Effect And Mechanism Of Shenfu Injection On Protecting LPS Injury Human Umbilical Vein Endothelial Cells By Regulating NOS/NO Signaling Pathway

Background and Objective:Thromboangiitis obliterans(TAO),also known as Buerger’s disease,mainly involves small and medium-sized arteries and veins of the extremities and presents segmental,occluded,thrombosis and non-atheromatous inflammatory diseases.Its symptoms include intermittent claudication,resting pain,ischemic ulcers,pyorrhoresis,Raynaud’s disease,and gangrene.Patients are at risk of amputation and death if not treated promptly or properly.The pathogenesis of TAO has not been fully elucidated until now,and no single viewpoint can explain all the clinical manifestations of TAO.At present,TAO is considered to be closely related to the damage of vascular endothelial cell(VEC).The therapy of thromboangiitis obliterans has been an important challenge in clinical research for many years,and traditional Chinese medicine(TCM)has made marked achievements for this disease.The treatment of combination of Chinese traditional and Western medicine can significantly improve the cure rate of TAO and reduce the recurrence rate.Overoxidation is one of the main factors causing VEC apoptosis,and the damaged VEC presented some changes in its morphology and function,which is an important mechanism to promote the formation of thrombosis in TAO.Lipopolysaccharide(LPS)was the main component of the cell wall of gram-negative bacilli,and was often used as an irritant to induce endothelial cell injury.At present,there is no fundamental cure for TAO at home and abroad.Based on the possible pathogenesis of TAO,more clinical treatment is just taken to reduce the pain of patients.Shenfu injection(SFI)is orginated from a Chinese herbal compound prescription,the main components of which includes ginsenoside and alkaloids.SFI has been proved to be capable of returning Yang to save Qi and replenishing blood in TCM.Our preliminary research found that SFI may strengthen the inhibition of platelet aggregation,improve the antithrombotic function of blood vessels,reduce the thrombosis of TAO model rats,and improve its pathological signs.Nitric oxide(NO)is a paracrine factor that affects vascular tension and platelet function,prevents white blood cell adhesion and reduces endometrial hyperplasia.Based on the NOS/NO signaling pathway,LPS-induced primary culture HUVECs was used as an isolated TAO model to further explore the mechanism underlying SFI’s antithromboocclusive vasculitis.Methods:1.Acquisition,culture,passage,cryopreservation and identification of primary HUVEC.2.MTT assay was used to detect and screen the damage degree of HUVEC caused by different concentrations of LPS to determine the appropriate concentration of LPS-induced HUVEC damage.3.MTT assay was used to detect the effects of different concentrations of SFI on the growth of LPS-damaged HUVEC,and the experimental groups were determined.4.The NO concentrations in HUVEC supernatant was detected by Griess method,and the influence of SFI on the m RNA expression of e NOS and i NOS in LPS-induced HUVEC was detected by Q-PCR.5.The effect of SFI on LPS-induced HUVEC cell apoptosis was detected by flow cytometry.6.Under fluorescence microscope,the mitochondrial membrane potential level of HUVEC was determined by RH-123 staining,and the reactive oxygen species level of HUVEC cells was evaluated by DHE staining.Results:1.HUVEC isolated from the umbilical cord was initially round or oval,and after1 d,it could fuse into loose monolayer cells and grow adherently,usually polygonal or short spindle.After 6 days,the cells were completely fused into a continuous monolayer,which was flat,polygonal or fusiform,with obvious nuclei,pebble-like Mosaic arrangement of cells with clear boundaries and no overlapping growth phenomenon.2.HUVEC 24 hours of LPS incubation at different concentrations(0-100(g/ml)showed LPS to cause VEC death,10?g/ml LPS reduced cell survival to 56.13%,and not significantly with increased concentration,so we selected a 10?g/ml LPS-induced VEC injury model for.3.Compared with the control group,the cell survival rate in the LPS model group was significantly lower(P<0.001).Compared with LPS model group,the survival rate of cells in low,medium and high dose SFI groups was significantly increased with the increase of dose(P<0.05),in a dose-dependent manner.4.Compared with the control group,NO content in LPS model was significantly increased(P<0.01);Compared with the LPS model group,the NO content in HUVEC supernatant in each dose SFI group was significantly decreased,with statistical significance(P<0.05).5.Compared with the control group,the m RNA expression of e NOS in LPS model group was significantly decreased(P < 0.01);Compared with the LPS model group,the m RNA expression of e NOS in the low-dose,medium-dose and high-dose SFI groups was significantly increased(P<0.05 or P< 0.01),and the m RNA expression of e NOS was up-regulated more obviously with the increase of SFI dose.6.Compared with the control group,the m RNA expression of i NOS in LPS model group was significantly increased(P<0.01);Compared with LPS model group,the m RNA expression of i NOS in low,medium and high dose SFI groups was significantly decreased(P<0.05 or P< 0.01),and the down-regulation of i NOS expression was more obvious with the increase of SFI dose.7.Scatter plot results showed that a small number of injured or early apoptotic cells were observed in the control group,and a large number of early apoptotic cells(Q4)and a small number of late apoptotic cells(Q2)were observed in the LPS model group compared with the control group(P<0.01).Compared with the LPS model group,the apoptosis rate was significantly decreased after being pretreated with low,medium and high doses of SFI(P<0.01 or P<0.05),and the apoptosis rate was the lowest in the medium dose group.The results showed that SFI significantly inhibited the damage of LPS to HUVEC and reduced the apoptosis rate of HUVEC cells.8.Compared with the control group,the yellow-green fluorescence was enhanced in the LPS model group,suggesting apoptosis of a large number of cells.Compared with the LPS model group,the yellow-green fluorescence decreased after low,medium and high dose SFI pretreatment,indicating a significant reduction in the number of cell apoptosis.Among them,the yellow-green fluorescence was the weakest after medium dose SFI pretreatment,indicating the least number of cell apoptosis.9.Compared with the control group,the red fluorescence in the LPS model group was stronger,indicating significantly higher levels of cell reactive oxygen species.Compared with the LPS model group,red fluorescence was weaker after low,medium and high dose SFI pretreatment,indicating that SFI reduced the level of cell reactive oxygen species.Moreover,with the increase of SFI dose,the ROS level of HUVEC cells decreased.Conclusions:1.SFI pretreatment significantly inhibited LPS-induced HUVEC injury and improved its survival rate,indicating that Shenfu injection produce a protective effect on LPS-induced HUVEC.2.SFI decreased the NO concentration in HUVEC supernatant,reduced the level of reactive oxygen species,and inhibited HUVEC apoptosis induced by LPS treatment,suggesting that the protective effect of Shenfu injection on LPS-induced HUVEC may be related to its regulatings NO production,reducting oxidative stress,and inhibiting HUVEC apoptosis.3.SFI up-regulated the m RNA expression of e NOS and down-regulated the m RNA expression of i NOS,suggesting that SFI may maintain the appropriate NO concentration through its regulating NOS/NO signaling pathway,thereby producing its protective effect on HUVEC induced by LPS.