Whole milk protein powder separated by low-temperature nanofiltration membrane administration alleviates sepsis-induced myopathy

Abstract

Sepsis-induced myopathy (SIM) has been recognized as a critical risk factor for the development of acquired muscle weakness among patients in the intensive care unit. These individuals frequently encounter inadequate dietary intake and malnutrition. With the aggravation of the severity of the person's condition, leading to increased skeletal muscle protein breakdown and reduced synthesis, which is an urgent problem to be solved in clinical nutritional treatment. Whole milk protein powder (WMPP) has promising bioactive nutrients and holds promising potential for enhancing skeletal muscle mass. The study was designed to delve into the potential effects and mechanisms of WMPP intervention for increaseing skeletal muscle mass on SIM mice. Our results clearly show that the intervention with WMPP can significantly improve the exercise capacity and skeletal muscle mass in SIM mice. It significantly increases the diameter and cross-sectional area (CSA) of skeletal muscle fibers, while effectively reducing the excessive aggregation of collagen fibers and the abnormal accumulation of adipose tissue in the skeletal muscle of SIM mice. Moreover, WMPP intervention also significantly alleviated the oxidative stress status of mitochondria, which subsequently enhanced the expression of mitochondrial metabolic enzymes. The mechanism may be associated with decreased AMPK phosphorylation in skeletal muscle tissue and simultaneously increased phosphorylation of mTOR, p70S6K1, and 4EBP-1 in SIM mice. In summary, the WMPP intervention significantly enhances exercise capacity and skeletal muscle mass while mitigating the oxidative stress status of mitochondria. Furthermore, it regulates skeletal muscle anabolism via the AMPK/mTOR signaling pathway in SIM mice.

Keywords: AMPK/mTOR signal pathway; Mitochondria function; Sepsis-induced myopathy; Whole milk protein powder.

Fig. 1

The animal treatment protocol is as follows: Mice were administered a single injection of LPS to simulate SIM, followed by gavage administration of WMPP for the prevention and alleviation of SIM. Beginning on the first day of LPS injection, daily monitoring of body weight, food intake, and water consumption was conducted until sample collection

Fig. 2

WMPP intervention improves skeletal muscle mass and function in SIM mice. (A) Body weight. (B) Weight of gastrocnemius (GAS), tibialis anterior (TA), quadriceps (QU), biceps brachii (BB), soleus (SOL) and extensor digitorum longus (EDL). (C) Skeletal muscle weight/body weight. (D) Representative images of GAS, BB, and tibialis anterior morphology in each group of mice. (E) Latency to fall time. (F) Climbing distance. (G) Turnaround and bottom arrival time. Data are presented as mean ± SD (n = 6). *p < 0.05 compared with the control group; #p < 0.05 compared with the SIM group; &p < 0.05 compared with the 1.5 g/kg WMPP group

Fig. 3

WMPP intervention mitigates skeletal muscle inflammation and muscle damage in SIM mice. (A) Serum levels of IL-1β. (B) Serum levels of TNF-α. (C) Relative expression levels of IL-1β mRNA in the GAS. (D) Relative expression levels of TNF-α mRNA in the GAS. (E) Serum levels of IL-4. (F) Serum levels of IL-13. (G) Levels of CK in the GAS. (H) Levels of MYO/MB in the GAS. (I) Levels of LDH in the GAS. Data are presented as mean ± SD (n ≥ 3). *p < 0.05 compared with the control group; #p < 0.05 compared with the SIM group; &p < 0.05 compared with the 1.5 g/kg WMPP group

Fig. 4

WMPP intervention mitigates skeletal muscle atrophy, collagen deposition, and lipid accumulation in SIM mice. (A) Representative images of HE staining in the GAS, frequency distribution of different average CSA of skeletal muscle fibers, average CSA of skeletal muscle fibers, and average CSA% (relative to the control group) (scale bar = 25 μm). (B) Representative images of HE staining in the TA, frequency distribution of different average CSA of skeletal muscle fibers, average CSA of skeletal muscle fibers, and average CSA% (relative to the control group) (scale bar = 25 μm). (C) Representative images of Masson staining in the GAS and quantification of collagen deposition in the GAS (scale bar = 100 μm). (D) Representative images of Oil Red O staining in the GAS and quantification of lipid deposition in the GAS (scale bar = 100 μm). Data are presented as mean ± SD (n ≥ 5). *p < 0.05 compared with the control group; #p < 0.05 compared with the SIM group; &p < 0.05 compared with the 1.5 g/kg WMPP group

Fig. 5

WMPP intervention attenuates oxidative stress levels and improves mitochondrial function in SIM mice. (A) Relative levels of ROS in the GAS. (B) MDA content in the GAS. (C) SOD activity in the GAS. (D) CAT activity in the GAS. (E) GSH-PX activity in the GAS. (F) AMP content in the GAS. (G) ATP content in the GAS. (H) AMP/ATP in the GAS. (I) Expression of SDHB protein in the SOL. (J) Mean optical density value of SDHB protein in the SOL. (K) Expression of NUDFB8 protein in the SOL. (L) Mean optical density value of NUDFB8 protein in the SOL. (M) Expression of UQCRC2 protein in the SOL. (N) Mean optical density value of UQCRC2 protein in the SOL. Data are presented as mean ± SD (n = 6). *p < 0.05 compared with the control group; #p < 0.05 compared with the SIM group; &p < 0.05 compared with the 1.5 g/kg WMPP group

Fig. 6

WMPP intervention improves the anabolism of skeletal muscle in SIM mice. (A) Expression levels of AMPK and p-AMPK proteins in the GAS. (B) Quantitative analysis of p-AMPK/AMPK protein expression in the GAS. (C) Serum TP levels. (D) Serum ALB levels. (E) Expression levels of mTOR, p70S6K1, and 4EBP-1 proteins and their phosphorylated proteins in the GAS. (F) Quantitative analysis of p-mTOR/mTOR protein expression in the GAS. (G) Quantitative analysis of p-p70S6K1/p70S6K1 protein expression in the GAS. (H) Quantitative analysis of p-4EBP-1/4EBP-1 protein expression in the GAS. (I) Immunofluorescence expression of MYH protein in the GAS. (J) Immunofluorescence expression of MYH protein in the TA. (K) Mean optical density values of MYH in the GAS. (L) Mean optical density values of MYH in the TA. Data are presented as mean ± SD (n ≥ 4). *p < 0.05 compared with the control group; #p < 0.05 compared with the SIM group; &p < 0.05 compared with the 1.5 g/kg WMPP group

Fig. 7

WMPP intervention alleviates the catabolism of skeletal muscle in SIM mice. (A) Expression levels of Atrogin-1 and MURF-1 mRNA in the GAS. (B) Expression levels of Atrogin-1 and MURF-1 proteins in the GAS. (C) Quantitative analysis of Atrogin-1 protein expression in the GAS. (D) Quantitative analysis of MURF-1 protein expression in the GAS. (E) Serum BUN levels. (F) Serum Cr levels. (G) Serum BUN/Cr levels. Data are presented as mean ± SD (n ≥ 3). *p < 0.05 compared with the control group; #p < 0.05 compared with the SIM group; &p < 0.05 compared with the 1.5 g/kg WMPP group