Dietary elaidic acid boosts tumoral antigen presentation and cancer immunity via ACSL5.

Immunomodulatory effects of long-chain fatty acids (LCFAs) and their activating enzyme, acyl-coenzyme A (CoA) synthetase long-chain family (ACSL), in the tumor microenvironment remain largely unknown. Here, we find that ACSL5 functions as an immune-dependent tumor suppressor. ACSL5 expression sensitizes tumors to PD-1 blockade therapy in vivo and the cytotoxicity mediated by CD8+ T cells in vitro via regulation of major histocompatibility complex class I (MHC-I)-mediated antigen presentation. Through screening potential substrates for ACSL5, we further identify that elaidic acid (EA), a trans LCFA that has long been considered harmful to human health, phenocopies to enhance MHC-I expression. EA supplementation can suppress tumor growth and sensitize PD-1 blockade therapy. Clinically, ACSL5 expression is positively associated with improved survival in patients with lung cancer, and plasma EA level is also predictive for immunotherapy efficiency. Our findings provide a foundation for enhancing immunotherapy through either targeting ACSL5 or metabolic reprogramming of antigen presentation via dietary EA supplementation.

Bufei Yishen Formula Inhibits the Cell Senescence in COPD by Up-Regulating the ZNF263 and Klotho Expression.

Background: Bufei Yishen formula (BYF) is an effective prescription for the clinical treatment of chronic obstructive pulmonary disease (COPD). However, the molecular mechanism by which it exerts its pharmacological effects remains to be explored. Methods: The human bronchial cell line BEAS-2B was treated with cigarette smoke extract (CSE). Cellular senescence markers were detected by Western blot and ELISA. Potential transcription factor of klotho was predicted using JASPAR and USCS databases. Results: CSE induced cellular senescence with intracellular accumulation of cellular senescence biomarkers (p16, p21 and p27) and increased secretion of senescence-related secretory phenotypic (SASP) factors (IL-6, IL-8, and CCL3). In contrast, BYF treatment inhibited CSE-induced cellular senescence. CSE suppressed the transcription, expression and secretion of klotho, whereas BYF treatment rescued its transcription, expression and secretion. CSE downregulated the protein level of ZNF263, whereas BYF treatment rescued the expression of ZNF263. Furthermore, ZNF263-overexpressing BEAS-2B cells could inhibit CSE-induced cellular senescence and SASP factor secretion by upregulating the expression of klotho. Conclusion: This study revealed a novel pharmacological mechanism by which BYF alleviates clinical symptoms of COPD patients, and regulating ZNF263 and klotho expression may be beneficial to the treatment and prevention of COPD.

Identification of key genes, biological functions, and pathways of empagliflozin by network pharmacology and its significance in the treatment of type 2 diabetes mellitus.

Background: To explore the key genes, biological functions, and pathways of empagliflozin in the treatment of type 2 diabetes mellitus (T2DM) through network pharmacology. Methods: The TCMSP (a traditional Chinese medicine system pharmacology database and analysis platform) was used to screen empagliflozin's active components and targets. The target genes of T2DM were screened according to the GeneCards and OMIM databases, and a Venn diagram was constructed to obtain the target for T2DM treatment. Cytoscape 3.7.2 software was adopted to construct the drug-component-target-disease network. Functional annotation of Gene Ontology (GO) and enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed using R software. Results: Target genes with a probability >0 were selected, among which Compound 012, Compound 060, Compound 093, Compound 111, and Compound 119 Swiss Target Prediction suggested that no similar active substances or predictable target genes were found. A "compound-target gene-disease" network was constructed, in which SLC5A2, SLC5A1, SLC5A4, SLC5A11, ADK, and ADORA2A were the core genes of T2DM. The key factors of the GO summary map included chemical reaction, membrane organelle, protein binding, and so on. The KEGG pathway summary map included the AMPK pathway, insulin resistance, the MAPK pathway, longevity-related pathway regulation, and so on. The top 10 pathways were endocrine resistance, the NF-κB signaling pathway, the HIF-1 signaling pathway, apoptosis, cell senescence, the Ras signaling pathway, the MAPK signaling pathway, the FoxO signaling pathway, the P13K-Akt signaling pathway, and the p53 signaling pathway. The binding of active compounds to key proteins was verified based on the Swiss Dock database, and the molecular docking of 193 bioactive compounds was finally verified. Among them, SLC5A2, SLC5A1, LDHA, KLK1, KLF5, and GSTP1 had better binding to the protein molecules. Conclusions: Empagliflozin may regulate the targets of SLC5A2, SLC5A1, LDHA, KLK1, KLF5, and GSTP1. There are numerous ways of treating T2DM with empagliflozin, including by regulating apoptosis, cell aging, as well as the NF-κB, HIF-1HIF-1, Ras, MAPK, FoxO, P13K-Akt, and p53 pathways.

Metabolomics and molecular docking-directed antiarthritic study of the ethyl acetate extract from Celastrus orbiculatus Thunb.

ETHNOPHARMACOLOGICAL RELEVANCE: Celastrus orbiculatus Thunb., an important folk medicine, has long been used for the treatment of rheumatoid arthritis and its ethyl acetate extract (COE) has been reported to possess anticancer, antiinflammation and antiarthritic effects. However, the therapeutic effect and mechanism of COE treatment in rheumatoid arthritis has been rarely studied especially from the perspective of metabolomics. AIM OF STUDY: To reveal the therapeutic effects of COE on adjuvant-induced arthritis (AIA) rats through histopathological analysis, non-targeted metabolomics, and molecular docking study. MATERIALS AND METHODS: Forty-three Wistar rats were randomly divided into normal group, AIA model group, methotrexate group, and COE groups (80 mg/kg, 160 mg/kg and 320 mg/kg of ethyl acetate extract). Paw swelling and arthritis score were monitored through the experiment. Serum levels of tumor necrosis factor α (TNF-α) and nitric oxide were determined and histopathological evaluation was performed. Furthermore, Ultra-high performance liquid chromatography-linear trap quadrupole-Orbitrap-based metabolomics was employed to characterize metabolic changes of AIA rats after COE treatment and molecular docking was performed to predict the potential phytochemicals of COE against TNF-α. RESULTS: COE at three dosages could significantly relieve paw swelling and reduce arthritis scores of AIA rat. Histopathological analysis revealed remarkable decrease in synovial inflammation and bone erosion after COE treatment, especially at middle and high dosage. Additionally, COE down-regulated serum levels of TNF-α and nitric oxide. Serum metabolomics showed that 22 potential biomarkers for the COE treatment of AIA rats were identified, which were closely related to fatty acid metabolism, glycerophospholipid catabolism, and tryptophan metabolism. The molecular docking models predicted that olean-type triterpenes in COE may contribute most to therapeutic effects of rheumatoid arthritis through targeting TNF-α. CONCLUSIONS: COE could significantly relieve the arthritic symptoms in AIA rats and the ultra-high performance liquid chromatography-mass spectrometry based metabolomics proved to be an efficient method to characterize subtle metabolic changes of AIA rats after COE treatment.

Effect of Enzyme-Assisted Extraction on the Chemical Properties and Antioxidant Activities of Polysaccharides Obtained from the Wood Ear Mushroom, Auricularia auricula (Agaricomycetes).

An effective method of polysaccharide extraction from Auricularia auricula (AAPs) by mannanase was developed and optimized by response surface methodology in which the ABTS+ [diammonium 2,2'-azino-bis(3-ethylben-zothiazoline-6-sulfonate radical] scavenging rate was the response. AAPs were graded by stepwise ethanol precipitation with concentrations of 5, 10, 15, and 20% ethanol successively. The fractions with a strong radical scavenging rate were obtained, and then their antioxidant stress effect was studied using Caenorhabditis elegans as a model organism. The ABTS+ scavenging rate of AAPs could reach 37.95 ± 0.53% at a temperature of 55°C, a time of 4 h, a liquid-to-material ratio of 58 mL/1 g, and an enzyme-to-substrate ratio of 2.97%. AAP-20 obtained by 20% ethanol with a strong radical scavenging rate was a heteropolysaccharide composed of mannose, glucose, galactose, xylose, and glucuronic acid. AAP-20 could significantly prolong the lifespan of C. elegans under oxidative stress conditions induced by methyl viologen or hydrogen peroxide, and it could also enhance the activity of antioxidant enzymes including catalase, glutathione reductase, and superoxide dismutase at 0.50 mg/mL (P < 0.05). These studies showed that AAPs prepared with mannanase had a significant protective effect against damage induced by intracellular radical generating agents.

Preparation of the Enzymatic Hydrolysates from Chlorella vulgaris Protein and Assessment of Their Antioxidant Potential Using Caenorhabditis elegans.

This study aimed to assess the antioxidant potential of Chlorella vulgaris protein-derived enzymatic hydrolysate using Caenorhabditis elegans. Protein extraction was performed using an alkali solution after complete C. vulgaris swelling and hydrolysis using four commercial proteases (alcalase, neutrase, protamex, and flavourzyme). The results showed that the flavourzyme hydrolysates exhibited the strongest antioxidant activity both in vitro and in vivo. Under the optimum conditions of the enzymatic hydrolysis, the half-maximal effective concentration of the hydrolysates for superoxide and hydroxyl radicals was 0.323 mg/mL and 0.139 mg/mL, respectively. The hydrolysates could significantly extend the lifespan, improve the resistance to methyl viologen-induced oxidative stress, reduce the levels of reactive oxygen species, and enhance the activity of catalase and superoxide dismutase in C. elegans.

Shi-Style Cervical Mobilizations Versus Massage for Cervical Vertigo: A Multicenter, Randomized, Controlled Clinical Trial.

Objectives: Large sample and high-quality evidence to evaluate the preliminary safety of the mobilizations and massage for cervical vertigo are not yet available. Thus, the present study aimed to investigate the comparative effectiveness and preliminary safety of Shi-style cervical mobilizations (SCM) compared with traditional massage (TM) in cervical vertigo patients. Design: A prospective, multicenter, open-label, randomized, controlled clinical trial with a 1:1 allocation ratio. Settings: Five academic medical centers. Subjects: A total of 360 adult patients with a diagnosis of cervical vertigo. Interventions: The patients were randomly allocated to either an SCM (n = 180) or TM (n = 180) group. The patients were treated during six sessions over 2 weeks. The primary outcome was the Dizziness Handicap Inventory (DHI) total scale score, and secondary outcomes included the DHI subscales, Chinese version of the Short-Form 36 Health Survey (CSF-36), and adverse events (AEs). Outcomes were assessed in the short term at 2 weeks, 1 month, and 3 months, and in the intermediate term at 6 months after randomization. Results: Significant changes were observed from the baseline in the DHI total scale and subscales at 2 weeks and 1, 3, and 6 months in both groups (all p < 0.05). However, the differences between the two groups were not significant (all p > 0.05). Furthermore, we noted significant changes from the baseline in SF-36 scores at 2 weeks in both groups (all p < 0.05), whereas CSF-36 scores were not significantly higher in the SCM group (all p > 0.05) compared with the TM group. No serious AEs were reported in either of the two groups. Conclusions: No differences in outcomes were detected between the SCM and TM groups in terms of treatment of cervicogenic dizziness. Efficacy trials are required to determine whether the improvement observed for each treatment was causally related to the interventions.

Evaluation of the antioxidant effects of acid hydrolysates from Auricularia auricular polysaccharides using a Caenorhabditis elegans model.

Caenorhabditis elegans is an important model organism for studying stress response mechanisms and identifying genetic pathways that influence longevity. The present study was designed to explore the in vivo-antioxidant potential and the probable mechanism of acid hydrolysates prepared from A. auricula polysaccharides (AAPHs-F) with the optimal acid hydrolysis conditions using Box-Behnken design, and C. elegans was used as a model organism. The effects of AAPHs-F on the locomotory behavior, lifespan, activities of antioxidant-related enzymes and levels of antioxidants in C. elegans were studied. In addition, the potential of AAPHs-F in up-regulating the expression of antioxidant-related genes in C. elegans, such as daf-16, skn-1, sod-1, sod-2 and sir-2.1, and the inhibition of cell apoptosis of C. elegans were also discussed. The results indicated that AAPHs-F could significantly increase the U-Turn frequency of nematodes, extend their lifespan, enhance antioxidant systems including superoxide dismutase (SOD) by 70.60%, catalase (CAT) by 73.45% and glutathione reductase (GR) by 258.68% (p < 0.01), increase the level of glutathione (GSH) by 110.22% (p < 0.01), and decrease the level of reactive oxygen species (ROS) and malondialdehyde (MDA) by 31.86% and 46.16% (p < 0.01), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that AAPHs-F could up-regulate mRNA expression levels of daf-16, skn-1, sir, sod-1 and sod-2 in wild-type C. elegans (>1.3 fold) when treated at a concentration of 0.1 mg mL-1 (p < 0.05 or p < 0.01). AAPHs-F was concluded to be heteropolysaccharides composed of mannose, glucose and galactose with a molar ratio of 12.7 : 3.25 : 1. The molecular weight of AAPHs-F was determined to be 885.37 Da. Furthermore, AAPHs-F is mainly formed of (1 → 3)-linked-α-d-glucopyranose, and carboxyl or acetamide is present in the molecule. In summary, our studies provide evidence that AAPHs-F helps improve the antioxidant defense system, and up-regulation of stress and longevity related genes suggests the possible involvement of these genes in the prevention of stress damage in C. elegans.

Huachansu Capsule inhibits the proliferation of human gastric cancer cells via Akt/mTOR pathway.

BACKGROUND: For decades, the traditional Chinese medicine preparation, Huachansu Capsule (HCS), has been applied to a variety of solid tumors and leukemias with significant curative effects. More importantly, HCS has few side effects on cardiovascular and gastrointestinal functions in patients. However, the potential mechanism of the anti-tumor activity of HCS has not been fully revealed. The current study investigated the in vivo and in vitro effects of HCS on the proliferation and apoptosis of human gastric cancer (GC) cells and explored the underlying mechanism. MATERIALS AND METHODS: HCS was first diluted to varying concentrations followed by the treatment to MGC-803 and BGC-823 GC cells. Cell proliferation was evaluated by Cell Counting Kit-8 assay. Cell invasion and migration were assessed using Transwell membrane chambers. Apoptosis and cell cycle arrest in GC cells induced by HCS were detected by flow cytometry. Western blotting assays were used to measure the influence of HCS on apoptosis-related proteins, including B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and Cleaved-Caspase-3. Additionally, mammalian target of rapamycin (mTOR) signaling pathway-related proteins such as phosphorylated (p)-Akt, p-mTOR and p-4E-BP1 were detected. Transmission electron microscopy was used to observe the microstructure of apoptotic cells. An animal imaging technique was used to analyze the influence of HCS on the growth of GC cells in vivo and immunohistochemistry assays were performed to investigate the signal transduction pathways influenced by HCS. RESULTS: HCS significantly inhibited the proliferation, invasion and migration of MGC-803 and BGC-823 GC cells. It also induced cell cycle arrest at the G2/M phase and increased the cell apoptotic rate. Additionally, the HCS treatment downregulated the protein levels of Bcl-2, but upregulated the protein expression of Bax and cleaved-caspase 3. Furthermore, HCS downregulated the levels of p-Akt, p-mTOR and p-4E-BP1, suggesting that HCS inhibited tumor growth of GC via suppressing the Akt/mTOR pathway. CONCLUSION: This study indicated that HCS has significant anti-proliferative and apoptotic effects both in vitro and in vivo, and that HCS can inhibit tumor growth of GC via suppressing the Akt/mTOR pathway and induce apoptosis through the intrinsic pathway. Our study provides a scientific basis for the clinical application of HCS.

RNase1 alleviates the Aeromonas hydrophila-induced oxidative stress in blunt snout bream.

RNase1 is an enzyme important in host defense in vertebrates where it degrades the RNA of bacteria and viruses. We evaluated the effect of RNase1 on the resistance to Aeromonas hydrophila infection in Megalobrama amblycephala. The fish were randomly divided into four groups: a blank group (none-treated M. amblycephala), a control group (injected PBS), a challenge group (A. hydrophila-injected) and a treatment group (pre-treated with RNase1 24 h before the A. hydrophila injection), and we collected five tissues of each group. Then we recorded changes in the levels of glutathione (GSH), oxidized glutathione (GSSG), hepatic catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and lysozyme; and the relative mRNA expression of catalase (CAT), selenium-dependent glutathione peroxidase (GPx), Cu/Superoxide dismutase (Cu/Zn-SOD), glutamate-cysteine ligase (GCLC), glutathione reductase (GR) and nuclear factor erythroid 2-related factor 2 (Nrf2) for four groups. The expression of six genes was highest in liver and blood of the blank group. It was significantly higher in the gut of the treatment group (compared to control and challenge groups) 12 h after the infection. The treatment group exhibited a significant increase in GSH, SOD and CAT activity, and a decrease in GSSG, MDA and lysozyme content (compared to the control and challenge groups) 6 and 12 h after infection. These results suggest that supplementation with RNase1 protein can enhance resistance against A. hydrophila infections in M. amblycephala.

Hypoglycemic Effect of the Degraded Polysaccharides from the Wood Ear Medicinal Mushroom Auricularia auricula-judae (Agaricomycetes).

Auricularia auricula-judae is an important culinary-medicinal mushroom. The A. auricula-judae polysaccharides (AAPs) were prepared from A. auricula-judae in the early stage through alkali extraction and deproteination with the Sevag method, and optimal acid hydrolysis conditions were established by Box-Behnken to prepare the degraded polysaccharides (AAPs-F) from AAPs. In this study, a nonenzymatic glycosylation reaction system was used for the evaluation of the inhibitory effects on the formation of advanced glycation end products (AGEs). In addition, high glucose resistance was assessed by glucose consumption of HepG2 cells and the lifespan of Caenorhabditis elegans under high sugar stress. It was found that both 0.5 mg·mL-1 AAPs and 0.2 mg·mL-1 AAPs-F could significantly inhibit AGE formation in short- and long-term glycosylation (P < .05) in a dose-dependent manner, determined by ultraviolet and fluorospectrophotometry. It indicated activity against AGE formation for different concentrations of AAPs and AAPs-F. AAPs-F at 0.5 mg·mL-1 significantly enhanced the glucose absorption of HepG2 cells by 24.4% (P < .05) in a dose-dependent manner at 24 h, and markedly extended the lifespan of C. elegans by 32.9% (P < 0.05) under high sugar stress conditions. This study demonstrated that the derived hydrolysates produced by the hydrolysis of acid had a prominent effect on the inhibition of AGE formation and relieved the stress state caused by high sugar levels.

Celastrus Orbiculatus Extract Potentiates the Sensitivity of Cisplatin Via Caspase-Depenent Apoptosis in Gastric Cancer.

BACKGROUND: Cisplatin-based treatment often leads to therapeutic failure because the acquisition of cisplatin resistance. The combination of cisplatin with other agents has been recognized as a promising strategy to overcome cisplatin resistance. OBJECTIVE: Celastrus orbiculatus is a traditional Chinese medicine from Celastraceae family with multiple pharmacological activities. We previously found that the ethyl acetate extract of Celastrus orbiculatus (COE) exhibited significant antitumor activity in gastric cancer. Here, we asked whether COE could increase the sensitivity of cisplatin. METHODS: We use CCK8 assay to show synergistic cytotoxicity of COE and cisplatin. Then, PI single staining and FITC-Annexin V/PI double staining were used to observe apoptotic cells through flow cytometry. The proteins of caspase signaling pathway were examined by Western blotting. RESULTS: COE and cisplatin showed synergistic cytotoxicity in a dose-dependent manner in BGC 823 and SGC 7901 gastric cancer cells, and COE could increase the number of apoptotic cells upon cisplatin treatment in vitro. Moreover, our results indicated that COE could enhance cisplatin-induced activation of caspase-8 or caspase- 9/caspase-3/PARP1 signaling pathways. The xenograft study further confirmed that COE increased the sensitivity of cisplatin in vivo. CONCLUSION: Our findings provided new evidence that COE could increase the sensitivity of cisplatin on the antitumor effect.

Icariin influences cardiac remodeling following myocardial infarction by regulating the CD147/MMP-9 pathway.

Objective We investigated the protective effect of icariin on myocardial infarction-induced cardiac remodeling. Methods A cardiac remodeling model was constructed by ligating rats' coronary artery. Different icariin and CD147 concentrations were administered in the model group, and echocardiography was used to detect systolic function, screening out ideal experimental concentrations. The ventricular systolic function, myocardial apoptosis rate, and expression of collagen type I (Col I), Col III, CD147, matrix metalloproteinase 9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) were detected by hematoxylin-eosin staining, TUNEL assay, and western blot. MMP-9 activity was evaluated by gelatin zymography. Results The expression of Col I, Col III, CD147, and MMP-9 was higher, the expression of TIMP-1 was lower, and the maximal rates of left ventricular pressure rise and fall (+dp/dtmax and -dp/dtmax, respectively) were lower in model than control rats. The expression of CD147, MMP-9, Col I, and Col III was lower, the expression of TIMP-1 was higher, and the +dp/dtmax and -dp/dtmax were higher in the icariin than model group. The apoptosis rate was lower in the icariin and icariin + CD147 groups than control group. Conclusion Icariin attenuated myocardial apoptosis following myocardial infarction by apoptosis rate reduction and CD147/MMP-9 pathway inhibition.

Yeast culture dietary supplementation modulates gut microbiota, growth and biochemical parameters of grass carp.

Gut microbiota contributes positively to the physiology of their host. Some feed additives have been suggested to improve livestock health and stimulate growth performance by modulating gut bacteria species. Here, we fed grass carp with 0 (control), 8% (Treat1), 10% (Treat2), 12% (Treat3) and 16% (Treat4) of yeast culture (YC) for 10 weeks. The gut microbiota was analysed by 16S rRNA gene V3-4 region via an Illumina MiSeq platform. PCoA test showed that gut bacterial communities in the control and Treat3 formed distinctly separate clusters. Although all the groups shared a large size of OTUs as a core microbiota community, a strong distinction existed at genus level. Treat3 contained the highest proportion of the beneficial bacteria and obviously enhanced the capacity of amino acid, lipid metabolism and digestive system. In addition, Treat3 significantly improved the fish growth and increased the liver and serum T-SOD activities while dramatically decreased the liver GPT and GOT. Collectively, these findings demonstrate the beneficial effects of YC feeding on gut microbiota, growth and biochemical parameters and Treat3 might be the optimal supplementation amount for grass carp, which opens up the possibility that a new feed additive can be developed for healthy aquaculture.

Supplementation with dietary linseed oil during peri-puberty stimulates steroidogenesis and testis development in rams.

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs), such as α-linolenic acid (ALA), eicosapentaenoic acid, and docosahexaenoic acid, are involved in male reproductive function. In this study, we investigated the effects of linseed oil (LO) as a source of ALA on the steroidogenesis and changes of testicular histology in rams. Sixteen 3-month old rams during peri-puberty were randomly assigned into two groups. Eight rams were assigned as the control group, and the other received LO (4% dry matter of total diet) as the LO treatment group. After an 81-day feeding trial, the rams were slaughtered and investigated. Results revealed that compared with control group, diet containing LO did not affect body weight (36.87 ± 0.53 kg vs. 37.65 ± 0.64 kg, respectively; P = 0.361), average daily gain (227.47 ± 5.82 g vs. 237.95 ± 9.22 g, respectively; P = 0.353) and epididymis weight (40.77 ± 4.41 g vs. 45.53 ± 4.01 g, respectively; P = 0.398), however, it up-regulated PUFAs metabolism and steroidogenesis-related genes mRNA expression (P < 0.05), and increased plasma estradiol concentration (14.88 ± 0.67 pg/mL vs. 19.50 ± 1.27 pg/mL, respectively; P < 0.05). Therefore, LO stimulated seminiferous tubule development and increased the number of Sertoli cells (19.17 ± 2.14 vs. 27.2 ± 2.39, respectively; P < 0.01), germ-cell layers, as well as testis weight (148.65 ± 22.66 g vs. 249.96 ± 30.63 g, respectively; P < 0.05). All these results suggested that LO can improve testis development during peri-puberty by regulating steroidogenesis in rams' testes.

Comparative analysis of two ferritin subunits from blunt snout bream (Megalobrama amblycephala): Characterization, expression, iron depriving and bacteriostatic activity.

Iron is an essential microelement for almost all living organisms, while an excess of iron is toxic, thus maintenance of iron homeostasis is vital. As iron storage protein, ferritin plays an important role in iron metabolism. In the present study, we cloned and characterized the ferritin H subunit from Megalobrama amblycephala, termed as MamFerH. An iron-responsive element (IRE) was predicted in the 5' untranslated region (UTR) of MamFerH, while its bulge structural was different from that of the reported ferritin M subunit (MamFerM). The MamFerH and MamFerM genes exhibited similar expression patterns during early development with specifically high expression post hatching, whereas their tissue expression patterns were different. Specifically, MamFerM was highly expressed in the spleen, liver and kidney, while MamFerH was predominantly expressed in the blood and brain, indicating their different functions. In addition, the expression of the two genes was induced upon Aeromonas hydrophila infection at both transcriptional and translational levels, and MamFerH was more efficient. Immunohistochemistry and immunofluorescence analysis confirmed their significant changes at protein level and distribution in the liver post infection, indicating their participation in host immune response. Furthermore, bacteriostatic experiment revealed that recombinant MamFerH displayed more significant inhibitory effect on the growth of A. hydrophila.

Characterization and expression analysis of an intelectin gene from Megalobrama amblycephala with excellent bacterial binding and agglutination activity.

Intelectin is a recently discovered lectin that plays vital roles in the innate immune response, iron metabolism and early embryogenesis. The structure, expression pattern and function of intelectin in mammals and amphibians have been well studied, while not well known in fish. In this study, we cloned a intelectin (MamINTL) gene from blunt snout bream (Megalobrama amblycephala), examined its expression patterns and explored its roles in innate immune response. The MamINTL cDNA encoded 312 amino acids, with a pro-protein of 34 kDa. Sequence analysis revealed the presence of a fibrinogen-related domain and eight conserved cysteine residues in the MamINTL. The MamINTL mRNA was detectable at various developmental stages, while it increased significantly post hatching. In healthy adult M. amblycephala, MamINTL was detected in various tissues with the highest expression in the liver. Upon challenge with Aeromonas hydrophila, significantly up-regulated expression of the MamINTL mRNA was observed in the liver, spleen, kidney, intestine and gill. In addition, increased level of MamINTL protein detected by Western Blotting was also observed in the liver, kidney and spleen, indicating the participation of MamINTL in the immune response. Immunohistochemistry analysis of the M. amblycephala liver sections showed significant changes in expression and location post infection. In addition, the recombinant MamINTL showed excellent binding and agglutination activity against GFP-expressed E. coli in a Ca2+-dependent manner. Generally, the present study provides clues for a better understanding of the characterization, expression patterns and functions of fish intelectins.

Rosiglitazone Promotes Bone Marrow Adipogenesis to Impair Myelopoiesis under Stress.

OBJECTIVE: The therapeutic use of thiazolidinediones (TZDs) causes unwanted hematological side effects, although the underlying mechanisms of these effects are poorly understood. This study tests the hypothesis that rosiglitazone impairs the maintenance and differentiation of hematopoietic stem/progenitor cells, which ultimately leads to hematological abnormalities. METHODS: Mice were fed a rosiglitazone-supplemented diet or a normal diet for 6 weeks. To induce hematopoietic stress, all mice were injected once with 250 mg/kg 5-fluorouracil (5-Fu) intraperitoneally. Next, hematopoietic recovery, hematopoietic stem/progenitor cells (HSPCs) subsets, and myeloid differentiation after 5-Fu treatment were evaluated. The adipogenesis induced by rosiglitazone was assessed by histopathology and oil red O staining. The effect of adipocytes on HSPCs was studied with an in vitro co-culture system. RESULTS: Rosiglitazone significantly enhanced bone marrow adipogenesis and delayed hematopoietic recovery after 5-Fu treatment. Moreover, rosiglitazone inhibited proliferation of a granulocyte/monocyte progenitor (GMP) cell population and granulocyte/macrophage colony-stimulating factor (GM-CSF) colonies, although the proliferation and mobilization of Lin-c-kit+Sca-1+ cells (LSK) was maintained following hematopoietic stress. These effects could be partially reversed by the selective PPARγ antagonist BADGE. Finally, we demonstrated in a co-culture system that differentiated adipocytes actively suppressed the myeloid differentiation of HSPCs. CONCLUSION: Taken together, our results demonstrate that rosiglitazone inhibits myeloid differentiation of HSPCs after stress partially by inducing bone marrow adipogenesis. Targeting the bone marrow microenvironment might be one mechanism by which rosiglitazone impairs stress-induced hematopoiesis.

Inhibition of Glycolate Oxidase With Dicer-substrate siRNA Reduces Calcium Oxalate Deposition in a Mouse Model of Primary Hyperoxaluria Type 1.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, metabolic disorder caused by mutations of alanine-glyoxylate aminotransferase (AGT), a key hepatic enzyme in the detoxification of glyoxylate arising from multiple normal metabolic pathways to glycine. Accumulation of glyoxylate, a precursor of oxalate, leads to the overproduction of oxalate in the liver, which accumulates to high levels in kidneys and urine. Crystalization of calcium oxalate (CaOx) in the kidney ultimately results in renal failure. Currently, the only treatment effective in reduction of oxalate production in patients who do not respond to high-dose vitamin B6 therapy is a combined liver/kidney transplant. We explored an alternative approach to prevent glyoxylate production using Dicer-substrate small interfering RNAs (DsiRNAs) targeting hydroxyacid oxidase 1 (HAO1) mRNA which encodes glycolate oxidase (GO), to reduce the hepatic conversion of glycolate to glyoxylate. This approach efficiently reduces GO mRNA and protein in the livers of mice and nonhuman primates. Reduction of hepatic GO leads to normalization of urine oxalate levels and reduces CaOx deposition in a preclinical mouse model of PH1. Our results support the use of DsiRNA to reduce liver GO levels as a potential therapeutic approach to treat PH1.

[Changed accumulation of active ingredient in different localities and growth period of Hemsleya zhejiangensis (Cucurbitaceae)].

In this paper, the content of moisture, ethanol-soluble extractives, total saponins and polysaccharide of different tuber samples of Hemsleya zhejiangensis, from different localities, years and seasons, were detected based upon Chinese Pharmacopoeia 2010 version. The samples of roots, stems and leaves in summer were detected as well. The results are mainly as follows. (1)With tuber quality increasing, the content of total saponins increased and then decreased. The individual quality of tubers getting 594.06 g, the content of total saponins reached the peak. (2) The content of active ingredients in different localities was significantly different, and the population of Wuyanling had the maximum content of total saponins and polysaccharide. (3) The content of active ingredients revealed stability between the years 2012 and 2013, but the content of polysaccharide was significantly different. The content in 2012 was higher than that of 2013. (4) The content of active ingredients reached the peak in autumn, which was the best harvest season. (5) Among different component content detection of nutritional organs, tubers had the maximum content of ethanol-soluble extractives, total saponins and polysaccharide. Leaves also contained higher content of ethanol-soluble extractives and total saponins than roots and stems. All of these provide theoretical basis for plant, harvest and production of H. zhejiangensis, which is an endemic, rare, and endangered medicinal plants.