Novel peptides from sea cucumber intestines hydrolyzed by neutral protease alleviate exercise-induced fatigue via upregulating the glutaminemediated Ca2+ /Calcineurin signaling pathway in mice.

Sea cucumber intestines are considered a valuable resource in the sea cucumber processing industry due to their balanced amino acid composition. Studies have reported that peptides rich in glutamate and branched-chain amino acids have anti-fatigue properties. However, the function of the sea cucumber intestine in reducing exercise-induced fatigue remains unclear. In this study, we enzymatically hydrolyzed low molecular weight peptides from sea cucumber intestines (SCIP) and administered SCIP orally to mice to examine its effects on exercise-induced fatigue using swimming and pole-climbing exhaustion experiments. The results revealed that supplementation with SCIP significantly prolonged the exhaustion time of swimming in mice, decreased blood lactate and urea nitrogen levels, and increased liver and muscle glycogen levels following a weight-loaded swimming test. Immunofluorescence analysis indicated a notable increase the proportion of slow-twitch muscle fiber and a significant decrease the proportion of fast-twitch muscle fiber following SCIP supplementation. Furthermore, SCIP upregulated mRNA expression levels of Ca2+ /Calcineurin upstream and downstream regulators, thereby contributing to the promotion of skeletal muscle fiber type conversion. This study presents the initial evidence establishing SCIP as a potential enhancer of skeletal muscle fatigue resistance, consequently providing a theoretical foundation for the valuable utilization of sea cucumber intestines.

Icariside II prevents kidney fibrosis development in chronic kidney disease by promoting fatty acid oxidation.

Renal interstitial fibrosis (RIF) is the main pathological basis for the progression of chronic kidney disease (CKD), however, effective interventions are limited. Here, we investigated the effect of Icariside II (ICA-II) on RIF and explored the underlying mechanisms. Rats receiving 5/6 ablation and infarction (A/I) surgery were gavaged with ICA-II (5 or 10 mg/kg) for 8 weeks. In vitro, TGF-ß1-stimulated NRK-52E cells were treated with ICA-II and (or) oleic acid, etomoxir, ranolazine, fenofibrate, and GW6471. The effects of ICA-II on RIF, fatty acid oxidation, lipid deposition, and mitochondrial function were determined by immunoblotting, Oil red O staining, colorimetric, and fluorometric assays. Using adeno-associated virus injection and co-culture methods, we further determined mechanisms of ICA-II anti-RIF. ICA-II ameliorated the fibrotic responses in vivo and in vitro. RNA-seq analysis indicated that ICA-II regulated fatty acid degradation and PPAR pathway in 5/6 (A/I) kidneys. ICA-II attenuated lipid accumulation and up-regulated expression of PPARα, CPT-1α, Acaa2, and Acadsb proteins in vivo and in vitro. Compared to ICA-II treatment, ICA-II combined with Etomoxir exacerbated mitochondrial dysfunction and fibrotic responses in TGF-ß-treated NRK-52E cells. Importantly, we determined that ICA-II improved lipid metabolism, fatty acid oxidation, mitochondrial function, and RIF by restoring PPARα. Co-culture revealed that ICA-II decreased the expression of Fibronectin, Collagen-I, α-SMA, and PCNA proteins in NRK-49F cells by restoring PPARα of renal tubular cells. ICA-II may serve as a promising therapeutic agent for RIF in 5/6 (A/I) rats, which may be important for the prevention and treatment of CKD.

Shen Shuai II Recipe inhibits hypoxia-induced glycolysis by preserving mitochondrial dynamics to attenuate kidney fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Shen Shuai II Recipe (SSR) is a traditional Chinese medicine prescription with significant clinical efficacy in chronic kidney disease (CKD) by invigorating Qi and resolving blood stasis, clearing away heat and dampness. Our previous studies demonstrated that SSR attenuated renal interstitial fibrosis (RIF) by improving hypoxia and mitochondrial dysfunction. AIM OF THE STUDY: The aim of this study was to investigate the potential mechanisms of SSR against RIF. MATERIALS AND METHODS: The CKD was established by 5/6 ablation/infarction (A/I) operation. After 4 weeks, rats were gavaged with SSR or Fenofibrate for 8 weeks. Hypoxia-treated NRK-52 E cells were treated with SSR and (or) glycolysis inhibitors, including GSK2837808 A (GSK) and 2-Deoxy-D-glucose (2-DG). In addition, Drp1-deficient or MFP-M1-treated NRK-52 E cells were treated with SSR under hypoxic conditions. The effects of SSR on fibrotic phenotype, glycolysis, mitochondrial dynamics and membrane potential in hypoxia-exposed NRK-52 E cells were examined by immunoblotting, colorimetric, and fluorometric methods. Furthermore, we constructed a lactic acid-induced activation model of NRK-49 F cells and a co-culture system. The activation of NRK-49 F cells was evaluated by immunoblotting method. RESULTS: Our findings indicated that SSR significantly attenuated abnormal glycolysis in vivo and in vitro, which was correlated with its renoprotective effect. Further studies revealed that improvement of mitochondrial dynamics could be one of the mechanisms by which SSR inhibits glycolysis to achieve anti-renal fibrosis. Furthermore, treatment with SSR significantly inhibited the lactic acid-induced activation of NRK-49 F cells. The co-culture results further highlighted that SSR inhibited activation of renal fibroblasts and deposition of extracellular matrix by reducing glycolysis in renal tubular cells. CONCLUSIONS: SSR alleviates RIF by inhibiting hypoxia-induced glycolysis through improvement of mitochondrial dynamics.

Novel Peptides Derived from Sea Cucumber Intestine Promotes Osteogenesis by Upregulating Integrin-Mediated Transdifferentiation of Growth Plate Chondrocytes to Osteoblasts.

The sea cucumber intestine is a major by-product of sea cucumber processing and contains high levels of protein. In this study, we isolated and identified 28 novel osteogenic peptides from sea cucumber intestinal hydrolysis by the activity-tracking method for the first time. In vitro experimental results showed that compared with high molecular weight, the peptides from sea cucumber intestine (SCIP) with molecular weight <3 kDa more significantly promoted the proliferation and mineralized nodules of MC3T3-E1 cell and exhibited potential integrin binding capacity. In vivo experimental results showed that the SCIP supplement significantly increased the longitudinal bone length and elevated the height of the growth plate (especially the hypertrophic zone, 37.2%, p < 0.01) in adolescent mice. Further, immunofluorescence labeling results indicated that the SCIP supplement increased chondrocyte transdifferentiate to osteoblast in the growth plate close to the diaphysis. Mechanistically, transcriptome analysis revealed that the SCIP supplement induced the dedifferentiation of chondrocyte to osteoprogenitor cell via integrin-mediated histone acetylation and then redifferentiated to osteoblast via integrin-mediated Wnt/ß-catenin signaling. These results reported for the first time that sea cucumber intestine had the potential to develop into a dietary supplement for promoting osteogenic, and provide new evidence for the mechanism of dietary promotes chondrocyte to osteoblast transdifferentiation.

Novel peptides from sea cucumber intestinal hydrolysates promote longitudinal bone growth in adolescent mice through accelerating cell cycle progress by regulating glutamine metabolism.

Sea cucumber intestines are recognized as a major by-product in the sea cucumber processing industry and have been shown to exhibit bioactive properties. However, whether the sea cucumber intestine is beneficial for osteogenesis remains unknown. In this study, low molecular weight peptides rich in glutamate/glutamine were obtained from sea cucumber intestines (SCIP) by enzymatic hydrolysis, and orally administered to adolescent mice to investigate the effects on longitudinal bone growth. The results showed that the SCIP supplement significantly increased the femur length and new bone formation rate by 9.6% and 56.3%, and elevated the levels of serum osteogenic markers alkaline phosphatase (ALP), Collagen I and osteocalcin (OCN). Notably, H&E staining showed that SCIP significantly increased the height of the growth plate, in which the height of the proliferation zone was elevated by 95.6%. Glutamine is a major determinant of bone growth. SCIP supplement significantly increased glutamine levels in the growth plate by 44.2% and upregulated the expression of glutamine metabolism-related enzymes glutaminase 1 (Gls1) and glutamate dehydrogenase 1 (GLUD1) in the growth plate. Furthermore, SCIP supplement upregulated growth plate acetyl coenzyme A levels to promote histone acetylation and accelerated cell cycle progression by upregulating Sox9 expression, thereby contributing to rapid chondrocyte proliferation. To the best of our knowledge, this is the first report where SCIP could enhance longitudinal bone growth via promoting growth plate chondrocyte proliferation. The present study will provide new ideas and a theoretical basis for the high-value utilization of sea cucumber intestines.

[Icariin improves renal interstitial fibrosis in a rat model of chronic renal failure by regulating mitochondrial dynamics].

This study aims to explore the effect of icariin(ICA) on mitochondrial dynamics in a rat model of chronic renal failure(CRF) and to investigate the molecular mechanism of ICA against renal interstitial fibrosis(RIF). CRF was induced in male Sprague-Dawley(SD) rats with 5/6(ablation and infarction, A/I) surgery(right kidney ablation and 2/3 infarction of the left kidney). Four weeks after surgery, the model rats were randomized into the following groups: 5/6(A/I) group, 5/6(A/I)+low-dose ICA group, and 5/6(A/I)+high-dose ICA group. Another 12 rats that received sham operation were randomly classified into 2 groups: sham group and sham+ICAH group. Eight weeks after treatment, the expression of collagen-Ⅰ(Col-Ⅰ), collagen-Ⅲ(Col-Ⅲ), mitochondrial dynamics-related proteins(p-Drp1 S616, p-Drp1 S637, Mfn1, Mfn2), and mitochondrial function-related proteins(TFAM, ATP6) in the remnant kidney tissues was detected by Western blot. The expression of α-smooth muscle actin(α-SMA) was examined by immunohistochemical(IHC) staining. The NRK-52 E cells, a rat proximal renal tubular epithelial cell line, were cultured in vitro and treated with ICA of different concentration. Cell viability was detected by CCK-8 assay. In NRK-52 E cells stimulated with 20 ng·mL~(-1) TGF-ß1 for 24 h, the effect of ICA on fibronectin(Fn), connective tissue growth factor(CTGF), p-Drp1 S616, p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 was detected by Western blot, and the ATP content and the mitochondrial morphology were determined. The 20 ng·mL~(-1) TGF-ß1-stimulated NRK-52 E cells were treated with or without 5 µmol·L~(-1) ICA+10 µmol·L~(-1) mitochondrial fusion promoter M1(MFP-M1) for 24 h and the expression of fibrosis markers Fn and CTGF was detected by Western blot. Western blot result showed that the levels of Col-Ⅰ, Col-Ⅲ, and p-Drp1 S616 were increased and the levels of p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 were decreased in 5/6(A/I) group compared with those in the sham group. The levels of Col-Ⅰ, Col-Ⅲ, and p-Drp1 S616 were significantly lower and the levels of p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 were significantly higher in ICA groups than that in 5/6(A/I) group. IHC staining demonstrated that for the expression of α-SMA in the renal interstitium was higher in the 5/6(A/I) group than in the sham group and that the expression in the ICA groups was significantly lower than that in the 5/6(A/I) group. Furthermore, the improvement in the fibrosis, mitochondrial dynamics, and mitochondrial function were particularly prominent in rats receiving the high dose of ICA. The in vitro experiment revealed that ICA dose-dependently inhibited the increase of Fn, CTGF, and p-Drp1 S616, increased p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6, elevated ATP content, and improved mitochondrial morphology of NRK-52 E cells stimulated by TGF-ß1. ICA combined with MFP-M1 further down-regulated the expression of Fn and CTGF in NRK-52 E cells stimulated by TGF-ß1 compared with ICA alone. In conclusion, ICA attenuated RIF of CRF by improving mitochondrial dynamics.

Shen Shuai â ¡ Recipe attenuates renal fibrosis in chronic kidney disease by improving hypoxia-induced the imbalance of mitochondrial dynamics via PGC-1α activation.

BACKGROUND: Shen Shuai Ⅱ Recipe (SSR) is an effective Chinese herbal formula for the treatment of patients with chronic kidney disease (CKD) in the clinic and significantly improves 5/6 ablation and infarction (A/I) surgery-induced renal interstitial fibrosis (RIF) and intrarenal hypoxia in rats. However, the underlying molecular mechanisms need further elucidation. PURPOSE: This study aims to investigate the renoprotective mechanisms of SSR in vivo and in vitro. METHODS: CKD model was induced in rats with 5/6 (A/I) surgery. 4 weeks later, rats were treated with vehicle or SSR or Fenofibrate by daily gavage. In vitro, HK2 cells exposed to hypoxia (1% O2) were treated with SSR in the presence or absence of 100 µM MitoTEMPO or 10 µM Mitochondrial Fusion Promoter M1. The effects of SSR on RIF, mitochondrial dynamics, oxidative metabolism, and mitochondrial ROS (mtROS) were determined by immunoblotting, colorimetric, and fluorometric assays according to the experimental protocols. Furthermore, to explore the mechanisms of SSR against RIF, HK2 cells of PGC-1α or MFN2 knockdown under hypoxic stimulation were treated with 400 µg/ml of SSR and (or) 1 µM of ZLN005. RESULTS: The results showed that treatment with SSR significantly improved mitochondrial morphology and function, up-regulated the expression of PGC-1α protein, and inhibited the production of mtROS in 5/6 (A/I) kidneys and hypoxia-treated HK2 cells, which may be closely correlated with its anti-RIF effect. In addition, compared to wild-type HK2 cells, the roles of SSR in improving mitochondrial dynamics and energy metabolism were greatly diminished in HK2 cells of PGC-1α knockdown under hypoxic exposure. More importantly, compared to ZLN005 or SSR combined with ZLN005 treatment, MFN2-deficient HK2 cells exhibited the increased protein levels of FN, α-SMA, TGF-ß1 and cleaved IL-1ß in response to hypoxic stimulation. CONCLUSION: SSR exerted the renoprotective effects by improving mitochondrial dynamics under hypoxic condition via PGC-1α activation.

Icariin attenuates renal fibrosis in chronic kidney disease by inhibiting interleukin-1ß/transforming growth factor-ß-mediated activation of renal fibroblasts.

Icariin (ICA) is a bioactive flavonoid extracted from Epimedium brevicornum Maxim and exhibits a variety of pharmacological activities including antiinflammatory and antioxidant effects. Recently, icariin has shown renoprotective role by inhibiting pathological matrix. However, the underlying mechanisms of the efficacy remain unknown. This study aimed to determine the effects of icariin on renal fibrosis and explore its molecular mechanisms. Chronic kidney disease (CKD) was induced in rats with 5/6 ablation and infarction (A/I) operation. Four weeks later, rats were treated with vehicle or 20 mg/kg (low dose) or 40 mg/kg (high dose) of icariin by daily gavage. Furthermore, to further elucidate the effect mechanisms of icariin, in vitro, NRK-49F cells stimulated by 8 ng/ml IL-1ß were treated with icariin in the presence or absence of SB431542 or the neutralizing antibody of transforming growth factor-ß (TGF-ß) for 24 h. We showed that icariin treatment for 8 weeks dose-dependently improved 5/6 (A/I)-induced kidney injury and fibrosis, and blocked the release of inflammatory cytokine IL-1ß. In vitro, icariin inhibited IL-1ß/TGF-ß-mediated activation of renal fibroblasts. In summary, anti-fibrotic effects of icariin were interconnected with the inhibition of renal fibroblast activation caused by IL-1ß/TGF-ß signaling.