| The traditional Chinese medicine Rhizoma Zedoariae has the effect of breaking blood stasis,relieving pain and eliminating accumulation,and is often used for chest arthralgia and heartache caused by qi stagnation and blood stasis,amenorrhea caused by blood stagnation,ruffian mass and injury caused by fall.Professor Shi Dazhuo believes that the formation of atherosclerosis(AS)belongs to blood disease.In clinical practice,zedoary turmeric combined with products for replenishing qi and activating blood circulation was used in AS-related diseases.It was found that this kind of prescription can relieve related clinical symptoms,and the mechanism is related to stabilizing plaque progression and reducing the incidence of stent restenosis.Previous basic studies have found that Zedoarondiol,the active component of Rhizoma Curcumae,can protect endothelial cells from oxidative stress,delay the progression of AS in vivo and reduce the load of inflammatory cells in atherosclerotic plaques.On the one hand,based on big data’s analysis of the compatibility of Professor Shi Dazhuo’s prescription containing zedoary turmeric in the treatment of atherosclerosis-related diseases,this paper provides ideas for the modernization of traditional Chinese medicine;on the other hand,it studies the effect and mechanism of Zedoarondiol mediating inflammatory cell transendothelial migration from the cellular and molecular level.Clinical ResearchBased on big data’s excavation and analysis of the compatibility of Professor Shi Dazhuo’s prescription containing zedoary turmeric in the treatment of atherosclerosis-related diseasesObjective:to analyze the prescription containing zedoary turmeric in Professor ShiDazhuo’s outpatient prescription for atherosclerosis-related diseases based on big data’s mining theory,so as to provide a new compatibility idea for the differential diagnosis and treatment of atherosclerosis with zedoary turmeric.Methods:a total of 220 prescriptions containing zedoary turmeric were selected from Professor Shi Dazhuo’s outpatient clinic for the treatment of atherosclerosis from June 2019 to March 2021,and the diagnosis and medication information were introduced into the TCM inheritance auxiliary platform(V2.5).Click the corresponding function button in turn for analysis,including drug frequency statistics,prescription rule analysis,including cluster analysis,extraction combination,network display and so on.The statistical analysis method is the mining algorithm of the software,including rule analysis,improved mutual information method and so on.Results:through "frequency statistics",it was found that the frequently used drugs were Salvia miltiorrhiza,Ligusticum chuanxiong,Radix Paeoniae Rubra,safflower,tangerine peel,Radix Astragali,Radix Scutellariae,Poria cocos;the four qi were mainly warm,followed by cold and flat;among the five flavors,bitterness,bitterness and sweetness were the main,and the meridians were mainly liver,spleen and heart.When the support degree is 30%,a total of 280 common core drug combinations are found,including 41 association rules for 2 drugs,78 association rules for 3 drugs,86 association rules for 4 drugs,55 association rules for 5 drugs,18 association rules for 6 drugs and 2 association rules for 7 drugs.When the support degree is set to 30%,Rhizoma Curcumae is compatible with Salvia miltiorrhiza,Coptis chinensis and Radix Astragali,the core combination is similar and higher,but when the support degree is 10%and 20%,the types of peripheral drugs are quite different.Conclusion:in the treatment of atherosclerosis,Professor Shi Dazhuo often combines zedoary with Salvia miltiorrhiza and safflower to enhance the power of promoting blood circulation and removing blood stasis,and with Huangqi to achieve the purpose of "tonifying qi to assist blood circulation".Compatibility with heat-clearing and detoxifying drug Huanglian to achieve the purpose of clearing blood stasis without stagnation.Experimental ResearchStudy 1 Effect of Zedoarondiol on atherosclerosis induced by high-fat diet in ApoE-\-miceObjective:To observe the effect of Zedoarondiol on atherosclerosis induced by highfat diet in ApoE-mice by regulating NF-κ B/adhesion molecule signal pathway to protect the function of vascular endothelial cells.Methods:Forty 8-week-old apolipoprotein E(apolipoproteinE,ApoE)gene deficient mice were fed with high fat diet to establish AS model.They were randomly divided into model group,low-dose Zedoarondiol group,high-dose Zedoarondiol group and Atto vastatin group.At the same time,10 homologous C57/BL mice were selected as blank control group and fed with common diet.The mice in the low-dose Zedoarondiol group were given Zedoarondiol 1 mg/kg/d,and the high-dose Zedoarondiol group was given Zedoarondiol 5 mg/kg/d.The model group and the blank control group were given an equal volume of normal saline by gavage for 12 weeks.After the experiment,blood serum and aortic tissue were taken,blood lipids were measured by biochemical method,inflammatory factor changes in serum and aortic tissue were measured by Elisa,and the aortic root section was stained with oil red O and modified Masson staining to observe the plaque size and collagen content Changes,immunofluorescence detection of inflammatory cell content in plaques,Western Blot detection of aortic VCAM-1,ZO-1 protein expression,RT-qPCR detection of VC AM-1 mRNA,ZO-1 transcription.Results:Compared with the normal group,the plaque area of the aortic root of the model group was significantly increased(P<0.01),the collagen content in the plaque was significantly reduced(P<0.01),and the area of CD68+in the plaque was significantly increased(P<0.01).The content of total lipid cholesterol,triglycerides,and low-density lipoprotein increased,and the content of low-density lipoprotein decreased significantly(P<0.01).Serum NO content and total SOD activity content were significantly reduced(P<0.01),and MDA content was increased(P<0.01);inflammatory cytokines IL-6 and MCP-1 content in aortic tissue were significantly increased(P<0.01);scanning electron microscopy results showed:The fissures of endothelial cells in the model group increased(P<0.01),monocytes and fat deposition increased significantly(P<0.01);Western Blot results showed that the expression of NF-κB p52,VCAM-1,and cofilin in the aortic tissues of the model group were Increased(P<0.01),while the level of ZO-1 decreased.RT-qPCR results showed that the transcription level of VCAM-1 mRNA in the model group increased,while the level of ZO-1 mRNA decreased(P<0.01).Compared with the model group,the oil red O staining area of mouse aortic root slices decreased(P<0.01),the collagen content inside the plaque increased(P<0.01),and the number of CD68-positive cells in the plaque decreased after the intervention of Zedoarondiol(P<0.01).Serum lipid detection results after the intervention of Zedoarondiol,the total cholesterol(P<0.01)and low-density lipoprotein levels(P<0.01)of mice decreased.Serum nitric oxide content and total SOD activity were significantly increased(P<0.01),and MDA content was reduced(P<0.01);the levels of inflammatory cytokines IL-6 and MCP-1 in aortic tissue were significantly reduced;scanning electron microscopy results showed:Zedoarondiol can reduce endothelial cell fissures(P<0.01),reduce monocytes and fat deposition(P<0.01);Western Blot results show that the expression of NF-κB p52,VCAM-1,and cofilin decreased after Zedoarondiol intervention(P<0.01),while the ZO-1 level has increased.RT-qPCR results showed that the transcription level of VCAM-1 mRNA decreased after Zedoarondiol intervention,while the level of ZO-1 RNA increased(P<0.01).Conclusion:1.Zedoarondiol can regulate the lipid metabolism of ApoE-/-mice induced by high-fat diet,reduce the area of atherosclerotic plaque,protect the barrier function of vascular endothelium,inhibit the adhesion of inflammatory cells,and reduce the load of inflammatory cells in the plaque.2.Zedoarondiol protects the ultrastructure of the inner wall of the aorta in ApoE-/mice induced by a high-fat diet.Its mechanism is related to the expression of adhesion molecules mediated by the NF-κB signaling pathway and the expression of ZO-1.Study 2 Zedoarondiol protects endothelial cell barrier and inhibits the migration of inflammatory cellsObjective:To observe the effects of Zedoarondiol on reducing hydrogen peroxideinduced endothelial cell injury,improving endothelial cell barrier dysfunction,inhibiting inflammatory cell adhesion and transendothelial migration.Methods:HUVECs were stimulated with 200 mM hydrogen peroxide solution for 12 h to establish an endothelial cell injury model.The MTT method was used to observe the protective effect of Zedoarondiol on the endothelial cells induced by hydrogen peroxide;the cell co-culture method was used to observe that Zedoarondiol inhibits the adhesion of THP-1 and endothelial cells under a laser confocal microscope,and the transwell experiment observed that Zedoarondiol inhibits THP-1 transendothelial Migration.The cytoskeleton was labeled with phalloidin,and the intercellular junction protein was labeled with immunofluorescence.The effect of Zedoarondiol on the cytoskeleton reorganization and the intercellular junction protein was observed.Western Blot was used to detect the intercellular tight binding protein ZO-1;RT-qPCR to detect and ZO-1 mRNA transcription levels.Results:Compared with the normal group,the adhesion of monocytes to HUVEC in the model group increased(P<0.01),and the number of monocytes that migrated across the endothelium also increased(P<0.01).The area covered by the bottom of Transwell upper chamber without HUVEC increased significantly(P<0.01).Cellular immunofluorescence results showed that hydrogen peroxide stimulation resulted in high expression of Cav-1 in the model group(P<0.01).Western Blot results showed that the expression of NF-κB,Cav-1,and VC AM-1 in the model group increased and ZO-1 expression(P<0.05).RT-qPCR results showed that the transcription level of NF-κB mRNA,Cav-1 mRNA,and VCAM-1 mRNA in the model group increased,and the transcription level of ZO-1 mRNA decreased(P<0.01).Compared with the model group,Zedoarondiol can reduce the adhesion of monocytes to HUVEC(P<0.01)and the number of monocytes that migrate across the endothelium(P<0.01).The area covered by the bottom of Transwell upper chamber without HUVEC was significantly reduced(P<0.01).Cellular immunofluorescence results show that Zedoarondiol can reduce the high expression of Cav-1 caused by hydrogen peroxide stimulation(P<0.01).Western Blot results show that Zedoarondiol can inhibit the increase of NF-κB,Cav-1,and VCAM-1 expression.1 expression(P<0.05).RT-qPCR results showed that Zedoarondiol can inhibit the expression levels of NF-κB mRNA,Cav-1 mRNA,and VCAM-1 mRNA,and increase the expression of ZO-1 mRNA(P<0.01).Conclusion:Zedoarondiol can reduce the oxidative stress damage of endothelial cells induced by hydrogen peroxide,protect the vascular endothelial barrier function,and inhibit the adhesion of inflammatory cells to endothelial cells and transendothelial migration.Its mechanism is related to the cytoskeleton of caveolin-1/NF-κB/adhesion molecule signaling pathway regulating the expression of the protein cofilin. |
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