Inflammatory cytokines may mediate the causal relationship between gut microbiota and male infertility: a bidirectional, mediating, multivariate Mendelian randomization study.

Introduction: Studies have shown that the gut microbiota is associated with male infertility (MI). However, their causal relationship and potential mediators need more evidence to prove. We aimed to investigate the causal relationship between the gut microbiome and MI and the potential mediating role of inflammatory cytokines from a genetic perspective through a Mendelian randomization approach. Methods: This study used data from genome-wide association studies of gut microbes (Mibiogen, n = 18, 340), inflammatory cytokines (NFBC1966, FYPCRS, FINRISK 1997 and 2002, n=13, 365), and male infertility (Finngen, n=120, 706) to perform two-way Mendelian randomization (MR), mediated MR, and multivariate MR(MVMR) analyses. In this study, the inverse variance weighting method was used as the primary analysis method, and other methods were used as supplementary analysis methods. Results: In the present study, two gut microbes and two inflammatory cytokines were found to have a potential causal relationship with MI. Of the two gut microorganisms causally associated with male infertility, Anaerotruncus increased the risk of male infertility (odds ratio = 1.81, 95% confidence interval = 1.18-2.77, P = 0.0062), and Bacteroides decreased the risk of male infertility (odds ratio = 0.57, 95% confidence interval = 0.33-0.96, P = 0.0363). In addition, of the two inflammatory cytokines identified, hepatocyte growth factor(HGF) reduced the risk of male infertility (odds ratio = 0.50, 95% confidence interval = 0.35-0.71, P = 0.0001), Monocyte chemotactic protein 3 (MCP-3) increased the risk of male infertility (odds ratio = 1.28, 95% confidence interval = 1.03-1.61, P = 0.0039). Mediated MR analysis showed that HGF mediated the causal effect of Bacteroides on MI (mediated percentage 38.9%). Multivariate MR analyses suggest that HGF may be one of the pathways through which Bacteroides affects MI, with other unexplored pathways. Conclusion: The present study suggests a causal relationship between specific gut microbiota, inflammatory cytokines, and MI. In addition, HGF may mediate the relationship between Bacteroides and MI.

Pesticide residue and dietary intake risk of vegetables grown in Shanghai under modern urban agriculture in 2018-2021.

Shanghai as an international metropolis is representative of modern urban agriculture in China, so it is of great significance to analyse the pesticide residue in vegetables grown in Shanghai. This study investigated the residue of 68 commonly used pesticides (divided into insecticides, fungicides, herbicides and plant growth regulators) in 7028 vegetable samples in Shanghai from 2018 to 2021, and estimated the dietary intake risk of these pesticides. These samples were divided into 6 categories. A total of 29.21% of vegetable samples had pesticide residues, and 0.47% of samples exceeded the maximum residue limits (MRLs) set by the national food safety standard of China. Leafy vegetables had the highest detection rate of pesticide residues (32.9%), multiple detection rate (12.2%), pesticide residue concentration (35.7 mg/kg), and the number of samples exceeding the MRL (30). There were 36 out of 68 pesticides detected in vegetables, and the top 3 were dimethomorph, propamocarb and acetamiprid. The target hazard quotient (THQ) and hazard index (HI) of these noticeablepesticides were all less than 1, illustrating that there may be no obvious health hazard for residents exposed to the pesticide levels. This study can promote the green development of the pesticide industry and provide important reference data for the monitoring of pesticide residues and their hazards under modern urban agriculture.

Inflammatory markers are associated with infertility prevalence: a cross-sectional analysis of the NHANES 2013-2020.

BACKGROUND: Inflammation exerts a critical role in the pathogenesis of infertility. The relationship between inflammatory parameters from peripheral blood and infertility remains unclear. Aim of this study was to investigate the association between inflammatory markers and infertility among women of reproductive age in the United States. METHODS: Women aged 20-45 were included from the National Health and Nutrition Examination Survey (NHANES) 2013-2020 for the present cross-sectional study. Data of reproductive status was collected from the Reproductive Health Questionnaire. Six inflammatory markers, systemic immune inflammation index (SII), lymphocyte count (LC), product of platelet and neutrophil count (PPN), platelet-lymphocyte ratio (PLR), neutrophil-lymphocyte ratio (NLR) and lymphocyte-monocyte ratio (LMR) were calculated from complete blood counts in mobile examination center. Survey-weighted multivariable logistic regression was employed to assess the association between inflammatory markers and infertility in four different models, then restricted cubic spline (RCS) plot was used to explore non-linearity association between inflammatory markers and infertility. Subgroup analyses were performed to further clarify effects of other covariates on association between inflammatory markers and infertility. RESULTS: A total of 3,105 women aged 20-45 was included in the final analysis, with 431 (13.88%) self-reported infertility. A negative association was found between log2-SII, log2-PLR and infertility, with an OR of 0.95 (95% CI: 0.78,1.15; p = 0.60), 0.80 (95% CI:0.60,1.05; p = 0.10), respectively. The results were similar in model 1, model 2, and model 3. Compared with the lowest quartile (Q1), the third quartile (Q3) of log2-SII was negatively correlation with infertility, with an OR (95% CI) of 0.56 (95% CI: 0.37,0.85; p = 0.01) in model 3. Similarly, the third quartile (Q3) of log2-PLR was negatively correlation with infertility, with an OR (95% CI) of 0.61 (95% CI: 0.43,0.88; p = 0.01) in model 3. No significant association was observed between log2-LC, log2-PPN, log2-NLR, log2-LMR and infertility in model 3. A similar U-shaped relationship between log2-SII and infertility was found (p for non-linear < 0.05). The results of subgroup analyses revealed that associations between the third quartile (Q3) of log2-SII, log2-PLR and infertility were nearly consistent. CONCLUSION: The findings showed that SII and PLR were negatively associated with infertility. Further studies are needed to explore their association better and the underlying mechanisms.

Study on anti-inflammatory effect of Shangkehuangshui in vitro and in vivo based on TLR4/TLR2-NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shangkehuangshui (SK) has been traditionally used to treat traumatic injury, soft tissue and bone injury in Foshan hospital of traditional Chinese medicine for more than 60 years, which composed of many Chinese herbs such as Coptis chinensis Franch., Gardenia jasminoides Ellis, Phellodendron chinense Schneid. and etc. SK exhibits heat-clearing and detoxifying, enhancing blood circulation to eliminate blood stasis properties, and demonstrates noteworthy clinical efficacy. Nevertheless, the underlying mechanism remains uncertain. AIM OF THE STUDY: The early study found that SK had good anti-inflammatory effects in acute soft tissue injury model. This research is to verify the anti-inflammatory properties of SK both in vitro and in vivo via TLR4/TLR2-NF-κB signaling pathway, to clarify the underlying mechanisms responsible for the curative effect of SK. METHODS: The RAW264.7 cells inflammatory model was established with lipopolysaccharide (LPS) in vitro. NO and TNF-α, IL-6, IL-1ß were determined with Griess method and ELISA method respectively. The mRNA and protein expression levels of TLR4/TLR2-NF-κB pathway were evaluated by qPCR and Western blot method. In vivo experiment, chronic soft tissue injury rat models were established by tracking gastrocnemius muscle with electrical stimulation, then local appearance and pathological changes were observed and recorded, the contents of inflammatory factors in serum and tissue were performed. Moreover, we also measured and contrasted the expression of TLR4/TLR2-NF-κB related factors. RESULTS: SK effectively inhibited the LPS-induced generation of inflammatory cytokines, including NO, TNF-α, IL-6 and IL-1ß in RAW264.7 cells, and significantly suppressed the expression of TLR4, TLR2, MyD88, IκB, and NF-κB. In vivo, SK remarkably decreased the damage appearance scores after 4 and 14 days of administration and inhibit the quantity of NO and leukocytes present in the serum. Additionally, the inflammatory infiltration in the pathological section was alleviated, myofibrillar hyperplasia and blood stasis were reduced. SK markedly downregulated NO, TNF-α, IL-6 and IL-1ß in injured tissues of rats, also declined the expression of TLR4, TLR2, MyD88, IκB, NF-κB, IL-6, TNF-α and IL-1ß. CONCLUSION: This study revealed that SK had obvious effects of anti-inflammatory actions in vivo and vitro, effectively reduced acute and chronic soft tissue injury in clinical, this might be attributed to inhibit the TLR4/TLR2-NF-κB pathway, further inhibit the expression of downstream relevant pro-inflammatory cytokines.

Study on skin infection model of Staphylococcus aureus based on analytic hierarchy process and Delphi method.

Purpose: Infectious skin diseases are a type of inflammatory skin lesions caused by pathogenic microorganisms. Because of the uncertainty of methodology, the skin infection model usually have low replication rate and lack of good evaluation system. We aimed to establish multi-index and comprehensive evaluation method for Staphylococcus aureus (S.aureus) skin-infection models through Analytic hierarchy process (AHP) and Delphi method, and screen high quality animal models through it. Materials and methods: Firstly, the evaluation indicators of skin infection were collected basing on literature research. The weight of the evaluation indicators were decided according to AHP and Delphi method. Then different ulcer models (mouse or rat) infected by S. aureus were selected as the research objects. Results: The evaluation indicators were classified into four groups of criteria (including ten sub-indicators) and given different weights, physical sign changes (0.0518), skin lesion appearance (0.2934), morphological observation (0.3184), etiological examination (0.3364). Through the evaluation system, we screened and found that the mouse ulcer model which caused by a round wound and 1.0 × 1010 CFU/mL (0.1 mL) bacterial concentration got the highest comprehensive score, and also found that the model which caused by a 1.5 cm-round wound and 1.0 × 1010 CFU/mL (0.2 mL) maybe the best rat ulcer model. Conclusions: This study has established an evaluation system based on AHP and Delphi method, also provided the best skin ulcer models selected by this system, the models are suitable for disease research and drug development research of skin ulcer.

16p11.2 CNV gene Doc2α functions in neurodevelopment and social behaviors through interaction with Secretagogin.

Copy-number variations (CNVs) of the human 16p11.2 genetic locus are associated with neurodevelopmental disorders, including autism spectrum disorders (ASDs) and schizophrenia. However, it remains largely unclear how this locus is involved in the disease pathogenesis. Doc2α is localized within this locus. Here, using in vivo and ex vivo electrophysiological and morphological approaches, we show that Doc2α-deficient mice have neuronal morphological abnormalities and defects in neural activity. Moreover, the Doc2α-deficient mice exhibit social and repetitive behavioral deficits. Furthermore, we demonstrate that Doc2α functions in behavioral and neural phenotypes through interaction with Secretagogin (SCGN). Finally, we demonstrate that SCGN functions in social/repetitive behaviors, glutamate release, and neuronal morphology of the mice through its Doc2α-interacting activity. Therefore, Doc2α likely contributes to neurodevelopmental disorders through its interaction with SCGN.

[Network Meta-analysis of single Hirudo prescriptions in treatment of ischemic cerebrovascular disease].

This study intended to evaluate the efficacy and safety of single Hirudo prescriptions in the treatment of ischemic cerebrovascular disease(ICVD) by frequency network Meta-analysis and traditional Meta-analysis. CNKI, Wanfang, VIP, SinoMed, PubMed, EMbase, and Cochrane Library databases were searched to collect the randomized controlled trial(RCT) of single Hirudo prescriptions for ICVD from the inception of the databases to May 2022. The quality of the included literature was evaluated by Cochrane risk of bias tool. Finally, 54 RCTs and 3 single Hirudo prescriptions were included. Statistical analysis was conducted by RevMan 5.3 and Stata SE 15. Network Meta-analysis showed that in terms of the clinical effective rate, the surface under the cumulative ranking curve(SUCRA) of intervention measures was as follows: Huoxue Tongmai Capsules+conventional treatment>Maixuekang Capsules+conventional treatment>Naoxuekang Capsules+conventional treatment>conventional treatment. Traditional Meta-analysis revealed that in terms of the safety of ICVD treatment, Maixuekang Capsules+conventional treatment had higher safety than conventional treatment alone. According to the network Meta-analysis and traditional Meta-analysis, it was found that conventional treatment combined with single Hirudo prescriptions improved the clinical efficacy of ICVD patients, and compared with that of conventional treatment alone, the incidence of adverse reactions of combined treatment was low and the safety was high. However, the methodological quality of the articles included in this study was generally low and there were large differences in the number of articles on the three combined medication. Therefore, the conclusion of this study needed to be confirmed by subsequent RCT.

Purification Process and In Vitro and In Vivo Bioactivity Evaluation of Pectolinarin and Linarin from Cirsium japonicum.

Pectolinarin and linarin are two major flavone O-glycosides of Cirsium japonicum, which has been used for thousands of years in traditional Chinese medicine. Pharmacological research on pectolinarin and linarin is meaningful and necessary. Here, a process for the purification of pectolinarin and linarin from C. japonicum was established using macroporous resin enrichment followed by prep-HPLC separation. The results show the purity of pectolinarin and linarin reached 97.39% and 96.65%, respectively. The in vitro bioactivities result shows the ORAC values of pectolinarin and linarin are 4543 and 1441 µmol TE/g, respectively, meanwhile their inhibition rate of BSA-MGO-derived AGEs is 63.58% and 19.31% at 2 mg/mL, which is 56.03% and 30.73% in the BSA-fructose system, respectively. The COX-2 inhibition rate at 50 µg/mL of linarin and pectolinarin reached 55.35% and 40.40%, respectively. Furthermore, the in vivo bioassay combining of histopathologic evaluation and biochemical analysis of liver glutamic oxaloacetic transaminase, serum creatinine and TNF-α show pectolinarin can alleviate lipopolysaccharide (LPS)-induced acute liver and kidney injury in mice. Metabolomics analysis shows that pectolinarin attenuates LPS-challenged liver and kidney stress through regulating the arachidonic acid metabolism and glutathione synthesis pathways. Collectively, our work presents a solid process for pectolinarin and linarin purification and has discovered a promising natural therapeutic agent-pectolinarin.

Biochanin A ameliorated oleate-induced steatosis in HepG2 cells by activating the SIRT3/AMPK/ULK-1 signaling pathway.

Biochanin A (Bio-A), an isoflavone abundant in chickpeas, possesses hypoglycemic, hypolipidemic, and anti-inflammatory effects. However, whether Bio-A has antihepatosteatosis effect remains unclear. This study aimed to evaluate the antihepatosteatosis effect of Bio-A on oleate (OA)-treated hepatocytes, and explore the underlying mechanism. When incubated with OA for 24 h, HepG2 cells were treated with various concentrations of Bio-A for 24 h to obtain an optimal antihepatosteatosis dose. HepG2 cells were treated with the AMP-activated protein kinase (AMPK) inhibitor Compound C, or the sirtuin-3 (SIRT3) inhibitor 3-TYP, and incubated with 50 µM Bio-A. The results indicated that 12.6% of lipid content, particularly 11.0% of triglyceride content, and the expression of adipocyte differentiation-related protein were significantly decreased in Bio-A-treated hepatosteatosis cells, followed by an increase in the expression of Beclin 1, phosphorylation of Unc-51-like kinase 1 (ULK-1), the microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio, and a decrease in expression of p62. The results indicated that Bio-A upregulated autophagosome formation and autophagy flux. In addition, Bio-A increased SIRT3 expression and AMPK phosphorylation in OA-treated HepG2 cells. Blockade of AMPK and SIRT3 blocked the antihepatosteatosis effect and ULK-1 activation by Bio-A. AMPK inhibition did not eliminate the activation of SIRT3 by Bio-A. AutoDock analysis demonstrated that interaction might exist between Bio-A and SIRT3. In conclusion, Bio-A reduced fat accumulation in OA-treated HepG2 cells by activating SIRT3/AMPK/ULK-1-mediated autophagy. The findings provide a theoretical basis for the effect of Bio-A on hepatic steatosis-related diseases. PRACTICAL APPLICATIONS: This study highlights the antihepatosteatosis effects of biochanin A (Bio-A) on oleate (OA)-treated hepatocytes. Bio-A, one of the isoflavones in Cicer arietinum Linn., possesses multiple bioactivities such as antiobesity, anti-inflammation, and hypoglycemic and hypolipidemic effects. This study provides a new application of Bio-A to treat hepatic steatosis, and revealed the underlying mechanism of Bio-A involved in the activation of the SIRT3/AMPK/ULK-1-mediated autophagy. The findings provide a theoretical basis for the application of Bio-A to hepatic steatosis-related diseases.

Isoflavaspidic Acid PB Extracted from Dryopteris fragrans (L.) Schott Inhibits Trichophyton rubrum Growth via Membrane Permeability Alternation and Ergosterol Biosynthesis Disruption.

Isoflavaspidic acid PB (PB), a phloroglucinol derivative extracted from aerial parts of Dryopteris fragrans (L.) Schott, had antifungal activity against several dermatophytes. This study was aimed at exploring the antifungal mechanism of PB against Trichophyton rubrum (T. rubrum). The effectiveness of PB in inhibiting T. rubrum growth was detected by time-kill kinetics study and fungal biomass determination. Studies on the mechanism of action were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), sorbitol and ergosterol assay, nucleotide leakage measurement, and UPLC-based test and enzyme-linked immunosorbent assay. Fungicidal activity of PB was concentration- and time-dependent at 2 × MIC (MIC: 20 µg/mL) after 36 h. The total biomass of T. rubrum was reduced by 64.17%, 77.65%, and 84.71% in the presence of PB at 0.5 × MIC, 1 × MIC, and 2 × MIC, respectively. SEM analysis showed that PB changed mycelial morphology, such as shrinking, twisting, collapsing, and even flattening. TEM images of treated cells exhibited abnormal distributions of polysaccharide particles, plasmolysis, and cytoplasmic content degradation accompanied by plasmalemma disruption. There were no changes in the MIC of PB in the presence of sorbitol. However, the MIC values of PB were increased by 4-fold with exogenous ergosterol. At 4 h and 8 h, PB increased nucleotide leakage. Besides, ergosterol content in T. rubrum membrane treated with PB at 0.5 × MIC, 1 × MIC, and 2 × MIC was decreased by 9.58%, 15.31%, and 76.24%, respectively. There was a dose-dependent decrease in the squalene epoxidase (SE) activity. And the reduction in the sterol 14α-demethylase P450 (CYP51) activity was achieved after PB treatments at 1 × MIC and 2 × MIC. These results suggest that PB displays nonspecific action on the cell wall. The membrane damaging effects of PB were attributed to binding with ergosterol to increase membrane permeability and interfering ergosterol biosynthesis involved with the reduction of SE and CYP51 activities. Further study is needed to develop PB as a natural antifungal candidate for clinical use.

Ketamine Promotes LPS-Induced Pulmonary Autophagy and Reduces Apoptosis through the AMPK/mTOR Pathway.

To explore the protective effect of ketamine on acute lung injury (ALI) in sepsis mice regarding the autophagy and apoptosis, lipopolysaccharide (LPS) was used to construct a sepsis-induced ALI model. In in vivo experiments, ketamine at a concentration of 20 mg/kg was injected before modeling. The serum levels of inflammatory factors IL-1ß, IL-6, and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA) kit. At the same time, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect apoptosis-related factors Bax and Bcl-2 and autophagy-related factors Beclin-1 and P62. In in vitro experiment, firstly, Cell Counting Kit-8 (CCK8) assay was used to detect the cell viability and identify optimal concentration of ketamine. TUNEL staining, Western blotting (WB), and qRT-PCR were used to detect alveolar type II epithelial cells (AEC II) AEC II cell apoptosis. The content of inflammatory factors in the cell supernatant was detected by kits and the autophagy intensity of AEC II cells was detected by PCR and WB. At the same time, the expression changes of AMPK/mTOR pathway were detected by WB technology. Compared with the Sham group, the dry-wet ratio of the lung tissue in the LPS group was obviously increased, the expression of inflammatory factors in the serum was upregulated, and apoptosis and autophagy activation occurred. In the LPS + ketamine group, ketamine significantly promoted autophagy intensity and inhibited inflammatory response, thereby reducing apoptosis. In vitro, 1 mmol/L ketamine can effectively improve the viability of AEC II cells after LPS treatment, promote autophagy, and decrease cell apoptosis. And we found that the above-mentioned effect of ketamine was by regulating the activation of AMPK/mTOR pathway. In this study, we demonstrated that LPS treatment can induce inflammation and autophagy and induce apoptosis in lung cells. In contrast, AMPK expression was activated after ketamine treatment, inhibiting the mTOR pathway and promoting autophagy, thereby alleviating the apoptosis of AEC II cells.

[Mechanism of Liangfu Pills in treatment of functional dyspepsia: based on network pharmacology and experimental verification].

This study aims to explore the potential mechanism of Liangfu Pills in the treatment of functional dyspepsia(FD) based on network pharmacology and molecular docking, and verify the mechanism by animal experiment. The active components of Liangfu Pills were screened from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the targets of Liangfu Pills were predicted by SwissTargetPrediction. The targets of FD were retrieved from GeneCards. On this basis, the common targets of the disease and the pills were yielded and the protein interaction was retrieved based on STRING. The core targets were screened out, followed by Gene Oncology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis with DAVID. Finally, molecular docking was carried out with the help of AutoDock Tools to predict the binding degree between the effective components of Liangfu Pills and core targets. A total of 19 active components of Liangfu Pills and 591 FD-related targets were screened out by network pharmacology, of which 253 were common targets of the disease and the prescription. Liangfu Pills was mainly involved in the biological processes of response to drug, negative regulation of transcription, positive regulation of apoptotic process, and cell surface receptor signaling pathway, and the KEGG pathways of hypoxia-inducible factor-1(HIF-1) signaling pathway, serotonergic synapse, tumor necrosis factor(TNF) signaling pathway, cyclic adenosine monophosphate(cAMP) signaling pathway, calcium signal pathway, and inflammatory mediator regulation of transient receptor potential(TRP) channels. The results of molecular docking showed that the key active components of Liangfu Pills had certain binding activity to the targets mitogen-activated protein kinase 1(MAPK1), protein kinase B(AKT1), transient receptor potential cation channel subfamily V member 1(TRPV1), 5-hydroxytryptamine receptor 1 A(HTR1 A), and 5-hydroxytryptamine receptor 2 A(HTR2 A). FD was induced in rats, and then Liangfu Pills was given to FD rats for 7 days. The results showed that Liangfu Pills could significantly relieve the symptoms of FD rats, significantly increase the expression of 5-hydroxytryptamine(5-HT), and down-regulate the expression of TRPV1. Through network pharmacology, molecular docking, and experimental verification, this study proved that Liangfu Pills improved FD through multiple components and multiple targets. The result lays a basis for further research on the mechanism and clinical application of Liangfu Pills in the treatment of FD.

Development and Validation of a Hypoxia-related Prognostic Model for Ovarian Cancer.

BACKGROUND: The high heterogeneity of ovarian cancer (OC) brings great difficulties to its early diagnosis and prognostic forecast. There is an urgent need to establish a prognostic model of OC based on clinicopathological features and genomics. METHODS: We identified hypoxia-related differentially expressed genes (DEGs) between OC tissues from The Cancer Genome Atlas (TCGA) and normal tissues from the Genotype-Tissue Expression (GTEx). LASSO Cox regression analysis was applied for building a prognostic model in the TCGA-GTEx cohorts, and its predictive value was validated in the GEO-OC cohort. Functional enrichment analysis was performed to investigate the underlying mechanisms. By constructing a hypoxia model of the SKOV3 cell line and applying qRT-PCR, we investigated the relationship between hypoxia with two novel genes in the prognostic model (ISG20 and ANGPTL4). RESULTS: Twelve prognostic hypoxia-related DEGs were identified, and nine of them were selected to establish a prognostic model. OC patients were stratified into two risk groups, and the high-risk group showed reduced survival time compared to the low-risk group upon survival analysis. Univariate and multivariate Cox regression analysis demonstrated that the risk score was an independent risk factor for overall survival. The biological function of the identified prognostic hypoxia-related gene signature was involved in immune cell infiltration. Low expression of ISG20 was observed in the CoCl2-mimicked hypoxic SKOV3 cell line and negatively correlated with HIF-1α. CONCLUSION: Our findings showed that this hypoxia-related gene signature could serve as a satisfactory prognostic classifier for OC and will be beneficial to the research and development of targeted therapeutic strategies.

[A new phloroglucinol from Dryopteris fragrans and its antibacterial activity in vitro].

A new phloroglucinol was isolated from 50% ethanol extract of Dryopteris fragrans by silica gel column chromatography, Sephadex LH-20 gel column chromatography, thin-layer chromatography(TLC), and preparative liquid column chromatography. On the basis of MS, ~1H-NMR, ~(13)C-NMR, and reference materials, compound 1 was identified as 2,5-cyclohexadien-1-one, 2-{[2,6-dihydroxy-4-methoxy-3-methyl-5-(1-isobutyl)phenyl]methyl}-3,5-dihydroxy-4,4-dimethyl-6-(1-oxobutyl)(1), and named disaspidin BB. Compound 1 was evaluated for its antibacterial activity. The experimental results showed that compared with the commonly used topical antibiotics erythromycin or mupirocin, disaspidin BB exhibited significant antibacterial activities against Staphylococcus epidermidis(SEP), S. haemolyticus(SHA), and methicillin-resistant S. aureus(MRSA)(P<0.05). Additionally, disaspidin BB was sensitive to ceftazidime-resistant SEP1-SEP4, SHA5-SHA7, MRSA8, and MRSA9. The MIC values of disaspidin BB against SEP and SHA were 1.67-2.71 µg·mL~(-1) and 10.00-33.33 µg·mL~(-1) respectively. Disaspidin BB has good antibacterial activities and deserves development as a new anti-infective drug for external use.

Anoectochilus roxburghii flavonoids extract ameliorated the memory decline and reduced neuron apoptosis via modulating SIRT1 signaling pathway in senescent mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Anoectochilus roxburghii (A. roxburghii) is a precious herb and folk medicine in many Asian countries. It has been used traditionally to treat diabetes, etc., and also used as a dietary therapy to delay senescence. AIM OF THE STUDY: This study was to evaluate the neuroprotective effects of A. roxburghii flavonoids extract (ARF) and whether its effects were due to the regulation of SIRT1 signaling pathway in senescent mice and in D-galactose (D-gal) induced aging in SH-SY5Y cells. MATERIALS AND METHODS: 18-month-old mice were randomly divided into senescent model, low-dose ARF, high-dose ARF and vitamin E group. 2-Month-old mice were as a control group. After 8 weeks treatment, Morris water maze (MWM) was performed. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), monoamine oxidase (MAO) and acetylcholinesterase (ACh-E) in the cortex were determined. Hippocampus morphologic changes were observed with haematoxylin and eosin (H&E), Nissl, senescence-associated-galactosidase (SA-ß-gal) and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining. Apoptosis-related molecular expressions in the hippocampus were performed by western blotting. Furthermore, after stimulated by EX527 (a SIRT1 inhibitor), the SIRT1-dependent neuroprotective effects of ARF were determined by measuring SRIT1 and p53 expression in SH-SY5Y aging cells induced by D-gal. RESULTS: ARF could significantly ameliorate memory decline in senescent mice and reduce the generations of ROS, MDA and the activities of MAO and ACh-E, while increasing SOD activities in the cortex of aging mice. ARF obviously improved hippocampus pathological alterations, increased the number of Nissl bodies, while reducing senescent and apoptotic cells in senescent mice hippocampus. Further, ARF positively regulated SIRT1 expression, and reduced apoptosis-related molecules p53, p21 and Caspase-3 expression, while increasing the ratio of Bcl-2/Bax. In D-gal-induced SH-SY5Y cells, the effects of ARF on SIRT1 and p53, and the ability of scavenging ROS were mostly abolished after incubation with the EX527. CONCLUSIONS: ARF, in a SIRT1-dependent manner, exerted neuroprotection via modulating SIRT1/p53 signaling pathway against memory decline and apoptosis due to age-induced oxidative stress damage in senescent mice.

Basolateral amygdala activation enhances object recognition memory by inhibiting anterior insular cortex activity.

Noradrenergic activation of the basolateral amygdala (BLA) by emotional arousal enhances different forms of recognition memory via functional interactions with the insular cortex (IC). Human neuroimaging studies have revealed that the anterior IC (aIC), as part of the salience network, is dynamically regulated during arousing situations. Emotional stimulation first rapidly increases aIC activity but suppresses it in a delayed fashion. Here, we investigated in male Sprague-Dawley rats whether the BLA influence on recognition memory is associated with an increase or suppression of aIC activity during the postlearning consolidation period. We first employed anterograde and retrograde viral tracing and found that the BLA sends dense monosynaptic projections to the aIC. Memory-enhancing norepinephrine administration into the BLA following an object training experience suppressed aIC activity 1 h later, as determined by a reduced expression of the phosphorylated form of the transcription factor cAMP response element-binding (pCREB) protein and neuronal activity marker c-Fos. In contrast, the number of perisomatic γ-aminobutyric acid (GABA)ergic inhibitory synapses per pCREB-positive neuron was significantly increased, suggesting a dynamic up-regulation of GABAergic tone. In support of this possibility, pharmacological inhibition of aIC activity with a GABAergic agonist during consolidation enhanced object recognition memory. Norepinephrine administration into the BLA did not affect neuronal activity within the posterior IC, which receives sparse innervation from the BLA. The evidence that noradrenergic activation of the BLA enhances the consolidation of object recognition memory via a mechanism involving a suppression of aIC activity provides insight into the broader brain network dynamics underlying emotional regulation of memory.

Pharmacological Mechanisms of Shangke Huangshui against Skin and Soft Tissue Infection.

BACKGROUND: Skin and soft tissue infections (SSTIs) are a group of common diseases, usually caused by bacteria. Shangke Huangshui (SK) has been widely used to treat various SSTIs diseases for many years, but its mechanism is unclear. Therefore, this study was designed to investigate the anti-infective effect of SK on different skin and soft tissue infection diseases and to explore its underlying mechanism. METHODS: The subcutaneous abscess mouse model, the dermal ulcer rat model, and the infectious soft tissue injury rat model were established by injection of Staphylococcus aureus to evaluate the anti-inflammatory and antibacterial effects of SK. Abscess volume, local appearance score and histological changes, bacterial contents, and hydroxyproline concentration in the skin or soft tissue were analyzed. The levels of NO, TNF-α, IL-1ß, and IL-8 in the serum and tissue were determined by ELISA method. The mRNA expression levels of TLR2, MyD88, TAK1, NF-κB, AP-1, and other genes were measured with qRT-PCR method, and the protein expression of TLR2, MyD88, TAK1, NF-κB, and AP-1 was detected by western blot method. RESULTS: SK had an obvious therapeutic effect on skin and soft tissue infections. It reduced the volume of abscess and promoted the healing of skin ulcer, improved pathological phenomena, such as inflammatory infiltration of skin and soft tissue, reduced the levels of white blood cells and NO in the blood, decreased bacteria contents in the skin and soft tissue. Furthermore, SK decreased the mRNA expression of TLR2, MyD88, TAK1, NF-κB and AP-1, and other related genes and also downregulated the protein expression of TLR2, MyD88, TAK1, NF-κB, and AP-1. CONCLUSION: The experiments provide evidence that SK can treat skin and soft tissue infection diseases based on its comprehensive antibacterial and anti-inflammatory effects. The therapeutic mechanism may be associated with the inhibition of TLR2/MyD88/NF-κB signaling pathway.

A High-Fat High-Fructose Diet Dysregulates the Homeostatic Crosstalk Between Gut Microbiome, Metabolome, and Immunity in an Experimental Model of Obesity.

SCOPE: Ample evidence supports the prominent role of gut-liver axis in perpetuating pathological networks of high-fat high-fructose (HFF) diet induced metabolic disorders, however, the molecular mechanisms are still not fully understood. Herein, this study aims to present a holistic delineation and scientific explanation for the crosstalk between the gut and liver, including the potential mediators involved in orchestrating the metabolic and immune systems. METHODS AND RESULTS: An experimental obesity-associated metaflammation rat model is induced with a HFF diet. An integrative multi-omics analysis is then performed. Following the clues illustrated by the multi-omics discoveries, putative pathways are subsequently validated by RT-qPCR and Western blotting. HFF diet leads to obese phenotypes in rats, as well as histopathological changes. Integrated omics analysis shows that there exists a strong interdependence among gut microbiota composition, intestinal metabolites, and innate immunity regulation in the liver. Some carboxylic acids may contribute to gut-liver communication. Moreover, activation of the hepatic LPS-TLR4 pathway in obesity is confirmed. CONCLUSION: HFF-intake disturbs gut flora homeostasis. Crosstalk between gut microbiota and innate immune system mediates hepatic metaflammation in obese rats, associated with LPS-TLR4 signaling pathway activation. Moreover, α-hydroxyisobutyric acid and some other organic acids may play a role as messengers in the liver-gut axis.

MiR-181a reduces radiosensitivity of non-small-cell lung cancer via inhibiting PTEN.

BACKGROUND: The aim of this study is to explore the effect of micro ribonucleic acid (miR)-181a on the radiosensitivity of non-small cell lung cancer (NSCLC) and its potential mechanism of action. METHODS: The differentially expressed miRNAs were screened in lung cancer tissues of radiotherapy-resistant and non-radiotherapy-resistant NSCLC patients, and verified via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Next, the effects of different miRNA expressions on patients' survival time were discussed, and target genes of miR-181a were predicted. The effect of miR-181a expression on radiosensitivity was determined using cell counting kit-8 (CCK-8) assay and flow cytometry. The direct target of miR-181a was verified via luciferase reporter assay. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) was overexpressed using lentiviruses, and then whether miR-181a reduces radiosensitivity via targeting PTEN was detected via CCK-8 assay and flow cytometry. Finally, Western blotting was performed to detect the protein expression of PTEN. RESULTS: The screening results of microarray expression profile assay revealed that 15 miRNAs had significant differences in lung cancer tissues of radiotherapy-resistant NSCLC patients compared with those in non-radiotherapy-resistant NSCLC patients. The results of RT-qPCR showed that hsa-miR-181a, hsa-miR-199b, hsa-miR-489 and hsa-miR-589 were significantly up-regulated in the lung cancer tissues of radiotherapy-resistant NSCLC patients compared with those in non-radiotherapy-resistant NSCLC patients. In addition, it was found that the survival time of NSCLC patients was obviously prolonged in hsa-miR-181a low-expression group and hsa-miR-589 high-expression group, but hsa-miR-489 and hsa-miR-199b had no significant influence on the survival time of NSCLC patients. According to KEGG enrichment analysis, the target genes of miR-181a were evidently enriched in the phosphatidylinositol 3-hydroxy kinase (PI3K)/protein kinase B (AKT) signaling pathway, NSCLC signaling pathway and other cancer signaling pathways. Under the radiation dose of 2, 4, 6 and 8 Gy, the survival rate of A549 cells rose in miR-181a mimic group, but declined in miR-181a inhibitor group. Moreover, compared with that in model group, the radiotherapy-induced apoptosis was markedly inhibited in miR-181a mimic group, but markedly promoted in miR-181a inhibitor group. It was also observed that the response of cells to radiotherapy-induced apoptosis was remarkably weakened in miR-181a mimic + PTEN overexpression group compared with that in miR-181a mimic group. Finally, miR-181a mimic group had a significantly lower protein expression of PTEN and significantly higher protein expressions of CXC chemokine receptor 4 (CXCR4), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), p-AKT1 and p-mammalian target of rapamycin (mTOR) than model group, while miR-181a inhibitor group had the opposite protein expressions. The protein expressions of CXCR4, p-STAT3, p-AKT1 and p-mTOR were obviously lower in miR-181a mimic + PTEN overexpression group than those in miR-181a mimic group. CONCLUSIONS: MiR-181a reduces the radiosensitivity of NSCLC via inhibiting PTEN expression.

3 CpG Methylation Biomarkers for the Diagnosis of Polycystic Ovary Syndrome (PCOS) in Blood Samples.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine disease in women that seriously interferes with patient's metabolic and reproductive functions. The current diagnostic criteria for PCOS are expert-based and still disputed. Previous studies have identified changes in DNA methylation in peripheral blood of women with PCOS, but their diagnostic potential for PCOS remains to be studied. OBJECTIVE: The present study aimed to identify potential methylation biomarkers for the diagnosis of PCOS in blood. METHODS: Methylation profiling of peripheral blood was downloaded from a public database, Gene Expression Omnibus (GEO), including 30 PCOS patients (diagnosed with the revised 2003 Rotterdam consensus criteria) and 30 age-matched healthy women recruited from Centre of Reproductive Medicine, Linyi People's Hospital, Shandong, China. Weighted gene co-expression network analysis (WGCNA) was utilized to identify PCOS-related co-methylation CpG sites (co- MPs). Functional enrichment analysis was performed on the localized genes of PCOS-related co- MPs. The least absolute shrinkage and selection operator (LASSO) regression was used to screen out CpG methylation signatures for PCOS diagnosis, and receiver operating characteristic (ROC) analysis was conducted to evaluate their diagnostic accuracy. To assess the accuracy of the combination of the investigated indicators, multivariate ROC analysis was performed on the predicted probability values obtained using binary logistic regression on the methylation levels of selected CpGs. RESULTS: Seven co-methylation modules were obtained, among which the turquoise module is the most relevant to PCOS, containing 194 co-MPs. The genes that these co-MPs located in were mainly associated with the immune-related pathway. According to LASSO regression, three Co- MPs (cg23464743, cg06834912, cg00103771) were identified as potential diagnostic biomarkers of PCOS. ROC analysis showed an AUC (area under curve) of 0.7556 (sensitivity 60.0%, specificity 83.3%) for cg23464743, 0.7822 (sensitivity 70.0%, specificity 80.0%) for cg06834912, and 0.7611 (sensitivity 63.3%, specificity 83.3%) for cg00103771. The diagnostic accuracy of the combination of these 3 indicators presented to be higher than any single one of them, with the AUC of 0.8378 (sensitivity 73.3%, specificity 93.3%). CONCLUSION: The combination of 3 CpG methylation signatures in blood was identified with a good diagnostic accuracy for PCOS, which may bring new insight into the development of PCOS diagnostic markers in the future.