| Background: Diabetes has become a serious global public health problem,especially in China.According to the Global Diabetes Map released by the 2017 International Diabetes Federation(IDF),the number of diabetes patients worldwide in 2017 was approximately 425 million,and the number of adults with diabetes in China reached114 million,ranking first in the world,and the number is expected to grow to 120 million in 2045.90%-95% of them are Type 2 diabetes(T2DM).Therefore,the prevention and treatment of T2 DM has become a current research focus.However,the mechanism of T2 DM has not yet been fully elucidated.Since one of the most typical manifestations of T2 DM is an abnormal increase in blood sugar,the development of related drugs focuses on lowering blood sugar.With the deepening of people’s understanding,it is found that simply lowering blood sugar can not solve the fundamental problem of T2 DM treatment.With the improvement of people’s living standards and the improvement of dietary standards,people also unconsciously eat excessively.Excessive calorie intake is one of the important reasons for the soaring prevalence of obesity and T2 DM in China in recent years.Therefore,controlling calorie intake through diet has become a research hotspot in the treatment of T2 DM and obesity.In the early stage,our research group conducted research on the decomposing of Banxia Xiexin Decoction.The study found that the sweet medicine group(ginseng,licorice,jujube,GWZ)can effectively inhibit the appetite of T2 DM rats modeled by STZ and improve insulin resistance and lower blood sugar at the same time.However the mechanism is not clear.This experiment intends to further explore the mechanism of GWZ appetite suppression on the basis of this research.Objections:Based on the previous research results of the research group,this project further clarified the appetite suppression effect of GWZ on T2 DM rats modeled by STZ,and focused on exploring whether GWZ can regulate peripheral appetite hormone to achieve the effect of appetite suppression.Using network pharmacology and molecular docking analysis technology to predict and analyze the specific mechanism of GWZ appetite suppression,conduct experimental verification,clarify the potential molecular mechanism of GWZ appetite suppression and screen potential effective ingredients.From the perspective of gastrointestinal appetite hormones,explore the theoretical connotation of GWZ "supporting the spleen and suppressing the stomach",and explore its dose-effect relationship to provide reference for clinical application.method:In this study,SD rats modeled by STZ were used as experimental subjects,and they were divided into(Normal Control group,NC),Sitagliptin group(T2DM rats with Sitagliptin group,TS),and high-dose sweet medicine group(T2DM rats with Highdose-GWZ group,HG),medium-dose sweet medicine group(T2DM rats with Highmiddle-GWZ group,MG),low-dose sweet medicine group(T2DM rats with Low-doseGWZ group,LG)and(Diabetes control group,DC).Observe and record the general conditions of the rat’s coat color,food intake,drinking water,and mental status.Evaluate the improvement of GWZ on the basic condition of T2 DM and evaluate T2 DM model through HE staining pathological sections,fasting insulin levels,fasting blood glucose levels,and insulin resistance index HOMA-IR.Elisa was used to detect peripheral appetite hormone PYY,NPY,Leptin,Ghrelin,GLP-1 and other indicators to assess the effect of GWZ on peripheral appetite-related hormones.Combined with the above experimental results,combined with type 2 diabetes and appetite-related targets,explore its possible potential mechanisms through network pharmacology techniques,and conduct laboratory tests to verify the conjectures of key molecules.results:1.The effect of GWZ on the basic condition of T2 DM ratsDC rats have poor coat color,fatigue,and sleepiness.After GWZ gavage,the above symptoms are significantly improved.The food intake of rats in the DC group was significantly higher than that in the NC group,while the food intake of rats was significantly reduced after GWZ gavage,suggesting that GWZ can significantly inhibit the appetite of T2 DM rats modeled by STZ and reduce food intake.In terms of body weight,the weight of the DC group rats showed a significant upward trend in the early stage of T2 DM modeling.The body weight of rats in DC group showed an obvious trend of increase in the early stage of T2 DM modeling.Since the state of hyperglycemia was not under control,the body weight began to decrease in the later stage.GWZ could significantly slow down the early weight rise of T2 DM rats and the trend of weight loss caused by hyperglycemia in the later stage,which may be attributed to the control of blood glucose by GWZ.In terms of fasting blood glucose,GWZ can effectively reduce the fasting blood glucose level of T2 DM rats,and there is an obvious dose-effect relationship,and the hypoglycemic effect is positively correlated with the dose;The results of pancreatic HE staining showed that GWZ could protect islet cells and improve the damage of islet cells caused by STZ.The protective effect of islet cells was positively correlated with the dose of GWZ.GWZ can significantly reduce the level of insulin resistance in T2 DM rats,but it has no significant effect on promoting insulin secretion.2.The influence of GWZ on the peripheral orexinAfter STZ modeling,the peripheral appetite hormone levels of rats in DC group were significantly changed compared with those in NC group,most of which were synchronized with the changes in appetite.Compared with NC group,the levels of GLP-1 and PYY in DC group were significantly decreased,while the levels of Ghrelin and NPY in DC group were significantly increased.Although LEP is an appetite suppressant hormone,the level of LEP in DC rats was significantly higher than that in DC rats,and the mechanism may be related to leptin resistance.GWZ significantly affected most of the appetite hormones.Compared with DC group,GWZ significantly increased the levels of GLP-1 and PYY,and decreased the levels of Ghrelin and LEP,but had no significant effect on the level of NPY.GWZ can significantly reduce the level of peripheral LEP,the mechanism may be related to the improvement of leptin resistance.3.Network pharmacology prediction resultsBy combining the related targets of the preliminary results of the combination with T2 DM and appetite related targets,the mechanism was explored.Through multiple databases,analysis platforms and software,GWZ contained 31 components related to appetite and T2 DM regulation,and a total of 43 targets were involved.Cluster analysis GO and KEGG analysis were carried out on the target,the results show that GLP-1,PYY,LEP,Ghrelin,NPY and their related targets are clustering to the same function module,the module of biology function mainly involved in appetite regulation and mechanism is mainly involved in the regulation of neuropeptide activity,signaling pathways mainly involves(3 ’,5 ’-cyclic adenosine monophosphate,cAMP)signaling pathway.4.Molecular mechanism verificationThe levels of cAMP and NPY in hypothalamus,and cAMP in stomach and colon were detected.The results showed that the level of hypothalamus cAMP was significantly decreased and the level of NPY was significantly increased in DC group compared with NC group.After gavage of GWZ,the level of cAMP in hypothalamus was significantly increased,while the level of NPY was significantly decreased.The level of cAMP in gastric tissue of DC group was significantly higher than that of NC group,and the level of cAMP in colon tissue of DC group was significantly lower than that of NC group.GWZ showed bidirectional regulation of cAMP in stomach and colon.The results showed that cAMP level in stomach tissue was significantly decreased compared with DC group,while cAMP level in colon tissue was significantly increased compared with DC group after gavage of GWZ.5.Screening of Potential Active IngredientsThe 31 potential components of GWZ were docked with adenylate cyclase,and the results showed that most of GWZ components could effectively bind to AC,among which quercetin,licorice O,olenolic acid and Kanzonol F had the strongest binding ability,that was worth further exploration.conclusion:1.After STZ modeling,the rats showed symptoms of spleen deficiency(discolored hair,mental tiredness,fatigue and tendency to lie down)and hyperactivity of stomach(hyperappetite),while GWZ could improve the hair color and mental state of the rats and inhibit the appetite of the rats by "supporting the spleen and suppressing the stomach".2.GWZ can reduce fasting blood glucose level and reduce insulin resistance in T2 DM rats modeled by STZ,but it does not show significant effect of promoting insulin secretion.The hypoglycemic mechanism may be mainly related to the inhibition of appetite and the improvement of insulin resistance.3.GWZ decoction can bidirectional regulation of gastrointestinal appetite hormone,increase the secretion of PYY and GLP-1 which is mainly secreted by colon,and decrease the secretion of Ghrelin which is mainly secreted by stomach.At the same time,the promotion and inhibition of the secretion of gastrointestinal appetite hormone were synchronized with the intracellular cAMP level,suggesting that the bidirectional regulation effect might be achieved by regulating intracellular cAMP level.Gastrointestinal secretion function is the embodiment of spleen and stomach function of TCM,and the mechanism of GWZ "supporting spleen and inhibiting stomach" may be related to the regulation of cAMP level changes.4.GWZ decoction had clear dose-effect difference in hypoglycemic and protective effects on islet cells,and the high-dose group had the most obvious hypoglycemic and protective effects.There was also a significant dose-effect relationship in the secretion of peripheral appetite hormone in GWZ.Compared with the low-dose group,the highdose group could increase the content of cAMP in colon cells and thus increase the secretion of GLP-1 and PYY more efficient.Compared with the low-dose group,the high-dose group of GWZ could decrease the level of peripheral LEP more efficient.GWZ showed no significant dose-effect relationship in inhibiting the secretion of Ghrelin,and there was no statistical difference between the traditional Chinese medicine intervention groups5.The molecular docking results showed that quercetin,oleanolic acid,glycyrrhizin O,and Kanzonol F have the strongest binding ability with AC,but there are few related experimental studies worthy of further exploration. |
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