The Effect Of Luofengning No.0 Scaffold Coating Complex On TLR4-MyD88 Signaling Pathway During HCASMC Inflammatory Activation And Its Possible Mechanism

BackgroundIn-stent restenosis and thrombosis after PCI is a huge challenge in the vascular interventional field.Although in-stent thrombosis rate has dropped to 0.2%in the new generation of DES,the rate of restenosis has not been effectively controlled,which seriously affected the quality of life and long-term prognosis of patients after intervention.Thus,how to minimize the risk of stent restenosis and thrombosis is always the focus and frontier area of balloon and stent research both at home and abroad.Based on the new theory of collateral wind syndrome which proposed by my mentor professor Wang xian,we innovate the way of drug delivery by extracting the active ingredients paclitaxel and hirudin from Traditional Chinese Medicine Taxus and leech,composed of "Luo Feng Ning No.0" eluted on the coronary stent/balloon surface.We have prepared this new type of Luo Feng Ning No.0 eluted stent/balloon for the treatment of cardiovascular emergencies with integrated Traditional Chinese and Western Medicine.At present,Luo Feng Ning 0 coated balloon has preliminarily proved its safety and effectiveness in miniature pig experiment,especially in the prevention and treatment of lumen restenosis and thrombosis.The advantage of this new balloon is still inspiring,but its microbiological mechanism still needs further exploration.Our previous study found that patients with endogenous collateral wind syndrome are in the state of high inflammation,modern research also showed that the inflammatory microenvironment after PCI is closely related with the formation of stent restenosis and thrombosis.Besides,proliferation and migration of inflammatory activated smooth muscle cells is known as a key link in restenosis process,the efficacy of anti-inflammation in the treatment of restenosis is effective,which provides us with the inspiration that the effect mechanism of Luo Feng Ning No.0 on the prevention of in-stent restenosis may be related to the anti-inflammatory.Then we use inflammation as an entry point and focus on the classic TLR4-MyD88 inflammatory pathway,taking HCASMC as the research tool to explore the effect and possible mechanism of Luo Feng Ning No.0 on the inflammatory pathway,and try to open a breakthrough for further research on the next step of in-stent restenosis.PurposeThis subject used LPS to induce inflammatory activation of HCASMC to observe the effect of Luo Feng Ning No.0 on the inflammatory pathway of TLR4/MyD88/NF-k B and try to find out its action target,explore the possible mechanism of anti stent restenosis of the stent/balloon eluted complex of the Luo Feng Ning No.0 from the angle of inflammation,then provide new ideas for the research and development of Traditional Chinese Medicine compound drug eluted interventional apparatus.Methods1.In vitro culture of HCASMCThe second generation of HCASMC was purchased with no antibiotic medium for standard resuscitation and generation,and a stable HCASMC for 4 to 6 generations was established as a tool for follow-up cell research.2.Establish LPS induced HCASMC inflammatory activated cell model and maximize the activation of TLR4/MyD88/NF-K B inflammatory pathway2.1 Finding the nontoxic concentration range of LPS induced model by MTT method.2.2 Using ELISA.Q-PCR,Western blot method to screen the best LPS concentration and stimulation time of HCASMC in high inflammatory activation state,which provides a platform for the study of the mechanism of Luo Feng Ning No.0.3.To explore the effect of Luo Feng Ning No.0 on the TLR4-MyD88 signaling pathway in the process of HCASMC inflammatory activation3.1 MTT method was used to screen the non-toxic concentration range of HCASMC,which kept the cell vitality more than 90%.3.2 ELISA,Q-PCR and Western blot were used to detect the changes of TLR4/MyD88/NF-k B inflammatory pathway and the expression of downstream inflammatory products after the treatment of Luo Feng Ning No.0 to predict the anti-inflammatory sites of the "Luo Feng Ning 0n recipe.4.To explore the possible mechanism of "Luo Feng Ning No.0" inhibited the TLR4-MyD88 signaling pathway in the inflammatory activation of HCASMC4.1 The HCASMC specific MyD88 siRNA was designed.and the MyD88 knockout cell model was constructed by liposome transfection.4.2 The knockdown of MyD88 expression or blocking MyD88 degradation by proteasome inhibitor Epoxomocin selectivity,explored whether Luo Feng Ning No.0 was by acting on TLR4/MyD88/NF-k B pathway in the MyD88 node,affected the downstream NF-k B p65 activity to down regulate the inflammatory reaction.Results1.LPS induced nontoxic concentration range of HCASMC inflammatory activated cell modelLow concentration(0-10 ? g/mL)LPS compared with the control group of HCASMC activity had no significant effect(cell viability remained above 90%),while high concentration(1000 ?g/mL)LPS had an impact on cell viability(cell viability 80.1%).Therefore,the non-toxic concentration of LPS to HCASMC is 0-10 ?g/mL.2.Optimal model conditions for LPS induced HCASMC inflammatory activation2.1 In light microscopy,the number of HCASMC increased in a dose-dependent manner,and the cell growth was the most active when the concentration of LPS was 0.5,1,10 ?g/mL.2.2 ELISA results showed that 0.5 and 1 ?g/mL LPS stimulated 48h could activate HCASMC to secrete IL-6,TNF-? and:IL-1 ?at the same time,which was significantly different from the blank control group(P<0.05).2.3 Q-PCR results showed that 1 ? g/mL LPS stimulated 48h could activate ?HCASMC at the same time,upregulated TLR4,MyD88.NF-? B p65,IL-6,TNF-?and IL-1 expression,compared with the blank control group,the difference was statistically significant(P<0.05).2.4 Western blot could be seen that 1 ? g/mL LPS stimulated 48h could simultaneously activate HCASMC to increase the expression of TLR4,MyD88 and NF-k B p65 protein.Therefore,the stimulation of HCASMC 48h with 1 ? g/mL LPS can ensure that HCASMC is in a high inflammatory activation state and can activate TLR4-MyD88 signaling pathway to the maximum extent,which can serve as the best model for HCASMC inflammatory activated cell model.3.Effect of Luo Feng Ning No.0 on TLR4-MyD88 signaling pathway in HCASMC inflammatory activation process3.1 The non-toxic concentration range of Luo Feng Ning No.0 was screened by MTT method.The high concentration group was 1?mol/L paclitaxel +0.2mg/mL hirudin(cell viability was 92%),and the low concentration group was 1?mol/L paclitaxel +0.0125mg/mL hirudin(cell viability was 100%).3.2 ELISA results showed that the expression of IL-6,TNF-a and IL-1? protein was down regulated by Luo Feng Ning No.O.The difference between the high and low concentration group of Luo Feng Ning No.0 was significantly different from that of the inflammatory activation model group(P<0.05),and the high concentration group of Luo Feng Ning No.0 had a more obvious anti-inflammatory effect.4.The possible mechanism of Luo Feng Ning No.0 to inhibit inflammatory activation of TLR4-MyD88 signaling pathway in HCASMC induced by LPS4.1 Q-PCR results showed that Epoxomicin did not affect the expression of MyD88 gene after the stimulation of Luo Feng Ning No.0(P>0.05),but high concentration of Epoxomicin(1 ?M)could reduce the inhibitory effect of Luo Feng Ning No.0 on downstream inflammatory factors IL-6,TNF-a and IL-1? gene expression by blocking MyD88 degradation compared with the control group and the inflammation model group were statistically significant(P<0.05).4.2 ELISA results showed that high concentration of Epoxomicin(1 ?M)could weaken the inhibitory effect of Luo Feng Ning No.0 on the expression of downstream inflammatory factors IL-6,TNF-a and IL-1 ? protein expression by blocking the degradation of MyD88.Compared with the blank control group,the difference was statistically significant(P<0.05),but there was no statistically significant difference compared with inflammatory model group(P>0.05).4.3 Western blot results showed that Epoxomicin could weaken the inhibitory effect of Luo Feng Ning No.0 on the expression of downstream inflammatory protein TLR4,MyD88,NF-k B p65 expression by blocking the degradation of MyD88,and the blocking effect of high concentration of Epoxomicin was more significant.Conclusion1.Luo Feng Ning No.0 has the effect of inhibiting the local vascular inflammatory reaction.2.Luo Feng Ning No.0 plays an anti-inflammatory effect by inhibiting the TLR4/MyD88/NF-k B signal pathway.3.Luo Feng Ning No.0 through the action of MyD88 node in TLR4/MyD88/NF-k B pathway,promotes MyD88 protein degradation and reduces the activity of TLR4,MyD88,NF-k B p65 to weaken the inflammatory reaction induced by LPS.