| Objective: Type 2 diabetes(T2D)is a chronic metabolic disease characterized by insulin resistance(IR)and abnormal lipid metabolism.At present,T2 D patients mainly maintain the stability of blood sugar through good lifestyle and drug treatment,and there is no radical cure.Long-term hyperglycemia will cause a variety of serious complications including stroke,diabetic retinopathy,etc.,affecting the quality of life of patients.Therefore,the prevention and treatment of diabetes,especially the prevention and treatment of T2 D,is particularly important.Eugenol(EU)exists in a variety of medicinal plants,and it has been confirmed that EU has anti-inflammatory,antioxidant,bacteriostatic and other pharmacological effects.However,the mechanism of EU improving IR and lipid metabolism of T2 D has not been reported,so this article aims to study the effect of EU on T2 D improvement,and explore the possible mechanism of EU anti-T2 D based on network pharmacology.Methods: Male SD rats at 5 weeks of age were randomly divided into blank group(CON),type II.diabetes group(T2D),EU low-dose group(EU10,10mg/kg),EU high-dose group(EU30,30mg/kg)and positive control metformin group(MET,180mg/kg),10 rats in each group,and the experimental group induced daily morning gavage after daily morning gavage after feeding high-fat feed combined with a single intraperitoneal injection of streptozotocin for 4 weeks,and treated for 6 weeks.The blood glucose concentration and fasting insulin concentration of rats in each group were detected,and the insulin resistance index(HOMA-IR)and insulin sensitivity index(HOMA-ISI)were calculated.The serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP)and lactate dehydrogenase(LDH)were detected in each group to evaluate liver function.HE staining observed liver pathological changes,oil red staining to detect liver lipid deposition.ELISA detects interleukin 6(IL-6)and tumor necrosis factor(TNF-α)content;kit detection of total cholesterol(TC),total triglycerides(TG),high-density lipoprotein(HDL-C)and low-density lipoprotein(LDL-C)content and oxidative stress level.In order to further study the mechanism of EU improvement of T2 D,the relevant targets of EU and T2 D diseases were obtained through TCMSP and TTD databases,and imported into STRING database for PPI network analysis,and normalized target names were imported into Metascape database for visual analysis and mapped in R language.Finally,according to the enrichment results,Real-Time PCR,Western Blot and immunohistochemistry were used to detect the m RNA and protein expression levels of PPARγ,IRS2 and GLUT2 in the livers of each group.Results: 1.EU significantly reduced the blood glucose level of T2 D rats:Compared with the CON group,the T2 D group had a significant decrease in weight gain(P<0.01),fasting blood glucose(FBG)value and glucose tolerance(AUC)value(P<0.01).Compared with the T2 D group,the weight gain factor of the EU high-dose group was significantly increased(P<0.05)compared with the T2 D group,and the blood glucose value and AUC value(P<0.05)were significantly reduced in the EU group,and the dose and time dependent,the MET group had the most significant hypoglycemic effect(P<0.001),but there was no statistically significant difference in weight gain multiple,the MET group decreased the AUC value(P<0.01)to the same extent as the EU high-dose group.2 EU improves fasting insulin levels and insulin resistance in T2 D rats: Compared with the CON group,fasting insulin(FINS)content and insulin resistance index(HOMA-IR)were significantly increased(P<0.01)and insulin resistance index(HOMA-ISI)(P<0.01)in the T2 D group;Compared with the T2 D group,the EU low-dose group significantly reduced HOMA-IR(P<0.05),and there was no significant difference(P<0.05)in FINS content and HOMA-ISI,while the EU high-dose group and MET group significantly reduced FINS and HOMA-IR(P<0.05)in T2 D rats and significantly increased HOMA-ISI(P<0.01).3.Enrichment analysis of EU and T2 D intersection targets: KEGG enrichment analysis was carried out on 101 targets co-regulated by EU and T2 D diseases through network pharmacology,and enriched to IR and lipid metabolism related to T2 D.4.EU improves liver function in T2 D rats: Compared with the CON group,the serum contents of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP)and lactate dehydrogenase(LDH)in the T2 D group increased significantly(P<0.01).Compared with the T2 D group,the serum content of ALT and ALP was reduced in the EU low-dose group(P<0.05),and the serum content of liver function-related enzymes in the EU high-dose group and the MET group was significantly reduced(P<0.05).5.EU improves liver pathological changes and inflammatory response in T2 D rats:(1)EU improves liver injury in T2 D rats: HE staining results showed that the T2 D group had disordered chordal cord and sinus arrangement,steatorosis,pathological changes of hepatocyte ballooning,and a little focal inflammatory infiltrate between the lobules.The arrangement of hepatic cords and sinuses in the MET group and the EU low-dose group was restored,and the morphology and structure of hepatocytes in the EU high-dose group were basically normal,and the steatosis and inflammatory infiltration were significantly reduced.(2)EU improves inflammation in T2 D rats: Compared with the CON group,the contents of interleukin-6(IL-6)and tumor necrosis factor(TNF-α)in blood in the T2 D group were significantly increased(P<0.01).Compared with the T2 D group,the content of IL-6 and TNF-α in serum(P<0.05)was significantly reduced in the EU high,low-dose and MET groups.6.EU improves liver lipid deposition and lipid metabolism in T2 D rats:(1)EU improves lipid deposition in T2 D rats.The results of oil red staining showed that the liver in the T2 D group had large areas of lipid droplets compared with the CON group;Compared with the T2 D group,the EU high-dose group and MET group were significantly reduced compared with the EU low-dose group.(2)EU improves lipid metabolism in T2 D rats.Compared with the CON group,the contents of total cholesterol(TC),total triglycerides(TG)and low-density lipoprotein(LDL-C)in the T2 D group increased significantly(P<0.01),and the content of high-density lipoprotein(HDL-C)decreased significantly(P<0.01).Compared with the T2 D group,the contents of TC,TG and LDL-C were significantly reduced in the EU and MET groups(P<0.05),and HDL-C in the EU high-dose group(P<0.05).7.EU improves oxidative stress levels in T2 D rats: Compared with the CON group,the contents of total superoxide dismutase(T-SOD)and reduced glutathione(GSH)in the T2 D group were significantly reduced(P<0.05),and the content of malondialdehyde(MDA)was significantly increased(P<0.01).Compared with T2 D,the EU low-dose group increased T-SOD content and decreased MDA content(P<0.05);The contents of T-SOD and GSH in the EU high-dose group and the MET group increased significantly(P<0.05),and the MDA content decreased significantly(P<0.01).8.EU activates the expression of PPARγ/IRS2/GLUT2 in the liver of T2 D rats:(1)EU up-regulates the expression of peroxisome proliferators-activated receptor γ(PPARγ)in T2 D rats: According to the PPI of the EU and T2 D intersection genes,PPARγ is associated with lipid metabolism and IR and the node interaction value is large.Further at the protein level,PPARγ expression in the T2 D group decreased(P<0.05)compared with the CON group;Compared with the T2 D group,PPARγexpression was significantly increased in the EU group(P<0.01),and there was no significant statistical difference in the MET group(P<0.05).Immunohistochemistry can observe the expression of the same trend.At the m RNA level,PPARγ expression was reduced in the T2 D group compared to CON(P<0.05);Compared with the T2 D group,the EU high-dose group(P<0.01)increased PPARγ expression(P<0.01)more significantly than the EU low-dose group(P<0.05),and there was no significant statistical difference(P<0.05)in the MET group.(2)EU upregulates the expression of Insulin receptor 2(IRS2)in T2 D rats: At the protein level,IRS2 expression in the T2 D group decreased compared with the CON group(P <0.01);Compared with the T2 D group,IRS2 expression was significantly increased in the EU group(P <0.05),and there was no significant difference in the MET group(P <0.05).Immunohistochemistry can also observe the expression of the same trend.At the m RNA level,IRS2 expression was reduced in the T2 D group compared to CON(P<0.05);Compared with the T2 D group,the EU high-dose group(P <0.01)significantly increased IRS2 expression,and there was no significant statistical difference between the EU low-dose group and the MET group(P <0.05).(3)EU upregulated the expression of Glucose transporter 2(GLUT2)in T2 D rats: At the protein level,GLUT2 expression was reduced in the T2 D group(P <0.05)compared with the CON group.Compared with the T2 D group,the EU high-dose group(P<0.01)increased GLUT2 expression more significantly than the EU low-dose group(P <0.05),and there was no significant statistical difference in the MET group(P<0.05).At the m RNA level,GLUT2 expression was reduced in the T2 D group compared to CON(P <0.01);Compared with the T2 D group,the expression of GLUT2 in the EU group was significantly increased(P <0.01)and the expression of GLUT2 in the MET group was significantly reduced(P <0.05),which was consistent with the immunohistochemical results of GLUT2 in the liver of animals in each group.Conclusion: EU can significantly reduce blood glucose levels and improve IR and lipid metabolism in T2 D rats,possibly by activating PPARγ and further activating IRS2 and GLUT2. |
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