Construction Of Diabetes Drug Screening Model And Protective Effect Of Hypericin On Pancreatic β Cells

PurposesTo establish an anti-diabetes drug screening model which used intracellular free calcium concentration as an indicator.To establish perfused dual-phase insulin secretion model by using islet tissues.To explore the potential role of hypericin on β-islet cells through investigating its impact on intracellular free calcium,insulin secretion and cell viability of β-islet cells,as well as studying its protective effect on high-glucose and high-fat induced damage on β-islet cells.Methods1.Intracellular calcium modeling:INS-1 cells was stimulated with high glucose and high potassium following incubation with the fluorescent probe fura 2-AM and then intracellular free calcium real-time concentration curve was measured with BD-pathway 855 HCS instruments.2.Perfused dual-phase insulin secretion model:Fresh pancreases were first isolated from C57 mice and then were shredded to get pancreatic islets through collagenase digestion.Discreted islets were put into hand-made perfusion machine and pretreated with low-glucose solution for 30min followed by sequential perfusion with low-glucose solution(3mM)for 10min and high-glucose(33mM)solution for 20min.Insulin levels of effluent per minute were detected by ELISA,rendering the pancreatic islets perfusion curve.3.Hypericin’s effect on pancreatic β cells(1)Effect on intracellular free calcium influx:INS-1 or RIN-m5f islet β cells were incubated in culture medium containing 20nM,200nM or 2μM hypericin for 24h,and then treated with high potassiμm solution to detect the change of intracellular free calcium.(2)Effect on cell viability:INS-1 or RIN-m5f islet β cells were incubated in culture medium containing 20nM,200nM or 2μM hypericin for 24h followed by cell viability measurement by using MTT.(3)Glucotoxicity protection experiments:INS-1 or RIN-m5f islet β cells were incubated in culture medium with normal glucose(17mM),high glucose(33mM)or high glucose plus hypericin(20nM,200nM or 2μM)for 72h and then cell viability was measured using MTT.(4)Lipotoxicity protection experiments:INS-1 or RIN-m5f islet β6 cells wereincubated in culture medium containing 0μM,100μM,200μM or 300μM palmitate(PA)with or without 20nM,200nM or 2μM hypericin for 24h and then cell viability was detected using MTT.(5)Effect on insulin secretion:INS-1 cells were incubated in culture medium containing OnM,20nM,200nM or 2μM hypericin respectively for 24h.The supernatant was discarded and the cells were replenished with fresh culture medium with normal glucose,high potassium(30mM),or high glucose and incubated for another 30min.Then supernatant was collected and subjected to insulin detection by ELISA.Results1.Intracellular calcium model:INS-1 or RIN-m5f islet β cells loaded with fura-2 probe showed increased fluorescence intensity at 340nm accompanied with decreased intensity at 380nm upon the potassium stimulation.The 380nm decrease was four times over 340nm rise and 340/380 ratio raised around 0.2 which suggests that the model was successfully constructed.2.Perfused dual-phase insulin secretion model:Shredded collagenase digestion method can obtain more complete dispersed better islets which were positively stained with dithizone staining.By using hand-made perfusion device,the islets showed an expected dual-phase insulin secretion curve:within the 1-2min of high glucose treatment,insulin level showed a rapid rise with a peak at 1.6 times higher than the baseline insulin level and then decreased to 1.4 times and remained at that level.3.Hypericin inhibited potassium-induced calcium influx in islet β cells:20nM,200nM or 2μM hypericin have no acute effect on calcium influx in INS-1 cells.But long term incubation for 24h showed that compared with the control group,200nM and 2μM hypericin significantly inhibited the increased concentration of intracellular calcium upon potassium stimulation and this inhibitory effect was in a dose dependent manner.The similar results were obtained in RIN-m5f cells:20nM,200nM or 2μM hypericin significantly inhibited the intracellular calcium rise induced by potassium.4.Hypericin increased the viability of islet β cells:24h incubation with 200nM or 2μM hypericin increased INS-1 cell viability up to 113%and 120%respectively compared with untreated control(set at 100%).Wherease in RIN-m5f cells,only 2μM hypericin increased the cell viability to 116%.5.Hypericin protected islet β-cells from high-glucose induced glucotoxicity:Under glucotoxic induction condition,200nM and 2μM hypericin could significantly promote the survival of INS-1 cells from 91%in high glucose to 103%and 110%respectively,with the survival rate of untreated cells set to 100%.In RIN-m5f cells,2μM hypericin could obviously promote the cell survival rate from 82%in high glucose to 91%.6.Hypericin protected islet β-cells from PA induced lipotoxicity:100μM,200μM,or 300μM PA dramatically reduced the survival rate of INS-1 cells to 97%,90%and 49%and decreased the survival rate of RIN-m5f cells to 93%,79%,46%respectively with the survival rate of untreated cells set to 100%.However,following 24h incubation with 20nM,200nM or 2μM hypericin,the cell survival rate increased from 97%to 116%,106%,135%under 100μM PA stimulation,or from 90%to 93%,100%,119%under 200μM PA stimulation.But under 300μM PA treatment,none of the three concentration of hypericin can exert protective effects.Similar results were also observed in RIN-m5f cells:20nM,200nM or 2μ M hypericin could increase the survival rate of cells from 93%to 90%,95%and 112%respectively when PA was 100μM or from 79%to 78%,84%and 102%respectively when PA was 200μM.None of the utilized hypericin could improve the cell viability when PA was 300μM.7.Hypericin inhibited insulin secretion in INS-1 β-cells:Under low-glucose condition,2μM hypericin showed an inhibitory effect on insulin secretion and inhibited 11mM and 17mM glucose-stimulated insulin secretion as well.Similarly,2μM hypericin also inhibited potassium induced insulin secretion.