[Metabolomics of interventional effects of Coptidis Rhizoma and its processed products on oral ulcer due to excess heat in rats].

Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF-MS) was employed to examine the impact of Coptidis Rhizoma(CR) and its processed products on the metabolism in the rat model of oral ulcer due to excess heat and to compare the effectiveness of CR and its three products. Male SD rats were randomly allocated to the sham-operation(Sham), model(M, oral ulcer due to excess heat), CR, wine/Zingiberis Rhizoma Recens/Euodiae Fructus processed CR(wCR/zCR/eCR), and Huanglian Shangqing Tablets(HST) groups. Except the Sham group, the other groups were administrated with Codonopsis Radix-Astragali Radix decoction by gavage for two consecutive weeks. The anal temperature and water consumption of rats were monitored throughout the modeling period of excess heat. Following the completion of the modeling, oral ulcer was modeled with acetic acid. Hematoxylin-eosin(HE) staining was employed to observe the mucosal pathological changes in oral ulcer. A colorimetric assay was employed to determine the serum level of glutathione peroxidase(GSH-Px). Enzyme-linked immunosorbent assay(ELISA) was conducted to determine the levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), interleukin-1ß(IL-1ß), superoxide dismutase(SOD), and malondialdehyde(MDA) in the serum. The non-targeted metabolomics analysis based on UPLC-Q/TOF-MS was conducted on the serum samples. Metabolic profiles were then built, and the potential biomarkers were screened by principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA). The Mev software was used to establish a heat map and conduct cluster analysis on the quantitative results of the markers. The online databases including MBRole, KEGG, and MetaboAnalyst were used for pathway enrichment analysis and metabolic network building. The experimental results showed that the modeling led to pathological damage to the oral mucosa, elevated serum levels of TNF-α, IL-6, IL-1ß, and MDA, and lowered levels of SOD and GSH-Px in rats. The drug administration recovered all the indices to varying extents, and wCR exhibited the best performance. Non-targeted metabolomics identified 48 differential metabolites including 27 metabolites in the positive ion mode and 21 metabolites in the negative ion mode. Five enriched pathways were common, including glycerophospholipid metabolism, linoleic acid metabolism, and tyrosine metabolism. Conclusively, CR and its three processed products could alleviate the inflammation and oxidative stress injury in rats suffering from oral ulcers due to excess heat by regulating lipid and amino acid metabolism. Notably, wCR demonstrated the most significant therapeutic effect.

Manganese Intoxication Recovery and the Expression Changes of Park2/Parkin in Rats.

Occupational overexposure to manganese (Mn) produces Parkinson's disease-like manganism. Acute Mn intoxication in rats causes dopaminergic neuron loss, impairment of motor activity and reduction of the expression of Park2/Parkin. The expression of Park2/Parkin is also reduced. Whether these changes are reversible after cessation of Mn exposure is unknown, and is the goal of this investigation. Adult male rats were injected with Mn2+ at doses 1 mg/kg and 5 mg/kg in the form of MnCl2·4H2O, every other day for one-month to produce acute Mn neurotoxicity. For a half of rats Mn exposure was suspended for recovery for up to 5 months. Mn neurotoxicity was evaluated by the accumulation of Mn in blood and brain, behavioral activities, dopaminergic neuron loss, and the expression of Park2/Parkin in the blood cells and brain. Dose-dependent Mn neurotoxicity in rats was evidenced by Mn accumulation, rotarod impairments, reduction of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra, decreased level of Park2 mRNA in the blood and brain, and decreased Parkin protein in the brain. After cessation of Mn exposure, the amount of Park2 mRNA in the blood started to increase one month after the recovery. After 5-month of recovery, blood and brain Mn returned to normal, rotarod activity recovered, the reduction of TH-positive dopaminergic neurons ameliorated, and the level of Park2 mRNA in the blood and Park2/Parkin in the midbrain and striatum were returned to the normal. Mn neurotoxicity in rats is reversible after cessation of Mn exposure. The level of Park2 mRNA in the blood could be used as a novel biomarker for Mn exposure and recovery.

Acrylamide biodegradation ability and plant growth-promoting properties of Variovorax boronicumulans CGMCC 4969.

Species of the genus Variovorax are often isolated from nitrile or amide-containing organic compound-contaminated soil. However, there have been few biological characterizations of Variovorax and their contaminant-degrading enzymes. Previously, we reported a new soil isolate, Variovorax boronicumulans CGMCC 4969, and its nitrile hydratase that transforms the neonicotinoid insecticide thiacloprid into an amide metabolite. In this study, we showed that CGMCC 4969 is able to degrade acrylamide, a neurotoxicant and carcinogen in animals, during cell growth in a mineral salt medium as well as in its resting state. Resting cells rapidly hydrolyzed 600 mg/L acrylamide to acrylic acid with a half-life of 2.5 min. In in vitro tests, CGMCC 4969 showed plant growth-promoting properties; it produced a siderophore, ammonia, hydrogen cyanide, and the phytohormone salicylic acid. Interestingly, in soil inoculated with this strain, 200 mg/L acrylamide was completely degraded in 4 days. Gene cloning and overexpression in the Escherichia coli strain Rosetta (DE3) pLysS resulted in the production of an aliphatic amidase of 345 amino acids that hydrolyzed acrylamide into acrylic acid. The amidase contained a conserved catalytic triad, Glu59, Lys 134, and Cys166, and an "MRHGDISSS" amino acid sequence at the N-terminal region. Variovorax boronicumulans CGMCC 4969, which is able to use acrylamide for cell growth and rapidly degrade acrylamide in soil, shows promising plant growth-promoting properties. As such, it has the potential to be developed into an effective Bioaugmentation strategy to promote growth of field crops in acrylamide-contaminated soil.

Biphasic Dose-Response of Mn-Induced Mitochondrial Damage, PINK1/Parkin Expression, and Mitophagy in SK-N-SH Cells.

Excessive manganese (Mn) exposure produces neurotoxicity with mitochondrial damage. Mitophagy is a protective mechanism to eliminate damaged mitochondria to protect cells. The aim of this study was to determine the dose-response of Mn-induced mitochondria damage, the expression of mitophagy-mediated protein PINK1/Parkin and mitophagy in dopamine-producing SK-N-SH cells. Cells were exposed to 0, 300, 900, and 1500 µM Mn2+ for 24 h, and ROS production, mitochondrial damage and mitophagy were examined. The levels of dopamine were detected by ELISA and neurotoxicity and mitophagy-related proteins (α-synuclein, PINK1, Parkin, Optineurin, and LC3II/I) were detected by western blot. Mn increased intracellular ROS and apoptosis and decreased mitochondrial membrane potential in a concentration-dependent manner. However, at the low dose of 300 µM Mn, autophagosome was increased 11-fold, but at the high dose of 1500 µM, autophagosome was attenuated to 4-fold, together with decreased mitophagy-mediated protein PINK1/Parkin and LC3II/I ratio and increased Optineurin expression, resulting in increased α-synuclein accumulation and decreased dopamine production. Thus, Mn-induced mitophagy exhibited a novel biphasic regulation: at the low dose, mitophagy is activated to eliminate damaged mitochondria, however, at the high dose, cells gradually loss the adaptive machinery, the PINK1/Parkin-mediated mitophagy weakened, resulting in neurotoxicity.

Coptis chinensis-Induced Changes in Metabolomics and Gut Microbiota in Rats.

Rhizoma coptidis (CR) is traditionally used for treating gastrointestinal diseases. Wine-processed CR (wCR), zingiber-processed CR (zCR), and evodia-processed CR (eCR) are its major processed products. However, the related study of their specific mechanisms is very limited, and they need to be further clarified. The aim of this study is to compare the intervening mechanism of wCR/zCR/eCR on rats via faecal metabolomics and 16S rDNA gene sequencing analysis. First, faecal samples were collected from the control and CR/wCR/zCR/eCR groups. Then, a metabolomics analysis was performed using UHPLC-Q/TOF-MS to obtain the metabolic profile and significantly altered metabolites. The 16S rDNA gene sequencing analysis was carried out to analyze the composition of gut microbiota and screen out the significantly altered microbiota at the genus level. Finally, a pathway enrichment analysis of the significantly altered metabolites via the KEGG database and a functional prediction of relevant gut microbes based on PICRUSt2 software were performed in combination. Together with the correlation analysis between metabolites and gut microbiota, the potential intervening mechanism of wCR/zCR/eCR was explored. The results suggested that wCR played a good role in maintaining immune homeostasis, promoting glycolysis, and reducing cholesterol; zCR had a better effect on protecting the integrity of the intestinal mucus barrier, preventing gastric ulcers, and reducing body cholesterol; eCR was good at protecting the integrity of the intestinal mucus barrier and promoting glycolysis. This study scientifically elucidated the intervening mechanism of wCR/zCR/eCR from the perspective of faecal metabolites and gut microbiota, providing a new insight into the processing mechanism research of Chinese herbs.

[Study on the changes of T-lymphocyte subsets in the patients with endometriosis].

OBJECTIVE: To investigate the function of T-lymphocyte subsets in patients with endometriosis. METHODS: The levels of interleukin-2 (IL-2) and interleukin-6 (IL-6) in the serum, and peritoneal fluid of 30 cases with endometriosis were detected using enzyme linked immunoabsorbent assay and compared with those of 20 non-endometriosis cases. The expression of IL-2 and IL-6 in ectopic endometrial tissue from the patients with endometriosis and the endometrial tissues of 10 normal women was investigated by immunohistochemistry method. RESULTS: Significantly elevated levels of IL-6 (cytokines of T help cell 2) were found in the serum and peritoneal fluid of patients with endometriosis (Median: 5.3 ng/L, 2.1 ng/L, P < 0.05) compared with that of non-endometriosis patients median: 2.5 ng/L, 0.9 ng/L). The level of IL-6 in the serum and peritoneal fluid of endometriosis patients in early stages (stages I, II) was 3.7 ng/L, 1.6 ng/L, significantly lower than those in advanced stages (stages III, IV) (13.6 ng/L, 4.1 ng/L) (P < 0.05). The ratio of IL-2/IL-6 in the serum and peritoneal fluid in patients with endometriosis (0.7, 1.1) was significantly lower than those in the control non-endometriosis group (0.8, 6.2, P < 0.05). The levels of IL-6 detected in the peritoneal fluid of patients with endometriosis had a positive correlation with those in the serum (r = 0.745, P < 0.01). The levels of IL-6 in serum and peritoneal fluid in the patients with endometriosis had a negative correlation with the IL-2/IL-6 ratios in the serum and peritoneal fluid respectively (r = -0.406, r = -0.480, P < 0.05). IL-2 and IL-6 were expressed in the interstitial cells of ectopic endometrial tissue, with an expression rate of 56.7%, and 60.0% respectively. There was significant difference in the expression of IL-2 and IL-6 between the ectopic endometrial tissue and normal endometrial tissue. CONCLUSIONS: The levels of IL-6 in the serum and peritoneal fluid of patients with endometriosis are increased, implying that IL-6 might play a role in the pathophysiology of endometriosis. The ratio of IL-2/IL-6 in the serum and peritoneal fluid was decreased in patients with endometriosis compared with the control group, suggesting shift of Th1 cell toward Th2 cell in patients with endometriosis. Stronger expression of IL-2 and IL-6 in the ectopic endometrial tissues may contribute to the disturbed immune regulation in patients with endometriosis.

Biotransformation of the neonicotinoid insecticide thiacloprid by the bacterium Variovorax boronicumulans strain J1 and mediation of the major metabolic pathway by nitrile hydratase.

A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analysis indicated the major pathway of thiacloprid (THI) metabolism by V. boronicumulans J1 involved hydrolysis of the N-cyanoimino group to form an N-carbamoylinino group containing metabolite, THI amide. Resting cells of V. boronicumulans J1 degraded 62.5% of the thiacloprid at a concentration of 200 mg/L in 60 h, and 98% of the reduced thiacloprid was converted to the final metabolite thiacloprid amide. A 2.6 kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an α-subunit of 213 amino acids and a ß-subunit of 221 amino acids. The nitrile hydratase gene was recombined into plasmid pET28a and overexpressed in Escherichia coli BL21 (DE3). The resting cells of recombinant E. coli BL21 (DE3)-pET28a-NHase with overexpression of nitrile hydratase transformed thiacloprid to its amide metabolite, whereas resting cells of the control E. coli BL21 (DE3)-pET28a did not. Therefore, the major hydration pathway of thiacloprid is mediated by nitrile hydratase.