| Background:The early energy metabolism substrate remodeling is seriously harmful for diabetic cardiomyopathy(DCM)patients,and its diagnosis and treatment are relatively difficult.There are few researchs on whether Celastrol has a protective effect on it.As well,it is not clear whether PET/CT myocardial metabolism imaging can play a role in diagnosis and efficacy evaluation.Objective:Based on Micro PET/CT myocardial metabolism imaging,combine with network pharmacology,bioinformatics and basic experimental verification,this research aims to study the diagnosis of early energy metabolism substrate remodeling in DCM and the efficacy evaluation and possible mechanism of Celastrol reverse regulation.Methods:DCM rat models were prepared,the main intervention method was oral gavage of common feed combined with different doses(50 vs 500 vs 1000 ug/kg/day)of Celastrol,and the rat models were divided into the Healthy control group,Cel Low-dose group(CL),Cel Medium-dose group(CM),Cel High-dose group(CH)and non-intervention group,and non-intervention group.clinical symptoms,blood biochemistry,echocardiography and pathological diagnostic indicators were used as comprehensive evaluation methods.18F-FDG and 18F-FTHA PET/CT myocardial metabolism imaging of model rats were completed before and after Celastrol intervention,meanwhile,the energy substrate flow index Influx was analyzed.Blood samples and myocardial tissue materials were obtained at the end of the experiment,HE staining and Masson were adopted to observe myocardial morphological changes and fibrosis degree.At the same time,the network pharmacology and bioinformatics methods were used to predict the key targets and related pathways of Celastrol on the energy remodeling and reverse regulation of DCM.Western blotting(WB)and real-time fluorescence quantitative PCR(RT-PCR)were used to verify specific pathway proteins.Results:1.Basic information of rats in each group:(1)General behavior and clinical symptoms: Before the intervention,compared with the Healthy control group,the rats in the model group were in poorer general states such as spirit,reaction,and coat color,and the water,food intake,and urine output were significantly higher.After the intervention,the rats in the non-intervention group had no significant improvement in the above conditions compared with the model group.In the CL group,the rats’ water,food intake,and urine output decreased to varying degrees,and the spirit,activity and hair were improved to varying degrees compared with the non-intervention group and the pre-intervention model group.The rats in the CM and CH intervention groups had different degrees of abdominal distension,slow movement,and died of unknown causes at different time points of the intervention.Dissection showed obvious intestinal flatulence,local intestinal adhesion to the abdominal wall,dark red lesions in the heart and kidney,and thoracic and abdominal effusion.(2)Blood biochemical indexes: Before the intervention,the fasting blood glucose,free fatty acid and serum insulin in the model group were significantly higher than those in the Healthy control group,and the differences between the two groups were statistically significant(P<0.05).After the intervention,the fasting blood glucose,triglyceride and free fatty acid in the non-intervention group were all higher than those in the pre-intervention model group,but the differences were not statistically significant(P>0.05).There was statistically significant difference between the non-intervention group and the pre-intervention model group in serum insulin(P<0.05).Compared with the non-intervention group and the pre-intervention model group,the levels of triglycerides,free fatty acids and serum insulin in the CL group were decreased to varying degrees;the differences were statistically significant with those of the non-intervention group(P<0.05),but the differences were not statistically significant with those of the pre-intervention model group(P>0.05).(3)Changes of body weight and heart weight:(1)Body weight of rats in each group: Before intervention,after DCM model was successfully made,the body weight of DCM model group was higher than that of Healthy control group,and the difference was statistically significant(P<0.05).After the intervention,the body weight of the CL group and the non-intervention group was lower than that of the Healthy control group,and the difference was statistically significant(P<0.05),but there was no statistically significant difference between the CL intervention group and the non-intervention group(P>0.05).(2)Heart weight and cardiac index of rats in each group: There was no statistically significant difference in heart weight and cardiac index of rats in each group(P>0.05).2.Micro PET/CT myocardial glucose and fatty acid metabolism imaging: Before intervention,the myocardial glucose metabolism Influx in the model group was significantly lower than that in the Healthy control group and the fatty acid metabolism Influx was significantly higher than that in the healthy control group,the differences were statistically significant(P<0.05).After the intervention,compared with the pre-intervention model group,the glucose metabolism Influx in the non-intervention group was increased,and the difference was statistically significant(P<0.05).There was no statistically significant difference in the fatty acid metabolism Influx between the two groups(P>0.05).Compared with the pre-intervention model group,the glucose metabolism Influx in the CL group was increased,and the fatty acid metabolism Influx was decreased,the differences were statistically significant(P<0.05).There was no statistically significant difference in glucose metabolism Influx between the CL group and the non-intervention group(P>0.05),but the mean descriptive statistics showed that the fatty acid metabolism Influx of the CL group was significantly higher than that of the non-intervention group(0.015±0.004 vs 0.009±0.002),and the difference was statistically significant(P<0.05);3.Rat echocardiography: Before intervention,compared with healthy rats,the left ventricular end-systolic diameters(LVIDs)in the model group increased,and the EF and left ventricular fractional shortening FS decreased,with statistical differences between the two groups(P<0.05).After intervention,there was no statistically significant differences in LVIDs,EF and FS between the non-intervention group and the pre-intervention model group(P>0.05).There was no statistically significant difference in each index between the CL group,the non-intervention group and the pre-intervention model group(P>0.05),but the mean descriptive statistics showed that the LVIDs in the CL group were lower than those in the non-intervention group(4.14±0.31 vs 4.52±0.27),while EF and FS increased(35.80±1.92 vs 30.33±6.11;71.20±2.77 vs 63.33±8.08).4.Histopathological analysis of the heart tissues:(1)Compared with the Healthy control group,the myocardial fibers in the DCM model group were relatively disordered,accompanied by myocardial fiber swelling and a certain degree of myocardial fibrosis;Compared with the model group,the interstitial fibrosis of the rat cardiomyocytes in the non-intervention group was not significantly relieved;In the CL group,compared with the model group,the cardiomyocytes were arranged in a relatively dense and orderly manner,and there was no obvious fibrosis.(2)Masson staining of rat heart in each group:cardiomyocytes were in red and collagen was in blue.There were no blue-green collagen fibers in the interstitium and surrounding blood vessels of cardiomyocytes in the Healthy control group;In the model group,the cardiomyocytes were loosely arranged,and there were a lot of blue-green collagen fibers in the myocardial interstitium and around the blood vessels;Compared with the model group,the blue-green collagen fibers in the non-intervention group were not significantly reduced;In the CL group,compared with the model group,the arrangement of cardiomyocytes was relatively dense,and the interstitial collagen was significantly reduced.5.Prediction results of network pharmacology and bioinformatics: Between Celastrol and DCM,five key targets were screened,i.e.IL6,VEGFA,TNF,CASP3,and MAPK8,all of which were involved in the regulation of AGE-RAGE,TNF,MAPK,and TOLL-like receptors,insulin resistance and other signaling pathways,and are closely related to adipocytokines,fatty acid metabolism,glycolipid biosynthesis,and glycosylphosphatidylinositol biosynthesis.6.Basic experimental verification results: Based on Western blotting and real-time fluorescence quantitative PCR(RT-PCR)methods,it was found that the ratio of P38 MAPK to GAPDHA in the myocardial tissue of the model group was significantly increased compared with that of the Healthy control group,and the difference was statistically significant(P < 0.01);Compared with the model group,the P38MAPK/GAPDHA in the CL group was significantly lower,and the difference was statistically significant(P<0.05).Conclusion:1.Celastrol has a certain protective effect on the early energy remodeling of DCM.50ug/kg/day Celastrol gavage,while correcting the state of myocardial energy remodeling in DCM model rats,can improve the general behavior,clinical symptoms,and abnormal serum glucose and lipid metabolism indexes of DCM rats,and delay the deterioration of cardiac function to a certain extent.Cardiac interstitial fibrosis.However,when Celastrol was administered at a dose of ≥500ug/kg/day,it may cause severe toxic side effects in DCM rats.2.Micro PET/CT myocardial glycolipid combined metabolic imaging can play a role in the diagnosis and efficacy evaluation of early energy remodeling in DCM.3.The abnormal P38 MAPK signaling pathway plays a certain role in the early energy remodeling of DCM,and may be an effective intervention target for Celastrol. |
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