| Type 2 diabetes(Type 2 diabetic mellitus,T2DM)is a chronic metabolic disease characterized by insulin resistance.As of 2014,China has become the country with the largest number of diabetic patients in the world,and about 90% of the diabetic patients are type 2 diabetes.Berberine is a quaternary ammonium alkaloid extracted from the traditional Chinese medicinal material Coptis chinensis Franch.It has a spectrum of pharmacological effects like antibacterial,anti-inflammatory,antitumor,antioxidant,anti-atherosclerosis andso on.Studies have shown that berberine also has a good effect of lowering blood glucose,which is recognized as the active ingredient of traditional Chinese medicine with exact hypoglycemic effect.However,due to its multi-target effect,its hypoglycemic mechanism has not been fully defined.Glycogen is a polysaccharide substance connected by glucose.Because of its structural similarity to amylopectin,it is also called animal starch.It is an effective form of energy storage for the body.The liver glycogen molecular structure is divided into three levels: the primary structure is a single-chain chain of glucose connected by α-1,4-glycosidic bonds;the secondary structure is formed by the glucose chain connected by α-1,6-glucosidic bonds,which has been called β particles;the tertiary structure is a polymer of β particles that are polymerized by glycogenin protein and hydrogen bonds,which has been called α particles.Glycogen in liver tissues is mostly stored in the form of α particles.Under certain physiological conditions,α particles will be degraded into β particles.At the same time,α particles and β particles can be degraded into glucose that could provide energy to the body.But under some pathological conditions,such as the diabetic state,the instability of the tertiary structure of glycogen may cause an abnormally high degradation rate of glycogen,which may lead to pathological features such as glucose metabolism disorders.Therefore,regulating blood glucose levels by improving glycogen structure may also be an effective strategy to alleviate diabetic conditions.Glycogen phosphorylase(GP)is a key rate-limiting enzyme in the glycogen metabolism of the liver.It can catalyze the breakdown of α-1,4-glycosidic bonds at the end of glycogen chains and is an effective target for relieving diabetes,but whether berberine could act on GP has not been further studied.Based on the two aspects of glycogen phosphorylase and liver glycogen structure,this study explored the mechanism of berberine in regulating liver glucose metabolism in diabetic mice.This could not only provide a theoretical basis for the further clinical application of berberine,and also provide a new target for the development of hypoglycemic agents.Part Ⅰ: Pharmacodynamics of berberine in relieving hyperglycemia in type 2 diabetic miceThe db/db mice were used as diabetic model mice,metformin(300 mg/kg)was used as a positive control agent,and berberine(300 mg/kg)was administered orally for 8 consecutive weeks for intervention to investigate the effects of berberine on diabetes symptoms.During the experiment period,fasting blood glucose,body weight,food and water intake of the mice were measured,and the changes of db/db mice under the intervention of berberine were evaluated.After the experiment,the serum glucose-regulating hormone levels were measured.The effect of berberine on the glucagon level and insulin sensitivity of db/db mice was evaluated.At the same time,the liver tissues of mice were taken,and the liver damage factors and glycogen content in the liver were measured.Combined with H&E and PAS staining of liver tissue,the effects of berberine on liver structure,function and glycogen content in db/db mice were evaluated.The results in this section indicate that berberine can effectively reduce the abnormally elevated blood glucose levels,glucagon levels,and insulin levels,increase insulin sensitivity,relieve insulin resistance and improve the symptoms of obesity,drink more,eat more and urinate in db/db mice.At the same time,berberine can also effectively reduce serum liver injury factor levels in db/db mice with diabetes,improve liver function in db/db mice,and reduce excessive fat and glycogen deposition in liver tissues.Part Ⅱ: The mechanism of berberine improving liver glucose metabolism in type 2 diabetic miceAfter the experiment,the liver tissues of mice were taken as test samples,and the activities and expression levels of glycogen catabolism,gluconeogenesis and related signaling pathway proteins in the liver tissues of mice were detected by ELISA and Western Blot to explore the mechanism of berberine improving liver glucose metabolism in db/db mice with diabetes.The results showed that berberine could effectively inhibit the activities of key rate-limiting enzymes,GP and GDBE,of the glycogen catabolism process of diabetes model db/db mice,and could effectively down-regulate key proteins on the c AMP-PKA signaling pathway in liver tissues and thereby inhibiting the protein expression of GP.In addition,berberine can effectively inhibit the activity of key rate-limiting enzymes,PEPCK and G-6-Pase,of the gluconeogenesis in db/db mice.Besides,it can also inhibit the expression of HNF-4α and p-AMPK to affect protein expression of PEPCK and G-6-Pase.The results in this section indicate that berberine can inhibit the catabolism of liver glycogen by down-regulating the c AMP-PKA signaling pathway,and at the same time can inhibit the gluconeogenesis by affecting the expression of HNF-4α and p-AMPK.Berberine can effectively improve glycometabolism,reduce blood glucose,and alleviate symptoms of diabetes model db/db mice by effectively inhibiting liver glycogen catabolism and gluconeogenesis.Part Ⅲ: The effect of berberine on the structure of liver glycogen in type 2 diabetic miceSucrose density gradient centrifugation was used to extract mouse liver glycogen,and the size distribution,density distribution,and chain length distribution of liver glycogen were measured by TEM,SEC and FACE methods.The effect of berberine on the liver glycogen structure in db/db mice was evaluated.The results showed that liver glycogen of all mice existed as α particles before DMSO treatment,while the db/db mouse liver glycogen was extremely susceptible to structural changes after DMSO treatment and was degraded into more β particles,showing a damaged and unstable glycogen structure.Berberine can effectively repair the damaged structure of liver glycogen in diabetic db/db mice,so that it still exists as intact alpha particles after DMSO treatment.In addition,there is a high proportion of long straight chains in the liver glycogen of db/db mice.The presence of a high proportion of long straight chains makes the secondary and tertiary structures not tightly connected and would be easily degraded by external forces.Berberine can significantly change the chain length distribution of liver glycogen in diabetic db/db mice,increase the proportion of its short-chain branches,and make its structure more compact.The results in this section indicate that berberine can change the chain length distribution of glycogen in diabetic db/db mice,repair the damaged structure of glycogen,and improve the structural stability of glycogen.This could make it not easy to degrade to glucose,improve its glucose metabolism,reduce abnormally elevated blood glucose in diabetic db/db mice,and alleviate its symptoms of diabetes.Part Ⅳ: The mechanism of berberine improving liver glucose metabolism in type 2 diabetic miceSucrose density gradient centrifugation was used to extract mouse liver glycogen.In order to evaluate the effect of berberine on liver glycogen degradation rate and its affinity to GP in db/db mice with diabetes,we simulated the in vivo degradation process of mouse liver glycogen in vitro to detect the degradation rate of different mouse liver glycogen under the action of GP.The results showed that the rate of liver glycogen degradation in diabetes model db/db mice is much higher than that of healthy mice,and the affinity with GP is much higher than that of healthy mice.Berberine can significantly reduce the degradation rate of liver glycogen in diabetic db/db mice and reduce its affinity with GP.The results in this section indicate that berberine can effectively reduce the affinity of liver glycogen and GP of the diabetic model db/db mice,reduce its degradation rate,improve the abnormal glucose metabolism of diabetic db/db mice,and reduce its excessively high blood glucose levels to relieve their diabetes symptoms. |
