| Objective: To study the traditional Chinese medicine(TCM)pathogenesis of hypertension complicated with atherosclerosis in more standardized and objective ways,as well as to provide the data support for making a better clinical diagnosis and treatment,we explored the distribution of TCM Syndromes of hypertension combined with carotid atherosclerosis by collecting the diagnostic information of patients and prescriptions.According to the characteristics of the clinical syndrome distribution and prescription rule of hypertension combined with carotid atherosclerosis,the experimental study was designed to construct an endothelial injury cellular model of human aortic endothelial cells(HAECs)with oxidized low density lipoprotein(ox-LDL),the lncRNA regulatory mechanisms of endothelial dysfunction induced by ox-LDL were studied by integrated analysis of mRNAs microarray and lncRNAs microarray.Tribulus terrestris was extracted and effective ingredients were identified,and then the best compatibility proportion of Tribulus terrestris and Eucommia ulmoides was determined.Thereafter,the target and molecular mechanism of Eucommia ulmoides-Tribulus terrestris herb couples on endothelial dysfunction induced by ox-LDL was studied.We hope to contribute to alleviating atherosclerosis and treating hypertension by improving endothelial function and provides laboratory support for the clinical application of Eucommia ulmoides and Tribulus terrestris herb couples.Methods:1.Study on the distribution of TCM Syndromes of hypertension combined with carotid atherosclerosis and the rules of prescriptions: the clinical data and prescriptions of 110 patients with hypertension combined with carotid atherosclerosis were collected and the frequency of symptoms and signs was counted,then cluster analysis was conduct by SPSS.The correlation was assessed between syndromes and intima-media thickness(IMT),brachial-ankle pulse wave velocity(baPWV),maximum plaque area and blood lipid level,so as to conclude the characteristics of each TCM syndromes.According to the concept and method of modern data mining,the rule of medication prescription of Professor Yang Chuanhua in treating hypertension combined with carotid atherosclerosis was analyzed.2.The screening and extraction of effective components of Tribulus terrestris: Tribulus terrestris was ultra-fine pulverized and soaked in 70% ethanol,then extracted with heating reflux method and the extracts were filtrated and combined with the soluble part.The crude extracts were concentrated by rotary evaporator,and then extracted and recovered under negative pressure.The effective ingredients of Tribulus terrestris was identified by LC-ESI-MS/MS.The effective parts of Tribulus terrestris and Eucommia ulmoides and the best compatibility proportion were screened by MTT method and Transwell migration experiment.3.Study on the mechanisms of endothelial dysfunction induced by ox-LDL: endothelial injury cellular model was induced by ox-LDL,proliferation,apoptosis,migration and energy metabolism of HAEC was studied by MTT method,flow cytometry,Transwell migration experiment and seahorse energy metabolism experiment.Different expressed(DE)lncRNAs of HAEC induced by ox-LDL was profiled by integrated analysis of mRNAs microarray and lncRNAs microarray and bioinformatics analysis.After the knockdown of representative lncRNA(NONHSAT061051),we verified its role in ox-LDL induced endothelial injury.4.Study on the pharmacology of Tribulus terrestris-Eucommia ulmoides herb couples on the endothelial protection: HAECs were induced by ox-LDL and then administrated by the extract of Tribulus terrestris-Eucommia ulmoides,proliferation,apoptosis,migration and energy metabolism were studied by MTT method,flow cytometry,Transwell migration experiment and seahorse energy metabolism experiment.Differentially expressed lncRNAs after Tribulus terrestris-Eucommia ulmoides administration was determined by integrated analysis of mRNAs microarray and lncRNAs microarray and bioinformatics analysis to study the mechanism of protective effect of Tribulus terrestris-Eucommia ulmoides on endothelial dysfunction induced by ox-LDL.Results:1.Study on the distribution of TCM Syndromes of hypertension combined with carotid atherosclerosis and the rules of prescriptions: 110 patients at the age of 47 to 80 years old(average age of 69.25±10.39 years old),through cluster analysis,110 patients were divided into 3 categories;while according to the clinical symptoms,they were divided into 5 categories: insufficiency of kidney and liver yin,hyperactivity of liver yang,block of blood stasis,retention of phlegm,block of blood stasis and phlegm,accounting for 43.64%,20.00%,15.45%,11.82%,9.09% respectively.The IMT of the carotid artery in the group of retention of phlegm and hyperactivity of liver yang was higher than that in the insufficiency of kidney and liver yin group(P<0.05).The baPWV values of all groups were higher than normal,and the baPWV level of insufficiency of kidney and liver yin group was significantly higher than that of other groups(P<0.05),suggesting their positively correlated relationship(r=0.469).The largest patch area in block of blood stasis and block of blood stasis and phlegm was higher than the other groups,according to statistical calculation,the largest patch area showed moderate positive correlation with block of blood stasis and phlegm(r=0.342),weak positive correlation with block of blood stasis(r=0.122);the TG and TC levels of retention of phlegm group and block of blood stasis and phlegm group were higher than insufficiency of kidney and liver yin and hyperactivity of liver yang group(P<0.05).TG levels were positively correlated with retention of phlegm syndrome and block of blood stasis and phlegm(r=0.306;r=0.329),weakly correlated with block of blood stasis syndrome(r=0.179).TC were weakly correlated with retention of phlegm syndrome,block of blood stasis syndrome and block of blood stasis and phlegm syndrome(r=0.254;r=0.156;r=0.254).The LDL-C levels of all groups were higher than the normal,showing no significant difference between all groups in HDL and LDL.In the prescription prescribed by superviser,Huangqi,Du Zhong,Robinia,Epimedium,Mulberry,Angelica,Rhizoma Chuanxiong,Achyranthes bidentata,Anemarrhenae,and Huangqi were the top 10 commonly used herbs for treating hypertension combined with carotid atherosclerosis.The herbs were mostly focused on the tonic medicine,heat-clearing medicine,Pinggan Xifeng medicine,and blood circulation and stasis medicine according to the classification of traditional Chinese medicine with a frequency of over 150 times.The most frequently used herb couples were Tribulus terrestris-Eucommia ulmoides.2.The screening and extraction of effective components of Tribulus terrestris: Firstly,Tribulus terrestris was primarily separated into four parts: light petroleum,acetic ether,n-butyl alcohol and water,in which the parts of acetic ether and n-butyl alcohol were more effective than the other two parts.Secondly,acetic ether part was separated into eighteen parts,and n-butyl alcohol was separated into eight parts.Ethyl acetate extracts Fr.15 of Tribulus terrestris showed more effective than other parts in cell viability and cell migration screening experiment.340 compounds were identified in Ethyl acetate extracts Fr.15 of Tribulus terrestris,among which 289 compounds were known.The compounds were mainly classified as choline,flavonoids,alkaloids,vitamins and organic acids,among which acetylcholine and nicotinic acid were closely related to endothelial function.Quercetin showed more effective in cell viability and cell migration than other ingredients of Eucommia ulmoides(P<0.05).When the proportion of quercetin and acetic ether Fr.15 was 3:1,the compatibility demonstrated the best effect(P<0.05).3.The mechanism study of endothelial dysfunction induced by ox-LDL: After 24 h ox-LDL treatment,the apoptosis rate of HAEC was increased,migration rate was decreased,basal respiration rate,ATP production and maximum respiratory value was decreased.532 DE lncRNAs were identified(among them,298 lncRNAs were up-regulated and 234 lncRNAs were down-regulated)and 532 DE mRNAs were identified(among them,195 mRNAs were up-regulated and 337 mRNAs were down-regulated)after ox-LDL exposure for 24 h(fold change ? 2.0,P < 0.05).The functions of DE lncRNAs were mainly focused on fatty acid metabolism,terpene skeleton synthesis,PPAR signaling pathway,NOD like receptor signal transduction pathway,fatty acid degradation and many other endothelial related pathways.LncRNA NONHSAT061051 was verified to upregulate the expression of ICAM-1 and ICAM-1 related protein,promoting the adhesion of mononuclear cells(THP-1 cells)to endothelial cells.4.The mechanism study of Tribulus terrestris-Eucommia ulmoides on the endothelial dysfunction induced by ox-LDL: Compared to ox-LDL-treated cells,the apoptosis rate of HAEC was decreased,cell migration improved,basal respiration value,the amount of ATP produced,maximum respiration increased,the adhesion to mononuclear cells(THP-1 cells)was reduced after Tribulus terrestris-Eucommia ulmoides herb couples administration(P<0.05).149 DE lncRNAs were identified(among them,69 lncRNAs were up-regulated and 80 lncRNAs were down-regulated)and 64 DE mRNAs were identified(among them,11 mRNAs were up-regulated and 53 mRNAs were down-regulated)after Tribulus terrestris-Eucommia ulmoides herb couples administration(fold change ? 2.0,P < 0.05).The functions of DE lncRNAs were mainly focused on glycerol metabolism,fatty acid metabolism,glycerol phospholipid metabolism,adhesion,JAK-STAT signaling pathway and cytokine receptor interaction.After Tribulus terrestris-Eucommia ulmoides administration,the expression of lnc-MAPK8-2:1,NONHSAT061051,lnc-MTHFD2L-1:1 and lnc-CXCL3-1:1 was downregulated and the expression of lnc-TK1-2:1,lnc-PKLR-1:1,lnc-TGFB3-5:1 and lnc-KLHL10-2:1 was upregulated,demonstrating the antagonism effect on the ox-LDL.In addition,Tribulus terrestris-Eucommia ulmoides downregulated the expression of ICAM-1 gene as well as ICAM-1 encoded protein by downregulating NONHSAT061051 expression.Conclusion: There was a certain relation between age and hypertension combined with carotid atherosclerosis.The syndrome of insufficiency of kidney and liver yin was the main syndrome of hypertension combine with carotid atherosclerosis,and it showed a positive correlation with baPWV value.Accompanied with the formation of plaques,dyslipidemia always occurred in patients with hypertension and carotid atherosclerosis.The levels of TG and TC were positively related to phlegm and blood stasis.Phlegm and blood stasis were the key pathological factors of hypertension combined with carotid atherosclerosis.To treat hypertension and atherosclerosis,Supervisor Yang usually used the methods of nourishing liver and kidney,activating blood circulation and removing blood stasis.Tribulus terrestris-Eucommia ulmoides was the most often used herb couples.From the perspectives of endothelial protection,ethyl acetate extracts Fr.15 of Tribulus terrestris was the most effective one,among which the main components of acetylcholine and nicotinic acid were predicted as the components closely related to endothelial function.The most effective part of Eucommia ulmoides was quercetin.When the proportion of quercetin and ethyl acetate extracts Fr.15 of Tribulus terrestris was 3:1(ie,3?mol/L Quercetin: 1.3mg/L Fr.15),the compatibility showed the best endothelial protective effect.Ox-LDL induced endothelial injury,increased apoptosis rate,decreased the migration ability,and damaged mitochondrial function,reduced the ability of ATP production and decreased the capacity of respiratory by regulating lncRNAs.Tribulus terrestris-Eucommia ulmoides improved endothelial dysfunction by promoting cell proliferation and migration,inhibiting cell apoptosis,and increasing cell energy metabolism by regulating lncRNA. |
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