| Obesity is the common pathological basis of many chronic metabolic diseases.With the increasing of obesity population and the trend of gradually younger age,obesity and its related diseases,including type-2 diabetes,coronary atherosclerosis,chronic kidney disease,high hypertension and various cancers are the main causes of death in obese patients up to now,and the potential harm of obesity is being paid more and more attention.Until now,the pathogenesis of obesity has not been clarified.It is generally believed that obesity is a systemic chronic inflammatory state in which insulin resistance participates.Insulin resistance is defined clinically as the inability of insulin to properly direct its physiological effects.Aurantio-obtusin(AUR)is one of the effective components in semen Cassiae(Sennatora(L.)Roxb)was included in the homologous species of medicine and food by the State Planning Commission in 2018.It has a long medicinal history and can be used in the treatment of glaucoma,hyperlipidemia and prevention of liver injury.In recent years,it has been found that orange cassia has the effects of anti-inflammation and regulating blood lipids,but its mechanism is not clear.In view of this,the purpose of this study is to carry out the mechanism of hesperidin in improving insulin resistance in vivo and in vitro from three aspects:whole animal,in vitro experiment and network pharmacology,so as to clarify the mechanism of AUR in improving insulin resistance.Therefore,the mechanism of improving insulin resistance by AUR is elucidated,which can provide scientific basis for the basic research and clinical application of AUR.Method:(1)The effect of hesperidin on glucose and lipid metabolism in C57BL/6J obese mice induced by high fatTwenty-four C57BL/6J mice were fed with high-fat diet to induce food-induced obesity.Then the mice were divided into normal group(NFD,n=8),model group(NFD,n=8),low concentration of AUR group(HFD+5AUR,n=8)and high AUR group of hesperidin(HFD+10AUR,n=8).The normal group and the model group were given the same dose of saline respectively,the low concentration group of AUR was given 5mg/kg AUR suspension,the high concentration group of AUR was given10 mg/kg AUR suspension.At the 6th,9th and 11th week,fasting blood glucose of mice was measured by tail-cutting method,and oral glucose tolerance of mice was measured at the 11th week.At the 12th week,the mice were killed by cervical vertebra dislocation after enucleation of eyeballs and taking blood.The liver and white adipose tissues were taken to weigh the wet weight of the organs,and the tissues were cryopreserved or fixed.Serum levels of TC,TG,HDL-C,LDL-C,ALT and AST were detected.The liver tissue was sectioned and treated with HE staining and oil red O staining.The morphological changes and lipid deposition were analyzed.(2)The mechanism of AUR on insulin resistance induced by high fat in C57BL/6J mice.Part of the frozen liver tissue was prepared into tissue homogenate,and the expression of TG and TC in liver tissue was determined by ELISA method.The white fat of liver and epididymis was weighed and added to the lysate to extract tissue protein to determine the protein concentration according to the ratio of tissue:protease inhibitor:lysate=10mg:1?L:100?L.The expression of PI3K,Akt,p-Akt and GLUT4 in liver and white adipose tissue,the expression of GLUT2 protein in liver and adipose tissue and the expression of PPAR?protein in adipose tissue were determined by Western blot.The sections of liver tissues of each group were taken and the expression of GLUT4 protein in liver tissue was detected by immunohistochemical technique.The expression of mRNA was extracted from white adipose tissue of liver and epididymis.The expression of PI3K,Akt,p-Akt,GLUT4,PPAR?and PPAR?genes in liver and white adipose tissue,and the expression of CPT-1,UCP-2,SREBP-1c,FAS and SCD-1 genes in adipose tissue were determined by fluorescence quantitative PCR.(3)To study the effect of hesperidin on the proliferation and differentiation of 3T3-L1 preadipocytes based on network pharmacology.The cells were blown and beaten to make cell suspension,the blank group had a complete culture medium of 100?L,and the remaining 100 ?L was about 5×10~3 cells per hole,and 6 multiple holes were repeated.When the cell density was 70%?80%,the blank group and the control group were added with 100?L DMEM,solution containing 0.8,1.6,3.1,6.2,12.5,25,50(?g/mL)AUR solution for 24 hours and 48 hours,respectively.The suitable concentration of AUR was screened from CCK-8.3T3-L1 cells were inoculated in 6-well plates according to the classical hormone cocktail to induce 3T3-L1 cell differentiation.During the induction of differentiation,AUR was continuously administered at doses of blank group and solvent control group:1%EtOH;0.8?g/mL AUR;3.1?g/mL AUR;6.2?g/mL AUR;12.5?g/mL AUR.The changes of cell morphology were recorded by light microscope.The differentiated cells were stained with oil red O,and the oil red O dye stained on the cells was collected to determine the absorbance.The effective compounds in semen Cassiae were screened through the pharmacological database and analysis platform of traditional Chinese medicine system.The target genes of the screened active components were matched with the disease genes,the effective compound-target gene-disease network was constructed,the protein interaction network was constructed,and the key target genes were analyzed by enrichment analysis and KEGG signal pathway analysis.Results:(1)AUR can significantly reduce the level of blood glucose and blood lipid in C57BL/6J obese mice,decrease the accumulation of lipid in liver tissue,improve the morphology of liver tissue cells,reduce the accumulation of collective white fat,and improvement of glucose and lipid metabolism disorder in C57BL/6J obese mice.AUR can improve insulin resistance by protecting liver tissue,improving body fat metabolism disorder and reducing the accumulation of white fat.(2)AUR can increase the consumption of glucose and decrease the blood glucose level of C57BL/6J obese mice through PI3K/Akt/GLUT4 insulin signal pathway.AUR can increase the expression of PPAR?and PPAR? in adipose tissue,promote the differentiation of adipocytes,increase the sensitivity of the body to insulin,promote the expression of CPT-1 and UCP-2,and promote the transformation of white fat.Inhibit the expression of SREBP-1c,FAS and SCD-1 genes in adipose tissue,inhibit adipose signal pathway,reduce lipid synthesis and improve insulin resistance.AUR promotes the expression of GLUT2 and PPAR?in liver tissue and increases the consumption of glucose in liver.AUR promotes the expression of adiponectin gene,inhibits the inflammatory pathway and then inhibits the expression of inflammatory factors,and improves the chronic inflammation caused by obesity.(3)AUR concentration of 0.8?g/mL;3.1?g/mL,1.6?g/mL,6.2?g/mL,12.5?g/mL,25?g/mL and 50?g/mL inhibited the proliferation of 3T3-L1 cells to a certain extent,and 25?g/mL and 50?g/mL had the most obvious inhibitory effect.Therefore,we chose concentration of 0.8?g/mL,3.1?g/mL,6.2?g/mL,12.5?g/mL respectively,which had certain inhibitory effect on 3T3-L1 cells,but there was no obvious cytotoxicity concentration as the drug concentration for the follow-up experiment.After induced differentiation to the 10th day,optical microscopic observation showed that the degree of cell differentiation after the intervention of AUR was higher than that of NC group and 1%EtOH group,and the higher the concentration,the greater the degree of cell differentiation.On the 11th day of induced differentiation,it was found that the lipid droplets distributed around the nucleus were stained orange-red by oil red O staining,and the intracellular lipid droplets stained by AUR increased significantly.The higher the concentration of AUR,the more the number of lipid droplets,the greater the degree of cell differentiation.Through the analysis of the results of the"drug-target-disease"network map constructed by network pharmacology,we found that the best binding effects of AUR and IR-related targets are PTGS2 and NCOA2,in which PTGS2 participates in PI3K/Akt pathway and plays a role in anti-inflammation and improving insulin resistance,while NCOA2 is a coactivator of adipocyte differentiation and has a close relationship with preadipocyte proliferation and differentiation.Conclusion:To sum up,AUR can reduce the accumulation of lipids in liver tissue,improve the morphology of liver cells,reduce the accumulation of white fat,and improve the disorder of glucose and lipid metabolism in C57BL/6J obese mice.AUR can increase the glucose transport of GLUT4and reduce the blood glucose level of C57BL/6J obese mice by activating PI3K/Akt/GLUT4 insulin signal pathway.By promoting the expression of adiponectin gene,improving the sensitivity of the body to insulin,inhibiting the response of inflammatory pathway and then inhibiting the expression of inflammatory factors,it can improve the chronic inflammation caused by obesity.AUR can promote the differentiation of3T3-L1 cells.The results of network pharmacology show that AUR can act on the two targets of PTGS2 and NCOA2 and play a role in anti-inflammation,promoting the differentiation of preadipocytes and improving insulin resistance. |
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