Astrgaloside ? Protects High Glucose Induced Cell Inflammatory Reaction Through The Inhibition Of The JNK Pathway In Human Umbilical Vein Endothelial Cells

Objective Through the establishment of in vitro high glucose induced human umbilical vein endothelial cells(HUVECs)injury model,the dissertation researches the protective effect of astragalside IV(AS-IV)on high glucose-induced HUVECs injury and its molecular mechanism,and provides experimental basis and new ideas for the prevention and treatment of diabetes mellitus and its complications.Methods1 Establishment of human umbilical vein endothelial cell model in vitro with high glucose.Culture HUVECs cell lines in vitro.According to the medium concentration of glucose concentration,the experiments are randomly divided into two groups: the normal control group(glucose concentration 5.55 mM),high glucose injury group(glucose concentration are 11.1,22.2,33.3,44.4 mM).The mannitol balances osmotic pressure between different groups.The cell morphology is observed by optical upside down microscope at 24 h,48h and 72 h after incubation.MTT assay is used to detect the cell viability.Hoechst 33258 staining is used to observe the apoptotic morphology.2 Protective effect of AS-IV on high glucose-induced inflammatory injury in human umbilical vein endothelial cells.The experiment is divided into five groups: The normal control group(glucose concentration 5.55 mM 48h);high glucose model group(glucose concentration 33.3mM 48h);JNK inhibitor group(glucose concentration 33.3 mM 48 h after adding JNK inhibitor SP600125 concentration 20 ?M);AS-IV group(glucose JNK inhibitor +AS-IV group(glucose concentration 33.3 mM 48 h,JNK inhibitor SP600125 concentration of 20 ?M pretreatment 2 h by adding 50 ?M AS-IV),The mannitol balance osmotic pressure between different groups.After incubation for 24 h,cells are harvested and Hoechst 33258 staining is used to observe the apoptotic morphology.Flow cytometry is used to detect the apoptotic rate.The levels of TNF-? and IL-1? in supernatant of HUVECs are detected by ELISA.The expression of TNF-? and IL-1? mRNA in HUVECs is detected by real-time PCR.The expression of JNK,p-JNK,ASK1,p-ASK1,Cyt-c,Caspase-3 and Caspase-9 protein in HUVECs are detected by Western blot.Results1 Establishment of human umbilical vein endothelial cell injury model induced by high glucose in vitro.(1)Effects of different concentrations and glucose on morphology of HUVEC at different times showe that: HUVECs were cultured in vitro.After 24,48 and 72 hours of culture,the cells in the normal control group are adherent and arranged in different sizes.The samples are similar in size and are in the form of pebbles.The edges are clear and the cells are interconnected.The nuclei are round or elliptical shape,located in the center of the cell.In the high glucose injury group,with the increase of glucose concentration,the cell morphology is obviously damaged,the disorder is disordered,the size of the nucleus increased,the aspect ratio and the ratio of nuclear to nuclear.When the glucose concentration is more than 33.3mM,the culture time is more than48 h,the cells appear different degrees of disorder,swelling round or fusiformity,the edge of the cell membrane is blurred,part of the cell membrane is not complete,damage rupture.(2)Effects of different concentrations of glucose on the viability of HUVECs cells at different concentrations show that: HUVECs culture in different concentrations of glucose for 24,48,72 h,with the gradual increase in glucose concentration,the cell survival rate decreases gradually,and shows time and concentration-dependent.(3)Hoechst 33258 staining was used to observe the apoptosis,The results show:In the normal control group,the nucleus of the glucose group is dim and blue,the nucleus is intact and the chromatin fluorescence is homogeneous.With the increase of glucose concentration,some nuclei shows concentrated fragments,the fluorescence shows blue and white dense dense staining,a small number of nuclei can be seen in nuclear chromatin condensation of apoptotic bodies,apoptotic damage morphology shows glucose concentration-dependent.2 Effect of AS-IV on Apoptosis of HUVECs Induced by High Glucose.(1)Effects of different concentrations of AS-IV on cell viability are shown: In a certain concentration range,with increasing concentrations of AS-IV has promote proliferation of HUVECs;when the concentration of AS-IV is 50 ?M,which promotes the most significant effect(P <0.01),when a high concentration of AS-IV At 50 u M,its promoting effect gradually weakened.(2)Effect of AS-IV on Apoptosis of HUVECs Induced by High Glucose,the results show: After high glucose treatment,HUVECs cells and nuclei show significant nuclear pyknosis,dissolution,fragmentation and other apoptotic characteristics.Hoechst 33258 stained HUVECs apoptotic cell morphology,showing that AS-IV can reduce the high glucose-induced apoptosis.Flow cytometry further validates the effect of AS-IV on the apoptosis rate of HUVECs induced by high glucose and the results of Hoechst 33258 staining.(3)Effects of AS-IV on TNF-? and IL-1? Contents in Supernatant of HUVECs in High Glucose Environment,the results show: Compared with the normal control group,the levels of TNF-? and IL-1? in the supernatant of the model group are significantly increased(P <0.01).The levels of TNF-? and IL-1? in supernatant of AS-IV group are significantly lower than those in model group(P <0.01).(4)Effects of AS-IV on TNF-? and IL-1? mRNA Expression in HUVECs Induced by High Glucose,the results show: Compared with the normal control group,the mRNA expression of TNF-? and IL-1? in model group HUVECs increased.Compared with the model group,the mRNA up-regulation of TNF-? and IL-1? in AS-IV group decreased(P <0.01).3 Protective Mechanism of AS-IV on High Glucose-induced Inflammatory Injury of HUVECs by JNK Pathway.Western Blot results show that: Compared with normal control group,high glucose model group,p-JNK,p-ASK1,Bax,Cyt-c,Caspase-9,Caspase-3 expression is significantly increased(P <0.01),Bcl-2 expression is significantly decreased(P<0.01),P-JNK/JNK,p-ASK1/ASK1,Bax /Bcl-2 increase(P <0.01).Compared with the high glucose model group,the expression of p-JNK,Bax,Cyt-c,Caspase-9 and Caspase-3 are decreased(P <0.05 or P <0.01),P-JNK/JNK,Bax/Bcl-2 values decrease.(P <0.05 or P <0.01).The expression of p-JNK,p-ASK1,Bax,Cytase-9 and Caspase-3 are decreased(P <0.05 or P <0.01),p-JNK / JNK,p-ASK1 / ASK1,Bax/Bcl-2 decreased(P <0.05 or P <0.01).AS-IV inhibitor group is the same results as AS-IV group.Compared with the inhibitor group,the expression of p-JNK in AS-IV group is higher than that in the inhibitor group(P <0.01)Conclusions1 In high glucose condition,the survival rate of HUVECs in vitro is decreased and the apoptosis is increased,and the time and concentration are decreased.Glucose concentration of 33.3mM for 48 hours may be HUVECs in vitro to establish a high glucose injury model of the appropriate experimental conditions.2 High glucose environment cell survival rate decreases,apoptosis increases.Inflammatory cytokines TNF-? and IL-1? gene expression and protein secretion increase.The mechanism may be related to the activation of JNK pathway in HUVECs by high glucose,the promotion of cell inflammatory response and induction of apoptosis.3 AS-IV has a protective effect on high glucose-induced HUVECs.The mechanism may be related to inhibition of JNK signaling pathway overexpression,reduction of cellular inflammatory response and reduction of apoptosis.