The Study On The Molecular Mechanism Of The Effect Of Berberine On Improving Insulin Resistance

Type 2 diabetes mellitus(T2DM) is a chronic inflammatory disease characterized by insulin resistance(IR) which means the capacity of insulin promoting the body glucose utilization decreased. Berberine is an isoquinoline alkaloid extracting from Rhizoma Coptidis. Previous studies have indicated that berberine has the hypoglycemic effect, but the molecular target and the pathway of its function are still unclear. Therefore, this study focused on the hypoglycemic mechanism of berberine and its target. Previous studies have found that berberine inhibits acetylcholinesterase(AC hE) activity, which is associated with the "cholinergic anti- inflammatory pathway" and "T2DM inflammation theory". So in our study, we investigate whether berberine improving insulin resistance, inhibiting inflammatory factors secretion is activating alpha7 nicotinic acetylcholine receptor(α7nAChR) in HepG2 cells, through the cholinergic anti- inflammatory pathway(CAP).This study has treated the human hepatoma cells as the object, established IR Hep G2 cell model in vitro and used nicotine as a positive control drug. Fluorescence spectroscopy method is used to detect the effect of berberine on glucose uptake in this study. According to the effect of berberine on the activity of AChE, the secretion of IL-6 and the related protein in CAP, the mechanism of berberine on insulin resistance and glucose uptake in HepG2 cells was investigated. The specific research contents are as follows:(1)Establishment of IR model in HepG2 cell: the IR model was induced by using high concentration of insulin in Hep G2 cell. 25 mmol/L glucose and 10-6 mol/L insulin was added in Hep G2 cells for 36 h. Glucose uptake obviously decreased in the 10-6 mol/L insulin, the IR model was successfully established in HepG2 cells.(2)Cytotoxicity test: when the concentration of berberine was more than 10 mol/L, the cell viability was significantly decreased, a nd the cell viability was significantly decreased when the concentration of nicotine was more than 0.05 mmol/L. So the final optimal concentration of the two drugs were 10 mol/L, 0.05 mmol/L.(3)The effect of berberine on glucose uptake in IR-Hep G2 cells: in order to study the effect of berberine on glucose uptake in HepG2 cells, 2-NBDG was used to investigate the effect of berberine on glucose uptake. The results showed t hat the optimal incubation concentration and incubation time of 2-NBDG were 200 μmol/L and 80 min, respectively. After the IR model was induced with high concentration of insulin, added 10 μmol/L or 50 μmol/L nicotine for 24 h, and then added 2-NBDG. The results showed that both berberine and nicotine improve the glucose uptake in model group. The fluorescence intensity of berberine group was dependent on the concentration of berberine, and the effect of 10 mol/L berberine was best.(4)The mechanism of berberine on insulin resistance in HepG2 cells: according to the effect of berberine on the activity of AChE, the secretion of IL-6 and the related protein in CAP, the mechanism of berberine on insulin resistance and glucose uptake in Hep G2 cells was investigated. An experimental have studied on the activity of AChE and the secretion of IL-6 and related proteins. The results show that the 10 μmol/L of berberine can inhibit AChE activity in the supernatant, which is consistent with the previous work on the subject.; but the effect of nicotine on AC hE activity had no obvious change, which is consistent with the theory nicotine directly acts on α7nAChR. Berberine, nicotine can also inhibit the secretion of inflammatory factors IL-6, thereby reducing the inflammatory response. The expression level of α7nAChR was significantly decreased in model group, the expression level of NF-κB p65 protein was significantly increased, and phosphorylation of IKKβ Ser181 phosphorylation was increased, which was consistent with the increase of IL-6 in the model group. The intervention of berberine and nicotine could reverse its effect, increase the expression of α7nAChR protein, decreased the expression of NF-κB p65 protein and the phosphorylation of IKKβ Ser181.These results suggested that, berberine improving IR, in HepG2 cells may be through the inhibition of AC hE activity in supernatant and reduce AC h decomposition, increasing both the level and duration of the neurotransmitter ACh. A nd AC h directly combined with α7nAChR and activated CAP which inhibiting the pro-inflammatory cytokines, which means berberine relieves insulin resistance and glucose uptake via CAP.