| Purpose:Animal experiments were conducted to observe the effect of Yitangkang on metabolism related indexes such as blood glucose,insulin,blood lipids,and the pathological changes of liver and skeletal muscle,pigment epithelium-derived factor,key proteins of PI3K/Akt and AMPK signaling pathway in type 2 diabetic rats.Cell experiments were conducted to observe the regulatory effects of medicated serum of Yitangkang on the morphology,viability,glucose consumption,PEDF and key proteins of PI3K/Akt and AMPK signaling pathways of Hep G2 cells and 3T3-L1 cells with insulin resistance.To study the effect of Yitangkang on glucose and lipid metabolism and the improvement of insulin resistance in diabetic rats,to preliminary explore its mechanism of action,aiming to provide experimental evidence for Chinese medicine to treat diabetes,and strive to find new therapeutic targets.Material and method:Paper one:60 male SPF Sprague-Dawley(SD)rats were divided into 15 in the normal control group and 45 in the high-fat diet group according to the random number table method.The rats in normal control group were fed by normal diet and the rats in high-fat diet group were fed by high-fat and high-sugar diet.After 8 weeks,fasting for 12 hours,the T2 DM rat model was established by streptozotocin intra-abdominal injection.Afert modeling,all rats were randomly divided into model control group(15),Yitangkang group(15),and metformin group(15).According to the equivalent dose conversion formula of rat and human,the gavage dosage of rat was calculated.According to the equivalent dose conversion formula of rat and human,the gavage dosage of rat was calculated.On the second day after the successful modeling,the rats in each group were given oral gavage,the normal control group and the model control group were given gavage of 0.9% normal saline 10ml/kg/d,the Yitangkang group was given gavage of 9.45ml/kg/d,and the metformin group was given gavage of 9ml/kg/d once a day for 8 weeks.Weight and blood sugar were measured every 2weeks.After the intervention,Hb A1 c in erythrocyte lysate and FINS in plasma were detected by ELISA,TC,TG,LDL-C,HDL-C in plasma were detected by colorimetric measure,liver and skeletal muscle morphological change were observed by HE staining,insulin resistance was detected by Hyperinsulinemic-Euglycemic Clamp.Paper two:The animals,medicines,feeding,grouping and administration methods were the same as paper one.ELISA was used to detect the content of PEDF in serum,liver and adiposes of rats in each group,western-blot method was used to detect the expression levels of PI3 K,Akt,GSK-3? protein and its phosphorylation in liver and adiposes,AMPK,ATGL protein and its phosphorylation in liver and skeletal muscle tissue of each group.Immunofluorescence detection was used to detect the phosphorylated expression of PI3 K,Akt,GSK-3?,AMPK,ATGL in liver tissues.Paper three:30 male SPF SD rats were adaptive feeding for one week,then divided the rats into 10 in the normal group,10 in the Yitangkang group and 10 in the metformin group according to the random number table method.Intragastrically with 10ml/kg saline,18.9ml/kg Yitangkang decoction,and 18ml/kg metformin tablet solutionblank,once a day for 7 days to prepare blank serum,medicated serum of Yitangkang and medicated serum of metformin.Insulin resistance model of Hep G2 cells and 3T3-L1 cells were established and divided into normal group,model group,Yitangkang group,metformin group,PEDF group,and different concentrations of drugs were used for intervention.The cell morphology was observed by inverted microscope,cell viability was measured by means of CCK-8,and glucose consumption was measured by means of Glucose Oxidase,PEDF expression in cells was measured by ELISA and immunofluorescence,expression levels of PI3 K,Akt,GSK-3,AMPK,ATGL protein and its phosphorylation expression in cells were measured by western-blot.Results:Paper one:1.Yitangkang has a regulating effect on food intake,water consumption,urine volume,etc of T2 DM rats.2.Compared with the normal control group,the weight of rats increased obviously ineach groups before intervention(P<0.01).The weight of rats in Yitangkang group and metformin group lossed significantly in the 2nd and 4th week after intervention compared with model control group(P<0.01).The weight of rats in model control group lossed more than Yitangkang group and metformin group in the 6nd and 8th week after intervention(P<0.01).With the increase of the intervention time,the weight loss of rats in Yitangkang group and metformin group gradually stabilized,there was no statistically difference between the two groups(P>0.05).3.Compared with the normal control group,the FBG of rats increased obviously in the model control group before intervention(P<0.01).The FBG decreased significantly in Yitangkang group and metformin group during after the 2th,4th,6th,8th week of intervention(P<0.01).After 6 and 8 weeks of intervention,the FBG in the Yitangkang group was higher than that in the metformin group,and the difference was statistically significant(P<0.05).4.Compared with the normal control group,the rats' Hb A1 c and FINS increased obviously in the model control group before administration(P<0.01).The levels of Hb A1 c and FINS decreased significantly in the Yitangkang group and metformin group,there was no statistically difference between the two groups(P>0.05).5.Compared with the normal control group,the rats' TC,TG and LDL-C increased obviously and HDL-C decreased obviously in the model control group(P<0.01).The TC,TG and LDL-C decreased obviously and HDL-C increased obviously in the Yitangkang group and metformin group compared with the model control group,decreased significantly with significiant differences(P<0.01).Compared with the Yitangkang group,the TC level in the metformin group was higher(P<0.01).There was no significant difference in the levels of TG,LDL-C,HDL-C between the Yitangkang group and the metformin group(P>0.05).6.Hyperinsulinemic-euglycemic clamp test showed,compared with the normal control group,the steady-state BG in the model control group was significantly increased,and the GIR was significantly decreased.Compared with the model control group,the rats in the two administration groups had a significant decrease in BG and a significant increase in GIR,but there was no significant difference between the two groups(P> 0.05).7.HE staining showed the liver cells of Yitangkang group and metformin group were arranged more regularly,and the pathological changes such as cell volume and lipid vacuoles were significantly reduced.,the damaged muscle fibers showed different degrees of improvement,and the nuclear disturbance and displacement were reduced.Paper two:1.The PEDF content in serum of model control group was significantly increased,while decreased significantly in liver and fat tissues compared with normal control group(P<0.01).The PEDF content in serum decreased while increased significantly in liver and fat tissues of Yitangkang group and metformin group compared with the model control group(P<0.01),there was no significant difference between the two treatment groups(P> 0.05).2.Western-blot and immunofluorescence results showed that,there were no significant differences in the expression levels of PI3 K,Akt,GSK-3? in the liver of the each groups compared with normal control group(P>0.05),while the expression of p-PI3K?p-Akt?p-GSK-3? decreased(P<0.01).The expression of p-PI3 K,p-Akt and p-GSK-3? increased in the liver significantly in Yitangkang group and metformin group compared with the model control group(P<0.01),there was no significant difference between the two treatment groups(P> 0.05).3.Western-blot and immunofluorescence results showed that,there were no significant differences in the expression levels of AMPK,ATGL in the liver and fat tissues of the each groups compared with normal control group(P>0.05),while the expression of p-AMPK,p-ATGL decreased(P<0.01).The expression of p-AMPK,p-ATGL increased in the liver and fat tissues significantly in Yitangkang group and metformin group compared with the model control group(P<0.01),there was no significant difference between the two treatment groups(P> 0.05).Paper three:1.Compared with the normal group,cell viability and glucose consumption in the model group of Hep G2 cells and 3T3-L1 cells were significantly decreased,the differences were statically significant(P<0.01),while the cell viability and glucose consumption of each groups in Hep G2 cells and 3T3-L1 cells increased differently compared with the model group(P<0.01),but there was no significant difference between the three groups(P> 0.05).2.Western-blot and immunofluorescence results showed that,Hep G2 cells: compared with the normal group,the PEDF level in the model group was significantly decreased(P<0.01),while PEDF level of each intervention groups increased differently compared with the model group(P<0.01).Among them,PEDF group was significantly higher than the Yitangkang group and the metformin group(P <0.01),and there was no statistical difference between the Yitangkang group and the metformin group(P> 0.05).3T3-L1 cells: compared with the normal group,the PEDF level in the model group was significantly decreased(P<0.01),the PEDF level of each intervention groups increased differently compared with the model group(P<0.01).Among them,the Yitangkang group and the PEDF group was significantly higher than the metformin group(P <0.05,P <0.01),there was no statistical difference between the Yitangkang group and the PEDF group(P> 0.05).3.Hep G2 cells: compared with the normal group,the expression levels of PI3 K,Akt and GSK-3? in the model group were not significantly different(P>0.05),while the levels of p-PI3 K,p-Akt and p-GSK-3? significantly decreased(P<0.01).The levels of p-PI3 K,p-Akt and p-GSK-3? in each intervention groups increased differently compared with the model group(P<0.01).Among them,the p-PI3 K level of Yitangkang group and PEDF group was higher than that of metformin group(P<0.01);The p-Akt level of PEDF group was higher than that of metformin group(P<0.01),there was no statistically significant difference between Yitangkang group and metformin group(P>0.05);There was no significant difference in p-GSK-3? levels between the three groups(P>0.05).3T3-L1 cells: compared with the normal group,the expression levels of PI3 K,Akt and GSK-3? in the model group were not significantly different(P>0.05),while the levels of p-PI3 K,p-Akt and p-GSK-3?significantly decreased(P<0.01).The levels of p-PI3 K,p-Akt and p-GSK-3? in each intervention groups increased differently compared with the model group(P<0.01).Among them,the p-PI3 K level of the Yitangkang group and the PEDF group was significantly higher than that of the metformin group(P<0.05,P<0.01);The p-Akt and p-GSK-3? levels of the PEDF group were significantly higher than those of metformin.Group(P<0.05),there was no significant difference between Yitangkang group and metformin group or PEDF group(P>0.05).4.Hep G2 cells: compared with the normal group,the expression levels of AMPK,ATGL in the model group were not significantly different(P>0.05),while levels of p-AMPK,p-ATGL significantly decreased(P<0.01).The levels of p-AMPK,p-ATGL in each intervention groups increased differently compared with the model group(P<0.01).Among them,the p-AMPK level in the PEDF group was significantly higher than that in the Yitangkang group and the metformin group(P<0.01).There was no statistical significance between the Yitangkang group and the metformin group(P>0.05);The p-ATGL level in PEDF group was higher than the metformin group(P<0.05),there was no statistical significance between the Yitangkang group and the metformin group or PEDF group(P>0.05).3T3-L1 cells: compared with the normal group,the expression levels of AMPK,ATGL in the model group were not significantly different(P>0.05),while levels of p-AMPK,p-ATGL significantly decreased(P<0.01).The levels of p-AMPK,p-ATGL in each intervention groups increased differently compared with the model group(P<0.01).Among them,the p-AMPK level of PEDF group was significantly higher than that of Yitangkang group and metformin group(P<0.01),there was no statistically significant difference between Yitangkang group and metformin group(P>0.05);There were no significant differences in p-ATGL levels between the three intervention groups(P>0.05).Conclusion:1.Yitangkang could reduce blood glucose levels,regulate glucose metabolism,and improve insulin resistance in T2 DM rats.2.Yitangkang could reduce blood lipid levels and improve lipid metabolism disorders in T2 DM rats.3.Yitangkang might improve insulin resistance in T2 DM rats by upregulating PEDF expression in liver and fat tissues and activating PI3K/Akt and AMPK signaling pathways. |
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