| BACKGROUNDIslets are highly vascularized micro-organs,islet microvascular endothelial cells (IMECs),which not only have general nature,such as angiogenesis,the role of hemostasis,but also feedβcells,the secretion functions of growth and development of signal to islet cells,are the main components of the vascular endothelial cells. Previous studies have proved that the depletion of beta cells is much more likely the representation of islet capillary disease.And an excess of islet microvasular endothelial cells apoptosis plays an important role in islet capillarys'disease.In normal situation,the proliferation and apoptosis of microvasular endothelial cells is in a dynamic balance,but for the type 2 diabetes patients who are in chronic low-grade sub-clinical inflammatory all time especially acute infection,the start of inflammatory cascade in short time can result in the apoptosis of microvascular IMECss,the disturbance of balance,the restraint growth of microvasulars,eventually causeβcell damage.Therefore,the in-depth study of islet endothelial cells in pathophysiological changes in the mechanism,and the establishment of an effective way to block the damage pathway,is of positive significance to protect islet cells and prevent diabetes.Inflammatory etiology theory has drawn great attention in recent years.The theory is that type 2 diabetes is a low-grade inflammatory disease.Inflammation has become an important pathogenesis of T2DM,the relationship between the international research inflammation and T2DM has become a hotspot in the past decades.Some evidence has proved that adipose tissue secretion dysfunction played an important role in inflammation-insulin resistance-diabetes -2 pathophysiological processes.Inflammatory cytokines TNF-α(tumor necrosis factor alpha,TNF-α),one of the fat-derived cytokines,is considered to participate in insulin resistance(IR) and eventually lead to type 2 diabetes.TNF-αis a cytokine which has broad biological activity.TNF-αmay induce apoptosis directly or indirectly,it is considered that the Cytological effect of TNF-αis mediated mainly by TNFR1.TNFR1 activates the apoptosis pathway,leading to cell apoptosis.The expression of TNF-αreceptor in vascular endothelial cell surface makes the vascular endothelial cells the target cells of TNF-α.In primary cultured bovine vascular endothelial cells,adding TNF-αlead to many cells apoptosis,and that there is dose-time-effect relationship.At T2DM such a low chronic inflammatory state,for the patients who suffer from microvascular lesions,their apoptosis of vascular endothelial cells is closely related to the high serum tumor necrosis factor. The injured endothelial cells have enhanced release of IL-1 and TNF-α,aggravated the injury in vascular endothelial cells again,both the effects are in a vicious cycle.In addition,scholars have found that TNF-αcause pancreaticβ-cell apoptosis through such as ways to the death receptor,oxidative stress.Research has found that the vascular endothelial cell apoptosis caused by TNF-αand is related to caspase-8 and caspase-3.Followed by reports from Chang and others,TNF-αcan produce the same biological effects in mitochondrial pathway and activates caspase-9,3,7.The research above indicate that TNF-αcan lead to the endothelial cells withβ-cell injury or apoptosis in a variety of ways.A lot of experiments showed that addition of RNA synthesis inhibitors Actinomycin D(ActD) or protein synthesis inhibitors cycloheximide makes the cells which aren't sensitive to TNF-αoriginally appeared apoptosis and the sensitive ones appeared stronger apoptotic effect.In order to observe the impact of inflammatory cytokines TNF-α,actinomycin D/TNF-αon IMECss,expression of TNFR1 on IMECss,as well as the mechanisms blocking apoptosis pathway.Based on the establishment of mouse IMECss apoptosis model,we take the IMECss as object, explore the relationship of inflammatory cytokines and apoptosis of IMECss,with a view to provide new evidence to the prevention and treatment of diabetes.OBJECTIVEStudy the expression of tumor necrosis factor type 1 receptor(TNFR1) in mice IMECss,and assay the stimulation to IMECss induced by TNF-α,ActD/TNF-αand its possible mechanism of injury,in order to provide intervention with diabetes and finding a new therapeutic target with pilot evidence.METHODS1.Chose the American Type Culture Collection,MS-1 cells as our experimental object than culture and induce apoptosis.Culture MS-1 cells in the DMEM high glucose medium that contain 5%newborn bovine serum,the condition of the evidence is 37℃,5%CO2.The cells grow adherent to the wall,when they covered it, digest they with 0.25%pancreatin and subcultures every 3 days.2.Cells climb and immunofluorescence:compound MS-1 cell suspension with 5%newborn bovine serum cells,inoculate cells in 6-well plates until the cell grow well.Rinse slides with PBS for 30minitus after 4%paraformaldehyde fixed.The cells was blockade with normal goat serum at working fluid followed 0.1%Triton X-100,drop the serum and add drops of rabbit polyclonal bodies against mouse TNFR1(TNF1-R),overnight add drops of goat anti-rabbit IgG,incubated at room temperalure for 2 hours.After mount observe the cells under the fluorescence microscope and photographed.3.TEM observation:The cells were randomly divided into 3 groups:①normal control group,②TNF-αgroup(TNF-αfinal concentration is 100ng/ml,the concentration within the following brackets refers to the final concentration),③ActD/TNF -αgroup(add TNF-αafter pretreatment 20ng/ml ActD for 15min).After 24 hours of drug stimulation,cells are digested with 0.25%pancreatin,rinsed with PBS,centrifuged and discarded the supernatant followed by fix with 4%,PH7.4 glutaraldehyde.Dual-lead stain under transmission electron microscope for observation.4.Cytotoxicity experiment divided into two parts:the cells are planted in 96-well plates,each well 100μl,cultivated in the condition of 37℃,5%CO2 for 24 hours,each eight-hole complex.After stimulation of different concentrations of TNF-α(each hole add 100μl) for 48 hours,add MTT solution(5g/l)20μl to each hole, and then incubate them in 37℃for 4 hours,absorb the medium and add 150μl DMSO to each hole,vibrate for 10minites,570nm wavelength absorbance measurements to determine cell viability.Meanwhile,set up blank holes based only add culture medium without cells,regulate to zero based on blank cells while comparing the colors.The colorimetric absorbance measured by MTT automated colorimetric assay reflects damage of the cells.4.1 Cytotoxic effect of TNF-αin vitro on MS-1Stimulate the MS-1 with different concentrations of TNF-α,and observe growth. Experimental Packet:control group,which cells without any treatment;TNF-αgroup by adding different concentrations of TNF-α(20,40,80,100 ng/ml);ActD/TNF-αgroup which add the corresponding concentrations of TNF-αin formal group followed by 20ng/ml ActD.24 hours later,test cytotoxicity by MTT assay.4.2 Ac-DEVD-CHO antagonist the effect of cytotoxicity on the IMECs induced by ActD/TNF-α.Test the effect of antagonism on the IMECs induced by Ac-DEVD-CHO. Experimental Packet:A group for the control group,cells without any treatment;B group add TNF-α(100ng/ml) only;C add TNF-α(100ng/ml) after the pretreatment of ActD(20ng/ml);D add Ac-DEVD-CHO(200μmol/L) 15min after the pretreatment of ActD / TNF-α,24 hours later,test each group's cytotoxicity by MTT assay.5.Test apoptosis by flow cytometry:The cells are planted in 6-well plates,and the test packet:TNF-αgroup,add only TNF-α(100ng/ml);Ac-DEVD-CHO group only add Ac-DEVD-CHO(200μmol / L);ActD/TNF-αgroup add TNF-α(100ng/ml) after the pretreatment of ActD(20ng/ml);ActD/TNF-α/Ac-DEVD-CHO group is divided into 3 groups,adding Ac-DEVD-CHO of different concentrations (50,100,,200μmol/L) respectively 15 minutes before adding ActD/TNF-α;control group for the cells without any treatment.After the 24-hours stimulation,digest each group with 0.25%pancreatin,rinse with ice PBS and then stain cells by adding Annexin V-FITC and PI,test them after the dark incubation at room temperature 10 min.Test 5 samples each experimental group,Cell fit software collect 10 000 cells, and the percentage of apoptosis is the Annexin V-FITC positive percentage.RESULTS1.After immunofluorescent TNFR1 in MS-1 cells membranes showed a strong yellow-green fluorescence,while the control ones is negative.2.Observation under the TEM shows that the control group does not instinct differ from TNF-αone in morphology,most the majority are normal cells.ActD/ TNF-αgroup for many apoptotic cells,which are vacuolated,shrink significantly,the cell structure and morphology are in chaos,endoplasmic reticulum and membrane merge and budding,clear nuclear membrane and a superfluous of apoptosis body.3.With 20,40,80,100 ng/ml TNF-αacting on the MS-1 cells for 24 hours,the results show that in 20,40,80,100 ng/ml TNF-αconcentrations,ActD/TNF-αgroup absorbance(OD value) were 0.42±0.04,0.31±0.04,0.20±0.06,0.09±0.06 respectively, and OD value of TNF-αgroups of the same concentrations of TNF-αwere 0.50±0.05,0.48±0.05,0.46±0.07,0.45±0.05 respectively.The former ones were significantly lower than the latter ones(in 20ng/mlTNF-αgroup,P=0.015;40,80,100 ng/ml TNF-αgroup,P=0.000).Control groups with OD value of 0.50±0.05,were significantly higher than those of ActD/TNF-αgroups with different concentrations (compare with 20ng/ml ActD/TNF-αgroups,P=0.025;compare with 40,80,109ng/ml ActD/TNF-αgroups,P=0.000).4.Comparison of four ActD/TNF-αgroups with different concentrations of TNF-α,shows a significant negative correlation between the OD value of MS-1 cells, which is sensitized by ActD,and the concentration of TNF-α(r=-0.923,P=0.000). There exists interaction effects between concentration of TNF-αand interfere factors (F=12.083,P=0.000). 5.In the study of Ac-DEVD-CHO antagonis cytotoxicity on the MS-1 cells induced by ActD/TNF-α,showing that after incubation for 24 hours,the of TNF-αgroup,with the OD value of 0.452±0.063,and the Ac-DEVD-CHO/ActD/TNF-αgroup,with the OD value of 0.438±0.083,compared with control group respectively, showing no significant difference(P=0.618,0.620).The ActD/TNF-αgroup,with the OD value of 0.175±0.110,significantly lower than the control group(P=0.000) and TNF-αgroupas well(P=0.000).6.The study of Ac-DEVD-CHO protect the IMS-1 cells from apoptosis which induced by ActD/TNF-α,showing that the apoptosis rate of Ac-DEVD-CHO group, TNF-αgroups were(11.13±1.13)%,(11.39±1.09)%respectively.The two groups compared with the control group,showing no significant difference(P=0.550,0.361). The apoptosis rate of ActD/TNF-αgroup and Ac-DEVD-CHO/ActD/TNF-αgroups with 50,100,200μmol/LAc-DEVD-CHO were(49.25±1.43)%,(28.56±1.44)%, (25.03±1.75)%,(18.26±0.64)%respectively.The apoptosis rate of ActD/TNF-αgroup,Ac-DEVD-CHO/ActD/TNF-αgroups with different concentration are significantly were higher than the control group significantly(P=0.000).7.Compared the apoptosis rate of ActD/TNF-α/Ac-DEVD-CHO groups three different concentrations,we found that there existing a significant negative inter-relationship between the apoptosis rate of MS-1 cells and the concentration of Ac-DEVD-CHO(r=-0.946,P=0.000).CONCLUSION1.TNF-αreceptor(TNFR1) were observed on the mouse islet microvascular endothelial cell line MS-1 for the first time,ActD/TNF-αinduced MS-1 to establish the apoptosis model of IMECs.2.Under only TNF-α,morphological changes did not occur in MS-1 cells,but after ActD pretreatment this occur.With a certain concentration range(20～100ng/ ml) TNF-α,The cell viability of MS-1 cells gradually decreased with the increase of TNF-α,and showed dose-effect relationship in a certain range.This suggest that under the condition of IMECss being sensitized or others,inflammatory cytokines TNF-αcan decrease the cell activity of MS-1,and even trigger apoptosis.3.Ac-DEVD-CHO can significantly antagonize apoptosis of MS-1 induced by ActD/TNF-α,and decrease the occurrence of apoptosis.Moreover,the antagonism was enhanced with the increase of Ac-DEVD-CHO dose,suggesting that ActD/ TNF-α-induced apoptosis in MS-1 is mediated by caspase-3.
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