NMR-Based Metabolic Profiling of the Effects of α-Ketoglutarate Supplementation on Energy-Deficient C2C12 Myotubes.

Skeletal muscle is closely linked to energy metabolism, but it is inevitably deprived of energy. Cellular differentiation is an essential and energy-demanding process in skeletal muscle development. Much attention has been paid to identifying beneficial factors that promote skeletal muscle satellite cell differentiation and further understanding the underlying regulatory mechanisms. As a critical metabolic substrate or regulator, α-ketoglutarate (AKG) has been recognized as a potential nutritional supplement or therapeutic target for skeletal muscle. We have previously found beneficial effects of AKG supplementation on the proliferation of C2C12 myoblasts cultured under both normal and energy-deficient conditions and have further elucidated the underlying metabolic mechanisms. However, it remains unclear what role AKG plays in myotube formation in different energy states. In the present study, we investigated the effects of AKG supplementation on the differentiation of C2C12 myoblasts cultured in normal medium (Nor myotubes) and low glucose medium (Low myotubes) and performed NMR-based metabonomic profiling to address AKG-induced metabolic changes in both Nor and Low myotubes. Significantly, AKG supplementation promoted myotube formation and induced metabolic remodeling in myotubes under normal medium and low glucose medium, including improved energy metabolism and enhanced antioxidant capacity. Specifically, AKG mainly altered amino acid metabolism and antioxidant metabolism and upregulated glycine levels and antioxidase expression. Our results are typical for the mechanistic understanding of the effects of AKG supplementation on myotube formation in the two energy states. This study may be beneficial for further exploring the applications of AKG supplementation in sports, exercise, and therapy.

NMR-based metabolomic analysis for the effects of moxibustion on imiquimod-induced psoriatic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Moxibustion is a traditional medical intervention of traditional Chinese medicine. It refers to the direct or indirect application of ignited moxa wool made of mugwort leaves to acupuncture points or other specific parts of the body for either treating or preventing diseases. Moxibustion has been proven to be effective in treating skin lesions of psoriasis. AIM OF THE STUDY: This study was performed to elucidate molecular mechanisms underlying the effects of moxibustion treatment on imiquimod-induced psoriatic mice. MATERIALS AND METHODS: We established an imiquimod (IMQ)-induced psoriatic mice (Model) and assessed the effects of moxibustion (Moxi) treatment on skin lesions of psoriatic mice by the PASI scores and expressions of inflammation-related factors relative to normal control mice (NC). We then performed nuclear magnetic resonance (NMR)-based metabolomic analysis on the skin tissues of the NC, Model and Moxi-treated mice to address metabolic differences among the three groups. RESULTS: Moxi mice showed reduced PASI scores and decreased expressions of the pro-inflammatory cytokines IL-8, IL-17A and IL-23 relative to Model mice. Compared with the Model group, the NC and Moxi groups shared 9 characteristic metabolites and 4 significantly altered metabolic pathways except for taurine and hypotaurine metabolism uniquely identified in the NC group. To a certain extent, moxibustion treatment improved metabolic disorders of skin lesions of psoriatic mice by decreasing glucose, valine, asparagine, aspartate and alanine-mediated cell proliferation and synthesis of scaffold proteins, alleviating histidine-mediated hyperproliferation of blood vessels, and promoting triacylglycerol decomposition. CONCLUSIONS: This study reveals the molecular mechanisms underlying the effects of moxibustion treatment on the skin lesions of psoriasis, potentially improving the clinical efficacy of moxibustion.

Filamentous GLS1 promotes ROS-induced apoptosis upon glutamine deprivation via insufficient asparagine synthesis.

GLS1 orchestrates glutaminolysis and promotes cell proliferation when glutamine is abundant by regenerating TCA cycle intermediates and supporting redox homeostasis. CB-839, an inhibitor of GLS1, is currently under clinical investigation for a variety of cancer types. Here, we show that GLS1 facilitates apoptosis when glutamine is deprived. Mechanistically, the absence of exogenous glutamine sufficiently reduces glutamate levels to convert dimeric GLS1 to a self-assembled, extremely low-Km filamentous polymer. GLS1 filaments possess an enhanced catalytic activity, which further depletes intracellular glutamine. Functionally, filamentous GLS1-dependent glutamine scarcity leads to inadequate synthesis of asparagine and mitogenome-encoded proteins, resulting in ROS-induced apoptosis that can be rescued by asparagine supplementation. Physiologically, we observed GLS1 filaments in solid tumors and validated the tumor-suppressive role of constitutively active, filamentous GLS1 mutants K320A and S482C in xenograft models. Our results change our understanding of GLS1 in cancer metabolism and suggest the therapeutic potential of promoting GLS1 filament formation.

Taurine Protects C2C12 Myoblasts From Impaired Cell Proliferation and Myotube Differentiation Under Cisplatin-Induced ROS Exposure.

In cancer patients, chemotherapeutic medication induces aberrant ROS (reactive oxygen species) accumulation in skeletal muscles, resulting in myofiber degradation, muscle weakness, and even cachexia, which further leads to poor therapeutic outcomes. Acting as an antioxidant, taurine is extensively used to accelerate postexercise muscle recovery in athletes. The antioxidant effects of taurine have been shown in mature myotubes and myofibers but not yet in myoblasts, the myotube precursor. The proliferation and differentiation ability of myoblasts play a very important role in myofiber repair and regeneration, which is usually impaired during chemotherapeutics in cancer patients as well. Here, we explored the effects of taurine supplementation on C2C12 myoblasts exposed to cisplatin-induced ROS. We found that cisplatin treatment led to dramatically decreased cell viability; accumulated ROS level; down-regulated expressions of MyoD1 (myoblast determination protein 1), myogenin, and MHC (myosin heavy chain); and impaired myotube differentiation in myoblasts. Significantly, taurine supplementation protected myoblasts against cisplatin-induced cell viability decrease, promoted cellular ROS clearance, and, most importantly, preserved the expressions of MyoD1, myogenin, and MHC as well as myotube differentiation ability. We further conducted NMR-based metabolomic analysis to clarify the underlying molecular mechanisms. We identified 14 characteristic metabolites primarily responsible for the discrimination of metabolic profiles between cisplatin-treated cells and normal counterparts, including increased levels of BCAAs (branched-chain amino acids: leucine and isoleucine), alanine, glycine, threonine, glucose, ADP (adenosine diphosphate), phenylalanine, and PC (O-phosphocholine), and decreased levels of lysine, ß-alanine, choline, GPC (sn-glycero-3-phosphocholine), and myo-inositol. Evidently, taurine supplementation partially reversed the changing trends of several metabolites (isoleucine, threonine, glycine, PC, ß-alanine, lysine, and myo-inositol). Furthermore, taurine supplementation promoted the proliferation and myotube differentiation of myoblasts by alleviating cellular catabolism, facilitating GSH (reduced glutathione) biosynthesis, improving glucose utilization and TCA (tricarboxylic acid) cycle anaplerosis, and stabilizing cellular membranes. Our results demonstrated the protective effects of taurine on cisplatin-impaired myoblasts and elucidated the mechanistic rationale for the use of taurine to ameliorate muscle toxicity in clinical chemotherapy cancer patients.

NMR-Based Metabolomic Analysis for the Effects of α-Ketoglutarate Supplementation on C2C12 Myoblasts in Different Energy States.

α-Ketoglutarate (AKG) is attracting much attention from researchers owing to its beneficial effects on anti-aging and cancer suppression, and, more recently, in nutritional supplements. Given that glucose is the main source of energy to maintain normal physiological functions of skeletal muscle, the effects of AKG supplementation for improving muscle performance are closely related to the glucose level in skeletal muscle. The differences of AKG-induced effects in skeletal muscle between two states of normal energy and energy deficiency are unclear. Furthermore, AKG-induced metabolic changes in skeletal muscles in different energy states also remain elusive. Here, we assessed the effects of AKG supplementation on mouse C2C12 myoblast cells cultured both in normal medium (Nor cells) and in low-glucose medium (Low cells), which were used to mimic two states of normal energy and energy deficiency, respectively. We further performed NMR-based metabolomic analysis to address AKG-induced metabolic changes in Nor and Low cells. AKG supplementation significantly promoted the proliferation and differentiation of cells in the two energy states through glutamine metabolism, oxidative stress, and energy metabolism. Under normal culture conditions, AKG up-regulated the intracellular glutamine level, changed the cellular energy status, and maintained the antioxidant capacity of cells. Under low-glucose culture condition, AKG served as a metabolic substrate to reduce the glutamine-dependence of cells, remarkably enhanced the antioxidant capacity of cells and significantly elevated the intracellular ATP level, thereby ensuring the normal growth and metabolism of cells in the state of energy deficiency. Our results provide a mechanistic understanding of the effects of AKG supplements on myoblasts in both normal energy and energy deficiency states. This work may be beneficial to the exploitation of AKG applications in clinical treatments and nutritional supplementations.

NMR-based metabolomic analysis for the effects of Huiyang Shengji extract on rat diabetic skin ulcers.

ETHNOPHARMACOLOGICAL RELEVANCE: Huiyang Shengji formula (HSF) is a compound Chinese herbal medicine prescription, and has long been used for treating chronic non-healing wounds. AIM OF THE STUDY: The purpose of this study was to provide new insight into molecular mechanisms of healing effects of the HSF treatments. MATERIALS AND METHODS: We established a rat diabetic skin ulcer (DSU) model, and assessed healing effects of four HSF treatments on DSUs by calculating wound healing rates and immunohistochemical detection of the expressions of angiogenesis-related factors in the model rats (Mod) relative to normal rats (Nor), including Huiyang extract (HE), Shengji extract (SE), Huiyang Shengji extract (HSE) and HSE associated with acupuncture (Ac-HSE). We then performed NMR-based metabolomic analyses on skin tissues of the Nor, Mod, HSE-treated, Ac-HSE-treated rats to address metabolic mechanisms underlying these effects. RESULTS: These treatments up-regulated expressions of two angiogenesis-related factors VEGF and CD31, and improved efficacy of healing DSUs, in which HSE and Ac-HSE exhibited the most significant effects. Compared with Mod, HSE and Ac-HSE groups shared four characteristic metabolites (lactate, histidine, succinate and acetate) and four significantly altered metabolic pathways with Nor. Both HSE and Ac-HSE treatments could partly reverse the metabolically disordered pathological state of DSUs to the normal state. They might improve wound healing through promoting glucose metabolism, BCAAs metabolism, and enhancing antioxidant capacity and angiogenesis in DSU tissues. Ac-HSE significantly enhanced wound healing rates compared to HSE, potentially owing to significant capacities of enhancing anti-oxidation and angiogenesis and interfering three more metabolic pathways. CONCLUSIONS: This work provides a mechanistic understanding of the healing effects of the HSE and Ac-HSE treatments on DSUs, is of benefit to improvements of the HSF treatments for clinically healing chronic non-healing wounds.

Serum metabolomics model and its metabolic characteristics in patients with different syndromes of dyslipidemia based on nuclear magnetic resonance.

Dyslipidemia is known as a common clinical disease that affects the health of millions of people around the world. The treatment of dyslipidemia with traditional Chinese medicine (TCM) is generally based on the accurate identification of disease syndromes. TCM syndromes are judged by traditional four-diagnosis method, which is subjective and fuzzy. Additionally, the judgment of TCM syndromes highly depend on doctors' own clinical experience. In this present study, we used nuclear magnetic resonance (NMR)-based serum metabolomics patterns to figure out the metabolic characteristics of different syndromes in patients with dyslipidemia. In total, we enrolled 60 patients with dyslipidemia (30 cases with Spleen and Kidney Yang Deficiency syndrome (SKYD) and 30 cases with Phlegm-Dampness Retention syndrome (PDR)) and 20 healthy controls. Based on NMR technique, the serum metabolomics patterns of patients with different syndromes and healthy controls were analyzed, in the hope of screening the different metabolites among different syndromes and the differential metabolic pathway, as well as exploring the changes of metabolic network among different syndromes of dyslipidemia. The results suggested that the serum metabolomics patterns based on NMR was used to identify serum metabolites in patients with dyslipidemia of SKYD and PDR as well as healthy controls. Besides, it was found that the metabolic patterns of these three groups can be distinguished well and the different metabolites between different syndromes can be screened. From the point of view of metabolites, the metabolic characteristics of the patients with PDR were mainly the accumulation of noxious metabolites, while the metabolic characteristics of the patients with SKYD were mainly the lack of metabolites with protective function. From the point of view of metabolic mode, there were different metabolic patterns in patients with different syndromes of dyslipidemia in liver metabolism, oxidation, inflammatory reaction as well as energy metabolism, which reflects the difference of syndromes from different angles. The differences in metabolic outcomes among patients with different syndromes of dyslipidemia had a close association with to the effects of multiple signaling pathways. This study identified the characteristics of serum metabolic model of patients with different syndromes of dyslipidemia and the potential differential metabolites and characteristic metabolic characteristics of syndromes in order to understand the biological characteristics of patients with dyslipidemia of SKYD and PDR.

Lead compounds and key residues of ribosomal protein S1 in drug-resistant Mycobacterium tuberculosis.

Ribosomal protein S1 (RpsA) has been identified as a novel target of pyrazinoic acid (POA), which is the active form of pyrazinamide (PZA), in vivo. RpsA plays a crucial role in trans-translation, which is widespread in microbes. In our investigation, we first described the discovery of promising RpsA antagonists for drug-resistant mycobacterium (MtRpsAd438A) and M. smegmatis, as well as wild-type M. tuberculosis. These antagonists were discovered via structure/ligand-based virtual screening approaches. A total of 21 targeted compounds were selected by virtual screening, combined scores, affinity, similarities and rules for potential as drugs. Next, the affinities of these compounds for three targeted proteins were tested in vitro by applying various technologies, including fluorescence quenching titration (FQT), saturation transfer difference (STD), and chemical shift perturbation (CSP) assays. The results showed that seven compounds had a high affinity for the targeted proteins. Our discovery set the stage for discovering new chemical entities (NCEs) for PZA-resistant tuberculosis and providing key residues for rational drug design to target RpsA.

[Effect of Electroacupuncture at "Neiguan"(PC 6) on Serum and Myocardial Metabolites in Rats with Myocardial Ischemia Reperfusion Injury Based on Nuclear Magnetic Resonance Spectroscopy].

OBJECTIVE: We have repeatedly demonstrated that electroacupuncture (EA) of "Neiguan"(PC 6) can improve myocardial ischemia in rats. The present study was designed to investigate the metabolomic profile of peripheral blood se-rum and myocardium involving EA-induced improvement of myocardial ischemia-reperfusion injury (MIRI) in rats by using nuclear magnetic resonance spectroscopy. METHODS: Thirty male SD rats were equally randomized into blank control, model and EA groups. Rats of the control group were only banded for 20 min, once a day for 7 days. The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 60 min, and rats of the model group were banded as those in the control group. EA (10 Hz/50 Hz, 1 mA) was applied to bilateral PC 6 for 20 min, once daily for 7 days. The blood samples and left ventricular myocardial tissues were collected for assaying the profiles of differential metabolites using 1H nuclear magnetic resonance (1H NMR) spectroscopy and multivariate statistical analysis such as the principal components analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) with SIMCA-P software 12.0. RESULTS: A total of 19 differential metabolites (17 down-regulated, 2 up-regulated) in the serum and 14 differential metabolites (13 down-regulated and 1 up-regulated) in the ischemic left myocardium were identified after MIRI. Of the 19 serum differential metabolites, amino acids (leucine, isoleucine, valine,alanine, lysine, glycine, glutamine), 3-hydroxy butyric acid (3-HB), lactic acid, acetate, N-acetyl glycoprotein (NAc), acetone, acetoacetate, succinate, polyunsaturated fatty acids (PUFA), creatine, glycerophosphocholine (GPC) were down-regulated; while low density lipoprotein (LDL), LDL/very low density lipoprotein(LDL/VLDL)and glucose obviously up-regulated. Of the 14 myocardial differential metabolites, amino acids (alanine, lysine, glutamate, glutamine, aspartate, taurine, glycine, threonine), GPC, creatine, lactic acid, adenosine monophosphate (AMP), nicotinamide adenine dinucleotide (NAD＋) were significantly decreased, and glucose was up-regulated. Following EA treatment, most of the decreased serum differential metabolites except acetone, acetoacetate and PUFA, and the increased serum LDL, LDL/VLDL and glucose recovered, basically close to the control level; and the decreased myocardial creatine, GPC and NAD＋ were also apparently up-regulated and the increased myocardial glucose was down-regulated. But, myocardial threonine and AMP still presented a decreasing state. Although the pattern of myocardial differential metabolites of the EA group had a trend to be close to the control group, the significant difference still existed, while the metabolic pattern of serum metabolites in the EA group was close to that of the control group. CONCLUSION: EA stimulation of PC 6 can regulate serum or/and myocardial metabolites as amino acids, carbohydrates, lipids, etc. in MIRI rats, of which both serum and myocardial creatine, GPC and glucose may be jointly confer a favorable potential for EA-induced improvement of MIRI.

NMR-based metabolomic analysis of the effects of alanyl-glutamine supplementation on C2C12 myoblasts injured by energy deprivation.

The dipeptide alanyl-glutamine (Ala-Gln) is a well-known parenteral nutritional supplement. The Ala-Gln supplementation is a potential treatment for muscle-related diseases and injuries. However, molecular mechanisms underlying the polyphenic effects of Ala-Gln supplementation remain elusive. Here, we performed NMR-based metabolomic profiling to analyze the effects of Ala-Gln, and the free alanine (Ala) and glutamine (Gln) supplementations on the mouse myoblast cell line C2C12 injured by glucose and glutamine deprivation. All the three supplementations can promote the differentiation ability of the injured C2C12 cells, while only Ala-Gln supplementation can facilitate the proliferation of the injured cells. Ala-Gln supplementation can partially restore the metabolic profile of C2C12 myoblasts disturbed by glucose and glutamine deprivation, and exhibits more significant effects than Ala and Gln supplementations. Our results suggest that Ala-Gln supplementation can promote MyoD1 protein synthesis, upregulate the muscle ATP-storage phosphocreatine (PCr), maintain TCA cycle anaplerosis, enhance the antioxidant capacity through promoting GSH biosynthesis, and stabilize lipid membranes by suppressing glycerophospholipids metabolism. This work provides new insight into mechanistic understanding of the polyphenic effects of Ala-Gln supplementation on muscle cells injured by energy deprivation.

NMR-based metabolomic analysis for the effects of creatine supplementation on mouse myoblast cell line C2C12.

Creatine (Cr) supplementation has drawn much attention from researchers owing to its widespread efficacy in sports, and more recently, in therapeutic fields. However, the underlying molecular mechanisms remain elusive. Here, we performed nuclear magnetic resonance-based metabolomic analysis to address the metabolic profile of aqueous extracts from the mouse myoblast cell line C2C12 exposed to 2 mM Cr for 24 h (the Cr-treated group). Results showed that Cr supplementation facilitated the proliferation of C2C12 myoblasts. Both pattern recognition and hierarchical cluster analyses demonstrated that the metabolic profiles of the Cr-treated and control groups were distinctly different. We identified 13 characteristic metabolites significantly responsible for the discrimination of metabolic profiles between the two groups, through orthogonal projection to latent structures discriminant analysis and independent samples t-test. We further verified the discrimination performances of these metabolites by conducting univariate receiver operating characteristic curve analysis. Compared with the control group, the Cr-treated group exhibited increased levels of Cr, phosphocreatine (PCr), glutathione (GSH), and glucose, but decreased levels of leucine, valine, isoleucine, phenylalanine, methionine, choline, O-phosphocholine, sn-glycero-3-phosphocholine, and glycerol. Our results demonstrated that Cr supplementation upregulated PCr and glucose, promoted trichloroacetic acid cycle anaplerotic flux and GSH-mediated antioxidant capacity, and stabilized lipid membranes through suppressing glycerophospholipid metabolism. Our work provides new clues to the molecular mechanisms underlying the pleiotropic effects of Cr in muscle cells.

Celastrol attenuates inflammatory and neuropathic pain mediated by cannabinoid receptor type 2.

Celastrol, a major active ingredient of Chinese herb Tripterygium wilfordii Hook. f. (thunder god vine), has exhibited a broad spectrum of pharmacological activities, including anti-inflammation, anti-cancer and immunosuppression. In the present study, we used animal models of inflammatory pain and neuropathic pain, generated by carrageenan injection and spared nerve injury (SNI), respectively, to evaluate the effect of celastrol and to address the mechanisms underlying pain processing. Intraperitoneal (i.p.) injection of celastrol produced a dose-dependent inhibition of carrageenan-induced edema and allodynia. Real-time PCR analysis showed that celastrol (0.3 mg/kg, i.p.) significantly reduced mRNA expressions of inflammatory cytokines, TNF-α, IL-6, IL-1ß, in carrageenan-injected mice. In SNI mice, pain behavior studies showed that celastrol (1 mg/kg, i.p.) effectively prevented the hypersensitivity of mechanical nociceptive response on the third day post-surgery and the seventh day post-surgery. Furthermore, the anti-hyperalgesic effects of celastrol in carrageenan-injected mice and SNI mice were reversed by SR144528 (1 mg/kg, i.p.), a specific cannabinoid receptor-2 (CB2) receptor antagonist, but not by SR141716 (1 mg/kg, i.p.), a specific cannabinoid receptor-1 (CB1) receptor antagonist. Taken together, our results demonstrate the analgesia effects of celastrol through CB2 signaling and propose the potential of exploiting celastrol as a novel candidate for pain relief.

Metabolomic analysis of anti-hypoxia and anti-anxiety effects of Fu Fang Jin Jing Oral Liquid.

BACKGROUND: Herba Rhodiolae is a traditional Chinese medicine used by the Tibetan people for treating hypoxia related diseases such as anxiety. Based on the previous work, we developed and patented an anti-anxiety herbal formula Fu Fang Jin Jing Oral Liquid (FJJOL) with Herba Rhodiolae as a chief ingredient. In this study, the anti-hypoxia and anti-anxiety effects of FJJOL in a high altitude forced-swimming mouse model with anxiety symptoms will be elucidated by NMR-based metabolomics. METHODS: In our experiments, the mice were divided randomly into four groups as flatland group, high altitude saline-treated group, high altitude FJJOL-treated group, and high altitude diazepam-treated group. To cause anxiety effects and hypoxic defects, a combination use of oxygen level decreasing (hypobaric cabin) and oxygen consumption increasing (exhaustive swimming) were applied to mice. After a three-day experimental handling, aqueous metabolites of mouse brain tissues were extracted and then subjected to NMR analysis. The therapeutic effects of FJJOL on the hypobaric hypoxia mice with anxiety symptoms were verified. RESULTS: Upon hypoxic exposure, both energy metabolism defects and disorders of functional metabolites in brain tissues of mice were observed. PCA, PLS-DA and OPLS-DA scatter plots revealed a clear group clustering for metabolic profiles in the hypoxia versus normoxia samples. After a three-day treatment with FJJOL, significant rescue effects on energy metabolism were detected, and levels of ATP, fumarate, malate and lactate in brain tissues of hypoxic mice recovered. Meanwhile, FJJOL also up-regulated the neurotransmitter GABA, and the improvement of anxiety symptoms was highly related to this effect. CONCLUSIONS: FJJOL ameliorated hypobaric hypoxia effects by regulating energy metabolism, choline metabolism, and improving the symptoms of anxiety. The anti-anxiety therapeutic effects of FJJOL were comparable to the conventional anti-anxiety drug diazepam on the hypobaric hypoxia mice. FJJOL might serve as an alternative therapy for the hypoxia and anxiety disorders.

Metabonomic analysis of the therapeutic effect of Zhibai Dihuang Pill in treatment of streptozotocin-induced diabetic nephropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhibai Dihuang Pill (ZDP) is one of ancient traditional Chinese medicines (TCMs), which is usually used for the treatment of kidney deficiency for thousands of years in China. AIM OF THE STUDY: Traditional Chinese medicines (TCMs) usually operate in vivo through multi-components, multi-ways and multi-targets. However, the molecular mechanisms of TCMs remain unclear. In the present work, nuclear magnetic resonance (NMR)-based metabonomic analysis was used to evaluate the therapeutic effect of Zhibai Dihuang Pill (ZDP) on diabetic nephropathy (DN) rats induced by streptozotocin and to address the underlying molecular mechanism. MATERIALS AND METHODS: Male rats were divided into three groups: control, DN and ZDP-treated DN (ZDP-DN), respectively. Based on (1)H NMR spectra of sera, urine and kidney extracts from the rats, principle component analysis (PCA) was performed to identify different metabolic profiles. Kidney portions and serum and urine samples were also subjected to histopathological or biochemical examination. RESULTS: PCA scores plots demonstrate that the cluster of DN rats is separated from that of control rats, while some of ZDP-DN rats are located close to control rats, indicating that metabolic profiles of these ZDP-DN rats are restored toward those of control rats. Our results illustrate that ZDP treatment could lower the levels of lipids and 3-hydrobutyrate, and raise the level of lactate in sera of DN rats. Moreover, ZDP treatment could also reduce the levels of glucose, 3-hydrobutyrate and lactate, enhance the level of betaine in kidney tissues. CONCLUSION: Our study indicates that ZDP treatment can ameliorate DN symptoms by intervening in some dominating metabolic pathways, such as inhibiting glucose and lipid metabolism, enhancing methylamine metabolism. Our work may be of benefit to both evaluation of the therapeutic effect of TCM and elucidation of the underlying molecular mechanism.

1H NMR spectroscopy analysis of metabolites in the kidneys provides new insight into pathophysiological mechanisms: applications for treatment with Cordyceps sinensis.

BACKGROUND: The number of patients with chronic kidney disease (CKD) is continuously growing worldwide. Treatment with traditional Chinese medicine might slow the progression of CKD. METHODS: In this study, we evaluated the renal protective effects of the Chinese herb Cordyceps sinensis in rats with 5/6 nephrectomy. Male Sprague-Dawley mice (weighing 150-200 g) were subjected to 5/6 nephrectomy. The rats were divided into three groups: (i) untreated nephrectomized group (OP group, n = 16), (ii) oral administration of C. sinensis-treated (4 mg/kg/day) nephrectomized group (CS group, n = 16) and (iii) sham-operated group (SO group, n = 16). The rats were sacrificed at 4 and 8 weeks after 5/6 nephrectomy, and the kidneys, serum and urine were collected for (1)H nuclear magnetic resonance spectral analysis. Multivariate statistical techniques and statistical metabolic correlation comparison analysis were performed to identify metabolic changes in aqueous kidney extracts between these groups. RESULTS: Significant differences between these groups were discovered in the metabolic profiles of the biofluids and kidney extracts. Pathways including the citrate cycle, branched-chain amino acid metabolism and the metabolites that regulate permeate pressure were disturbed in the OP group compared to the SO group; in addition, these pathways were reversed by C. sinensis treatment. Biochemistry and electron microscopic images verified that C. sinensis has curative effects on chronic renal failure. These results were confirmed by metabonomics results. CONCLUSION: Our study demonstrates that C. sinensis has potential curative effects on CKD, and our metabonomics results provided new insight into the mechanism of treatment of this traditional Chinese medicine.

Potential anti-cancer activities of Furanodiene, a Sesquiterpene from Curcuma wenyujin.

Furanodiene is a sesquiterpene extracted from the essential oil of the rhizome of Curcuma wenyujin Y.H. Chen et C. Ling (Wen Ezhu). Furanodiene is the primary component in Wen Ezhu's essential oil, accounting for more than 20% by weight. In vitro, MTT assay was used to compare the inhibitory effects of furanodiene and Wen Ezhu's essential oil on 11 human cancer cell lines. Compared to the essential oil, furanodiene showed stronger growth inhibitions on Hela, Hep-2, HL-60, PC3, SGC-7901 and HT-1080 cells with IC(50) between 0.6-4.8 microg/ml. In vivo, furanodiene was also found to exhibit inhibitory effects on the growth of uterine cervical (U14) and sarcoma 180 (Sl80) tumors in mice. Our data suggests that furanodiene, an active component from the essential oil of Wen Ezhu, possesses efficacy against uterine cervical cancer.

Odorranalectin is a small peptide lectin with potential for drug delivery and targeting.

BACKGROUND: Lectins are sugar-binding proteins that specifically recognize sugar complexes. Based on the specificity of protein-sugar interactions, different lectins could be used as carrier molecules to target drugs specifically to different cells which express different glycan arrays. In spite of lectin's interesting biological potential for drug targeting and delivery, a potential disadvantage of natural lectins may be large size molecules that results in immunogenicity and toxicity. Smaller peptides which can mimic the function of lectins are promising candidates for drug targeting. PRINCIPAL FINDINGS: Small peptide with lectin-like behavior was screened from amphibian skin secretions and its structure and function were studied by NMR, NMR-titration, SPR and mutant analysis. A lectin-like peptide named odorranalectin was identified from skin secretions of Odorrana grahami. It was composed of 17 aa with a sequence of YASPKCFRYPNGVLACT. L-fucose could specifically inhibit the haemagglutination induced by odorranalectin. (125)I-odorranalectin was stable in mice plasma. In experimental mouse models, odorranalectin was proved to mainly conjugate to liver, spleen and lung after i.v. administration. Odorranalectin showed extremely low toxicity and immunogenicity in mice. The small size and single disulfide bridge of odorranalectin make it easy to manipulate for developing as a drug targeting system. The cyclic peptide of odorranalectin disclosed by solution NMR study adopts a beta-turn conformation stabilized by one intramolecular disulfide bond between Cys6-Cys16 and three hydrogen bonds between Phe7-Ala15, Tyr9-Val13, Tyr9-Gly12. Residues K5, C6, F7, C16 and T17 consist of the binding site of L-fucose on odorranalectin determined by NMR titration and mutant analysis. The structure of odorranalectin in bound form is more stable than in free form. CONCLUSION: These findings identify the smallest lectin so far, and show the application potential of odorranalectin for drug delivery and targeting. It also disclosed a new strategy of amphibian anti-infection.

Identification of a substrate recognition site on Ubc9.

Human Ubc9 is homologous to ubiquitin-conjugating enzymes. However, instead of conjugating ubiquitin, it conjugates a ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), also known as UBL1, GMP1, SMTP3, PIC1, and sentrin. The SUMO-1 conjugation pathway is very similar to that of ubiquitin with regard to the primary sequences of the ubiquitin-activating enzymes (E1), the three-dimensional structures of the ubiquitin-conjugating enzymes (E2), and the chemistry of the overall conjugation pathway. The interaction of substrates with Ubc9 has been studied using NMR spectroscopy. Peptides with sequences that correspond to those of the SUMO-1 conjugation sites from p53 and c-Jun both bind to a surface adjacent to the active site Cys93 of human Ubc9, which has been previously shown to include residues that demonstrate the most significant dynamics on the microsecond to millisecond time scale. Mutations in this region, Q126A, Q130A, A131D, E132A, Y134A, and T135A, were constructed to evaluate the role of these residues in SUMO-1 conjugation. These alterations have significant effects on the conjugation of SUMO-1 with the target proteins p53, E1B, and promyelocytic leukemia protein and define a substrate binding site on Ubc9. Furthermore, the SUMO-1 conjugation site of p53 does not form any defined secondary structure when either free or bound to Ubc9. This suggests that a defined secondary structure at SUMO-1 conjugation sites in target proteins is not necessary for recognition and conjugation by the SUMO-1 pathway.