| ObjectivesType 2 diabetes Mellitus is a kind of inherited disease characterized by hyperglycemia and insulin resistance (IR) in peripheral tissues such as adipose tissues and skeletal muscles. Although Chinese herbal medicines have been widely used to treat this disease, little is known about the underlying mechanisms. In the study, we investigated the influence of a Chinese herbal formula, Tangnaikang (TNK, containing Prunella vulgaris, Guava leaf, Saururus Chinensis, Lingustrum lucidum and Ginseng) on the expression of adipose-derived proteins in T2DM and IR .We investigated the effect of TNK on the adipocyte-derived protein,SOD and IDE in Type 2 diabetic KKAy mice and 3T3-L1 adipocytes induced with dex and insulin, in order to supply the basic theory and experimental data for researching the mechanism of action of the secretation function of the adipocyte in IR and T2DM and for the mechanism of action of recipe-in-TCM Tangnaikang across the board .Methods1 Experiment in vivoThe 40 maleness KKAy mice were divided randomly into four groups:model-group western-medicine-group,TNKlow-dosis and high-dosis groups; every group included 10 mice; and 10 C57BL/6J mice were normo-group. model-group and normo-group were intragastric administrated with sterile water; western-medicine-group were intragastric administrated with Pioglitazone ,and low-dosis and high-dosis groups were intragastric administrated with TNK 1.67g/kg/d and 6.56g/kg/d separately .After treated correspondingly separately for 8 weeks, then all mice were killed to collect the blood and the adipocyte.Blood glucose were detected by test-paper method; IL-6,Leptin,resistin and adiponectin were detected by ELISA; TNF-αwas detected by ELISA,Western blotting and immunohistochemistry method.2 Experiment in vitroThe ripe 3T3-L1 adipocytes were divided into seven groups: normo-group,model-group,western-medicine-group(with Pioglitazone)and TNK four different dosis groups. After being changed into IR model, they were treated with drug-contained serum correspondingly separately for 72h.Then the cells and their supernatant were collected to be detected. TNF-α,IL-6,Leptin,Resistin,Adiponectin,AdipoR1 and PPARγin the supernatant were detected by ELISA. TNF-αand adiponectin in cells were detected by Western blotting; TNF-αmRNA,AdiponectinmRNA and adipoR1 mRNA were detected by RT-PCR; adiponectin and TNF-αimmumofluorescence method.Results 1 Experiment in vivo1.1 When they grow up to about 13 weeks ,most KKAy mice have been very fat and high level of blood glucose.1.2 There are much adiponectin ,and a certain levels of TNF-α,IL-6,resistin and leptin in adipocytes of healthy mice; the level of TNF-αin adipocytes is about 1/5 of in serum, the level of IL-6 is about 1/3 of in serum. The levels of them in adipocytes of KKAy mice are both about 4.5 times of those of the healthy mice.And there is positive correlation between TNF-αand FPG of KKAy mice.1.3 TNK could improve the state of higher levels of blood glucose of KKAy mice , the same as Pioglitazone could.Obviously compared with normal group, the levels of IL-6 and TNF-αof model-group were higher significantly, whether in blood or in adipocyte. And the levels of leptin,resistin and adiponectin in adipocytes were higher, too. TNK can decrease the levels of TNF-α,IL-6,leptin,resistin and adiponectin significantly , almost the same as the Pioglitazone group.2 Experiment in vitro2.1 Compared with the norm-group, the levels of IL-6 and TNF-αin the supernatant of the model group are higher significantly. TNK low-dosis and most- dosis groups could decrease IL-6 significantly;both of them had no significant contrast . The TNK more-dosis and most-dosis groups could decrease the level of TNF-αsignificantly.The results by every detection method was consistent.2.2 Compared with the model group, the level of adiponectin in the supernatant of the TNK more-dosis and middle-dosis groups decreased significantly; the levels of resistin of the TNK more-dosis and middle-dosis groups increased significantly,but compared with the normo-group,they had no significant contrast; the level of leptin decreased significantly, the level of leptin of the TNK low-dosis,middle-dosis and more-dosis groups could increase significantly,but the level of leptin of the most-times-dosis groups could decrease significantly, and the western-medicine group had no significant effect.The levels of AdipoR1 of mormo-group and model-group had no significant contrast; compared with the model group, the level of AdipoR1 of the TNK low-dosis group increased significantly. Compared with the model group, the level of PPARγof mormo-group and the TNK more-dosis and most-dosis groups increased significantly, all of them had no significant contrast. There was inverse correlation between the level of PPARγand TNF-αin the supernatant.2.3 Compared with the norm-group, the level of SOD in the supernatant of the model group had a degrading trend; the TNK low-dosis,more-dosis and more-dosis groups increased it to some extent,but the TNK most-dosis group decreased it to some extent. Compared with the norm-group, the level of IDEmRNA in the supernatant of the model group increased a little; the western medicinegroup could not decrease it, the TNK more-dosis group decreased it obviously.Conclusions The animal research indicated that the herbal formula, TNK can decrease sig- nificantly the levels of TNF-α,IL-6,Leptin and Resistin,and increase significantly the level of adiponectin in mouse with T2DM. And TNK may regulate the insulin signal transduction pathway by PPARγmediated.The vitro research indicated the TNK can de-crease significantly the levels of TNF-αand IL-6,and increase significantly the level of adiponectin,Leptin,Resistin and PPARγin cells with IR,and can inhi-bit free radical and decrease the level of IDE at a certain degree.In a word, Our results demonstrate that TNK can heal T2DM with obesity, and the effectiveness of it may involve the molecular mecheniams that regulate the secretation function of the adipocyte in IRand T2DM. |
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