2.1 Animals
Male C57BL/6J mice (20-22g) were obtained from the Animal Experimental Center of Zhejiang University, and the mice were housed in an SPF environment. All mice have free access to water and food.
The mice were randomly divided into the following groups: CON group, DM group, and 2-AG group. Mice in the DM and 2-AG groups were fasted overnight and injected intraperitoneally with 100 mg/kg streptozotocin (STZ) (dissolved in 100 mM citrate buffer, pH 4.5, purchased from Sigma, USA). Mice in the CON group received an injection of the same volume of citrate buffer. On the 3rd and 7th day, two consecutive fasting (for 8 h) blood glucose measurements were obtained by the tail vein. Mice with 8 h fasting-blood glucose >11.1 mM were considered diabetic and continued feeding for 12 weeks. After 12 weeks, the mice in the 2-AG group were intraperitoneally injected with 2-AG (dissolved in physiological saline, purchased from Tocris, USA) at 1 µg/kg for 4 consecutive weeks, and blood glucose and body weight were measured weekly during the administration. After 4 weeks, the mice were sacrificed by an overdose of 100 mg/kg ketamine hydrochloride (Ketanest, Pfizer, Germany) and 16 mg/kg xylazine hydrochloride (Rompun 2%, Bayer, Germany). Cardiac function was measured, the heart samples were weighed, and serum and heart tissue were collected for further analysis.
2.2 Echocardiographic evaluation
Cardiac function was determined noninvasively by transthoracic echocardiography before sacrifice[13].Doppler analysis was performed using a SONOS 5500 ultrasound (Philips Electronics, Amsterdam, The Netherlands) with a 15 MHz linear array ultrasound transducer to determine cardiac function.
2.3 Detection of serum lipids
Blood samples were collected and centrifuged at 3000 rpm/min for 15 minutes at 4 °C to separate the supernatant (serum). The levels of TG, T-CHO, LDL-C, and HDL-C in the serum were measured according to the kit instructions (Nanjing Jiancheng, China).
2.4 Histological analysis
The heart tissue was fixed in 4% paraformaldehyde overnight, dehydrated and embedded in paraffin, sectioned at 5-μm thickness, and mounted on glass slides. Pathological lesions were assessed by hematoxylin and eosin staining (H&E Assay Kit, Beyotime, China). Fibrosis was assessed using Sirius red staining (Sirius Heart Stain Kit, Solarbio, China). Images were observed and acquired using a Nikon microscope (Nikon, Japan).
2.5 Immunohistochemical analysis
Thee sections were deparaffinized, rehydrated in gradient xylene and ethanol, antigen repair was performed by microwaving in 0.1 mol/L citrate buffer (pH 6.0), and the sections were then allowed to stand in a 3% hydrogen peroxide solution. After blocking with 2.5% BSA (Sigma, USA), the sections were incubated with anti-Collagen I (1:200, Abcam, UK) at 4 °C overnight and then secondary antibody (1:200, Santa Cruz, USA), colored by 3,3′-diaminobenzidine tetrahydrochloride (DAB; ZSGB-Bio, China), followed by sealing in neutral resin after dehydration in ethanol xylene Images were taken under a Nikon microscope.
2.6 Western blot analysis
The myocardial tissue was homogenized and lysed, and the total protein concentration was quantified by using a BCA protein assay kit (Thermo, USA). A total of 30-50µg of protein was loaded and separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE). Then, the protein was transferred from the gel to a polyvinylidene fluoride membrane. After blocking with 5% skim milk and 0.05% Tween 20, the membrane was incubated overnight in primary antibody (TGF- β1(1:1000,Abcam:92486,UK),Smad2/3(1:1000,CST:8685,USA),p-Smad2/3(1:1000,CST:8828,USA),Collagen I(1:1000,Abcam:34701,UK),and β-actin(1:1000,CST:4970,USA)). Bands were detected with a specific horseradish peroxidase-conjugated secondary antibody (CWBIO) (1:10000, Biosharp, China) and visualized by enhanced chemiluminescence reagents (Thermo, USA). Protein expression was quantified using ImageJ software.
2.7 Statistical analysis
All data are expressed as the mean ± standard deviation, and statistical analysis was performed using GraphPad Pro Prism 7.0. One-way ANOVA was used, and then multiple comparison tests with a Tukey correction were performed, with p < 0.05 considered a significant difference.