Research progress on the treatment of perimenopausal insomnia with Chaihu Jia Longgu Muli decoction based on brain-intestine-bacteria axis: A review.

With the progress and rapid societal development, women are confronted with multifaceted pressures in their lives, encompassing familial and other domains. Furthermore, during the perimenopausal phase, endocrine equilibrium is disrupted, leading to the emergence of psychological and physiological health challenges. Insomnia is a prevalent symptom among perimenopausal individuals. The brain-gut-bacteria axis assumes a pivotal role in the prevention, diagnosis, and management of perimenopausal insomnia. Chaihu Jia Longgu Muli decoction is a commonly prescribed remedy for addressing perimenopopausal insomnia. Consequently, this paper aims to investigate the interplay between the brain-gut-bacteria axis, intestinal microbiota, and the pathogenesis of perimenopausal insomnia. The study focuses on examining the regulatory effects of Chaihu Jia Longgu Muli decoction on the nervous system, intestinal microbiota, and the hypothalamus-pituitary-adrenal axis. Additionally, it explores the mechanisms underlying Hujia Longgu Muli decoction in mitigating perimenopausal insomnia.

Study on mechanism of Zhenwu Decoction in treatment of heart failure based on network pharmacology: A review.

To explore the mechanism of Zhenwu Decoction (ZWD) in the treatment of heart failure (HF) by network pharmacology analysis, so as to provide a basis for the innovation and application of drugs. The effective components and targets of 5 Chinese herbal medicines in ZWD were retrieved by TCM Pharmacology Database and Analysis Platform (TCMSP).Gene card, OMIM and TTD databases were used to obtain the disease targets of HF, and the intersection with the targets of ZWD was obtained. We used Cytoscape3.9.1 software to construct a drug-active component-disease-target interaction network for ZWD treatment of HF, and performed protein-protein interaction (PPI) network and topology analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed. Fifty-nine effective components and 229 targets of ZWD were screened. Among them, ZWD for HF has 27 active components and 38 common targets, and the core targets of PPI are IL-6, ATK1 and TNF. Pathway enrichment analysis included lipid and atherosclerotic and TNF signaling pathways. This study preliminarily clarified the main active components, targets and related pathways of ZWD in the treatment of HF, and laid a foundation for further study of the pharmacological effects of ZWD.

Study on the active ingredients and mechanism of Jiaotai Pill in the treatment of primary insomnia based on network pharmacology and GEO statistics: A review.

OBJECTIVE: To explore the active components and mechanism of Jiaotai Pill (JTP) in the treatment of primary insomnia (PI) based on gene expression omnibus. METHODS: The main active components of Jiaotai Pills were obtained by TCMSP and literature mining, and the targets of the active components of Jiaotai Pills were predicted. The targets were verified and standardized by Uniprot database. PI-related targets were obtained from GeneCards, OMIM, DrugBank, PharmGKB, and TTD databases. Obtaining an intersection action target point of the Jiaotai pill and the PI by using a Venny diagram; Gene chip data (GSE208668) was downloaded from gene expression omnibus database, and then gene probe enrichment analysis (GSEA) was used to screen the differentially expressed genes between PI patients and normal controls, and molecular docking was used to virtually verify the screened differentially expressed genes with potential active compounds. RESULTS: 21 active components and 263 potential targets of Jiaotai Pill were screened by database analysis and literature mining, 112 of which were intersected with PI. Molecular docking results showed that quercetin, EGCG, kaempferol, R-kanatin, stigmasterol, berberine and other core active components had good docking activity with related differential genes. CONCLUSION: Jiaotai Pill can regulate the release of inflammatory factors through multiple active ingredients, multiple disease targets, multiple biological pathways and multiple pathways to achieve the purpose of treating PI, which provides a theoretical basis for the clinical treatment of PI and broadens the clinical use of Jiaotai Pill.

Clinical study of "Jiaotai Pill" combined with head massage with 5-tone rhythm on insomnia patients of heart-kidney disharmony type.

OBJECTIVE: To explore the curative effect of "Jiaotai Pill" combined with head rhythmic massage consistent with 5-tone rhythm on insomnia of heart-kidney disharmony type. METHODS: Sixty patients with insomnia in massage clinic and ward were randomly divided into treatment group A (30 cases) and treatment group B (30 cases). Patients in group A were treated with traditional head massage combined with oral estazolam tablets. Group B was treated with "Jiaotai Pill" combined with head rhythmic massage therapy consistent with 5-tone rhythm. After 2 weeks of treatment, the scores of Hamilton Anxiety Scale, Pittsburgh Sleep Quality Index, Insomnia Severity Index and Traditional Chinese Medicine Symptom Scale, as well as the expression changes of interleukin (IL)-6 and IL-8 in serum were compared between the 2 groups before and after treatment. RESULTS: After 2 weeks of treatment, the total effective rate of group B was 93. 33%, which was significantly higher than that of group A (66. 67%) (P < .05). After treatment, the scores of Hamilton Anxiety Scale, PQSI, insomnia severity index and traditional Chinese medicine symptom scores were significantly decreased in both groups, and the decrease in group B was more significant than that in group A (P < .05). After treatment, the serum levels of IL-6 and IL-8 were significantly decreased in both groups, and the decrease in group B was greater than that in group A, the difference was statistically significant (P < .05). CONCLUSION: The overall efficacy of Jiaotai Pill combined with head massage therapy consistent with 5-tone rhythm is significantly better than that of traditional massage combined with 5-element music therapy for insomnia patients with heart-kidney disharmony.

Two Liberibacter Proteins Combine to Suppress Critical Innate Immune Defenses in Citrus.

We adopted a systems-based approach to determine the role of two Candidatus Liberibacter asiaticus (CLas) proteins, LasP 235 and Effector 3, in Huanglongbing (HLB) pathogenesis. While a published work suggests the involvement of these CLas proteins HLB pathogenesis, the exact structure-based mechanism of their action has not been elucidated. We conducted the following experiments to determine the structure-based mechanisms of action. First, we immunoprecipitated the interacting citrus protein partners of LasP 235 and Effector 3 from the healthy and CLas-infected Hamlin extracts and identified them by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Second, we performed a split green fluorescent protein (GFP) assay in tobacco to validate that the interactions observed in vitro are also retained in planta. The notable in planta citrus targets of LasP 235 and Effector 3 include citrus innate immune proteins. Third, in vitro and in planta studies were performed to show that LasP 235 and Effector 3 interact with and inhibit the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species, blocking of bactericidal lipid transfer protein (LTP), and induction of premature programed cell death (PCD), all of which are beneficial to CLas lifecycle and HLB pathogenesis. Finally, we performed molecular dynamics simulations to visualize the interactions of LasP 235 and Effector 3, respectively, with LTP and Kunitz protease inhibitor. This led to the design of an LTP mimic, which sequestered and blocked LasP 235 and rescued the bactericidal activity of LTP thereby proving that LasP 235 , indeed, participates in HLB pathogenesis.

An in vitro protocol for rapidly assessing the effects of antimicrobial compounds on the unculturable bacterial plant pathogen, Candidatus Liberibacter asiaticus.

BACKGROUND: Most bacteria are not culturable, but can be identified through molecular methods such as metagenomics studies. Due to specific metabolic requirements and symbiotic relationships, these bacteria cannot survive on typical laboratory media. Many economically and medically important bacteria are unculturable; including phloem-limited plant pathogens like Candidatus Liberibacter asiaticus (CLas). CLas is the most impactful pathogen on citrus production, is vectored by the Asian citrus psyllid (ACP, Diaphorina citri), and lacks an effective treatment or resistant cultivars. Research into CLas pathogenicity and therapy has been hindered by the lack of persistent pure cultures. Work to date has been mostly limited to in planta studies that are time and resource intensive. RESULTS: We developed and optimized an in vitro protocol to quickly test the effectiveness of potential therapeutic agents against CLas. The assay uses intact bacterial cells contained in homogenized tissue from CLas-infected ACP and a propidium monoazide (PMA) assay to measure antimicrobial activity. The applicability of PMA was evaluated; with the ability to differentiate between intact and disrupted CLas cells confirmed using multiple bactericidal treatments. We identified light activation conditions to prevent PCR interference and identified a suitable positive control for nearly complete CLas disruption (0.1% Triton-X 100). Isolation buffer components were optimized with 72 mM salt mixture, 1 mM phosphate buffer and 1% glycerol serving to minimize unwanted interactions with treatment and PMA chemistries and to maximize recovery of intact CLas cells. The mature protocol was used to compare a panel of peptides already under study for potential CLas targeting bactericidal activity and identify which were most effective. CONCLUSION: This psyllid homogenate assay allows for a quick assessment of potential CLas-disrupting peptides. Comparison within a uniform isolate largely eliminates experimental error arising from variation in CLas titer between and within individual host organisms. Use of an intact vs. disrupted assay permits direct assessment of potential therapeutic compounds without generating pure cultures or conducting extensive in planta or field studies.

Hexaacetyl-chitohexaose, a chitin-derived oligosaccharide, transiently activates citrus defenses and alters the feeding behavior of Asian citrus psyllid.

Plants have a perception system triggered by pathogen and pest signals to initiate defense. These signals include evolutionarily conserved molecules from microbes and insects termed pathogen/herbivore-associated molecular patterns (PAMPs/HAMPs). Here we showed that hexaacetyl-chitohexaose (HC), an oligosaccharide from chitin, a structural component in insect exoskeletons and fungi cell walls, upregulated defense-associated genes WRKY22, GST1, RAR1, EDS1, PAL1 and NPR2, and downregulated ICS1 at 1 h after HC treatment in Sun Chu Sha mandarin leaves. The effect was transient as defense gene transcriptional changes were not observed at 18 h after the treatment. Electrical penetration graph (EPG) recordings were used to study the feeding behavior of Asian citrus psyllid (ACP) following the HC treatment. ACP is the hemipteran vector of Candidatus Liberibacter asiaticus (CLas), the pathogen associated with huanglongbing (HLB). Adult ACP displayed reduced intercellular probing, reduced xylem feeding count and duration, and increased non-probing activity on HC-treated citrus compared to controls. During an 18-h recording, percentage for total duration of xylem ingestion, phloem ingestion, intercellular probing were lower, and the percentage of non-probing behavior was higher in HC-treated leaves than in controls. In host-selection behavior studies, HC treatment did not alter the attractiveness of citrus leaves under light or dark conditions. In addition, ACP feeding on HC-treated leaves did not show differences in mortality for up to 10 day of exposure. In summary, we report that HC induced a transient defense in citrus and an inhibitory effect on ACP feeding but did not affect host selection or the insect fitness under the tested conditions.

Temporal and spatial detection of Candidatus Liberibacter asiaticus putative effector transcripts during interaction with Huanglongbing-susceptible, -tolerant, and -resistant citrus hosts.

BACKGROUND: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.

Identification of Gene Candidates Associated with Huanglongbing Tolerance, Using 'Candidatus Liberibacter asiaticus' Flagellin 22 as a Proxy to Challenge Citrus.

The 22-amino acid (flg22) pathogen-associated molecular pattern from the flagellin of Xanthomonas citri subsp. citri has been shown to induce defense responses correlated with citrus canker resistance. Here, flg22 of 'Candidatus Liberibacter asiaticus', the putative causal agent of Huanglongbing (HLB), elicited differential defense responses that were weaker than those from Xcc-flg22, between those of the HLB-tolerant mandarin cultivar Sun Chu Sha and susceptible grapefruit cultivar Duncan. Transcriptomics was used to compare the effect of CLas-flg22 and Xcc-flg22 between the citrus genotypes and identified 86 genes induced only by CLas-flg22 in the tolerant mandarin. Expression of 16 selected genes was validated, by reverse transcription-quantitative polymerase chain reaction, and was evaluated in citrus during 'Ca. L. asiaticus' infection. Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with 'Ca. L. asiaticus', suggesting their involvement in HLB tolerance. In addition, several genes were similarly regulated by CLas-flg22 and 'Ca. L. asiaticus' treatments, while others were oppositely regulated in the tolerant mandarin, suggesting similarity and interplay between CLas-flg22 and 'Ca. L. asiaticus'-triggered defenses. Genes identified are valuable in furthering the study of HLB tolerance mechanisms and, potentially, for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy.

Comparative transcriptome analysis during early fruit development between three seedy citrus genotypes and their seedless mutants.

Identification of genes with differential transcript abundance (GDTA) in seedless mutants may enhance understanding of seedless citrus development. Transcriptome analysis was conducted at three time points during early fruit development (Phase 1) of three seedy citrus genotypes: Fallglo (Bower citrus hybrid (Citrus reticulata×C. reticulata×C. paradisi)×Temple (C. reticulata×C. sinensis)), grapefruit (C. paradisi), Pineapple sweet orange (C. sinensis), and their seedless mutants. Seed abortion in seedless mutants was observed at 26 days post anthesis (Time point 2). Affymetrix transcriptomic analysis revealed 359 to 1077 probe sets with differential transcript abundance in the comparison of seedless versus seedy fruits for each citrus genotypes and time points. The GDTA identified by 18 microarray probe sets were validated by qPCR. Hierarchical clustering analysis revealed a range of GDTA associated with development, hormone and protein metabolism, all of which may reflect genes associated with seedless fruit development. There were 14, 9 and 12 genes found exhibiting similar abundance ratios in all three seedless versus seedy genotype comparisons at time point 1, 2 and 3, respectively. Among those genes were genes coding for an aspartic protease and a cysteine protease, which may play important roles in seedless fruit development. New insights into seedless citrus fruit development may contribute to biotech approaches to create seedless cultivars.

A survey of FLS2 genes from multiple citrus species identifies candidates for enhancing disease resistance to Xanthomonas citri ssp. citri.

Pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) is an important component of plant innate immunity. In a previous study, we showed that the PAMP flg22 from Xanthomonas citri ssp. citri (Xflg22), the causal agent of citrus canker, induced PTI in citrus, which correlated with the observed levels of canker resistance. Here, we identified and sequenced two bacterial flagellin/flg22 receptors (FLS2-1 and FLS2-2) from 'Duncan' grapefruit (Citrus paradisi, CpFLS2-1 and CpFLS2-2) and 'Sun Chu Sha' mandarin (C. reticulata, CrFLS2-1 and CrFLS2-2). We were able to isolate only one FLS2 from 'Nagami' kumquat (Fortunella margarita, FmFLS2-1) and gene flanking sequences suggest a rearrangement event that resulted in the deletion of FLS2-2 from the genome. Phylogenetic analysis, gene structure and presence of critical amino acid domains all indicate we identified the true FLS2 genes in citrus. FLS2-2 was more transcriptionally responsive to Xflg22 than FLS2-1, with induced expression levels higher in canker-resistant citrus than in susceptible ones. Interestingly, 'Nagami' kumquat showed the highest FLS2-1 steady-state expression levels, although it was not induced by Xflg22. We selected FmFLS2-1, CrFLS2-2 and CpFLS2-2 to further evaluate their capacity to enhance bacterial resistance using Agrobacterium-mediated transient expression assays. Both FmFLS2-1 and CrFLS2-2, the two proteins from canker-resistant species, conferred stronger Xflg22 responses and reduced canker symptoms in leaves of the susceptible grapefruit genotype. These two citrus genes will be useful resources to enhance PTI and achieve resistance against canker and possibly other bacterial pathogens in susceptible citrus types.

Responsiveness of different citrus genotypes to the Xanthomonas citri ssp. citri-derived pathogen-associated molecular pattern (PAMP) flg22 correlates with resistance to citrus canker.

The bacterial agent of citrus canker disease (Xanthomonas citri ssp. citri, Xcc) has caused tremendous economic losses to the citrus industry around the world. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is important to plant immunity. In this study, we compared the defence responses of citrus canker-resistant and citrus canker-susceptible genotypes to the Xcc-derived PAMP flg22 (Xflg22) by analysing the expression of 20 citrus defence-associated genes. We showed that, in the most resistant genotype, 'Nagami' kumquat, there was significant induction of several defence genes (EDS1, NDR1, PBS1, RAR1, SGT1, PAL1, NPR2 and NPR3) as early as 6 h and up to 72 h after Xflg22 treatment. At the other end of the spectrum, highly susceptible 'Duncan' grapefruit showed no induction of the same defence genes, even 120 h after treatment. Citrus genotypes with partial levels of resistance showed intermediate levels of transcriptional reprogramming that correlated with their resistance level. Xflg22 also triggered a rapid oxidative burst in all genotypes which was higher and accompanied by the induction of PTI marker genes (WRKY22 and GST1) only in the more resistant genotypes. Pretreatment with Xflg22 prior to Xcc inoculation inhibited bacterial growth in kumquat, but not in grapefruit. A flagellin-deficient Xcc strain (XccΔfliC) showed greater growth increase relative to wild-type Xcc in kumquat than in grapefruit. Taken together, our results indicate that Xflg22 initiates strong PTI in canker-resistant genotypes, but not in susceptible ones, and that a robust induction of PTI is an important component of citrus resistance to canker.