| BackgroundDiabetes is a common disease caused by a metabolic disorder,and patients often die from its serious complications.Among them,diabetic cardiomyopathy(DCM)is one of the most important complications,and its main manifestations are myocardial hypertrophy and cardiac fibrosis.Docosahexaenoic acid(DHA),as an essential polyunsaturated fatty acid to the human body,has a very good protective effect on the cardiovascular and cerebrovascular systems.At the same time,many studies have shown that it has a preventive effect on organ fibrosis,However,it is unclear whether it is effective for cardiac fibrosis caused by DCM.Organ fibrosis is a disease caused by the synthesis/degradation of extracellular matrix,differentiation/de-differentiation of fibroblasts,and the imbalance of apoptosis/autophagy.In this process,it is often accompanied by an imbalance of cell metabolism.Hyperglycemia is the main cause of diabetes and its complications,as well as the main factor of cell metabolism disorders,but at present,the mechanism of hyperglycemia affecting cardiac fibroblast metabolism and fibrosis is not clear.DHA can regulate cell metabolism disorders,and cell metabolism disorders can promote organ fibrosis.Therefore,this topic will explore whether DHA can reduce cardiac fibrosis by regulating the metabolic disorders caused by hyperglycemia,thereby improving diabetic cardiomyopathy.Methods(1)Explore the effect of DHA on cardiac fibrosis in diabetic mice.Mice were received streptozotocin at the dose of 50 mg/kg by intraperitoneal(i.p.)injection to induce type I diabetes model.For successful diabetes mice,the algae oil(1.5g/kg/d,DHA～600mg/kg/d)was delivered by gavage daily,blood taken from the tail vein every 4 weeks was used to monitor blood glucose levels.Cardiac functions of mice were assessed using an echocardiography system in the last week before sacrififice,and blood was taken to test the serum biochemical indicators.After the experiment,the heart tissue sections were stained with Sirius red and immunostained to detect the improvement of cardiac fibrosis by DHA.(2)Explore the mechanism of DHA in improving cardiac fibrosis caused by hyperglycemia in vitro.Primary cardiac fibroblasts were isolated from the hearts of adult rats and cultured with a high concentration of glucose to simulate the diabetic environment in vitro,and DHA was added into the medium at the same time.MTT experiment,immunofluorescence and Western Blot were used to detect the effects of DHA on the differentiation and collagen synthesis of myofibroblasts.Flow cytometry,Western Blot and lyso-Tracker red were used to detect apoptosis and autophagy sensitivity.Labelfree quantitative analysis of proteomics was carried out by mass spectrometry to investigate the mechanism of DHA to improve the fibrosis caused by high-glucose.The detection of lactate level and Seahorse XF 96 were used to study the effects of high concentrations of glucose and DHA on glycolysis and fatty acid oxidation in cardiac fibroblasts.And then we treated fibroblasts with rosiglitazone and etomoxir,RT-PCR and Western Blot were used to explore the effect of DHA on cardiac fibrosis by enhancing or inhibiting fatty acid oxidation.Finally,immunofluorescence of heart tissue sections was stained with Vimentin and p-PPARγ or CD36 to detect the effect of DHA on fatty acid oxidation in situ.Results(1)DHA relieved diabetic cardiomyopathy in model mice by improving fibrosisBlood biochemical tests showed that DHA improved blood lipid levels,but can not reduce blood glucose levels.Echocardiographic results indicate that DHA significantly improved the cardiac function of diabetic mice.Cardiac tissue section staining showed that DHA improved diabetic cardiac fibrosis and inhibited the proliferation of cardiac fibroblasts.(2)DHA inhibited the development of fibrosis in HG-cultured fibroblasts in vitroMTT experiment and immunofluorescence for Ki67 staining show that DHA inhibited the proliferation of fibroblasts cultured in HG.Immunofluorescence and Western Blot show that DHA inhibited HG-induced myofibroblast differentiation and collagen synthesis.Flow cytometry and Western Blot showed that DHA promoted the apoptosis of HG-cultured cardiac fibroblasts.Western Blot and lyso-Tracker red showed that DHA promoted autophagy.(3)Proteomics analysis revealed that the occurrence of fibrosis may be related to the transformation of fibroblast metabolismLabel-free proteomics quantitative analysis showed that HG significantly causes the metabolic remodeling of cardiac fibroblasts and the synthesis of collagen,which is mainly manifested by enhancing glycolysis and inhibiting fatty acid oxidation.DHA significantly inhibited glycolysis,enhanced fatty acid oxidation,and reduced collagen synthesis.(4)DHA relieved the development of fibrosis by promoting fatty acid oxidationThe test of lactic acid content in the cell culture supernatant showed that DHA inhibited the production of lactic acid in HG cultured cells.Seahorse XF96 tests showed that HG significantly increased the level of glycolysis in fibroblasts,but reduced the level of fatty acid oxidation,while the DHA group was the opposite.RTPCR and Western Blot results showed that enhanced fatty acid oxidation can promote apoptosis and autophagy of fibroblasts and alleviate fibrosis,but inhibition of fatty acid oxidation blocked the improvement effect of DHA on fibrosis.Tissue section staining also confirmed the above results.ConclusionDHA can inhibit the proliferation,differentiation and collagen synthesis of cardiac fibroblasts through enhancing the fatty acid oxidation and promoting apoptosis and autophagy of cardiac fibroblasts,eventually improve cardiac fibrosis caused by hyperglycemia. |
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