Nutr Res Pract.  2022 Apr;16(2):147-160. 10.4162/nrp.2022.16.2.147.

Ursolic acid improves the indoxyl sulfate-induced impairment of mitochondrial biogenesis in C2C12 cells

• Sasaki Y
• Kojima-Yuasa A
• Tadano H
• Mizuno A
• Kon A
• Norikura T

• KMID: 2527807
• DOI: http://doi.org/10.4162/nrp.2022.16.2.147

Abstract

BACKGROUND/OBJECTIVES
Patients with chronic kidney disease (CKD) have a high concentration of uremic toxins in their blood and often experience muscle atrophy. Indoxyl sulfate (IS) is a uremic toxin produced by tryptophan metabolism. Although an elevated IS level may induce muscle dysfunction, the effect of IS on physiological concentration has not been elucidated. Additionally, the effects of ursolic acid (UA) on muscle hypertrophy have been reported in healthy models; however, it is unclear whether UA ameliorates muscle dysfunction associated with chronic diseases, such as CKD. Thus, this study aimed to investigate whether UA can improve the IS-induced impairment of mitochondrial biogenesis.
MATERIALS/METHODS
C2C12 cells were incubated with or without IS (0.1 mM) and UA (1 or 2 µM) to elucidate the physiological effect of UA on CKD-related mitochondrial dysfunction and its related mechanisms using real-time reverse transcription-polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay.
RESULTS
IS suppressed the expression of differentiation marker genes without decreasing cell viability. IS decreased the mitochondrial DNA copy number and ATP levels by downregulating the genes pertaining to mitochondrial biogenesis (Ppargc1a, Nrf1, Tfam, Sirt1, and Mef2c), fusion (Mfn1 and Mfn2), oxidative phosphorylation (Cycs and Atp5b), and fatty acid oxidation (Pdk4, Acadm, Cpt1b, and Cd36). Furthermore, IS increased the intracellular mRNA and secretory protein levels of interleukin (IL)-6. Finally, UA ameliorated the IS-induced impairment in C2C12 cells.
CONCLUSIONS
Our results indicated that UA improves the IS-induced impairment of mitochondrial biogenesis by affecting differentiation, ATP levels, and IL-6 secretion in C2C12 cells. Therefore, UA could be a novel therapeutic agent for CKD-induced muscle dysfunction.

Keyword

Chronic kidney disease; indoxyl sulfate; ursolic acid; mitochondrial biogenesis

Figure

• 

  Fig. 1 Effects of IS and UA on the cell viability and differentiation of C2C12 cells. C2C12 cells were treated with or without IS (0.1 mM) and UA (1 or 2 µM) for 6 days. (A) Cell viability was assessed using a neutral red assay. (B) The mRNA expression of differentiation marker genes (Myod1 and Myog) was measured by real-time RT-PCR. Data are expressed as mean ± SD (n = 6).IS, indoxyl sulfate; UA, ursolic acid; RT-PCR, reverse transcription-polymerase chain reaction.Each group was compared with the IS-treated group by one-way analysis of variance followed by Dunnett's test, *P < 0.05 and **P < 0.01.

• 

  Fig. 2 Effects of IS and UA on ERK1/2 and AKT phosphorylation.Cells were treated with or without IS (0.1 mM) and UA (1 or 2 µM) for 1 h. The levels of ERK1/2, AKT, and their phosphorylated states were assessed by western blotting. b-actin was used as the internal control. Results are expressed as mean ± SD (n = 3).IS, indoxyl sulfate; UA, ursolic acid; ERK, extracellular signal-regulated kinase; p-, phospho-.Each group was compared with the IS-treated group by one-way analysis of variance followed by Dunnett's test, **P < 0.01.

• 

  Fig. 3 Effects of IS and UA on mtDNA copy number and mitochondrial biogenesis and dynamics in C2C12 cells. C2C12 cells were treated with or without IS (0.1 mM) and UA (1 or 2 µM) for 6 days. (A) Using real-time PCR, the mtDNA copy number, defined as the ratio of mitochondrial DNA-encoded ND1 to nDNA-encoded b-actin, was determined. (B, C) The mRNA expression levels of mitochondrial fusion (Mfn1 and Mfn2) and fission (Drp1 and Fis1) marker genes were measured by real-time RT-PCR. (D) The mRNA expression levels of Ppargc1a, Nrf1, Tfam, Sirt1, and Mef2c were measured by real-time RT-PCR. Results are expressed as mean ± SD (n = 6).IS, indoxyl sulfate; UA, ursolic acid; mtDNA, mitochondrial DNA; PCR, polymerase chain reaction; nDNA, nuclear DNA; RT-PCR, reverse transcription-polymerase chain reaction.Each group was compared with the IS-treated group by one-way analysis of variance followed by Dunnett's test, *P < 0.05 and **P < 0.01.

• 

  Fig. 4 Effects of IS and UA on IL-6 expression in C2C12 cells. Cells were treated with or without IS (0.1 mM) and UA (1 or 2 µM) for 4 days. (A) IL-6 mRNA expression was measured using real-time RT-PCR. (B) Secretion levels of the IL-6 protein in the culture media were measured by enzyme-linked immunosorbent assay. (C) The quantified levels of p-STAT3 were normalized to that of STAT3. b-actin was used as the internal control. Data are expressed as mean ± SD (n = 3).IS, indoxyl sulfate; UA, ursolic acid; RT-PCR, reverse transcription-polymerase chain reaction; p-, phospho-.Each group was compared with the IS-treated group by one-way analysis of variance followed by Dunnett's test, *P < 0.05 and **P < 0.01.

• 

  Fig. 5 Effects of IS and UA on ATP levels in C2C12 cells. Cells were treated with or without IS (0.1 mM) and UA (1 or 2 µM) for 6 days. (A, B) The mRNA expression levels of Pdk4, Acadm, Cpt1b, Cd36, Cycs, and Atp5b were measured by real-time RT-PCR. (C) The ATP levels in C2C12 cells were assessed using the CellTiter-Glo® 2.0 assay. Data are expressed as mean ± SD (n = 6).IS, indoxyl sulfate; UA, ursolic acid; RT-PCR, reverse transcription-polymerase chain reaction.Each group was compared with the IS-treated group by one-way analysis of variance followed by Dunnett's test *P < 0.05 and **P < 0.01.

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