| Post-menopausal women’s risk of atherosclerosis is significantly increased due to estrogen deficiency.With the aging of the population,post-menopausal atherosclerosis has become one of the major public health issues that threaten the health of elderly women."Vessel collateral theory" and " qi collateral theory " are two branches of the collateral disease theory.The former is often used for vessel collateral problems such as cardio-cerebrovascular diseases,and the latter is often used for qi collateral problems such as disease of the nerve,endocrine and immunology.It is obvious that atherosclerosis is a typical vascular disease and belongs to the vessel collaterals.A significant increase in the incidence of atherosclerosis in post-menopausal women is related to estrogen deficiency,which is associated with dysfunction of the qi collaterals.Bazi Bushen Capsule(BZBS)is a traditional Chinese medicine preparation under the guidance of the theory of collateral disease.It has the functions of nourishing kidney essence,regulating yin and yang,tonifying kidney qi,and clearing blood circulation,and its fingerprints have identified a variety of phytoestrogens.Phytoestrogens have estrogen-like effects,however,the effect of BZBS on post-menopausal atherosclerosis is unclear.Objective:This subject was studied from two aspects of theoretical discussion and experimental research.The theoretical part was to explore the guiding value of collateral disease theory in the prevention and treatment of post-menopausal atherosclerosis.In the experimental part,through the establishment of a postmenopausal atherosclerosis mouse model and an inflammation-induced endothelial cell damage model,the effects of BZBS on the treatment of postmenopausal atherosclerosis from different levels of metabolomics,whole animals,tissues and organs.The focus is on the mechanism of action of estrogen membrane receptors,inflammation,apoptosis.This study provides theoretical and experimental data support for collateral disease theory to guide the prevention and treatment of post-menopausal atherosclerosis.Methods:(1)Theoretical research:This research systematically collated the related literatures of vessel collaterals,qi collaterals,Qi-blood and tonifying kidney essence method,and summarized the research progress of BZBS containing phytoestrogens.From the perspective of qi and blood related to the collateral disease theory,to explore post-menopausal atherosclerosis was associated with both vessel collaterals and qi collaterals.These studies provided a theoretical basis for the collateral disease theory to guide the prevention and treatment of post-menopausal atherosclerosis,and discussed the material basis of BZBS in the treatment of post-menopausal atherosclerosis from the perspective of modern pharmacology.(2)Experimental research:Part 1:Using Waters H-class UPLC chromatographic analyzer and ultraviolet detector,the fingerprint of BZBS was studied experimentally.Chromatographic conditions:Column ACQUITY UPLC BEH C18 1.7μ.m,2.1×100mm;set the following parameters as initial conditions,flow rate:0.3mL/min;wavelength:full wavelength scan;column temperature:30℃;injection volume:1μL;mobile phase:Acetonitrile-acid water(0.1%phosphoric acid aqueous solution).Sample processing method:Screen the dominant components that can be identified by the higher content of the reference substance,take the sum of their peak areas as indicators,and optimize the extraction conditions through orthogonal experiments to determine the number of extractions,extraction solvent,ultrasound duration,and solvent volume.Sixty female homozygous ApoE-/-mice(of C57BL/6J background)and fifteen female C57BL/6J control mice(6-8 weeks old and weighing 18-22 g)were purchased from GemPharmatech Co.,Ltd.After a three-day adaptation period,all ApoE-/-mice were subjected to ovary ligation and bilateral ovariectomy(Ovx)under sterile conditions to induce surgical menopause(Ovx/ApoE-/-mice),whereas sham surgery(needle threading)was performed in the C57BL/6J mice.The animals were allowed to recover for 7 days,eliminating the retention of sex hormones in the body.The Ovx/ApoE-/-mice were then randomly assigned into 4 groups(n=15 per group)namely:the model group(high-fat diet+CMC via intragastric injection),the G1 treatment group(high-fat diet+G1 0.2 μig/day via subcutaneous injection),the high-dose BZBS(HD-BZ)group(high-fat diet+2.8 g/kg/day BZBS via intragastric injection),and the low-dose BZBS(LD-BZ)group(high-fat diet+1.4 g/kg/day BZBS via intragastric injection).The 15 C57BL/6J mice were used as the control group(normal diet+CMC via intragastric injection).After 12 weeks of the experiment,the mice were weighed and then sacrificed.The serum,aortic root,aortic arch,whole thoracoabdominal aorta and uterus of each group of mice were collected,and relevant indicators were detected.① Weigh the weight of each group of mice and the weight of the uterus to calculate the uterine atrophy index.② The serum estradiol(E2)content of mice was measured by radioimmunoassay.③ Metabolomics was used to detect the changes of serum metabolites in each group of mice.④ The lipid levels of each group were measured using a full-automatic biochemical analyzer.⑤ Oil red O staining technique was used to observe the formation of atherosclerotic plaques in the thoracoabdominal aorta of each group of mice.⑥Observe the lipid deposition in the aortic roots of mice in each group by H&E staining and oil red O staining.Part 2:In vivo experiments:Sixty female homozygous ApoE-/-mice(of C57BL/6J background)and fifteen female C57BL/6J control mice(6-8 weeks old and weighing 18-22 g)were purchased from GemPharmatech Co.,Ltd.After a three-day adaptation period,all ApoE-/-mice were subjected to ovary ligation and bilateral ovariectomy(Ovx)under sterile conditions to induce surgical menopause(Ovx/ApoE-/-mice),whereas sham surgery(needle threading)was performed in the C57BL/6J mice.The animals were allowed to recover for 7 days,eliminating the retention of sex hormones in the body.The Ovx/ApoE-/-mice were then randomly assigned into 4 groups(n=15 per group)namely:the model group(high-fat diet+CMC via intragastric injection),the G1 treatment group(high-fat diet+G1 0.2 μg/day via subcutaneous injection),the high-dose BZBS(HD-BZ)group(high-fat diet+2.8 g/kg/day BZBS via intragastric injection),and the low-dose BZBS(LD-BZ)group(high-fat diet+1.4 g/kg/day BZBS via intragastric injection).The 15 C57BL/6J mice were used as the control group(normal diet+CMC via intragastric injection).After the experiment,the mice were sacrificed,and the serum,aortic arch,whole thoracoabdominal aorta of mice were collected,and the morphological changes of inflammation and apoptosis and related molecular biological indicators were detected.①Observe the inflammatory cell aggregation in the aortic arch atherosclerotic lesion area of mice by immunofluorescence method.② Apoptosis of endothelial cell in vascular atherosclerotic plaques in each group was detected by TUNEL apoptosis.③ ELISA was used to detect the secretion levels of serum adhesion factors VCAM-1 and ICAM-1 in each group of mice.④ The qRT-PCR method was used to detect the expression of VCAM-1,ICAM-1,Bcl-2 and Bax genes in the aorta of each group of mice.⑤ Western blot was used to detect the expression of aortic inflammation-related proteins p-IκBα,IκBα,p-p65,p65,VCAM-1,ICAM-1,and apoptosis-related proteins Bcl-2,Bax,cleaved caspase3,and caspase3 in each group of mice.In vitro experiments:Human umbilical vein endothelial cells(HUVECs)were cultured in vitro,and oxidized low-density lipoprotein(Ox-LDL)was used to construct injury models.The experiment was randomly divided into 5 groups:the normal group(Control):serum-free medium,the model group(Model):serum-free medium+Ox-LDL,positive control group(G1):G1(pre-dose 4h)+none Serum medium+Ox-LDL,the high-dose BZBS(HD-BZ)group:HD-BZ(pre-dose 4h)+serum-free medium+Ox-LDL and the low-dose BZBS(HD-BZ)group(LD-BZ):LD-BZ(pre-dose 4h)+serum-free medium+Ox-LDL.①The MTS method was used to detect the changes in cell survival activity of each group,and the modeling conditions and G1，HD-BZ and LD-BZ doses were determined.② The VCAM-1 and ICAM-1 contents in the cell supernatant of each group were detected by ELISA.③ Flow cytometry was used to detect the apoptosis rate of endothelial cells.④ The expression levels of VCAM-1,ICAM-1,Bcl-2 and Bax genes in each group of cells were detected by qRT-PCR technology.⑤ Western blot was used to detect the expression of inflammation-related proteins p-IκBα,IκBα,p-p65,p65,VCAM-1,ICAM-1 and apoptosis-related proteins Bcl-2,Bax,cleaved caspase3 and caspase3 in each group.Part 3:HUVECs were cultured in vitro,and GPER1 gene silencing endothelial cell model was constructed using transient transfection technology.HUVECs were divided into 6 groups:the negative control group(siNC-Control),the negative model group(siNC-Model),the negative BZBS group(siNC-BZBS),the GPER1 gene silencing control group(siGPERl-Control),and the GPER1 gene silencing Model group(siGPER1-Model),GPER1 gene silencing BZBS group(siGPER1-BZBS).① Observe the expression of red fluorescent substance under the fluorescence microscope and identify the GPER1 silencing efficiency by Western Blot technology.② The VCAM-1 and ICAM-1 contents in the cell supernatant of each group were detected by ELISA.③ Flow cytometry was used to detect the apoptosis rate of transfected endothelial cells.④The apoptosis rate of each group was detected by TUNEL apoptosis.⑤ Western Blot technology was used to detect the expression of p-IκBα,IκBα,p-p65,p65,VCAM-1,ICAM-1 and apoptosis-related proteins Bcl-2,Bax,cleaved caspase3 and caspase3 in each group.Results:1.Theoretical research:① The combination of vessel collateral theory and qi collateral theory is a new theory to guide post-menopausal atherosclerosis.② Deficiency of kidney essence leaded to deficiency of vital energy and lack of vitality,leading to loss of regulation of Ying and Wei.Disturbances in Ying and Wei caused abnormal movement and transformation of Qi,resulting in stagnation and deficiency stagnation of collateral qi,phlegm and blood stasis,and vessel collateral stasis resistance.These are the important pathogenesis of vessel collateral injury in elderly women.Replenish kidney essence is essential for atherosclerosis in post-menopausal women.③ BZBS not only tonified kidney essence,kidney Yin,kidney Yang and kidney qi,but also dredge blood vessels,providing a new treatment method for the clinical prevention and treatment of post-menopausal atherosclerosis.2.Experimental research:Part 1:①UPLS fingerprints identified that Bazi Bushen capsules contained 14 compounds,namely:neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,isoquercitrin(ISO),Hyperoside(Hyp),Verbascoside(VB),Epimedin A(epimedin A),Epimedin(icariin,ICA),Baohuoside I(baohuoside I),Imperatorin(Imperatorin,IMP),osthole(ost),catalpol(CP),schizandrin A(deoxyschizandrin),schisandrin B(schisandrin B).Among them,except for neochlorogenic acid,chlorogenic acid,and cryptochlorogenic acid,the other 11 are PEs.②The results of uterine atrophy index showed that G1 and BZBS had no obvious stimulating effect on uterine hyperplasia.③ The results of E2 showed that compared with the Model group,the E2 level of G1 group had no significant change,while the E2 concentration of HD-BZ group and LD-BZ group increased.④ Serum metabolomics results showed that the OPLS-DA model successfully distinguished the population with better predictive power and revealed the trend of their separation.Among the four compounds with the highest contents identified,G1 and BZBS increased the content of serum docosahexaenoic acid,3-hydroxybutyric acid and 5(Z),8(Z),11(Z)-eicosaenoic acid,and reduced the content of lysophosphatidylethanolamine(18:0).⑤Blood biochemical results showed that compared with the Model group,G1 and BZBS significantly reduced the serum TC,TG,LDL-C levels,and increase the HDL-C levels.① The oil red O staining of the aorta showed that G1 and BZBS could reduce the formation of atherosclerotic plaques.⑦ The results of H&E staining of aortic arch and oil red O staining of aortic root showed that G1 and BZBS could reduce the lipid deposition of aortic root and aortic arch.Part 2:In vivo:① Immunofluorescence results show that G1 and BZBS reduced the aggregation of inflammatory cells in the aortic arch atherosclerotic lesion area of mice.② TUNEL apoptosis results showed that G1 and BZBS reduced endothelial cell apoptosis in vascular atherosclerotic plaques in mice.③ ELISA results showed that G1 and BZBS reduced the secretion levels of serum adhesion factors VCAM-1 and ICAM-1 in mice.④ The results of qRT-PCR showed that G1 and BZBS could reduce the expression of VCAM-1,ICAM-1,Bcl-2 and Bax mRNA in mouse aorta.⑤ Western blot results showed that G1 and BZBS inhibited the phosphorylation of IκBα and NFκB p65 in the aorta of mice,reduced the p-IκBκ/IκBα and p-NFκB p65/NFκB p65 ratios,and further inhibited the downstream VCAM-1 and ICAM-1 expression G1 and BZBS can also up-regulate the anti-apoptotic protein Bcl-2 and down-regulate the expression of the pro-apoptotic proteins Bax and cleaved caspase3.In vitro:① MTS method results showed that Ox-LDL 120μg/mL is the modeling concentration,G1 10 nmol/L,HD-BZ 400 μg/mL and LD-BZ 100 μg/mL are the subsequent experimental concentrations.① ELISA results showed that G1 and BZBS could reduce the content of VCAM-1 and ICAM-1 in the cell supernatant.③ The results of flow cytometry showed that G1 and BZBS reduced the apoptosis rate.④ The results of qRT-PCR showed that G1 and BZBS reduced the expression levels of VCAM-1,ICAM-1,Bcl-2 and Bax mRNA in cells.⑤ Western blot results showed that G1 and BZBS inhibited the NF-κB pathway,reduced the p-κBα/IκBαand p-NFκB p65/NFκB p65 ratios,and inhibited the expression of VCAM-1 and ICAM-1.G1 and BZBS also up-regulated the anti-apoptotic protein Bcl-2,down-regulated the apoptotic protein Bax and inhibited the cleavage and activation of caspase3.Part 3:① Observation under the fluorescence microscope showed that the transfection rate of the cells was over 80%;Western Blot results showed that compared with the siNC-Control group,the expression of GPER1 in the successfully transfected cells was significantly reduced.② The ELISA results showed that the levels of VCAM-1 and ICAM-1 protein in the siNC-Model group and siGPER1-Model group increased significantly,and the siGPER1-Model group was more significant.Compared with the siNC-Model group,the siNC-BZBS group significantly inhibited the expression of adhesion molecules;compared with the siGPER1-Model group,the siGPER1-BZBS group inhibited the expression of adhesion molecules.③ The results of flow cytometry showed that the apoptosis rate of siNC-Model group and siGPER1-Model group increased significantly,and that of siGPER1-Model group was more significant.Compared with the siNC-Model group,the siNC-BZBS group significantly inhibited apoptosis;compared with the siGPER1-Model group,the anti-apoptotic effect of the siGPER1-BZBS group was weakened.④ The apoptosis results of TUNEL showed that the apoptosis rate of siNC-Model group and siGPER1-Model group increased significantly,and that of siGPER1-Model group was more significant.Compared with the siNC-Model group,the siNC-BZBS group significantly inhibited apoptosis;compared with the siGPER1-Model group,the anti-apoptotic effect of the siGPER1-BZBS group was weakened.⑤ Western Blot results showed that the expressions of NF-κB signaling pathway-related proteins(p-IκBα/IκBα,p-p65/p65)and downstream VCAM-1 and ICAM-1 were significantly up-regulated in the siNC-Model and siGPER1-Model groups.The siGPER1-Model group was more significant.Compared with the siNC-Model group,the expression level of inflammation-related proteins in the siNC-BZBS group was significantly reduced;compared with the siGPER1-Model group,the inhibitory effect of siGPER1-BZBS group on inflammation was significantly reduced.In the siNC-Model and siGPER1-Model groups,the levels of Bcl-2 were significantly reduced,while the levels of Bax and cleaved-caspase3 were significantly increased,and the siGPER 1-Model group was more significant.Compared with the siNC-Model group,the siNC-BZBS group significantly inhibited apoptosis;compared with the siGPER1-Model group,the anti-apoptotic effect of the siGPER1-BZBS group was significantly weakened.Conclusions:1.The relevant characteristics of qi and blood of collateral disease theory suggest that post-menopausal atherosclerosis is associated with both vessel collaterals and qi collaterals.Deficiency of kidney essence,deficiency of collateral qi and stasis of the veins are the main pathogenesis of the disease,and supplementation of kidney essence is a method of treating the disease.BZBS has the effects of supplementing kidney essence and adjusting yin and yang,and has become an effective drug for the treatment of the disease.2.Experiments have confirmed that BZBS nourishes the kidney,thereby invigorating qi,supplementing phytoestrogen,activating estrogen receptors,systematically regulating the disorder of serum metabolite spectrum,blocking vascular inflammation.BZBS also regulates the perfusing blood,nourishing metabolic functions of vessel collaterals,reduces inflammation-mediated apoptosis of vascular endothelial cells,inhibits atherosclerotic plaque formation,and provides experimental support for the treatment of postmenopausal atherosclerosis... |
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