Metabolic Mechanism Of Cell Glucose Consumption Induced By Hypoxia And Intervention Of Gegen Qinlian Decoction Containing Serum

Objective Metabolic mechanism of cell glucose consumption induced by hypoxia and intervention of Gegen Qinlian Decoction containing serum.Methods1.Preparation and quality control of GQD by clinical and classical decocting methods:based on HPLC,the contents of isoflavones,flavonoids,diterpenoids and alkaloids related to the efficacy of the two decoctions were investigated.2.Quality control of decoction GQD containing serum: based on AB LC-MS,the contents of isoflavones,flavonoids,diterpenes and alkaloids in decoction GQD containing serum were investigated.3.Establishment of the model of reducing oxygen-glucose consumption and the effect of different factors on hypoxia: 1% oxygen concentration was used,glucose consumption and cell viability in supernatant of HepG2 hepatoma cells and L-02 hepatocytes were measured at 6,12,18,24 and 48 hours of hypoxia,respectively,the best time point was chosen to establish the model of reducing oxygen and glucose consumption.Under hypoxia condition,palmitic acid(free fatty acid),dexamethasone(glucocorticoid)and TNF-α(inflammatory factor)were used to investigate the interaction of different factors on the model of reducing oxygen-glucose consumption.4.GQD pharmacodynamics and intervention mechanism under hypoxia condition: the cells were divided into normal control group,hypoxia group,metformin group,GQD low,middle,high(5,10,15%)dose group.The normal control group was cultured with normal oxygen,the anoxic group was cultured with 1% oxygen concentration;the glucose consumption and cell viability were measured at the time point of successful modeling.After administration,cell samples were collected and analyzed by metabonomics technique,potential biomarkers associated with hypoxia and GQD were identified,and the change trend and pathway were analyzed.Results1.The contents of GQD active ingredients prepared by the two methods were different.Prolonging the decocting time of radix puerariae is helpful to promote the dissolution of flavonoids.2.The results of the determination of GQD drug-containing serum by LC-MS showed that the specificity and stability of the method met the requirements of biological sample determination.3.(1)HepG2 cells:glucose consumption at different time points of 1 % oxygen concentration: vs normal control group,glucose consumption at 6 h after hypoxia was not significantly changed,and glucose consumption at 12 and 18 h after hypoxia was significantly reduced(P< 0.01);The glucose consumption increased significantly at 24 and 48 h after hypoxia(P< 0.01).1% oxygen concentration at different time points: vs the normal control group,cell viability increased after 48 hours of hypoxia(P< 0.01),and cell viability was not affected after 6,12,18 and 24 h of hypoxia.(2)L-02 cells: glucose consumption was measured at different time points of 1%oxygen concentration.Results: vs normal control group,glucose consumption at 6 h was not significantly changed,at 12,18,24 and 48 h was significantly decreased(P<0.01);1% oxygen concentration at different time points: vs the normal control group,the cell viability decreased significantly after 48 h of hypoxia(P<0.01),and the cell viability did not change significantly after 6,12,18 and 24 h of hypoxia.(3)The interaction of palmitic acid,dexamethasone and TNF-α with the model of decreasing glucose consumption in HepG2 cells: glucose consumption: vs normal control group,glucose consumption decreased significantly after 18 h of hypoxia(P<0.01);vs the hypoxic group,the glucose consumption of the administration group decreased significantly(P<0.01).Cell viability: vs normal control group,hypoxia for 18 h had no effect on cell viability.(4)Effects of palmitic acid,dexamethasone acid and TNF-α on the model of decreasing glucose consumption in L-02 cells: glucose consumption: vs normal control,the glucose consumption decreased significantly after 18 h of hypoxia(P<0.05);vs the hypoxic group,the glucose consumption of the palmitic acid group decreased significantly(P<0.01),the glucose consumption of the TNF-α and the dexamethasone group did not change significantly,and the cell viability: vs the normal control group,after 18 hours of hypoxia,palmitic acid group showed a significant decrease(P<0.01),and the cell viability of the other groups was not affected.4.Results of pharmacodynamics of GQD intervention on HepG2 cells: glucose consumption: vs normal control group,glucose consumption decreased significantly in hypoxia 18 h(P<0.01);vs hypoxia group,glucose consumption in GQD low,middle and high groups were improved(P<0.01);Cell viability: vs the normal control group,there was no effect on cell viability in each group after 18 h of hypoxia.The results of metabonomics showed that there were 93 biomarkers related to hypoxia,and 28 metabolic pathways were involved.GQD could regulate 12 of them effectively by affecting the metabolism of phospholipids,amino acids and nucleotides.5.GQD intervention L-02 cell pharmacodynamics results: glucose consumption: vs normal control group,glucose consumption of hypoxia 18 h was significantly reduced(P<0.01);vs hypoxia group,glucose consumption of GQD-L、M and H group was significantly increased(P<0.01),GQD-H group had no significant changes in glucose consumption and cell viability: vs normal control group,there was no significant change in each group.The results of metabonomics showed that there were 14 biomarkers associated with hypoxia and 8 metabolic pathways involved.GQD effectively interfered with 3biomarkers,and played a role by affecting the metabolism of phospholipids and sphingolipids.ConclusionBy analyzing the effective components of decoction and serum of GQD prepared by different decocting methods,the prolonged decocting time of Pueraria Montana was beneficial to the dissolution and entry of flavonoids into the blood.HepG2 and L-02 cell were cultured at 1% oxygen concentration,and it was found that hypoxia could decrease glucose consumption of the cells,which was related to the time of hypoxia.The results showed that the serum containing GQD could regulate the metabolism of phospholipids and sphingomyelins in two cells.