[Anti-aging effect and molecular mechanism of Xiyangshen Sanqi Danshen Granules based on metabolomics and bioinformatics].

This study investigated the anti-aging mechanism of Xiyangshen Sanqi Danshen Granules based on metabonomics, network pharmacology, and molecular docking. The aging mice model was induced by intraperitoneal injection of D-galactose(D-gal). Mice were randomly divided into a control group, model group, melatonin group(MT group), and low, medium, and high dose groups of Xiyangshen Sanqi Danshen Granules(XSD-L, XSD-M, and XSD-H). An open-field experiment was conducted, and the expression of cell cycle arrest proteins(p16) and phosphorylated histone family 2A variant(γH2AX) in the brain tissue was detected by immunofluorescence. The expression of interleukin-1ß(IL-1ß) and interleukin-6(IL-6) in the brain tissue was detected by enzyme-linked immunosorbent assay(ELISA). Metabolomics analysis was performed on the serum of mice in control, model, and XSD-H groups to obtain metabolic processes and metabolites. The effective chemical components and potential targets of Xiyangshen Sanqi Danshen Granules were predicted through network pharmacology, and the network diagram of "drug-effective chemical components-key targets" was constructed. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis were carried out, and a protein-protein interaction(PPI) network was constructed to clarify the anti-aging mechanism of Xiyangshen Sanqi Danshen Granules. The results showed that the Xiyangshen Sanqi Danshen Granules could significantly improve the aging degree of D-gal mice, significantly improve the total motion distance and the mean motion speed of D-gal mice, and reduce the rest time. In addition, Xiyangshen Sanqi Danshen Granules could significantly reduce the protein levels of IL-6 and IL-1ß and the expression of p16 and γH2AX in D-gal mice. Compared with the model group, 66 differential metabolites(DMs) were significantly up-regulated, and 91 DMs were down-regulated in the XSD-H group. Moreover, four key metabolic pathways(tryptophan metabolism, glycerophospholipid metabolism, pyrimidine metabolism, and lysine degradation) and 16 biomarkers(lysine, tryptophan, indoleacetaldehyde, PCs, LysoPCs, 3-hydroxyanthranilic acid, melatonin, etc) were screened out. 58 main active components and 62 key targets of Xiyangshen Sanqi Danshen Granules were screened by network pharmacology. The GO functional enrichment analysis found the positive regulation of gene expression, drug response, etc. KEGG pathway enrichment screening involved diabetic complications-related AGE-RAGE signaling pathway, hypoxia inducible factor-1 signaling pathway, etc. Through the PPI network and molecular docking, six potential core targets of STAT3, MAPK1, MAPK14, EGFR, FOS, and STAT1 were screened.

[Exposure Level and Risk Impact Assessment of Pesticides and Veterinary Drugs in Aquaculture Environment].

To explore the exposure level of pesticides and veterinary drugs in an aquaculture environment and its impact on the ecological environment, this study took the aquaculture environment in Shanghai as an example, and samples of water, sediment, and inputs from 40 major aquaculture farms were collected from July to September 2022. The types and contents of pesticides and veterinary drugs were screened using high-performance liquid chromatography-electrostatic field orbital ion trap mass spectrometry, and the risk quotient (RQ) method was used to assess the ecological risk of pesticide contamination in water and sediment. The results showed that 13 drugs were screened out from 204 samples (72 samples of water, 72 samples of mud, and 60 samples of input), namely, chlorpromazine, carbendazim, thiophanate, diazepam, florfenicol, simazine, amantidine, diazepam, trimethoprim, ciprofloxacin, ofloxacin, mebendazole, and enrofloxacin. Among them, 12 species were found in water samples with concentrations ranging from 0.016 µg·L-1 to 2.084 µg·L-1. The concentrations of seven species in the mud samples ranged from 0.018 µg·kg-1 to 23.101 µg·kg-1. The results showed that there were four types of inputs, ranging from 1.979 µg·kg-1 to 101.940 µg·kg-1. Seven drugs were found in both water and sediment. The risk quotient (RQ) results showed that there were some high and middle risks in both water and sediment samples of aquaculture farms, and the ecological risks of carbendazim were the highest in both water and sediment samples of aquaculture farms; the RQ values were 3.848 and 1.580, respectively, indicating high risk. It is suggested to strengthen the control and management of exogenous pesticides and veterinary drugs in aquaculture environments to protect the ecosystem health of the aquaculture environment.

Multi-omics reveals Dengzhan Shengmai formulation ameliorates cognitive impairments in D-galactose-induced aging mouse model by regulating CXCL12/CXCR4 and gut microbiota.

Dengzhan Shengmai (DZSM), a traditional Chinese medicine formulation, has been administered extensively to elderly individuals with cognitive impairment (CI). However, the underlying mechanisms by which Dengzhan Shengmai improves cognitive impairment remains unknown. This study aimed to elucidate the underlying mechanism of the effect of Dengzhan Shengmai on aging-associated cognitive impairment via a comprehensive combination of transcriptomics and microbiota assessment. Dengzhan Shengmai was orally administered to a D-galactose-induced aging mouse model, and evaluation with an open field task (OFT), Morris water maze (MWM), and histopathological staining was performed. Transcriptomics and 16S rDNA sequencing were applied to elucidate the mechanism of Dengzhan Shengmai in alleviating cognitive deficits, and enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (PCR), and immunofluorescence were employed to verify the results. The results first confirmed the therapeutic effects of Dengzhan Shengmai against cognitive defects; specifically, Dengzhan Shengmai improved learning and impairment, suppressed neuro loss, and increased Nissl body morphology repair. Comprehensive integrated transcriptomics and microbiota analysis indicated that chemokine CXC motif receptor 4 (CXCR4) and its ligand CXC chemokine ligand 12 (CXCL12) were targets for improving cognitive impairments with Dengzhan Shengmai and also indirectly suppressed the intestinal flora composition. Furthermore, in vivo results confirmed that Dengzhan Shengmai suppressed the expression of CXC motif receptor 4, CXC chemokine ligand 12, and inflammatory cytokines. This suggested that Dengzhan Shengmai inhibited CXC chemokine ligand 12/CXC motif receptor 4 expression and modulated intestinal microbiome composition by influencing inflammatory factors. Thus, Dengzhan Shengmai improves aging-related cognitive impairment effects via decreased CXC chemokine ligand 12/CXC motif receptor 4 and inflammatory factor modulation to improve gut microbiota composition.

Single-cell RNA sequencing reveals that VIM and IFITM3 are vital targets of Dengzhan Shengmai capsule to protect against cerebral ischemic injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke is one of the leading causes of mortality, but therapies are limited. Dengzhan Shengmai capsule (DZSM) was included by the Chinese Pharmacopoeia 2020 and has been broadly used for the treatment of ischemic stroke. However, the mechanism of DZSM against ischemic stroke is unclear. AIM OF THE STUDY: This study used RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) to investigate the mechanism of action of DZSM against ischemic stroke. MATERIALS AND METHODS: The rats were randomly divided into six groups: the Sham, I/R (water), I/R + DZSM-L (0.1134g/kg), I/R + DZSM-H (0.4536g/kg), I/R + NMDP (20mg/kg), and I/R + Ginaton (20mg/kg). The rats were administrated drugs for 5 days then followed by the ischemic brain injury caused by middle cerebral artery occlusion (MCAO). The neuroprotective effect was assessed by infraction rate, neurological deficit scores, regional cerebral blood flow (rCBF), hematoxylin and eosin (H&E) staining, and Nissl staining. Based on RNA-seq and scRNA-seq, the vital biological processes and core targets of DZSM against cerebral ischemia were revealed. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) staining were used to investigate the vital biological processes and core targets of DZSM against ischemic stroke. RESULTS: Administration of DZSM significantly reduced the infarction rate and Zea Longa score, Garcia JH score, and ameliorated the reduction in rCBF. And alleviated the neuronal damage, such as increased neuronal density level and Nissl bodies density level. RNA-seq analysis revealed that DZSM played important roles in inflammation and apoptosis. ELISA and IF straining validation confirmed that DZSM significantly decreased the expression of IL-6, IL-1ß, TNF-α, ICAM-1, IBA-1, MMP9, and Cleaved caspase-3 in MCAO rats. ScRNA-seq analysis identified 8 core targets in neurons including HSPB1, SPP1, MT2A, GFAP, IFITM3, VIM, CRIP1, and GPD1, and VIM and IFITM3 was verified to be decreased by DZSM in neurons. CONCLUSION: Our study illustrates the neuroprotective effect of DZSM against ischemia stroke, and VIM and IFITM3 were identified as vital targets in neurons of DZSM in protecting against MCAO-induced I/R injury.

Integrating RNA-sequencing and network analysis to explore the mechanism of topical Pien Tze Huang treatment on diabetic wounds.

Introduction: Diabetic ulcers have become one of the major complications of diabetes mellitus (DM) and are a leading cause of death and disabling disease. However, current therapies are not effective enough to meet clinical needs. A traditional Chinese medicine (TCM) formula, Pien Tze Huang (PZH), is known as a medicine that is used to treat diabetic ulcers. Methods: In this study, PZH (0.05 g/cm2 and 0.15 g/cm2) and the positive drug-rhEGF were topically administered in a high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic full-thickness incisional wounds, respectively. Wound healing was assessed by wound closure rate, two-photon microscope (SHG), staining with Hematoxylin and eosin (H&E), and Masson's trichrome (MTC). Then, RNA sequencing (RNA-seq) analysis, Enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence (IF), network analysis, were performed. Results and discussion: The results showed that PZH significantly accelerated wound healing, as well as enhanced the expression of collagen. RNA-seq analysis showed that PZH has functions on various biological processes, one of the key biological processes is inflammatory response. Tlr9, Klrk1, Nod2, Tlr2, and Ifng were identified as vital targets and the NF-κB signaling pathway was identified as the vital pathway. Additionally, PZH profoundly reduced the levels of Cleaved caspase-3 and promoted the expression of CD31 and TGF-ß1. Mechanically, PZH significantly decreased expression of NKG2-D, NOD2, and TLR2, and further inhibited the activation of downstream NF-κB signaling pathway and inhibited expression of inflammatory factors (IFN-γ and IL-1ß). Importantly, we found that several active ingredients may play a significant role in diabetic wound healing, including Notoginsenoside R1, Deoxycorticosterone, Ursolic acid, and 4-Methoxyphenol. In summary, our study sheds light on the complicated mechanisms underlying the promising anti-diabetic wounds of PZH and provides the discovery of agents treating diabetic ulcers.

Simulation experiment on OH-PCB being ingested through daily diet: Accumulation, transformation and distribution of hydroxylated-2, 2', 4, 5, 5'-pentachlorobiphenyl (OH-PCB101) in mice.

Animals exposure to polychlorinated biphenyls (PCBs) may result in retention of hydroxylated PCBs (OH-PCBs). OH-PCBs can be accumulated in animals, including humans, through the transmission of food chain. However, there are few studies on the accumulation and metabolism of OH-PCBs exposed to the body through daily diet. Therefore, this study was conducted to investigate the fate of OH-PCBs after being ingested through dietary intake. By adding 3-OH-PCB101 and 4-OH-PCB101 to the edible tissue of crucian carp, which were used as raw materials to prepare mouse feed, with an exposure concentration of 2.5 µg/kg ww. The exposure experiment lasted for a total of 80 days. The blood, feces and 11 tissues of mice at different times were analyzed qualitatively and quantitatively. It was found that major OH-PCB101 were accumulated in intestine or excreted with feces. A small part was accumulated in heart, lung and spleen. For the first time that the conversion from OH-PCB101 to PCB101 in mice was discovered, which shows from another perspective that persistent organic pollutants are difficult to be completely degraded in the environment. 4-MeO-PCB101, 3-MeSO2-PCB101, and 4-MeSO2-PCB101 were also found in various tissues. The results of this study show that after OH-PCBs accumulated in animals re-enter the organism through the food chain, they can be metabolized again and may be reversely transformed into the parent compounds. The present research shed new light on simulating the metabolic transformation process of OH-PCBs exposed to mammals through ingestion of fish. Available data show that second-generation persistent organic pollutants in the environment still need to be continuously concerned.

Identification of 2,2',4,5,5'-Pentachlorobiphenyl (PCB101) metabolites and their transmission characteristics in silver crucian carp (Carassius auratus gibelio).

Polychlorinated biphenyls (PCBs) have been attracting global concern due to their persistence and toxicity. However, the study on the metabolites of PCBs in freshwater fish is limited. In this study, the metabolites of 2,2',4,5,5'-Pentachlorobiphenyl (PCB101) in silver crucian carp (Carassius auratus gibelio) were identified for the first time. After intraperitoneal injection of PCB101 (2 mg/kg), the results showed that it could be metabolized to at least three types of metabolites, including hydroxylated (OH-), methoxylated (MeO-) and methyl sulfonated (MeSO2-) PCB101. The OH- metabolites identified in most tissues were 3-OH-PCB101and 4-OH-PCB101, such as liver, gallbladder, blood and muscle. MeSO2- metabolites identified in gallbladder, blood and brain were 3-MeSO2-PCB101 and 4-MeSO2-PCB101. Meanwhile, the MeO- metabolite identified in liver, gallbladder, blood and spleen of silver crucian carp was 4-MeO-PCB101. The investigation of the types and structures of PCB101 and its metabolites, as well as the tissue distribution and accumulation characteristics in silver crucian carp are beneficial to understand the transformation and metabolic mechanisms of PCBs in aquatic organisms. It is of great significance to identify potential pollution hazards of precursor compounds and their metabolites on aquatic products and ensure the quality and safety of aquatic products.

Integrated transcriptomics and metabolomics analysis reveals that C3 and C5 are vital targets of DuZhi Wan in protecting against cerebral ischemic injury.

BACKGROUND/AIMS: Duzhi Wan (DZW) has been extensively used in the prevention and treatment of ischemic stroke, but the mechanisms underlying its effects remain unclear. In this study, a combination of transcriptomics, metabolomics and network analysis was applied to identify the preventive mechanism of DZW in middle cerebral artery occlusion (MCAO)-induced ischemia/reperfusion (I/R) injury. METHODS: The mice were divided into five groups: the sham group, I/R group, I/R + Ginaton group, I/R+DZW-L group, and I/R+DZW-H group. Neurological deficit scores and regional cerebral blood flow (rCBF), hematoxylin and eosin (H&E) and Nissl staining results were evaluated. Transcriptomics analysis and metabolomics analysis were applied to identify the key genes and metabolites, and qRT-PCR, ELISA, and immunofluorescence were applied to verify the key targets. RESULTS: DZW significantly decreased the infarction size and neurological deficit scores, increased the rCBF percentage and neuronal number and improved neuronal morphology after MCAO. Transcriptomics and metabolomics analysis revealed that C3 and C5ar1 were core targets of DZW and indirectly regulated downstream purine metabolism, the pentose phosphate pathway, and glycerophospholipid metabolism-associated pathways via inflammatory cells. Moreover, ELISA, qRT-PCR, and immunofluorescence further confirmed that DZW significantly decreased the expression of C3, C5ar1, C5 and downstream inflammatory cytokines, including IL-6, IL-1ß and MMP-9, at the gene and protein levels, suggesting that DZW decreased neuroinflammation and inhibited related metabolic pathways. CONCLUSION: C3 and C5 play important roles in the neuroprotective and antineuroinflammatory effects of DZW in protecting against cerebral I/R. This study provides novel insights into the neuroprotective effects of DZW and its clinical application.

An Effective and Efficient Sample Preparation Method for 2-Methyl-Isoborneol and Geosmin in Fish and Their Analysis by Gas Chromatography-Mass Spectrometry.

The intensive aquaculture strategy and recirculating aquaculture system often lead to the production of off-flavor compounds such as 2-methyl-isoborneol (2-MIB) and Geosmin (GSM). The regular purge and trap extraction followed by analysis with gas chromatography-mass spectrometry (GC-MS) usually involve a complicated assembly of facilities, more working space, long sample preparation time, and headspace solid-phase microextraction (SPME). In this work, a method with easier sample preparation, fewer and simplified facilities, and without SPME on GC-MS analysis is developed for the determination of 2-MIB and GSM in fish samples. Unlike previous methods, solvent extract from samples, QuEChERS-based cleanup, and solid-phase extraction for concentration are applied. The LOD (S/N > 3) and LOQ (S/N > 10) of this method were validated at 0.6 µg/kg and 1.0 µg/kg for both 2-MIB and GSM, which are under the sensory limit (1 µg/kg). Application of this method for incurred fish samples demonstrated acceptable analytical performance. This method is suitable for large-scale determination of 2-MIB and GSM in fish samples, owing to the use of simple facility and easy-to-operate procedure, rapid sample preparation, and shorter time for GC-MS analysis without SPME.

Detection of Total and Pathogenic Vibrio parahaemolyticus in Shellfish Growing along the South Yellow Sea and the East China Sea.

This study was conducted to monitor the densities of total and pathogenic Vibrio parahaemolyticus in 300 samples of nine shellfish species harvested from the coasts of the South Yellow Sea and the East China Sea (N 23° to 34°, E 116° to 124°), People's Republic of China, between May and October 2015. Total V. parahaemolyticus densities were measured, and V. parahaemolyticus isolates were biochemically identified with probes for the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh). We found that 202 of the 300 samples were positive for V. parahaemolyticus from all the sites: 58 of the 100 samples from the Fujian province, 71 of the 100 samples from the Zhejiang province, and 73 of the 100 samples from the Jiangsu province. In most (170) of the 300 samples, V. parahaemolyticus densities were 0.3 to 10 most probable number (MPN)/g; five lots exceeded 110 MPN/g, and two lots were estimated at 110 MPN/g. Among the 202 V. parahaemolyticus strains, only one was trh positive. Densities of V. parahaemolyticus in these shellfish were temperature dependent, with highest densities in June and July. Among the nine mollusk species, V. parahaemolyticus was most abundant in the agemaki clam (Sinonovacula constricta). The highest and lowest V. parahaemolyticus prevalences were found in oriental cyclina (Cyclina sinensis, 93.8%) and mussels (Mytilus edulis, 28.1%), respectively. Overall, although V. parahaemolyticus is widely distributed in marine environments, the density of V. parahaemolyticus was low and the prevalence of the main virulence factor was very low in shellfish along the coasts of the South Yellow Sea and East China Sea, which is important from a public health perspective. Data presented here will be useful for correlational research and can be utilized for developing risk management plans that establish food safety guidelines for V. parahaemolyticus in Chinese shellfish.