2,3-Butanediol from the leachates of pine needles induces the resistance of Panax notoginseng to the leaf pathogen Alternaria panax.

Compared with the use of monocultures in the field, cultivation of medicinal herbs in forests is an effective strategy to alleviate disease. Chemical interactions between herbs and trees play an important role in disease suppression in forests. We evaluated the ability of leachates from needles of Pinus armandii to induce resistance in Panax notoginseng leaves, identified the components via gas chromatography-mass spectrometry (GC-MS), and then deciphered the mechanism of 2,3-Butanediol as the main component in the leachates responsible for resistance induction via RNA sequencing (RNA-seq). Prespraying leachates and 2,3-Butanediol onto leaves could induce the resistance of P. notoginseng to Alternaria panax. The RNA-seq results showed that prespraying 2,3-Butanediol onto leaves with or without A. panax infection upregulated the expression of large number of genes, many of which are involved in transcription factor activity and the mitogen-activated protein kinase (MAPK) signaling pathway. Specifically, 2,3-Butanediol spraying resulted in jasmonic acid (JA) -mediated induced systemic resistance (ISR) by activating MYC2 and ERF1. Moreover, 2,3-Butanediol induced systemic acquired resistance (SAR) by upregulating pattern-triggered immunity (PTI)- and effector-triggered immunity (ETI)-related genes and activated camalexin biosynthesis through activation of WRKY33. Overall, 2,3-Butanediol from the leachates of pine needles could activate the resistance of P. notoginseng to leaf disease infection through ISR, SAR and camalexin biosynthesis. Thus, 2,3-Butanediol is worth developing as a chemical inducer for agricultural production.

Treatment with Commelina communis Extract Exerts Anti-inflammatory Effects in Murine Macrophages via Modulation of the Nuclear Factor-κB Pathway.

The incidence of severe inflammatory diseases caused by chronic inflammation has increased owing to unprecedented changes brought about by industrialization. In this study, we aimed to assess the effect of treatment of lipopolysaccharide- (LPS-) induced murine macrophages with Commelina communis Linne extract (CCE) on synthesis of nitric oxide (NO), hypersecretion of proinflammatory cytokines, intranuclear transition of the p65 subunit of nuclear factor- (NF-) κB, and degradation of the NF-κB inhibitor IκBα. Notably, CCE treatment did not affect cell viability even at a final concentration of 1.5 mg/mL. At a high concentration of CCE, the LPS-induced high levels of NO, tumor necrosis factor-α, interleukin- (IL-) 1ß, and IL-6 were decreased via downregulation of inducible NO synthase and proinflammatory cytokine mRNA expression. Furthermore, phosphorylation of IκBα was significantly decreased upon CCE treatment, and the intranuclear transition of NF-κB p65 triggered by LPS was inhibited at a high concentration of CCE. Polyphenols and flavonoids, secondary metabolites in CCE that regulate the NF-κB pathway, may be responsible for its anti-inflammatory activity. We suggest that CCE has anti-inflammatory effects related to suppression of the NF-κB pathway and can be used to treat chronic inflammation.

First Report of Leaf Spot on Panax notoginseng Caused by Caryophylloseptoria pseudolychnidis in Yunnan, China.

Panax notoginseng is a unique traditional medicinal plant in China, which has the effects of improving myocardial ischemia, protecting liver and preventing cardiovascular diseases (Jiang, 2020). In July 2021, gray-brown round spots were found on the leaves of P. notoginseng in the plantations of Lincang City (23º43´10˝N, 100º7´32˝E). By September, the symptoms were observed on more P. notoginseng plants, with incidence reaching 31%. Initial symptoms on leaves were small, brown spots that expanded, with black granular bulges on the lesions, often surrounded with yellow halo. As the disease progressed, multiple lesions merged, leaves became yellow, and abscission occurred. To isolate the causal pathogen, twelve symptomatic leaves were randomly obtained from twelve P. notoginseng plants. Small pieces of infected leaf tissues (about 5 mm2) were disinfected with 75% ethanol for 30 s, soaked in 2% sodium hypochlorite for 3 min, and then rinsed 3 times with sterile water and blotted dry. Sample tissues were plated on potato dextrose agar (PDA) plates incubated at 25℃ for 5 days with 12 h light/dark photoperiod. Hyphal-tips from the growing edge of colonies were transferred to fresh PDA to obtain pure cultures. Eight isolates were obtained with similar colony morphology, gray (top view) or black (back view) coloration, with a villous surface, and slow-growing on PDA. Conidia were hyaline, slender and obtuse to subobtuse at both ends, 10.3 to 52.62 (av. 25.2) µm × 1.4 to 4.0 (av. 2.4) µm (n=200) in size. Characteristics of the colonies and conidia were consistent with Caryophylloseptoria pseudolychnidis as described by Quaedvlieg et al. (2013) and Verkley et al. (2013). Genomic DNA of three representative isolates (LINC-4 to LINC-6) was extracted, and the rDNA-ITS region, ACT, and LSU gene regions were amplified and sequenced using the primer pairs ITS4/ITS5, 512F/783R, and LSU1Fd/LR5, respectively. Sequences have been deposited in GenBank (OK614104-OK614106 for ITS, OK614109-OK614111 for LSU, OK628350-OK628352 for ACT). BLAST search showed that all sequences were 98% to 100% homology with the corresponding sequences of C. pseudolychnidis. ITS sequences of the three isolates (LINC-4 to LINC-6) showed 99.21% identity (500/504 bp) to C. pseudolychnidis strain CBS 128630 (GenBank accession no. NR156266). LSU sequences of the three isolates showed 99.76% identity (823/825 bp) to C. pseudolychnidis strain CBS 128630 (MH876481). For ACT sequences, LINC-4 and LINC-5 showed 98.53% identity (201/204 bp) to C. pseudolychnidis strain 128614 (KF253599); LINC-6 showed 99.02% identity (202/204 bp) to C. pseudolychnidis strain 128614 (KF253599). Further, the neighbor-joining and maximum-likelihood method were used for multilocus phylogenetic analysis of the obtained sequences using MEGA-X (Kumar et al. 2018). The three isolates were clustered in the same clade with two C. pesudolychidis from database. Three isolates (LINC-4 to LINC-6) were tested for pathogenicity to confirm Koch's postulates. Annual potted P. notoginseng was inoculated with spore suspension (105 spores.mL-1). Each isolate was inoculated onto two leaves each of five P. notoginseng plants. The controls were similarly mock-inoculated with sterile water. To maintain high humidity (>90% RH), all plants were placed in transparent plastic boxes in a greenhouse at 25℃ with a 12 h light/dark photoperiod. Fifteen days post-inoculation, inoculated leaves showed similar symptoms to those observed in the field, and control plants remained healthy. The pathogen were reisolated from symptomatic leaf spots, and the colony characteristics were the same as those of the original isolates. Morphological characteristics, molecular data, and Koch's postulates tests confirmed C. pseudolychnidis as the cause of P. notoginseng leaf spot disease. To our knowledge, this is the first report of C. pseudolychnidis causing leaf spot on P. notoginseng in Yunnan, China. The spread of this disease might pose a serious threat to the production of P. notoginseng. The occurrence and spread of this pathogen should be further studied in order to formulate reasonable control measures.

Evodiae fructus Extract Inhibits Interleukin-1ß-Induced MMP-1, MMP-3, and Inflammatory Cytokine Expression by Suppressing the Activation of MAPK and STAT-3 in Human Gingival Fibroblasts In Vitro.

Periodontitis is a Gram-negative bacterial infectious disease. Numerous inflammatory cytokines, including interleukin-1ß (IL-1ß), regulate periodontitis pathophysiology and cause periodontal tissue destruction. In human gingival fibroblasts (HGFs), IL-1ß stimulates the production of matrix metalloproteinases (MMPs) and proinflammatory cytokines via various mechanisms. Several transcription factors, such as signal transducer and activator of transcription 3 (STAT-3), activator protein 1 (AP-1), and nuclear factor-κB (NF-κB), regulate gene expression. Mitogen-activated protein kinases (MAPKs) regulate these transcription factors. However, the MAPK/STAT-3 activation signal in HGFs is unknown. We investigated the potential inhibitory effects of the extract of Evodiae fructus (EFE), the dried, ripe fruit of Evodia rutaecarpa, on MMP and proinflammatory cytokine expression in IL-1ß-stimulated HGFs. EFE inhibited the expression of MMP-1, MMP-3, and proinflammatory cytokines (TNF-α, IL-6, and IL-8) in IL-1ß-stimulated HGFs through the inhibition of IL-1ß-induced MAPK/STAT-3 activation. Also, these results suggest that the EFE may be a useful for the bioactive material for oral care.

Aqueous extract of Chrysanthemum morifolium Ramat. inhibits RANKL-induced osteoclast differentiation by suppressing the c-fos/NFATc1 pathway.

OBJECTIVE: The flower of chrysanthemum, used worldwide as a medicinal and edible product, has shown various bioactivities, such as anti-inflammatory, antioxidant, anti-tumorigenic, and hepatoprotective activities, as well as cardiovascular protection. However, the effect of Chrysanthemum morifolium Ramat. on the regulation of osteoclast differentiation has not yet been reported. In this study, we aimed to investigate the inhibitory effect of Chrysanthemum morifolium Ramat. water extract (CME) on RANKL-induced osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). STUDY DESIGN: Bone marrow-derived macrophages (BMMs) isolated from the C57BL/6 J mice. The viability of BMMs was detected with MTT assays. Inhibitory effects of CME on osteoclast differentiation and bone resorption was measured by TRAP staining and Pit assay. Osteoclast differentiation-associated gene expression were assessed by Real-time quantitative polymerase chain reaction. Intracellular signaling molecules was assessed by western blot. RESULTS: CME significantly inhibited osteoclast differentiation in BMMs without cytotoxicity, besides inhibiting MAPK/c-fos and PLCγ2/CREB activation. The inhibitory effects of CME on differentiation-related signaling molecules resulted in significant repression of NFATc1 expression, which is a key transcription factor in osteoclast differentiation, fusion, and activation. CONCLUSION: Our results confirmed the inhibition of RANKL-induced PLCγ2/CREB/c-fos/NFATc1 activation by CME during osteoclast differentiation. The findings collectively suggested CME as a traditional therapeutic agent for osteoporosis, RA, and periodontitis.

Petchiether A attenuates obstructive nephropathy by suppressing TGF-ß/Smad3 and NF-κB signalling.

Obstructive nephropathy is the end result of a variety of diseases that block drainage from the kidney(s). Transforming growth factor-ß1 (TGF-ß1)/Smad3-driven renal fibrosis is the common pathogenesis of obstructive nephropathy. In this study, we identified petchiether A (petA), a novel small-molecule meroterpenoid from Ganoderma, as a potential inhibitor of TGF-ß1-induced Smad3 phosphorylation. The obstructive nephropathy was induced by unilateral ureteral obstruction (UUO) in mice. Mice received an intraperitoneal injection of petA/vehicle before and after UUO or sham operation. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin-1ß and tumour necrosis factor-α) and reducing extracellular matrix deposition (α-smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF-κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down-regulated the expression of the fibrotic marker collagen I in TGF-ß1-treated renal epithelial cells. Further, we found that petA dose-dependently suppressed Smad3-responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF-ß/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF-ß/Smad3 signalling.

Artemisia princeps Inhibits Growth, Biofilm Formation, and Virulence Factor Expression of Streptococcus mutans.

This study was designed to determine whether the ethanol extract of Artemisia princeps could inhibit the cariogenic activity of Streptococcus mutans. The increase in acid production and biofilm formation by S. mutans were evaluated. The expression levels of virulence factor genes were determined by performing the real-time polymerase chain reaction (PCR). The bactericidal effect was tested by confocal laser scanning microscopy. The A. princeps extract was observed to inhibit the growth of S. mutans at concentrations >0.05 mg/mL (P < .05). After using the safranin staining method, we found that the A. princeps extract had an inhibitory effect against biofilm formation at a concentration of >0.05 mg/mL. These experimental results were similar to that observed with the scanning electron microscopy. The results of the confocal microscopy revealed that the A. princeps extract at high concentrations of 0.4-3.2 mg/mL showed a bactericidal effect in a concentration-dependent manner. According to the results of the real-time PCR analysis, it was observed that the A. princeps extract inhibited the expression of virulence factor genes. These results suggest that A. princeps may inhibit the cariogenic activity of S. mutans, and may be useful as an anticariogenic agent.

Monitoring and Analysis of Chinese Chronic Myeloid Leukemia Patients Who Have Stopped Tyrosine Kinase Inhibitor Therapy.

Discontinuation of tyrosine kinase inhibitor (TKI) therapy after achieving a persistent deep molecular response (DMR) is an urgently needed treatment goal for chronic myeloid leukemia (CML) patients and has been included in the National Comprehensive Cancer Network (NCCN) guidelines (version 2.2017) for CML. Indeed, various studies have confirmed the feasibility of discontinuing TKI therapy. In this study, we analyzed data from 45 CML patients who had discontinued TKI therapy. Univariate analysis was performed to predict factors that were potentially related to treatment-free remission (TFR) and identify the differences between early relapse and late relapse. Out of the 45 patients, 20 exhibited molecular relapse after a median follow-up of 18 months (range, 1-54 months), and the estimated TFR at 24 months was 40%. The univariate analysis revealed that a high Sokal score and interruptions or dose reductions during TKI treatment were the only baseline factors associated with poor outcomes. Our results indicate that TKI discontinuation could be successfully put into practice in China.

Evaluation of the ratio of the estimated area under the concentration-time curve to minimum inhibitory concentration (estimated AUIC) as a predictor of the outcome for tigecycline treatment for pneumonia due to multidrug-resistant bacteria in an intensive care unit.

OBJECTIVES: Based on pharmacokinetics/pharmacodynamics (PK/PD) and the minimum inhibitory concentration (MIC) of tigecycline (TGC), dose increases have been advocated to maximize the efficacy against pneumonia that is suspected to be due to multidrug-resistant (MDR) bacteria in an intensive care unit. This practice-based study explored the relationship between the predicted PK parameter, the ratio of the area under the concentration-time curve to the 24 h of dosing/minimum inhibitory concentration (AUC0-24/MIC or AUIC), and the clinical and microbiological outcomes in critically ill patients with pneumonia due to MDR bacteria. METHODS: We conducted a prospective cohort study of the treatment of pneumonia due to MDR bacteria in an intensive care unit. The study patients were recruited and assigned to either TGC standard dose (SD, 50 mg q12 h) or high dose (HD, 100 mg q12 h) for the treatment of pneumonia due to MDR bacteria depending on the doctors' decisions. The relationships between the PK/PD parameters and outcomes were examined. RESULTS: Over the study period, 105 patients were included in the study. Whereas C1/2, Cmin, MIC and AUC were dramatically higher in the HD group than in the SD group (all P < 0.05), the Cmax and AUIC had no difference in both groups (all P > 0.05). The patients in the HD group had a higher clinical cure rate than those in the SD group (P = 0.029), but the bacterial eradication rate and survival rate of the patients in the HD group were not better than those in SD group (P = 0.279 and 0.416, respectively). The Cmax, C1/2, Cmin and AUC in the cured group were higher than those in failure group (all P < 0.05). The MICs were dramatically higher in the failure group than those in cure group (P = 0.0001), which led to significantly lower AUICs (P = 0.0001). In the ROC analysis, the areas of Cmax, C1/2, Cmin, AUC, negative-MIC and AUIC under the ROC curve were 0.64, 0.69, 0.67, 0.66, 0.73 and 0.82, respectively. The sensitivity was ascertained to be 75% and the specificity was 89% when the AUIC cut-off value was considered to be 10.12. Moreover, the sensitivity was ascertained to be 63% and the specificity was 80% when the MIC cut-off value was considered to be 1.75 mg/L. CONCLUSIONS: The AUIC and MIC are associated with tigecycline treatment outcomes in pneumonia due to MDR bacteria, and aiming to achieve an individualized AUIC ≥ 10.12 when MIC < 1.75 mg/L could improve outcomes.

Traditional Chinese herbal medicine for vascular dementia.

BACKGROUND: Traditional Chinese herbal medicine (TCHM) is widely used for treating vascular dementia (VaD) in China. Recent studies of a number of TCHMs have demonstrated in vitro biological activity and therapeutic effects in animals, but the published clinical evidence has not been systematically appraised. OBJECTIVES: To evaluate the efficacy and safety of TCHMs listed in either the Chinese Pharmacopoeia (CP) or the Chinese National Essential Drug List (NEDL) that are used to treat VaD. A secondary aim was to identify promising TCHMs for further clinical research. SEARCH METHODS: We searched ALOIS, the Cochrane Dementia and Cognitive Improvement Group's Specialised Register (on 14 March 2018) and also several Chinese biomedical databases: the Chinese Biological Medicine Database (January 1979 to May 2015), Wanfang database (January 1998 to May 2015), Chongqing VIP Information Co. Ltd or Weipu (January 1998 to May 2015) and the Chinese National Knowledge Infrastructure (January 1979 to May 2015). SELECTION CRITERIA: We included randomised controlled trials (RCTs) of TCHMs compared to placebo, to Western medicine (WM) or to routine therapy for VaD risk factors. Eligible participants were men and women aged 18 years and above, diagnosed with VaD by any of the following four criteria: (1) Diagnostic and Statistical Manual of Mental Disorders (DSM) versions III, III-R, IV, IV-TR; (2) National Institute of Neurological Disorders and Stroke (NINDS-AIREN); (3) International Classification of Diseases 9 or 10; (4) the Hachinski or the Modified Hachinski Ischaemic Score. We required the use of an imaging technique to differentiate VaD from other dementias. We excluded (1) trials with participants diagnosed with mixed dementia or those that did not use an imaging technique to ascertain VaD; (2) trials of NEDL-listed Gingko biloba or Huperzine A as experimental interventions, to avoid duplication of existing Cochrane Reviews; (3) trials using acupuncture alone as the experimental intervention; (4) trials using another CP- or NEDL-listed TCHM (except for Huperzine A and Gingko which are popular in Western practice) as the control intervention; and (5) trials using purely non-pharmacological interventions as the control intervention unless explicitly described as 'routine therapy for VaD risk factors'. DATA COLLECTION AND ANALYSIS: We assessed the risks of bias using the Cochrane 'Risk of bias' tool and adapted the Outcome Reporting Bias in Trials (ORBIT) classification system for outcome reporting bias. We assessed TCHM effects on five clinically important outcomes: cognition, global performance, safety, activities of daily living and behaviour and summarised the effects using mean differences for continuous outcomes and risk ratios or risk differences for binary outcomes. We stratified the studies into those that estimated the TCHM versus 'no treatment' effect and those that estimated the TCHM versus the WM effect, with further stratification by the specific TCHM tested or by one of the four modes of action. We pooled using a random-effects model. Due to substantial clinical and design heterogeneity, we did not estimate an 'overall TCHM effect'. MAIN RESULTS: We only found studies (47 studies, 3581 participants) for 18 of the 29 eligible TCHMs as defined by our inclusion criteria. All were superiority trials conducted in China between 1997 and 2013, with most employing a two-arm parallel design with sample sizes ranging from 26 to 240 and a median treatment duration of 12 weeks (range: 2 to 24 weeks).We found that reporting and trial methodology were generally poor; in particular, there was a lack of information on randomisation, an absence of blinding of participants and outcome assessors and incomplete reporting of adverse events (AEs). None of the 30 trials published from 2007 onwards adopted the CONSORT recommendations for reporting RCTs of herbal interventions.We found seven TCHMs which each had potentially large benefits in studies estimating the TCHM versus 'no treatment' effect and in studies estimating the TCHM versus the WM effect. Two TCHMs (NaoXinTong and TongXinLuo) were common to both groups. Three of these TCHMs - Nao XinTong, NaoMaiTai and TongXinLuo - had the strongest evidence to justify further research. Two TCHMs (NaoMaiTai and TongXinLuo) had a 5% or more increased risk of AEs compared to the 'no Treatment' control, but the quality of this evidence was poor. AUTHORS' CONCLUSIONS: We found moderate- to very low-quality evidence of benefit and harm of TCHMs for VaD. Methodological inadequacies need to be addressed by better conducted and reported trials. We identified NaoMaiTai, NaoXinTong and TongXinLuo as warranting special research priority.

Apelin-13 Upregulates BDNF Against Chronic Stress-induced Depression-like Phenotypes by Ameliorating HPA Axis and Hippocampal Glucocorticoid Receptor Dysfunctions.

Localization of apelin and its receptor APJ in limbic structures such as the hippocampus suggests potential involvement of apelin/APJ signaling in stress-related emotional responses. We have recently reported that apelin-13 exerts antidepressant-like actions in acute stressed rats, and that the hippocampus is a critical brain region mediating its actions. However, the neural mechanism underling antidepressant-like actions of apelin-13 is still largely unknown. The aim of the present study is to determine whether apelin-13 ameliorates chronic water-immersion restraint stress (CWIRS)-induced depression-like phenotypes and its neural mechanism in rats. Here, we report that CWIRS exposure leaded to upregulation of apelin/APJ signaling in the hippocampus. Apelin-13 ameliorated CWIRS-induced depression-like phenotypes including hedonic-like deficit and behavioral despairs. Moreover, apelin-13 ameliorated hypothalamic-pituitary-adrenal (HPA) axis hyperactivity, and hippocampal BDNF expression deficit and glucocorticoid receptor (GR) nucleus translocation hypoactivity in chronic stressed rats. Finally, apelin-13-mediated effects were blocked by the selective TrkB receptor antagonist ANA-12. These results suggest that apelin-13 upregulates BDNF against chronic stress-induced depression-like phenotypes by ameliorating HPA axis and hippocampal GR dysfunctions.

The Hippocampus is a Critical Site Mediating Antidepressant-like Activity of Apelin-13 in Rats.

The peptide apelin and its receptor APJ are found to express in multiple brain regions, especially in the regions such as the hippocampus and hypothalamus that play important roles in stress and depression. The distribution of apelin and APJ suggests that the apelinergic signaling may be a key mediator in the development of stress-related depressive behavior. We recently demonstrated that intracerebroventricular (i.c.v) injection of apelin-13 exerts an antidepressant-like activity in the rat forced swimming test (FST). However, the possible brain region mediating apelin-13's antidepressant-like activity remains unclear. In the present study, we determined whether the hippocampus and hypothalamus are the possible regions mediating antidepressant-like activity of apelin-13. We found that forced swimming exposure upregulated apelin and APJ protein expression levels in the hippocampus but not hypothalamus in rats. Further, intrahippocampal injection of apelin-13 exerted an antidepressant-like activity (as indicated by a decreased immobility behavior), and intrahippocampal infusion of APJ receptor antagonist F13A blocked the antidepressant-like activity produced by i.c.v injection of apelin-13 in the FST. Moreover, intrahypothalamic injection of apelin-13 did not affect the immobility behavior in the FST. These findings suggest that the hippocampus, but not hypothalamus, is a critical site mediating antidepressant-like activity of apelin-13 in rats.

Comparing the Effectiveness of Electroacupuncture with Different Grades of Knee Osteoarthritis: A Prospective Study.

BACKGROUND: The electroacupuncture (EA) with different number of points significantly affected its efficacy on knee osteoarthritis (KOA), and the severity of KOA also influenced its response to treatments. Hence, we prospectively compared the clinical efficacy of EA on KOA with different severities. METHODS: A total of 132 KOA patients recruited from 181st Central Hospital of The Chinese People's Liberation Army between March 2014 and March 2015 were classified into 4 KOA stages according to Kellgren Lawrence grading scale. They were allocated into three treatment groups, including two-point group, four-point group and six-point group. Patients in the six-point group received treatment at six-points including ST34, SP10, SP9, ST36, ST35 and EX-LE4. Patients in the four-point group received treatment at ST34, SP10, ST35 and EX-LE5, while patients in the two-point group received treatment at ST35 and EX-LE4. Visual Analog Scale (VAS), McMaster Universities Osteoarthritis Index (WOMAC) and self-assessment questionnaire of patients were assessed after treatment. RESULTS: Three kinds of EA treatments all have significant clinical effects on KOA patients with down-regulated scores of VAS and WOMAC. Regarding post-treatment efficacy, the six point group exhibited lower VAS score and higher WOMAC score compared with the other two groups. For patients with different KOA grades, patients with higher KOA grades were associated with lower grade of treatment efficacy. CONCLUSIONS: Patients with KOA, especially those with lower KOA stages, could gain beneficial efficacies from EA treatments with two, four and six points, respectively.

Chamaecyparis obtusa Suppresses Virulence Genes in Streptococcus mutans.

Chamaecyparis obtusa (C. obtusa) is known to have antimicrobial effects and has been used as a medicinal plant and in forest bathing. This study aimed to evaluate the anticariogenic activity of essential oil of C. obtusa on Streptococcus mutans, which is one of the most important bacterial causes of dental caries and dental biofilm formation. Essential oil from C. obtusa was extracted, and its effect on bacterial growth, acid production, and biofilm formation was evaluated. C. obtusa essential oil exhibited concentration-dependent inhibition of bacterial growth over 0.025 mg/mL, with 99% inhibition at a concentration of 0.2 mg/mL. The bacterial biofilm formation and acid production were also significantly inhibited at the concentration greater than 0.025 mg/mL. The result of LIVE/DEAD® BacLight™ Bacterial Viability Kit showed a concentration-dependent bactericidal effect on S. mutans and almost all bacteria were dead over 0.8 mg/mL. Real-time PCR analysis showed that gene expression of some virulence factors such as brpA, gbpB, gtfC, and gtfD was also inhibited. In GC and GC-MS analysis, the major components were found to be α-terpinene (40.60%), bornyl acetate (12.45%), α-pinene (11.38%), ß-pinene (7.22%), ß-phellandrene (3.45%), and α-terpinolene (3.40%). These results show that C. obtusa essential oil has anticariogenic effect on S. mutans.

Composition Analysis and Inhibitory Effect of Sterculia lychnophora against Biofilm Formation by Streptococcus mutans.

Pangdahai is a traditional Chinese drug, specifically described in the Chinese Pharmacopoeia as the seeds of Sterculia lychnophora Hance. Here, we separated S. lychnophora husk and kernel, analyzed the nutrient contents, and investigated the inhibitory effects of S. lychnophora ethanol extracts on cariogenic properties of Streptococcus mutans, important bacteria in dental caries and plaque formation. Ethanol extracts of S. lychnophora showed dose-dependent antibacterial activity against S. mutans with significant inhibition at concentrations higher than 0.01 mg/mL compared with the control group (p < 0.05). Furthermore, biofilm formation was decreased by S. lychnophora at concentrations > 0.03 mg/mL, while bacterial viability was decreased dose-dependently at high concentrations (0.04, 0.08, 0.16, and 0.32 mg/mL). Preliminary phytochemical analysis of the ethanol extract revealed a strong presence of alkaloid, phenolics, glycosides, and peptides while the presence of steroids, terpenoids, flavonoids, and organic acids was low. The S. lychnophora husk had higher moisture and ash content than the kernel, while the protein and fat content of the husk were lower (p < 0.05) than those of the kernel. These results indicate that S. lychnophora may have antibacterial effects against S. mutans, which are likely related to the alkaloid, phenolics, glycosides, and peptides, the major components of S. lychnophora.

Vitamin D Protects Against Atherosclerosis via Regulation of Cholesterol Efflux and Macrophage Polarization in Hypercholesterolemic Swine.

OBJECTIVE: Prevalence of vitamin D (VD) deficiency and its association with the risk of cardiovascular disease prompted us to evaluate the effect of VD status on lipid metabolism and atherosclerosis in hypercholesterolemic microswine. APPROACH AND RESULTS: Yucatan microswine were fed with VD-deficient (0 IU/d), VD-sufficient (1000 IU/d), or VD-supplemented (3000 IU/d) high-cholesterol diet for 48 weeks. Serum lipids and 25(OH)-cholecalciferol levels were measured biweekly. Histology and biochemical parameters of liver and arteries were analyzed. Effect of 1,25(OH)2D3 on cholesterol metabolism was examined in human hepatocyte carcinoma cell line (HepG2) and human monocytic cell line (THP-1) macrophage-derived foam cells. VD deficiency decreased plasma high-density lipoprotein levels, expression of liver X receptors, ATP-binding membrane cassette transporter A1, and ATP-binding membrane cassette transporter G1 and promoted cholesterol accumulation and atherosclerosis in hypercholesterolemic microswine. VD promoted nascent high-density lipoprotein formation in HepG2 cells via ATP-binding membrane cassette transporter A1-mediated cholesterol efflux. Cytochrome P450 (CYP)27B1 and VD receptor were predominantly present in the CD206(+) M2 macrophage foam cell-accumulated cores in coronary artery plaques. 1,25(OH)2D3 increased the expression of liver X receptors, ATP-binding membrane cassette transporter A1, and ATP-binding membrane cassette transporter G1 and promoted cholesterol efflux in THP-1 macrophage-derived foam cells. 1,25(OH)2D3 decreased intracellular free cholesterol and polarized macrophages to M2 phenotype with decreased expression of tumor necrosis factor-α, interleukin-1ß, interleukin-6 under lipopolysaccharide stimulation. 1,25(OH)2D3 markedly induced CYP27A1 expression via a VD receptor-dependent c-Jun N-terminal kinase (JNK) 1/2 signaling pathway and increased 27-hydroxycholesterol levels, which induced liver X receptors, ATP-binding membrane cassette transporter A1, and ATP-binding membrane cassette transporter G1 expression and stimulated cholesterol efflux that was inhibited by VD receptor antagonist and JNK1/2 signaling inhibitor in THP-1 macrophage-derived foam cell. CONCLUSIONS: VD protects against atherosclerosis in hypercholesterolemic swine via controlling cholesterol efflux and macrophage polarization via increased CYP27A1 activation.

Chamaecyparis obtusa Essential Oil Inhibits Methicillin-Resistant Staphylococcus aureus Biofilm Formation and Expression of Virulence Factors.

The emergence of antibiotic-resistant bacteria has caused difficulty in treating infectious diseases. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most commonly recognized antibiotic-resistant bacteria. Novel antibiotics are urgently required to treat these bacteria. Raw materials derived from natural sources can be used for the development of novel antibiotics, such as Chamaecyparis obtusa (C. obtusa), which has been traditionally used in treating asthmatic disease. In this study, the antibacterial activity of the essential oil (EO) extracted from C. obtusa leaves against MRSA was investigated. MRSA growth and acid production from glucose metabolism were inhibited at concentrations greater than 0.1 mg/mL C. obtusa EO. MRSA biofilm formation was observed using scanning electron microscopy and safranin staining. C. obtusa EO inhibited MRSA biofilm formation at concentrations greater than 0.1 mg/mL. Using real-time polymerase chain reaction, mRNA expression of virulence factor genes, sea, agrA, and sarA, was observed. agrA expression was inhibited with C. obtusa EO concentrations greater than 0.2 mg/mL, whereas inhibition of sea and sarA expression was also observed at a concentration of 0.3 mg/mL. C. obtusa EO was analyzed by gas chromatography (GC) and GC coupled for mass spectrometry, which identified 59 constituents, accounting to 98.99% of the total EO. These findings suggest that C. obtusa EO has antibacterial effects against MRSA, which might be associated with the major components of C. obtusa EO, such as sabinene (19.06%), α-terpinyl acetate (16.99%), bornyl acetate (10.48%), limonene (8.54%), elemol (7.47%), myrcene (5.86%), γ-terpinene (4.04%), and hibaene (3.01%).

Inhibitory Effects of Chrysanthemum boreale Essential Oil on Biofilm Formation and Virulence Factor Expression of Streptococcus mutans.

The aim of the study was to evaluate the antibacterial activity of essential oil extracted from Chrysanthemum boreale (C. boreale) on Streptococcus mutans (S. mutans). To investigate anticariogenic properties, and bacterial growth, acid production, biofilm formation, bacterial adherence of S. mutans were evaluated. Then gene expression of several virulence factors was also evaluated. C. boreale essential oil exhibited significant inhibition of bacterial growth, adherence capacity, and acid production of S. mutans at concentrations 0.1-0.5 mg/mL and 0.25-0.5 mg/mL, respectively. The safranin staining and scanning electron microscopy results showed that the biofilm formation was also inhibited. The result of live/dead staining showed the bactericidal effect. Furthermore, real-time PCR analysis showed that the gene expression of some virulence factors such as gtfB, gtfC, gtfD, gbpB, spaP, brpA, relA, and vicR of S. mutans was significantly decreased in a dose dependent manner. In GC and GC-MS analysis, seventy-two compounds were identified in the oil, representing 85.42% of the total oil. The major components were camphor (20.89%), ß-caryophyllene (5.71%), α-thujone (5.46%), piperitone (5.27%), epi-sesquiphellandrene (5.16%), α-pinene (4.97%), 1,8-cineole (4.52%), ß-pinene (4.45%), and camphene (4.19%). These results suggest that C. boreale essential oil may inhibit growth, adhesion, acid tolerance, and biofilm formation of S. mutans through the partial inhibition of several of these virulence factors.

Chemical and biological insights into uranium-induced apoptosis of rat hepatic cell line.

Uranium release into the environment is a threat to human health, and the mechanisms of cytotoxicity caused by uranium are not well-understood. To improve our understanding in this respect, we herein evaluated the effects of uranium exposure on normal rat hepatic BRL cells. As revealed by scanning electron microscopy and transmission electron microscope analysis, uranyl nitrate was found to be transformed into uranyl phosphate particles in the medium and taken up by BRL cells in an endocytotic uptake manner, which presumably initiates apoptosis of the cell, although soluble uranyl ion may also be toxic. The apoptosis of BRL cells upon uranium exposure was also confirmed by both the acridine orange and ethidium bromide double staining assay and the Annexin V/propidium iodide double staining assay. Further studies revealed that uranium induced the loss of mitochondrial membrane potential in a dose-dependent manner. Moreover, the uranium-induced apoptosis was found to be associated with the activation of caspase-3, caspase-8 and caspase-9, indicating both a mitochondria-dependent signaling pathway and a death receptor pathway by a crosstalk. This study provides new chemical and biological insights into the mechanism of uranium toxicity toward hepatic cells, which will help seek approaches for biological remediation of uranium.