Polygonati Rhizoma Prevents Glucocorticoid-Induced Growth Inhibition of Muscle via Promoting Muscle Angiogenesis Through Deoxycholic Acid

Abstract

Background: Glucocorticoids are commonly used in clinical treatments but can cause muscle growth inhibition and weakness at high doses. The mechanisms and treatments for glucocorticoid-induced muscle growth inhibition remain poorly understood. This study aims to investigate the anti-atrophic effects of Polygonati Rhizoma (PR) and a mixture of low-dose fructose and glucose (MFG, an active component mimic in PR) on skeletal muscle.

Methods: Male C57BL/6 mice (3-week-old, n = 8) were gavaged with aqueous extract of PR (AEPR). MFG was used to gavage normal male C57BL/6 mice (3-week-old, n = 10) and male C57BL/6 mice with dexamethasone (DEX)-induced muscle growth inhibition (3-week-old, n = 7). After 2 weeks of gavage, the body weight and muscle mass of the mice were measured. Intestinal content was collected, the concentration of deoxycholic acid (DCA) was analysed and gut microbiota changes were assessed through 16S rRNA gene sequencing. Muscle angiogenesis was examined through the expression of vascular endothelial growth factors (VEGFs), focusing on the DCA-activated TGR5/cAMP/PKA/pCREB pathway.

Results: AEPR significantly increased the body weight (22.90 ± 0.90 vs. 21.83 ± 0.87 g, *p < 0.05) and grip strength (1.32 ± 0.11 vs. 1.04 ± 0.12 N, ***p < 0.001) of mice. MFG (0.5 g/kg body weight) also significantly elevated the body weight (21.44 ± 0.71 vs. 20.14 ± 0.82 g, **p < 0.01) and muscle mass (0.37 ± 0.018 vs. 0.33 ± 0.035 g, **p < 0.01) of mice. In the DEX group, MFG restored the DCA level (log₂[intensity]) in intestinal content (25.41 ± 1.64 vs. 22.69 ± 0.74, *p < 0.05) and increased the abundance of Collinsella aerofaciens as measured by DNA concentration (0.80 ± 0.64 vs. 0.24 ± 0.09 pg/μL, p = 0.096). Mechanistically, MFG upregulated VEGFs expression and promoted muscle angiogenesis via the TGR5/cAMP/PKA/pCREB pathway.

Conclusions: This study demonstrates that AEPR and its active component mimic MFG can promote muscle growth and MFG mitigates muscle growth inhibition by modulating gut microbiota and enhancing muscle angiogenesis. These findings suggest that fructose-containing treatments are novel strategies to address skeletal muscle dysfunction.

Keywords: Polygonati Rhizoma; angiogenesis; deoxycholic acid; fructose; skeletal muscle.

FIGURE 1

AEPR promotes growth and force of skeletal muscle. (A) Study design with different concentrations of AEPR, n = 8 biological replicates in each group and body weight curve under different treatments; (B) forced swimming times of mice and grip strength of mice; (C) representative images of TA, Qu and GAS muscles in the CON, LAEPR and HAEPR mice and muscle mass; (D and E) representative H&E staining of myofibre cross‐section of GAS and cross‐sectional area (CSA) distribution and average CSA of muscle fibre. Data information: t test was used in this figure where error bars represent SD; and *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

MFG promotes muscle development. (A) Study design of MFG treatment, n = 10 biological replicates in each group and body weight curve under different treatments; (B) TA, GAS and Qu muscle mass; (C) representative H&E staining of myofibre cross section of TA, GAS and Qu and the CSA distribution and average CSA of muscle fibre; (D) western blot and quantification of MyoD, MyoG and MRF4; Data information: t test was used in this figure where error bars represent SD; and *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

MFG alleviates glucocorticoid‐induced muscle growth inhibition. (A) Study design of the DEX‐induced muscle growth inhibition model, n = 7 biological replicates in each group; (B) body weight curve under different treatments; (C) body weight; (D) TA, GAS, Qu and total muscle mass; (E) representative H&E staining of myofibre cross section of TA, GAS and Qu and CSA distribution and average CSA of muscle fibre; (F) western blot and quantification of MyoD, MyoG and MRF4. Data information: t test was used in this figure where error bars represent SD; and *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

MFG promotes muscle angiogenesis via VEGFs to alleviate glucocorticoid‐induced muscle growth inhibition. (A) Groups of mRNA sequencing, n = 3 biological replicates in each group and work flow of selection strategy of mRNA sequencing; (B) GO pathway enrichment analyses of the upregulated DEGs in MFG versus CON and DEX‐MFG versus DEX, the dot size represents the number of DEGs and the dot colour represents the corresponding p value; (C) heatmap of representative DEGs in DEX‐MFG versus DEX and representative significantly upregulated GO terms with genes in DEX‐MFG versus DEX; (D) RPKM levels of representative genes in the control (CON), DEX and DEX‐MFG and mRNA levels of angiogenesis and contractility genes in DEX‐MFG group; (E and F) representative images and quantification of CD31 immunofluorescence staining in Qu muscle of DEX‐MFG group and MFG group. Data information: t test was used in this figure where error bars represent SD; and *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

MFG promotes muscle angiogenesis through increasing intestine‐derived DCA production. (A) Groups of non‐targeted metabonomic sequencing, n = 3 biological replicates in each group; (B) heatmaps of representative metabolites in ileal contents in mice of the DEX versus control (CON) groups, and DEX‐MFG versus DEX groups; (C) Venn plot of common and distinct differential metabolites of DEX versus CON and DEX‐MFG versus DEX and relative enrichment of common differential metabolites measured by LC–MS/MS; (D) body weight curve under different treatments, n = 8 biological replicates in each group and muscle mass; (E) representative H&E staining of myofibre cross section of TA, GAS and Qu and CSA distribution and average CSA of muscle fibre; (F) representative images and quantification of CD31 immunofluorescence staining in Qu muscle of DCA group. Data information: t test was used in this figure where error bars represent SD; and *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

DCA is produced by C. aerofaciens in response to MFG supplementation. (A) Groups of 16S rRNA gene high‐throughput sequencing, n = 4 biological replicates in each group; (B) α‐diversity analysis of gut microbes reflected by Chao1 and Simpson indices; (C) gut microbiota abundance at the phylum level and comparison of phyla in the CON and MFG groups; (D) principal component analysis plot of the colonic contents in the CON and MFG groups; (E) bile acid profile measurement, n = 3 biological replicates in each group and DCA synthesis in vitro; (F) standard curve of C. aerofaciens and DNA concentrations of C. aerofaciens in DEX‐MFG group, n = 6 biological replicates in each group; t test was used in this figure where error bars represent SD; and *p < 0.05.

FIGURE 7

TGR5/cAMP/PKA/pCREB pathway accounts for the upregulation of VEGFs in muscle angiogenesis. (A) Study design of the C2C12 cells with DCA treatment, n = 3 biological replicates in each group and mRNA levels of genes under treatments with different concentrations of DCA; (B) cAMP concentration of culture medium; (C) Western blot of p‐CREB under treatments with different concentrations of DCA; (D) western blot and quantification of p‐CREB and TGR5 in DCA treatment groups; (E) western blot and quantification of p‐CREB under treatments with DCA and inhibitors; (F) luciferase activity with DCA treatment, n = 4 biological replicates in each group and luciferase activity under DCA and inhibitor treatments. Data information: t test was used in this figure where error bars represent SD; and *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 8

Mechanism diagram for MFG to ameliorate glucocorticoid‐induced skeletal muscle growth inhibition in mice.