Inhibitory Effects of Crocetin on High Glucose-induced Apoptosis in Cultured Human Umbilical Vein Endothelial Cells and Its Mechanism
|Keywords||High-sugar Endothelial cells Apoptosis Crocetin|
BackgroundCardiovascular complications are the major cause of morbidity and mortality in patients with diabetes mellitus.The mechanism of diabetic complications may be associated with intracellular formation of AGEs,polyol pathway flux,activation of protein kinase C,flux through the hexosamine pathway and oxidative stress,etc. Endothelial dysfunction is a pivotal early event in diabetic atherosclerosis,and cell apoptosis plays an important role in endothelium dysfunction.Cell apoptosis,also named programmed cell death,is a kind of physiological or pathological death process different from the cell necrosis,involving a series of activation,express and modulation of related genes.Emerging evidences suggest that high glucose would induce generation of reactive oxygen species(ROS)in endothelial cells,which can cause cellular dysfunction,and even cell death.Many recent studies suggested that glucose induced apoptosis of human umbilical vein endothelial cells.Therefore,prevention of glucose-mediated endothelial cell apoptosis may be effective on preventing diabetes-associated vascular complications by pharmacological method.It is well known that through activation of the downstream serine/threonine kinase Akt,PI-3K plays an important role in preventing cell death produced by many death stimuli.Akt activation has been reported to promote endothelium cell survival. Akt can activate endothelial nitric oxide synthase(eNOS),and thereby results in nitric oxide(NO)production,which prevents superoxide anion from forming its dismutation product.Therefore,PI-3K/Akt/NO pathway plays a key role in preventing ROS-induced endothelial cell injury.Nuclear factor-κB(NF-κB)is an important regulator factor of transcription,and participates in various important processes,including inflammatory response,cell proliferation,and atherosclerosis pathogenesis.Several lines of evidence indicated that high glucose would induce NF-κB activation in endothelial cells,mesangial cells, vascular smooth muscle cells and monocytic cells.Recent studies found that NF-κB activation can down-regulate proapoptotic JNK signaling,and thus preventing cell apoptosis.However,in endothelial cells,NF-κB activation has been found to produce cell apoptosis instead of preventing the cells from apoptosis.ROS-dependent activation of NF-κB plays a major role in cell apoptosis induced by high glucose. Therefore,the antioxidant may play a beneficial effect on ROS-mediated apoptosis through the inhibition of NF-κB pathway.Crocetin,an extract from Crocus sativus L.and Gardenia jasminoides Ellis, has antiatherosclerosis,strong antioxidation and effect of protection on myocardial cells,etc.Recent research found that crocin(an analog of crocetin)inhibited human endothelial cell apoptosis induced by trisol and prevented endothelial dysfunction. Other studies showed that crocetin,possibly through intracellular ROS inhibition and [Ca2+]i stabilization,prevented the bovine aortic endothelium cell apoptosis induced by OX-LDL or AGEs(advanced glycation end products,AGEs).However,whether crocetin inhibits HUVECs remains unclear.These findings called us to test the hypothesis that crocetin may exert a beneficial effect in preventing glucose-induced endothelial cell apoptosis through the PI-3K/Akt/eNOS pathway and/or NF-κB pathway.In the present study,we use the cultured HUVECs in vitro to investigate the antiapoptotic effect of crocetin and its possible mechanism to provide theoretic evidences for application of crocetin on diabetes patients.Part 1.Inhibitory effects of crocetin on high glucose-induced apoptosis in cultured human umbilical vein endothelial cellsObjetive:To observe the inhibitory effect of crocetin on high glucose-induced apoptosis in HUVECS.Methods:Human umbilical vein endothelial cells(HUVECs)were obtained by collagenase treatment of umbilical cord veins.These cells were tested positive for factorⅧantigen by immunocytochemical examination.HUVECs were incubated at 37℃in a humidified atmosphere of 95%air-5%carbon dioxide in RPMI-1640 medium supplemented with 10%FBS.HUVECs in passage 3 to 5 were used for experiments. In experiments,HUVECs cultured in normal glucose(5.5 mM)served as normal control group(NG group).HUVECs were incubated with high glucose(15,33 mM, HG group)for different intervals(24-48 h).To observe the effect of crocetin on high glucose-mediated apoptosis,HUVECs were pretreated with crocetin(0.01,0.1 and 1.0μM;C1 group,C2 group,C3 group,respectively)for 18 h before incubation with high glucose(33 mM).HUVECs apoptosis was evaluated by cell chromatin staining with Hoechst 33258,cell death detection and caspase-3 activity examination with ELISA.The identification of HUVECs and the morphological assessment of apoptotic endothelial cells:Human umbilical vein endothelial cells(HUVECs)were obtained and tested positive for factorⅧantigen with immunocytochemical examination. HUVECs growing up and being adherent in culture flask showed typical appearance of cobblestone.In apoptotic cells,nuclear condensation and chromatin fragmentation as one of the most important and specific morphological change of cell apoptosis was shown by Hoechst 33,258 under phase contrast microscope and fluorescence microscope.In contrast,nuclei of normal HUVECs stained light and homogeneous without nuclear condensation and chromatin fragmentation.Results:Compared with normal glucose,morphologically,much more nuclei in HUVECs after exposure to high glucose(15,33mM)showed chromatin condensed,and multiple chromatin fragments were shown by Hoechst 33,258 under phase contrast microscope and fluorescence microscope.Results showed high glucose induced apoptosis in a dose-dependent manner,and apoptosis was not evident until 36 h of high glucose treatment.Pretreatment of crocetin(1.0μM)made the chromatin not even bright and fewer chromatin fragments were seen as compared to high glucose treatment,which showed that crocetin inhibited high glucose-induced apoptosis.Quantitatively,to determine high glucose-induced apoptosis in HUVECS and inhibitory effect of crocetin,the cell death assay kit and ELISA for caspase-3 activity were used.Results of the cell death assay showed high glucose-induced apoptosis was markedly increased at 36 h and 48 h(vs.HG at 0 h or at 24 h,p＜0.01),and high glucose induced apoptosis in a time-dependent manner(apoptosis at 48 h compared with at 36 h,p＜0.05);however,crocetin(1.0μM)pronouncedly inhibited high glucose induced-apoptosis compared with the high glucose treatment(p＜0.05,at 36h; p＜0.01,at 48h).Furthermore,the antiapoptotic effect of crocetin increased with the increase of concentration of crocetin(crocetin 1.0μM vs.crocetin 0.1μM,p＜0.05), indicating its dose-dependent manner of antiapoptosis.Crocetin(0.01μM)showed no significant difference compared with high glucose group.The high-glucose induced increase of caspase-3 activity was also decreased by crocetin,being similar to the findings described above.Conclusions:1.High glucose for 36-48h may induce a pronounced increase of apoptosis in a dose-dependent manner compared with normal glucose(5.5mM)。2.Crocetin inhibits high glucose-induced HUVECs apoptosis in a dosedependent manner.Part 2.Mechanism involved in the inhibitory effects of crocetin on high glucose-induced apoptosis in cultured human umbilical veinendothelial cellsObjective:To explore the mechanism involved in the inhibitory effect of crocetin on high glucose-induced apoptosis in HUVECs.Methods:HUVECs cultured in normal glucose(5.5 mM)served as normal control group (NG group).HUVECs were incubated with high glucose(33 mM,HG group)for 48h. To observe the antioxidant effect of crocetin on high glucose- induced apoptosis, HUVECs were pretreated with crocetin(1.0μM)for 18 h before incubation with high glucose,then the MDA and SOD content in medium and ROS in HUVECs was evaluated.To further determine the effect of crocetin on the role of the PI-3K/Akt/eNOS or NF-κB/JNK pathway,other groups of HUVECs were pretreated with PI-3K inhibitor LY294002(10μM),or an eNOS inhibitor NG-nitro-arginine methyl ester(L-NAME or LN;100μM)or NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC,10μM)for 30 min before crocetin was added.The cells were harvested at the predetermined time to evaluate cell apoptosis by cell death ELISA kit and the activation of Akt and eNOS was determined by western blot. Similarly,to explore the effect of NF-κB/JNK pathway,IκB and p-JNK protein was determined by western blot,and NF-κB activity was evaluated by electrophoretic mobility shift assays(EMSA).The effect of exogenous and endogenous NO on activity of NF-κB was evaluated to explore the relation between the two pathways of NF-κB/JNK/Caspase-3 and PI-3K/Akt/eNOS.Results:Crocetin markedly increased SOD content,decreased MDA content in medium and depressed ROS level in HUVECs compared with high glucose treatment,showing strong antioxidant effect.Exposure to high glucose(33mM)for 48h induced a pronounced increase in apoptosis compared with normal glucose(5.5mM).Exposure to high glucose for 48h attenuated activation of Akt and endothelial nitric oxide synthase(eNOS);Also,high glucose suppressed the expression of IκB protein, enhanced the NF-κB activity and increased the expression of p-JNK protein. Pretreatment with crocetin for 18 h up-regulated the activation of Akt,eNOS and increased NO production;however,pretreatment with PI-3K inhibitor LY294002 or eNOS inhibitor L-NAME inhibited crocetin’s effect on p-Akt or eNOS and decreased the protection of crocetin from apoptosis,respectively.As for NF-κB/JNK,high glucose markedly increased the activity of NF-κB,followed by the increase of p-JNK and Caspase-3;NF--κB inhibitor PDTC(10μM)significantly suppressed protein expression of p-JNK and Caspase-3 activity;crocetin increased the expression of IκB, inhibited NF-κB activity and down-regulated p-JNK protein expression.Both exogenous and endogenous NO markedly inhibited the activity of NF--κB,showing the existence of feed-back of PI3K/Akt/eNOS to NF-κB/JNK/Caspase-3 pathway.Conclusions:1.High glucose-mediated HUVECs apoptosis is through ROS-dependent NF-κB /JNK/Caspase-3 activation and Akt/e-NOS/NO inactivation.2.Crocetin has strong antioxidant activities.3.For the first time,we find crocetin inhibits high glucose -induced apoptosis,at least partly,via the enhancement of Akt/eNOS/NO and the inhibition of NF-κB/JNK/Caspase-3 in HUVEC.Furthermore,crocetin has an effect of feed-back inhibition on NF-κB/JNK/Caspase-3 pathway through PI3K/Akt/eNOS pathway.4.This study may have important theoretical and practical implications for the research and development of new medicine in preventing and treating diabetesassociated cardiovascular complications.Innovative points and significanceThe present study found that high glucose may induce apoptosis in HUVECs,in which ROS plays a pivotal role.Crocetin,an extract from Gardenia jasminoides Ellis, have has strong antioxidant activities.Crocetin may block NF-κB,followed by the decrease of JNK/Caspase-3 activity;and crocetin enhances the pathway of Akt/eNOS/NO,by which,at least partly,crocetin inhibits the glucose-induced apoptosis in HUVECs.This study may have important implications for the research and development of new medicine in preventing and treating diabetes-associated cardiovascular complications.