Protective Efficacy of Lactobacillus plantarum Postbiotic beLP-K in a Dexamethasone-Induced Sarcopenia Model

Abstract

Sarcopenia is characterized by a reduction in muscle function and skeletal muscle mass relative to that of healthy individuals. In older adults and those who are less resistant to sarcopenia, glucocorticoid secretion or accumulation during treatment exacerbates muscle protein degradation, potentially causing sarcopenia. This study assessed the preventive effects and mechanisms of heat-killed Lactobacillus plantarum postbiotic beLP-K (beLP-K) against dexamethasone (DEX)-induced sarcopenia in C2C12 myotubes and Sprague-Dawley rats. The administration of beLP-K did not induce cytotoxicity and mitigated cell damage caused by DEX. Furthermore, beLP-K significantly reduced the expression of forkhead box O3 α (FoxO3α), muscle atrophy f-box (MAFbx)/atrogin-1, and muscle RING-finger protein-1 (MuRF1), which are associated with muscle protein degradation. DEX induced weight loss in rats; however, in the beLP-K group, weight gain was observed. Micro-computed tomography analysis revealed that beLP-K increased muscle mass, correlating with weight and grip strength. beLP-K alleviated the DEX-induced reduction in grip strength and increased the mass of hind leg muscles. The correlation between beLP-K administration and increased muscle mass was associated with decreased expression levels of muscle degradation-related proteins such as MAFbx/atrogin-1 and MuRF1. Therefore, beLP-K may serve as a treatment for sarcopenia or as functional food material.

Keywords: FoxO3α; Lactobacillus plantarum; MAFbx/atrogin-1; MuRF1; beLP-K; dexamethasone; postbiotic; sarcopenia.

Figure 1

Cytotoxic effects of beLP-K in C2C12 myoblasts. (A) C2C12 myoblasts were treated with beLP-K at concentrations of 0, 50, 100, 200, and 500 μg/mL, and cultured for 24 and (B) 48 h. Cytotoxicity was assessed using the MTT assay. Results are expressed as cell viability relative to the control group. MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 2

Cytotoxic effects of beLP-K in C2C12 myotubes. (A) C2C12 myotubes were treated with beLP-K at concentrations of 50–500 μg/mL and cultured for 24 and (B) 48 h. Cytotoxicity was assessed using the MTT assay. Results are presented as cell viability relative to the control group. Data are presented as the mean ± SEM (n = 4); *** p < 0.001 compared to the control group. MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 3

Effects of beLP-K on the viability of DEX-induced cell damage in C2C12 myotubes. Cell viability was assessed using MTT assay. Data are presented as the mean ± SEM (n = 4); *** p < 0.001 compared to the control group. # p < 0.05 compared to the DEX treatment group. DEX: dexamethasone, MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 4

Effects of beLP-K against DEX-induced cell damage in C2C12 myotubes and morphological analysis. (A) Depiction of stained C2C12 myotubes after 24 h of treatment with 100 μM DEX to induce cell damage (scale bar: 20 μm). (B–D) Comparative analysis of myotube diameter, length, and area between the beLP-K treatment groups following treatment with DEX. All images were randomly selected from three independent experiments and analyzed using ImageJ software (version 1.8.0.170). Data are presented as the mean ± SEM (n = 15 per group); *** p < 0.001 compared with the control group; # p < 0.05, ## p < 0.01 and ### p < 0.001 compared with the DEX treatment group. DEX: dexamethasone.

Figure 5

Effects of beLP-K-mediated E3-ubiquitin proteasome pathway inhibition on DEX-induced damage in C2C12 myotubes. (A) Western blotting of FoxO3α, MAFbx/atrogin-1, MuRF1, and GAPDH. (B) Quantitative analysis of FoxO3α and GAPDH expression (n = 4). (C) Quantitative analysis of MAFbx/atrogin-1 and GAPDH expression (n = 7). (D) Quantitative analysis of MuRF1 and GAPDH expression (n = 5). GAPDH expression remained stable across all groups and was used to normalize the expression levels of each protein. Results are expressed as multiple changes compared to the DEX treatment group, which was set to 1. Data are presented as the mean ± SEM; *** p < 0.001 compared to the control group. ## p < 0.01 and ### p < 0.001 compared to the DEX treatment group. DEX: dexamethasone, FoxO3α: forkhead box o3 α, MAFbx: muscle atrophy f-box, MuRF1: muscle ring finger protein-1, and GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Figure 6

Effects of beLP-K on DEX-induced sarcopenia in SD rats. (A) Schematic for drug administration and experimentation on male SD rats. (B) Alterations in the body weights of the experimental animals were monitored following beLP-K and DEX administration. (C) Changes in muscle volume were assessed thrice through micro-CT scanning. Muscle volume was measured using CT Analyzer software (v1.11). Data are expressed as the mean ± SEM (n = 6). Statistical significance was determined using two-way ANOVA; ## p < 0.01 and ### p < 0.001 compared with the DEX treatment group. DEX: dexamethasone, SD: Sprague–Dawley, CT: computed tomography, GA, gastrocnemius, TA, tibialis anterior, GS: grip strength, L-beLP-K: low-dose beLP-K, H-beLP-K: high-dose beLP-K, I.P.: intraperitoneal, and P.O., per os.

Figure 7

Effects of beLP-K on functional decline in DEX-induced sarcopenia model SD rats. (A) Alterations in rat GS prior to DEX administration. (B) Alterations in rat GS prior to sacrifice, following DEX administration. GS changes were evaluated and documented 5 d pre- and post-DEX administration, subsequent to oral treatment with beLP-K. GS for each group was assessed thrice per rat. Data are expressed as the mean ± SEM (n = 6 per group); *** p < 0.001 compared to the CON group. ### p < 0.001 compared to the DEX treatment group. DEX: dexamethasone, SD: Sprague–Dawley, DEX: dexamethasone, GS: grip strength, CON: control, L-beLP-K: low-dose beLP-K, and H-beLP-K: high-dose beLP-K.

Figure 8

Effects of beLP-K on total tibial muscle weight in DEX-induced sarcopenia model SD rats. (A) Comparative depiction of tibial muscles in each group post-sacrifice. (B) Comparative analysis of the weights of the extracted tibial muscles. DEX-induced sarcopenia induced significantly lower muscle weight in the beLP-K treatment group than in the DEX treatment group. Data are expressed as the mean ± SEM (n = 6 per group); ** p < 0.01 compared to the CON group. # p < 0.05 compared to the DEX treatment group. DEX: dexamethasone, SD: Sprague–Dawley, DEX: dexamethasone, GS: grip strength, CON: control, L-beLP-K: low-dose beLP-K, and H-beLP-K: high-dose beLP-K.

Figure 9

Effects of beLP-K on GA and TA muscle weight in DEX-induced sarcopenia model SD rats. Rats in each experimental group were administered beLP-K for 2 weeks, then administered DEX for 5 d. Muscle tissue form and weight changes were determined for the (A) GA and (B) TA muscles after the sacrifice of each experimental animal. Data are presented as the mean ± SEM (n = 6 per group); * p < 0.05 and *** p < 0.001 compared to the CON group. # p < 0.05 compared to the DEX treatment group. DEX: dexamethasone, SD: Sprague–Dawley, DEX: dexamethasone, GS: grip strength, CON: control, L-beLP-K: low-dose beLP-K, and H-beLP-K: high-dose beLP-K.

Figure 10

beLP-K attenuates sarcopenia by suppressing the expression of muscle degradation-related proteins in the DEX-induced SD rat model. (A) Representative Western blot analysis of MAFbx/atrogin-1, MuRF1, and β-actin in GA. (B) Comparison of MAFbx/atrogin-1 protein expression in the GA group. (C) Comparison of MuRF1 protein expression levels in each group. Protein expression levels were normalized to β-actin for quantification. Data are presented as the mean ± SEM (n = 3); * p < 0.05, compared to the CON group. # p < 0.05 and ## p < 0.01 compared to the DEX group. DEX: dexamethasone, SD: Sprague–Dawley, DEX: dexamethasone, GA: gastrocnemius, MAFbx: muscle atrophy f-box, MuRF1: muscle ring finger protein 1. CON: control, L-beLP-K: low-dose beLP-K, and H-beLP-K: high-dose beLP-K.

Figure 11

Schematic diagram of the mechanisms of beLP-K in modulating sarcopenia in DEX-induced C2C12 myotubes and SD rats. Red arrows indicate the effects of beLP-K; yellow arrows indicate DEX administration in vivo and in vitro; and gray arrows represent signaling pathways downstream of glucocorticoid receptor activation. DEX: dexamethasone, I.P.: intraperitoneal, P.O.: per os, SD: Sprague–Dawley, FoxO3α: forkhead box o3 α, MAFbx: muscle atrophy f-box, and MuRF1: muscle ring finger protein-1.