2.1 Animals and surgical operation

Six-week-old male Wistar rats were purchased from SPF Biotechnology Co., Ltd. (Beijing, China) and housed at the Animal Center of the Qilu Hospital of Shandong University. Rats were raised in a specific pathogen-free environment with constant temperature and humidity under a 12-h light/dark cycle and were given normal water and feed. According to a previous study by our group, 10 rats were used in each group. After 1 week of acclimatization, 10 rats were fed with normal diet continuously as the control (CON) group (n = 10). At the same time, other rats were fed a HFD (60% calories as fat, Xietong Biological Company, Nanjing, China) for 4 weeks to induce obesity and insulin resistance and then intraperitoneally injected with STZ (Sigma, Shanghai, China) at a dose of 35 mg/kg after 12 h of fasting to establish the type 2 diabetic rat model. One week after STZ injection, the measurement of fasting blood glucose (FBG) level (≥11.1 mmol/L) showed that the model of type 2 diabetic rats was successful. Continuous HFD feeding for another 4 weeks to increase heart damage was employed to establish the diabetic cardiomyopathy rats. Twenty diabetic cardiomyopathy rats were randomly allocated to the sham (n = 10) and DJB (n = 10) groups. A total of 30 rats were included herein. The researchers were not blinded to the group allocation during the experiment. All procedures for animals were reviewed and approved by the Animal Experimental Ethics Committee of Qilu Hospital of Shandong University, and all animal studies were conducted in accordance with the relevant ethical regulations of animal testing and research.

After fasting for 12 h, the rats in SHAM and DJB were anesthetized with 3% isoflurane, and sham and DJB operations were performed in the corresponding groups in strict accordance with the operation requirements. DJB and SHAM operations were performed according to standard procedures.^28-30

DJB: First, a median incision was made in the upper abdomen, the duodenum was cut 1 cm away from the distal pylorus of the stomach, and the stump of the duodenum was sealed. The jejunum was then cut off at approximately 10 cm at the distal end of the ligament of Treitz, and the distal limb of the jejunum was anastomosed with the proximal duodenum. Finally, at the distal 15 cm of the duodenojejunostomy, the cholangiopancreatic limb was anastomosed end to side with the jejunum.

SHAM: The incisive and transected site was similar to that of the DJB group, and the reanastomosis was performed in situ. The operation time was the same as that in the DJB group.

After the operation, rats were fed in the cage individually, and all rats were given subcutaneous saline injection immediately, given water 24 h later, fed a liquid diet from 3 days to 1 week, and fed HFD 1 week later. Vital signs and operation-related complications were strictly examined within 1 week. The relevant indices were recorded in accordance with the determined time. Twelve weeks after the surgery, the animals were euthanized after fasting for 12 h.

2.2 Physiological index

Body weight and food intake were measured before surgery and at 1, 2, 3, 4, 6, 8, 10, and 12 weeks after surgery.

2.3 Blood biochemistry

(FBG was measured at the tip of the tail using a blood glucose meter (Abbott, Shanghai, China) after fasting for 12 h. Before the operation and at 1, 2, and 3 months after the operation, blood was collected from the lateral canthus vein of rats under 3% isoflurane anesthesia after fasting for 12 h. Serum insulin concentration was determined using an Insulin ELISA Kit (Elabscience, Wuhan, China).

2.4 Oral glucose tolerance test and homeostasis model assessment of insulin resistance index

Before surgery and 3 months after the operation, oral glucose tolerance test (OGTT) was performed after fasting for 12 h. Additionally, insulin resistance homeostasis model assessment of insulin resistance (HOMA-IR) was performed.

2.5 Histology and immunohistochemistry

After euthanizing the rats in each group, heart samples were taken to weigh and measure the percentage of heart mass. The paraffin sections of myocardial tissue were stained with hematoxylin and eosin, Masson's trichrome, Sirius red, wheat germ agglutinin (WGA), and col-1 (abcam, 1:500) and col-3 (abcam, 1:500) were detected by immunohistochemistry (Servicebio). Next, cardiomyocyte size, morphology, collagen distribution, and fibrosis were assessed. Further, fresh frozen tissue sections were stained for ROS using dihydroethidium (Sigma, Shanghai, China). Images were obtained using an Axio Scope A1 microscope (Zeiss, Oberkochen, Germany). ImageJ was used to analyze the results with the same parameters.

2.6 H9C2 cell culture

The H9C2 rat embryonic cardiomyocyte line was donated by the Key Laboratory of Cardiovascular Remodeling and Function of Shandong University (Jinan, China) and verified using short tandem repeat analysis. H9C2 cells were cultured in complete low-glucose Dulbecco's modified Eagle's medium (Gibco, Shanghai, China) and were placed in a humidified incubator at 37°C and 5% CO₂. To simulate the diabetic environment in vitro, cells were stimulated with HG + PA comprising 33.3 mM D-glucose (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) and 0.4 mmol/L PA (Aladdin, Shanghai, China) for 48 h.^{31, 32} Moreover, H9C2 cells were treated with a selective AMPK agonist (20 μmol/L, A-769662, MCE, China) for 48 h to determine the effect of activation of the AMPK pathway. Sera (10%) from the CON, SHAM, and DJB groups 12 weeks after surgery were added to H9C2 cells for 48 h to explore the effects of other improvements in the eNOS/NO pathway and antioxidant system, with the exception of changes in blood glucose levels to some extent. In addition, we used the selective AMPK inhibitor dorsomorphin (10 μmol/L, Selleck, Shanghai, China) to further verify the role of the AMPK pathway in the improvement of the eNOS/NO pathway and antioxidant system. All the simulations were performed after 12 h of starvation.

2.7 Protein extraction and western blot

Heart tissue proteins were extracted using a total protein extraction kit for muscles (Invent Biotechnologies, Beijing, China). Radioimmunoprecipitation assay lysate (Meilunbio, Dalian, China) was mixed with protease and phosphatase inhibitors (Solarbio, Beijing, China) to extract proteins from H9C2 cells. The concentration of extracted proteins was measured using a bicinchonic acid protein assay kit (Millipore, Billerica, MA, USA). After mixing the protein with the loading buffer (Solarbio) at 4:1, boiled protein solutions were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (Meilunbio) and were transferred to a 0.22 μm polyvinylidene difluoride membrane (Millipore). After 2 h of blocking with 5% skim milk powder (Beyotime, Shanghai, China), the membranes were incubated with the following primary antibodies on a 4°C shaker overnight: anti-ACC (Proteintech, 1:1000), anti-p-ACC (CST, 1:1000), anti-AMPK (CST, 1:1000), anti-p-AMPK (CST, 1:1000), anti-SIRT1 (Abcam, 1:1000), anti-β-actin (Invitrogen, 1:2000), anti-eNOS (Proteintech, 1:1000), anti-p-eNOS (CST, 1:1000), anti-NRF2 (Proteintech, 1:4000), anti-HO-1 (Proteintech, 1:2000), and anti-SOD2 (Proteintech, 1:2000). Next, they were incubated with the secondary antibody (ZSGB-Bio, Beijing, China) at room temperature for 2 h. After adding the mixed hypersensitive electrochemiluminescence solution (Millipore), proteins were detected using a LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, United States). ImageJ was for quantitative analysis of western blot results.

2.8 Detection of NO in myocardial tissue and H9C2 cells

Fresh myocardial tissue (20 mg) was homogenized to extract the tissue lysate, which was centrifuged at 12000 × g for 5 min at 4°C. The myocardial NO content was detected using a NO detection kit (Nanjing Jiancheng Biological Co., Ltd., Nanjing, China).

The NO content of H9C2 cells was detected using a NO detection kit (Nanjing Jiancheng Biological Co., Ltd., Nanjing, China).

The NO levels were normalized to the total protein concentration.

2.9 ROS assay of H9C2 cells

The ROS levels of H9C2 cells were detected using the Reactive Oxygen Species Assay Kit (Meilunbio, Dalian, China). Images were acquired using an Axio Scope A1 microscope (Zeiss).

2.10 RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-PCR)

An RNA extraction kit (Fastagen, Shanghai, China) was used to extract RNA from rat myocardium and H9C2 cells, and the RNA concentration was measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, United States). cDNA was synthesized using the ReverTra Ace qPCR RT kit (TOYOBO, Osaka, Japan), and RT-PCR was performed on a Roche LightCycler480 II (Roche, Basel, Switzerland) using SYBG Green real-time fluorescence quantitative PCR premixture (TOYOBO, Osaka, Japan). Relative mRNA quantification was performed using the ΔΔCt method, and β-actin was used as the internal reference. The sequences of the RT-PCR primers (Sangon, Shanghai, China) were as follows:

2.11 Statistical analysis

The data are shown as mean ± SD and were analyzed by GraphPad Prism 9 (California, USA). Two-way analysis of variance (ANOVA) followed by Tukey's correction between-group analysis was used to compare variations in FBG, weight, food intake, and OGTT (A: Treatment, B: Time, AB: Intervention on rats × Time). One-way ANOVA followed by Bonferroni correction between-group analysis was used to compare variations in other data. p < .05 indicated statistical significance. Statistical details are included in the respective figure legends.

3.1 DJB improves general metabolic characteristics and cardiac hypertrophy and fibrosis in diabetic cardiomyopathy rats

As shown in the curve, the body weight of rats in the DJB and SHAM groups began to decrease after surgery and began to rise from the second week (Figure 1B). There was no significant difference in the increase in body weight between the DJB and SHAM groups. Similarly, the food intake of rats in the DJB and SHAM groups decreased significantly 1 week after surgery and then gradually returned to the preoperative level (Figure 1C). The FBG levels in rats in the DJB group decreased almost to levels in rats in the CON group approximately 1 month after surgery and then remained stable, whereas there was no downward trend in FBG level in the SHAM group (Figure 1D). The OGTT showed that the glucose tolerance of the DJB group was significantly improved 12 weeks after the operation compared with that of the SHAM group, and the area under the curve decreased significantly (Figure 1E–H). Meanwhile, the HOMA-IR values decreased after DJB compared with those in the SHAM group, suggesting that insulin sensitivity was effectively improved (Figure 1I). These results suggest that DJB can effectively improve metabolic state and glucose homeostasis.

Cardiac function of diabetic cardiomyopathy rats was reportedly damaged but was improved after DJB in previous studies by our research group.^23-25 In the present study, Hype staining and WGA staining showed that the SHAM group showed cardiomyocyte hypertrophy compared with the control group; however, hypertrophy was alleviated after DJB (Figure 2A,F,G). Heart mass as a percentage of body weight increased in diabetic cardiomyopathy rats but decreased after DJB (Figure 2H). Further, Sirius red, Masson, and immunohistochemistry staining of collagen fibers suggested a significant increase in the level of cardiac fibrosis in diabetic cardiomyopathy rats, whereas DJB effectively improved this cardiac fibrosis and reduced collagen deposition (Figure 2B–E). These results suggest that DJB can effectively improve cardiac hypertrophy and fibrosis in diabetic cardiomyopathy rats.

3.2 DJB ameliorates the cardiac AMPK/eNOS pathway and antioxidant system in diabetic cardiomyopathy rats and improves myocardial oxidative stress

To detect oxidative stress pressure, we performed ROS fluorescence staining in the myocardium of the three groups of rats (Figure 3A,B). The oxidative stress pressure of diabetic cardiomyopathy rats was higher than that of normal rats, and this condition was effectively improved after DJB. Moreover, western blot results showed that the expression of AMPK pathway-related molecules was significantly improved after DJB (Figure 3D), and the eNOS phosphorylation level (Figure 3F,G) and NO content were significantly increased (Figure 3H). Additionally, the SOD2 and NRF2/HO-1-related pathways, which play an important role in ROS removal, were significantly improved after DJB, which promoted the scavenging ability for ROS and improved cardiac oxidative stress (Figure 3C). Real-time RT-PCR showed that the relative transcriptional levels of Sirt1, Nrf2, Homx-1, and Sod2 were consistent with their corresponding protein levels (Figure 3E). These results showed that DJB can improve oxidative stress in the heart of diabetic cardiomyopathy rats by ameliorating the AMPK/eNOS pathway and antioxidant system.

3.3 Activation of the AMPK pathway can improve eNOS activity and antioxidant system of H9C2 cell line in the HG + PA environment

To verify the function of the AMPK pathway, HG + PA was used to stimulate H9C2 cells to simulate the HG and high-fat environment of diabetic cardiomyopathy. A-769662 was used to stimulate H9C2 cells for 48 h to activate the AMPK pathway. ROS staining showed that ROS activity in H9C2 cells increased significantly in the HG + PA group but decreased significantly after AMPK pathway activation (Figure 4A). Western blotting showed that in the HG + PA environment, the activity of the AMPK pathway decreased (Figure 4D) and eNOS phosphorylation decreased, decreasing the activity of eNOS (Figure 4E). The NO detection results showed that the NO content decreased (Figure 4F). Further, the antioxidant system, such as the SOD2 and NRF2/HO-1 pathways, also decreased (Figure 4B). Real-time RT-PCR showed that the relative transcriptional levels of Nrf2, Homx-1, and Sod2 were consistent with their corresponding protein levels (Figure 4C). After activation of the AMPK pathway, western blotting showed that the protein levels of AMPK/eNOS were significantly increased (Figure 4D,E), and the NO content was significantly increased (Figure 4F). The antioxidant system-related protein SOD2 and NRF2/HO-1 pathways were also significantly increased (Figure 4B). Real-time RT-PCR showed that the relative transcriptional levels of Nrf2, Homx-1, and Sod2 were consistent with their corresponding protein levels (Figure 4C). The eNOS phosphorylation level and the antioxidant system were damaged in the HG + PA environment, while activation of the AMPK pathway reversed this. The above results verified the function of the AMPK pathway and related molecules in vitro.

3.4 Effects of CON, SHAM, and DJB group rat sera on AMPK/eNOS pathway and antioxidant system of H9C2 cells in the HG + PA environment

To further verify the connection between DJB and the AMPK pathway, eNOS phosphorylation and the antioxidant system were assessed. Rat sera from the CON, SHAM, and DJB groups were used to stimulate H9C2 cells in the HG + PA environment. The external HG + PA environment almost eliminated the changes in blood glucose levels in the serum of rats in the different groups. The results showed that under stimulation with the SHAM group serum, the oxidative stress of H9C2 cells in the HG + PA environment did not improve and was even aggravated (Figure 5A). Meanwhile, western blotting showed that the levels of AMPK pathway-related proteins also did not improve (Figure 5B). The eNOS phosphorylation level and those of antioxidant system-related pathway proteins were similar to those in the HG + PA group, with no significant improvement (Figure 5E). Further, the NO content decreased (Figure 5F). Real-time RT-PCR showed that the relative transcriptional levels of Nrf2, Homx-1, and Sod2 were consistent with their corresponding protein levels (Figure 5C). However, under stimulation with the DJB and CON group sera, oxidative stress resulting from the HG + PA environment significantly improved. ROS staining showed that the level of ROS was decreased (Figure 5A). Western blotting showed that the AMPK pathway was activated (Figure 5B), and the eNOS phosphorylation level increased (Figure 5E). The NO content also increased (Figure 5F). Simultaneously, antioxidant system-related proteins were significantly improved (Figure 5D). Real-time RT-PCR showed that the transcriptional levels of Nrf2, Homx-1, and Sod2 were consistent with the corresponding protein levels (Figure 5C).

3.5 Improvement of oxidative stress in H9C2 cells in HG + PA environment by serum of DJB rats depends on activation of the AMPK pathway

To verify the effect of the AMPK pathway, the AMPK inhibitor dorsomorphin combined with DJB group serum and AMPK agonist combined with SHAM group serum were used to stimulate the AMPK pathway in H9C2 cells in the HG + PA environment. The results showed that the reduction in ROS as well as the improvement in the AMPK/eNOS pathway, NO content, and antioxidant system was inhibited after adding the AMPK inhibitor (Figure 6A,C–F). By contrast, the decrease in ROS and the improvement of the AMPK/eNOS pathway and antioxidative system were further improved after adding the AMPK agonist combined with the SHAM group serum in H9C2 cells in the HG + PA environment (Figure 6A,C–F). Real-time RT-PCR showed that the relative transcriptional levels of Nrf2, Homx-1, and Sod2 were consistent with their corresponding protein levels (Figure 6B).