In vivo analysis and animal modeling

In the current in vivo study, Swiss male mice 6–8 weeks old were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China) and maintained in a clean environment with constant temperature and humidity. Mice were grouped correspondingly (20 mice/group); the normal group was on a regular diet, and the T2D modeling groups were fed a high-fat and high-sugar diet for 3–4 months and then injected with streptozotocin (STZ) (Sigma Aldrich, USA) at 60 mg/kg for 7–10 days until T2D was achieved. The successful modeling of T2D was determined by measuring the animals’ fasting blood glucose and fasting insulin content. All procedures were performed in accordance with ethical guidelines for animal experimentation to ensure minimal animal suffering. Diabetic animals were divided into two groups, T2D (diabetic mice with no further treatment), and fABT + T2D (diabetic mice subjected to fresh autologous blood transfusion).

All animal experiments were performed according to the guidelines of the animal ethical organization and obtained the permission of the Shanghai Gongli Hospital, Naval Military Medical University

Preparation of fresh autologous blood transfusion fluid

Fresh autologous blood was obtained from healthy Swiss mice and was subjected to certain preservatives such as mannitol to improve RBCs’ membrane stability, reduce sugar accumulation, and improve their oxygen-carrying capacity. Blood samples were transfused forthwith.

Analysis of physiological erythrocyte index

After treating the animals accordingly, animals were sacrificed, and the blood samples were collected from the abdominal aorta and stored at 4°C in tubes containing ethylenediaminetetraacetic acid (EDTA) and defoaming agent (10 µl) until further analysis. Simultaneously, we stimulated the arterial oxygen partial pressure as previously described [22]. Briefly, the test parameters were as follows: O₂ = 16 ml/min, CO₂ = 3 ml/min, N₂ = 120 ml/min, flow rate: 100 ml/min, 37°C to sample inflatable 9 min. One milliliter was used for blood gas analysis as previously described [23]. The determination of the erythrocytes’ oxygen-carrying capacity was done using the Q value following this formula: Q = 20 × (S1 – S2) ml; S1 is the oxygen saturation of Hb after the oxygen partial pressure reached 100 mm Hg and stabilized, while S2 is the oxygen saturation of Hb after the oxygen partial pressure of mixed gas was 40 mm Hg. The erythrocytes’ oxygen consumption rate was determined as previously described [24].

For osmotic fragility testing, the saline dilution technique was performed as previously described [25]. Briefly, a phosphate buffer stock solution containing 10% NaCl was prepared and then diluted using distilled water until it reached 1% NaCl solution. Each treatment group included sixteen serial dilutions containing 0.85 to 0.0% NaCl. Blood samples (20 µl) were added to each tube, mixed by inversion, and then left to settle at room temperature for 30 min. Next, all samples were centrifuged at 2,000 g for 10 min. Then, the supernatant’s optical density was determined at 540 nm using a spectrophotometer. At 0% NaCl we assumed 100% hemolysis. The percentage of erythrocytes exposed to each saline concentration was determined from the supernatant at 540 nm compared to the 0.0% NaCl supernatant for each series of dilutions.

Flow cytometry

Blood samples were diluted at a 1 : 3 ratio using PBS solution containing heparin. Then, 3 ml of Ficoll-Hypaque solution (Beyotime Co., Ltd., Shanghai, China) was added slowly to each tube, followed by centrifugation twice at 2500 g for 15 min at 4°C. Supernatants were transferred to new tubes containing Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal calf serum (FCS). Initially, we analyzed the percentage of macrophages after staining the monocytes with allophycocyanin (APC)-conjugated monoclonal antibody against mouse CD32 (Thermo Scientific, Waltham, USA); subsequently, the phycoerythrin (PE)-conjugated monoclonal antibody against mouse CD206 (eBioscience, USA) was separately stained with the monocytes for 1 h at 4°C. Subsequently, cells were washed with PBS and incubated with the secondary antibody – anti-mouse (IgG). Finally, the monocytes were fixed with paraformaldehyde for 30 min and analyzed using FACSCalibur (eBioscience).

Oxidative stress and mitochondrial function analysis

Mitochondrial protein concentration was assessed as previously described [26]. The activity of superoxide dismutase (SOD) in erythrocytes was determined as previously described [27]; the activity of SOD is expressed in nitrite units (NU) per ml (NU/ml). Malondialdehyde (MDA) levels in erythrocytes were determined as previously described [28]. The assay was performed using a reaction with thiobarbituric acid and analyzed using a spectrophotometer. The results are expressed in µmol/l.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from blood samples was extracted using Trizol reagent (Takara, Japan). Following the manufacturer’s protocol, the corresponding complementary DNA (cDNA) was synthesized using a High Capacity cDNA Reverse Transcription kit (Promega, Kansas, USA). The RT-qPCR experiment was performed using SYBR Green Master mix (Applied Biosystems, USA) and StepOne Real-Time PCR System (Applied Biosystems, USA).

Western blotting

Proteins from blood samples were extracted using RIPA buffer (Beyotime, China), and their concentration was determined as indicated in the BCA Kit (Thermo, USA). Each protein sample was added to the sample buffer and boiled for 10 min at 95°C. 30 µg of each sample was loaded and electrophoresed using 10% polyacrylamide gel electrophoresis (PAGE), then transferred onto polyvinylidene fluoride (PVDF) membranes. Next, membranes were blocked in 5% bovine serum albumin (BSA) for 1 h at room temperature and subsequently incubated with each corresponding primary antibody at 4°C overnight. The antibodies were as follows: IGF2R (1 : 1000, Santacruz, USA); PI3K (1 : 1000, Cell Signaling, USA); Akt (1 : 1000, Cell Signaling, USA); PKM2 (1 : 1000, Cell Signaling, USA); β-actin (Beyotime, China). The next day, membranes were rinsed with TBST three times, then incubated with the secondary antibody and incubated at room temperature for 1 h, followed by washing three more times. A chemiluminescence reagent was used for the visualization of protein bands. All target protein bands were analyzed using Image J software (ImageJ NIH, USA).

Statistical analysis

All data analyses were conducted using Graph-Pad Prism software (San Diego, CA, USA). Data are presented as the mean ± standard deviation. Comparisons between two groups were analyzed using an unpaired t-test. Multiple group analysis was conducted using two-way analysis of variance (ANOVA). Statistical significance was considered when p < 0.05.

Fresh autologous blood transfusion (fABT) reduced M2 macrophage polarization and enhanced hepatic mitochondrial metabolism

Although some studies have reported no significant adverse effects of transfusing red blood cells in patients undergoing treatment or operations, recent studies have indicated the adverse impact of old red blood cell transfusions [8, 29]. We tested the effect of fABT in diabetic mice and found that it reduced M2 macrophage polarization over M1 compared to the negative control and diabetes groups (Figure 1 A). Noting that different fluid resuscitation impacts hepatic mitochondria [15], we found that fABT significantly improved hepatic mitochondria in diabetic mice compared to the negative control and diabetes groups (Figure 1 B).

Figure 1

The effect of fresh autologous blood transfusion (fABT) on macrophage polarization. A – FACS analysis of fABT on CD16, CD32, and CD206 cells. B – Mitochondrial functional analysis in T2D animal hepatocytes. Data are presented as mean ± SD

fABT alleviates oxidative stress in diabetic mice

It is known that storing red blood cells alters their oxidative processes [29, 30]. Such processes have unique markers to determine their oxidative stress status, superoxide dismutase (SOD) and malondialdehyde (MDA) [31]. The results showed that fABT decreased MDA levels (Figure 2 A) and improved the level of SOD (Figure 2 B) compared to the T2D and NC groups. These results suggest that fABT is advantageous for sustaining protection against red blood cell oxidative stress.

Figure 2

The effect of fABT on hepatic oxidative stress levels. A – MDA content in mitochondria of T2D animal hepatocytes. B – Mitochondrial SOD activity in T2D animal hepatocytes

fABT modulates the IGF2R/PI3K signaling pathway in diabetic mice

Based on the results mentioned above, we further investigated the underlying mechanism by which fABT affects biological processes in diabetic mice. Our results showed that fABT promoted the IGF2R/PI3K signaling pathway, evidenced by the increased expression of IGF2R, PI3K, AKT, and PKM2 proteins compared to the T2D group (Figure 3). These data indicated that fABT potentially exerts its effect in diabetic mice through the IGF2R/PI3K signaling pathway.

Figure 3

The effect of fABT on the IGF2/PI3K signaling pathway. A – Western blotting analysis of the IGF2/PI3K signaling in T2D animal hepatocytes. B – Protein expression levels in relation to β-actin

fABT promoted erythrocyte functions

The abovementioned results confirmed the vital role of fABT in the biological functions in diabetic mice; therefore, we determined the erythrocyte functions in diabetic mice after receiving fABT. Our data showed that fABT enhanced the oxygen-carrying capacity of erythrocytes (Figure 4 A) and reduced oxygen consumption (Figure 4 B) and osmotic fragility (Figure 4 C) compared to the T2D group. These results confirmed the vital effects of fABT on erythrocyte functions and various biological processes, such as in diabetic mice.

Figure 4

The effect of fABT on erythrocytic functionality. A – The effective oxygen-carrying capacity in T2D animals. B – Oxygen consumption rate in T2D animals. C – Osmotic fragility of red blood cells in T2D animals