| Objective:In order to provide a scientific basis for the promotion and utilization of fish oil in type 2diabetes mellitus（T2DM）patients with atherosclerosis（AS）and to slow down the progress of T2DM,this study investigated the association between AS development and some indexes in patients with T2DM by cross-sectional survey,and animal experiments were conducted to study the possible mechanism of fish oil on lipid metabolism and oxidative stress in T2DM combined with AS model rats.Methods:（1）The population cross-sectional survey:research subjects were recruited from outpatient clinics and inpatients of the Endocrinology Department of Lianshui County People’s Hospital in Huaian City,Jiangsu Province.We surveyed general clinical data through questionnaires and examined carotid artery lesions of the subjects by ultrasonography.We used a fully automated biochemical analyzer to measure blood samples,and the AS-1000 pulse wave velocity analyzer to detect the degree of arteriosclerosis in the extremities.The body compositions of the subjects were detected using body composition meters.We used the 3d 24h dietary recall survey to investigate the dietary intake of the subjects.We used one-way analysis of variance,independent sample t-test,logistic regression analysis,Kendall’s tau-b correlation analysis,receiver operating characteristic curve and other statistical methods to analyze the correlation between the level of indicators in T2DM patients and carotid artery disease.（2）Animal experiments:We involved 12 Wistar male rats in the normal control group and these rats were fed with normal feed.The T2DM model rats were developed by fed with T2DM high-sugar and high-fat diet combined with giving streptozotocin injected intraperitoneally.After the successful establishment of the T2DM model,we randomly selected 16 T2DM rats as the T2DM model control group,and fed them T2DM high-sugar and high-fat diets.We injected vitamin D3 intraperitoneally into the remaining T2DM rats and fed them with AS high-fat diet to construct a T2DM&AS model.After the successful establishment of the T2DM&AS model,the T2DM&AS model rats were divided randomly into T2DM combined AS model control group（16 rats）,low-dose fish oil group（15 rats）,medium-dose fish oil group（15 rats）,high-dose fish oil group（15 rats）,and simvastatin group（15rats）.The LFO group,the MFO group and the HFO group were given fish oil by gavage at the daily doses of 250 mg/kg,500 mg/kg and 750 mg/kg respectively.The SIM group was given simvastatin by gavage at a daily dose of 10 mg/kg.During the experiment,we weighed the fasting body weight and measured the fasting blood glucose on a fixed day once a week.At the end of 12 weeks of intervention,after fasting all rats for one night,the rats were anesthetized,the blood samples were collected from the femoral artery.The liver tissues of rats were isolated.We stored serum and liver in the-80°C ultra-low temperature freezer.We used an automatic biochemical analyzer to measure fasting blood glucose,serum total cholesterol,triglyceride,low density lipoprotein and high-density lipoprotein levels.We used enzyme linked immunosorbent assay to measure serum catalase,glutathione peroxidase,malondialdehyde levels.We used quantitative real-time polymerase chain reaction to measure the m RNA expression of some factors in the liver including adenosine monophosphate-dependent protein kinase,peroxidase proliferator-activated receptor alpha,carnitine palmitoyltransferase-1,nuclear erythroid 2-related factor 2,Kelch-like ECH-associated protein 1,Heme oxygenase 1.We determined the protein expression levels of the above factors by Western blotting.Results:（1）The analysis of the correlation between the level of related indicators and carotid lesions in patients with T2DM:the serum LDL level in the normal carotid group was significantly lower than that in the carotid stenosis group（P<0.05）.The serum LDL level in the carotid thickening group was significantly lower than that in the carotid plaque group（P<0.05）and the carotid stenosis group（P<0.05）.The serum Apo-B level in the carotid artery stenosis group was significantly higher than that in the carotid artery normal group（P<0.05）and the carotid artery thickening group（P<0.05）.The risk of carotid artery lesions in patients with T2DM is increased compared with those with normal ankle brachial index（OR=3.739,95%CI=1.369～10.227）.Carotid plaque formation was positively correlated with waist to hip ratio（R=0.191,P=0.002）and visceral fat area（R=0.179,P=0.003）.ROC analysis showed that waist to hip ratio（AUC=0.632,P=0.002,95%CI=0.551～0.714）and visceral fat area（AUC=0.628,P=0.003,95%CI=0.546～0.710）predicted value is higher.Dietary vitamin C intake was a protective factor for carotid plaque formation（OR=0.986,95%CI=0.975～0.998）.For every 1 mmol/L increase in serum LDL level,the risk of carotid plaque formation in T2DM patients increased by 3.139 times（OR=4.139,95%CI=1.900～9.016）.（2）Effects of fish oil on lipid metabolism in rats with T2DM and AS through AMPK/PPARα/CPT1 pathway:compared with the CON group,the fasting blood glucose,serum TG and TC levels in the T2DM group and the T2DM&AS group were significantly increased（P<0.05）.After the intervention,compared with the T2DM&AS group,the following situations occurred.Serum TG levels in SIM group were significantly decreased（P<0.05）.The levels of fasting blood glucose,serum TG,TC and HDL in the MFO group were significantly decreased（P<0.05）.Compared with the CON group,the m RNA expression of CPT1 in the liver of the T2DM group was significantly down-regulated（P<0.05）,the m RNA expression of PPARαand CPT1 in the liver of the T2DM&AS group was significantly down-regulated（P<0.05）,and the m RNA expression of CPT1 in the liver between the two model groups was significantly different（P<0.05）.After the intervention,compared with the T2DM&AS group,the m RNA expressions of AMPK,PPARαand CPT1 in the liver of the SIM group,the HFO group and the MFO group were significantly increased（P<0.05）.Compared with the CON group,the expression levels of PPARαprotein in the liver of the two model groups were significantly down-regulated（P<0.05）.After the intervention,compared with the T2DM&AS group,except for the LFO group,the expression levels of AMPK protein in the liver of the other intervention groups were significantly increased（P<0.05）,but the p AMPK protein expression levels in the liver of the other intervention groups were significantly down-regulated（P<0.05）.After the intervention,compared with the T2DM&AS group,the expression levels of PPARαprotein in the liver of the LFO group and the MFO group were significantly increased（P<0.05）.After the intervention,compared with the T2DM&AS group,the expression level of CPT1protein in the liver of each intervention group was significantly increased（P<0.05）.（3）Effects of fish oil on oxidative stress in rats with T2DM and AS through Nrf2/Keap1/HO1pathway:compared with the CON group,the CAT and GSH-Px levels in the serum in the two model groups were decreased,but only the T2DM group had no significant decrease in the serum CAT level（P>0.05）,and the MDA levels in the serum in the two model groups were significantly increased（P<0.05）.After the intervention,compared with the T2DM&AS group,the levels of CAT in the serum in the SIM group,the LFO group and the HFO group were significantly increased（P<0.05）and the levels of MDA in serum in the above three groups were significantly decreased（P<0.05）,and the level of GSH-Px in serum in the SIM group was significantly increased（P<0.05）.RT-q PCR results indicated that compared with the CON group,the m RNA expression of liver Nrf2 in the two model groups was significantly down-regulated（P<0.05）.After the intervention,compared with the T2DM&AS group,the m RNA expression of Nrf2 in the liver of the LFO group was significantly increased（P<0.05）,and the m RNA expression of Keap1 in the liver of the MFO group and the HFO group was significantly decreased（P<0.05）.WB showed the following results.Compared with the CON group,the expression levels of Nrf2 protein in the liver of the two model groups were significantly down-regulated（P<0.05）,and the protein expressions of Keap1 and HO1 were significantly increased（P<0.05）.After the intervention,compared with the T2DM&AS group,except for the SIM group,the expression level of Nrf2 protein in the liver of fish oil groups was significantly increased（P<0.05）.After the intervention,compared with the T2DM&AS group,except for the LFO group,the Keap1 protein expression in the liver of the other intervention groups was significantly down-regulated（P<0.05）.After the intervention,compared with the T2DM&AS group,the expression level of HO1 protein in the liver of each intervention group was significantly increased（P<0.05）.Conclusion:Carotid artery disease in patients with T2DM has a certain relationship with serum LDL levels,Apo-B levels,whether ABI normal,waist to hip ratio levels,visceral fat area levels,and dietary vitamin C intakes.Fish oil can reduce fasting blood glucose,serum TG and TC levels and activate the AMPK/PPARα/CPT1 pathway in the liver and have beneficial effects on lipid metabolism of rats with T2DM and AS.Fish oil can also activate the Nrf2/Keap1/HO1 pathway and reduce the production of peroxides in the liver and improve the antioxidant capacity of rats with T2DM and AS. |
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