Based On The PI3K/AKT/mTOR Autophagy Signal Pathway, The Effect Of Luofengning No. 0 On The Proliferation Of HCASMC Was Explored

BackgroundIn-stent restenosis(ISR)is a difficult problem in the interventional field of coronary heart disease.Drug-coated balloons(DCB)and new-generation drug-eluting stents(DES)still have certain limitations and shortcomings.As the fourth revolution in the field of percutaneous coronary intervention(PCI),Biodegradable stents(BRS)has brought good news to patients.In the early days,Based on the new theory of "Endogenous Collaterals Wind",combined with the principle of "enhancing effect and reducing toxicity" in tranditional Chinese medicine prescriptions and modern pharmacological research,my mentor Pro.Wang Xian made a prescription-Luofengning No.0,which includes the active ingredients paclitaxel and hirudin from Traditional Chinese Medicine Taxus and leech,for the development of agents coating of DES/DCB.So far,we have developed new type of Luofengning No.0 eluted stent/balloon successfully.Now the optimal Luofengning No.0(paclitaxel and bivalirudin)will be used for the development of biodegradable stents.PurposeEstablishing a proliferation model of human coronary artery smooth muscle cells(HCASMC).Using autophagy as an entry point and focusing on the PI3K/AKT/mTOR signaling pathway to explore the possible mechanism of anti stent restenosis of the stent eluted complex of the Luofengning No.0,then providing new ideas for the research and development of Chinese Medicine compound drug eluted bioabsorbable stents.Methods1.Invitro culture of HCASMCThe second generation of HCASMC was purchased with matched medium for standard resuscitation and generation,and a stable HCASMC for the 4th to 6th generation was established as a tool for follow-up cell research.2.Effect of the stent eluted complex of the Luofengning No.0 on HCASMC proliferation induced by Thrombin(1)Establish the proliferation model of HCASMC induced by thrombinThe 4th to 6th generation HCASMC was selected as research platforms.After intervention with different concentrations of thrombin(0.01 NIH units/mL,0.05 NIH units/mL,0.1 NIH units/mL,0.5 NIH units/mL,1 NIH units/mL,5 NIH units/mL,10 NIH units/mL,and 20 NIH units/mL)for different periods(24 h,48 h,and 72 h),CCK-8 assay was used to detect the cell viability after intervention.To find out the thrombin concentration and intervention time to promote HCASMC proliferation to the maximum extent.(2)To explore the non-toxic concentration range of Luofengning No.0 stent coating complex to interfere with HCASMCThe 4th to 6th generation HCASMC was selected as the research platforms.Using different concentrations of Luofengning No.0 stent coating complex,that is,1 ?mol/L of paclitaxel and different concentrations of bivalirudin(1/256 mg/mL,1/128 mg/mL,1/64 mg/mL,1/32 mg/ml,1/16 mg/mL,1/8 mg/mL,1/4 mg/mL,1/2 mg/mL,1 mg/mL)interfere HCASMC for 4 h,using The CCK-8 assay detects the cell viability after intervention,and finding out the concentration ratio of Luofengning No.0 that keeps cell viability above 90%and has no obvious inhibitory effect on cells.(3)To Screen the most effective concentration of Luofengning No.0 stent coating complex to inhibit the proliferation of HCASMC induced by thrombinThe 4th to 6th generation HCASMC was selected as research platforms.First,establish a thrombin-induced HCASMC proliferation model according to the modeling conditions explored in the previous experiment,and then use the non-toxic concentration range of Luofengning No.0 that was explored in the previous experiment to intervene the model,and use the CCK-8 assay to test the cell viability after intervention.The test was conducted to screen out the concentration ratio of Luofengning No.0 that can inhibit the proliferation of HCASMC induced by thrombin to the greatest extent.3.To explore the effect of Luofengning No.0 stent coating complex on autophagy and PI3K/AKT/mTOR signaling pathway during the proliferation of HCASMC induced by thrombinThe most effective concentration of Luofengning No.0 stent coating complex obtained in the previous experiment was used to interfere with the thrombin-induced HCASMC.Western blot was used to detect the protein expression changes of LC3,Beclinl,p62,PI3K,AKT,and mTOR.RT-qPCR assay was used to detect the mRNA expression changes of PI3K,AKT and mTOR.Results1.The HCASMC that has been subcultured to 4-6 generations grows well and can be used for subsequent experiments.2.Effect of the stent eluted complex of the Luofengning No.0 on HCASMC proliferation induced by thrombin(1)Establish the proliferation model of HCASMC induced by thrombinThe results of CCK-8 after 24 hours of intervention with different concentrations of thrombin in HCASMC showed that with the increase of thrombin concentration,cell viability gradually increased,and the two were positively correlated.When the thrombin concentration reached 0.05 NIH units/mL,it started to affect the cells.It showed obvious proliferation effect,and the difference was statistically significant compared with the control group(P<0.05);The results of CCK-8 after the intervention of HCASMC with different concentrations of thrombin for 48 h showed that the cell viability was enhanced compared with the control group.With the increase of thrombin concentration,starting from the concentration of 0.01 NIH units/mL,it showed a significant proliferation effect on cells,and the difference was statistically significant compared with the control group(P<0.05).When the thrombin concentration reaches 1 NIH units/mL,the cell viability reaches the strongest.Subsequently,the cell viability decreases as the thrombin concentration increases;The results of CCK-8 after 72 h of HCASMC intervention with different concentrations of thrombin showed that after 72 h of different concentrations of thrombin,the cells showed obvious proliferation effect on the cells,and the difference was statistically significant compared with the control group(P<0.05).As the thrombin concentration increases,the cell viability first increases and then decreases.When the thrombin concentration is 1 NIH units/mL,the cell viability reaches the strongest.Taking into account that subsequent experiments need to be further implemented on the basis of the model,1 NIH units/mL of thrombin to HCASMC for 48 h can be used as a modeling condition for thrombin to maximize the proliferation of HCASMC.(2)The non-toxic concentration range of Luofengning No.0 stent coating complex to interfere with HCASMCThe Luofengning No.0 stent coating complex with different concentration ratios interfered with HCASMC for 4 h,which inhibited cell viability to varying degrees.The cell viability decreased with the increase of bivalirudin concentration,and the two were negatively correlated.When 1 ?mol/L paclitaxel was mixed with 1/64 mg/mL bivalirut,Luofengning No.0 stent coating complex began to show a significant inhibitory effect on the growth of HCASMC.And the difference was statistically significant compared with the control group(P<0.05).When the concentration of bivalirudin in Luofengning No.0 is not greater than 1/128 mg/mL,it has no obvious inhibitory effect on the growth of HCASMC and can maintain cell viability above 90%.At this time,paclitaxel(1 ?mol/L)and bivalirudin(0 mg/mL-1/128 mg/mL)can be used as the non-toxic concentration range of Luofengning No.0 stent coating complex to interfere with HCASMC.(3)The most effective concentration of Luofengning No.0 stent coating complex to inhibit the proliferation of HCASMC induced by thrombinDifferent concentrations of Luofengning No.0 stent coating complex inhibited the proliferation of HCASMC induced by thrombin to varying degrees.Compared with the model group,the cell viability decreased with the increase of bivalirudin concentration,and the two were negatively correlated..When the ratio of 1 ?mol/L paclitaxel to 1/256 mg/mL bivalirut,Luofengning No.0 stent coating complex began to significantly inhibit the proliferation of HCASMC induced by thrombin.Compared with the model group,the difference is statistically significant.Academic significance(P<0.05).Compared with Luofengning No.0 with a bivalirudin concentration of 1/256 mg/mL,Luofengning No.0 with a bivalirudin concentration of 1/128 mg/mL has a better inhibitory effect on the proliferation of HCASMC induced by thrombin.Significant,the difference is statistically significant(P<0.05).it can be used as the most effective concentration of Luofengning No.0 stent coating complex to inhibit the proliferation of HCASMC induced by thrombin,that is,1 ?mol/L paclitaxel+1/128 mg/mL of bivalirudin.3.The effect of Luofengning No.0 stent coating complex on autophagy and PI3K/AKT/mTOR signaling pathway during the proliferation of HCASMC induced by thrombinWestern blot results showed that compared with the control group,the ratio of LC3?/? and the protein expression of Beclinl in the thrombin model were significantly reduced,and the difference was statistically significant(P<0.05).The expression proteins of p62,PI3K,AKT and mTOR was significantly increased,and the difference was statistically significant(P<0.05);compared with the model group,the ratio of LC3?/? and the protein expression of Beclinl in the Luofengning No.0 group were significantly increased,and the difference was statistically significant(P<0.05),the protein expression of p62,PI3K,AKT and mTOR was significantly reduced,and the difference was statistically significant(P<0.05).The RT-qPCR results showed that compared with the control group,the mRNA expression of PI3K,AKT and mTOR in the thrombin model was significantly higher,and the difference was statistically significant(P<0.05).Compared with the model group,the mRNA expression of PI3K,AKT and mTOR in Luofengning No.0 group was significantly reduced,and the difference was statistically significant(P<0.05).Conclusion1.Thrombin may inhibit autophagy by up-regulating the PI3K/AKT/mTOR signaling pathway,thereby promoting the proliferation of HCASMC.2.Luofengning No.0 stent coating complex may promote autophagy by down-regulating the PI3K/AKT/mTOR signaling pathway,thereby inhibiting the proliferation of HCASMC induced by thrombin and exerting an anti-stent restenosis effect.