Curcumin ameliorates CKD-induced mitochondrial dysfunction and oxidative stress through inhibiting GSK-3β activity
Curcumin alleviated CKD-induced mitochondrial oxidative damage and mitochondrial dysfunction via inhibiting GSK-3β activity in skeletal muscle
- This study suggested that curcumin could exert beneficial effects, including weight maintenance, improved muscle function, increased mitochondrial biogenesis, and alleviated mitochondrial dysfunction by increasing adenosine triphosphate levels.
Key Ideas
- Muscle atrophy is characterized by the loss of muscle proteins, with relation to chronic kidney disease (CKD), disuse, aging, cancer and several other disease states
- Currently, oxidative stress and the subsequent mitochondrial dysfunction are distinguished widely as a vital contributing factors in the process of muscle atrophy.
- Oxyidative stress originated from an imbalance between oxidant and antioxidant species
- Recent report highlights GSK-3β as a determinant of muscle mass because of its involvement in both protein biosynthesis and myuclear turnover [18]
- Several evidences have suggested that mitochondria play an important role in muscle metabolism, from which majority of adenosine triphosphate (ATP) and one of the reactive oxygen species (ROS) are generated
- Identification of the underlying molecular mechanisms that regulate mitochondrial function and oxidative stress, contributing to the development of preventive and curative strategies, might be beneficial for the treatment of muscular atrophy in CKD
Cell culture and myotube analysis
- C2C12 cells were cultured and differentiated as described in our previous study [30] and treated with the following conditions for 24 h: fresh culture medium alone, 100 μM H2O2, and 5 μM curcumin (purity >98%; CAS #458-37-7, Sigma-Aldrich, St. Louis, MO, USA)
Animals
- Male C57BL/6 mice, 6 to 7 weeks old, were purchased from Guangdong Medical Laboratory Animal Center (GDMLAC, Guangzhou, China).
- All mice were randomly assigned to either the CKD group or the sham-operated control group.
- After a week, the sham and CKD mice received either a control diet of normal mouse chow (AIN-76A Rodent Diet) or an identical diet containing 0.04% (w/w) curcumin.
Immunofluorescence staining and myofibers size measurement
- The frozen sections of the tibialis anterior (TA) muscle (5 μm) were fixed with 4% formaldehyde.
- After washing, the slide was blocked by protein blocking for 20 min, labeled with a rabbit polyclonal anti-dystrophin antibody (H-300), and revealed with an anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody (Dako, Glostrup, Denmark).
- All samples were double-labeled with a rat monoclonal antibody for laminin-2 followed by anti-rat tetramethylrhodamino amyloid-19 antibody as internal control.
Evaluation of lean muscle mass
- Mice could be adopted to assess the efficacy of curcumin to increase lean mass mass.
Measurement of proteasome activities
- Proteasome activity was assayed by using the Suc-LLVY-AMC (Enzo Life Sciences, #P802) and Z-ARR-AMM (EMD Millipore, #539149), respectively, as described previously [36].
- The measurements were carried out in 50 μg proteasom extract together with 50 mM EDTA and 50 μM fluorogenic substrates in a total volume of 200 μl of ATP/DTT lysis buffer at 37°C.
Estimation of lipid peroxide, antioxidant and antioxidant enzyme activities
- Quad muscle tissue was homogenized in 4 vol of homogenization buffer (10 mM KH2PO4, 30 mM HCl, 1 mM EDTA, pH 7.4) and centrifuged at 10,000g for 30 min, and the protein content of the homogenate was estimated
- SOD and catalase activities were determined by detecting the inhibition rate of the auto-oxidation of epinephrine and the rate of decomposition of H2O2
- The antioxidant status of the skeletal muscle was evaluated based on the GSH level using Ellman's reagent
- GSH concentration was measured in muscle tissue using the method of Sedlak and Lindsay
Measurement of mitochondrial ETC enzyme activities
- All assays were performed at 30°C with a Shimadzu UV-1601 spectrophotometer.
Determination of mitochondrial O2
- Dihydroethidium (DHE, #MX4812-25MG, MAOKANG Bioengineering Co., Shanghai, China) is an ethidium-based, redox-sensitive fluorescent probe shown to be oxidized by O2.− to form 2-hydroxyethidium.
Mitochondrial oxygen consumption rate (OCR)
- The basal OCR (State 4) was measured by using the Seahorse XFe24 Extracellular Flux Analyzers (Seahorse Bioscience, Billerica, MA, USA).
- Briefly, 10 μg isolated muscle mitochondria (3-6 μl) was dripped into the center of the XF24 cell culture microplates on ice and mixed carefully with 50 μl of the substrates, followed by a solution of mitochondrial assay solution (MAS, 70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4,5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, and 0.2% BSA, pH 7.4).
Transmission electron microscopy (TEM)
- The detailed procedures for TEM for muscle were reported previously by our group
Determination of GSK-3β activity
- ADP-Glo kinase assay kit (Promega, Madison, WI, USA)
Quantitative real-time PCR
- Real-time reverse transcription (RT)-PCR) was performed in a 20-ml reaction mixture containing 10 ml of SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA), 10 pmol of forward primer, 10 amol of reverse primer and 1 μg of cDNA by using a Stratagene Mx3005P QPCR System.
Western blotting
- Tissues or cells were lysed in RIPA buffer containing protease inhibitor cocktail. Protein concentration was determined by using BCA protein assay kit (#P0010S, Beyotime Biotechnology, Haimen, China).
- The antibodies of NRF-1, PGC-1α, GSK-3β, and GAPDH were purchased from Santa Cruz Biotechnology and Proteintech Group, Inc. respectively.
Statistical Analysis
- All statistical analyses were carried out by two-way analysis of variance to compare differences among multiple groups, followed by Bonferroni post hoc analysis to specifically test individual differences between groups.
Changes in renal function and food intake
- CKD mice showed increase levels of serum creatinine (SCr) and urea nitrogen (BUN) in accordance with extent of body weight loss (Fig. 1A) when compared with those of the sham mice, and curcumin did not reverse these parameters.
- Additionally, there was an obvious decrease of food intake in the sham group compared to the CKD group.
- Meanwhile, the mice of cKD-treated CKD displayed greater food intake than those in the normal group.
Curcumin improves muscle atrophy and muscle function
- After treatment for 4 weeks, the change in body weight of the sham group was more significant than that in the CKD group, with this difference increasing in magnitude during the subsequent experiment (Fig. 1A).
- The decrease in weight of gastrocnemius, TA and soleus muscles evoked by CKD was fully reversed by curcumin treatment.
- Detailed quantitative analyses of muscle sections demonstrated that the improvement of muscle mass was confirmed by an increased CSA of the myofibers.
Curcumin improves mitochondrial function
- Mice in the CKD group showed a remarkable generation of mitochondrial O2.− in skeletal muscle, which can be attenuated by curcumin treatment (Fig. 2A).
Curcumin attenuates CKD-induced disruption of mitochondrial ETC enzyme activities.
- There was a significant decrease in the activities of enzymes of mitochondria ETC complexes, i.e., complex I, II, III, and IV, in CKD mice treated with curcumin compared to untreated controls.
Curcumin increases mitochondrial biogenesis
- The deleterious alterations of morphology of mitochondria in the CKD group were observed by TEM, mainly including such indices as fractured or dissolved cristae, myofibril breakage, nuclear membrane shrinkage, dilated sarcotubular system and perinuclear space widening and which could be inhibited largely by curcumin treatment.
- In addition, we observed that the expressions of TFAM, PGC-1α and NRF-1 were significantly diminished in the muscle of CKD mice.
Ameliorates oxidative stress
- MDA, an indicator of lipid peroxidation, was also elevated in CKD mice, consistent with the change of mitochondrial O2 production (Fig. 5C).
Curcumin inhibits GSK-3β expression in vivo and in vitro.
- Our results showed that the level of activity was elevated in CKD skeletal muscle (Fig. 6A-C) and in H2O2-induced C2C12 myotubes, as well as the expression levels of its mRNA and protein, but both of these increases were inhibited by curcumin treatment in vivo (Figure 6D-F).
- In addition, immunofluorescence staining with an MHC-specific antibody revealed that exposure to 100 μM H 2O2 caused a reduction of the diameter of myolids, which was attenuated by cucumin treatment.
GSK-3β KO prevents CKD-induced muscle atrophy.
- The body weight of KO mice was found to maintain higher level than that of WT mice after 5/6 Nx and to increase steadily with the same trend as that of the WT mice (Fig. 8A).
- Similarly, increases were observed in the weights of the Gastroc, TA and soleus of mGSK-303β KO mice compared to WT mice as well as increases in expression of MAFbx and MuRF-1 mRNA.
GSK-3β KO ameliorates CKD-induced mitochondrial dysfunction
- An apparent preventive effect on reduction of mitochondrial OCR, mtDNA copy number and mitochondrial ATP production was observed.
GSK-3β KO prevents CKD-induced decreased mitochondrial ETC enzyme activities and antioxidant defenses
- The activities of complexes I, II, III, and IV tended to increase in muscle from mGSK-KO compared to the WT mice after 5/6 Nx (Fig. 10A-D).
Discussion:
- The research of pharmaceutical is indispensable for preventive and therapeutic strategy of CKD-induced muscle atrophy.
- In the present study, we demonstrated a natural product, curcumin, as a promising candidate to prevent CKD.
- The inhibition of mitochondrial dysfunction, deficient mitochondrial biogenesis, impaired mitochondrial oxidative metabolism, as well as inhibition of activated GSK-3β in skeletal muscle was demonstrated by the use of the drug.
- In addition, the knockout of GSK3β protected against CKD and decrements in muscle function through inhibition of ubiquitin-proteasome system (UPS), in accordance with evidence from in C2C12 myotubes exposed to H2O2.
- Our findings suggest that the reduction in muscle mitochondria content elicited by CKD can be prevented by cucumin treatment.
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