The Mechanism Of Catalpol In Protecting β-cell Function And Improving Hepatic Insulin Resistance In Vitro

ObjectiveTo investigate the hypoglycemic effects and mechanism of catalpol,as the active ingredient of Gegen Qinlian Decoction(GQD),for restoring β-cell function and improving insulin resistance(IR).It would provide a theoretical and scientific basis for the action mechanisms of GQD components and their combinations in the treatment of type 2 diabetes mellitus(T2DM).Methods1.Molecular docking study of the active ingredients and target proteins of GQD interventions in the insulin signal pathway of T2DM,focusing on the potential of docking catalpol with endoplasmic reticulum stress protein.(1)The core set of GQD active ingredients were obtained and their structures were downloaded from TCM-related databases and academic research literature.(2)Molecular docking predicted the binding ability of GQD core pharmaco-dynamic components in insulin hypoglycemic pathway and target proteins involving in β-cell function,then,the component with better scores was selected.(3)Catalpol was docking with target proteins involving in endoplasmic reticulum stress.2.To establish streptozotocin(STZ)-damaged INS-1 cells as in vitro model.(1)2 mmol·L-1 STZ was applied to INS-1 cells with different time treatment(3h,6h,9h,12h),and the cell survival rate was detected by CCK-8 method for the optimal time.(2)The CCK-8 method was used to determine the cell viability of different concentrations of catalpol(0.05,0.2,1,5,12.5,25,50 μmol·L-1)for 24h treatment and glibenclamide(0.05,0.1,0.5,1,2.5,5,10 μmol·L-1)for 12h treatment.(3)The CCK-8 method was used to detect cell proliferation viability in 96-well plates,and different concentrations of catalpol(0.2,1,5,12.5,25 μmol·L-1)were set up for 12h pre-treatment,followed by normal control group,STZ damaged group,N-acetylcysteine group(NAC,200 μmol·L-1),glibenclamide(GLI,2.5 μmol·L-1)group,except for the normal control group,which was added with equal amount of sodium citrate buffer,and the rest of the groups were added with 2 mmol·L-1 STZ.3.The anti-apoptosis effects of catalpol on the STZ damaged INS-1 cells:(1)Normal control group,STZ-damaged group,NAC group(200 μmol·L-1),GLI group(2.5 μmol·L-1)group,and catalpol(1,5 μmol·L-1)group were set up,and the cell viability was measured by CCK-8 method.(2)Microscopy and crystalline violet staining were performed to show cell apoptosis.(3)Flow cytometry was used to detect for the apoptosis rate.(4)WB assay was performed for the protein levels of important apoptosis protein BAX and BCL2.4.The anti-endoplasmic reticulum stress effects of catalpol on the STZ-damaged INS-1 cells:(1)Intracellular calcium content was detected by calcium content detection kit.(2)Endoplasmic reticulum stress proteins IRE1α and GRP78 were detected by WB.5.The anti-endoplasmic reticulum stress effect of catalpol on the STZ-damaged INS-1 cells:(1)Intracellular ROS and mitochondrial activity in INS-1 cells were detected by DCFH-DA fluorescent probe combined with mitochondrial fluorescent probe.(2)Total antioxidant capacity(T-AOC)were detected by T-AOC assay kit.(3)Superoxide dismutase(SOD)activity were measured by SOD detection kit.(4)Malondialdehyde(MDA)was measured by MDA detection kit for the cellular lipid peroxidation.(5)Catalase(CAT)was measured by detection kit.6.Effects of catalpol on the function markers of INS-1 cells:(1)Nuclear transcription factor PDX-1 was dectected by IF.(2)Insulin(Ins)was dectected by IF.(3)PDX-1 and Oct4,a key protein for self-renewal and pluripotency,was dectected by WB.(4)PDX-1,GLUT2,Insl,Ins2 and FoXO1 mRNA,was dectected by qPCR.(5)ELISA detected the insulin secretion content of each group under the conditions of 2.8mmol·L-1 basal glucose(BIS)and 33.3 mmol·L-1 high glucose culture medium(GSIS).7.Establishment of IR-HepG2 cell model induced by glucosamine(GlcN):(1)HepG2 cells were induced by 20mmol·L-1 GlcN with different time,and the GOD-POD method was used to detect the extracellular glucose content and optimize the modeling time.(2)CCK-8 method was used to detect the viability of HepG2 cells with different concentrations of catalpol intervention for 24h.8.Action and mechanism of catalpol on IR-HepG2 cell model:(1)Normal control group,GlcN-induced IR group(20 μmol·L-1),positive group(metformin:1mmol·L-1 Met),and different concentrations of catalpol(1,5,25 μmol·L-1)were set up for 18h and 24h.The extracellular fluid glucose content was measured to optimize the dose and time of administration.(2)Periodate-Scherff(PAS)staining was performed for glycogen contents in IR-HepG2 cells(3)2-NDBG fluorescent probe to detect glucose uptake in IR-HepG2 cells(4)Adiponectin(ADPN)and O-glycosyltransferase(OGT)were dectected by WB in IR-HepG2 cells.Results1.Molecular docking showed good binding ability of catalpol in GQD in insulin signal,β-cell function markers and endoplasmic reticulum stress protein(1)10 active chemical components(baicalin,baicalin,berberine,catalpol,penetrating spongestrol,soybean sapogenins,pharmacogenin,kaempferol,gerberin and baicalin)were obtained from for molecular docking simulation validation(InsR,IRS1,IRS2,PI3K p85,AKT,GSK3β,FOXO1,PDX-1,SOX9 and MAFA).(2)Catalpol showed high binding activity with endoplasmic reticulum-related proteins(IRE1α,PERK,GRP78,ATF4,FAM134B,CCPG1,ERO-1 and PDI)by molecular docking.2.To establish STZ-damaged INS-1 cells in vitro model and safe concentrations of catalpol and glibenclamide(GLI):(1)Compared to control group,cell viability were87.86%± 0.034,46.67%±0.027,24.46%± 0.014,14.18%± 0.079 at 3h,6h,9h,12h with STZ intervention.Thus,2 mmol·L-1 STZ intervention for 6h was used as the most appropriate modeling concentration.(2)Cell viability was not significantly affected by 0.05 mmol·L-1 to 50 mmol·L-1 catalpol for 24 h.Compared with the model group,5 and 10 μmol·L-1 GLI produced significant toxic effects on INS-1 cells(P<0.01)when pretreated with 0.05 mmol·L-1 to 10 mmol·L-1 GLI for 12 h.3.The anti-apoptosis effect of catalpol in protecting STZ-damaged INS-1 cells:(1)The catalpol groups(0.2,1,5,12.5,25 μmol·L-1)were pre-treament for 12 h and then set up the control group,STZ group,NAC group(200 μmol·L-1)and GLI group(2.5 μmol·L-1)were set up for 6 h.Compared with STZ group,cell viability was increased in the NAC,GLI and 1μmol·L-1 catalpol groups(P<0.001)and 5μmol·L-1 catalpol group(P<0.01).(2)Microscopy and crystalline violet staining showed a large number of apoptotic cells in the STZ-damaged group and more rounded cell morphology,while NAC,GLI and catalpol groups showed different degrees of remission with obvious clusters.(3)Compared with the control group,the STZ-damaged group showed a statistically significant increase in cell apoptosis in(P<0.001)whereas NAC,GLI,1 and 5 μmol·L-1 catalpol groups decreased compared with the STZ-damaged group(P<0.001)by flow cytometry with Annexin V-FITC/PI staining.(4)WB results showed that the pro-apoptotic factor BAX was highly expressed in the STZ-damaged group(P<0.001)and decreased in NAC,GLI,1μmol·L-1 and 5μmol·L-1 catalpol groups(P<0.01、P<0.05);the anti-apoptotic factor BCL2 was down-regulated in the STZ-damaged groupand increased in NAC,GLI and 1μmol·L-1 catalpol groups(P<0.001).(5)Compared with control group,BCL2 was down-regulated and Chop was up-regulated in STZ-damaged group(P<0.001),BCL2 was up-regulated in 1μmol·L-1 catalpol group(P<0.05)and Chop was down-regulated(P<0.001);In the 5μmol·L-1 catalpol group,Chop was significantly down-regulated(P<0.001)and BC12 was tend to up-regulated.4.Catalpol protected endoplasmic reticulum in the STZ-damaged INS-1 cells:(1)The results showed that intracellular calcium ion contents were significantly decreased in the STZ-damaged group(P<0.001).Compared with the STZ-damaged group,intracellular calcium ion contents were significantly increased in NAC group(P<0.01)and GLI group(P<0.001),1μmol·L-1 and 5μmol·L-1 catalpol groups(P<0.05).(2)WB results showed that IRE1α,a key endoplasmic reticulum stress-relative protein,was upregulated in the STZ-damaged group(P<0.001)and decreased in the NAC,GLI,1μmol·L-1 and 5μmol·L-1 catalpol groups compared to the STZ-damaged group(P<0.01、P<0.05).Glucose-regulated protein 78(GRP78)was upregulated in the STZ-damaged group and decreased in the NAC,GLI,and 1μmol·L-1 catalpol groups(P<0.001、P<0.01).5.Catalpol reduced oxidative stress in the STZ-damaged INS-1 cells:(1)The STZ-damaged INS-1 cells showed a large amount of ROS generation accompanied by severe mitochondrial damage and cell reduction.Moreover ROS in the NAC group,GLI group and catalpol 1μmol·L-1 group were significantly reduced and mitochondria were restored.(2)T-AOC assay showed that the total antioxidant capacity was severely impaired in the STZ-damaged group(P<0.001)whereas T-AOC was significantly increased in the NAC,1μmol·L-1 and 5μmol·L-1 catalpol groups(P<0.001)and GLI group(P<0.01).(3)The results showed that the SOD content in the STZ-damaged group decreased significantly(P<0.001);the NAC group significantly increased the SOD activity(P<0.001),GLI group and 5μmol·L-1 catalpol group significantly increased(P<0.01),and 1μmol·L-1 catalpol group also showed an increase(P<0.05).(4)The results showed that the MDA content was significantly increased in the STZ-damaged group(P<0.001);MDA production was significantly reduced in the NAC,GLI,5μmol·L-1 and 1μmol·L-1 catalpol groups(P<0.001).(5)The results showed that the CAT was significantly reduced in the STZ-damaged group(P<0.001);CAT was significantly increased in the NAC,GLI,5μmol·L-1 and 1μmol·L-1 catalpol groups(P<0.001、P<0.01、P<0.05).(6)Compared with control group,Nrf2 was down-regulated in STZ-damaged group(P<0.001).Compared with STZ-damaged group,Nrf2 was up-regulated in 1μmol·L-1 and 5μmol·L-1 catalpol group(P<0.05).5.Catalpol improved β-cell function markers on the STZ-damaged INS-1 cells:(1)IF results showed that PDX-1,a key transcription factor for β-cell development and maturation was scattered in the STZ-damaged group with lower number of blue fluorescence,indicating that PDX-1 expression was downregulated;NAC group,the GLI group and catalpol 1μmol·L-1 group showed high PDX-1 expression and more cells.(2)IF results showed that the STZ-damaged group showed weak green fluorescence and weak of blue fluorescence for the decrease of Ins were decreased whereas NAC,GLI and 1μmol·L-1 catalpol groups showed an increase of Ins amount.(3)WB result showed that PDX-1 was low expressed in the STZ-damaged group(P<0.001)and increased in NAC,GLI,1μmol·L-1 and 5μmol·L-1 catalpol groups(P<0.001、P<0.01、P<0.05).Oct4,a protein involving of self-renewal and differentiation potential,was significantly decreased in the STZ-damaged group whereas the administered groups showed no significant change.It indicated that the differentiation ability of INS-1 cells was inhibited in STZ-damaged INS-1 cells,which was difficult to be reversed by drug administration.(4)Compared with normal control group,Nrf2 in STZ injury group was down-regulated(P<0.001);PDX-1,GLUT2,Insl,FoXO1 in 1μmol·L-1 catalpol group was down-regulated significantly(P<0.001,P<0.01)compared with 5μmol·L-1 catalpol group and normal control group.Ins2 showed a down-regulated trend;Catalpol up-regulated PDX-1,GLUT2,Ins1,Ins2,Nrf2,Bcl2 and FoXO1,all showing statistical differences(P<0.001,P<0.01,P<0.05).(5)In BIS state,1μmol·L-1 catalpol significantly increased Ins secretion compared with STZ group(P<0.001).In GSIS condition,1μmol·L-1 and 5μmol·L-1 catalpol increased Ins secretion compared with STZ group(P<0.001,P<0.05).6.In vitro model of IR-HepG2 with safe concentration of catalpol:(1)Compared with control group,20 mmol·L-1 concentration of GlcN stimulated HepG2 cells at 12h did not obvious changet;the difference appeared statistically significant at 18h(P<0.05)and 24h(P<0.001).(2)The cell viability show no significant difference at all concentrations(1-50μmol·L-1)of catalpol groups on HepG2 cells for 24h treament.7.Action mechanism of catalpol intervention in GlcN-induced IR-HepG2:(1)The extracellular glucose consumption in the GlcN group showed a highly significant increase(P<0.001);the extracellular glucose content were decreased in the Met and 1 μmol·L-1 catalpol groups was decreased(P<0.001);the glucose content in the 5 μmol·L-1 and 25 μmol·L-1 catalpol groups was also downregulated(P<0.01).(2)The GlcN group decreased glycogen content whereas Met group and the 1μmol·L-1 catalpol group increased glycogen content by PAS staining.(3)The GlcN group showed inhibited glucose uptake of HepG2 cells whereas Met group,1 μmol·L-1 and 5 μmol·L-1 catalpol groups increased glucose uptake of HepG2 cells by 2-NBDG fluorescence probe detection.(4)ADPN was downregulated in the GlcN group compared with the normal control group(P<0.001);ADPN was upregulated in the Met,5 μmol·L-1 and 25μmol·L-1 catalpol groups compared with the GlcN group(P<0.001,P<0.01).OGT was downregulated in the GlcN group compared with the normal control group(P<0.001),and OGT was upregulated in the Met and 1μmol·L-1 catalpol groups compared with the GlcN group(P<0.01,P<0.05).ConclusionCatalpol effectively alleviated STZ-damaged INS-1 cells and exerted its pharmacological effects at multiple levels from anti-apoptosis,alleviating endoplasmic reticulum stress,and improving oxidative stress,increasing insulin synthesis and secretion.Moreover,catalpol could effectively increased hepatic glycogen content,and upregulate the glucose uptake to enhance glucose consumption to improve IR on the GlcN-induced IR-HepG2 cell.In summary,catalpol had dual action mechanism of catalpol in protecting β-cell function and improving hepatic insulin resistance.