| BackgroundCurrently,the incidence of carotid atherosclerosis(CAS)in China shows an upward trend day by day.Many studies have shown that the incidence of carotid artery plaque(CAP)in cerebrovascular patients reaches 63%.CAS is characterized by intima-media thickening at the initial stage,and then develops into CAP.When the CAP exceeds 50%of the lumen diameter,it can lead to severe carotid stenosis.Inflammation is regarded as the main factor of CAS occurrence and development.In recent years,with the in-depth study of CAS mechanism,macrophage polarization has attracted wide attention.Under the influence of different environments,macrophages can be divided into M1 type and M2 type.Generally speaking,M1 type is a pro-inflammatory subtype,which secretes pro-inflammatory factors and can promote the development of CAS,while M2 type is an anti-inflammatory subtype and delay the development of CAS.At present,drug intensive treatment of carotid endarterectomy and carotid stent implantation are the main treatment methods for CAP.Among them,statins are the core of intensive drug therapy,and the ultimate goal is to reverse or subside plaque.In the past decades,although the guidelines recommend statins as the cornerstone drugs for lipid reduction and widely publicize the availability of statins,the low-density lipoprotein cholesterol(LDL-C)of many patients has not dropped to the target level.Statins are affected by dose-dependent response,liver and kidney dysfunction and other factors,and some clinical adverse reactions may occur,including fibrous tissue injury,skeletal muscle system injury,liver function injury and so on."Management of Dyslipidemia in 2020:Prospects for Diagnosis and Treatment" points out that relying solely on statins can no longer meet the increasingly low lipid-lowering requirements.Dyslipidemia belongs to the categories of "blood turbidity","fat people" and "ointment people" in traditional Chinese medicine.Traditional Chinese medicine has certain advantages in treating dyslipidemia.There are five different subtypes of bispecific tyrosine phosphorylation-regulated kinases(DYRK)in mammals,including Class Ⅰ(Dyrk1α,DYRK 1β)and Class Ⅱ(DYRK 2,DYRK 3,DYRK 4).At present,it has been found that dual-specific tyrosine phosphorylation-regulated kinase IA(Y)-phosphorylation-regulated kinase 1α,Dyrk1α)is highly expressed and abnormally activated in neuropathy,tumor and diabetes.Dyrk1β mutation is directly related to human genetic diseases,and its 102-site mutation can accurately predict early-onset coronary artery disease,concentric obesity,hypertension,and diabetes,revealing the important role of Dyrk1β in energy metabolism and cardiovascular diseases.Studies have shown that Dyrkl a can participate in the formation of inflammatory microenvironment.The balance between Th17 and T-regulated(Treg)cells plays a key role in regulating immune homeostasis,and insufficient Treg response can lead to inflammatory diseases.Dyrk1α is a physiological regulator of Treg cell differentiation,which can balance immune homeostasis,and related DYRK family members play a wide role in immune homeostasis.Clinical database shows that SNP mutation of Dyrkl a plays an important role in vascular remodeling.However,there are still few studies on whether Dyrk1αcan treat atherosclerosis by regulating inflammatory microenvironment.Traditional Chinese medicine and natural medicine are the important material basis and precious starting point of pharmaceutical chemistry,which have unique chemical structure and biological activity diversity.With the in-depth study of the biological function of Dyrk1α,its inhibitors have been further developed.Several types of Dyrk1α inhibitors have been reported over the years,some of which have been used as pharmacological tools.According to their sources,Dyrkla inhibitors can be divided into two categories:compounds derived from traditional Chinese medicine and natural products,and compounds synthesized by chemistry.With the continuous exploration of traditional Chinese medicine and natural medicine,natural products have played an irreplaceable role in the treatment of various diseases.HZTZ granules are an effective prescription for treating atherosclerosis developed.Previous studies have shown that HZTZ granules can reduce LDL-C level,increase ankle-brachial index,reduce arm-ankle pulse wave velocity,and improve arterial elasticity.Based on the above research,this study intends to explore whether HZTZ granules regulate macrophage polarization through Dyrk1α,to achieve the effect of treating CAP.Objective1.To investigate the effect of HZTZ granules on carotid intima-media thickness(IMT),plaque area,plaque total score and hemodynamics in patients with CAP syndrome of phlegm and blood stasis.2.To establish the CAP model of Apoe-/-mice by feeding high fat diet and carotid cannula surgery and study the effects of HZTZ granules on plaque vulnerability and macrophage polarization in Apoe-/-mice.3.To construct the model of macrophage polarization by using the mouse monocyte macrophage leukemia cell line(RAW264.7),and further explore the mechanism of HZTZ granules regulating macrophage polarization by cell transfection technology.MethodsStudy I Randomized controlled trial of HZTZ granules in improving carotid intima-media thickness64 patients with CAP syndrome of phlegm and blood stasis were randomly assigned to HZTZ granules group,atorvastatin group and Xuezhikang group according to 1:1:1.The treatment period was 8 weeks.The main outcome indexes were bilateral carotid IMT,plaque area,plaque total score and hemodynamics.Study II Study on the Effective Components of HZTZ granules Regulating Dyrkla Based on Virtual ScreeningThe main chemical constituents of HZTZ granules were identified by LC-MS/MS,and the chemical constituents of HZTZ granules were searched by TCMSP,HERB and ETCM databases;ADMET lab 2.0 was used to evaluate the drug formation of the compounds,and then AutoDock software was used to dock the screened compounds with the key target Dyrk1α,and the target binding stability of the compounds was further verified by molecular dynamics.Study Ⅲ Effects of HZTZ granules on stability and inflammation of CAP50 male Apoe-/-mice aged 6-8 weeks were randomly divided into 5 groups:high fat group(n=10),surgical group(high fat diet+operation model,n=10),HZTZ granules group(HZTZ granules+high fat diet+operation model,n=10),enterolactone group(enterolactone+high fat diet+operation model,n=10),atorvastatin group(atorvastatin+high fat diet+operation model,n=10).All animals were fed with high-fat diet in the whole process,and the other four groups were treated with carotid cannula surgery based on high-fat diet to induce common CAS.After 8 weeks of administration,hemodynamic examination was performed,blood lipid level was detected,and polarization of macrophages in spleen and blood was detected by flow cytometry.Frozen sections of mouse carotid arteries were used for pathological examination,and aortic gross oil red staining,frozen oil red staining,HE staining,Sirius red staining and immunofluorescence staining were performed to observe CAP area,lipid deposition,plaque morphology,positive staining area of macrophages and smooth muscle cells,and calculate plaque vulnerability index.Study Ⅳ Effects of HZTZ granules on macrophage phenotype based on DyrklaM1 and M2 macrophage models were induced by LPS(100ng/ml)+IFN-γ(1Ong/ml),IL-4(20ng/ml)+TGF-β(10ng/ml)for 48h.The expression of CD86,iNOS,CD206,Argl and other marker genes and proteins related to macrophage polarization phenotype were analyzed by q-PCR and Western Blot.After successful modeling,we set up blank control group,model group(M1,M2),HZTZ granules serum(low,medium,high dose)intervention group.In addition to the blank control group,model group and HZTZ granules-containing serum intervention group were used to replicate the models.q-PCR and Western Blot were used to analyze the expression of CD86,iNOS,CD206 and Argl genes and proteins.To observe the effect of Dyrk1α on the polarization of macrophages,Dyrk1α was knocked down and overexpressed in RAW264.7 macrophage line to observe the polarization of macrophages.The serum containing HZTZ granules(low,middle,and high doses)was used for intervention,and the expression of CD86,iNOS,CD206 and Argl genes and proteins were analyzed by q-PCR and Western Blot.ResultsStudy Ⅰ Randomized controlled trial of HZTZ granules in improving carotid intima-media thickness1.IMTInter-group comparison:The left IMT at the end of the treatment between the three groups were different(P=0.01),the comparison between HZTZ granules group,atorvastatin group and Xuezhikang group were different(P=0.000,P=0.004).Therefore,in the left carotid IMT improvement,HZTZ granules group,atorvastatin group is better than Xuezhikang group.The left IMT difference(P=0.042)was obtained by calculating the difference between three groups of different subjects.There were differences among HZTZ granules group,atorvastatin group and Xuezhikang group(P=0.002,P=0.002),which proved that HZTZ granules group was superior to Xuezhikang group and equivalent to atorvastatin group in improving left IMT.2.Carotid short axis plaque areaComparison between groups:The difference of right carotid artery short axis plaque area was calculated after the difference calculation among three groups of different subjects(P=0.034),and there was a difference between HZTZ granules group and Xuezhikang group at the end of treatment(P=0.035).It is concluded that the effect of HZTZ granules group is better than that of Xuezhikang group and the same as that of atorvastatin group in improving the difference of plaque area in the right carotid artery short axis.3.Maximum cross-sectional length of CAPInter-group comparison:The maximum CAP cross-sectional length at the end of the treatment between the three groups compared(P=0.002),with statistical significance.At the end of treatment,there were differences among HZTZ granules group,Xuezhikang group and atorvastatin group(P=0.004,P=0.038).Therefore,in the improvement of the maximum CAP cross-sectional length,the efficacy of HZTZ granules group and Xuezhikang group is better than atorvastatin group.4.Left end-diastolic minimum blood flow velocityInter-group comparison:The left end diastolic minimum blood flow velocity at the end of treatment was different among the three groups(P=0.035).At the end of treatment,there were differences between HZTZ granules group,Xuezhikang group and atorvastatin group(P=0.023,P=0.030),which indicated that the efficacy of HZTZ granules group was better than atorvastatin group and equivalent to Xuezhikang group in improving the left end-diastolic minimum blood flow velocity.Study Ⅱ Study on the Effective Components of HZTZ granules Regulating Dyrkla Based on Virtual ScreeningIn this study,394 chemical constituents of HZTZ granules were identified by LCMS/MS,TCMSP database and HERB website,and 165 chemical constituents were obtained by combining them and removing duplicate values.The drug properties of the compounds were evaluated by AD MET lab 2.0,and 11 compounds were screened out.The results of molecular docking showed that the main compounds of HZTZ granules,such as tangerinin,gardenin B and salvia saponin,had good binding activity with Dyrk1α.Study Ⅲ Effects of HZTZ granules on stability and inflammation of CAP1.Effects of HZTZ granules on hemodynamics in Apoe-/-miceCompared with the surgical group,HZTZ granules group significantly reduced PSV(P<0.01),which indicated that HZTZ granules could significantly reduce the maximum systolic velocity of carotid artery.2.Effects of HZTZ granules on four items of blood lipid in Apoe-/-miceIn terms of TG,compared with the surgical group,the Enterolactone group can significantly reduce the TG level(P<0.01);In the aspect of LDL-C,compared with the high fat group,the LDL-C level in the surgical group was significantly increased(P<0.05),and the LDL-C level in the HZTZ granules group was significantly decreased(P<0.0001,P<0.0001)compared with the surgical group,the enteral lipid group and the HZTZ granules group.3.Effects of HZTZ granules on total aortic lesion area in Apoe-/-miceAfter 8 weeks,the aorta of mice was carefully dissected,and the plaque lesion area in the whole aorta of different groups of mice was observed by oil red O staining.Compared with the surgical group,the intervention of enterolactone and HZTZ granules significantly reduced the plaque area of the whole aorta(P<0.01,P<0.05).4.Effects of HZTZ granules on CAP area in Apoe-/-miceThe CAP of Apoe-/-mice was stained with oil red O,and obvious plaque was formed in the proximal part of the right carotid cannula of mice.The results showed that compared with the high fat group,the CAP area in the surgical group was significantly increased(P<0.0001),which indicated that the CAS model was successfully established by carotid cannula operation.Compared with high fat and surgical groups,enterolactone and HZTZ granules significantly reduced the area of CAP(P<0.0001,P>0.05;P<0.05,P<0.05).5.Effects of HZTZ granules on morphological structure of Apoe-/-mouse plaqueThe results of HE staining and pathological section of aortic root plaque in Apoe/-mice showed that compared with high fat group and surgical group,enteral lipid group and HZTZ granules group could significantly increase the proportion of fibrous cap area(P<0.001,P<0.001;P<0.01,P<0.01).Compared with surgical group,enterolactone group and HZTZ granules group could significantly reduce the proportion of necrotic core(P<0.05,P<0.05);In terms of cap/nucleus ratio,the ratio of fibrous cap area in enterolactone group and HZTZ granules group was significantly increased compared with that in high fat group and surgical group(P<0.001,P<0.001;P<0.001,P<0.001).The results of HE staining and pathological sections of CAP of Apoe-/-mice showed that compared with high fat group,enterolactone group and HZTZ granules group could significantly increase the proportion of fibrous cap area(P<0.01,P<0.01).Compared with high fat group and surgical group,enteral lipid group and HZTZ granules group significantly reduced the proportion of necrotic core(P<0.01,P<0.05;P<0.0001,P<0.001);In terms of cap/nucleus ratio,the ratio of fibrous cap area in enterolactone group and HZTZ granules group was significantly increased compared with that in high fat group and surgical group(P<0.0001,P<0.001;P<0.0001,P<0.001).Compared with high fat group and surgical group,the fibrous cap thickness in enterolactone group and HZTZ granules group increased(P>0.05,P<0.01;P<0.01,P<0.001).6.Effects of HZTZ granules on collagen content in Apoe-/-mouse plaqueThe pathological sections of aortic root plaques in Apoe-/-mice were stained with Sirius red to evaluate the collagen deposition level at the plaques.The results showed that compared with the high fat group,the collagen content in the surgical group decreased significantly(P<0.05).Compared with high fat group and surgical group,HZTZ granules group significantly increased the collagen content in plaque(P<0.001,P<0.0001).Compared with the surgical group,the Enterolactone group can increase the collagen content in plaque(P<0.001).Pathological sections of CAP in Apoe-/-mice were stained with Sirius red to evaluate collagen deposition level in plaque.The results showed that compared with the high fat group and the surgical group,the collagen content in the plaque was significantly increased in the enteral lipid group and the HZTZ granules group(P<0.0001,P<0.0001;P<0.0001,P<0.0001).7.Effects of HZTZ granules on inflammatory level of CAPThe inflammatory level of macrophages in plaque was evaluated by immunofluorescence co-staining of macrophage marker CD68 with pro-inflammatory factors IL-1β,CD86,iNOS and anti-inflammatory factors CD206,Argl,respectively.Compared with the surgical group,the intervention of Enterolactone and HZTZ granules significantly reduced the co-expression area of CD68 with IL-1β,CD86(P<0.0001,P<0.0001)and iNOS(P<0.05,P<0.0001),and increased the co-expression area of CD68 with CD206(P<0.01,P<0.01);Compared with high fat group,the intervention of Enterolactone and HZTZ granules increased the co-expression area of CD68 and Arg1(P<0.001,P<0.05).8.Effects of HZTZ granules on CAP vulnerability in Apoe-/-miceIn the aspect of plaque vulnerability index HZTZ granules group significantly decreased the plaque vulnerability index(P>0.05,P<0.01;P<0.05,P<0.01)compared with high fat surgical group and enterolactone group.Study IV Effect of HZTZ granules on macrophage phenotype based on Dyrkla1.HZTZ granules promote the fine polarization of M1 macrophages to M2 macrophagesCompared with the M1 model group,the expression of CD86(P<0.001,P<0.0001,P<0.0001),iNOS(P<0.0001,P<0.0001,P<0.0001)mRNA gene and protein level were decreased after the intervention of HZTZ granules with low,medium and high doses;Up-regulation of TGF-β(P<0.0001,P<0.0001,P<0.01),Arg1(P<0.0001,P<0.0001,P<0.0001)mRNA gene and protein expression levels;Compared with the M2 model group,the mRNA expression of TGF-β increased significantly after HZTZ granulescontaining serum intervention(P<0.01),while the mRNA expression of Arg1 did not increase significantly after HZTZ granules-containing serum intervention(P<0.05),while the mRNA expression of IL-10 increased significantly after HZTZ granulescontaining serum intervention(P<0.05).2.Dyrkla regulates macrophage polarizationCompared with the control group,the overexpression of Dyrkla significantly increased the mRNA expression of iNOS and CD86(P<0.0001,P<0.01);After Dyrk1αwas knocked down by transfected siRNA,the mRNA expression levels of TGF-β and Argl were significantly increased by si-Dyrkla compared with that of blank control group(P<0.01,P<0.01);The expression of TGF-β and IL-10 in si-Dyrk1α group was significantly increased.3.Effects of HZTZ granules on the regulation of macrophage polarization by DyrklaCompared with the control group,the expression of iNOS and CD86 mRNA in the Dyrkla overexpression group was significantly up-regulated(P<0.05,P<0.001).Compared with the Dyrkla overexpression group,the expression of iNOS and CD86 mRNA in the low,middle,and high doses of HZTZ granules serum was significantly reduced(P<0.05,P<0.05,P<0.05;P<0.0001,P<0.05,P<0.05).Compared with the control group,the mRNA expression level of Arg1 was significantly up-regulated in the Si-Dyrkla group(P<0.0001).Compared with the SiDyrk1α group,the mRNA expression level of Argl was more significantly up-regulated after the intervention of low,medium,and high doses of HZTZ granules(P<0.0001,P<0.0001,P<0.0001).The mRNA expression of TGF-β and Arg1 was significantly increased in M2 model group(P<0.0001,P<0.01)compared with the control group.The mRNA expression of TGF-β(P<0.0001)and Arg1(P>0.05)was down-regulated in Dyrk1αoverexpression group.However,the mRNA expression of TGF-β and Arg1 was significantly increased after HZTZ granules-containing serum(P<0.0001,P<0.05).The mRNA expression of TGF-β and Arg1 was significantly increased after HZTZ granulescontaining serum was administered(P<0.0001,P<0.05),especially in low dose group(P<0.0001,P<0.01).Conclusions1.HZTZ granules are superior to atorvastatin in improving the maximum CAP cross-sectional length and the minimum left end diastolic blood flow velocity;It is superior to Xuezhikang in improving the difference between left IMT and right carotid short axis plaque area.2.The main compounds of HZTZ granules,such as tangerinin,gardenin B and salvianin,have good binding activity with Dyrk1α,which proves that Dyrkla may be the key target of the compound in regulating atherosclerosis.3.HZTZ granules and enterolactone can increase the stability of CAP,which may be related to the polarization of M1 macrophages to M2 macrophages in CAP of Apoe/-mice.4.Dyrkla may bidirectionally regulate macrophage polarization.HZTZ granules can inhibit the M1 polarization induced by Dyrk1α overexpression and promote the M2 polarization induced by Dyrkla knock-down. |
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