3.4 GA protects MIN-6 cells from apoptosis under h...
Figure 3 GA I induces autophagy in MIN-6 cells under high glucose stress
(a)MIN-6 cells were cultured with or without 33.3mmol/L glucose，10mmol/L 3-MA，33.3mmol/L glucose+10mmol/L 3-MA，5μg/mL GA I，33.3mmol/L glucose+5μg/mL GA I，33.3mmol/L glucose 5μg/mL GA I+10mmol/L 3-MA for 36 hr and stained with an anti-LC3 antibody(scale bar=5μm).(b)Cells were treated as(a)and the umber of autophagosome per cell was counted.At least 30 cells were analyzed and three repeats were performed.**p＜0.01 and*p＜0.05，compared with the control group by one-way analysis of variance；##p＜0.01，compared with the high glucose plus GA I group by Student's t test.(c)Cells were treated as(a)and total cell extracts wereimmunoblotted with antibodies against LC3 and GAPDH.(d)MIN-6 cells were cultured with or without 33.3mmol/L glucose，5μg/mL GA I，33.3mmol/L glucose+5μg/mL GA I for 36 hr and total cell extracts were immunoblotted with antibodies against mTOR or phospho-mTOR.(e)MIN-6 cells were cultured with 33.3mmol/L glucose，5μg/mL GA I+33.3mmol/L glucose，33.3mmol/L glucose+50μM chloroquinine，33.3mmol/L glucose+50μM chloroquinine+5μg/mL GA I for 36 hr and total cell extracts were immunoblotted with antibodies against LC3 or GAPDH.GA I：gymnemic acid I；GAPDH：glyceraldehyde 3-phosphate dehydrogenase；LC：light chain［Color figure can be viewed at wileyonlinelibrary.com］
Our previous results showed that GA I treatment decreased the apoptosis rate ofβcells under high glucose stress(from 17.37%to 10.5%，Figure 1b).When 3-MA was added，the apoptotic rate ofβ-cells was increased from 9.93%to 32.58%(Figure 4a).The activity of apoptosis-related proteins caspase 3 was consistent with these results(Figure 4b).It indicated that GA I protected MIN-6 cells from apoptosis under high glucose stress(Figure 4b).without 33.3mmol/L lucose，33.3mmol/L glucose+10mmol/L 3-MA，33.3mmol/L glucose+5μg/mL GA I+10mmol/L 3-MA for 36 hr and the activity of caspase-3 was tested.Error ars，SD；**p＜0.01 compared with control cells by one-way analysis.At least three replicates were performed.FITC：fluorescein isothiocyanate；GA I：gymnemic acid I［Color figure can be viewed at wileyonlinelibrary.com］(https://www.daowen.com)
Figure 4 GA I protects MIN-6 cells from apoptosis under high glucose stress
(a)The MIN-6 cells were treated with 33.3mmol/L glucose，33.3mmol/L glucose+10mmol/L 3-MA，33.3mmol/L glucose+5μg/mL GAⅠ，33.3mmol/L glucose+10mmol/L 3-MA+5μg/mL GA I.The apoptosis rate of MIN-6 cells is tested by flow cytometry.(b)MIN-6 cells were ultured with or without 33.3mmol/L lucose，33.3mmol/L glucose+10mmol/L 3-MA，33.3mmol/L glucose+5μg/mL GA I+10mmol/L 3-MA for 36 hr and the activity of caspase-3 was tested.Error ars，SD；**p＜0.01 compared with control cells by one-way analysis.At least three replicates were performed.FITC：fluorescein isothiocyanate；GA I：gymnemic acid I［Color figure can be viewed at wileyonlinelibrary.com］