| Object ive:1.In order to explore the possibility that phellodendrine,one of the main components of the hypoglycemic traditional chinese medicine Cortex Phellodendri,reduces postprandial blood sugar by inhibiting α-glucosidase activity,the inhibitory effect and the inhibition type of phellodendrine on α-glucosidase in vitro and the molecular mechanism of its function were studied.2.The insulin resistance(IR)model of HepG2 cells was established to investigate the effect of phellodendrine on improving glycolipid metabolism disorder in IR-HepG2 cells and to further explore its regulation on IRS-1/PI3K/Akt insulin signaling pathway for the possible mechanism of phellodendrine on improving insulin resistance in HepG2 cells,providing experimental basis for further study of the hypoglycemic effect of phellodendrine in vivo.Methods:1.Using 4-Ntrophenyl-α-D-glucopyranoside(PNPG)as substrate and acarbose as positive control,an inhibitor screening model was established in vitro to examine the inhibition rate of phellodendrine on α-glucosidase.The inhibition type was investigated by kinetic method.The three-dimensional structure of α-glucosidase was constructed by homology modeling method,and the potential inhibition molecular mechanism of phellodendrine on α-glucosidase was analyzed by molecular docking technology.2.The cytotoxicity of phellodendrine on HepG2 cells was detected by MTT method after administration of different dosage of phellodendrine(6.25,12.5,25,50,100,200,400 μM)for 48 h.3.The insulin resistance model of HepG2 cells was established by inducing HepG2 cells with 1 mM palmitic acid and 2 mM oleic acid for 24 hours.IR-HepG2 cells consumption of glucose in cell culture medium was detected by Glucose oxidase method after treatment of different dosage of phellodendrine(6.25,12.5,25,50,100 μM)for 12,24 and 48 h,respectively.4.The cytotoxicity of berberine on HepG2 cells was detected by MTT method after administration of different dosage of phellodendrine(6.25,12.5,25,50,100,200,400 μM)for 24 h.5.IR-HepG2 cells consumption of glucose in cell culture medium was detected by Glucose oxidase method after treatment of different dosage of berberine(6.25,12.5,25,50,100 μM)for 24 h.6.The contents of TG,TCH,LDL-C and HDL-C in IR-HepG2 cells were determined respectively by using triglyceride(TG),cholesterol(TCH),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)detection kits after treatment of low,middle and high dosage of phellodendrine(25,50,100 μM)for 24 h,and the berberine group was set up for comparison.7.The mRNA expression of INSR,IRS-1,PI3K,Akt2 and GSK-3β of IRS-1/PI3K/Akt insulin signaling pathway in IR-Hepg2 cells was detected by RT-qPCR after treatment of phellodendrine for 24 h,and the berberine group was set up for comparison.8.The phosphorylation levels of p-IRS-1(Ser307),p-Akt(ser473)and p-GSK-3 β(ser9),as well as the expression of PI3K-p85 protein of IRS-1/PI3K/Akt insulin signal pathway in IR-Hepg2 cells were measured by Western blot after treatment of phellodendrine for 24 h,and the berberine group was set up for comparison.Results:1.Phellodendrine displayed obvious inhibitory activity on a-glucosidase with an IC50 value of 126.36 mg·L-1,in a concentration-dependent manner.The results of enzyme kinetics indicated that the reaction rate was lowered and Vmax、Km were decreased with the concentration increasing of phellodendrine.The results of molecular docking showed that the binding energy of phellodendrine to α-glucosidase site 5 was the lowest,and its optimal docking conformation binding energy was-7.5 kcal·mol-1.Phellodendrine may exert its inhibitory activity by forming hydrogen bonds with amino acid residues such as LYS15、SER295、HIS258,hydrophobic interaction with ALA289 and n-anion interaction with GLU10.2.When the dosage of phellodendrine ranged from 6.25 to 100 μ M,it had no cytotoxicity on HepG2 cells after administration for 48 h(P>0.05).3.IR-HepG2 cells consumption of glucose in cell culture medium increased in different degrees and in a dose-dependent manner compared with the model group after treating with different dosage of phellodendrine for 12 hours and 24 hours,of which 25,50 and 100 μM dosages had the strongest improvement effect(P<0.05 or P<0.01 or P<0.001);After treating with phellodendrine for 48 hours,the glucose consumption of the model group had no significant change compared with the normal group(P>0.05),even some dosages of phellodendrine was slightly higher than the model group in terms of the glucose consumption,there was no significant difference(P>0.05).Therefore,25,50 and 100 μM were selected as the low,middle and high dosages,and 24 hours as treatment time.The treatment time of berberine was also set as 24 hours.4.When the dosage of berberine ranged from 6.25 to 100 μM,it had no cytotoxicity on HepG2 cells after administration for 24 h(P>0.05).5.After treating with different dosage of berberine,IR-HepG2 cells consumption of glucose in cell culture medium was significantly higher than that of the model group(P<0.05 or P<0.001),Considering the cytotoxicity of berberine on HepG2 cells and the promotion effect on IR-HepG2 cells consumption of glucose in cell culture medium,6.25,12.5 and 25 uM were selected as the low,middle and high dosages.6.The content of TG,TCH and LDL-C in HepG2 cells increased significantly(P<0.01)and the content of HDL-C decreased significantly(P<0.01)after induction of 1 mM palmitic acid with 2 mM oleic acid for 24 h.The content of TG,TCH and LDL-C in IR-HepG2 cells were decreased significantly after administration of the high、middle and low dosages of phellodendrine(P<0.05 or P<0.01 or P<0.001)and the content of HDL-C increased significantly after administration of the high and middle dosages of phellodendrine(P<0.05 or P<0.001).Among them,the effect of high(100μM)or middle(50 μM)dosage of phellodendrine is more obvious,slightly better than berberine.7.The mRNA expression of INSR,IRS-1,PI3K,Akt2 and GSK-3 β of IRS-1/PI3K/Akt signaling pathway in HepG2 cells was significantly down-regulated(P<0.05 or P<0.01)after induction of 1 mM palmitic acid with 2 mM oleic acid.Phellodendrine could up-regulate the mRNA expression of INSR,IRS-1,PI3K,Akt2 and GSK-3β of IRS-1/PI3K/Akt signaling pathway in IR-HepG2 cells.Among them,the effect of high(100 μM)or middle(50μM)dosage of phellodendrine is more significant(P<0.05 or P<0.01 or P<0.001).8.1 mM palmitic acid combined with 2 mM oleic acid significantly increased the phosphorylation level of p-IRS-1(Ser307)protein(P<0.01),decreased the expression of PI3K-p85 protein and the phosphorylation level of p-Akt(Ser473)and p-GSK-3β(Ser9)protein in HepG2 cells(P<0.05).Phellodrine could down-regulate the phosphorylation level of p-IRS-1(Ser307),up-regulate the phosphorylation level of p-Akt(Ser473),p-GSK-3β(Ser9)and the expression of PI3K-p85 protein(P<0.05).Among them,the effect of high(100 μM)dosage of phellodendrine is more significant(P<0.05 or P<0.01 or P<0.001).Conclusion:Phellodendrine could obviously inhibit the activity of α-glucosidase in vitro and the inhibitory type is uncompetitive inhibition.The inhibitory activity of phellodendrine on α-glucosidase may be mainly realized by the formation of Hydrogen Bond,Hydrophobic interaction and π-anion interaction between phellodendrine and α-glucosidase.Phellodendrine can improve the glycolipid metabolism disorder in IR-HepG2 cells,and its mechanism may be related to regulate the mRNA expression of INSR,IRS-1,PI3K,Akt,GSK-3 βof IRS-1/PI3K/Akt signaling pathway and the phosphorylation level of p-IRS-1(Ser307),p-Akt(ser473)and p-GSK-3β(ser9),as well as the expression of PI3K-p85 protein. |
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