To Explore The Mechanism Of Mulberry Leaf In The Treatment Of Type 2 Diabetes Based On Proteomic And Transcriptomic Technology

Background:Type 2 diabetes mellitus(T2DM)is a metabolic disease characterized by absolute or relative insufficiency of insulin secretion and decreased sensitivity of target organs to insulin.Clinical manifestation of T2DM is "three more and less",which means drinking more,eating more,urinating more and weight loss.Skeletal muscle is one of the main target organs for the body to take up and utilize glucose and fatty acids,an important peripheral target organ for insulin action,and one of the target organs damaged by hyperglycemia.With improvement of modern living standards,the incidence of T2DM is increasing.Its pathogenesis is complex,characterized by network and multi-channel.Common chemical drugs have hypoglycemic effect.However,the target is relatively single,and long-term medication is accompanied by adverse reactions.Traditional Chinese medicine(TCM)has the characteristics of "multi-channel,multi-component,multi-target" comprehensive effect,matching with the complex pathological mechanisms of disease,can regulate the body from the overall level,and has significant advantages in the treatment of T2DM.Mulberry leaf is the dry leaf of Morus alba L.,which is sweet,bitter,cold in nature,and located at lungs and liver meridian.It has functions of dispersing wind and heat,clearing lungs and moisturizing dryness,and clearing liver.It is used to deal with wind-cold,lung heat,dry cough,dizziness,headache and other symptoms.Mulberry leaf,as a common medicinal and food homologous medicine used to treat T2DM,is rich in alkaloids,flavonoids,polysaccharides and other active ingredients for lowering blood glucose.Modern pharmacological studies have shown that mulberry leaf has effects of lowering blood glucose and lipids,antioxidant,preventing and treating macro vascular complications.However,the mechanisms of how mulberry leaf and mulberry leaf water extracts exert their hypoglycemic effects have not been clarified.In recent years,with the development of systems biology,omics technology has become more and more popular,which can reflect changes in the body from multiple levels and has unique advantages in exploring the mechanism of disease and drug treatment.Therefore,in this study,skeletal muscle was taken as research object,and transcriptomics and proteomics techniques were used to explore the pathological mechanism of T2DM and the mechanism of mulberry leaf in the treatment of T2DM at RNA and protein molecular level,in order to find potential targets of mulberry leaf hypoglycemic effect.Methods:This subject used mulberry leaf water extract as test drug,special pathogen free(SPF)grade male Sprague-dawley(SD)rats as test subject.The rats were fed high-sugar and high-fat diet for four weeks and then intraperitoneal injected 35 mg/kg streptozotocin(STZ)to establish T2DM rat model.Rats with blood glucose≥11.1 mmol/L were included in the experiment,and the rest were excluded.The model rats were randomly divided into model group,metformin(0.2 g/kg)group,and mulberry leaf(4.0 g/kg,best dose of efficacy)group according to blood glucose and body weight.At the same time,a normal control group was set up,with 9 rats in each group.Rats in each group were given intragastric administration as well as normal diet for 8 weeks.During the administration period,basic indicators such as fasting blood glucose,body weight,food and water intake were measured at a fixed time point every week.Oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)were conducted at the 7th and 8th week respectively.At the end of the experiment,blood serum,skeletal muscle and other tissues of rats were collected to determine the levels of glycosylated hemoglobin,insulin,free fat acid(FFA),leptin(LEP),adiponectin(ADP),total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C);the pathological changes of rat skeletal muscle were observed by HE staining;the skeletal muscle of rat was taken for transcriptomics and proteomics analysis,and the results of proteomics were verified by Western blot.Results:The pharmacodynamic experiment results of mulberry leaf treatment in T2DM rats showed that compared with normal group,rats in the model group always maintained a state of hyperglycemia,indicating that the T2DM model was established successfully and stably.Compared with the model group,fasting blood glucose in the mulberry leaf group was significantly reduced at 3 rd and 5th to 8th weeks(P<0.05 or P<0.01).Water intake of mulberry leaf group was significantly reduced at the first week of administration,and the food intake was significantly reduced at the 7th week of administration(P<0.05).There was no difference in the other weeks,and no significant change was found in body weight.Compared with the model group,the OGTT blood glucose of mulberry leaf group was significantly decreased at 0 and 15 min(P<0.05 or P<0.01),and there was no difference at other time points.The area under curve(AUC)of mulberry leaf group was significantly reduced(P<0.01).Compared with the model group,glycosylated hemoglobin of mulberry leaf group was significantly reduced(P<0.01).Compared with the model group,fasting insulin level in the mulberry leaf group was decreased,but there was no statistical difference.While the homeostasis model assessment of insulin resistance(HOMA-IR)value was significantly reduced(P<0.01).Compared with the model group,levels of TC,TG,LDL-C,FFA and LEP in the mulberry leaf group were significantly decreased(P<0.05 or P<0.01),while the contents of HDL-C and ADP were not significantly different.Compared with model group,blood glucose and AUC of mulberry leaf group at each time node of ITT showed a decreasing trend,but there was no statistical difference.Compared with model group,skeletal muscle fibers of mulberry leaf group were arranged more neatly,and muscle fiber gaps became smaller,showing a better effect of improving muscle atrophy.The results showed that mulberry leaf could improve blood glucose metabolism,insulin sensitivity,glucose and lipid metabolism disorder as well as muscle atrophy.Results of proteomics research showed that the pathogenesis of T2DM and hypoglycemic mechanism of mulberry leaf were related to lipid metabolism.Western blot was used to verify the results of proteomics.The results showed that there was lipid metabolism disorder in T2DM,and Apoal as well as Apoa4 may be the key targets for pathogenesis of T2DM and treatment of T2DM by mulberry leaf.Transcriptomics research results showed that Cox8b,Cox7a1,Cox5b,Ppp2r2d,Nr4a1,Ywhaz,and Foxo3 may be potential targets for the pathogenesis of T2DM,and Stard3,Abcal,CD36,Aplnr as well as Prkag3 may be potential targets for the treatment of T2DM by mulberry leaf.Combined analysis of omics found that the pathogenesis of T2DM was related to lipid metabolism and insulin signal transduction.And mulberry leaf treated T2DM by improving lipid transport and regulating lipid metabolism.Conclusions:Mulberry leaf had a good hypoglycemic effect,which can improve fasting blood glucose,food and water intake,glucose tolerance,glycosylated hemoglobin,insulin sensitivity,blood lipid metabolism and skeletal muscle fiber arrangement of T2DM rats;its hypoglycemic mechanism may be related to lipid regulation transport and improvement of lipid metabolism.Apoal,Apoa4,Stard3,Abcal,CD36,Aplnr and Prkag3 may be the key targets for mulberry leaf to exert a hypoglycemic effect.