| Purpose:Through Chinese herbal slices,Chinese herbal components and monomers,the effects of Radix Astragali,Pueraria Lobata and their compatibility on adipokines and AMPactivated protein kinase(AMPK)signaling pathway in adipose tissue were studied by the theory of diabetes caused by spleen deficiency.The mechanism and active components of Radix Astragali,Pueraria lobata and their compatibility regulating glucose and lipid metabolism of diabetes were studied.Material and method:Part One: Literature ResearchThe relationship between spleen and diabetes was discussed with etiology,pathogenesis and treatment of diabetes by reviewing TCM books and modern medical studies.The relationship between spleen and lipid was also discussed with etiology,pathogenesis and treatment of lipid.Part two: Experiment ResearchExperiment One:86 Male SD rats were randomly divided into normal group,model group,Jinqi Jiangtang tablet group,Radix Astragali group,Pueraria lobata group and Radix Astragali-Pueraria lobata Compatibility group.There were 11 rats in normal group and 15 rats in the other groups.Diabetic model was induced by intraperitoneal injection of STZ once time.On the same day,rats were given drugs 30 days continuously.The rats were given 10 m L/kg distilled water in normal group and model group.The rats were given 1.47 g/kg Jinqi Jiangtang tablet suspension in Jinqi Jiangtang tablet group.The rats were given 2.7 g/kg Radix Astragali in Radix Astragali group.The rats were given 1.35 g/kg Pueraria lobata in Pueraria lobata group.The rats were given 4.05 g/kg Huangqi Gegen Decoction in Radix Astragali-Pueraria lobata Compatibility group.Blood glucose,triglyceride(TG)and cholesterol(CHO)in serum were detected by biochemical method.The contents of adiponectin(APN),leptin(LEP),tumor necrosis factor α(TNF-α)and glucose transporter 4(GLUT-4)were detected by enzyme linked immunosorbent assay(ELISA).The relative expression of Chemerin,Chem R 23,AMPK,GLUT-4 and β-actin m RNA was detected by Real-time PCR.Experiment Two:146 Male SD rats were randomly divided into normal group,model group,Jinqi Jiangtang tablet group,Astragalus Flavonoids(AF)group,Astragalus Saponins(AS)group,Pueraria lobate Flavonoids(PF)group,compatibility of AF and AS(AF+AS)group,compatibility of AF and PF(AF+PF)group,compatibility of AS and PF(AS+PF)group,compatibility of AF,AS and PF(AF+AS+PF)group.There were 11 rats in normal group and 15 rats in the other groups.Diabetic model was induced by intraperitoneal injection of STZ once time.On the same day,rats were given drugs 30 days continuously.The rats were given 10 m L/kg distilled water in normal group and model group.The rats were given 1.47 g/kg Jinqi Jiangtang tablet suspension in Jinqi Jiangtang tablet group.The rats were given 0.039 g/kg AF in AF group.The rats were given 0.104 g/kg AS in AS group.The rats were given 0.036 g/kg PF in PF group.The rats were given 0.143 g/kg AF and AS in AF+AS group.The rats were given 0.075 g/kg AF and PF in AF+PF group.The rats were given 0.140 g/kg AS and PF in AS+PF group.The rats were given 0.179 g/kg AF,AS and PF in AF+AS+PF group.Blood glucose,TG and CHO in serum were detected by biochemical method.The contents of APN,LEP,TNF-α and GLUT-4 were detected by ELISA.The relative expression of Chemerin,Chem R 23,AMPK,GLUT-4 and β-actin m RNA was detected by Real-time PCR.Experiment Three:3T3-L1 preadipocytes were cultured with 50 μM,100 μM,200 μM,400 μM calycosin-7-glucoside complete medium and 0.5 μM,1 μM,2 μM,4 μM,8 μM puerarin complete medium in 24 h,48 h,72 h.The optimum administration time and concentration were selected by MTT to detect 3T3-L1 preadipocytes survival rate.3T3-L1 preadipocytes were cultured with 100 μM calycosin-7-glucoside and 2 μM puerarin complete medium,200 μM calycosin-7-glucoside and 2 μM puerarin complete medium,400 μM calycosin-7-glucoside and 2 μM puerarin complete medium,100 μM calycosin-7-glucoside and 4 μM puerarin complete medium,200 μM calycosin-7-glucoside and 4 μM puerarin complete medium,400 μM calycosin-7-glucoside and 4 μM puerarin complete medium in 48 h.The optimum concentration was selected by MTT to detect 3T3-L1 preadipocytes survival rate.3T3-L1 preadipocytes were divided into 5 group.3T3-L1 preadipocytes were cultured with complete medium in normal group and model group.3T3-L1 preadipocytes were cultured with 200 μM calycosin-7-glucoside complete medium in calycosin-7-glucoside group.3T3-L1 preadipocytes were cultured with 4 μM puerarin complete medium in puerarin group.3T3-L1 preadipocytes were cultured with 200 μM calycosin-7-glucoside and 4 μM puerarin complete medium in calycosin-7-glucoside and puerarin compatibility group.All the cells were cultured in 48 h.Except normal group,the other group were given Inducer A(1 μg/m L IBXM,1 μM DEX and 1 μg/m L INS in complete medium)and respective treatment drugs in 48 h.Except normal group,the other group were given Inducer B(1 μg/m L INS in complete medium)and respective treatment drugs in 48 h,given twice.3T3-L1 preadipocytes differentiation into adipocytes were detected by Oil Red O.The contents of TG,CHO,APN,LEP,TNF-α and GLUT-4 were detected by ELISA.The content of 2-NBDG was detected by flow cytometry.GLUT-4 was detected by Immunofluorescence method.The contents of AMPK,p-AMPK and β-actin were detected by Western Blot.Results:Part One: Literature Research1.The relationship between spleen and diabetes:Diabetes was related to spleen deficiency.Deficiency of spleen qi could not dispersed fluid to body.And deficiency of spleen yin also the pathogenesis of diabetes.The treatment of diabetes could base on spleen.The prescription for benefiting qi for strengthening spleen could be selected.2.The relationship between spleen and fat:In modern anatomy fat and muscle belong to the category of “muscle” of TCM,which was governed by spleen.When the diet stagnation damaged the spleen would lead to spleen deficiency or deficiency of spleen yang.Which could not spread fine to body would lead to fat formation.The prescription for strengthening spleen could be selected.Part Two: Experiment ResearchIn Vivo Experiment(Experiment One and Two):1.Effects on blood glucose and blood lipids in diabetic rats:(1)Effects on blood glucose in diabetic rats: On the 30 th day,the blood glucose in the model group was significantly higher than that in the normal group.The difference was statistically significant(P<0.05).Compared with the model group,Radix Astragali,Pueraria lobate,Radix Astragali-Pueraria lobata compatibility and Astragalus Flavonoids could significantly reduce blood glucose.The difference was statistically significant(P<0.05).(2)Effects on blood lipids in diabetic rats: Compared with the normal group,the levels of serum CHO and TG in the model group were significantly higher.The difference was statistically significant(P<0.05).Compared with the model group,Radix Astragali,Pueraria lobate,Radix Astragali-Pueraria lobata compatibility,AF,AS,PF,AF+AS,AF+PF,AS+PF and AF+AS+PF could reduce the content of CHO and TG(except AF+AS+PF)in serum of rats.The difference was statistically significant(P<0.05).At the level of Chinese herbal slices,the combination of Radix Astragali and Pueraria lobata could reduce TG synergistically,best.At the level of Chinese herbal components,single AS had the best effect on reducing CHO,and single AF had the best effect on reducing TG.2.Effects on adipokines in diabetic rats:(1)Effects on APN,LEP and TNF-α in diabetic rats: Compared with the normal group,the expression of APN and LEP was significantly lower,and the level of TNF-α was significantly higher in the model group.The difference was statistically significant(P<0.05).Compared with the model group,Radix Astragali,Radix Astragali-Pueraria lobata compatibility,AF+AS,AF+PF,AS+PF and AF+AS+PF could increase the content of APN in diabetic rat adipose tissue.Compared with the model group,Radix Astragali and Radix Astragali-Pueraria lobata compatibility could increase the content of LEP in diabetic rat adipose tissue.Compared with the model group,Radix Astragali,Radix Astragali-Pueraria lobata compatibility,AF,AS,PF,AF+AS,AF+PF,AS+PF and AF+AS+PF could reduce the content of TNF-α in diabetic rat adipose tissue.The difference was statistically significant(P<0.05).At the level of Chinese herbal slices,the effect of single Radix Astragali on reducing TNF-α was better than Radix Astragali-Pueraria lobata compatibility.At the level of Chinese herbal components,AF and AS compatibility could reduce TNF-α synergistically,and the combination effect was the best.(2)Effects on Chemerin and Chem R23 m RNA in diabetic rats: Compared with the normal group,the relative expression of Chemerin and Chem R23 m RNA was significantly higher.The difference was statistically significant(P<0.05).Compared with the model group,Radix Astragali,Pueraria lobate,AF,AS,AF+AS could reduce the relative expression of Chemerin m RNA in diabetic rat adipose tissue.Compared with the model group,Radix Astragali,Pueraria lobate,Radix Astragali-Pueraria lobate compatibility,AF,AS,PF,AF+AS,AF+PF,AS+PF and AF+AS+PF could reduce the relative expression of Chem R23 m RNA in diabetic rat adipose tissue.The difference was statistically significant(P<0.05).At the level of Chinese herbal slices,single Radix Astragali could reduce the relative expression of Chemerin m RNA best.At the level of Chinese herbal components,AF reduced the relative expression of Chemerin and Chem R23 m RNA best.3.Effects on AMPK and GLUT-4 in diabetic rats:(1)Effects on AMPK and GLUT-4 m RNA in diabetic rats: Compared with the normal group,the relative expression of AMPK and GLUT-4 m RNA was significantly lower in model group.The difference was statistically significant(P<0.05).Compared with the model group,Radix Astragali,Pueraria lobate,Radix Astragali-Pueraria lobate compatibility,AF,and AF+AS+PF could increase the relative expression of AMPK m RNA in diabetic rat adipose tissue.Compared with model group,Radix Astragali,Pueraria lobate,Radix Astragali-Pueraria lobate compatibility,AF,AF+PF and AF+AS+PF could increase the relative expression of GLUT-4 m RNA in diabetic rat adipose tissue.The difference was statistically significant(P<0.05).At the level of Chinese herbal slices,Radix Astragali and Pueraria lobata could reduce the relative expression of GLUT-4 m RNA synergistically,and the combination effect was the best.At the level of Chinese herbal components,AF reduced the relative expression of AMPK and GLUT-4 m RNA best.(2)Effects on the content of GLUT-4 in diabetic rats: Compared with normal group,the content of GLUT-4 was significantly lower in model group.The difference was statistically significant(P<0.05).Compared with model group,Radix Astragali,Radix Astragali-Pueraria lobate compatibility,AF,AF+AS,AF+PF,AS+PF and AF+AS+PF could increase the content of GLUT-4 in diabetic rat adipose tissue.The difference was statistically significant(P<0.05).At the level of Chinese herbal components,AF+AS reduced the content of GLUT-4 best.In vitro Experiment:1.Effects on adipogenesis and differentiation of 3T3-L1 preadipocytes:(1)Effects on the survival rate of 3T3-L1 preadipocytes: After 48 hours of culture,100 μM,200 μM and 400 μM calycosin-7-glucoside could promote the proliferation of 3T3-L1 preadipocytes,2 μM and 4 μM puerarin could promote the proliferation of 3T3-L1 preadipocytes,and 200 μM calycosin-7-glucoside combined with 4 μM puerarin could promote the proliferation of 3T3-L1 preadipocytes.(2)Effects on adipogenesis of 3T3-L1 preadipocytes: Under microscope,there were a large number of mature adipocytes in calycosin-7-glucoside group,puerarin group and their compatibility group.The number of adipocytes in calycosin-7-glucoside group,puerarin group and their compatibility group was significantly more than that in normal group and model group.The results of oil red O absorbance showed that there was no significant difference between the model group and the normal group(P≥0.05).Compared with the model group,calycosin-7-glucoside,puerarin and their compatibility could significantly increase the absorbance of oil red O,and the difference was statistically significant(P<0.05).Calycosin-7-glucoside and puerarin could promote the formation of adipocytes synergistically,and the combination effect was best.2.Effects on glucoses and lipids in the insulin resistant 3T3-L1 adipocytes:(1)Effects on the uptake of 2-NBDG in the insulin resistant 3T3-L1 adipocytes: Compared with the normal group,the content of 2-NBDG was decreased in the model group,and the difference was statistically significant(P<0.05).Compared with the model group,calycosin-7-glucoside and calycosin-7-glucoside-puerarin compatibility could significantly increase the content of 2-NBDG in IR 3T3-L1 adipocytes.(2)Effects on CHO and TG in the insulin resistant 3T3-L1 adipocytes: Compared with the normal group,the levels of CHO and TG in the model group were significantly higher and the difference was statistically significant(P<0.05).Compared with the model group,calycosin-7-glucoside,puerarin and their compatibility could significantly reduce the levels of CHO and TG in IR adipocytes,and the difference was statistically significant(P<0.05).3.Effects on adipokines in the insulin resistant 3T3-L1 adipocytes: Compared with the normal group,the levels of APN and LEP in the model group were decreased significantly,and the level of TNF-α was increased in the model group.And the difference was statistically significant(P<0.05).Compared with the model group,calycosin-7-glucoside,puerarin and their compatibility could significantly increase the levels of APN and LEP,while decrease the level of TNF-α in the insulin resistant 3T3-L1 adipocytes.And the difference was statistically significant(P<0.05).Calycosin-7-glucoside and puerarin could increase the levels of APN,LEP and decrease the level of TNF-α synergistically,and the combination effect was the best.4.Effects on AMPK and GLUT-4 in the insulin resistant 3T3-L1 adipocytes:(1)Effects on the AMPK in the insulin resistant 3T3-L1 adipocytes: Compared with the normal group,the level of p-AMPK/AMPK was significantly lower in the model group,and the difference was statistically significant(P<0.05).Compared with the model group,calycosin-7-glucoside,puerarin and their compatibility could significantly increase the level of pAMPK/AMPK in the insulin resistant 3T3-L1 adipocytes,and the difference was statistically significant(P<0.05).(2)Effects on GLUT-4 in the insulin resistant 3T3-L1 adipocytes: Compared with the normal group,the level of GLUT-4 was decreased in the model group significantly.Calycosin-7-glucoside,puerarin and their compatibility could increase the GLUT-4 content in the insulin resistant 3T3-L1 adipocytes.Conclusion:1.“Spleen deficiency” is the main pathogenesis of diabetes and fatty formation.Diabetes may be treated from the spleen.2.AF and calycosin-7-glucoside are the active components of Radix Astragali in lowering blood glucose and blood lipids(CHO and TG).3.PF and puerarin are the active components of Pueraria lobate in lowering blood lipids(CHO and TG).4.Radix Astragali-Pueraria lobate compatibility and calycosin-7-glucoside-puerarin compatibility can reduce blood glucose,and the combination effect was the best.The mechanism of Radix Astragali-Pueraria lobate compatibility and calycosin-7-glucosidepuerarin compatibility to reduce blood glucose may be related to regulate adipocytokines and AMPK/GLUT-4 singaling passway.5.Radix Astragali-Pueraria lobate compatibility,AF+PF and calycosin-7-glucoside-puerarin compatibility can reduce TG and CHO,and the combination effect was the best(except AF+PF).The mechanism of Radix Astragali-Pueraria lobate compatibility,AF+PF and calycosin-7-glucoside-puerarin compatibility to reduce blood lipid may be related to regulate adipocytokines and AMPK singaling passway. |
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