[Effects of Biochar Application Two Years Later on N2O and CH4 Emissions from RiceVegetable Rotation in a Tropical Region of China].

The effects of biochar application on soil nitrous oxide （N2O） and methane （CH4） emissions in a typical rice-vegetable rotation system in Hainan after two years were investigated. The aim was to clarify the long-term effects of biochar on greenhouse gas emissions under this model， and it provided a theoretical basis for N2O and CH4 emission reduction in rice-vegetable rotation systems in tropical regions of China. Four treatments were set up in the field experiment， including no nitrogen fertilizer control （CK）； nitrogen， phosphorus， and potassium fertilizer （CON）； nitrogen， phosphorus， and potassium fertilizer combined with 20 t·hm-2 biochar （B1）； and nitrogen， phosphorus， and potassium fertilizer combined with 40 t·hm-2 biochar （B2）. The results showed that： ① compared with that in the CON treatment， the B1 and B2 treatments significantly reduced N2O emissions by 32% and 54% in the early rice season （P < 0.05， the same below）， but the B1 and B2 treatments significantly increased N2O emissions by 31% and 81% in the late rice season. The cumulative emissions of N2O in the pepper season were significantly higher than those in the early and late rice seasons， and the B1 treatment significantly reduced N2O emissions by 35%. There was no significant difference between the B2 and CON treatments. ② Compared with that in the CON treatment， B1 and B2 significantly reduced CH4 emissions by 63% and 65% in the early rice season， and the B2 treatment significantly increased CH4 emissions by 41% in the late rice season. There was no significant difference between the B1 and CON treatments. There was no significant difference in cumulative CH4 emissions between treatments in the pepper season. ③ The late rice season contributed to the main global warming potential （GWP） of the rice-vegetable rotation system， and CH4 emissions determined the magnitude of GWP and greenhouse gas emission intensity （GHGI）. After two years of biochar application， B1 reduced the GHGI of the whole rice-vegetable rotation system， and B2 increased the GHGI and reached a significant level. However， the B1 and B2 treatments significantly reduced GHGI in the early rice season and pepper season， and only the B2 treatment increased GHGI in the late rice season. ④ Compared with that in the CON treatment， the B1 and B2 treatments significantly increased the yield of early rice by 33% and 51%， and the B1 and B2 treatments significantly increased the yield of pepper season by 53% and 81%. In the late rice season， there was no significant difference in yield except for in the CK treatment without nitrogen fertilizer. The results showed that the magnitude of greenhouse gas emissions in the tropical rice-vegetable rotation system was mainly determined by CH4 emissions in the late rice season. After two years of biochar application， only low biochar combined with nitrogen fertilizer had a significant emission reduction effect， but high and low biochar combined with nitrogen fertilizer increased the yield of early rice and pepper crops continuously.

Substance basis and pharmacological mechanism of heat-clearing herbs in the treatment of ischaemic encephalopathy: a systematic review and network pharmacology.

BACKGROUND AND OBJECTIVE: Ischaemic encephalopathy is a common cerebrovascular disease caused by insufficient blood supply to the cerebral vessels. The ischaemic encephalopathy is closely associated with the development of many chronic diseases such as obesity, hypertension and diabetes. Neurotrophic therapy has become the main therapeutic strategy for ischaemic encephalopathy. However, neurotrophic drugs only slightly recover the neurological function of patients, and their long-term efficacy is uncertain. Previous reports revealed that the active ingredients of natural medicines play important roles in the treatment of cerebral ischemia. In this study, we reviewed clearing herbs with anti-ischaemic encephalopathy functions using the data from quantitative statistical and network pharmacological exploration methods. We also discussed the different bioactive components and pharmacological effects of these herbs. METHODS: First, we collected Chinese herbal prescriptions against ischaemic encephalopathy in four databases. Then, we statistically analysed the frequency of application of heat-clearing herbs to obtain the commonly used heat-clearing herbs against ischaemic encephalopathy, and classified them according to their efficacy according to the statistical results, to summarize the mechanism of anti-ischaemic effects of different bioactive components; Second, the network database was used to obtain the above components of heat-clearing Chinese medicines and their corresponding targets of action, disease targets of ischaemic stroke; Venny 2.1.0 was used to obtain component-disease target intersections; Cytoscape was used to construct the 'Drug-Active Ingredient-Target Network Graph '; DAVID was used for GO and KEGG enrichment analysis. RESULTS: Literature and database screening involved 149 prescriptions, with a total of 269 flavours of Chinese medicines and 20 flavours of single-flavour heat-clearing Chinese medicines; The top nine in terms of frequency of use were Radix Paeoniae Rubra、Rehmanniae Radix Praeparata、Figwort Root、Cortex Moutan、Scutellariae Radix、Coptidis Rhizoma、Gardeniae Fructus、Cassiae Semen、Lonicerae Japonicae Flos. The common components obtained from network pharmacology were beta-sitosterol, quercetin, and stigmasterol, which mainly act on key targets such as RELA, AKT1, JUN, PRKACA, PTGS2, RAF1 and CHUK; and their active ingredients are mainly involved in signalling pathways such as Calcium, PI3K-Ak, MAPK, cAMP, IL-17, HIF-1, TNF, T-cell receptor, NF-kappa B and JAK-STAT. CONCLUSIONS: Heat-clearing herbs are useful and promising for the protection against and prevention of ischemic encephalopathy. The results of the network pharmacological studies are similar to the mechanisms of anti-ischemic encephalopathy of the active ingredients of the purgative herbs we have listed; Thin either directly protects cerebrovascular tissues by improving vascular permeability and reducing the area of infarcted tissues, or produces protective effects through molecular signaling pathways. It can be seen that the components of heat-clearing Chinese medicines can exert cerebroprotective effects through multiple pathways, which provides us with a reference for further development and study of heat-clearing Chinese medicines in the treatment of ischemic cerebrovascular diseases.

Network-based identification and mechanism exploration of active ingredients against Alzheimer's disease via targeting endoplasmic reticulum stress from traditional chinese medicine.

Alzheimer's disease is a neurodegenerative disease that leads to dementia and poses a serious threat to the health of the elderly. Traditional Chinese medicine (TCM) presents as a promising novel therapeutic therapy for preventing and treating dementia. Studies have shown that natural products derived from kidney-tonifying herbs can effectively inhibit AD. Furthermore, endoplasmic reticulum (ER) stress is a critical factor in the pathology of AD. Regulation of ER stress is a crucial approach to prevent and treat AD. Thus, in this study, we first collected kidney-tonifying herbs, integrated chemical ingredients from multiple TCM databases, and constructed a comprehensive drug-target network. Subsequently, we employed the endophenotype network (network proximity) method to identify potential active ingredients in kidney-tonifying herbs that prevented AD via regulating ER stress. By combining the predicted outcomes, we discovered that 32 natural products could ameliorate AD pathology via regulating ER stress. After a comprehensive evaluation of the multi-network model and systematic pharmacological analyses, we further selected several promising compounds for in vitro testing in the APP-SH-SY5Y cell model. Experimental results showed that echinacoside and danthron were able to effectively reduce ER stress-mediated neuronal apoptosis by inhibiting the expression levels of BIP, p-PERK, ATF6, and CHOP in APP-SH-SY5Y cells. Overall, this study utilized the endophenotype network to preliminarily decipher the effective material basis and potential molecular mechanism of kidney-tonifying Chinese medicine for prevention and treatment of AD.

Rhynchophylline inhibits methamphetamine dependence via modulating the miR-181a-5p/GABRA1 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Uncaria rhynchophylla (Miq.) Miq. ex Havil. is a plant species that is routinely devoted in traditional Chinese medicine to treat central nervous system disorders. Rhynchophylline (Rhy), a predominant alkaloid isolated from Uncaria rhynchophylla (Miq.) Miq. ex Havil., has been demonstrated to reverse methamphetamine-induced (METH-induced) conditioned place preference (CPP) effects in mice, rats and zebrafish. The precise mechanism is still poorly understood, thus further research is necessary. AIM OF STUDY: This study aimed to investigate the role of miRNAs in the inhibitory effect of Rhy on METH dependence. MATERIALS AND METHODS: A rat CPP paradigm and a PC12 cell addiction model were established. Microarray assays were used to screen and identify the candidate miRNA. Behavioral assessment, real-time PCR, dual-luciferase reporter assay, western blotting, stereotaxic injection of antagomir/agomir and cell transfection experiments were performed to elucidate the effect of the candidate miRNA and intervention mechanism of Rhy on METH dependence. RESULTS: Rhy successfully reversed METH-induced CPP effect and the upregulated miR-181a-5p expression in METH-dependent rat hippocampus and PC12 cells. Moreover, suppression of miR-181a-5p by antagomir 181a reversed METH-induced CPP effect. Meanwhile, overexpression of miR-181a-5p by agomir 181a in combination with low-dose METH (0.5 mg/kg) elicited a significant CPP effect, which was blocked by Rhy through inhibiting miR-181a-5p. Finally, the result demonstrated that miR-181a-5p exerted its regulatory role by targeting γ-aminobutyric acid A receptor α1 (GABRA1) both in vivo and in vitro. CONCLUSION: This finding reveals that Rhy inhibits METH dependence via modulating the miR-181a-5p/GABRA1 axis, which may be a promising target for treatment of METH dependence.

Dendrobium nobile protects against ovalbumin-induced allergic rhinitis by regulating intestinal flora and suppressing lung inflammation.

Antibiotic exposure-induced dysbiosis of the intestinal flora increases the risk of developing allergic rhinitis. Hence, regulating the balance of intestinal flora may be useful for preventing and treating allergic rhinitis. However, the underlying mechanism is unclear. Dendrobium nobile (Shihu) exhibits anti-inflammatory and immune activities. Hence, in this study, we investigated the mechanism via which Shihu may improve allergic rhinitis. Mouse models of allergic rhinitis with intestinal flora dysbiosis (Model-D, antibiotics induce intestinal flora dysbiosis with ovalbumin-induced allergy) and normal intestinal flora with allergic rhinitis (Model-N, ovalbumin-induced allergy) were established. The effect of Shihu on intestinal flora and inflammation caused during allergic rhinitis were analyzed. Allergic symptoms, infiltration of hematoxylin and eosin in the lungs and nose, and the release of various factors [interleukin (IL)-2, IL-4, IFN-γ, IL-6, IL-10, and IL-17] in the lungs were evaluated. The results indicate that intestinal flora dysbiosis exacerbated lung and nose inflammation in allergic rhinitis. However, treatment with the Shihu extract effectively reversed these symptoms. Besides, the Shihu extract inhibited the PI3K/AKT/mTOR pathway and increased the level of Forkhead box protein in the lungs. Additionally, the Shihu extract reversed intestinal flora dysbiosis at the phylum and genus levels and improved regulator T cell differentiation. Furthermore, in the Model-D group, the Shihu extract inhibited the decrease in the diversity and abundance of the intestinal flora. Screening was performed to determine which intestinal flora was positively correlated with Treg differentiation using Spearman's correlation analysis. In conclusion, we showed that Shihu extract restored the balance in intestinal flora and ameliorated inflammation in the lungs of allergic rhinitis mice and predicted a therapeutic new approach using Traditional Chinese Medicine to improve allergic rhinitis.

Luteoloside protects the vascular endothelium against iron overload injury via the ROS/ADMA/DDAH II/eNOS/NO pathway.

Iron overload injury is considered to be a part of blood stasis syndrome of arthralgia in traditional Chinese medicine. Its primary therapies include clearing heat and detoxification, activating blood circulation, and removing blood stasis. Lonicera japonica flos (LJF) has long been known as an excellent antipyretic and antidote. Luteoloside (Lut) is one of the main components of LJF and exhibits antioxidant, anti-inflammatory, and cytoprotective properties. However, the protection of Lut against iron overload injury and its underlying mechanisms remain unclear. Therefore, HUVECs were exposed to 50 µmol·L-1 iron dextran for 48 h to establish an iron overload damage model and the effects of Lut were assessed. Our results showed that 20 µmol·L-1 Lut not only increased cell viability and weakened LDH activity, but also significantly up-regulated DDAHⅡ expression and activity, increased p-eNOS/eNOS ratio and NO content, and reduced ADMA content in HUVECs exposed to iron overload. Furthermore, Lut significantly attenuated intracellular/mitochondrial ROS generation, improved SOD, CAT, and GSH-Px activities, reduced MDA content, maintained MMP, inhibited mPTP opening, prevented cyt c from mitochondria released into cytoplasm, reduced cleaved-caspase3 expression, and ultimately decreased cell apoptosis induced by iron overload. The effects of Lut were similar to those of L-arginine (an ADMA competitive substrate), cyclosporin A (a mPTP blocker agent), and edaravone (a free radical scavenger) as positive controls. However, addition of pAD/DDAH II-shRNA adenovirus reversed the above beneficial effects of Lut. In conclusion, Lut can protect HUVECs against iron overload injury via the ROS/ADMA/DDAH II/eNOS/NO pathway. The mitochondria are the target organelles of Lut's protective effects.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Friedelin via Genetic Manipulation.

Friedelin, the most rearranged pentacyclic triterpene, also exhibits remarkable pharmacological and anti-insect activities. In particular, celastrol with friedelin as the skeleton, which is derived from the medicinal plant Tripterygium wilfordii, is a promising drug due to its anticancer and antiobesity activities. Although a previous study achieved friedelin production using engineered Saccharomyces cerevisiae, strains capable of producing high-level friedelin have not been stably engineered. In this study, a combined strategy was employed with integration of endogenous pathway genes into the genome and knockout of inhibiting genes by CRISPR/Cas9 technology, which successfully engineered multiple strains. After introducing an efficient TwOSC1T502E, all strains with genetic integration (tHMG1, ERG1, ERG20, ERG9, POS5, or UPC2.1) showed a 3.0∼6.8-fold increase in friedelin production compared with strain BY4741. Through further double knockout of inhibiting genes, only strains GD1 and GD3 produced higher yields. Moreover, strains GQ1 and GQ3 with quadruple mutants (bts1; rox1; ypl062w; yjl064w) displayed similar increases. Finally, the dominant strain GQ1 with TwOSC1T502E was cultured in an optimized medium in shake flasks, and the final yield of friedelin reached 63.91 ± 2.45 mg/L, which was approximately 65-fold higher than that of the wild-type strain BY4741 and 229% higher than that in ordinary SD-His-Ura medium. It was the highest titer for friedelin production to date. Our work provides a good example for triterpenoid production in microbial cell factories and lays a solid foundation for the mining, pathway analysis, and efficient production of valuable triterpenoids with friedelin as the skeleton.

Danggui-Shaoyao-San improves cognitive impairment through inhibiting O-GlcNAc-modification of estrogen α receptor in female db/db mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine formula Danggui-Shaoyao-San (DSS) has been reported to show therapeutic effect on dementia. AIM OF THE STUDY: The present study aims to investigate whether DSS treatment could alleviate diabetes-induced cognitive dysfunction, and explores its neuroprotective mechanism on db/db mice. MATERIALS AND METHODS: The female db/db mice were randomly divided into model group, DSS low-dose group and DSS high-dose group. Homologous female db/m mice were used as the control group. DSS was intragastric administrated for 15 weeks. Glucose tolerance, insulin tolerance, blood glucose and blood lipid levels were measured. Morris water maze was used to measure spatial learning and memory ability in mice. Nissl staining and Tunel staining were used to measure the changes of brain neurons, and ELISA kits were used to measure levels of inflammatory mediators (PGE2, TXB2 and LTB4). The kits detected oxidative stress (MDA, SOD, CAT, GSH-PX), nitrosative stress (NO, iNOS, TNOS) and glucose metabolism (LDH, PK, HK) levels. Western blot and immunofluorescence detected neurotrophic factors (PSD95, BDNF, NGF and SYN), apoptosis (Bcl-2, Bax, Bcl-xl, Caspase-3) and changes of ERα, O-GlcNAc, OGT, OGA levels. RESULTS: Morris water maze results showed that DSS could improve the learning and memory abilities of female db/db mice. Nissl staining showed that DSS could relieve hippocampal neurons damage of db/db mice. In addition, the serological tests showed that DSS could improve the impaired glucose tolerance and insulin resistance, while reduce hyperlipemia in db/db mice. Besides, DSS treatment increased the activities of SOD, GSH-PX, and CAT, and reduced MDA, NO, iNOs, tNOS, PGE2, TXB2 and LTB4 levels. Western blot and immunofluorescence results of PSD95, BDNF, NGF, and SYN showed that DSS could improve the expressions of neurotrophic factors. Meanwhile, Tunel staning and Western blot (Bcl-2, Bax, Bcl-xl, Caspase-3) results indicated that DSS could reduce neuronal apoptosis. Finally, Western blot (ERα, O-GlcNAc, OGA, and OGT) and immunofluorescence (ERα and O-GlcNAc) results indicated that DSS could increase the levels of ERα and OGA, decrease the levels of O-GlcNAc and OGT. CONCLUSION: DSS alleviate DE might be related to improve the abnormal O-GlcNAc-modification of ERα.

[Discussion of optimization of sham acupuncture setting based on a clinical trial about acupuncture treatment of migraine published in British Medical Journal].

In the randomized controlled clinical trials of acupuncture, the setting of the control group (sham acupuncture) directly affected the interpretations about their outcomes (beyond placebo), and has been being the hot spot and difficult problem. In the present paper, we discussed various types of sham acupuncture (invade and non-invade needling) commonly used nowadays and made an in-depth analysis on the factors contributing to the successful blinding to patients with episodic migraine without aura in a clinical study published in British Medical Journal (2020, 368:m697). Moreover, we put forward some thoughts on how to optimize the setting of sham acupuncture in the treatment of pain diseases. These thoughts are 1) setting different placebo control group for different types of pain, 2) selecting conventional acupoints not associated with the disease, 3) selecting the most sui-table type of placebo acupuncture through pre-tests, 4) choosing the distal non-meridian and non-acupoint not in the same neuronal segment with the pain locus when using non-invade consolation needling, 5) trying best to reduce the patients' doubts about placebo acupuncture operation, 6) selecting subjects with little or without acupuncture experience for multicenter studies, and 7) trying best to select objective indicators and to avoid the subjects' report bias when evaluating the effects of acupuncture and consolation acupuncture.

Chocolate consumption and all-cause and cause-specific mortality in a US population: a post hoc analysis of the PLCO cancer screening trial.

Few studies with mixed results have examined the association between chocolate consumption and mortality. We aimed to examine this association in a US population. A population-based cohort of 91891 participants aged 55 to 74 years was identified. Chocolate consumption was assessed via a food frequency questionnaire. Cox regression was used to estimate risk estimates. After an average follow-up of 13.5 years, 19586 all-cause deaths were documented. Compared with no regular chocolate consumption, the maximally adjusted hazard ratios of all-cause mortality were 0.89 [95% confidence interval (CI) 0.84-0.94], 0.84 (95% CI 0.79-0.90), 0.86 (95% CI 0.81-0.93), and 0.87 (95% CI 0.82-0.93) for >0-0.5 servings/week, >0.5-1 serving/week, >1-2 servings/week, and >2 servings/week, respectively (Ptrend = 0.009). A somewhat stronger inverse association was observed for mortality from cardiovascular disease and Alzheimer's disease. A nonlinear dose-response pattern was found for all-cause and cardiovascular mortality (all Pnonlinearity < 0.01), with the lowest risk observed at chocolate consumption of 0.7 servings/week and 0.6 servings/week, respectively. The favorable associations with all-cause and cardiovascular mortality were found to be more pronounced in never smokers than in current or former smokers (all Pinteraction < 0.05). In conclusion, chocolate consumption confers reduced risks of mortality from all causes, cardiovascular disease, and Alzheimer's disease in this US population.

Fabrication of a Polylactide-Glycolide/Poly-ε-Caprolactone/Dextran/Plastrum Testudinis Extract Composite Anti-Inflammation Nanofiber Membrane via Electrospinning for Annulus Fibrosus Regeneration.

Tissue engineering is a promising approach for the treatment of chronic lower back pain (LBP) caused by intervertebral disc degeneration (IDD) resulting from degeneration and inflammation of annulus fibrosus (AF) tissue. However, scaffold with an anti-inflammatory effect on AF cells has not been reported. In this study, we fabricated a polylactide-glycolide (PLGA)/poly-ε-caprolactone (PCL)Zdextran (DEX) composite membrane loaded with plastrum testudinis extract (PTE), a Traditional Chinese Medicine herbal extract, via electrospinning. The membranes were characterized by mechanical measurements and scanning electron microscopy (SEM). Using an in vitro inflammation model induced by interleukin (IL)-1ß, the cytocompatibility and anti-inflammatory effects of the composites were investigated by CCK-8 assay and flow cytometry. Potential regulatory mechanisms were examined by RT-qPCR and Western blotting. The results showed that the P10P8D2 (PLGA 10 g, PCL 8 g, DEX 2 g) composite nanofiber membrane exhibited the most uniform diameter distribution, best mechanical properties, a moderate degradation rate, and the best cytocompatibility characteristics. The optimal concentration of PTE was 120 µg/mL. Importantly, P10P8D2 combined with PTE exhibited anti-inflammatory and cell proliferation promotion effects. Moreover, the NF-κBB/NLRP3/IL-ß signaling pathway was inactivated. Our findings suggested that the nanofiber membrane composed of P10P8D2 and PTE has anti-inflammatory and pro-proliferation effects on AF cells. It may provide an effective strategy for AF tissue regeneration.

One-step removal of harmful algal blooms by dual-functional flocculant based on self-branched chitosan integrated with flotation function.

Harmful algal blooms induce severe environmental problems. It is challenging to remove algae by the current available treatments involving complicate process and costly instruments. Here, we developed a CaO2@PEG-loaded water-soluble self-branched chitosan (CP-SBC) system, which can remove algae from water in one-step without additional instrumentation. This approach utilizes a novel flocculant (self-branched chitosan) integrated with flotation function (induced by CaO2@PEG). CP-SBC exhibited better flocculation performance than commercial flocculants, which is attributed to the enhanced bridging and sweeping effect of branched chitosan. CP-SBC demonstrated outstanding biocompatibility, which was verified by zebrafish test and algae activity test. CaO2@PEG-loaded self-branched chitosan can serve as an "Air flotation system" to spontaneous float the flocs after flocculation by sustainably released O2. Furthermore, CP-SBC can improve water quality through minimizing dissolved oxygen depletion and reducing total phosphorus concentrations.

Explore of the beneficial effects of Huang-Lian-Jie-Du Decoction on diabetic encephalopathy in db/db mice by UPLC-Q-Orbitrap HRMS/MS based untargeted metabolomics analysis.

Diabetic encephalopathy (DE) is a severe diabetic complication with cognitive dysfunction. Huang-Lian-Jie-Du Decoction (HLJDD), a famous traditional Chinese formula, is effective for the treatment of diabetes mellitus and Alzheimer's disease in clinical practices, however, the therapeutic effects and the underlying mechanisms of HLJDD on DE is unclear yet. With this purpose, behavior test, brain histological and biochemical analysis were estimated to assess the beneficial effects of HLJDD on DE. Plasma samples were collected for metabolomics analysis based on UPLC-Q-Orbitrap HRMS/MS and chemometric analysis. As a result, morris water maze test revealed that HLJDD could effectively improve the learning and memory abilities in db/db mice. Brain histological and biochemical analysis indicated that HLJDD could protect against neurodegeneration and oxidative stress in db/db mice. Meanwhile, a total of 21 potential biomarkers with significant differences were identified between Model group and Control group using untargeted metabolomics strategy. Among them, 11 metabolites showed a trend towards the normal levels after HLJDD intervention. These metabolites principally involved in glycerophospholipid metabolism, fatty acid ß-oxidation, linoleic acid metabolism, glucose metabolism and glutathione metabolism based on the metabolic pathway analysis, which were regulated in DE model mice after HLJDD intervention. Generally, the results demonstrated that HLJDD had beneficial effects on DE, which could be mediated via ameliorating the metabolic disorders.

Screened antipsychotic drugs inhibit SARS-CoV-2 binding with ACE2 in vitro.

AIM: The coronavirus disease 2019 (COVID-19) pandemic has swept the globe and no specific effective drug has been identified. Drug repurposing is a well-known method to address the crisis in a time-critical fashion. Antipsychotic drugs (APDs) have been reported to inhibit DNA replication of hepatitis B virus, measles virus germination, and HIV infection, along with replication of SARS-CoV and MERS-CoV, both of which interact with host cells as SARS-CoV-2. METHODS: Nineteen APDs were screened using ACE2-HEK293T cell membrane chromatography (ACE2-HEK293T/CMC). Cytotoxicity assay, coronavirus spike pseudotype virus entry assay, surface plasmon resonance, and virtual molecular docking were applied to detect affinity between ACE2 protein and drugs and a potential antiviral property of the screened compounds. KEY FINDINGS: After the CMC screening, 8 of the 19 APDs were well-retained on ACE2-HEK293T/CMC column and showed significant antiviral activities in vitro. Three quarters of them belong to phenothiazine and could significantly inhibit the entrance of coronavirus into ACE2-HEK293T cells. Aother two drugs, aripiprazole and tiapride, exhibited weaker inhibition. We selected five of the drugs for subsequent evaluation. All five showed similar affinity to ACE2 and virtual molecular docking demonstrated they bound with different amino acids respectively on ACE2 which SARS-CoV-2 binds to. SIGNIFICANCE: Eight APDs were screened for binding with ACE2, five of which demonstrated potential protective effects against SARS-CoV-2 through acting on ACE2. Although the five drugs have a weak ability to block SARS-CoV-2 with a single binding site, they may provide a synergistic effect in adjuvant therapy of COVID-19 infection.

Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus.

BACKGROUND: The novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019 and there is no sign that the epidemic is abating . The major issue for controlling the infectious is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been reported to treat the disease, but the underlying mechanism remains controversial. PURPOSE: The objective of this study is to investigate whether CQ and HCQ could be ACE2 blockers and used to inhibit 2019-nCoV virus infection. METHODS: In our study, we used CCK-8 staining, flow cytometry and immunofluorescent staining to evaluate the toxicity and autophagy of CQ and HCQ, respectively, on ACE2 high-expressing HEK293T cells (ACE2h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking and surface plasmon resonance (SPR) assays, 2019-nCoV spike pseudotyped virus was also used to observe the viropexis effect of CQ and HCQ in ACE2h cells. RESULTS: Results showed that HCQ is slightly more toxic to ACE2h cells than CQ. Both CQ and HCQ could bind to ACE2 with KD = (7.31 ± 0.62)e-7 M and (4.82 ± 0.87)e-7 M, respectively. They exhibit equivalent suppression effect for the entrance of 2019-nCoV spike pseudotyped virus into ACE2h cells. CONCLUSIONS: CQ and HCQ both inhibit the entrance 2019-nCoV into cells by blocking the binding of the virus with ACE2. Our findings provide novel insights into the molecular mechanism of CQ and HCQ treatment effect on virus infection.

Yan-Hou-Qing formula attenuates ammonia-induced acute pharyngitis in rats via inhibition of NF-κB and COX-2.

BACKGROUND: Yan Hou Qing (YHQ) is a Chinese medicinal formula designed to alleviate sore throat symptoms, but underlying mechanism of YHQ treatment for pharyngitis is poorly defined up to now. METHODS: In this study, the modulation of YHQ on pharyngitis is investigated in ammonia-induced acute pharyngitis rat models. After treatment with YHQ or dexamethasone respectively for five consecutive days, all rats were sacrificed for biomolecular and histopathologic study. Protein expressions of MAPKs, NF-κB, COX-2 and 5-LOX in pharyngitis tissue were evaluated by western blot analysis and the levels of TNF-α, IL-6, prostaglandin (PG) E2, leukotrienes (LT)-B4 and LT-D4 in pharyngeal tissue were measured via ELISA assay. Evans blue (EB) dye exudation test was performed parallelly to assess the integrity of pharyngeal tissue. RESULTS: Compared with normal control group, EB dye exudation, and inflammatory cytokines in the model group were significantly increased, and the pharynx tissue was obviously infiltrated by inflammatory cells. YHQ treatment improved the inflammatory infiltrate in pharyngeal tissue, and reduced EB dye exudation in AP rat models. The up-regulated TNF-α and IL-6 in pharyngeal tissue of AP were significantly reduced by YHQ through inhibition of phosphorylation of p38, Erk and NF-κB. YHQ treatment also reversed the increased level of PGE2 through down-regulation of COX-2. CONCLUSIONS: YHQ formula attenuated the pharyngitis related symptoms via suppression of COX-2 and phosphorylation of p38, Erk and NF-κB (p65).

[Chinese medicine master SHENG Xie-sun's creative viewpoint on acupuncture-moxibustion and acupoints of Zhejiang school].

The creative viewpoint of SHENG Xie-sun, the master of Zhejiang school of Chinese medicine is introduced regarding the theories of acupuncture-moxibustion and acupoints. It is stated by master SHENG that the five-tissue needling methods are applicable not only for physical disorders, but also for zangfu disorders. Of these methods, the nearby bone needling method presents an instructive significance in treatment of pain disorder. The "reinforcing in the upper and reducing in the lower" needling method supplements the reinforcing and reducing theory of acupuncture. Master SHENG has put forward the theory of flexible selection of acupoint, the phenomenon of acupoint groups and the application of transverse acupoint composition, as well as the measures for intensifying needling induction, promoting qi regulation of acupuncture and improving clinical effect. Besides, the seven Chinese characters are used to summarize acupoint composition, meaning "main point, supplementary point, nearby point, corresponding point, back-shu point, front-mu point and extra point" separately. This creative viewpoint provides a clearer and more feasible mode in treatment with acupuncture-moxibustion and is instructive proactively in clinical practice.

[Discussion on research idea of quality marker of Salvia miltiorrhiza based on biosynthetic pathway of tanshinone compounds].

Based on the theory of Q-marker, the hairy root of Salvia miltiorrhiza and S. miltiorrhiza in many provinces were studied. The relative expressions of SmCPS, SmKSL and CYP76AH1 genes in hairy roots were detected by real-time fluorescence quantitative PCR and the contents of tanshinoneⅡ_A, cryptotanshinone, tanshinoneⅠ, 1,2-dihydrotanshinone, ferruginol and miltiradiene were detected by UPLC and GC-MS, respectively. Statistical analysis shows as fllows: in the hairy root of S. miltiorrhiza, the content of miltiradiene and ferruginol is positively correlated with the content of tanshinone compounds in the downstream, and the relative expression of important genes in the biosynthetic pathway of tanshinone can reflect the content of tanshinone compounds to a certain extent; in many provinces of S. miltiorrhiza, the content of ferruginol and tanshinone compounds can also be found that there is a positive correlation between the contents. Based on the biosynthetic pathway of tanshinone compounds, which is a special index component in S. miltiorrhiza, this study focused on the important relationship between the upstream gene, the middle intermediate compound and the downstream tanshinone compound content of the biosynthetic pathway, and explored the possible research ideas of improving the quality marker system of S. miltiorrhiza, and then provided the possible research ideas for understanding and studying the quality marker of traditional Chinese medicine from the biosynthetic pathway.

Isolation and characterization of a glycosyltransferase with specific catalytic activity towards flavonoids from Tripterygium wilfordii.

Flavonoids are important secondary metabolites that exist in many medicinal plants. Flavonoid glycosyltransferases can transfer sugar moieties to their parent rings, producing various flavonoid glycosides with significant pharmacological activities. Here, we report the molecular cloning of the O-glycosyltransferase TwUGT2 from Tripterygium wilfordii and its catalytic activity was explored by heterologous expression in E. coli. The results showed that TwUGT2 has specific glycosyltransferase activity towards C-3 and 7 hydroxyl groups of flavonoids, thereby converting quercetin and pinocembrin into isoquercitrin and pinocembrin 7-O-beta-D-glucoside, respectively. The identification of TwUGT2 will provide a useful molecular tool for synthetic biology and contribute to drug discovery.[Formula: see text].

[Effect of GR24 on accumulation of diterpenoids in Tripterygium wilfordii suspension cells].

Terpenoids are main bioactive components in Tripterygium wilfordii,but the contents of some terpenoids are relatively low. In order to provide scientific evidence for the regulation of terpenoids in T. wilfordii,this research explored the effect of GR24 on accumulations of four diterpenoids( triptolide,tripterifordin,triptophenolide,and triptinin B) in T. wilfordii suspension cells by biological technology and UPLC-QQQ-MS/MS. The results indicated that 100 µmol·L-1 GR24 inhibited the accumulations of triptolide,tripterifordin,triptophenolide,and triptinin B to different degrees. Compared with the control group,the contents of 4 diterpenoids( in the induced group) were down to 96.59%,63.80%,61.02% and 33.59% in 240 h,respectively. Among them,the accumulation of triptinin B iswas significantly inhibited. In addition,the key time point of inhibitory effect was 120 h after induction with GR24 in some diterpenoids. This is the first systematic study focusing on the effect of GR24 on the accumulations of diterpenoids in T. wilfordii suspension cells. The dynamic accumulation ruleregularity of four diterpenoids after induced by GR24 was summarized,which laid a foundation for further study on the chemical response mechanism of terpenoids to GR24.