Study On The Antidiabetic Activity And Mechanism Of Action Of The Ethnic Medicine Morus Nigra L.

This study of Morus nigra L.is an important folk medicine for Uyghur people and the preferred health food for special people with high blood lipids,with a long history in folklore.The leaves,bark,branches and fruits of Morus nigra L.have good medicinal functions and therapeutic effects;however,the material basis of its medicinal effects and its mechanism of action have not been elucidated.In this study,we investigated the therapeutic effects and mechanisms of Morus nigra L.branches and leaves on high-fat diet(HFD)combined with streptozotocin(STZ)induced type 2 diabetes mellitus(T2DM)mice to provide scientific basis for their rational and effective development and utilization.Object: In this study,we investigated the antidiabetic activity of Morus nigra L.branch ethyl acetate extract(MNT-EA)and Morus nigra L.leaves(MNL-EA),the chemical composition of the active parts of MNT-EA and MNL-EA,the antidiabetic activity of Morus nigra L.at the molecular,cellular and animal levels,and the elucidation of its pharmacological substance basis and its mechanism of action,to provide a scientific basis for the comprehensive evaluation of its medicinal value.Methods: Study on the anti-diabetic activity and mechanism of MNT-EA:(1)rapid acquisition of information on the main chemical components of MNT-EA using UPLC-Q-TOF/MS method in combination with UNIFI information management platform;(2)molecular docking simulation of 21 compounds in MNT-EA with Glucose transporter 4(GLUT4)targets using Auto Dock Tools 1.5.7 software;(3)In vitro activity studies of MNT-EA were performed using confocal microscopy to detect stable expression of rat skeletal muscle L6 cells based on the fluorescent protein IRAP-m Orange,combined with glucose uptake as well as an activity screening system by Western blot technique;(4)The HFD combined with STZ-induced T2 DM mice model was used to study the role of MNT-EA in the treatment of T2 DM and its mechanism.Study on the anti-diabetic activity and mechanism of MNL-EA:(1)rapid acquisition of information on the main chemical components of MNL-EA using the UPLC-Q-TOF/MS method in combination with the UNIFI information management platform;(2)study of the therapeutic effect of MNL-EA on T2 DM by HFD combined with STZ-induced T2 DM mouse model;(3)histopathological examination of the effects of MNL-EA on liver and adipose tissue;(4)Detection of the expression levels of PPARγ and C/EBP-α,Beclin-1,LC3 II/I and P62 in the liver and the effects on AMPK/m TOR pathway by Western blot technique.Result: Study on the anti-diabetic activity and mechanism of MNT-EA:(1)it was found that there were 21 main chemical constituents in MNT-EA,including 14 kinds of flavonoids and 7 kinds of stilbenes;(2)the interaction of the compounds with the proteins was dominated by both hydrogen bonding and hydrophobic interactions,with most of the hydroxyl positions(including the isopentene group)in the compound structure bound to the active site;(3)MNT-EA significantly up-regulated glucose uptake and GLUT4 protein expression in L6 cells and promoted GLUT4 translocation in IRAP-m Orange L6 cells.p-AMPK and p-PKC protein expression was also up-regulated by MNT-EA in L6 cells,and AMPK and PKC were inhibited in L6 cells using Compound C and G?6983 inhibitors,respectively.The effect of MNT-EA in promoting GLUT4 protein expression was found to be blocked;(4)fetal bovine serum(FBG)level in mice was significantly decreased and insulin resistance(IR)was significantly improved.serum levels of total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),and Free fatty acids(FFA)in T2 DM mice were significantly decreased after MNT-EA administration.Histopathological examination revealed that MNT-EA reduced hepatic steatosis and improved adipocyte hypertrophy and islet lesions in T2 DM mice.In addition,MNT-EA significantly upregulated p-AMPK,p-PKC and GLUT4 protein expression in liver,adipose and skeletal muscle.Study on the anti-diabetic activity and mechanism of MNL-EA:(1)it was found that there were 13 main chemical constituents in MNL-EA,including 8 kinds of flavonoids;(2)MNL-EA promoted the decrease of FBG level and significant improvement of glucose loading capacity in T2 DM mice,and the serum levels of TC and TG in T2 DM mice were significantly decreased and HDL-C level was increased.The serum levels of alanine aminotransferase(ALT)and glutamate aminotransferase(AST)in the MNL-EA low-dose group were significantly decreased;(3)Histopathological examination revealed that MNL-EA improved hepatic steatosis and adipocyte hypertrophy in T2 DM mice;(4)MNL-EA down-regulated the expression levels of PPARγ and C/EBP-α in liver tissues,significantly increased Beclin-1 levels and LC3 II/I ratio,decreased p62 levels,significantly up-regulated p-AMPK expression as well as inhibition of p-m TOR expression.Conclusion: This study for the first time confirmed that MNT-EA,rich in flavonoids and stilbene analogues,rich in flavonoids and streptavidin compounds,exerted therapeutic effects on T2 DM by activating AMPK and PKC signaling pathways in liver,adipose and skeletal muscle,promoting GLUT4 expression and translocation,and improving glucolipid metabolism.MNL-EA regulated hepatic lipid metabolism by down-regulating PPARγ and C/EBP-α expression,and AMPK/m TOR pathway induced hepatic autophagy,thereby improving hepatic steatosis,providing a basis for the development of new anti-diabetic drugs.