| Objective:Baiyimucao is the aboveground part of Panzeria alaschanica,is a characteristic Mongolian medicine with anti-inflammatory,promoting blood circulation,regulating menstruation,clearing heat and diuresis effects.In order to reveal the pharmacological activity of Baiyimucao,expand its clinical use,and lay a foundation for pharmacological research of baiyimucao preparation development.This study was carried out at the cellular and animal levels.Firstly,a stable method of isolation and culture of VSMCs was established,and the differences of morphological,growth and phenotypic characteristic protein expression between tissue adherence method and mixed enzyme digestion method were compared,which laid a foundation for the stable and controllable expression of VSMCs in the experiment.The effects of PDGF-BB,Ang II,TNF-a and LPS on VSMCs were evaluated from the aspects of inducing VSMCs proliferation and phenotypic transformation.The best inducing factors and inducing conditions were screened,and reliable drug evaluation indexes for screening and treating vascular proliferative diseases in vitro were established.To evaluate the effects of Leonurine and stachydrine on the proliferation and phenotypic transformation of VSMCs induced by PDGF-BB and their potential molecular mechanisms.To establish a model of femoral artery intimal hyperplasia in mechanically stripped mice,and to evaluate the effects of alkaloids,flavonoids,stachydrine and Leonurine on vascular intimal hyperplasia,and to clarify the possible mechanisms,so as to provide new research directions and ideas for the treatment of vascular hyperplasia diseases.Methods:1.Phenotypic characteristics of VSMCs isolated and cultured by patch method and enzymatic digestion method:consulting data,establishing tissue patch adherence method and enzymatic digestion method to obtain VSMCs stably.VSMCs obtained by two methods were subcultured in vitro.Cell morphology of VSMCs subcultured to 3rd and 7th generations was observed under microscope,and contractile protein SM a-actin of cells subcultured to 3rd and 7th generations was detected by immunofluorescence.The expression of SM actin and the cell morphology of the cells obtained by the two different methods were evaluated.2.Establish the induction conditions of VSMCs:screen four prnmary factors involved in atherosclerosis and vascular hyperplasia,including PDGF-BB,Ang ?,TNF-a and LPS,and establish the appropriate concentration scale of each stimulating factor.CCK-8 was used to detect the proliferation of VSMCs at different concentrations and at different time points.The stimulating factors at different time points were detected by immunofluorescence.To establish a reliable cell model of vascular proliferative disease in vitro by combining the two indicatorsS.To observe the toxicity of stachydrine,Leonurine and berberine to VSMCs:Referring to the literature,the commonly used dosage and administration conditions of the three drugs,the concentration scale of each drug was established.The VSMCs were treated with stachydrine,Leonurine and berberine at different concentrations for 24 h and 48 h respectively.CCK-8 was used to detect the cell viability.To evaluate the cytotoxicity of stachydrine,Leonurine and berberine on VSMCs.4.To observe the effects of stachydrine,Leonurine and berberine on phenotypic transformation of VSMCs induced by PDGF-BB:Referring to the above-mentioned in vitro modeling conditions of vascular proliferative diseases and toxicity experiments of drugs,the optimal inducing factors and induction time were determined,and the appropriate amount of three drugs were selected,and evaluated by CCK-8,immunofluorescence and western blot,respectively.Intervention of drugs on PDGF-BB-induced VSMCs proliferation,contractile-specific proteins(SM a-actin,SM MHC),synthetic marker proteins(SPP-1,CCL-2)and NOX4 protein expression.5.Establish a model of femoral artery intimal hyperplasia in mechanically stripped mice:male C57BL/6 mice were randomly divided into 7 groups:sham-operated group,28-day model group,21-day model group,14-day model group,7-day model group,3-day model group,1-day model group,7 rats in each group.After 24 h,the femoral artery was separated and collected at the end of the experiment.The pathological sections of the femoral artery were made and stained with H&E and Masson to observe the changes of vascular tissue structure and fibrosis.SM caactin,CCL-2 and NOX4 protein were labeled by tissue immunofluorescence,and fluorescence images were collected by laser scanning confocal microscopy to observe the three proteins of SM a-actin,CCL-2 and NOX4 in the blood vessels.Trend changes in the occur and progression of proliferative diseases.6.Effects of stachydrine,Leonurine,berberine,alkaloids and flavonoids ofBaiyimucao ineof ine on vascular injury in mice:Male C57BL/6 mice were randomly divided into seven groups:sham-operated group,model group,Leonurine group,stachydrine group,berberine group,alkaloids group and flavonoids group.After 24 h of operation,the mice were given pure water or drugs by intra gavage.The pathological sections of femoral artery were made and stained with H&E and Masson.The efifects of drugs on intimal hyperplasia were observed.The expressions of SM a-actin,CCL-2 and NOX4 were detected by immunofluorescence.The fluorescence images were collected by laser scanning confocal microscopy.The effects of drugs on the expressions of SM a-actin,CCL-2 and NOX4 were evaluated.Results:1.The phenotypic characteristics of VSMCs isolated and cultured by patching and enzymatic digestion were compared.The results showed that the expression of SM a-actin,a characteristic protein of primary VSMCs obtained by mixed enzymatic digestion,was significantly better than that of tissue patching.With the passage times increasing,VSMCs from the mixed enzymatic digestion retained some of the contractile phenotypic characteristics in the seventh generation,while which from the tissue patching method lost in the seventh generation and into a synthetic phenotype.2.Establishing a reliable cell model of vascular proliferative diseases in vitro showed that PDGF-BB and Ang ? could promote the proliferation of VSMCs more effectively than TNF-a and LPS.PDGF-BB could significantly promote the proliferation of VSMCs at 60 ng/ml for 24 h,while Ang ? at the scale of concentration of 0-6 nmol/L could effectively promote the proliferation of VSMCs and dependent on time.The results of immunofluorescence showed that PDGF-BB could significantly inhibit the expression of SM a-actin,a contractile marker protein,compared with Ang ? in VSMCs.Therefore,the final cell model condition was 60 ng/mL PDGF-BB,which lasted 24 h or 48 h.3.The toxicity of stachydrine,Leonurine and berberine to VSMCs was studied.The results showed that CCK-8 could detect the effects of stachydrine(0??100?M),berberine(0?100 ?m)and Leonurine(0?200?M)at different concentrations on VSMCs for 24 h and 48 h respectively.There was no significant difference between stachydrine and berberine and the control group(p>0.05).Leonurine had a certain inhibitation on the activity of VSMCs(p>0.05).However,there was no obvious cell death,so the three drugs had no obvious toxicity to VSMCs.4.The results of the intervention effects of stachydrine,Leonurine and berberine on phenotypic transformation of VSMCs induced by PDGF-BB showed that stachydrine,Leonurine and berberine did not improve the proliferation of VSMCs induced by PDGF-BB for 24 h.48 h later,stachydrine(0.1,1,10,20,40 VM),berberine(0.1,1 ?M)and berberine(0.1,1?M)did not improve the proliferation of VSMCs induced by PDGF-BB.The results of immunofluorescence showed that stachydrine(1?M),leonurine(10?M)and berberine(1?M)could significantly improve the decrease of SM a-actin contractile protein induced by PDGF-BB at 24 h,and western blot experiment showed that leonurine(1,10?M),stachydrine(1,10?M)and berberine(1,10?M)definitely increased the expression of ?-actin and MHC compared with model group,and the expression of synthetic marker protein SPP-1,chemokine CCL-2 and NOX4 was significantly decreased.5.Model results of femoral artery intimal hyperplasia in mechanically stripped in mice:H&E and Masson staining results showed that after mechanical injury of femoral artery intima in mice,inflammation was characterized in the early stage.Inflammatory cells accumulated in the intima and peritubule.With the prolongation after the injury time,smooth muscle cells migrated to the intima,and collagen fibers deposited in the intima,media and adventitia of the vessels,resulting in vascular reconstructure.The degree of vascular wall fibrosis was positively correlated with time,and the infiltration of inflammatory cells was most obvious on the third day,then the inflammation gradually subsided.The results of fluorescence labeling showed that the expression of SM a-actin,a contractile marker protein,decreased gradually with the prolongation of time.NOX4 was mainly distributed in the endothelial cell layer and the periphery of vascular wall,after sham-operation group and vascular injury repair.The expression of CCL-2 was the highest on the first day of modeling,and then gradually decreased,indicating that the inflammatory infiltration gradually subsided after the third day,corresponding to the results of H&E staining.6.The effects of stachydrine,Leonurine,berberine,alkaloids and flavonoids Baiyimucao on vascular injury in mice were studied by H&E and Masson staining.Fluorescence labeling results showed that the expression of SM a-actin in vascular wall of alkaloids and berberine group was similar to that of sham-operated group,showing a bright red color,while the fluorescence intensity of flavonoids group and stachydrine group was slightly darker;the endothelial cells of flavonoids,stachydrine and berberine group were NOX4 green.The colour fluorescence showed a complete ring,but no NOX4 endothelial cell ring was found in alkaloid group.CCL-2 expression was not found in all groups,which could effectively interfere with the phenotypic transformation and inflammatory reaction of VSMCs after injury,but may not be related to NOX4.Conclusion:1.Enzymatic digestion has more advantages in obtaining stable contractile phenotype VSMCs than tissue patch adherence method.Each laboratory should select accurately according to its own experimental conditions and design.2.Compared with Ang II,LPS and TNF-a,60 ng/mL PDGF-BB can effectively promote the proliferation and phenotypic transformation of VSMCs at 24 h or 48 h.3.Stachydrine(0?100?M),berberine(0?100?M)and Leonurine(0?200?M)acted on VSMCs for 24 h and 48 h respectively,without obvious toxicity to VSMCs.4.Low concentration of stachydrine(1,10 ?M),Leonurine(1,10 ?M)and berberine(1,10?M)could effectively interfere with phenotypic transformation of VSMCs induced by PDGF-BB for 24 h.5.Mechanical stripped the femoral artery in mice can significantly lead to intimal hyperplasia,vascular wall fibrosis,VSMCs phenotype transformation(contraction to synthesis),and is positively correlated with time.In addition,NOX4 is not expressed in vascular smooth muscle cells,which is closely related to the integrity of vascular endothelial cell layer;it also participates in the repair of peripheral vascular cells.6.Alkaloids and flavonoids of Baiyimucao,Leonurus alkaloids,stachydrine and berberine may partially inhibit VSMCs phenotype transformation induced by vascular injury through NOX4,and improve intimal proliferation and vascular fibrosis induced by mechanical injury. |
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