Soluble epoxide hydrolase (sEH) has been identified as an attractive target for anti-inflammatory drug design in recent years. Picomolar level compound G1 against sEH was obtained by introducing the hydrophilic group homopiperazine and hydrophobic fragment propionyl onto the structure of lead compound A. G1 showed good microsomal stability, a moderate plasma protein binding rate, and good oral bioavailability and was well tolerated in rats. G1 has significant analgesic effects on CFA-induced AIA mice, ameliorated the pancreatic injury in acute pancreatitis induced by l-arginine, reversed pancreatic injury, edema, and neutrophil infiltration, and increased the survival time of C57BL/6 mice in a lipopolysaccharide (LPS)-induced sepsis model. Moreover the expression levels of sEH, COX-2, NOS-2, vascular cell adhesion molecule (VCAM), IL-6, MCP-5, and tumor necrosis factor alpha (TNF-alpha) were measured by Western blot or enzyme-linked immunosorbent assay (ELISA), with varying degrees of decrease. These results suggested that G1 is a drug candidate worthy of further evaluation for the treatment of inflammation-induced diseases such as arthritis, acute pancreatitis, and sepsis.
Plant tannases (TAs) or tannin acyl hydrolases, a class of recently reported carboxylesterase (CXE) in tannin-rich plants, are involved in the degalloylation of two important secondary metabolites: flavan-3-ol gallates and hydrolyzable tannins (HTs). In this paper, we have made a new progress on the function of Camellia sinensis (Cs) TA-it is a hydrolase with promiscuous acyltransferase activity in vitro and in vivo experiments and promotes the synthesis of simple galloyl glucoses and flavan-3-ols gallates in plants. We gained the new understanding to the functions of CsTA through enzyme analysis, protein mass spectrometry identification, metabolic analysis of plants by genetic modification. Firstly, CsTA was proved that it is not only a hydrolase but also an acyltransferase. In the two-step covalent catalytic reaction, when CsTA hydrolyzes the galloylated compounds epigallocatechin-3-gallate (EGCG) or 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) into their degalloylated forms, a long-lived Ser159-linked galloyl-enzyme covalent intermediate is also formed. Under nucleophilic attack, the galloyl group on the intermediate is transferred to the nucleophilic acyl acceptors (including water, methanol, flavan-3-ols and simple galloyl glucoses). Then, metabolic analysis suggested that transiently overexpression of TAs in young strawberry fruits, young leaves of tea plants and young leaves of Chinese bayberry actually increased the total content of simple galloyl glucoses and flavan-3-ol gallates. Overall, these findings provide new insights into the promiscuous acyltransferase activity of plant tannase.
Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom.
Plant tannins, including condensed tannins (CTs) and hydrolyzable tannins (HTs), are widely distributed in the plant kingdom. To date, tannase (TA) - is a type of tannin acyl-hydrolase hydrolyzing HTs, CT monomer gallates and depsides - has been reported in microbes only. Whether plants express TA remains unknown. Herein, we report plant TA genes. A native Camellia sinensis TA (CsTA) is identified from leaves. Six TAs are cloned from tea, strawberry (Fragariasxsananassa, Fa) and four other crops. Biochemical analysis shows that the native CsTA and six recombinant TAs hydrolyze tannin compounds, depsides and phenolic glycosides. Transcriptional and metabolic analyses reveal that the expression of CsTA is oppositely associated with the accumulation of galloylated catechins. Moreover, the transient overexpression and RNA interference of FaTA are positively associated with the accumulation of ellagitannins in strawberry fruit. Phylogenetic analysis across different kingdoms shows that 29 plant TA homologs are clustered as a plant-specific TA clade in class I carboxylesterases. Further analysis across the angiosperms reveals that these TA genes are dispersed in tannin-rich plants, which share a single phylogenetic origin c. 120 million yr ago. Plant TA is discovered for the first time in the plant kingdom and is shown to be valuable to improve tannin compositions in plants.
Title: Study on the Regulation of Earthworm Physiological Function under Cadmium Stress Based on a Compound Mathematical Model Zhou H, Zhang T, Zhuang J, Xu M, Liu X, Shi Q, Zhou D Ref: Environ Toxicol Pharmacol, :103499, 2020 : PubMed
A cadmium (Cd) stress test was carried out on Eisenia fetida in artificial soil. Six Cd concentration gradient solutions (0, 50, 100, 125, 250 and 500 mg/kg) were prepared. Two treatment groups, short-term stress and long-term stress, were established. The former lasted for 10 days, and the latter lasted for 30 days. The Biolog ECO-microplate culture method was used to determine the utilization of the 31 carbon sources by the microbes in earthworm homogenate. The total protein content (TP), peroxidase activity (POD), catalase activity (CAT), superoxide dismutase activity (SOD), glutathione peroxidase activity (GPX), glutathione-S-transferase activity (GST), malondialdehyde content (MDA) and acetylcholinesterase activity (AChE) in earthworm were determined in order to investigate the regulation of oxidative stress and the functional diversity of microbial communities in earthworms under Cd stress. By combining the entropy weight method (EW) and the technique for order preference by similarity to an ideal solution model (TOPSIS), the physiological functional indices of earthworms were assessed objectively and scientifically, and the physiological changes under the different stress periods were evaluated. The results showed that a Cd-tolerant dominant population appeared in the microbial community under Cd stress. In the short-term test, oxidative stress were more effective in coping with Cd stress than the microbial community, and oxidative stress regulated the microbial community functional diversity. Under long-term Cd stress, the regulatory effect was weak or non-existent. In this study, a new evaluation model was established to explore the regulation process of earthworm on its oxidation stress and the functional diversity of microbial communities under Cd stress, and provide a theoretical basis for revealing the detoxification mechanism of earthworms.
The lack of pharmaceutical antidotes for deadly toxicants has motivated tremendous research interests in seeking synthetic nanoscavengers to absorb and neutralize harmful biological or chemical agents. Herein, we report a cell-membrane-cloaked oil nanosponge formulation capable of dual-modal detoxification. The biomimetic oil nanosponge consists of an olive oil nanodroplet wrapped by a red blood cell membrane. In such a construct, the oil core can nonspecifically soak up toxicants through physical partition and the cell membrane shell can specifically absorb and neutralize toxicants through biological binding. The dual-modal detoxification capability of the oil nanosponges was validated using three distinct organophosphates (OPs), including paraoxon, diisopropyl fluorophosphate, and dichlorvos. By inhibiting acetylcholinesterase, OPs cause the accumulation of acetylcholine, which leads to neuromuscular disorders and even death. In mouse models of OP poisoning, the oil nanosponges reduced clinical signs of OP intoxication, lowered OP concentration in tissues, and greatly enhanced mouse survival in both the therapeutic regimen and the prophylactic regimen. Overall, oil nanosponges combine the merits of both cell membrane and oil nanodroplets for safe and effective detoxification, which also serve as a prototype of multimodal detoxification platforms.
Title: Selenepezil, a Selenium-Containing Compound, Exerts Neuroprotective Effect via Modulation of the Keap1-Nrf2-ARE Pathway and Attenuates Abeta-Induced Cognitive Impairment in Vivo Yan J, Pang Y, Zhuang J, Lin H, Zhang Q, Han L, Ke P, Huang X Ref: ACS Chem Neurosci, 10:2903, 2019 : PubMed
Oxidative stress is a major risk factor for neurodegenerative disease. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is one of the most potent defensive systems against oxidative stress. Selenepezil, a selenium-based compound, was previously found to exhibit excellent acetylcholinesterase (AChE) inhibition, to mimic endogenous glutathione peroxidase (GPx) activity, and to exhibit scavenging activity for hydrogen peroxide in vitro. However, none of these activities have been evaluated in a cellular model, and detailed molecular mechanisms are not elucidated. Moreover, whether selenepezil ameliorates memory deficits in vivo remains unknown. This study validated the cytoprotective effect of selenepezil against 6-hydroxydopamine (6-OHDA)- or H2O2-induced SH-SY5Y cell damage via alleviation or neutralization of intracellular microtubule disorder, reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and cell apoptosis. Our study clearly demonstrated that selenepezil pretreatment exhibited remarkable cytoprotective effect in a Nrf2-dependent manner via activating the Keap1-Nrf2-ARE pathway and stimulating the transcription of Nrf2-ARE-regulated cytoprotective genes. Moreover, selenepezil.HCl exerts neuroprotective effect via attenuating Abeta-induced cognitive impairment in Alzheimer's disease (AD) rat and was more active than the reference drug donepezil. In summary, selenepezil deserves further consideration for AD therapy.
Hepatic expression profiling has revealed miRNA changes in liver diseases, while hepatic miR-155 expression was increased in murine non-alcoholic fatty liver disease, suggesting that miR-155 might regulate the biological process of lipid metabolism. To illustrate the effects of miR-155 gain of function in transgenic mouse liver on lipid metabolism, transgenic mice (i.e., Rm155LG mice) for the conditional overexpression of mouse miR-155 transgene mediated by Cre/lox P system were firstly generated around the world in this study. Rm155LG mice were further crossed to Alb-Cre mice to realize the liver-specific overexpression of miR-155 transgene in Rm155LG/Alb-Cre double transgenic mice which showed the unaltered body weight, liver weight, epididymal fat pad weight and gross morphology and appearance of liver. Furthermore, liver-specific overexpression of miR-155 transgene resulted in significantly reduced levels of serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL), as well as remarkably decreased contents of hepatic lipid, TG, HDL and free fatty acid in Rm155LG/Alb-Cre transgenic mice. More importantly, microarray data revealed a general downward trend in the expression profile of hepatic genes with functions typically associated with fatty acid, cholesterol and triglyceride metabolism, which is likely at least partially responsible for serum cholesterol and triglyceride lowering observed in Rm155LG/Alb-Cre mice. In this study, we demonstrated that hepatic overexpression of miR-155 alleviated nonalcoholic fatty liver induced by a high-fat diet. Additionally, carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) was identified as a direct miR-155 target gene that is potentially responsible for the partial liver phenotypes observed in Rm155LG/Alb-Cre mice. Taken together, these data from miR-155 gain of function study suggest, for what we believe is the first time, the altered lipid metabolism and provide new insights into the metabolic state of the liver in Rm155LG/Alb-Cre mice.
Title: A critical role of acute bronchoconstriction in the mortality associated with high-dose sarin inhalation: Effects of epinephrine and oxygen therapies Gundavarapu S, Zhuang J, Barrett EG, Xu F, Russell RG, Sopori ML Ref: Toxicol Appl Pharmacol, 274:200, 2014 : PubMed
Sarin is an organophosphate nerve agent that is among the most lethal chemical toxins known to mankind. Because of its vaporization properties and ease and low cost of production, sarin is the nerve agent with a strong potential for use by terrorists and rouge nations. The primary route of sarin exposure is through inhalation and, depending on the dose, sarin leads to acute respiratory failure and death. The mechanism(s) of sarin-induced respiratory failure is poorly understood. Sarin irreversibly inhibits acetylcholine esterase, leading to excessive synaptic levels of acetylcholine and, we have previously shown that sarin causes marked ventilatory changes including weakened response to hypoxia. We now show that LD50 sarin inhalation causes severe bronchoconstriction in rats, leading to airway resistance, increased hypoxia-induced factor-1alpha, and severe lung epithelium injury. Transferring animals into 60% oxygen chambers after sarin exposure improved the survival from about 50% to 75% at 24h; however, many animals died within hours after removal from the oxygen chambers. On the other hand, if LD50 sarin-exposed animals were administered the bronchodilator epinephrine, >90% of the animals survived. Moreover, while both epinephrine and oxygen treatments moderated cardiorespiratory parameters, the proinflammatory cytokine surge, and elevated expression of hypoxia-induced factor-1alpha, only epinephrine consistently reduced the sarin-induced bronchoconstriction. These data suggest that severe bronchoconstriction is a critical factor in the mortality induced by LD50 sarin inhalation, and epinephrine may limit the ventilatory, inflammatory, and lethal effects of sarin.
Title: Effect of early feed restriction on hepatic lipid metabolism and expression of lipogenic genes in broiler chickens Yang X, Zhuang J, Rao K, Li X, Zhao R Ref: Res Vet Sci, 89:438, 2010 : PubMed
The study was conducted to investigate the effect of early feed restriction (ER) on lipid metabolism and mitochondrial function in the liver of broiler chickens. Newly hatched broiler chickens were randomly allocated into control and ER group which was subjected to feed restriction with feed provided on alternate days from hatch to 14 days of age (14 d), followed by ad libitum feeding until the end of the experiment on 63 d. ER group exhibited significantly lower body weight throughout the experiment. Serum concentrations of total cholesterol (TC) and high density lipoprotein cholesterol (HDLC) were significantly higher in ER group at 14 d (P<0.05), and the higher serum TC level in ER group was also observed at 63 d. In contrast, the contents of triglyceride (TG), TC and lipoprotein lipase (LPL) activity in liver were significantly lower in ER group at 14 d (P<0.05). At 14 d no significant difference was detected for the mRNA expression of the acetyl-CoA carboxylase-alpha (ACC-alpha), carnitine palmitoyltransferase I (CPT-I), sterol regulatory element binding protein-1c (SREBP-1c) or peroxisome proliferator-activated receptors alpha (PPAR-alpha) between control and ER group. At 63 d ACC-alpha mRNA expression was significantly down-regulated accompanied with a significantly up-regulated CPT-ImRNA and a decreased tendency of SREBP-1c mRNA expression in ER group (P=0.09). Swollen mitochondria with fragmented and reduced cristae were observed in liver of ER group at 14 d. Meanwhile the inner mitochondria membrane viscidity increased and hepatic mitochondrial superoxide dismutase (SOD) activity decreased at 14 d. The results suggest that feed restriction at early postnatal stage may produce long-term effect on lipid metabolism of broiler chicken, probably through, at least in part, alterations in mitochondria morphology and function.
Title: Effect of tanshinone on the levels of nitric oxide synthase and acetylcholinesterase in the brain of Alzheimer's disease rat model Yin Y, Huang L, Liu Y, Huang S, Zhuang J, Chen X, Zhang L, Wu H, Shao F, Zhao Z Ref: Clinical Investigation Med, 31:E248, 2008 : PubMed
PURPOSE: To determine the influence of tanshinone on the levels of nitric oxide synthase (NOS) and acetylcholinesterase (AChE) in the brain of an Alzheimer's Disease (AD) rat model and on its potential therapeutic mechanism. METHODS: 100 Male Sprague Dawley rats were divided into three groups: control group, model group and tanshinone treatment group. 10 microg A beta 1-42 was injected bilaterally into the dorsal lateral region of the dentate gyrus in the hippocampus of rats in the model and tanshinone treatment groups to prepare the AD models. 24h after modeling, tanshinone, 50mg/kg, was administered by gastric perfusion to rats in the tanshinone treatment group. Later, immunohistochemical assay and Western blot analysis were used to detect expression of neuronal NOS (nNOS) and inducible NOS (iNOS) in the rat hippocampus. Activity of AChE in each subregion (CA1 approximately CA4) of rats' hippocampus was determined by a histochemical technique. RESULTS: Expression of nNOS in the model group was down-regulated whereas iNOS was up-regulated. After A beta 1-42 injection, the number of AChE positive fibers in each subregion (CA1 approximately CA4) of the hippocampus was decreased compared with controls. With tanshinone administration, the changes were improved to varying degrees. CONCLUSION: Tanshinone modulates AChE and NOS proteins concentrations in the hippocampus of AD rats. This may have therapeutic potential in AD rats.
