Atractylenolide III Attenuates Muscle Wasting in Chronic Kidney Disease via the Oxidative Stress-Mediated PI3K/AKT/mTOR Pathway

Abstract

Oxidative stress contributes to muscle wasting in advanced chronic kidney disease (CKD) patients. Atractylenolide III (ATL-III), the major active constituent of Atractylodes rhizome, has been previously reported to function as an antioxidant. This study is aimed at investigating whether ATL-III has protective effects against CKD-induced muscle wasting by alleviating oxidative stress. The results showed that the levels of serum creatinine (SCr), blood urea nitrogen (BUN), and urinary protein significantly decreased in the ATL-III treatment group compared with the 5/6 nephrectomy (5/6 Nx) model group but were higher than those in the sham operation group. Skeletal muscle weight was increased, while inflammation was alleviated in the ATL-III administration group compared with the 5/6 Nx model group. ATL-III-treated rats also showed reduced dilation of the mitochondria, increased CAT, GSH-Px, and SOD activity, and decreased levels of MDA both in skeletal muscles and serum compared with 5/6 Nx model rats, suggesting that ATL-III alleviated mitochondrial damage and increased the activity of antioxidant enzymes, thus reducing the production of ROS. Furthermore, accumulated autophagosomes (APs) and autolysosomes (ALs) were reduced in the gastrocnemius (Gastroc) muscles of ATL-III-treated rats under transmission electron microscopy (TEM) together with the downregulation of LC3-II and upregulation of p62 according to Western blotting. This evidence indicated that ATL-III improved skeletal muscle atrophy and alleviated oxidative stress and autophagy in CKD rats. Furthermore, ATL-III could also increase the protein levels of p-PI3K, p-AKT, and p-mTOR in skeletal muscles in CKD rats. To further reveal the relevant mechanism, the oxidative stress-mediated PI3K/AKT/mTOR pathway was assessed, which showed that a reduced expression of p-PI3K, p-AKT, and p-mTOR in C2C12 myoblast atrophy induced by TNF-α could be upregulated by ATL-III; however, after the overexpression of Nox2 to increase ROS production, the attenuated effect was reversed. Our findings indicated that ATL-III is a potentially protective drug against muscle wasting via activation of the oxidative stress-mediated PI3K/AKT/mTOR pathway.

Figure 1

ATL-III prevents CKD-induced muscle atrophy and reduces the levels of inflammatory factors in 5/6 Nx model rats. The animals were sacrificed after treatment for 5 weeks, and the gastrocnemius (Gastroc) and tibial anterior (TA) muscles were observed by a stereoscope to obtain images (n = 6/group) (a). The proportion of Gastroc (b) and TA (c) muscle masses normalized to total body weight was quantified and displayed. The volume of transverse muscle fibers was evaluated by H&E staining (d). Representative images of tissue sections from rats in the sham, model, and ATL-III groups are shown by the microscope (400×). The red arrows indicate myofibers affected by atrophy. The average muscle fiber area (μm²) of different groups was compared by one-way ANOVA. Inflammatory factors TNF-α, CRP, IL-1β, and IL-6 in serum and gastrocnemius muscle tissue were detected by ELISA (e) and qPCR analysis (f). Data are presented as the mean ± SD for three independent experiments. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 2

Altered mitochondrial morphology and peroxidase activity in gastrocnemius muscle tissue and serum in CKD rats. Rat gastrocnemius tissue obtained from the sham, model, and ATL-III groups was subjected to transmission electron microscopy (TEM) analysis, and the mitochondrial structure was compared. Representative electron micrographs showed changes in mitochondrial morphology of CKD. Mitochondria were swollen and showed a disordered arrangement and membrane ruptures or large vacuoles. The red arrows represent typical swollen mitochondria (a). Higher magnification views of the corresponding area are shown in (b). The mitochondrial average Feret diameter (c) and area (d) in gastrocnemius muscles from different groups were calculated and displayed on the right side. The activity of SOD, MDA, and GSH-Px and the level of CAT in muscle (e) and serum (f) are shown in the histograms. ^∗∗ P < 0.01 and ^∗∗∗ P < 0.001. SOD: superoxide dismutase; MDA: malondialdehyde; GSH-Px: glutathione peroxidase; CAT: catalase.

Figure 3

Typical autophagosomes and autolysosomes of gastrocnemius slices under TEM. The skeletal muscle of rats was obtained after 5 weeks of gavage. Autophagosomes and autolysosomes of gastrocnemius were observed by TEM. Representative TEM images showing autophagosome structures (denoted by black triangles) and autolysosome structures (denoted by red triangles) (a). The number of autophagosomes and autolysosomes was significantly higher in the model group than in the sham group. However, after ATL-III treatment, the numbers of both autophagosomes and autolysosomes were decreased (b). Western blotting analysis showed that the LC3-II expression was significantly higher in the model group than in the sham group (P < 0.001) but was decreased in the ATL-III-treated group (P < 0.05); the expression of P62 followed an opposite trend (c). The expression of p-PI3K, p-AKT (Ser473), and p-mTOR in muscle lysates of different groups by Western blots. The results showed that the expression levels of these three proteins decreased significantly in CKD rats compared to the sham group but could be restored by ATL-III treatment (d). ^∗ P < 0.05 and ^∗∗∗ P < 0.001.

Figure 4

Effects of ATL-III on apoptosis and autophagy in vitro. The effect of different concentrations of ATL-III on the viability of C2C12 myoblasts was tested by MTT, which indicated that 50 μM was the optimal concentration of the drug. Toxicity test of ATL-III showed that low concentration (20, 30, and 50) has no cytotoxicity to C2C12 myoblasts. The values are presented as the means ± SD of five independent experiments. ^∗ P < 0.05 (a). The cell proliferation assay showed that ATL-III at 50 μM displayed the best effect in proproliferation than 20 and 30 μM for C2C12 myoblasts incubated with TNF-α (20 ng/ml) (b). ATL-III displayed its antiapoptotic effect in C2C12 myoblasts in the presence or absence of TNF-α (20 ng/ml) in a dose-dependent manner (c). The expression of LC3 (d) by immunofluorescence (200×) in different groups was displayed with DAPI, LC3, and MERGE, respectively. Relative fluorescence intensity of LC3 (e) was compared between groups. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 5

ATL-III inhibits TNF-α-induced oxidative stress in C2C12 myoblasts. C2C12 myoblasts were treated with ATL-III in the presence or absence of TNF-α (20 ng/ml) for 24 h and then loaded with H2DCF-DA for 30 min and observed under a fluorescence microscope; the quantification showed that the much higher level of ROS induced by TNF-α was decreased dramatically by ATL-III in C2C12 myoblasts (a). After trypsin digestion, the cells were centrifuged at room temperature for 1000 rpm/min for 10 minutes; then, the supernatant was removed, leaving cell sedimentation for detection. The activity of SOD, MDA, and GSH-Px and the level of CAT in cells are shown in the histograms (b). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 6

ATL-III ameliorated TNF-α-induced apoptosis in C2C12 myoblasts through the PI3K/AKT/mTOR pathway. C2C12 myoblasts were incubated with ATL-III in the presence or absence of TNF-α (20 ng/ml) for 24 h. After treatment and protein quantification, the expression levels of p-PI3K, p-AKT (Ser473), p-mTOR, LC3, and P62 were tested by western blotting. GAPDH was used as a loading control. The signaling pathway was explored again after overexpression of Nox2 (a). The densitometric analysis of images is shown in (b). ^∗∗ P < 0.01, all compared with the A group; ^## P < 0.01, all compared with the B group; ^☆ P < 0.01, all compared with the B group.