Part 2 | A Chinese Herbal Formula, Tonic The Kindney, Improves Muscle Atrophy Via Regulating Mitochondrial Quality Control Process in 5/6 Nephrectomised Rats
Mar 10, 2022
Dongtao Wang1,3, Jianping Chen2, Xinhui Liu1, Ping Zheng1, Gaofeng Song1, Tiegang Yi1,2 & Shunmin Li1
Muscle atrophy is one of the serious complications of chronic kidney disease (CKD). Dysregulation of the mitochondrial quality control (MQC) process, including decreased mitochondrial biogenesis, impair mitochondrial dynamics and induce activation of mitophagy, play an important role in mediating muscle wasting. This study aimed to observe the effects of Jian-Pi-Yi-Shen (JPYS) decoction on muscle atrophy in CKD rats and explore its possible mechanism on the regulation of MQC processes. The 5/6 nephrectomized rats were randomly allocated into 2 groups: CKD group and JPYS group. Besides, sham-operated rats were a sham group. All rats were treated for 6 weeks. Results showed that administration of JPYS decoction prevented body weight loss, muscle loss, muscle fiber size decrease, muscle protein degradation, and increased muscle protein synthesis. In addition, JPYS decoction increased the mitochondrial content and biogenesis proteins and down-regulated the autophagy and mitophagy proteins. Furthermore, JPYS decoction increased mitochondrial fusion proteins, while decreasing mitochondrial fission proteins. In conclusion, JPYS decoction increased mitochondrial content and biogenesis, restored the balance between fission and fusion, and inhibited the autophagy-lysosome pathway (mitophagy). Collectively, our data showed that JPYS decoction is beneficial to muscle atrophy in CKD, which might be associated with the modulation of MQC process.
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Discussion
Many crude extracts and isolated active compounds from TCM have been identified and shown excellent efficacy, especially in anti-inflammation and metabolic disorder improvement for various types of kidney diseases. Muscle atrophy is a serious complication of CKD patients, which is characterized by progressive loss of muscle proteins. This adverse outcome substantially reduces the quality of life and survival16, 34. The most important features of muscle atrophy are a significant reduction in body weight and loss of muscle mass, implying a CKD-associated metabolic condition that specifically targets muscle. As a TCM, JPYS decoction has emerged as a potential therapeutic agent to treat muscle atrophy and increase muscle mass. To investigate the anti-muscle atrophy effect of JPYS decoction and its possible mechanism, in the present study, 5/6nephrectomy-induced CKD rats were performed, and results have shown that JPYS decoction considerably prevented body weight loss, muscle mass loss, muscle fiber size decrease, and muscle protein proteolysis, along with inhibition of UPS and FoxO3a. Moreover, JPYS decoction could increase mitochondrial content and biogenesis, restore the balance between fission and fusion, and block the activation of autophagy and mitophagy. Skeletal muscle mass depends upon a dynamic balance between protein synthesis and degradation. And the two processes are tightly interrelated35. The present results showed that JPYS decoction was able to increase protein synthesis and concomitantly inhibit the breakdown of muscle in CKD rats. To investigate the underline mechanisms of delaying protein degradation by JPYS decoction, we examined the pathways of protein degradation.
Increased Atrogin-1 and MuRF-1 promoted the ubiquitination and 26 S proteasome-mediated degradation of structural proteins, which increased muscle protein degradation and thus contributed to muscle wasting in our previous studies36, 37. Ubiquitinated proteins are rapidly degraded by the 20S proteasome including chymotrypsin and trypsin-like activities, which lead to Ub-conjugated proteins into small peptides38. In this study, our results showed that JPYS decoction prevented the elevated atrogin-1 and MuRF-1 proteins and chymotrypsin- and trypsin-like activities in CKD muscle. Previous studies identified that TCM (Zhimu-Huangbai Herb-Pair) treatment inhibited the Atrogin-1and MuRF1 expression in cancer-induced cachexia in mice muscle. These findings suggested that JPYS decoction could inhibit muscle protein degradation by inhibiting the activation of UPS in CKD rats.
FoxO3a can be phosphorylated by Akt at several sites, which functions as a scaffold within the cytoplasm, and are sequestered within the cytosol, rendering them unable to bind to the promoters of their target genes in the nucleus to regulate their transcription39. A previous study showed that the activation of FoxO3a in muscle leads to increased transcription of these retrogenes such as Atrogin-1 and MuRF1 and stimulates proteolysis to affect muscle atrophy40. Our result showed that JPYS decoction significantly increased the phosphorylation levels of FoxO3a, attenuated the level of total FoxO3a protein in the muscle of CKD rats. A previous study reported that TCM (Zhimu-Huangbai Herb-Pair) treatment reduced the expression of total FoxO3 protein in diabetic muscle26. It was reported that constitutively active FoxO3a induced atrogin-1 transcription and muscle atrophy, whereas inhibiting of FoxO3a activation blocked muscle atrophy in vivo and in vitro40. Combining with the results we concluded that JPYS decoction could efficiently reduce protein degradation of skeletal muscle, possibly through inhibition of FoxO3a transcription factors.
The ability of the skeletal muscle to adapt to cellular perturbations is highly dependent on mitochondrial biogenesis. It has recently been shown that muscle mitochondrial amount declines with CKD41, 42, which was consistent with our results, and the reduction was inhibited by JPYS decoction. The major steps of the mitochondrial biogenesis process include signaling events leading to transcriptional regulation of nuclear genes, such as NRF1, mainly mediated by PGC-1α43. Our results showed that mitochondrial biogenesis proteins appeared to be down-regulated in CKD muscle as indicated by the lower PGC-1α and its target proteins NRF-1 content, which was inhibited by JPYS decoction. Overexpression of PGC-1α in skeletal muscle increases mitochondrial content and oxidative capacity through its modulation of a large group of genes involved in metabolism44, 45. Moreover, PGC-1a levels tend to be reduced in muscle wasting conditions46, 47 and muscle-specific overexpression of PGC-1a has been shown to attenuate this muscle loss48. Collectively, our data implied that it is possible that JPYS decoction promotes the expression of PGC-1α/NRF1 and subsequent mitochondrial biogenesis in CKD muscle.
Autophagy/mitophagy is a highly conserved homeostatic mechanism that is used for the degradation and recycling, through the lysosomal machinery of bulk cytoplasm, long-lived proteins, mitochondria, and organelles49. The selectivity of mitophagy is controlled by the proteins PINK1, Parkin, and BNIP3L. PINK1 phosphorylates ubiquitin at Ser65 of ubiquitinated outer mitochondrial membrane (OMM) proteins and the ubiquitin-like domain of Parkin. Once phosphorylated, Parkin enhances the mitophagy signal by generating more ubiquitin chains on OMM proteins that can be further substrates for PINK1. BNIP3L is stabilized on the OMM, interacts with processed LC3II, which can promote sequestration of mitochondria within the autophagosome for degradation9. In our study, the results showed that the autophagic markers LC3II and p62, and mito-phagic markers PINK1 and Parkin were significantly increased in CKD muscle, and this was retarded by JPYS decoction. Recent studies have demonstrated that autophagy, including mitophagy, is often stimulated in multiple models of muscle atrophy, such as denervation and CKD37, 50. Collectively, our results suggested that JPYS decoction improved muscle atrophy through inhibition of the autophagy and mitophagy pathways.
Mitochondria are reported to be highly dynamic organelles that undergo constant movement through fission and fusion51. Mitochondrial fusion is thought to allow the exchanging of their content including the mitochondrial DNA (mtDNA) and proteins, thus maintaining mitochondrial quality and mtDNA integrity, Mitochondrial fusion is thought to prevent the accumulation of damaged and defective components through redistributing their content including the mitochondrial DNA (mtDNA), lipids, metabolites, and proteins, whereas mitochondrial fission allows mitochondria for segregation of severely damaged and dysfunctional mitochondria by mitophagy52. In the present study, we demonstrated that the fusion protein Mfn-2 and OPA-1 were down-regulated in CKD muscle, and this change was prevented by JPYS decoction. Mfn2 and OPA-1 play an important role in maintaining mtDNA integrity, and their down-regulation could induce mitochondrial fission as well as mitochondrial fragmentation and mitophagy53. In accordance with a role in mitochondrial fusion, fission has been linked to
the removal of severely damaged mitochondria through induction of mitophagy. In fact, our results showed that Fis-1 and Drop-1 expression were increased CKD muscle and this was prevented by JPYS decoction, which was consistent with our results50, 54. Therefore, the results of the present study indicate that JPYS decoction modulated mitochondrial dynamics of fusion and fission by increasing Mfn2 and OPA-1 expression, meanwhile decreasing Fis-1 and Drop-1 expression.
In conclusion, the present study shows that a 6-week JPYS decoction treatment preserved the body weight, prevent muscle mass loss and muscle fiber size decrease, and muscle protein degradation, along with inhibition of FoxO3a and UPS in CKD rats. Furthermore, JPYS decoction attenuated the CKD-induced disturbances of QMC processes, by increasing mitochondrial biogenesis, restoring the balance between fission and fusion, and inhibiting the autophagy-lysosome pathway (mitophagy). This finding suggests a promising strategy that improving the dysregulation of MQC processes might prevent and treat muscle atrophy in CKD.
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Materials and Methods
Composition of JPYS decoction.
Plant materials: Astragali Radix (Lot. 150621; roots of Astragalus membranaceus (Fisch.) Bge. var. Mongolic (Bge.) Hsiao), Atractylodis Macrocephalae Rhizoma (Lot. 141230; rhizomes of Atractylodes macrocephala Koidz.), Dioscoreae Rhizoma (Lot. 150615; rhizomes of Dioscorea opposite Tunb.), Cistanches Herba (Lot. 150621; herbs of Cistanche deserticola Y.C. Ma), Amomi Fructus Rotundus (Lot. 150617; fruits of Amomum kravanh Pierre ex Gagnep.), Salviae Miltiorrhizae Radix et Rhizoma (Lot. 150626; roots and rhizomes of Salvia miltiorrhiza Bge.), Rhei Radix et Rhizoma (Lot. 150104; roots and rhizomes of Rheum palmatum L.), and Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (Lot. 150615; roots and rhizomes of Glycyrrhiza uralensis Fisch.) were purchase from Shenzhen Huahui Pharmaceutical Co., Ltd (Shenzhen, China). Te plant materials were authenticated by Dr. Jianping Chen based on their morphological characteristics. The voucher specimens were kept at Pharmaceutical Department, Shenzhen Traditional Chinese Medicine Hospital with numbers 2010015Z, 2010024ZZ, 2010037Z, 2040056Z, 202086Z, 2010006Z, 2010040Z, and 2010008ZZ, respectively. Assurance of quality control for all the materials was validated according to the Chinese Pharmacopeia (China Pharmacopoeia Committee, 2015). Astragali Radix (30 g), Atractylodis Macrocephalae Rhizoma (10 g), Dioscoreae Rhizoma (30 g), Cistanches Herba (10 g), Amomi Fructus Rotundus (10 g), Salviae Miltiorrhizae Radix et Rhizoma (15 g), Rhei Radix et Rhizoma (10 g), and Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (6 g) were weighed and extracted in boiling water (1.2 L) twice for 1 h. After centrifugation, the supernatant was dried under reduced pressure to powder, and it was stored at −80 °C. Before the treatment, the powder was re-dissolved with Milli-Q water and vortexed at room temperature to obtain JPYS extract.
Before the treatment of extract onto animals, JPYS extract was chemically standardized. An HPLC fingerprint at 260nm was developed for the JPYSF extract (Fig. 8): An individual reference standard was employed to confirm numerous chemical components should be identified from the extract by HPLC analysis, such as sodium danshensu, echinacoside, acteoside, calycosin 7-O-b-glucoside, salvianolic acid B, formononetin and rhein. Besides, the minimal requirement for the amounts of echinacoside, salvianolic acid B, and rhein should be no less than 1.2mg/g, 5.7mg/g, and 0.2mg/g of the dried extract. The yield of the extraction was less than 32.59 ±1.1% (w/w, Mean±SD, n=3). The extract being used here reached the aforesaid requirements.
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Experimental animals.
The experimental and feeding protocols were in accordance with National Health guidelines and were approved by the Guangzhou University of Chinese Medicine Institutional Animal Care and Use Committee. Male Sprague–Dawley rats were purchased from Guangdong Medical Laboratory Animal Center (GDMLAC, China), Permission No. SCXK (YUE) 2013-0002 weighing 190–220 g. The animals were under controlled room temperature (20±1°C) and with humidity with 12/12-hour light-dark cycle, and had to access to water and food ad libitum. CKD was induced by a two-step 5/6 nephrectomy as described previously36. Briefly, the first renal surgery involved electrocautery of the left kidney except for a 2-mm area around the hilum. A second renal surgery was performed one week later by double ligation of the renal hilum with silk suture and surgical excision of the right kidney. Sham surgery consisted of an anesthetic, a fank incision exposing the kidney, and closure of the abdominal wall.
Administration of drugs.
At 16 weeks after the operation, the levels of Scr of the 5/6 nephrectomy group were significantly higher than those of the sham group (p<0.05). Ten, the 5/6 nephrectomy group was randomly divided into two groups: CKD group (5/6 NX, n=10): CKD rats were treated with distilled water and JPYS group (5/6 NX+JPYS decoction, n=10): CKD rats that were orally administrated a dose of 10.89 mg/kg of JPYS decoction daily. The sham-operated rats were also treated with distilled water. Te drugs were administered for 6 weeks. All rats used in this study received humane care.
Biochemical parameters.
After 6 weeks of treatment, the rats were sacrificed with sodium pentobarbital and blood samples were collected immediately. Serum biochemical indexes Scr, BUN, and ALB were detected using a Roche automatic biochemical analyzer.
Morphological studies (HE, SDH staining). Te transverse paralyzed muscle sections (6 mm) were stained with hematoxylin and eosin (HE) in line with standards. Muscle fiber cross-sectional area (CSA) was then measured in the way as our previously reported37. Fiber cross-sectional area was measured for approximately 100 adjacent muscle fibers in each section for each mouse using Image J 1.32 j software (NIH, Bethesda, MD, USA).
The frozen sections of the TA muscle were stained with succinate dehydrogenase (SDH, complex II of the respiratory chain) for measurements of SDH activity and classification of fiber type into I (slow oxidative), IIa (fast oxidative glycolytic), or IIb (fast glycolytic) in accordance with a previously described protocol41. Briefly, sections were first allowed to reach room temperature and were rehydrated with PBS (pH 7.4). Sections were
then incubated in a solution containing nitroblue tetrazolium (1.5mM), sodium succinate (130mM), phenazine methosulphate (0.2 mM), and Sodium azide (0.1 mM) for 60 min. Cross-sections were then washed 3 times in PBS, dehydrated in 75% (30 s), 90% (30 s), and 100% (10min) ethanol and coverslipped with a mixture of 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine in 0.01M PBS. Images of muscle were captured using a microscope (Nikon Eclipse Ti-SR, Japan) and were digitized as gray-level images on a computer-assisted NIS-Elements imaging software Version 4.10 (Eclipse Ti-SR, Nikon Corporation, Tokyo, Japan). A gray level value of zero was equivalent to 100% transmission of light (%T), and that of 255 was equivalent to 0%T. The optical density value of all the muscle fibers was determined based on the gray-level images (Scion Image, Scion, Frederick, MD) and classified into three groups, I (%T: 100–80%), IIa (%T: 60–40%) and IIb (%T: 20–0%).
Ultrastructural analysis (Transmission Electron Microscopy, TEM).
Detailed procedures of TEM for muscle were as our previously reported55. Briefly, sections of TA muscle 1mm3 in volume were fixed in 2.5% glutaraldehyde followed by post-fixation in 1% osmic acid for the assays of electron microscopy. Image J software was used to analyze images collected by EM (JEM-1400, JEOL Ltd., Tokyo, Japan) under x12,000 magnification. Mitochondrial content was determined by quantifying the number and the size (minimum diameter) of each mitochondrion per field. A total of 20 fields per condition were analyzed by taking advantage of the Image J sofware56.
Protein synthesis and protein degradation.
Protein synthesis and protein degradation were measured in vitro using the incorporation of 14-C phenylalanine (Phe) and tyrosine release as previously described57–59.
Measurement of proteasome activities.
Te chymotrypsin- and the trypsin-like activity of the 20 S proteasome were measured in vitro in the gastrocnemius muscle as our previously described36.
Western blotting.
Snap-frozen quadriceps muscle tissues were homogenized in lysis buffer as our previously reported36. Cytosolic proteins were separated on a 10% SDS-PAGE gel and then transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, U.S.). The membrane’s nonspecific binding sites were blocked at room temperature for 1 h with 5% non-fat powdered milk in Tris-buffered saline with tween (TBST) and then incubated overnight at 4 °C with primary antibodies. After washing with TBST, the membranes were incubated with secondary antibodies for 1h at room temperature with shaking. After washing, protein bands were detected and analyzed using a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, CA, U. S.). Results were expressed as the integrated optical density relative to GAPDH. p-AMPKα (1:1000, #2535), p-FoxO3a (1:1000, #13129), SQSTM1/p62 (1:1000, #5114), Mitofusin-2 (1:1000, #9482), FoxO3a (1:1000, #2497), DRP1 (1:1000, #8570), Cox IV (1:1000, #4844), BNIP3L/Nix (1:1000, #12396) Beclin-1(1:1000, #3495T) and AMPKα (1:1000, #5831) antibody were from Cell Signaling Technologies (Danvers, MA, U.S.). LC3 I/II (1:1000, ab58610), Parkin (1:1000, ab77924) and PINK1 (1:1000, ab23707) antibody were from Abcam (Cambridge, U.K.). ATP5B (1:1000, ARP48185_T100) antibody was from Aviva Systems Biology (San Diego, CA, U.S.). MuRF1 (1:1000, GTX110475) antibody was from Gene Tex (San Antonio, TX, U.S.). Fis1 (1:100, sc-98900) and NRF-1 (1:100, sc-33771) antibodies were from Santa Cruz Biotechnology (CA, U.S.). OPA1 (1:1000, 612606) was from BD Biosciences (San Jose, CA, U.S.). Atrogin-1 (1:1000, AP2041) was from ECM Biosciences (Versailles, KY, U.S.). PGC-1α (1:2000, NBP1-04676) was from Novus Biological (Colorado, U.S.). GAPDH (1:1000, 60004-1-Ig) was from Proteintech (Chicago, IL, U.S.).
Statistical analysis.
Data were analyzed with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Results are shown as mean ± SD. Normally distributed data were analyzed by one-way ANOVA followed by the least-significant difference (LSD) test, while data without normal distribution were analyzed using the Games-Howell test. Differences were considered statistically significant for P < 0.05.
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