| 1.Background and objective"Cerebral stroke",also known as "stroke",is the second major cause of death and disability in the world.From 1990 to 2019,the disease burden increased significantly,as the number of stroke patients increased by 70.0%,and the number of deaths caused by stroke increased by 43.0%.The number of stroke patients in China ranks first in the world and has become the leading cause of death and disability among adult residents.Prevention and treatment remain a long and arduous task.Previous studies of our group have confirmed that nasal administration of Artesunate-Tetramethylpyrazine compound has a tangible proof of effect on cerebral ischemic nerve injury caused by plasmodium parasites in experimental cerebral malaria(ECM),as it could significantly increases the survival rate of ECM mice,improves the neurological symptoms such as ataxia and mobility disorder,and significantly enhances the autonomous activity and exploration ability of ECM mice.The effects and functions mainly include promoting axon growth,increasing angiogenesis,reducing vascular obstruction,and increasing cerebral blood flow.The combination of Artesunate-Tetramethylpyrazine for the treatment of cerebral malaria has been authorized by domestic and international patents.During the onset of cerebral malaria,the tissue ischemia and hypoxic damage caused by the blockage of cerebral microvessels by red blood cells infected with malaria are different from ischemic damage caused by stroke.Although the etiology is different,the pathological mechanism of local ischemia in the lesion is similar.From the perspective of traditional Chinese medicine,both belong to the category of "blood stasis obstructing the meridians".The purpose of this study was to explore whether nasal administration of artesunate-Tetramethylpyrazine combination has protective effect on ischemic stroke and its possible molecular mechanism using big data analysis and prediction and animal model experimental verification,so as to provide certain experimental support for the development and application of artesunateTetramethylpyrazine combination.Preventing and treating ischemic stroke through drug administration and dispensing,can provide experimental basis for expanding the value chain of application of artemisinin and its derivatives.2.Methods2.1 Big data prediction analysis of the effect of Artesunate-Tetramethylpyrazine compound on ischemic strokePreliminary prediction of the mechanism of artesunate ligustrazine combination therapy for ischemic stroke based on network pharmacology.Retrieved and summarize the open database of human diseases related to ischemic stroke and performed cluster analysis.Clinical studies related to ischemic stroke were retrieved,and the highly identifiable biomarkers reported in clinical reports were integrated and clustered.Based on GEO database of NCBI and NCBI,Web of science and China National Knowledge Network,transcriptomic and proteomic sequencing studies of animal ischemic stroke induced by middle cerebral artery occlusion(MCAO)model were retrieved respectively.FC>1or<-1 was used as the standard to screen differential genes and differential proteins,and the key factors and important biological processes and pathways were obtained by GO and KEGG analysis.The mechanism of Artesunate-Tetramethylpyrazine compound in the treatment of ischemic stroke was preliminarily predicted based on network pharmacology.Firstly,the targets of Artesunate and Tetramethylpyrazine monomer were collected and predicted.Secondly,the related targets of ischemic stroke were searched.At the same time,the targets in related literature were collected and combined with them.The two monomer and the disease targets were corrected and analyzed,and the important targets were defined as the key targets by PPI analysis.The key targets were defined as the key targets by GO and KEGG analysis,and the network relationship of artesunate-Tetramethylpyrazine compounds,key targets and key pathways was constructed.2.2 Effect and mechanism of Artesunate-Tetramethylpyrazine compound on ischemic brain injury in rats with MCAO2.2.1 Effects of Artesunate-Tetramethylpyrazine compound on overall state and survival rate of MCAO ratsThe animals were divided into Sham group,Model group,ArtesunateTetramethylpyrazine compound low-dose(AT-Low),Artesunate-Tetramethylpyrazine high-dose(AT-High)and positive drug groups.Sham operation was performed in the Sham group,and MCAO was performed in the other animals to establish the rat model of ischemic stroke.The body weight of the animals was recorded at 24 h,48 h and 72 h after surgery,and the general external morphology such as hair,mental state and motor flexibility were observed.The survival rate of each group was recorded.2.2.2 Effect of Artesunate-Tetramethylpyrazine compound on neural function scores of MCAO ratsThe buckling,tension and movement trajectory of rats were scored and recorded at 24 h,48 h and 72 h after the MCAO operation,and the unmodelled animals were excluded and grouped according to the score in 24 h.The difference between 72 h and 24 h scores of each group was calculated according to the average score,and the total score of behavioral science was calculated at each time point.2.2.3 Effect of Artesunate-Tetramethylpyrazine compound on brain tissue injury in MCAO ratsThe animals were anesthetized 72 h after operation and the brain tissue was taken by perfusion of pre-cooled normal saline.A part of the brain tissue was placed in the mold,2 mm coronal slices were cut,TTC staining was performed,the infarct area was quantified after fixation in 4%paraformaldehyde,and the infarct rate of the brain tissue was calculated.Another part of the brain tissues in 4%paraformaldehyde were fixed for 24 h,and embedded by paraffin,before and after the fontanel 3 mm of the region to do continuous coronal section(thickness of about 5 μm),coronal section was performed after HE staining.Cerebral cortex,hippocampus CA 1 region,CA 3 region,DG region pathological changes were observated under the light microscope.2.2.4 Effect of Artesunate-Tetramethylpyrazine compound on blood-brain barrier injury in MCAO ratsThe animals were injected with 2%Evans blue into the caudal vein 70 h after surgery.The animals were anesthetized 72 h after surgery and irrigated with precooled normal saline.Brain tissue was taken and the ischemic lateral hemisphere was isolated.Tight junction related proteins ZO-1 and occludin in brain tissue were located and quantified by immunohistochemistry.The same position of cortex was selected under microscope,and the number of positive cells was observed and counted.2.2.5 Effect of Artesunate-Tetramethylpyrazine compound on brain apoptosis in MCAO ratsAfter 72 h anesthesia animals and precooled saline perfusion,take brain tissue,placed in 4%paraformaldehyde fixed 24 h,paraffin embedding,before and after the fontanel 3mm area to do continuous coronal section(thickness of about 5 μm),TUNEL staining.The same area of cortex was selected under the microscope,and the number of positive cells was observed and counted.Western Blot was used to detect the expression levels of related proteins Bax and Bcl-2 in infarct side cortex tissue.2.3 Effect of Artesunate-Tetramethylpyrazine compound on expression of key target-related factors in serum and brain tissue of MCAO ratsThe expression levels of TIMP-1 and MMP-9 in animal serum and infarct side cortex tissue were detected by ELISA,and the ratio of them was calculated.The mRNA and protein expression levels of TIMP-1 and MMP-9 were detected by rtqPCR and Western Blot,and the ratio of MMP-9 to TIMP-1 was calculated.Immunohistochemistry was used to locate and quantify TIMP-1 and MMP-9.2.4 Study on the protective effect and mechanism of ArtesunateTetramethylpyrazine compound on oxygen sugar deprivation injury model of nerve cells2.4.1 Effects of different concentrations of Artesunate-Tetramethylpyrazine combination on cell survival rateThe survival rate of cells treated with Artesunate,Tetramethylpyrazine and their combination for 12 h was determined by CCK-8 method to ensure that the drug concentration used in subsequent experiments was within the safe range.2.4.2 Protective effect of Artesunate-Tetramethylpyrazine on OGD injuryThe appropriate OGD model was established by observing the cell morphology and CCK-8 detection results,and the appropriate concentration of ArtesunateTetramethylpyrazine compound was explored based on the stable OGD model.2.4.3 The effect of Artesunate-Tetramethylpyrazine combination on mRNA expression of TIMP-1 and MMP-9 in cellsCollected total cell RNA after 4 hours of OGD,quantified the mRNA expression of TIMP-1 and MMP-9,and calculated the MMP-9/TIMP-1 ratio.2.5 Small interfering RNA(siRNA)transfection2.5.1 Confirm the safety range of transfection reagents24 well plate with 5000 to 7500 cells per well,culture until the density reaches 60%to 80%,and add 1,2,3,4,and 5 cells per well,respectively μ After 24 hours of treatment with L transfection reagent Lipofectamine 2000,the cell survival rate was detected using CCK8.2.5.2 Cell transfectionCultivate cells on a 24 well plate until the density reaches 60%-80%,and then change the medium to 400 for each well μL complete culture medium.Cells were divided into Control group,negative control group,positive control group,TIMP interference group 1,TIMP interference group 2,and TIMP interference group 3.The control group was cultured in an incubator throughout the entire process.The negative control group was added with Negative Control RNA oligo,the positive control group was added with GAPDH Positive Control RNA oligo,and the TIMP interference groups were added with corresponding TIMP RNA oligo.Add 500 to the centrifuge tube μ L serum-free medium,add 2 μ L Lipofectamine 2000,gently mix well and let stand at room temperature for 5 minutes.Add 500 to another centrifuge tube μ L serum-free medium,add 40 p mol of corresponding RNA oligo,gently mix well,and let stand at room temperature for 5 minutes.Drop the mixture containing Lipofectamine 2000 into the mixture containing RNA oligo,gently mix well,let it stand at room temperature for 15 minutes,and add it to each group of cells cultured on a 24 well plate.The final system for each well is 500 μL.Gently shake the culture plate and place it in the incubator for 6 hours.Replace it with a complete culture medium and continue to culture for 24 hours.Extract mRNA for Rt-qPCR detection.2.5.3 Observing the protective effect of artesunate tetramethylpyrazine after siRNA interference based on OGD model96 well plate cells were divided into Control group,Model group,TIMP interference group,and artesunate ligustrazine low-dose group(75 μM)Artesunate ligustrazine high-dose group(150 μM)After transfection with the method described in 2.11.2 for 6 hours,the supernatant was replaced with drug containing culture medium and continued to be cultured for 24 hours.After 4 hours of OGD,the cell survival rate was detected using CCK-8.The same procedure was used for 24 well plate cells,and cell RNA was collected after 4 hours of OGD.3.Result3.1 Prediction of the effect of Artesunate-Tetramethylpyrazine compound on ischemic stroke based on big data analysisBy clustering clinical biomarkers,it was found that the biological processes mainly involved included hypoxia response such as NOS,inflammatory response such as IL-6 and CCL-2,angiogenesis such as VEGFA and MMP,and extracellular matrix decomposition such as TIMP-1,MMP-9 and MMP-2.A total of 35 key differential genes were obtained by analyzing transcriptome data,including 5 up-regulated genes such as Itgb6 and 30 down-regulated genes such as Timpl,Ccl2 and S100a8,which are mainly involved in IL-17 signaling pathway,TNF signaling pathway,ECM receptor interaction and apoptosis.Analysis of proteomic data reveals key differential proteins.Major biological processes include regulation of neurotransmitter secretion,development of dendrites,regulation of cell maturation,presynaptic endocytosis,and internalization of postsynaptic neurotransmitter receptors.A total of 1826 targets related to ischemic stroke,31 targets of Tetramethylpyrazine(TMP)and 105 targets of Artesunate(AS)were obtained from the database and literature.After analysis,78 candidate targets were obtained,and 25 core targets such as VEGFA,MMP9 and MMP-2 were screened,which were mainly related to apoptosis,cell proliferation and vascular smooth muscle cell proliferation,focusing on VEGF,estrogen,TNF,PI3K-Akt and other signaling pathways.3.2 Efficacy evaluation of Artesunate-Tetramethylpyrazine compound in improving cerebral ischemic injury in rat model MCAO3.2.1 Effects of Artesunate-Tetramethylpyrazine compound on overall state and survival rate of MCAO ratsThe efficacy of Artesunate-Tetramethylpyrazine compound was evaluated based on stable MCAO model and no effect on the pathological changes of nasal mucosa.The end point was 72 h after MCAO operation.The survival rate of Model group was 60%,that of Artesunate-Tetramethylpyrazine compound low-dose group was 69%,that of Artesunate-Tetramethylpyrazine compound high-dose group was 73%,and that of positive drug group was 71%.3.2.2 Effect of Artesunate-Tetramethylpyrazine compound on neural function scores of MCAO ratsBehavioral scores showed that at the end point of the experiment(72 h after MCAO operation),the Model group showed significant neurological injury,and the total score of behavioral scores was significantly higher than that of the Sham group(P<0.001),indicating abnormal behavior and nerve injury after cerebral ischemia;Compared with Model group,the total score of each administration group was significantly decreased(P<0.01),suggesting that all drugs significantly reduced the nerve injury after MCAO operation.The effect of Artesunate-Tetramethylpyrazine compound on the neurobehavior of ischemic stroke rats was more directly observed by the difference of behavioral scores between 72 h and 24 h.Forelimb flexion and muscle tension injury were better than those in Model group at 24 h,48 h and 72 h.3.2.3 Effect of Artesunate-Tetramethylpyrazine compound on brain tissue injury in MCAO ratsTTC staining and infarct size quantification showed that compared with Sham group,infarct size in Model group was significantly increased(P<0.001);Compared with Model group,the cerebral infarction size of rats in high-dose and positive drug groups was signifi cantly reduced(P<0.05,P<0.01),indicating that ischemic injury was relieved after drug treatment.HE staining results showed that the cortical structure of Model group rats was seriously damaged and the number of cells was reduced,while the drug group showed different degrees of improvement.3.2.4 Effect of Artesunate-Tetramethylpyrazine compound on BBB injury in MCAO ratsThe results of Evans Blue showed that compared with Sham group,the absorbance of Model group was significantly higher(P<0.01),suggesting that the blood-brain barrier was destroyed after MCAO operation.Compared with Model group,the absorbance of each administration group was significantly decreased(P<0.05,P<0.01,P<0.01),suggesting that the destruction of blood-brain barrier was alleviated after treatment with various drugs.Immunohistochemical results showed that the brown positive areas of ZO-1 and occludin in the Sham group distributed around the blue nuclei,while the cell gaP in the Model group increased,while the positive areas decreased.The positive area of Artesunate-Tetramethylpyrazine in low and high dose groups increased slightly,and the cell gaP decreased.3.2.5 Effect of Artesunate-Tetramethylpyrazine compound on brain apoptosis in MCAO ratsTUNEL staining was used to observe the morphology,number and apoptosis of nerve cells in CA 1,CA 3 and DG regions of cortex and hippocampus.There were few necrotic or apoptotic nerve cells in Sham group.A large number of apoptotic neuron cells(brown)were observed in each area of the infarct hemisphere in the Model group,and the apoptotic phenomenon was the most obvious in the cortical area.The apoptotic phenomenon was less in each area of the administration group,and the apoptotic phenomenon was mostly manifested as necrosis of nuclear condensation(dark blue).The number of nerve cells was quantified and counted in this part.Compared with Sham group,the number of cells in cortex,hippocampal CA 1,hippocampal CA 3 and hippocampal DG regions in Model group were significantly decreased(P<0.001,P<0.001,P<0.001,P<0.001),cell necrosis and apoptosis were obvious.Compared with Model group,the number of cells in each administration group increased significantly.Western Blot results showed that compared with Sham group,the expression of Bax protein in infarct side cortex of Model group was significantly increased(P<0.05);Compared with Model group,Bax protein expression was significantly decreased in all administration groups(P<0.05,P<0,005,P<0.05).Compared with the Sham group,the expression of Bcl-2 in the cortex of the Model group showed a downward trend,and the expression of Bcl-2 in each administration group showed a downward trend.Compared with Sham group,Bax/Bcl-2 ratio in Model group was significantly increased(P<0.01),indicating severe apoptosis;Compared with Model group,the ratio of each administration group was adjusted back(P<0.01,P<0.005,P<0.05).3.3 Study on mechanism of Artesunate-Tetramethylpyrazine compound in treatment of experimental cerebral ischemiaBased on better efficacy and big data analysis and prediction,the mechanism of Artesunate-Tetramethylpyrazine compound in the treatment of experimental cerebral ischemia was prelim natively explored.ELISA results showed that compared with Sham group,the expression of TIMP1 in Model group was significantly decreased(P<0.01),the expression level of MMP-9 was significantly increased(P<0.05),the proportion of MMP-9/TIMP-1 was significantly increased(P<0.01).Compared with the Model group,the expression level of TIMP-1 in each administration group was significantly increased and tended to be normal(P<0.05,P<0.01,P<0.05),the expression level of MMP9 was significantly decreased(P<0.001,P<0.001,P<0.001),MMP-9/TIMP-1 ratio was significantly decreased(P<0.005,P<0.005,P<0.005).ELISA results showed that compared with Sham group,the expression level of TIMP-1 in Model group was significantly decreased(P<0.05),the expression level of MMP-9 was significantly increased(P<0.005),MMP-9/TIMP-1 ratio was significantly increased(P<0.005).Compared with Model group,the expression level of TIMP-1 at each administration was significantly increased and tended to be normal,but there was no significant difference.The expression level of MMP-9 in high-dose and positive drug groups was significantly decreased(P<0.01,P<0.01),the low dose group had a downward trend but no significant difference.The ratio of MMP-9 to TIMP-1 was significantly decreased(P<0.05,P<0.005,P<0.01).Immunohistochemical results showed that there was almost no brown positive area of MMP-9 in the Sham group,and the positive area was increased and distributed in the intercellular space in the Model group.Compared with Model group,the positive area of Artesunate-Tetramethylpyrazine low dose,high dose and positive drug groups was slightly reduced,and the cell space was reduced.The brown positive area of TIMP-1 surrounded the blue cells in the Sham group,while the gap between cells in the Model group was enlarged with almost no positive area.Compared with Model group,the positive area of Artesunate-Tetramethylpyrazine in low dose,high dose and positive groups increased and the cell space decreased.The results of rt-qPCR and Western Blot showed the same trend.The results showed that HIF-1α expression in Model group was significantly increased compared with Sham group.Compared with Model group,HIF-1α expression in all administration groups was significantly decreased.Compared with Sham group,the expression of VEGFA in Model group was significantly decreased.Compared with Model group,the VEGFA expression level in all drug administration groups was significantly increased.Compared with Sham group,the expression level of TIMP-1 in Model group was significantly decreased,the expression level of MMP-9 was significantly increased,and their ratio was significantly increased.Compared with Model group,the expression level of TIMP-1 in each administration group was significantly increased,and the expression level of MMP-9 was significantly decreased,and the ratio of MMP-9/TIMP-1 was significantly decreased,and the ratio of MMP-9/TimP-1 tended to be normal.3.4 Preliminary study on the protective effect of AS-TMP combination on oxygen glucose deprivation injury in nerve cells3.4.1 Effects of different concentrations of artesunate and ligustrazine on the survival rate of SK-N-BE cellsIn order to determine whether the drug itself causes damage to cells in the experiment and determine a reasonable concentration range for drug administration,CCK-8 was used to measure the cell survival rate after 12 hours of drug treatment.The results show 10-1 000 μM AS injection and 10-1000 μM TMP injection both have no significant effect on cell viability after 12 hours of treatment,and can be used for subsequent experiments.3.4.2 Establishment of OGD modelThe cell morphology and CCK-8 result of the Control group and OGD after 4 and 6 hours were determined,indicating that the distribution of cells in the Control group is uniform and the morphology is plump.After 4 hours of OGD,the number of cells significantly decreased and their morphology shrunk.Compared with the Control group,the survival rate was significantly reduced,reaching 48.91%.After 6 hours of OGD,the number of cells significantly decreased and their shape was abnormal.Compared with the Control group,the survival rate was significantly reduced,reaching 20.59%.Select OGD 4h for subsequent experiments.3.4.3 Protective effect of artesunate ligustrazine combination on cell OGD damageThe cell morphology and CCK-8 result after 4 and 6 hours of OGD were determined.It can be seen that the control group cells are evenly distributed and have full morphology,while the model group cells have a significant decrease in survival rate and shrinkage after 4 hours of OGD.Compared with the Model group,the survival rate of cells combination used of artesunate and ligustrazine was at 50,75,and 150μM significantly increased(P<0.05,P<0.001,P<0.001),300 μM dose group showed an increasing trend,but there was no significant difference.The cell morphology of each treatment group tended to be normal to varying degrees.3.4.4 Protective effect of artesunate ligustrazine combination on mRNA expression of key cytokines in cellsCollect total cell RNA after 4 and 6 hours of OGD,quantify the mRNA expression of TIMP-1 and MMP-9,and calculate MMP-9/TIMP-1.Compared with the Control group,the Model group showed a significant decrease in TIMP-1 mRNA expression(P<0.001),a significant increase in MMP-9 mRNA expression(P<0.001),and a significant increase in MMP-9/TIMP-1 ratio(P<0.001).Compared with the Model group,the AT-50 group showed a significant increase in TIMP-1 mRNA expression and a significant decrease in MMP-9 mRNA expression,but there was no significant difference.The MMP-9/TIMP-1 ratio significantly decreased(P<0.05);The expression of TIMP-1 mRNA significantly increased(P<0.01),MMP-9 mRNA significantly decreased(P<0.05),and the MMP-9/TIMP-1 ratio significantly decreased(P<0.01)in the AT-75 group;The expression of TIMP-1 mRNA was significantly increased(P<0.005),MMP-9 mRNA expression was significantly reduced(P<0.005),and the MMP-9/TIMP-1 ratio was significantly reduced(P<0.001)in the AT-100 group.3.5 The effect of TIMP interference on the protective effect of artesunate ligustrazine combined with OGD injury3.5.1 Confirm the safety range of transfection reagentsCompared to the Control group,using 1μL Lipofectamine 2000 showed no significant decrease in cell survival rate,2-5 μ Lipofectamine 2000 of L can significantly reduce the cell survival rate to varying degrees(P<0.05,P<0.01,P<0.001,P<0.001).3.5.2 Detection of siRNA transfection efficiencyCompared with the Control group,there was no significant change in the expression level of TIMP in the negative control group,while the expression level of GAPDH mRNA in the positive control group was significantly reduced.The expression level of TIMP-1 mRNA in cells treated with TIMP oligo 1,2,and 3 was significantly reduced to varying degrees(P<0.01,P<0.05,P<0.05),with oligo 1 being the most effective knockdown treatment.The experiment was continued with oligo 1.3.5.3 Observing the Effect of Artesunate Ligustrazine on Cell Survival after Interference with TIMP-1 Based on OGD ModelCompared with the Control group,the cell survival rate of the Model group significantly decreased after OGD(P<0.001).Compared with the Model group,the TIMP gene significantly decreased the cell survival rate after interfering with OGD(P<0.001).Compared with the TIMP interference group,the cell survival rate of the low-dose and high-dose groups of artesunate ligustrazine was significantly increased(P<0.001,P<0.001).3.5.4 Observing the Effect of Artesunate Ligustrazine on TIMP-1 and MMP-9 mRNA Expression Levels after Interference with TIMP-1 Based on OGD Model Compared with the Control group,the Model group showed a significant decrease in TIMP-1 mRNA expression level(P<0.001),a significant increase in MMP-9 mRNA expression level(P<0.001),and a significant increase in MMP-9/TIMP-1 ratio(P<0.05).Compared with the Model group,the TIMP interference group showed a significant decrease in TIMP-1 mRNA expression level(P<0.005),a significant increase in MMP-9 mRNA expression level(P<0.005),and a significant increase in MMP-9/TIMP-1 ratio(P<0.001).Compared with the TIMP interference group,the expression levels of TIMP-1 mRNA in the low-dose and high-dose groups of artesunate tetramethylpyrazine were significantly increased to varying degrees(P<0.001,P<0.001),MMP-9 mRNA expression levels were significantly reduced to varying degrees(P<0.001,P<0.001),and the MMP-9/TIMP-1 ratio was significantly reduced to varying degrees(P<0.001,P<0.001).4.ConclusionThe combination of artesunate and ligustrazine administered through nasal cavity can improve the overall state and survival rate of MCAO model rats,reduce infarction rate,alleviate brain tissue pathological damage BBB damage,and cortical nerve cell apoptosis,alleviate animal neurological function damage,and have a protective effect on ischemic stroke.Regulating the expression and ratio of MMP-9 and TIMP-1 may be one of the core mechanisms by which artesunate ligustrazine combination protects the integrity of the blood-brain barrier and related proteins alleviate ischemic damage in stroke. |
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