatory factors.7.Effects of Shenhuo moxibustion of yao medicine on cell apoptosisIn this study,Tunel positive cells were significantly increased in the model group after create a cerebral ischemia reperfusion model compared with the sham group,indicating a significant increase in apoptosis.When moxibustion and shenhuo moxibustion was used as an intervention,Tunel positive cells decreased significantly,showing a significant statistical difference compared with the model group(P<0.01),suggesting that Shenhuo moxibustion treatment can reduce the apoptosis of nerve cells in the ischemic brain tissue area to a certain extent,and play a protective role in I/R injury.However,when NAC was injected into the lateral ventricle and NAC was combined with Shenhuo moxibustion therapy,the apoptosis of nerve cells in the brain tissue of ischemia reperfusion rats was significantly reduced compared with the model group(P<0.01),suggesting that NAC could significantly reduce the apoptosis of neurons.Compared with the shenhuo moxibustion group,the effect of NAC and NA+shenhuo moxibustion on the apoptosis rate was not significant(P>0.05).8.Effects of Shenhuo moxibustion of yao medicine on NF-κB path-related proteins and downstream apoptosis factors(Cox-2,iNOS,NF-κB/p65、BAX、Bcl-2、Caspase-3)8.1 Cox-2 and iNOS expression in brain tissues(immunohistochemistry,WB,qPCR);The results suggested that,after ischemia reperfusion injury,compared with Sham group,the immunohistochemistry showed that Cox-2 and iNOS positive expression neurons increased in the brain tissue of the model group,Cox-2 and iNOS protein content increased in the brain tissue,and cox-2mRNA and iNOSmRNA content increased,suggesting that Cox-2 and iNOS were involved in the process of ischemia reperfusion injury.Compared with the model group,the expression of Cox-2 immunohistochemical positive cells and Cox-2 protein in the moxibustion group were significantly decreased(P<0.01),the content of Cox-2 mRNA was decreased(P<0.05),the expression of iNOS positive cells was decreased(P<0.05),and the content of iNOS protein and iNOSmRNA was significantly decreased(P<0.01).Compared with model group,shenhuo moxibustion treatment can obviously reduce the Cox-2 and iNOS positive cells expression,to reduce the expression of Cox-2 and iNOS protein in the brain and the brain Cox-2,iNOSmRNA content,the results are statistically significant differences(P<0.01),suggesting shenhuo moxibustion and moxibustion with inhibition of Cox-2 and iNOS can protect the brain tissue,and compared with shenhuo group moxibustion,the expression of iNOS positive cells increased significantly in moxibustion group(P<0.01),iNOS protein expression also increased(P<0.05),It suggested that shenhuo moxibustion had better inhibitory effect on iNOS expression than moxibustion group.However,for NAC and NAC+Shenhuo moxibustion treatment group,the expression of iNOS and Cox-2 positive neurons decreased,Cox-2 and iNOS protein content in brain tissue reduced,Cox-2mRNA and iNOSmRNA content reducing is more obvious in the model group(P<0.01),suggesting the NAC(NF-K b inhibition)can significantly reduce the positive expression of Cox-2 and iNOS,reverse cerebral ischemic injury,promote restoration of the neural function.8.2 Expression of NF-κB/p65 in brain tissues(immunohistochemistry,WB,qPCR);After cerebral ischemia reperfusion 14d,immunohistochemical results show that ischemia brain tissues NF-κB p65 positive expression increased in model group,illustrate the NF-κB p65 is activated,at the same time the number of positive cells which NF-κB p65 translocate into the nucleus increased,WB prompts the NF-κB p65 protein expression increases,qPCR suggests that NF-κB p65mRNA is up-regulated,it indicates that the NF-κB p65 pathway is activated.Compared with the model group,it was found that moxibustion could reduce the expression of NF-κB/p65 positive cells obviously(P<0.05)and decrease the expression of NF-κB/p65 protein in the brain(P<0.01),and significantly reduce the expression of NF-κB/p65mRNA in the brain(P<0.01),while the Shenhuo moxibustion could significantly reduce the expression of NF-x B/p65 positive cells,protein and NF-κB/p65 mRNA in the brain(P<0.01).It is suggested that the treatment of Shenhuo moxibustion and moxibustion may play an important role in reducing the brain injury of I/R in MACO rats by inhibiting the expression of NF-κB/p65 protein in ischemic brain tissue.In terms of decreased expression of NF-κB/p65 protein in brain,shenhuo moxibustion group was superior to moxibustion group(P<0.05).However,NAC and NAC+Shenhuo moxibustion could significantly reduce the expressions of NF-κB p65 positive cells、proteins and NF-κB/p65 mRNA in the brain(P<0.01),moreover,the effect of NAC+shenhuo moxibustion treatment on NF-K B/p65 was significantly better than that of single shenhuo moxibustion(P<0.01).8.3 expression of Bax、Bcl-2 and Caspase-3 in brain tissues(immunohistochemistry,WB,qPCR);In this study,I/R rats were detected by immunohistochemistry,Western blot and qPCR,the Bax and Caspase-3 were significantly increased and Bcl-2 levels were significantly decreased in MCAO group compared with Sham group(P<0.01).Compared with the model group,the expression of Bax positive cells in the moxibustion treatment group was significantly decreased(P<0.01),the increase of bcl-2 positive cells and the decrease of caspase-3 positive cells were not statistically significant(P>0.05),the expression of Bax and caspase-3 protein in the brain was decreased(P<0.01),the expression of Bax mRNA in the brain was decreased(P<0.05),and the expression of caspase-3 mRNA was significantly decreased(P<0.01).There was no significant increase of Bcl-2 protein in brain(P>0.05),and the level of Bcl-2 mRNA increased(P<0.05).It indicates that moxibustion has a certain effect on the regulation of Bax,Caspase-3 and Bcl-2,but the effect is not stable and the results are different.However,Compared with the model group,the expression of Bax and caspase-3 positive cells in shenhuo moxibustion group was decreased(P<0.01),and the expression of Bcl-2 positive cells was increased(P<0.05).Bax and caspase-3 protein expression decreased(P<0.01),Bcl-2 protein expression increased(P<0.01),Bax mRNA and caspase-3 mRNA expression decreased significantly in the brain(P<0.01),and Bcl-2 mRNA expression increased in the brain(P<0.01).This suggests that Shenhuo moxibustion therapy can influence apoptosis by affecting the levels of intracellular apoptotic factors such as Bcl-2,Bax and Caspase-3.Compared with the moxibustion group,shenhuo moxibustion had a better effect on reducing the expression of Bax and caspase-3 protein(P<0.01).For NAC group and Shenhuo moxibustion+NAC,Caspase3,Bax positive cells expression、protein levels in the brain and mRNA levels decreased more significantly(P<0.01),the Bcl-2 positive cells expressionp、rotein levels in the brain and mRNA levels significantly increased(P<0.01),suggesting NAC factor influence the apoptosis of antiapoptotic effect.And combine with Shenhuo moxibustion has more stronger effect on cell apoptosis pathway.Compared with single shenhuo moxibustion treatment,the difference was statistically significant(P<0.01),combination therapy can play a strong neuroprotective effect against apoptosis.Conclusion1、Shenhuo-moxibustion of yao medicine improved the behavioral performance of nerve function defects in rats with cerebral ischemia reperfusion,reduced the morphological and pathological changes of brain tissue,and it has neuroprotective effect on ischemia reperfusion rats.2、Shenhuo-moxibustion of yao medicine on neuroprotective effect of cerebral ischemia-reperfusion injury in rats may be related to the mechanism of anti-inflammation,anti-oxidation and anti-apoptosis.3、The neuroprotective mechanism of Shenhuo-moxibustion of yao medicine may be to exert anti-inflammatory and anti-apoptotic effects by inhibiting the inflammatory pathway of NF-κB. |
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