Dietary Supplementation with Bupleuri Radix Reduces Oxidative Stress Occurring during Growth by Regulating Rumen Microbes and Metabolites.

Oxidative stress (OS) in ruminants is closely associated with disease; thus, improving antioxidant capacity is an important strategy for maintaining host health. Bupleuri Radix (BR) could significantly improve host health and stress levels. However, the clear antioxidant mechanism of the function of BR remains unknown. In the current study, LC-MS metabolomics combined with 16S rRNA gene sequencing was employed to explore the effects of BR on rumen microbiota and metabolites in Shanbei Fine-Wool Sheep (SFWS), and Spearman correlation analyses of rumen microbiota, metabolites, and OS were performed to investigate the mechanism of antioxidant function of BR. Our results indicated that as SFWS grows, levels of OS and antioxidant capacity increase dramatically, but providing BR to SFWS enhances antioxidant capacity while decreasing OS. Rumen microbiota and OS are strongly correlated, with total antioxidant capacity (T-AOC) showing a significant negative correlation with Succiniclasticum and a positive correlation with Ruminococcus. Importantly, the Chao1 index was significantly negatively correlated with malondialdehyde (MDA) and positively correlated with superoxide dismutase (SOD) and T-AOC. Two biomarkers connected to the antioxidant effects of BR, 5,6-DHET and LPA (a-25:0/0:0), were screened according to the results of metabolomics and Spearman analysis of rumen contents, and a significant relationship between the concentration of rumen metabolites and OS was found. Five metabolic pathways, including glycerolipid, glutathione, nucleotide, D-amino acid, and inositol phosphate metabolism, may have a role in OS. The integrated results indicate that rumen microbiota and metabolites are strongly related to OS and that BR is responsible for reducing OS and improving antioxidant capacity in post-weaned SFWS. These findings provide new strategies to reduce OS occurring during SFWS growth.

Quercetin inhibits the amphiregulin/EGFR signaling-mediated renal tubular epithelial-mesenchymal transition and renal fibrosis in obstructive nephropathy.

Quercetin is a widely distributed, bioactive flavonoid compound, which displays potential to inhibit fibrosis in several diseases. The purpose of our study was to determine the effect of quercetin treatment on renal fibrosis and investigate the mechanism. Human proximal tubular epithelial cells (HK-2) stimulated by transforming growth factor-ß1 (TGF-ß1) and a rat model of unilateral ureter obstruction (UUO) that contributes to fibrosis were used to investigate the role and molecular mechanism of quercetin. PD153035 (N-[3-Bromophenyl]-6,7-dimethoxyquinazolin-4-amine) was used to inactivate EGFR (epidermal growth factor receptor). The level of fibrosis, proliferation, apoptosis, and oxidative stress in HK-2 were measured. All data are presented as means ± standard deviation (SD). p-value < .05 was considered statistically significant. In UUO rats, quercetin reduced the area of fibrosis as well as inflammation, oxidative stress, and cell apoptosis. In cultured HK-2 cells, quercetin significantly ameliorated the EMT induced by TGF-ß1, which was accompanied by increased amphiregulin (AREG) expression. Moreover, quercetin inhibited AREG binding to the EGFR receptor, thereby further affecting other downstream pathways. Quercetin may alleviate fibrosis in vitro and in vivo by inhibiting the activation of AREG/EGFR signaling indicating a potential therapeutic effect of quercetin in renal fibrosis.

Analysis of the genetic diversity and population structure of Monochasma savatieri Franch. ex Maxim using novel EST-SSR markers.

BACKGROUND: Monochasma savatieri Franch. ex Maxim is a medicinally valuable herb. However, the collection and protection of the wild germplasm resources of M. savatieri are still insufficient, and their genetic diversity and population structure have been poorly studied. RESULTS: We collected and examined 46 M. savatieri individuals from Fujian, Hunan, Jiangxi, and Zhejiang provinces for genetic diversity and population structure, using 33 newly developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. Applying these markers, we detected a total of 208 alleles, with an average of 6.303 alleles per locus. The polymorphic information content varied from 0.138 to 0.884 (average: 0.668), indicating a high level of polymorphism. At the population level, there was a low degree of genetic diversity among populations (I = 0.535, He = 0.342), with Zhejiang individuals showing the highest genetic diversity among the four populations (Fst = 0.497), which indicated little gene flow within the M. savatieri populations (Nm = 0.253). Mantel test analysis revealed a significant positive correlation between geographical and genetic distance among populations (R2 = 0.3304, p < 0.05), and structure and principal coordinate analyses supported classification of populations into three clusters, which was consistent with the findings of cluster analysis. CONCLUSIONS: As a rare medicinal plants, the protection of M. savatieri does not look optimistic, and accordingly, protective efforts should be beefed up on the natural wild populations. This study provided novel tools and insights for designing effective collection and conservation strategies for M. savatieri.

Glaucocalyxin A suppresses inflammatory responses and induces apoptosis in TNF-a-induced human rheumatoid arthritis via modulation of the STAT3 pathway.

The pathogenesis of rheumatoid arthritis (RA) is characterized by synoviocyte hyperplasia and proinflammatory cytokine secretion, as well as the destruction of cartilage and bone. Glaucocalyxin A (GLA) is an alkaloid derived from a Chinese medicinal plant that exhibits anti-inflammatory, anti-tumor and neuroprotective properties. We investigated the effects of GLA on RA-fibroblast-like synoviocytes (FLS cells), and collagen-induced arthritis (CIA), and further explored the underlying mechanisms. GLA inhibited TNF-a-induced RA-FLS proliferation, increased apoptotic ratios and upregulated levels of caspase-3, cleaved PARP, and Bax. GLA also inhibited the expression of IL-10, IL-1ß, and IL-6 in vitro. Levels of p-STAT3 were downregulated in a dose-dependent manner. Over-expression of STAT3 partly neutralized the GLA-mediated elevation of caspase-3 and cleaved PARP levels as well as the downregulation of IL-10, IL-1B and IL-6 expression levels. This suggests that GLA inactivated the STAT3 pathway. Furthermore, the production of inflammatory cytokines in RA-FLS and a CIA rat model were inhibited effectively by GLA. Taken together, our data suggest that GLA is a potential long-term therapeutic agent for patients with RA.

Serum protein complex profiling reveals heterogeneity of Balanced constitutional population in traditional Chinese medicine through blue native polyacrylamide gel electrophoresis.

BACKGROUND: Pulse-taking is widely used for diagnosis and treatment in traditional Chinese medicine (TCM), and protein complexes in serum perform various biological functions. The Balanced constitution is one of the major constitutions in TCM, people with Balanced constitution can also share some common characteristics with unbalanced constitution types. METHODS: Blue native polyacrylamide gel electrophoresis (BN-PAGE) was applied to the serum of 25 people with balanced constitutions. The patterns of the protein complexes could be recognized according to the number, molecular weight, and intensity of the gel bands. All of the individual bands from these patterns were cut and in-gel-digested with trypsin, followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis for protein identification and biological function analysis. RESULTS: The protein complex patterns were roughly categorized as type A and B with high stability and reproducibility, and there were 15 and 16 gel bands in type A and type B, respectively. Among the 25 serum samples, 14 belonged to type A, and 11 belonged to type B. High-abundance proteins significantly decreased from 99% to 44% after BN-PAGE separation. The unique proteins in type A were mainly related to lipid metabolism, while the unique proteins in type B were involved in biological processes related to immune response and inflammatory regulation. The Qi-deficiency constitution converted score of type A was higher than that of type B, while the Damp-heat constitution converted score of type A was lower than that of type B. CONCLUSIONS: Our study provided an objective reference for diagnosis and prognosis, which might lay a foundation for establishing the characteristic protein complex spectra of all of the TCM constitutions.

Effects of earthworms and effective microorganisms on the composting of sewage sludge with cassava dregs in the tropics.

The present study revealed the role of earthworm-effective microorganisms (EM) in converting sewage sludge and cassava dregs into a valuable product. Sewage sludge was toxic to earthworm, therefore it was mixed with cassava dregs in 80:20 proportions (dry weight). Treatments included mixed substrate inoculated versus not inoculated with EM and treated with or without earthworms. The pH, total organic carbon, total nitrogen, and C:N ratio decreased from the initial measurements in the range of 17.43-18.46%, 25.48-33.82%, 19.60-25.37%, and 6.68-14.05% respectively; but electrical conductivity and available phosphorus increased in the range of 113.47-158.16% and 42.42-57.58%, respectively. In addition, they interactively increased total phosphorus from 19.84-63.01% and potassium from 16.41-50.78%, and decreased the polycyclic aromatic hydrocarbons content of substrate from 21.17% to 32.14% with an increase in earthworms from 51.71 to 57.69, respectively. Earthworms and EM could be used together as an efficient method for co-composting sewage sludge plus cassava dregs in the tropics. This could be expected to result in stabilization of waste, increase in nutrients, and reduction of pollutant content. Implications: The first reports of interaction of earthworms and effective microorganisms in the treatment of sewage sludge and cassava dregs in the tropics. Co-composting was an efficient technology for treating sewage sludge and cassava dregs at the same time, in the tropics. The survival rate of the earthworms both> 95%, the highest number of cocoons (640.33) and hatchlings (4694.33) both in EW+EM (Earthworms added and EM inoculated) treatment. Earthworms and EM (Only EM inoculated) interactively increased total phosphorus and potassium content, and decreased the PAH content of substrate with increase in earthworms.

Traditional herbal medicine-derived sulforaphene promotes mitophagic cell death in lymphoma cells through CRM1-mediated p62/SQSTM1 accumulation and AMPK activation.

Sulforaphene (LFS-01) is the major chemical constituent of Raphanus sativus, a medicinal herb used for over a thousand years in traditional Chinese medicine. Here we identified that LFS-01 can selectively eradicate lymphoma cells while sparing normal lymphocytes by triggering concomitant mitophagy and apoptosis. We demonstrated that LFS-01 can retain Nrf2 in the nucleus by covalently modulating CRM1 and consequently upregulate p62/SQSTM1, an essential structural component of the autophagosomes during mitophagic process. We found that LFS-01 treatment also stimulated AMPK and thereby inhibited the mTOR pathway. On the contrary, we revealed that AMPK inhibition can severely impair the LFS-01-mediated mitophagy. Transcriptomic studies confirmed that 15 autophagy-associated genes such as p62/SQSTM1, VCP and BCL2 were differentially expressed after LFS-01 treatment. Furthermore, protein interactome network analysis revealed that the events of apoptosis and the assembly of autophagy vacuole were significant upon LFS-01 exposure. Lastly, we found that LFS-01 exhibited strong efficacy in xenograft mouse model yet with the lack of apparent toxicity to animals. We concluded that LFS-01 triggered mitophagic cell death via CRM1-mediated p62 overexpression and AMPK activation. Our findings provide new insights into the mechanism of action for LFS-01 and highlight its potential applications in treating major human diseases.

Effects of tussah immunoreactive substances on growth, immunity, disease resistance against Vibrio splendidus and gut microbiota profile of Apostichopus japonicus.

Tussah immunoreactive substance (TIS) comprises a number of active chemicals with various bioactivities. The current study investigated the effects of these substances on the sea cucumber Apostichopus japonicus. The specific growth rate (SGR) of TIS-fed sea cucumbers was significantly enhanced, whereas no significant difference in SGR was observed between those soaked in antibiotics and those fed with basal diet only. TIS also improved the immune response of the animals when given at a dose of 1.0% or 2.0%, as shown by increased phagocytic, lysozyme, superoxide dismutase, alkaline phosphatase, acid phosphatase, and catalase activities following injection with live Vibrio splendidus. At a dose of 1.0% or 2.0%, TIS significantly enhanced the immune ability (P < 0.05) of the sea cucumbers, but except for lysozyme activity, other immune indices were reduced one day after the animals were injected with Vibrio splendidus. However, the values of these immune indexes were still significantly higher compared to those of the control groups (P < 0.05). Intestinal micro flora counts and high-throughput sequencing showed that dietary TIS could improve the amount of probiotic bacteria, yielding a 6-fold increase in Bacillus and 10-fold increase in Lactobacillus for sea cucumbers fed with 2.0% TIS diet compared to the control. Furthermore, TIS-containing diet also greatly reduced the number of harmful bacteria, with the number of Vibrio in sea cucumbers fed with 1%TIS diet decreased by 67% compared to the control. The results thus indicated that TIS increased the growth of sea cucumbers and enhanced their resistance to V. splendidus infection by improving the immunity of the animals. TIS also improved the gut microbiota profiles of the animals by increasing the probiotics and reducing the harmful bacteria within their guts.

Effects of dietary cadmium on growth, antioxidants and bioaccumulation of sea cucumber (Apostichopus japonicus) and influence of dietary vitamin C supplementation.

The effects of dietary cadmium (Cd) supplementation on growth, antioxidant capacity and accumulation of Cd in tissues (body wall, digestive tracts, and respiratory tree) of sea cucumber, Apostichopus japonicus, exposed to sub-chronic concentrations (0, 10, 50, 100, and 500mg Cd/kg dry weight) of Cd were investigated. In addition, the potential protective effects of vitamin C (L-ascorbic acid, AsA) against the effects of Cd on sea cucumbers were investigated. Sea cucumbers were exposed to dietary Cd for 30 days, after which another group of healthy sea cucumbers was supplied diet supplemented with mixed Cd and AsA for another 30 days. Cd exposure for 30 days resulted in increased Cd accumulation in tissues of sea cucumbers with exposure time and concentration. The order of Cd accumulation in organs was digestive tracts>respiratory tree>body wall. On day 30, the body weight gain (BWG) and specific growth rate (SGR) decreased significantly (P<0.05) in the 500mg Cd/kg treatment. Superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity and catalase (CAT) activity in the coelomic fluid of sea cucumbers decreased with increasing dietary Cd concentration, but malondialdehyde (MDA) content in the coelomic fluid increased. Providing diet supplemented with Cd and AsA indicated that although sea cucumbers exhibited signs of Cd toxicity, no death occurred in response to 50mg Cd/kg for 30 days. Based on these findings, five treatments were provided: 50mg Cd/kg+0mg AsA/kg, 50mg Cd/kg+ 3000mg AsA/kg, 50mg Cd/kg+ 5000mg AsA/kg, 50mg Cd/kg+10,000mg AsA/kg, and 50mg Cd/kg+15,000mg AsA/kg. The BWG and SGR of sea cucumbers fed the AsA supplemented diet mixed with Cd increased. Additionally, MDA levels in coelomic fluid were negatively correlated with dietary AsA levels, while antioxidant capacities (SOD, GSH-Px and CAT) were positively correlated with dietary AsA levels. Moreover, Cd accumulation in tissues decreased in response to dietary AsA supplementation of treatments. Overall, antioxidant capacity and bioaccumulation in sea cucumber was found to decrease and be induced in response to Cd, but vitamin C mitigated these effects, with 5000mg AsA/kg providing the optimum protection against 50mg/kg Cd.

Discovery of serum proteomic biomarkers for prediction of response to moxibustion treatment in rats with collagen-induced arthritis: an exploratory analysis.

OBJECTIVE: To examine the possible impact of moxibustion on the serum proteome of the collagen-induced arthritis (CIA) rat model. MATERIALS AND METHODS: Thirty-six male Sprague-Dawley rats were included in this experiment. The CIA animal model was prepared by injection of type II bovine collagen in Freund's adjuvant on the first and seventh day. The 36 rats were randomly divided into two groups: the untreated CIA group (control), and the CIA plus treatment with moxibustion (CIA+moxi) group. Moxibustion was administered daily at ST36 and BL23 for 7, 14 or 21 days (n=12 rats each). Arthritis score was used to assess the severity of arthritis. At the end of each 7 day treatment, blood samples from the control group and the CIA+moxi group were collected. After removal of high abundance proteins from serum samples, two-dimensional gel combined with matrix-assisted laser desorption ionisation time-of-flight MS/MS (MALDI-TOF-MS/MS) techniques were performed to examine serum protein expression patterns of the CIA rat model with and without moxibustion treatment. In addition, the relevant proteins were further analysed with the use of bioinformatics analysis. RESULTS: Moxibustion significantly decreased arthritis severity in the rats in the CIA+moxi group, when compared with the rats in the CIA group 35 days after the first immunisation (p=0.001). Seventeen protein spots which changed >1.33 or <0.77 at p<0.05 using Bonferonni correction for multiple testing were found to be common to all three comparisons, and these proteins were used for classification of functions using the Gene Ontology method. Consequently, with the use of the Ingenuity Pathway Analysis, the top canonical pathways and a predicted proteomic network related to the moxibustion effect of CIA were established. CONCLUSIONS: Using the proteomics technique, we have identified novel candidate proteins that may be involved in the mechanisms of action underlying the beneficial effects of moxibustion in rats with CIA. Our findings suggest that immune responses and metabolic processes may be involved in mediating the effects of moxibustion. Moreover, periodxiredoxin I (PRDX1) and inositol 1,4,5-triphosphate receptor (IP3R) may be potential targets.

The effects of dietary lead on growth, bioaccumulation and antioxidant capacity in sea cucumber, Apostichopus japonicus.

Three different diets amended with lead nitrate [Pb(NO3)2] (100, 500 and 1000mg Pb/kg dry weight) and a Pb-free control diet (1.03mg Pb/kg dry weight) were fed to sea cucumber (Apostichopus japonicus) for 30 days. The patterns of Pb accumulation over time were determined in various tissues (body wall, intestine and respiratory tree), as well as growth performance and antioxidant enzymes activities. Pb accumulation in body wall and intestine increased with time in all dietary Pb treatments. When fed the highest Pb diet, the body wall exhibited the greatest Pb burden (16.37mg Pb/kg tissue wet weight), while Pb content in the intestine (2.68mg Pb/kg tissue wet weight) and the respiratory tree (1.78mg Pb/kg tissue wet weight) were lower than Pb content in the body wall by day 30. The body weight gain (BWG), specific growth rate (SGR) and survival rate (SR) had not been affected by 30 days oral administration of Pb supplemented diet. However, the antioxidant enzymes activities [superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)] of test groups were lower than control group in body wall and malondialdehyde (MDA) concentration in the body wall was opposite after 30 days in sea cucumbers. In summary, this work reports toxic effects in sea cucumber, A. japonicus, after dietary exposure to Pb.

Polycyclic aromatic hydrocarbons in surface sediment from Yangpu Bay, China: Distribution, sources and risk assessment.

The study investigated the occurrence of polycyclic aromatic hydrocarbons (PAHs) in the surface sediment from eleven sites in Yangpu Bay, China in December 2013 (winter) and July 2014 (summer). The 16 US EPA priority PAHs were found in the range of 1583.2-5701.7 ng/g dry weights with an average of 3134.7 ± 1241.3 ng/g in winter and ranged from 2161.8 to 4527.2 ng/g with an average of 3016.6 ± 748.0 ng/g in summer, respectively. The concentrations of the PAHs tended to be relatively high in comparison with other areas from the literatures. The identification using molecular indices analysis indicated that the PAHs originated mainly from pyrogenic and petrogenic sources in most of the sites. According to principle component analysis-multiple linear regression (PCA/MLR) for their source apportionment, the main sources of PAHs were vehicle emissions, petroleum products and biomass combustion. The risk assessment using international sediments quality guidelines and sediments quality criteria indicated that several PAHs, such as Nap, Flu, Phe, Ace, Acy and BghiP in most of the sites would potentially affect organisms in Yangpu Bay.

Effects of dietary vitamin C supplementation on lead-treated sea cucumbers, Apostichopus japonicus.

This study aimed to determine the effects of dietary supplementation with vitamin C (l-ascorbic acid, AsA) on lead (Pb) accumulation and toxicity in sea cucumber, Apostichopus japonicus. Three hundred sea cucumbers (10.02±0.02g) fed a basal diet containing 100mg Pb/kg were subjected to 5 levels of l-ascorbate-2-polyphosphate (LAPP) supplementation to achieve 5 AsA treatment levels of 0, 2727, 4630, 9171, 13,893mg AsA/kg. After 30 days, specific growth rate (SGR) and body weight gain (BWG) of supplementation groups were significantly (P<0.05) higher than the control group. Pb contents of the intestine significantly (P<0.05) decreased from 20 days to 30 days in all supplementation groups. Pb contents of the respiratory tree in sea cucumbers in the 2727 and 4630mg AsA/kg treatment groups significantly (P<0.05) decreased from 10 days to 30 days. Superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity and total antioxidant capacity (T-AOC) of the sea cucumber body wall in supplementation groups increased after 30 days. Malondialdehyde (MDA) content of the body wall significantly (P<0.05) declined with increasing AsA level. Therefore, it can be demonstrated that vitamin C could mitigate the effects of Pb on sea cucumber and the optimum levels ranged from 2727mg AsA/kg to 4630mg AsA/kg when Pb levels were 100mg/kg.

[Differential serum proteomic analysis of rheumatoid arthritis patients of cold-dampness arthralgia spasm syndrome].

OBJECTIVE: To preliminarily study the essence of rheumatoid arthritis (RA) patients of cold-dampness arthralgia spasm syndrome (CDASS) at the protein expression level. METHODS: Totally 24 RA patients were recruited from Department of Rheumatology, Affiliated Hospital of Nanjing University of Chinese Medicine from July 2009 to September 2010. They were assigned to the CDASS group and the dampness-heat arthralgia spasm syndrome (DHASS) group according to Chinese medicine syndrome typing, 12 in each group. The normal control group consisted of 12 healthy volunteers from the Health Examination Center, Affiliated Hospital of Nanjing University of Chinese Medicine. The serum proteins were compared between the CDASS group and the normal control group/the DHASS group respectively using two-dimensional gel electrophoresis. The common differential protein spots of CDASS were analyzed by mass spectrometry. The SwissProt database was inquired using Mascot Software to identify differential proteins. RESULTS: There were 81 differential protein spots between the CDASS group and the normal control group. There were 45 differential protein spots between the CDASS group and the DHASS group. Thirteen protein spots were found to be higher or lower in protein expression quantity of the CDASS group when compared with those of the other two groups. Nine differential protein spots were identified by mass spectrometry and database retrieval. It's suggested that these proteins were most likely to be related with inhibition of cellular events, such as cell proliferation, cell differentiation, and so on. CONCLUSION: 4.1 protein and DLC-1 protein were of potential significance in the diagnosis, prognostic markers, or treatment targets of RA patients of CDASS, which also provided evidence for further studies on the essence of CDASS.

[Establishment of a cell-based high-throughput screening model for PPARdelta agonists].

To establish a new high-throughput screening model for the agonist of PPARdelta, PPARdelta gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and subcloned to pGEM-T Vector for sequencing, then the PPARdelta fragment was excised by restriction enzymes, and inserted into pTARGET Vector to construct expression vector pTARGET-ppARdelta. Insert three copies of PPRE into pGl3-promoter vector to construct expression vector pGl3-PPRE x 3-luc. The vector pTARGET-ppARdelta was transiently cotransfected with pGl3-PPRE x 3-luc into different cell lines to assay the expression levels of luciferase. The PPARdelta agonist screening model was established and optimized. Bezafibrate and linoleic acid can induce the expression of luciferase significantly and in a dose-dependent manner. This method can be used for high throughput screening for the agonist of PPARdelta, which might become lead compounds for new anti-atheroscleriosis or anti-adiposity drugs.