Total Flavonoids From Ziziphora Clinopodioides Lam Pre-vents Atherosclerosis By Protecting Endothelial Cells And Inhibiting Inflammatory Reaction And The Underlying Mechanisms

Objective:Atherosclerosis?AS?is the pathologic basis of cardiovascular disease,and seriously pose a risk to human health.In the early stage of the research group,the curative effect of Tianxiangdan on cardiovascular diseases was confirmed.Ziziphora clinopodioides Lam?ZCL?,a common medicinal material in Xinjiang province in China,is the main drug of Tianxiangdan,and has long been used in the treatment of cardiovascular diseases.Modern pharmacological studies have shown that Ziziphora clinopodioides Lam.have the effects of lowering blood pressure,protecting the cardiovascular system and anti-atherosclerosis.Flavonoids are the main active ingredient of ZCL.However,the effect and mechanism in anti-atherosclerosis are unclear.Vascular endothelial cell injury and inflammation are important pathogenesis,and involves in the occurrence and development of atherosclerosis.Protecting vascular endothelial cells and inhibiting inflammation are important targets for prevention and treatment of atherosclerosis.In this study,preparation technology of total flavonoids from Ziziphora clinopodioides Lam?TFZC?was optimized and component was analyzed.Furthermore,In vitro test,the model of human umbilical vein endothelial cells?HUVECs?induced by hydrogen peroxide?H₂O₂?and macrophages 264.7?RAW264.7?induced by lipopo-lysaccharide?LPS?were established.After the intervention of TFZC,We explore its effect and mechanism in anti-atherosclerosis,so as to seek therapeutic targets and provide experimental basis for prevention and treatment of early atherosclerosis.Methods:Single factor experiment was used to investigate the effect of various extraction factors?alcohol volume fraction,ratio of liquid and material,extraction time and extraction temperature?on the content of TFZC.Orthogonal experiment was used to determine the better method of ultrasonic assisted extraction.Four types of macroporous resins?AB-8,D101,HPD-100,and HP-20?were screened,and the best resin of purifying TFZC was determined on the basis of its adsorption rate and analytic rate.The optimum purification process was determined by examining the effects of various factors?concentration of the supernatant,pH value,diameter-height ratio,flow rate of the supernatant,concentration of the eluent,velocity of elution,and volume of elution?on the total flavonoids adsorption rate and analytical rate of TFZC.UPLC-Q-TOF-MS/MS was used to analyze the chemical components of TFZC in the positive and negative ion mode of electrospray ionic source?ESI?.?2?The cell model of HUVECs damaged by H₂O₂ was established,and TFZC and vitamin C?positive drug?were administered,which were divided into normal group,model group,the low,medium and high dose group of TFZC and vitamin C group.The change of cell survival rate in each group was determined by CCK-8method,and the optimum concentration and time were determined.The contents of Superoxide dismutase?SOD?,malondialdehyde?MDA?,lactate dehydrogenase?LDH?and reduced glutathione?GSH?in the supernatant of each group were determined with the kit.Apoptosis was detected by Hoechst 33342 fluorescent staining and Annexin V-FITC/7-AAD double staining in flow cytometry.Mitochondrial membrane potential and ROS were detected by flow cytometry.The changes of autophagy body with Cyto-ID?Green were observed by laser confocal autophagy.The protein expression level of Bcl-2,Bax,cleaved caspase-3,LC-3 and beclin-1 was detected by Western blot.?3?The inflammatory cell model was constructed by LPS acting RAW264.7 cell line,TFZC,atorvastatin and TAK-42?specific blockers of TLR-4?were given,which was divided into the normal group,the model group,atorvastatin calcium group?positive drug?,the low,medium and high dose group of TFZC,and TAK-42group.MTT assay was used to detect the effect of TFZC on the activity of RAW.264.7 cells.Nitrogen oxides?NO?in the supernatant was determined by Griess method,and tumor necrosis factor-alpha?TNF-???interleukin 1 beta?IL-1??and interleukin-6?IL-6?in the supernatant were detected by enzyme-linked immuno–sorbent assay?ELISA?.The mRNA and protein expression levels of TLR4,MyD88,TRAF-6 and NF-?B p65 were measured by quantitative real-time PCR?qRT-PCR?and Western blot.Results:?1?The ultrasonic extraction process of TFZC was optimized,and the optimal extraction technology is as follows:ethanol volume fraction is 70%,the ratio of liquid and material is 1:50,extracting time is 50min,twice,extraction temperature is 60?.?2?HP-20macroporous resin for purification of TFZC was confirmed,and the optimal purification process is as follows:quantity of sample loading is 3mg/ml,pH value is 3,The ratio of diameter and height ratio is 1:10,the flow velocity of the sample is 3BV/h,and the maximum sample loading quantity is 7 BV.TFZC was eluted by water first,followed by5BV,50%ethanol elution,and the elution velocity is 3BV/h.Finally,the total flavonoids content of TFZC is 63.1%.?3?18 compounds were identified or estimated using UPLC-Q-TOF-MS/MS.There are 16 flavonoids:baicalin?1?,Kaempferol-3-O-?-D-galactoside-7-O-?-L-rhamnoside kaicalin?2?,quercetin?4?,Kaempferol 3,7-O-?-L-dirhamnoside?5?,hypericin?6?,rutin?7?,Kaempferol-7-O-neohesperidoside?8?,Quadranoside III?9?,Kaempferol-3-O-neohesperidoside?10?,Apigenin7-O-neohesperi-doside?11?,diosmin?12?,Chrysin-7-O-neohesperidoside?13?,5,7-dihydroxy flavonoids-7-O-neohesperidoside?14?,Linarin?15?,Kaempferol?16?,chrysin?17?.one of coumarin compound is scopolamine?3?.one of sesquiterpenoids is ent-16?,17-Dihydroxy-19-kauranoic acid?18?.Compounds 2,3,5,8-11,13,14,18have not been reported.?4?After intervention of 800?mol/L H₂O₂ for 4h,the cells became round and the edge of the cell membrane was not clear.The cell survival rate of HUVECs was54.38±3.61%,and the model induced by H₂O₂ was established.?5?TFZC had no significant effect on the survival rate of HUVECs in the range of 1¹00?g/ml.The low,medium and high dose group of TFZC and vitamin C group can increase the survival rate,SOD activity and GSH content,and reduce the content of MDA and LDH?P<0.05 or P<0.01?.?6?The low,medium and high dose of TFZC groups and vitamin C group could alleviate the apoptosis of HUVECs induced by H₂O₂ in different degrees.The apoptosis rate was detected by flow cytometry.Compared with the apoptosis rate in the normal group,The model group significantly increased?29.30±4.59%Vs 2.90±0.07%?)?P<0.01?.The apoptosis rate of low,medium and high dose of TFZC group and vitamin C group significantly decreased?respectively 18.20±1.87%,12.85%±1.28%,9.87±0.63%,14.13±1.63%??P<0.01?.?7?Mitochondrial membrane potential and ROS were detected by flow cytometry.Mitochondrial membrane potential decreased significantly in the model group,The mitochondrial membrane potential of the TFZC significantly increased in TFZC group and the vitamin C group.ROS results showed that the fluorescence intensity of intracellular ROS in the model group increased,In addition,the levels of intracellular ROS in the low,medium and high dose group and the vitamin C group were decreased significantly?P<0.01?.?8?After Cyto-ID?Green dye,there are less fluorescent Green dot in the normal control group and model group using the laser confocal,green dot fluorescent increased in TFZC groups with a dose-dependent manner.?9?TFZC and vitamin C groups significantly inhibited the up-regulation of Bax and cleaved caspase-3 protein expression in HUVECs induced by H₂O₂ and the down-regulation of Bcl-2 expression.Its raised protein expression of LC3-?and Bec1in-1?P<0.05 or P<0.01?.?10?The model of inflammatory cells was successfully constructed in RAW264.7 induced by LPS?1?g/ml?for 24h.?11?TFZC had no effect on the activity of RAW264.7 in the concentration range of 1¹00?g/ml.?12?The low,medium,high doses of TFZC and the atorvastatin group significantly inhibited the release of NO in RAW264.7 cell induced by LPS?P<0.01?,respectively,The inhibition rates is 19.12%,28.71%,32.05%and 30.21%,and TAK-42 can reduce the inhibitory effect of TFZC on the release of NO?inhibition rates is 9.52%?.The secretion of TNF-??IL-1?and IL-6 in RAW.264.7 induced by LPS can be down-regulated in the low,medium,high doses of TFZC and the atorvastatin group?P<0.05 or P<0.01?.?14?The mRNA and protein expression levels of TLR4,MyD88,TRAF-6 and NF-?B p65 in RAW.264.7induced by LPS are significantly reduced in the medium and high dose of TFZC and atorvastatin group?P<0.05 or P<0.01?,compared with the model group,but there are no significant in the low dose group?P>0.05?,among which atorvastatin has the best effect.TAK-42 can partially counteract the effect of TFZC on reducing the mRNA and protein expression of TLR4,MyD88,TRAF-6 and NF-?B p65.Conclusions:?1?The method of ultrasonography extraction and purification of TFZC by HP-20 is stable,and its chemical composition is preliminarily analyzed by ultra-high liquid mass spectrometry,which provides a basis for the research on the material basis of anti-atherosclerosis.?2?TFZC have protective effects on HUVECs induced by H₂O₂,and the mechanism is related to antioxidant,inhibition of apoptosis and promotion of autophagy.?3?TFZC can inhibit the inflammatory response of macrophages,and the mechanism is related to the inhibition of TLR4/MyD88/NF-?B signaling pathway.This study provides a theoretical basis for the anti-atherosclerosis effect of TFZC.