Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.

Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae.

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

Reduced SARS-CoV-2 infection risk is associated with the use of Seven-Flavor Herb Tea: A multi-center observational study in Shanghai, China.

OBJECTIVE: Omicron, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, is responsible for numerous infections in China. This study investigates the association between the use of Seven-Flavor Herb Tea (SFHT) and the risk of SARS-CoV-2 infection to develop precise and differentiated strategies for control of the coronavirus disease 2019 (COVID-19). METHODS: This case-control study was conducted at shelter hospitals and quarantine hotels in China. A total of 5348 laboratory-confirmed COVID-19 patients were enrolled between April 1 and May 31, 2022, while 2190 uninfected individuals served as healthy controls. Structured questionnaires were used to collect data on demographics, underlying diseases, vaccination status, and use of SFHT. Patients were propensity-score-matched using 1:1 nearest-neighbor matching of the logit of the propensity score. Subsequently, a conditional logistic regression model was used for data analysis. RESULTS: Overall, 7538 eligible subjects were recruited, with an average age of [45.54 ± 16.94] years. The age of COVID-19 patients was significantly higher than that of uninfected individuals ([48.25 ± 17.48] years vs [38.92 ± 13.41] years; t = 22.437, P < 0.001). A total of 2190 COVID-19 cases were matched with uninfected individuals at a 1:1 ratio. The use of SFHT (odds ratio = 0.753, 95% confidence interval: 0.692, 0.820) was associated with a lower risk of SARS-CoV-2 infection compared to untreated individuals. CONCLUSION: Our findings suggest that taking SFHT reduces the risk of SARS-CoV-2 infection. This is a useful study in the larger picture of COVID-19 management, but data from large-sample multi-center, randomized clinical trial are warranted to confirm the finding. Please cite this article as: Zhang SX, Chen XX, Zheng Y, Cai BH, Shi W, Ru M, Li H, Zhang DD, Tian Y, Chen YL. Reduced SARS-CoV-2 infection risk is associated with the use of Seven-Flavor Herb Tea: A multi-center observational study in Shanghai, China. J Integr Med. 2023; 21(4):369-376.

Functional Axis of PDE5/cGMP Mediates Timosaponin-AIII-Elicited Growth Suppression of Glioblastoma U87MG Cells.

Glioblastoma (GBM) is the most aggressive brain tumor, with high mortality. Timosaponin AIII (TIA), a steroidal saponin isolated from the medicinal plant Anemarrhena asphodeloides Bge., has been shown to possess anticancer properties in various cancer types. However, the effect of TIA on GBM is unknown. In this study, we reveal that TIA not only inhibited U87MG in vitro cell growth but also in vivo tumor development. Moreover, we found that the cause of TIA-induced cell growth suppression was apoptosis. When seeking to uncover antitumor mechanisms of TIA, we found that TIA diminished the expression of cGMP-specific phosphodiesterase 5(PDE5) while elevating the levels of guanylate cyclases (sGCß), cellular cGMP, and phosphorylation of VASPser239. Following the knockdown of PDE5, PDE5 inhibitor tadalafil and cGMP analog 8-Bro-cGMP both inhibited cell growth and inactivated ß-catenin; we reason that TIA elicited an antitumor effect by suppressing PDE5, leading to the activation of the cGMP signaling pathway, which, in turn, impeded ß-catenin expression. As ß-catenin is key for cell growth and survival in GBM, this study suggests that TIA elicits its anti-tumorigenic effect by interfering with ß-catenin function through the activation of a PDE5/cGMP functional axis.

Comparative Analysis of Aristolochic Acids in Aristolochia Medicinal Herbs and Evaluation of Their Toxicities.

Aristolochic acids (AAs) are a group of nitrophenanthrene carboxylic acids present in many medicinal herbs of the Aristolochia genus that may cause irreversible hepatotoxicity, nephrotoxicity, genotoxicity and carcinogenicity. However, the specific profile of AAs and their toxicity in Aristolochia plants, except for AAs Ι and ΙΙ, still remain unclear. In this study, a total of 52 batches of three medicinal herbs belonging to the Aristolochia family were analyzed for their AA composition profiles and AA contents using the UPLC-QTOF-MS/MS approach. The studied herbs were A. mollissima Hance (AMH), A. debilis Sieb.etZucc (ADS), and A. cinnabaria C.Y.Cheng (ACY). Chemometrics methods, including PCA and OPLS-DA, were used for the evaluation of the Aristolochia medicinal herbs. Additionally, cytotoxicity and genotoxicity of the selected AAs and the extracts of AMH and ADS were evaluated in a HepG2 cell line using the MTT method and a Comet assay, respectively. A total of 44 AAs, including 23 aristolochic acids and 21 aristolactams (ALs), were detected in A. mollissima. Moreover, 41 AAs (23 AAs and 18 ALs) were identified from A. debilis Sieb, and 45 AAs (29 AAs and 16 ALs) were identified in A. cinnabaria. Chemometrics results showed that 16, 19, and 22 AAs identified in AMH, ADS, and ACY, respectively, had statistical significance for distinguishing the three medicinal herbs of different origins. In the cytotoxicity assay, compounds AL-BΙΙ, AAΙ and the extract of AMH exhibited significant cytotoxicities against the HepG2 cell line with the IC50 values of 0.2, 9.7 and 50.2 µM, respectively. The results of the Comet assay showed that AAΙ caused relatively higher damage to cellular DNA (TDNA 40-95%) at 50 µM, while AAΙΙ, AMH and ADS extracts (ranged from 10 to 131 µM) caused relatively lower damage to cellular DNA (TDNA 5-20%).

Antiliver Fibrosis Formula of Fuzheng Huayu Alleviates Inflammatory Response.

Fuzheng Huayu's (FZHY) formula ameliorated liver fibrosis in clinical and experimental practice. Based on the close link between fibrosis and inflammation, its anti-inflammatory effect and related mechanisms were explored in this present study. With the aid of the inflammatory macrophage model, FZHY significantly blocked nitrite accumulation without observable cytotoxicity due to its suppression of inducible nitric oxide synthase (iNOS) gene and protein expressions in a concentration-depended manner. Proinflammatory mediators including IL-6, CD86, and CD40 were also restrained by FZHY. Interestingly, FZHY induced anti-inflammatory mediators heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) expressions simultaneously. Downregulation of iNOS and miR-155 and upregulation of PPAR-γ were also observed in CCl4-induced liver fibrosis mice upon FZHY administration. Mechanically, FZHY strikingly eliminated the phosphorylation of STAT1 and MAPK. Taken together, FZYH regulated the balance of proinflammatory and anti-inflammatory mediators partially via modulating STAT1/MAPK pathways and the miR-155/PPAR-γ axis.

[Pharmacodynamic material basis and anti-inflammatory mechanism of Chrysanthemum morifolium cv. Fubaiju based on UPLC-Q-TOF-MS/MS combined with network pharmacology].

The chemical components in rats after oral administration of the water extract of Chrysanthemum morifolium cv. Fubaiju(CMF) were analyzed by UPLC-Q-TOF-MS/MS technique. Forty-four compounds were identified from the water extract of CMF and 11 components were identified from the rat serum. A total of 264 potential anti-inflammatory targets were identified by network pharmacology based on serum components. The "component-target" network and protein-protein interaction(PPI) network were constructed, and GO function enrichment and KEGG pathway enrichment analyses were performed. The molecular docking was carried out to validate the results of network pharmacology. The results showed that CMF might act on AKT1, TNF, TP53, IL6, INS, and other core targets through apigenin, luteolin, acacetin, diosmetin, 3,4-O-dicaffeoylquinic acid, and other active components, and exert anti-inflammatory effects by regulating PI3 K-AKT signaling pathway, FoxO signaling pathway, cAMP signaling pathway, Ras signaling pathway, and other pathways. The pharmacodynamic materials basis of CMF was identified by UPLC-Q-TOF-MS/MS technology, and the core anti-inflammatory targets and the underlying mechanism of action were analyzed by network pharmacology and molecular docking, which provided a reference for comprehensively clarifying the pharmacodynamic materials basis and quality control of CMF.

Poria cocos water-soluble polysaccharide modulates anxiety-like behavior induced by sleep deprivation by regulating the gut dysbiosis, metabolic disorders and TNF-α/NF-κB signaling pathway.

Poria cocos (P. cocos) has been traditionally used as folk medicine and functional food in China for more than 2000 years. The water-soluble polysaccharide is the main component of P. cocos decoction. The effects and mechanisms of the water-soluble polysaccharide from P. cocos (PCWP) were investigated in chronic sleep deprivation (CSD)-induced anxiety in rats. CSD induced anxiety, gut dysbiosis, and inflammatory responses, and reduced neurotransmitter levels, whereas PCWP intervention ameliorated anxiety-like behaviors, increased the levels of 5-hydroxytryptamine, dopamine, norepinephrine, and γ-aminobutyric acid in the hypothalamus, regulated gastrointestinal peptide levels, reduced inflammatory factors, and inhibited the tumor necrosis factor (TNF)-α/nuclear factor (NF)-κB signaling pathway in rats with CSD. The changes in the intestinal flora composition were determined using 16S rDNA sequencing, and indicated that PCWP significantly improved species richness and diversity in the intestinal flora of rats with anxiety, and adjusted the abundance of the following dysregulated bacteria closer to that of the normal group: Rikenellaceae_RC9_gut_group, Ruminococcus, Prevotellaceae_UCG-001, Prevotellaceae_NK3B31_group, Fusicatenibacter. Metabolomics was used to analyze fecal samples to identify significantly altered metabolites in the PCWP-treated groups. Thirty-eight PCWP-related metabolites and four metabolic pathways such as sphingolipid metabolism, taurine and hypotaurine metabolism, vitamin B6 metabolism, and glycerophospholipid metabolism were explored. The results of serum metabolomics showed that 26 biomarkers were significantly changed after PCWP intervention compared with the model group. The regulatory effects of metabolic pathway enrichment on sphingolipid, phenylalanine, and taurine and hypotaurine metabolism, and validation results showed that PCWP intervention regulated the activity of enzymes involved in the above metabolic pathways. A strong correlation between intestinal bacteria and potential biomarkers was found. Our findings present new evidence supporting the potential effect of PCWP in preventing the progression of anxiety by inhibiting the TNF-α/NF-κB signaling pathway, alleviating metabolic disorders, and ameliorating the gut microflora imbalance.

[Mechanism of timosaponin Aâ ¢ in regulation of metabolism against glioblastoma growth].

Non-targeted metabonomics was used to investigate the metabolite changes in the glioblastoma orthotopic tumor-bearing mice after timosaponin AⅢ(TIA) intervention to explore the metabolic relevant mechanism of glioblastoma and TIA intervention. The mice were randomly divided into a blank group, a model group, and a TIA group. HPLC-LTQ-Orbitrap Elite liquid chromatography-mass spectrometry was used to detect the metabolite changes in the serum of rats in the three groups after treatment for 4 weeks. Principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were performed on the metabolites, and the differential metabolites were selected based on VIP values and P values(P<0.05). The results showed that TIA significantly inhibited the in vivo glioblastoma growth, but it had limited influence on body weight. Serum samples were clearly distinguishable among groups. As compared with the blank group, six metabolites including ceramide, succinic acid, α-ketoglutarate acid(αKG), citric acid, indophenol sulfate, and 3 a, 6 b, 7 b-trihydroxy-5 b-cholic acid in the model group significantly decreased. As compared with the model group, five metabolites except phenol sulfate, PC[20:4(5Z,7E,11Z,14Z)-OH(9)/diMe(9,3)], o-palmitoyl carnitine, α-ketoglutarate acid, and citric acid in the TIA group significantly increased. According to the MetaboAnalyst enrichment analysis, the metabolic pathways were enriched in the tricarboxylic acid cycle, and alanine, aspartic acid, and glutamate metabolism. These results show that during the glioblastoma growth process, the metabolites including αKG and citric acid are down-regulated, and TIA exerts the anti-glioblastoma growth effect through the regulation of tricarboxylic acid cycle, and alanine, aspartic acid, and glutamate metabolism to elevate the levels of αKG, citric acid, and other metabolites.

Xinglou Chengqi Decoction improves neurological function in experimental stroke mice as evidenced by gut microbiota analysis and network pharmacology.

The current study was designed to explore the brain protection mechanism of Xinglou Chengqi Decoction (XCD) based on gut microbiota analysis and network pharmacology. A transient middle cerebral artery occlusion (MCAO) model of mice was established, followed by behavioral evaluation, TTC and TUNEL staining. Additionally, to investigate the effects of gut microbiota on neurological function after stroke, C57BL/6 mice were treated with anti-biotic cocktails 14 days prior to ischemic stroke (IS) to deplete the gut microbiota. High-throughput 16S rDNA gene sequencing, metabonomics technique, and flow multifactor technology were used to analyze bacterial communities, SCFAs and inflammatory cytokines respectively. Finally, as a supplement, network pharmacology and molecular docking were applied to fully explore the multicomponent-multitarget-multichannel mechanism of XCD in treating IS, implicated in ADME screening, target identification, network analysis, functional annotation, and pathway enrichment analysis. We found that XCD effectively improved neurological function, relieved cerebral infarction and decreased the neuronal apoptosis. Moreover, XCD promoted the release of anti-inflammatory factor like IL-10, while down-regulating pro-inflammatory factors such as TNF-α, IL-17A, and IL-22. Furthermore, XCD significantly increased the levels of short chain fatty acids (SCFAs), especially butyric acid. The mechanism might be related to the regulation of SCFAs-producing bacteria like Verrucomicrobia and Akkermansia, and bacteria that regulate inflammation like Paraprevotella, Roseburia, Streptophyta and Enterococcu. Finally, in the network pharmacological analysis, 51 active compounds in XCD and 44 intersection targets of IS and XCD were selected. As a validation, components in XCD docked well with key targets. It was obviously that biological processes were mainly involved in the regulation of apoptotic process, inflammatory response, response to fatty acid, and regulation of establishment of endothelial barrier in GO enrichment. XCD can improve neurological function in experimental stroke mice, partly due to the regulation of gut microbiota. Besises, XCD has the characteristic of "multi-component, multi-target and multi-channel" in the treatment of IS revealed by network pharmacology and molecular docking.

Rhizosphere Microbiomes of Potato Cultivated under Bacillus subtilis Treatment Influence the Quality of Potato Tubers.

Plants serve as a niche for the growth and proliferation of a diversity of microorganisms. Soil microorganisms, which closely interact with plants, are increasingly being recognized as factors important to plant health. In this study, we explored the use of high-throughput DNA sequencing of the fungal ITS and bacterial 16S for characterization of the fungal and bacterial microbiomes following biocontrol treatment (DT) with Bacillus subtilis strain Bv17 relative to treatments without biocontrol (DC) during the potato growth cycle at three time points. A total of 5631 operational taxonomic units (OTUs) were identified from the 16S data, and 2236 OTUs were identified from the ITS data. The number of bacterial and fungal OTU in DT was higher than in DC and gradually increased during potato growth. In addition, indices such as Ace, Chao, Shannon, and Simpson were higher in DT than in DC, indicating greater richness and community diversity in soil following the biocontrol treatment. Additionally, the potato tuber yields improved without a measurable change in the bacterial communities following the B. subtilis strain Bv17 treatment. These results suggest that soil microbial communities in the rhizosphere are differentially affected by the biocontrol treatment while improving potato yield, providing a strong basis for biocontrol utilization in crop production.

Tongluo Huatan capsule improves cognitive function by regulating the endocytosis of N-methyl-D-aspartic acid receptors mediated by clathrin in a rat model of vascular dementia.

OBJECTIVE: To explore the neuroprotective mechanisms of Tongluo Huatan capsule (THC) in a rat model of vascular dementia (VD). METHODS: A rat model of VD was established by repeated clamping of bilateral common carotid arteries with the intraperitoneal injection of sodium nitroprusside solution. VD rats were administered THC, memantine hydrochloride, or distilled water daily for 14 d after operation. Learning and memory abilities were assessed using the step-down passive avoidance test, novel object recognition (NOR) test, and Morris water maze (MWM) test. Pathological changes in the hippocampus were observed through hematoxylin and eosin and Nissl staining. The expression levels of clathrin, RAB5B, and N-methyl-D-aspartic acid receptor 1 (NMDAR1) were measured by immunohistochemistry staining, real-time quantitative polymerase chain reaction and Western blot. RESULTS: Rats in VD group showed impaired learning and memory abilities (step-down passive avoidance, NOR, and MWM) and abnormalities in neuronal morphology (light microscopy) in the hippocampus. The mRNA or protein expression levels of clathrin and RAB5B were decreased, and NMDAR1 was increased in hippocampal tissues (P < 0.05). Administration of THC promoted the learning and memory abilities and the morphological structure of hippocampal neurons in VD rats. Besides, THC enhanced mRNA or protein expression levels of clathrin and RAB5B, and decreased NMDAR1 (P < 0.05). CONCLUSION: THC may improve cognitive functions by regulating the endocytosis of NMDA receptors mediated by clathrin.

Hedyotis diffusa plus Scutellaria barbata Suppress the Growth of Non-Small-Cell Lung Cancer via NLRP3/NF-κB/MAPK Signaling Pathways.

Hedyotis diffusa (HD) plus Scutellaria barbata (SB) have been widely used in antitumor clinical prescribes as one of herb pairs in China. We investigated the effect of aqueous extract from Hedyotis diffusa plus Scutellaria barbata at the equal weight ratio (HDSB11) in inhibiting the growth of murine non-small-cell lung cancer cell (NSCLC) line LLC in vivo and in vitro in this study. Compared with other aqueous extracts, HDSB11 showed the lowest IC50 in inhibiting cell proliferation at 0.43 mg/ml. Besides, HDSB11 effectively suppressed colony formation and induced cell apoptosis. The further assessment of HDSB11 on the murine Lewis-lung-carcinoma-bearing mouse model showed it significantly inhibited tumors' bioluminescence at the dose of 30 g crude drug/kg. Mechanistically, HDSB11 attenuated the expressions of NLRP3, procaspase-1, caspase-1, PRAP, Bcl-2, and cyclin D1 and downregulated the phosphorylation levels of NF-κB, ERK, JNK, and p38 MAPK. In conclusion, HDSB11 could alleviate cell proliferation and colony formation and induce apoptosis in vitro and tumor growth in vivo, partly via NF-κB and MAPK signaling pathways to suppress NLRP3 expression.

Ethyl Acetate Fraction from Hedyotis diffusa plus Scutellaria barbata Exerts Anti-Breast Cancer Effect via miR-200c-PDE7B/PD-L1-AKT/MAPK Axis.

BACKGROUND: Hedyotis diffusa (HD) Willd. and Scutellaria barbata (SB) D. Don in different ratios have been frequently used to treat various cancers in clinical Traditional Chinese Medicine prescriptions. However, the optimal ratio, active fraction, and molecular mechanisms associated with the anti-breast cancer role of this herbal couplet have not been elaborated. METHODS: To screen out the optimal ratio of this herbal couplet, we compare aqueous extracts of HD, SB, or HD plus SB in different weight ratios (HS11, HS12, HS21) for their anticancer effects on murine breast cancer 4T1 cells in vitro and in vivo. EA11, the ethyl acetate fraction from HS11 (the aqueous extract of the couplet at an equal weight ratio), is further assessed for its antiproliferative effect as well as the antitumorigenic impact with the aid of immunocompetent mice. Colony formation, flow cytometry, western blot, ELISA, and qRT-PCR are used to elucidate mechanisms underlying EA11-led effects. RESULTS: HS11 presents the most potential suppression of 4T1 cell proliferation and tumor growth among these aqueous extracts. The comparison results show that EA11 is more effective than HS11 in vitro and in vivo. EA11 inhibits colony formation and induces apoptosis in a concentration-dependent manner. EA11 reduces the protein expressions of PDE7B, PD-L1, ß-catenin, and cyclin D1 while elevating the concentration of cellular cAMP and miR-200c expression in 4T1 cells. Additionally, EA11 exerts its anticancer effect partially via the inactivation of MAPK and AKT signaling pathways. CONCLUSIONS: This study implicates that EA11 prevents breast tumor development by interfering with the miR-200c-PDE7B/PD-L1-AKT/MAPK axis. EA11 may represent a potential therapeutic candidate for breast cancer.

Ethyl Acetate Fraction from Hedyotis diffusa plus Scutellaria barbata Suppresses Migration of Bone-Metastatic Breast Cancer Cells via OPN-FAK/ERK/NF-κB Axis.

Hedyotis diffusa plus Scutellaria barbata is a couplet of medicinal that has been commonly used to treat inflammation-related diseases and various types of tumors. However, the effect of this couplet on tumor cell migration has not been elucidated. With the aid of MCF-7-BOM, a bone-metastatic subline of ER + breast cancer MCF-7, we showed that ethyl acetate fraction extracted at an equal weight ratio of Hedyotis diffusa plus Scutellaria barbata (EA11) inhibited cell migration of MCF-7-BOM in a concentration-dependent manner. To define the underlying molecular mechanism, we revealed that EA11 reduced the expression of osteopontin (OPN) and interfered with the FAK/ERK/NF-κB signaling pathways, which are both critical for breast cancer bone metastasis. This study strongly suggested EA11 may represent a potential therapeutic agent against bone metastasis of breast cancer.

[Spectrum-effect relationship between UPLC fingerprint of Smilax china and anti-pelvic inflammation in rats].

To study the correlation between ultra high performance liquid chromatography( UPLC) fingerprint of Smilax china and its anti-pelvic inflammatory effect,and to explore the pharmacodynamic material basis of S. china against pelvic inflammatory disease.UPLC fingerprints of 10 batches of S. china from different habitats were established,and the values of SOD,MDA,TNF-α,and IL-6 in rats with pelvic inflammation were measured. The weight of each single pharmacodynamics index to the total efficacy was determined by analytic hierarchy process,and the contribution of each peak in fingerprints to the each single pharmacodynamics index and total efficacy was analyzed by the grey relational analysis. Then the structures of chemical constituents at the identified peaks were confirmed by comparing with the reference substance. The 27 common characteristic peaks of UPLC fingerprints were all related to the anti-pelvic inflammation effect of S. china,of which 13 peaks were identified as peak 2( 3,5-dihydroxy-2-methylbenzoic acid-3-O-glucoside),peak 3( chlorogenic acid),peak 5( 2,7,4-trihydroxydihydroflavone-5-O-glucoside),peak 6( 7,4-dihydroxydihydroflavonol-5-O-glucoside),peak 7( taxifolin-7-O-glucoside),peak 9( taxifolin),peak 10( polydatin),peak 11( oxyresveratrol),peak 12( astilbin),peak15( resveratrol),peak 16( quercitrin),peak 18( engeletin) and peak 24( kaempferol). The correlation degree of 21 peaks and the total efficacy was greater than 0. 8,and the top 10 ranked by correlation degree were as follows: peak 1,3,7,19,18,17,4,11,16,and 21. The results showed that the anti-pelvic inflammation effect of S. china was achieved by the combined action of pharmacodynamic substances. In order to control the quality of S. china and its prepared slices more effectively,the index components of content detection should be selected reasonably.

Screening Five Qi-Tonifying Herbs on M2 Phenotype Macrophages.

Tumor-associated macrophages (TAMs) with M2 phenotype play an essential role in tumor microenvironment (TME) during the progression and development of numerous cancers and associated with poor prognosis. Thus, regulation of TAMs polarization emerged as a new strategy for tumor immune therapy. According to Traditional Chinese Medicine (TCM) theory, herbs with Qi-tonifying character are involved in improving the defense capacity of immune system. In this study, we screened extracts and ingredients from five Qi-tonifying herbs exhibiting an inhibitory effect on M2 polarization of murine macrophages RAW264.7 induced by IL-4 and IL-13. Among these candidates, total flavonoids from Glycyrrhiza Radix et Rhizoma (TFRG) and ethanol extract of Ginseng Radix et Rhizoma significantly inhibited the expression of Arginase-1 (Arg-1) (above 90% at 100µg/mL), one of the phenotype markers of M2 macrophages. The inhibition of total saponins of Ginseng Radix et Rhizoma, ethanol extract of Cordyceps, ethanol extract of Acanthopanacis senticosi Radix et Rhizoma Seu caulis, and ethanol extract of Astragali Radix reached above 50% at 100µg/mL. The inhibition of ingredients including glabridin, isoliquiritin apioside, lysionotin, cordycepin, astragaloside IV, and calycosin reached above 50% at 50µM. Then, we investigated the molecular mechanisms of TFRG. TFRG abolished the migration of murine breast cancer 4T1 stimulated by the conditioned medium from M2 macrophages (M2-CM). In addition to Arg-1, TFRG also antagonized the IL-4/13-mediated mRNA upregulation of the M2 markers including found in inflammatory zone 1 (FIZZ1), chitinase-3-like protein 3 (YM1), and mannose receptor (CD206) and upregulated the expression of inducible nitric oxide synthase (iNOS), one of the M1 markers. The further exploration showed that TFRG decreased the phosphorylation of STAT6 and increased the expression of miR-155. Our study provides a series of potential immune regulating natural products from five Qi-tonifying herbs on M2 phenotype. For instance, TFRG suppressed M2 polarization of macrophages partly by inactivating STAT6 pathway and enhanced the level of miR-155 to regulate the expressions of M1 and M2 markers.

[Regulatory effect and relevant mechanisms of fraction from heat-clearing and detoxifying herb couplet on macrophage M1/M2 phenotypes].

To explore the regulatory effect and relevant mechanisms of the fraction of Hedyotis diffusa and Scutellaria barbata herb couple(YDW11) on polarization of macrophage between M1/M2 phenotypes.RAW264.7 cells were induced with LPS/IFN-γ or IL-4/IL-13 to establish M1 or M2 macrophage cell model. MTT assay was used to measure the cell cytotoxicity of YDW11. Griess reaction was used to detect the changes of nitrite accumulation in the cell supernatant. Trans-well assay was used to measure the migration capability. QRT-PCR was used to assay mRNA expressions of iNOS, IL-1ß, Arg-1 and MR. Western blot was used to detect the effect of YDW11 on iNOS and Arg-1 protein expressions. Taqman MicroRNA RT-PCR was used to detect the effect of YDW11 on miR155 expression under M1 and M2 phenotype conditions. In addition, MS-UPLC assay was carried out to identify the constituents in YDW11. The results showed that the ethyl acetate of H. diffusa and S. barbata extracted in 1:1 ratio with water (YDW11) showed the activity in suppressing the nitrite content in M1 macrophages without cytotoxicity. YDW11 also inhibited the migration of breast cancer cells with the help of M2 macrophages by blocking their polarization towards M2. YDW11 decreased iNOS, IL-1ß, Arg-1and MR mRNA expressions and iNOS and Arg-1 protein expressions. YDW11 down-regulated miR155 expression in M1 phenotype, and up-regulated miR155 expression in M2 phenotype. Based on MS-UPLC,four compounds were identified in YDW11, including 4'-hydroxyacetophenone, scutellarin, luteolin and apigenin. YDW11 inhibited M1/M2 phenotypes of macrophages by regulating the expression of miR155.

Metabonomics delineates allergic reactions induced by Shuang-huang-lian injection in rats using ultra performance liquid chromatography-mass spectrometry.

Shuang-huang-lian Injection (SHLI) is the first successfully developed drug from traditional Chinese medicine (TCM) powder for injection, since its use for the treatment of acute respiratory tract infection, pneumonia, influenza, etc. At the same time, its allergic reactions have also emerged, which limits clinical applications. However, few scholars pay attention to the mechanism of allergic reactions. In this present study, metabonomics technology was used to explore the changes in endogenous metabolites in urine of the rat model of SHLI induced allergic reaction; we and analyzed the metabolites, metabolic pathway, and the mechanism which were closely related to the allergic reactions. The levels of serum histamine and tryptase were examined and changes in histomorphology were also observed. Based on the UPLC-Q-TOF/MS metabonomics, we carried out the pattern recognition analysis, selected potential biomarkers associated with allergic reactions, and explored the pathological mechanism for SHLI induced allergic reaction, which laid the foundation for the safety research of SHLI. Our results showed that SHLI increased the levels of serum histamine and tryptase in rats with allergic reaction; we determined 15 biomarkers in rat allergic reaction model induced by SHLI and found multiple metabolic pathways involved, such as metabolism of linolenic acid, phenylalanine, amino acid, 2-oxo acid, and purine and other metabolic pathways.

Total Flavonoids from Radix Glycyrrhiza Exert Anti-Inflammatory and Antitumorigenic Effects by Inactivating iNOS Signaling Pathways.

Inducible nitric oxide synthase (iNOS) plays an important role in inflammation, which has also been considered as a major driver of breast cancer disease progression. Radix Glycyrrhiza (RG) has been broadly used for its anti-inflammatory and antitumorigenic effects. However, the mechanisms of regulation of iNOS in inflammation and cancer have not been fully explored. Total flavonoids isolated from RG (TFRG) exhibited anti-inflammatory activity through the regulation of ERK/NF-κB/miR-155 signaling and suppression of iNOS expression in LPS/IFN-γ stimulated RAW264.7 macrophages without cytotoxicity. TFRG also markedly reduced tumor mass of breast cancer cell MDA-MB-231 xenografts with suppression of iNOS expression, formation of 3-nitrotyrosine (3-NT), and inactivation of protumorigenic JAK2/STAT3 signaling pathway. These results suggested that TFRG limited the development of breast cancer and inflammation due to its property of iNOS inhibition.