Intestinal microbiota analysis and network pharmacology reveal the mechanism by which Lianhua Qingwen capsule improves the immune function of mice infected with influenza A virus.

Objective: Lianhua Qingwen capsule (LHQW) can attenuate lung injury caused by influenza virus infection. However, it is unclear whether the intestinal microbiota plays a role in LHQW activity in ameliorating viral infectious pneumonia. This study aimed to investigate the role of intestinal microbiota in LHQW activity in ameliorating viral infectious pneumonia and its possible mechanisms. Research design and methods: A mouse model of influenza A viral pneumonia was established by intranasal administration in BALB/c mice. Detection of influenza virus in the lungs, pathological examination of the lungs and small intestine, and biochemical detection of inflammatory indices were performed. The effects of LHQW on intestinal microbiota were evaluated by 16S rRNA gene sequencing. The key components and targets of LHQW were screened via network pharmacology and verified through molecular docking, molecular dynamics simulation, and free binding energy calculations. Results: Body weight decreased, inflammatory factor levels were disturbed, and the lung and intestinal mucosal barriers were significantly injured in the infected group. The alpha diversity of the intestinal microbiota decreased, and the abundance of Bacteroidetes, Muribaculaceae_unclassified, and Streptococcus decreased significantly. LHQW treatment reduced the viral load in the lungs, rescued body weight and survival, alleviated lung and intestinal mucosal barrier injury, reversed the reduction in the intestinal microbiota alpha diversity, and significantly increased the abundance of Bacteroidetes and Muribaculaceae. Network pharmacological analysis showed that six active herbal medicinal compounds from LHQW could regulate the intestinal microbiota and inhibit the immune-inflammatory response through the Toll-like receptor (TLR) and nuclear factor-κB (NF-κB) signalling pathways in the lungs. Conclusion: These results suggest that LHQW is effective for treating influenza A virus infectious pneumonia, and the mechanism is associated with the regulation of the TLR4/NF-κB signalling pathway in the lungs by restoring intestinal microbiota and repairing the intestinal wall.

Rapid and convenient detection of SARS-CoV-2 using a colorimetric triple-target reverse transcription loop-mediated isothermal amplification method.

Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 poses a significant threat to global public health. Early detection with reliable, fast, and simple assays is crucial to contain the spread of SARS-CoV-2. The real-time reverse transcription-polymerase chain reaction (RT-PCR) assay is currently the gold standard for SARS-CoV-2 detection; however, the reverse transcription loop-mediated isothermal amplification method (RT-LAMP) assay may allow for faster, simpler and cheaper screening of SARS-CoV-2. In this study, the triple-target RT-LAMP assay was first established to simultaneously detect three different target regions (ORF1ab, N and E genes) of SARS-CoV-2. The results revealed that the developed triplex RT-LAMP assay was able to detect down to 11 copies of SARS-CoV-2 RNA per 25 µL reaction, with greater sensitivity than singleplex or duplex RT-LAMP assays. Moreover, two different indicators, hydroxy naphthol blue (HNB) and cresol red, were studied in the colorimetric RT-LAMP assay; our results suggest that both indicators are suitable for RT-LAMP reactions with an obvious color change. In conclusion, our developed triplex colorimetric RT-LAMP assay may be useful for the screening of COVID-19 cases in limited-resource areas.

Analysis of the Molecular Mechanism of Punicalagin in the Treatment of Alzheimer's Disease by Computer-Aided Drug Research Technology.

The objective of this work is to explore the effect and potential mechanism of Punicalagin (Pun) in managing Alzheimer's disease (AD) based on computer-aided drug technology. The following methods were used: the intersection genes of Pun and AD were retrieved from the database and subjected to PPI analysis, GO, and KEGG enrichment analyses. Preliminary verification was performed by molecular docking, molecular dynamics (MD) simulation, and combined free energy calculation. The motor coordination and balance ability, anxiety degree, spatial learning, and memory ability of mice were measured by a rotating rod fatigue instrument, elevated cross maze, and Y maze, respectively. The amyloid ß protein (Aß) in the hippocampus was examined by immunohistochemistry, and the phosphorylation of serine at position 404 of the tau protein (Tau-pS404) was examined by western blot in the mouse brain. The PPI network of Pun showed that the intersection genes were closely related and enriched in muscle cell proliferation and the response to lipopolysaccharide. Results of molecular docking, MD simulations, and MM-GBSA demonstrated that Pun was closely bound to the target protein. Pun could improve the cognitive function of AD mice, as well as reduce Aß1-42 deposition and Tau phosphorylation in the brain (P < 0.05, P < 0.01). It can be concluded that Pun holds great promise in improving the cognitive function of AD mice. Mechanistically, Pun potentially acts on ALB, AKT1, SRC, EGFR, CASP3, and IGF-1 targets and mediates proteoglycan, lipid, and atherosclerosis in cancer, so as to reduce the accumulation of neurotoxic proteins in the brain.

Ephrin-B2 inhibits Aß25-35-induced apoptosis by alleviating endoplasmic reticulum stress and promoting autophagy in HT22 cells.

Previous studies have shown that Erythropoietin-producing hepatocellular carcinoma receptor B2 (EphB2) inhibited Aß-induced neuron apoptosis and improved cognition in AD mice, but the role of which ligand Ephrin B2 (Ephrin-B2, efnB2) is not clear. The aim of this study was to investigate the effect of the efnB2-activated Eph/efn forward signaling pathway on Aß-induced HT22 hippocampal cell apoptosis using recombination mouse Ephrin B2-Fc chimera protein (efnB2-Fc). We found that non-toxic concentrations of efnB2-Fc decreased the release of lactate dehydrogenase (LDH) in HT22 cells and reduced cell apoptosis in a dose-dependent manner. Further studies revealed that efnB2-Fc alleviated Aß-induced ER stress and decreased the expression of ER-stress-related transcriptional factor C/EBP homologous protein (CHOP), binding immunoglobulin protein (Bip) and phosphorylated eukaryotic translation initiation factor 2 subunit α (p-eIF2α) in HT22 cells. These effects of efnB2-Fc were related to the inhibition of Akt/mTOR signal transduction, an increase in the level of microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ), and the activation of the autophagy pathway. Our results indicate that the efnB2-mediated forward signaling pathway may activate the autophagy pathway to alleviate the Aß-induced ER stress and apoptosis in the HT22 cell.

Activation of NRF2/ARE by isosilybin alleviates Aß25-35-induced oxidative stress injury in HT-22 cells.

Aß-mediated oxidative stress damage is considered a direct cause of Alzheimer's disease (AD). Therefore, drugs that have been developed to block oxidative stress are considered effective for AD treatment. Isosilybin is a flavonoid compound extracted from Silybum marianum, and it has been confirmed to have many pharmacological activities. This study aimed to verify that isosilybin could alleviate the Aß25-35-induced oxidative stress damage in HT-22 hippocampal cells and to investigate the specific targets of isosilybin. A non-toxic dose of isosilybin significantly inhibited the production of reactive oxygen species (ROS), the release of malondialdehyde (MDA) and lactate dehydrogenase (LDH), and the Aß25-35-stimulated reduction in total antioxidant capacity (T-AOC). Subsequent studies showed that isosilybin significantly increased the protein and mRNA expression of antioxidases, including heme oxygenase-1 (HO-1), glutathione S-transferase (GST), and aldo-keto reductases 1C1 and 1C2 (AKR1C2). Moreover, isosilybin stimulated the activity of an antioxidant-response element (ARE)-driven luciferase reporter gene. Further studies showed that isosilybin induced the expression of NFR-2 in a time- and dose-dependent manner and promoted its translocation to the nucleus. This result indicated that the antioxidant function of isosilybin might be achieved through the activation of NRF2/ARE signalling. Subsequent studies showed that the NRF2-specific agonist t-BHQ effectively inhibited ROS, MDA and LDH release and T-AOC reduction under Aß25-35 stimulation. In addition, t-BHQ induced the expression of HO-1, GST, and AKR1C2, as well as the activity of ARE luciferase reporter plasmids. NRF2 siRNA blocked the antioxidative stress damage function of isosilybin. Therefore, NRF2 is likely to be a key mediator of isosilybin's anti-Aß25-35-mediated oxidative stress damage function. Overall, our results confirmed that isosilybin regulates the expression of HO-1, GST, and AKR1C2 through the activation of NRF2/ARE signalling, inhibiting ROS accumulation and ultimately alleviating Aß25-35-induced oxidative stress damage in HT-22 cells.

[Peoniflorin activates Nrf2/ARE pathway to alleviate the Abeta(1-42)-induced hippocampal neuron injury in rats].

This study was to investigate the effect of peoniflorin on the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream signal molecules in the hippocampus of Alzheimer's disease (AD) rats for exploring the mechanism of peoniflorin protecting hippocampal neurons. AD model rats were established by bilateral intrahippocampal injection of beta-amyloid(1-42) (Abeta(1-42)) and divided randomly into 3 groups: AD model group, peoniflorin low-dose (15 mg x kg(-1)) group and peoniflorin high-dose (30 mg x kg(-1)) group. The vehicle control rats were given bilateral intrahippocampal injection of solvent with the same volume. After peoniflorin or saline was administered (ip) once daily for 14 days, the hippocampuses of all animals were taken out for measuring the expressions of Nrf2, heme oxygenase-1 (HO-1) and gamma-glutamylcysteine synthethase (gamma-GCS) mRNA by reverse transcription PCR, determining the contents of glutathione (GSH), malondialdehyde (MDA) and carbonyl protein (CP) using colorimetric method, and for assaying the expressions of neuronal apoptosis inhibitory protein (NAIP) and Caspase-3 by immunohistochemical staining method. The results showed that peoniflorin markedly increased the expressions of Nrf2, HO-1 and gamma-GCS mRNA, enhanced the level of GSH and decreased the contents of MDA and CP in the hippocampus, as compared with the model group. Peoniflorin also improved the NAIP expression and reduced the Caspase-3 expression in the hippocampus neurons. In conclusion, peoniflorin protects against the Abeta(1-42)-mediated oxidative stress and hippocampal neuron injury in AD rats by activating the Nrf2/ARE pathway.

[Protective effect of paeonol on neurotoxicity induced by Abeta1-42 and underlying mechanisms].

OBJECTIVE: To investigate the protective effect of paeonol on amyloid beta1-42 (Abeta1-42)-induced neurotoxicity and its mechanism. METHOD: Hippocampal neurons of well-grown newborn SD rats and differentiated SH-SY5Y cell lines were cultured with various concentrations of paeonol (1, 5, 10 micromol x L(-1), respectively) for 6 hours and then incubated with Abeta1-42 oligomer (30 micromol x L(-1)) for 24 hours and 48 hours, respectively. The neuron apoptosis was observed by Heochst33258. Annexin V/PI double stain flow cytometry assay was adopted for determining SH-SY5Y cell apoptosis rate. And the expression of BDNF and Bcl-2 mRNA was detected by RT-PCR. RESULT: Compared with the model group, various concentrations of paeonol (1, 5, 10 micromol x L(-1)) significantly reduced the hippocampal neurons karyopycnosis, decreased the rate of SH-SY5Y cell apoptosis to 22.4%, 18.1% and 16.4%, respectively, and improved the expressions of BDNF and Bcl-2 mRNA. CONCLUSION: Paeonol relieves Abeta1-42 oligomer-induced neuron injury by increasing BDNF and Bcl-2 expressions.

[Anti-inflammation effect of danggui shaoyao san on Alzheimer's diseases].

OBJECTIVE: To study the mechanisms of Danggui Shaoyao San (DSS) on Alzheimer's diseases (AD) focusing on anti-inflammation. METHOD: AD rats were established by intrahippocampal bilateral injection of Abeta1-42 protein. The AD rats were randomly divided into three groups: AD model group, DSS high-dose group, DSS low-dose group. Vehicle group rats were intrahippocampal bilateral injection of solvent with the same dose. The learning ability and memory of rats was investigated in step-down passive avoidance test and Morris water maze test, expression of IL-1beta, IL-6, TNF-alpha mRNA were observed by reverse transcriptase PCR (RT-PCR), levels of NO was measured by colorimetric method and neuron apoptosis in the hippocampus was investigated by tag method of TdT-mediated end-labeling of fragmented DNA (TUNEL). RESULT: DSS significantly reduced the escape latency and increased the time that rats spent in the target quadrant in Morris water maze test, shortened the responsive latency and decreased the error numbers in step-down passive avoidance test, reduced the expression of the IL-1beta, IL-6, TNF-alpha mRNA, and the level of the NO depressed the neuron apoptosis in the hippocampus. CONCLUSION: DSS improving cognition of the rats might be related to attenuate inflammatory reaction and reduce cell apoptosis in the hippocampus.

Peoniflorin attentuates Abeta((1-42))-mediated neurotoxicity by regulating calcium homeostasis and ameliorating oxidative stress in hippocampus of rats.

Peoniflorin (PEF), a monoterpene glycoside isolated from the aqueous extract of the dry root of Paeonia, possesses wide pharmacological effects in nervous system. In this study, by using a developed rat model of hippocampal dysfunction induced by intrahippocampal injection of Abeta((1-42)) oligomers, we investigated whether PEF exerted protection against Abeta-induced neurotoxicity. A stereotactic intrahippocampal bilateral injection of Abeta((1-42)) (5 microg per side) was performed in Sprague-Dawley rats (250-280 g). Abeta((1-42))-exposed rats showed remarkable memory impairment in Morris water maze test and neuronal apoptosis by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling in hippocampus. Chronic treatment with PEF (7.5, 15 and 30 mg/(kg day), for 20 days, intraperitoneally) significantly and dose-dependently attenuated cognitive deficit, ameliorated cell apoptosis in Abeta((1-42))-treated rats. The neuroprotective effect of PEF was closely associated with its activities of maintenance of [Ca(2+)](i) homeostasis, increase of reduced glutathione (GSH) content, suppression of NOS activity and nitric oxide (NO) level, decrease of carbonyl protein (CP) and malondialdehyde (MDA) levels. These results suggested that PEF possessed the activity of prevention of the neurotoxicity induced by Abeta((1-42)) and might exert beneficial action for the treatment of Alzheimer's disease (AD).

Paeonol attenuates neurotoxicity and ameliorates cognitive impairment induced by d-galactose in ICR mice.

In the present study, we examined the supplementation of paeonol extracted from Moutan cortex of Paeonia suffruticosa Andrews (MC) or the root of Paeonia lactiflora Pall (PL) on reducing oxidative stress, cognitive impairment and neurotoxicity in d-galactose (D-gal)-induced aging mice. The ICR mice were subcutaneously injected with D-gal (50 mg/(kg day)) for 60 days and administered with paeonol (50, 100 mg/(kg day)) simultaneously. The results showed that paeonol significantly improved the learning and memory ability in Morris water maze test and step-down passive avoidance test in D-gal-treated mice. Further investigation showed that the effect of paeonol on improvement of cognitive deficit was related to its ability to inhibit the biochemical changes in brains of D-gal-treated mice. Paeonol increased acetylcholine (Ach) and glutathione (GSH) levels, restored superoxide dismutase (SOD) and Na(+), K(+)-adenosine triphosphatase (Na(+), K(+)-ATPase) activities, but decreased cholinesterase AChe activity and malondialdehyde (MDA) level in D-gal-treated mice. Furthermore, paeonol ameliorated neuronal damage in both hippocampus and temporal cortex in D-gal-treated mice. These results suggest that paeonol possesses anti-aging efficacy and may have potential in treatment of neurodegenerative diseases.