Whey protein isolate and soybean protein isolate were purchased from Hilmar Ingredients (Livingston, CA, USA) and Shaanxi Pioneer Biotech Co. Ltd. (Xi’an, Shaanxi, China), respectively. Normal saline was procured from Shijiazhuang No.4 Pharmaceutical Co. Ltd. (Shijiazhuang, Hebei, China). The antibodies against CD68 (ab125212) and MPO (ab208670) were purchased from Abcam (Cambridge, MA, USA).

Male SD rats (6-wk-old, 180–220 g weight) were housed at room temperature (22–24°C) with a 12 h day-night cycle. All studies adhered to procedures consistent with the International Guiding Principles for Biomedical Research Involving Animals issued by the Council for the International Organizations of Medical Sciences (CIOMS) and were approved by the Animal Care and Use Committee of the Fourth Medical Center of PLA General Hospital. After adaptive feeding for 1 wk, the rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium salt. A full thickness burn model with 30% total body surface area (TBSA) was conducted as previously described [1819]. Briefly, an electric hair cutter was used to shave the dorsal hair. The burn rat model was established by placing the back skin in hot water (94°C) for 12 s; rats in the sham group were placed in warm water (37°C) for 12 s. The wounds were swabbed with 1% tincture of iodine to prevent infection. Fluid infusion was administered via an immediate intraperitoneal injection of balanced salt solution (40 mg/kg·body weight) for the prevention of shock.

After successful modeling, all rats were administered by gavage once a day for 3, 7 or 14 days. Equivalent whey protein and soybean protein (whey:soybean = 1:1) were directly dissolved in normal saline. Burn rats were randomly divided into 2 groups: (1) Burn+BP, burn rats were administered 2 mL blended protein dissolved in normal saline (200 mg/kg • body weight) by gavage, as described previously [6]; (2) Burn+S (burn rats) and Sham+S (sham rats) were administered 2 ml normal saline by gavage. After the respective treatment of rats in the three treatment groups, the samples were collected at indicated times (Fig. 1).

At the indicated times, the rats were anesthetized, and blood samples were collected from the abdominal aorta using the procoagulant blood vessel collection (yellow) tube and the anticoagulant blood vessel collection (purple) tube. Liver and kidney tissue was also collected and placed in paraformaldehyde for tissue fixation (Servicebio, Wuhan, Hubei, China).

Whole blood was collected using anticoagulant blood vessel collection, and routine blood examination was performed on the Sysmex K4500 hematology analyzer (Kobe, Japan).

Blood samples were collected from the treated animals using procoagulant blood vessel collection tubes, and various indices, including liver function and renal function, were measured by the KHB-ZY1280 (Shanghai Kehua Bio-Engineering Co. Ltd., Shanghai, China).

After treatment for 14 days, the liver and kidney tissue samples were harvested from the three treatment groups and subsequently fixed in 4% paraformaldehyde for 24 h at room temperature. Samples were then embedded in paraffin and sliced into 5 mm thick perpendicular plane sections, and immunohistochemical analysis was done following the manufacturer’s instruction kit from Servicebio. The antibodies against CD-68 and MPO, and the HRP labeled corresponding secondary antibody were applied. Treated sections were finally stained by exposure to 3,3’-diaminobenzidine (DAB), and the images were microscopically captured.

Rat Chemokine/Cytokine Panel 22plex (eBioscience, Vienna, Austria) was used to measure the concentration of plasma cytokines, as described in our previous publication [17]. Levels of interleukin (IL)-4, IL-6, IL-10, IL-13, macrophage chemoattractant protein-1 (MCP-1), Eotaxin, and regulated upon activation normal T cell expressed and secreted factor (RANTES) were analyzed using the Luminex™ 200 (Luminex, Shanghai, China).

All data are expressed as the mean ± SD and the differences are analyzed using the analysis of variance assay. The difference is considered to be statistically significant at P ≤ 0.05.

During an inflammatory reaction, there is a dramatic increase in the number of immune related cells, especially white blood cells. This is because the first line of defense in our body, viz., skin integrity, is broken after burn injury, leading to a pathogen invasion and subsequent excessive increment of white blood cells [17]. Therefore, the white blood cell count is one of the primary indicators for evaluating inflammation [20]. Effective treatment for reducing the white blood cell number might help ease the inflammatory reaction. Results of the current study revealed that the number of white blood cells significantly increases on days 3, 7, and 14 after burn injury (Fig. 2A), indicating that an inflammatory reaction is significantly promoted after burn injury. Previous studies have reported that protein nutritional support plays an important role in reducing inflammatory reactions [421]. In the current research, blended protein as described in “MATERIALS AND METHODS” was used to treat the burned rats. Compared with rats in the Burn+S group, blended protein supplementation remarkably alleviated the increment of white blood cells on days 3, 7, and 14 (Fig. 2A), thereby indicating that blended protein treatment has the potential to reduce burn-induced inflammation. White blood cells consist of monocytes, granulocytes (neutrophils, eosinophils, and basophils), and lymphocytes. The number of monocytes and neutrophils in different groups was measured, and similar to the variation tendency of white blood cells, the number of monocytes also increased dramatically after burn injury, and significantly reduced with blended protein administration (Fig. 2B). Burn injury also evoked the increment of neutrophils on days 3, 7, and 14. However, the number of neutrophils in the Burn+S group was comparable with those in the Burn+BP group on days 3 and 7, which subsequently decreased dramatically on day 14 (Fig. 2C). The data of the lymphocyte count indicates that burn injury results in a dramatic increase in the number of lymphocytes on days 3, 7, and 14, but blended protein treatment significantly reduces the lymphocyte number on day 14, as compared to numbers obtained in the Burn+S group (Fig. 2D). Taken together, our results indicate that blended protein ingestion might be beneficial in reducing burn-induced increment of inflammation related cells with simultaneous attenuation of the inflammatory reaction.

Cytokines are usually secreted by inflammation related cells such as monocytes, lymphocytes, and neutrophils, and exert important roles in regulating the inflammatory reaction. Subsequent to a burn injury, pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and IL-6 are usually overexpressed, and the level of anti-inflammatory factor IL-10 is down-regulated [151617]. Chemokines play important roles in recruiting inflammatory cells and promoting cell infiltration to the inflammatory lesions [22]. In order to detect the influence of blended protein administration on the expression of cytokines after burn injury, we measured the levels of pro-inflammatory factors, anti-inflammatory factors, and chemokines, using the Rat Chemokine/Cytokine Panel 22plex kit. As shown in Figure 3, the expression of pro-inflammatory factors TNF-α and IL-6 were significantly increased after burn injury, compared to levels obtained in the Sham+S group, on days 3, 7, and 14; moreover, the blended protein ingestion significantly down-regulated the burn-induced TNF-α and IL-6 overexpression on days 7 and 14 (Fig. 3A and B). Compared to the Sham+S group, expression of the anti-inflammatory factor IL-10 was dramatically decreased after burn injury on days 7 and 14, which recovered after the blended protein treatment (Fig. 3C). IL-4 and IL-13 are other important anti-inflammatory factors and attenuate the inflammatory reaction via regulation of macrophage polarization [23]. Consistent with the tendency of IL-10, secretion of IL-4 and IL-13 was also obviously suppressed after burn injury on days 7 and 14, but significantly increased in the blended protein supplementation group (Fig. 3D and E). These results indicate that a burn injury results in elevating the expressions of pro-inflammatory factors (TNF-α and IL-6) and reducing the levels of anti-inflammatory factors (IL-4, IL-10, and IL-13), but administration of blended protein remarkably reverses this phenomenon. This might be a partial mechanism of blended protein attenuating burn-induced inflammation. Administration of blended protein also exerted an effect on regulating chemokine expression. MCP-1/chemokine (C-C motif) ligand 2 (CCL2) regulates the release of monocytes from the bone marrow and mediates migration to the inflamed sites [24]. RANTES/CCL5 enhances chemotaxis of immune cells and is involved in trafficking them to the inflammatory lesions [25]. Eotaxin, a potent chemoattractant of eosinophils, stimulates eosinophils migration [26]. Hence, all chemokines effectively regulate the inflammatory reaction via mediating chemotaxis of the inflammatory cells. Our results indicate that burn injury promotes the expressions of MCP-1, RANTES, and Eotaxin on days 3, 7, and 14, but blended protein ingestion significantly reduces the expressions on days 7 and 14 (Fig. 3F-H), thereby exhibiting the potential role of blended protein treatment in reducing burn-induced inflammatory cell infiltration. Taken together, our results indicate the beneficial role of blended protein nutritional support in alleviating an inflammatory reaction and reducing inflammatory cells infiltration, thereby playing an essential role in protecting organ function after burn injury.

Organ dysfunction is a common adverse effect observed in patients with burn injury. Kidney and liver functions are usually impaired after major burn injuries, partially due to the excessive inflammatory reaction and inflammatory cells infiltration [2728]. To determine the function of blended protein in regulating inflammatory cells infiltration, we evaluated the expressions of CD68 (macrophage marker) and MPO (neutrophil marker) by subjecting the kidney and liver tissues to immunohistochemical assays. As presented in Fig. 4, the significantly increased expressions of CD68 and MPO in the kidney tissue after burn injury were effectively reduced after blended protein supplementation, in comparison to the expressions determined in the Burn+S group (upper panel). This suggests that blended protein treatment contributes to reducing the macrophage and neutrophil cells infiltration of kidney tissue. Similar results were obtained in the liver tissue; macrophage and neutrophil cells infiltration was promoted after burn injury, but was remarkably inhibited after blended protein supplementation (Fig. 4, lower panel). Previous publications have reported that attenuating macrophage and neutrophil cells infiltration results in ameliorating kidney and liver damage [2930]. Therefore, inhibiting inflammatory cells infiltration might be a key strategy for protecting the liver and kidney functions after burn injury. In summary, blended protein ingestion exerts an important role in reversing burn-induced increment of inflammatory cells infiltration, and potentially attenuates damage to the kidney and liver.

To further analyze the kidney function of rats in different groups, the levels of serum uric acid (UA), blood urea nitrogen (BUN), and creatinine (CRE) were measured. It has previously been demonstrated that consuming a high protein diet risks increasing the kidney burden and elevates serum UA levels [3132]. Assessing the UA level is generally applied as an early clue for judging kidney injury [33]. Especially in burn patients, gastrointestinal congestion leads to impaired functions of digestion and absorption, which further augments the concern of protein nutrition increasing kidney burden [34]. However, results from a clinical trial indicate that isolated soybean protein with flaxseed oil or corn oil nutritional support has no significant effect on increasing the kidney burden [35]. In the current study, we observed slightly increased UA levels after burn injury on days 3, 7, and 14, which were further elevated on days 7 and 14 after blended protein administration, but with no significant difference observed between the three groups (Fig. 5A). This indicates that a blended protein diet has no risk of increasing the kidney burden, which is probably because whey and soybean proteins are known to be easily digested and absorbed [36]. Previous studies also reported that soybean protein consumption slows the decline of the estimated glomerular filtration rate and significantly improves proteinuria in patients with nephropathy [37]. Other parameters such as BUN and CRE, which reflect the renal filtration function, are critical indicators of kidney function. Our results demonstrate that compared to the Burn+S group, burn injury led to a remarkable increment of BUN levels on days 3, 7, and 14, with noticeably reduced levels obtained after blended protein administration (Fig. 5B). Although the CRE level was comparable in all three groups on day 3, a similar tendency with BUN levels was observed on days 7 and 14, being dramatically up-regulated in the Burn+S group and down-regulated in the Burn+BP group (Fig. 5C). These results indicate that burn injury elevates the levels of BUN and CRE, implying the possibility of kidney function impairment. However, blended protein consumption effectively reduces the levels of BUN and CRE after burn injury, and might exert an essential role in protecting kidney function.

The liver is also one of the organs liable to be damaged after burn injury. In order to evaluate liver function, related indices were evaluated, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH). As shown in Fig. 6A, a significant increase was observed in the ALT level after burn injury on days 3, 7, and 14 compared to the Sham+S group, which were dramatically down-regulated after blended protein treatment. The level of AST was comparable among the three groups on day 3, but was obviously elevated after burn injury on days 7 and 14, and dramatically down-regulated in the Burn+BP group (Fig. 6B). Similar results were obtained for ALP and LDH levels; burn injury led to significant increment of ALP and LDH levels compared to the Sham+S group on days 3, 7, and 14. However, blended protein ingestion remarkably reduced the ALP and LDH expressions on day 14 (Fig. 6C and D). ALT and AST are mainly synthesized by liver cells, and their high levels in serum imply the occurrence of liver injury [38]. ALP and LDH are widely distributed in the liver, skeletal muscle, and kidney, and are therefore also important indices to determine liver injury [39]. Our results indicate that burn injury induces liver injury, while blended protein supplementation contributes to improving the liver function. Biosynthesis is one of the important functions of the liver, and is an essential site for protein synthesis in our body. Thus, the levels of total protein (TP) and albumin (ALB) are also important for evaluating liver function. The serum levels of TP and ALB were therefore measured in all three groups. Our results show no significant change in the total protein level among the three groups on days 3, 7, and 14 (Fig. 6E). Although the level of ALB was comparable among the 3 groups on day 3, a dramatic reduction of ALB level was observed after burn injury on days 7 and 14 (Fig. 6F), implying that ALB synthesis is suppressed after burn injury. Compared to the value obtained in the Burn+S group, the ALB level was remarkably restored after blended protein administration (Fig. 6F), indicating that blended protein nutritional intervention helps to improve the liver capacity for ALB synthesis. Taken together, all the results (including ALT, AST, ALP, LDH, and ALB) provide compelling evidence that blended protein consumption diminishes burn-induced liver injury and exerts an essential role in liver protection.