Effect Of Shenfu Injection On Protecting Human Umbilical Vein Endothelial Cell From H₂O₂Oxidative Stress

Background and Objective: Thromboangiitis obliterans (TAO) is a chronicnonatherosclerotic segmental occlusive vasculitis with arterial lumen thrombosis,which most commonly involves the small-and medium-sized arteries and veins of theextremities and usually leads to gangrene and tissue loss. Its pathogenesis has notbeen fully elucidated until now.Evidences from Chinese and western medicinesuggest that there is a close relationship between TAO and damaged Vascularendothelial cells (VEC).Excessive oxidation is one of the main factors that causeVEC apoptosis, in which the morphology and function of VEC were damaged. It isthe most important reason to promote intravascular thrombosis of TAO.Shenfuinjection (SFI) which originated from Shenfu Tang, a traditional Chinese formulationrescuing the patients from collapse by restoring Yang, is mainly composed of theextract of radix ginseng and radix aconitum carmichaeli root. Our previous resultsfirst suggested that SFI can cause a significant therapeutic effect on experimentalTAO model rats by its inhibiting platelet aggregation and enhancing anti-thrombusfunction of vessel endothelia.Using a damaged VEC model induced by H2O2oxidative stress, the present experiments were designed to further invertigated theprotection effect and its molecular mechanism of SFI on the damaged VEC in vitro. Itwill provide scientific basis in the clinical application of SFI treating TAO disease.Methods:1. Human umbilical vein endothelial cell line (ECV304) which purchased fromShanghai institute of cell biology in Chinese academy of sciences, were cultivated invitro in present studies, using immunochemical staining and immunofluorescencestaining for identification.2. The cell viability was examined by MTT assay and the cell morphologicalteration was detected under optical microscope. Normal cultured ECV304cellswere incubated several time periods (1.5h,3h,6h,12h,24h,48h) with variousconcentrations of SFI (5,10,20,30,40,80,160μl/ml) respectively, these results wasemployed to screen the appropriate concentration and duration of SFI action on ECV304cells. Like this, the results that the normal cultured ECV304cells incubatedseveral time periods (3h,6h,12h,24h) with various concentrations of H2O2(50,100,200,300,400,800μmol/L) respectively, were used to determine the appropriateconcentration and time of the H2O2oxidative stress in ECV304cells.3.According to the above results of preliminary experiment,normal cultured24hECV304were randomly divided into the following experimental groups:(1) controlgroup；(2) H2O2oxidative stress model group(last concentration of H2O2is300μmol/L)；(3) low dose SFI group（20μl/ml SFI+300μmol/L H2O2）；(4) mediumdose SFI group（30μl/ml SFI+300μmol/L H2O2）；(5) high dose SFI group（40μl/mlSFI+300μmol/L H2O2）.In H2O2group, ECV304cells were firstly incubated withserum-free medium for3h, then incubated with300μmol/L H2O2for another3h. Incontrol group, ECV304cells were treated just like in H2O2group but H2O2werereplaced by PBS. In low, medium and high dose SFI groups, ECV304cells werefirstly incubated with20,30or40μl/ml SFI for3h respectively，then incubated with300μmol/L H2O2for another3h. Cell morphology was observed by invertedmicroscope, cell inhibition rate was detected by method of MTT, the activities ofSOD and GSH-PX and the content of MDA in the supernatants of the cultures wereassayed by colorimetry method, cell apoptosis was detected by Flow cytometry andAO/EB double staining methods. The protein expressions of Bcl2、Bax and Caspase-3were detected by Western blotting. The levels of TXB2and6-K-PGF1α were assayedby radioimmunoassay. The mRNA expression of TXAS was detected by RT-qPCR.Results:1.The appraisal results of immunohistochemical staining and immunofluores-cence staining showed that more than95%cells appeared be positive tan andgreenfluorescent particles in the cytoplasm,which confirmed that these cultured cells isVECs.2.(1) The concentrations of SFI (5,10,20,30,40μl/ml) which incubated withECV304cells for different time periods (1.5h,3h,6h,12h,24h,48h) failed to damageECV304cells (P>0.05); while80,160μl/ml SFI induced a traumatic morphologicalchange in the ECV304cells (P <0.05, P <0.01). Considering the effects of SFI onECV304cells and its clinical application, the concentrations of20,30and40μl/ml SFI incubated with ECV304cells for3h can reach for the appropriate conditions andwere used in these experiments.(2) the concentration of H2O2(50,100,200,300,400,800μmol/L) incubated with ECV304cells for different timeperiod(3h,6h,12h,24h) damaged ECV304cells in dose-and time-dependent manner.300μmol/L H2O2incubated with ECV304cells for3h was shown to be a suitablecondition to make a oxidative-stress damaged ECV304cells model with its50%cellular proliferation inhibition rate (P <0.01).3.(1)When compared with control group, abnormal morphological changes andlower survival rate of cells in ECV304cells was observed in H2O2group（P<0.01）；The low, medium and high dose SFI improved ECV304cells morphological changesand increased cell survival rate of ECV304cells under H2O2oxidative-stressconditions(P<0.05,P<0.01）.(2) The activities of SOD and GSH-PX were higher andthe content of MDA was lower in H2O2model group than those in control group.These effects of H2O2on SOD, GSH-PX activities and MDA content were reversedby the low, medium and high dose SFI（P<0.05,P<0.01）.4. According to Flow cytometry tests, we found that the ECV304cells apoptosisrate of H2O2model group was55.4%±1.28%, much higher than9.6%±0.401incontrol group(P<0.01),low,medium and high dose SFI pretreatment made the cellapoptosis rate reduced respectively to47.5%±0.654,41.8%±0.734、34.5%±0.92,therewere much different from H2O2model group(P<0.05,P<0.01).(2) AO-EB dublestaining results indicated that no apoptosis of ECV304cells in control group, thereare a large number of typical morphological changes of apoptosis presented inECV304cells by H2O2oxidative stress, however, fewer apoptosis morphologicalchanges existed in Low, medium and high dose SFI pretreatment ECV304cells.(3)Compared with the control group, the protein expressions of caspase-3and Bax inECV304cells of H2O2model group increased significantly, while the proteinexpression of Bcl2decreased distinly (P<0.01);when compared with H2O2modelgroup,the protein expression of caspase3of low dose SFI group has no obviouschange(P>0.05),but the protein expression of caspase-3in medium and high dose SFIgroups significant decreased(P<0.05); in addition,Bcl2protein expressions inLow,medium and high dose SFI groups were higher than H2O2model group, with Bax protein expression much lower than H2O2model group(P<0.05,P<0.01).5.(1)Compared with control group,the levels of TXB2and6-K-PGF1αin H2O2model group increased significantly（P<0.01,P<0.05）;When compared with H2O2model group,the values of TXB2and TXB2/6-K-PGF1α(T/K) in low,medium andhigh dose SFI groups were visibly reduced（P<0.01）,but only in high dose SFI group6-K-PGF1α decreased obviously（P<0.05）.(2) TXAS mRNA expression in H2O2model group is much more than the normal group(P<0.01);Compared with H2O2model group,TXAS mRNA expression were significantly lower in low,medium andhigh dose SFI groups（P<0.05,P<0.01）.Conclusions:1. The concentrations of SFI(5,10,20,30,40μl/ml) cause no damage onVEC,higher dose SFI(80,160μl/ml)can induce a traumatic morphological change inthe VEC.2. SFI plays a protective role in the damage of VEC caused by H2O2oxidativestress, whose protection mechanism has close relation to the improving activities ofSOD, GSH-px and reducing MDA content as well as decreasing membrane lipidperoxidation of VEC.3. SFI can inhibit the VEC apoptosis induced by H2O2oxidative stress, itsmechanisms underlie improving the survival rate of VEC, raising the antiapoptoticBcl2protein’s expressions, and reduce the expressions of apoptosis protein Bax andCaspase3in the VEC.4. SFI can protect the VEC anticoagulant activity from H2O2oxidative stress,itsunderlying mechanism involved in reducing TXB2level,balancing TXB2/6-K-PGF1α ratio and downregulating TXAS mRNAexpression.5.Our findings suggested that the SFI could prevent and cure TAO by enhancingthe VEC antioxidant enzyme activities,reducing the membrane lipid peroxidation;improving the survival rate of VEC, upregulating antiapoptotic proteins and downreg-ulating apoptosis protein expressions;reducing the mRNA expressions of VECTXAS,balancing TXB2/6-K-PGF1α to protect its anticoagulant activity and so on.