Part 1 | A Chinese Herbal Formula, Tonic The Kindney, Improves Muscle Atrophy Via Regulating Mitochondrial Quality Control Process in 5/6 Nephrectomised Rats

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

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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Chronic kidney disease (CKD) is characterized by a progressive loss in renal function over a period of months or years. Enormous studies have been demonstrated that its action mechanism is related to the excessive accumulation of extracellular matrix and podocyte loss and inflammation as well as the dysfunctions of lipid metabolism and amino metabolism1–6. A few reports indicated that CKD is associated with muscle atrophy, which directly correlates with mortality and morbidity7. Potential stimuli of muscle atrophy in CKD include acidosis, angiotensin II production, inflammation, up-regulation of the ubiquitin-proteasome, and autophagy-lysosome systems (UPS and ALS), and dysregulation of mitochondrial quality control (MQC) processes8, 9. Unfortunately, preventive and therapeutic interventions that block muscle atrophy are still at the initial stages of development. More and more patients accepted complementary or alternative therapies such as traditional Chinese medicine (TCM)10–14. We examined whether a traditional Chinese medicine, Jian-Pi-Yi-Shen (JPYS) decoction, would prevent muscle atrophy by modulating the MQC process.

The UPS is recognized as the major contributor to muscle proteolysis, responsible for 50% or more of total protein degradation in skeletal muscle. The UPS is the major intracellular protein degradation pathway, which 1Department of Nephrology, Shenzhen Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Shenzhen, 518033, China. 2Shenzhen Key Laboratory of Hospital Chinese Medicine Preparation, Shenzhen Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Shenzhen, 518033, China. 3Department of Nephrology, Ruikang Affiliated Hospital, Guangxi University of Chinese Medicine, Nanning, 530011, China. Dongtan Wang and Jianping Chen contributed equally to this work. Correspondence and requests for materials should be addressed to D.W. can degrade the myofibril proteins into their components (actin, myosin, troponin, and tropomyosin)15. These proteins are targeted and degraded by two muscle-specific E3 ubiquitin(Ub) ligases, muscle atrophy F-Box (MAFbx/ Atrogin-1) and muscle-specific RING finger protein (MuRF1)16, 17. Parallel to the above pathway, ALS is believed to target and cleave long-lived proteins, bulk cytoplasm, and organelles through the lysosomal machinery18. Te activation of UPS and ALS-related genes is normally blocked by Akt through negative regulation of Forkhead box O (FoxO) transcription factors, including FoxO1, FoxO3a, and FoxO4. Te translocation and transcriptional activity of FoxO members is sufficient to increase atrogin-1 and MuRF1 expression, and cause muscle atrophy.

MQC processes are tightly regulated by several processes, e.g. biogenesis, fusion, fission, and mitophagy. It is reported that chronic diseases activate a mitochondrial response that ameliorates the “quality” of skeletal muscle mitochondria cells at different molecular levels: (i) biogenesis through the action of the key regulators' peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor 1/2 (NRF-1/2), adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPKα), and ATP synthesis; (ii) dynamics by the mitochondrial remodeling GTPase proteins such as mitofusin-2 (Mfn-2) and optic atrophy 1 (OPA-1) for fusion and dynamin-related protein 1 (DRP-1) and fission 1 (Fis-1) for fission; (iii) turnover of damaged mitochondria by mitophagy through PTEN induced putative kinase 1(PINK1), Parkin and Bnip3/Nix (BNIP3L); and (iv) quality control by degradation of misfolded proteins or again portion of damaged mitochondria by the proteolytic system with chaperones and proteases20. It has been reported that mitochondrial biogenesis was decreased involved in muscle atrophy, which was promoted by PGC-1α and AMPKα21, 22. However, the process of MQC including mitochondrial fusion, fission, biogenesis, and mitophagy in CKD muscle atrophy is still unclear.

TCM has been reported to be effective for the treatment of muscle atrophy23–26. However, there was little information available in the literature about whether Chinese herbal medicine with anti-muscle atrophy effect could affect MQC process in CKD. JPYS decoction has been widely used in treating malnutrition with spleen and kidney qi deficiency syndrome in CKD for many years. However, further study of its detailed anti-malnutrition and reversing muscle atrophy mechanism is still needed. Here, we aimed to examine how dysregulation of MQC process induces muscle wasting and whether JPYS decoction inhibits muscle atrophy through modulating the MQC process effectively.

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Results

Changes in renal function.

At the end of the study, the CKD group displayed significantly higher serum creatinine (Scr) and blood urea nitrogen (BUN) levels compared with the sham group. Interestingly, JPYS decoction was found to reduce the levels of Scr and BUN. However, the level of serum albumin (ALB) did not differ significantly between all groups (Table 1).

JPYS decoction improves muscle atrophy.

The bodyweight of the CKD group was significantly lower than that of the sham group at the beginning of the treatment; however, there were no differences in body weight between CKD and JPYS groups. Interestingly, the JPYS group showed obvious improvement of bodyweight on the treatment of 5 and 6 weeks when compared with the CKD group (Fig. 1a). The increase in body weight from adding JPYS included an increase in the weight of gastrocnemius (Gastroc) and tibialis anterior (TA) muscles in the CKD group (Fig. 1b,c). Te improved muscle mass in the JPYS group was confirmed by an increase in the average cross-sectional area of myofibers in TA muscles in the CKD group (Fig. 1d,e).

JPYS decoction increases protein synthesis and suppresses protein degradation.

The protein synthesis rate was lower in the CKD group than that of the sham group, and JPYS decoction was found to increase the rate of protein synthesis (Fig. 2a). On the other hand, the protein degradation rate was higher in the CKD group than in the sham group, which was completely inhibited by JPYS decoction (Fig. 2b).

JPYS decoction inhibits ubiquitin-proteasome system and FoxO3a activation.

Protein markers in the muscle ubiquitin-proteasome system and FoxO3a are presented in Fig. 3a. CKD group displayed an increase in the expression of Atrogin-1 and MuRF-1, and these changes were abolished by JPYS decoction (Fig. 3b,c). Also, to confirm the relevance of the changes in muscle proteolysis seen in the CKD rats, we measured the activities of the 20 S proteasome. Both chymotrypsin- and trypsin-like activities were higher in the CKD group than that of the sham group, while were weakened with JPYS decoction (Fig. 3g,h). Additionally, the phosphorylated (p)-FoxO3a and p-FoxO3a/FoxO3a ratio were decreased in the CKD group, while JPYS decoction trigger an increase as compared to the CKD group (Fig. 3d,f). As shown in Fig. 3e, the basal protein content of FoxO3a was up-regulated in CKD group, and this was also prevented by JPYS decoction.

JPYS decoction improves muscle mitochondrial content and aberrant muscle morphological features.

Mitochondrial content was assessed using SDH staining of Gastroc muscles (Fig. 4a). The SDH activity, which represents the mitochondrial amount, was markedly reduced in the CKD group and JPYS decoction antagonized this response (Fig. 4b). On the other hand, SDH staining revealed that type I (slow oxidative) and I (fast oxidative glycolytic) muscle fibers, which are mitochondria-rich fibers, were significantly decreased, and type IIb (fast glycolytic) fibers were increased in the CKD group. Quite interestingly, JPYS decoction displayed a shift in muscle fiber type in the CKD group, characterized by a reduction in type IIb fibers and a significant increase in type I and IIa muscle fibers (Fig. 4c). Consistent with the decrease in SDH activity, the TEM morphologic also revealed major alterations at the sarcomeric level, with abnormalities consistent with fewer and smaller mitochondria (black arrows; Fig. 4e,f) and with markedly thinner Z-lines (white arrows; Fig. 4d) in CKD group. Similarly, the I-band, mainly constituted of thin actin filaments, appeared thinner or completely absent in the CKD group (brackets; Fig. 4d). Importantly, these changes were prevented by JPYS decoction treatment.

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JPYS decoction increases mitochondrial biogenesis.

Protein markers in muscle mitochondrial biogenesis are presented in Fig. 5a. Te Cox IV protein can be used effectively as a mitochondrial loading control and represent mitochondrial content, which was consistent with the results of SDH activity (Fig. 5b). Additionally, mitochondrial biogenesis proteins NRF-1 and PGC-1α were also decreased in the CKD group and these changes were reversed with JPYS decoction (Fig. 5c–e). However, the protein levels for the ATP5B and p-AMPKα/ AMPKα ration were unchanged from all groups (Fig. 5d–f).

JPYS decoction inhibits autophagy and mitophagy pathway.

Te autophagy and mitophagy-related proteins were detected by Western blotting (Fig. 6a). Te level of Beclin-1 protein and LC3II/LC3I ration were up-regulated in the CKD group and this was retarded by JPYS decoction (Fig. 6b,c). The contribution of the autophagy adaptor p62 has been found to be dispensable for mitophagy, which was increased in the CKD group and attenuated by JPYS decoction (Fig. 6d). Interestingly, the levels of PINK1 and Parkin proteins were significantly increased in the CKD group, and these changes were prevented by JPYS decoction (Fig. 6f,g). However, there were no differences in BNIP3L expression between all groups (Fig. 6e).

JPYS decoction decreases mitochondrial fission and increases mitochondrial fusion.

Te mitochondrial fusion and fission-related proteins were detected by Western blotting (Fig. 7a). Te key fission proteins Fis-1 and Drop-1 levels were significantly higher in the CKD group, while these changes were impeded by JPYS treatment (Fig. 7b,c). However, the key fusion proteins OPA-1 and Mfn-2 levels were decreased in the CKD group, and this reduction was prevented by JPYS treatment (Fig. 7d,e).

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