Based On AMPK/mTOR Autophagy Signal Pathway, The Mechanism Of Luofengning No.0 Prescription Against The Proliferation Of HCASMC Was Explored

BackgroundIn recent years,the morbidity and mortality of cardiovascular diseases are high in China.Acute coronary syndrome seriously affects the survival of patients due to its acute onset and critical condition.Early active percutaneous coronary intervention(PCI)has become the standard and routine treatment for acute coronary syndromes.However,the restenosis problem that is hard to ignore after PCI still brings pain to patients,and the stent that has been repeated for several times because of restenosis problem cannot be fully satisfied.Further development of stent materials and technical innovation,and further exploration of anti-PCI restenosis solutions require for joint efforts of clinical and scientific research.Our team has been committed to the combine traditional Chinese and western medicine treatment of acute coronary syndrome,tutor Wang reveals the combination of theory and clinical experience,putting forward "collateral wind" pathogenesis theory in acute coronary syndrome innovatively,addressing Luofengning Formula series under the guidance of " curing collateral wind" treatment.Luofengning Formula 0 consists of yew and leeches,used to be traditional Chinese medicine monomer compound on stent,contributing to the prevention and treatment of PCI postoperative restenosis.Luofengning Formula 0 has been experimentally proved to inhibit the proliferation of smooth muscle cells and resist restenosis of the tube wall after PCI.However,the mechanism of this action at the cellular and molecular level is still unclear and needs to be further explored.Modern studies have shown that the level of autophagy affects the growth of smooth muscle cells.In this study,AMPK/mTOR,a classical autophagy signaling pathway,was selected as the entry point to explore the possible mechanism of Luofengning Formula 0 against the proliferation of human coronary artery smooth muscle cells(HCASMC).PurposeTo explore the probable mechanism of complex Luofengning Formula 0 against HCASMC proliferation.Focus on AMPK/mTOR autophagy signaling pathway,contrasting critical site protein in AMPK/mTOR autophagy signaling pathway to analyze the effect that Luofengning Formula 0 might have during its proliferation resistance and to discuss the possible mechanism of the formula.To contribute to new-drug exploring of traditional Chinese medicine,and further clinical prevention and treatment of acute diseases using Chinese medicine.Methods1.In vitro culture of HCASMC:Cell culture,liquid exchange,passage and cryopreservation were carried out from the primary HCASMC,and 4-6 generations of stable growth HCASMC were cultured as experimental cells.2.Establishment of HCASMC proliferation model induced by platelet-derived growth factor(PDGF):The cell proliferation effect was analyzed by MTT assay,and the appropriate PDGF-induced proliferation scheme was selected.3.Grope for Luofengning Formula 0 "Paclitaxel-Bivalirudin" non-toxic concentration range on HCASMC growth:Paclitaxel,the monomer component of Taxus Chinensis and bivalirudin were made into Luofengning Formula 0.MTT assay was used to screen the range of drug concentration with cell viability above 90%;4.Determine of the effective ratio concentration of "Paclitaxel-Bivalirudin" compound for anti-HCASMC proliferation:On the basis of smooth muscle cell proliferation prompted by PDGF,the effective ratio concentration of "Paclitaxel-Bivalirudin" compound within the range of non-toxic ratio concentration of PDGF was determined;5.To explore the "Paclitaxel-Bivalirudin" compound in the process of PDGF induced HCASMC proliferation AMPK/mTOR autophagy signaling pathway:Contrast the change of the key sites of protein signaling pathway,trying to analyze the possible mechanism of Luofengning Formula 0" Paclitaxel-Bivalirudin " compound by Western Blot assay.Results1.PDGF-induced HCASMC proliferation model:MTT assay was used to analyze the proliferation rate of cells stimulated by different concentrations of PDGF,and it had been found that the proliferation rate of smooth muscle cells reached more than 15%when the concentration of PDGF was 40ng/ml,which was significantly different from the blank control group(P<0.05).The PDGF concentration of 40ng/ml and stimulation time of 24h were selected as the modeling scheme of HCASMC proliferation model.2.The non-toxic concentration range for HCASMC growth of Luofengning Formula 0"Paclitaxel-Bivalirudin" compound:MTT assay was used to found that the activity of cells in paclitaxel 1umol/L+bivalirudin 1-5ug/L was 90%,which was significantly different from the blank control group(P<0.05),and was considered as the non-toxic range of HCASMC growth.3.The effective proliferation resistance concentration of Luofengning Formula 0"Paclitaxel-Bivalirudin":MTT assay was used to found that when paclitaxel was 1umol/L and bivalirudin was 2ug/L,Luofengning Formula 0 showed the strongest inhibitory effect on PDGF proliferation.Compared with the model group,the difference was statistically significant(P<0.05),and was selected as the effective ratio concentration of anti-HCASMC proliferation.4.Possible effect on AMPK/mTOR autophagy signaling pathway during Luofengning Formula 0 "Paclitaxel-Bivalirudin" inhibiting HCASMC proliferation induced by PDGF:The inhibiting effect was observed by microscope after "Paclitaxel-Bivalirudin" compound was used.Western Blot results showed that the detection protein content in the control group,the modeling group and the experimental group were different,and the difference was statistically significant(P<0.05).In terms of protein content changes,AMPK,ULK1,LC3B and Beclin 1 showed a consistent change trend.Compared with the control group,the content of protein in the modeling group decreased,while the content of the experimental group increased compared with the modeling group,but was lower than that in the control group.mTOR showed a contrary changing trend with the above detection protein.Compared with the control group,the content of modelling group increased,while the content of the experimental group decreased compared with that of the model group,but was higher than that of the control group.In this experiment,AMPK and ULK1 were inhibited after modeling,while mTOR function was promoted,and autophagy level was decreased,as a result that cell proliferation was promoted.On the contrary,AMPK and ULK1 functions were promoted,while mTOR was inhibited,and autophagy level was increased,as a result that cell proliferation was inhibited.Conclusion1.Luofengning Formula 0 "Paclitaxel-Bivalirudin" compound can effectively inhibit the proliferation of human coronary artery smooth muscle cells.2.Luofengning Formula 0 "Paclitaxel-Bivalirudin" compound may regulate the level of autophagy through AMPK/mTOR autophagy signaling pathway and play an anti-proliferation role.3.Luofengning Formula 0 "Paclitaxel-Bivalirudin" compound may act on AMPK/mTOR autophagy signaling pathway,up-regulating AMPK and ULK1 activity,down-regulating mTOR activity,improving the level of autophagy,and inhibiting cell proliferation initiated by PDGF.