Gentiopicroside improves NASH and liver fibrosis by suppressing TLR4 and NLRP3 signaling pathways.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) and liver fibrosis are progressive conditions associated with non-alcoholic fatty liver disease (NAFLD), characterized by hepatocyte pyroptosis and hepatic stellate cell (HSC) activation. Gentiopicroside (GPS) has emerged as a potential treatment for NASH, yet its underlying mechanism remains unclear. AIM: To confirm that GPS can improve NASH and liver fibrosis by blocking the NLRP3 signaling pathway STUDY DESIGN: Initially, different animal models were used to study the effects and mechanisms of GPS on NASH and fibrosis. Subsequent in vitro experiments utilized co-cultures and other techniques to delve deeper into its mechanism, followed by validation of the findings in mouse liver tissues. METHODS: C57BL/6 mice were fed high-fat, high-cholesterol (HFHC), or methionine-choline-deficient (MCD) diets to induce NASH and fibrosis. RAW264.7 cells and born marrow bone marrow-derived macrophages (BMDMs) were stimulated with LPS and ATP to induce inflammation, then co-cultured with primary hepatocytes and HSCs, treated with GPS, and its efficacy and mechanism were analyzed. RESULTS: In vivo, GPS alleviated NASH and liver fibrosis by inhibiting the NLRP3 pathway. In vitro, GPS attenuated inflammation induced by BMDMs by inhibiting TLR4 and NLRP3 signaling pathways, and Co-culture studies suggested that GPS reduced hepatocyte pyroptosis and HSC activation, which was also confirmed in liver tissues CONCLUSION: GPS improves NASH and liver fibrosis by inhibiting the TLR4 and NLRP3 signaling pathways. The specific mechanism may be related to the suppression of macrophage-mediated inflammatory responses, thereby reducing hepatocyte pyroptosis and HSC activation.

Ursolic acid targets secreted phosphoprotein 1 to regulate Th17 cells against non-alcoholic fatty liver disease.

Background/Aims: Non-alcoholic fatty liver disease (NAFLD) has become an increasingly important health challenge, with a substantial rise linked to changing lifestyles and global obesity. Ursolic acid, a natural pentacyclic triterpenoid, has been explored for its potential therapeutic effects. Given its multifunctional bioactive properties, this research further revealed the pharmacological mechanisms of ursolic acid on NAFLD. Methods: Drug target chips and bioinformatics analysis were combined in this study to explore the potential therapeutic effects of ursolic acid on NAFLD. Molecular docking simulations, surface plasmon resonance analyses, pull-down experiments, and co-immunoprecipitation assays were used to verify the direct interactions. Gene knockdown mice were generated, and high-fat diets were used to validate drug efficacy. Furthermore, initial CD4+ T cells were isolated and stimulated to demonstrate our findings. Results: In this study, the multifunctional extracellular matrix phosphorylated glycoprotein secreted phosphoprotein 1 (SPP1) was investigated, highlighting its capability to induce Th17 cell differentiation, amplifying inflammatory cascades, and subsequently promoting the evolution of NAFLD. In addition, this study revealed that in addition to the canonical TGF-ß/IL-6 cytokine pathway, SPP1 can directly interact with ITGB1 and CD44, orchestrating Th17 cell differentiation via their joint downstream ERK signaling pathway. Remarkably, ursolic acid intervention notably suppressed the protein activity of SPP1, suggesting a promising avenue for ameliorating the immunoinflammatory trajectory in NAFLD progression. Conclusions: Ursolic acid could improve immune inflammation in NAFLD by modulating SPP1-mediated Th17 cell differentiation via the ERK signaling pathway, which is orchestrated jointly by ITGB1 and CD44, emerging as a linchpin in this molecular cascade.

Gut microbiota-derived butyrate restores impaired regulatory T cells in patients with AChR myasthenia gravis via mTOR-mediated autophagy.

More than 80% of patients with myasthenia gravis (MG) are positive for anti-acetylcholine receptor (AChR) antibodies. Regulatory T cells (Tregs) suppress overproduction of these antibodies, and patients with AChR antibody-positive MG (AChR MG) exhibit impaired Treg function and reduced Treg numbers. The gut microbiota and their metabolites play a crucial role in maintaining Treg differentiation and function. However, whether impaired Tregs correlate with gut microbiota activity in patients with AChR MG remains unknown. Here, we demonstrate that butyric acid-producing gut bacteria and serum butyric acid level are reduced in patients with AChR MG. Butyrate supplementation effectively enhanced Treg differentiation and their suppressive function of AChR MG. Mechanistically, butyrate activates autophagy of Treg cells by inhibiting the mammalian target of rapamycin. Activation of autophagy increased oxidative phosphorylation and surface expression of cytotoxic T-lymphocyte-associated protein 4 on Treg cells, thereby promoting Treg differentiation and their suppressive function in AChR MG. This observed effect of butyrate was blocked using chloroquine, an autophagy inhibitor, suggesting the vital role of butyrate-activated autophagy in Tregs of patients with AChR MG. We propose that gut bacteria derived butyrate has potential therapeutic efficacy against AChR MG by restoring impaired Tregs.

Gentiopicroside improves non-alcoholic steatohepatitis by activating PPARα and suppressing HIF1.

Gentiopicroside (GPS) is a highly water-soluble small-molecule drug and the main bioactive secoiridoid glycoside of Gentiana scabra that has been shown to have hepatoprotective effects against non-alcoholic steatohepatitis (NASH), a form of non-alcoholic fatty liver disease (NAFLD) that can progress to cirrhosis and hepatocellular carcinoma. However, the effects of GPS on NASH and the underlying mechanisms remain obscure. Firstly, a high-fat, high-cholesterol (HFHC) diet and a high-sugar solution containing d-fructose and d-glucose were used to establish a non-alcoholic steatohepatitis (NASH) mice model. Secondly, we confirmed GPS supplementation improve metabolic abnormalities and reduce inflammation in NASH mice induced by HFHC and high-sugar solution. Then we used metabolomics to investigate the mechanisms of GPS in NASH mice. Metabolomics analysis showed GPS may work through the Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway and glycine, serine, and threonine metabolism. Functional metabolites restored by GPS included serine, glycine, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Western blot and qRT-PCR analysis confirmed GPS improve NASH by regulating PPARα and Hypoxia-Inducible Factor-1α (HIF-1α) signaling pathways. In vitro, studies further demonstrated EPA and DHA enhance fatty acid oxidation through the PPARα pathway, while serine and glycine inhibit oxidative stress through the HIF-1α pathway in palmitic acid-stimulated HepG2 cells. Our results suggest GPS's anti-inflammatory and anti-steatosis effects in NASH progression are related to the suppression of HIF-1α through the restoration of L-serine and glycine and the activation of PPARα through increased EPA and DHA.

Efficacy and safety of Shenbai Granules for recurrent colorectal adenoma: A multicenter randomized controlled trial.

BACKGROUND: Colorectal adenoma is benign glandular tumor of colon, the precursor of colorectal cancer. But no pharmaceutical medication is currently available to treat and prevent adenomas. PURPOSE: To evaluate efficacy of Shenbai Granules, an herbal medicine formula, in reducing the recurrence of adenomas. STUDY DESIGN: This multicenter, randomized, double-blind, placebo-controlled clinical trial was conducted by eight hospitals in China. METHODS: Patients who had received complete polypectomy and were diagnosed with adenomas within the recent 6 months were randomly assigned (1:1) to receive either Shenbai granules or placebo twice a day for 6 months. An annual colonoscopy was performed during the 2-year follow-up period. The primary outcome was the proportion of patients with at least one adenoma detected in the modified intention-to-treat (mITT) population during follow-up for 2 years. The secondary outcomes were the proportion of patients with sessile serrated lesions and other specified polypoid lesions. The data were analyzed using logistic regression. RESULTS: Among 400 randomized patients, 336 were included in the mITT population. We found significant differences between treatment and placebo groups in the proportion of patients with at least one recurrent adenoma (42.5 % vs. 58.6 %; OR, 0.47; 95 % CI, 0.29-0.74; p = 0.001) and sessile serrated lesion (1.8 % vs. 8.3 %; OR, 0.20; 95 % CI, 0.06-0.72; p = 0.01). There was no significant difference in the proportion of patients developing polypoid lesions (70.7 % vs. 77.5 %; OR, 1.43; 95 % CI, 0.88-2.34; p = 0.15) or high-risk adenomas (9.0 % vs. 13.6 %; OR, 0.63; 95 % CI, 0.32-1.25; p = 0.18). CONCLUSION: Shenbai Granules significantly reduced the recurrence of adenomas, indicating that they could be an effective option for adenomas. Future studies should investigate its effects in larger patient populations and explore its mechanism of action to provide more comprehensive evidence for the use of Shenbai Granules in adenoma treatment.

Berberine Enhances Intestinal Mucosal Barrier Function by Promoting Vitamin D Receptor Activity.

OBJECTIVE: To evaluate if berberine can act on vitamin D receptors (VDR) and thereby regulate the expression of tight junction proteins (TJPs) in irritable bowel syndrame-diarrhea-predominant (IBS-D) rats. METHODS: The newborn rats were induced into IBS-D rat model via neonatal maternal separation combined with acetic acid chemical stimulation. After modeling, the model was evaluated and rats were divided into the control group and berberine treatment groups (0.85, 1.7 and 3.4 mg/kg, once a day for 2 weeks). The distal colon was obtained and colonic epithelial cells (CECs) were isolated and cultured after IBS-D model evaluation. The vitamin D receptor response element (VDRE) reporter gene was determined in the CECs of IBS-D rats to analyze the effect of berberine on the VDRE promoter. VDR overexpression or silencing technology was used to analyze whether VDR plays a role in promoting intestinal barrier repair, and to determine which region of VDR plays a role in berberine-regulated intestinal TJPs. RESULTS: The IBS-D rat model was successfully constructed and the symptoms were improved by berberine in a dose-dependent manner (P<0.05). The activity of VDRE promoter was also effectively promoted by berberine (P<0.05). Berberine increased the expression of TJPs in IBS-D CECs (P<0.05). VDR expression was significantly increased after transfection of different domains of VDR when compared to normal control and basic plasmid groups (all P<0.05). RT-qPCR and Western blot results showed that compared with the blank group, expressions of occludin and zonula occludens-1 were significantly higher in VDR containing groups (all P<0.05). Berberine plus pCMV-Myc-VDR-N group exerted the highest expression levels of occludin and zonula occludens-1 (P<0.05). CONCLUSION: Berberine enhances intestinal mucosal barrier function of IBS-D rats by promoting VDR activity, and the main site of action is the N-terminal region of VDR.

Explore the active ingredients and potential mechanisms of JianPi QingRe HuaYu Methods in the treatment of gastric inflammation-cancer transformation by network pharmacology and experimental validation.

BACKGROUND: JianPi QingRe HuaYu Methods (JQH) have been long used to treat chronic atrophic gastritis (CAG) and precancerous lesions of gastric cancer (PLGC). However, whether JQH can inhibit the transformation of gastritis to gastric cancer (GC) remains unclear. METHODS: Herein, we first retrieved the active ingredients and targets of JQH from the TCMSP database and the targets related to the gastric inflammation-cancer transformation from public databases. Differentially expressed genes (DEGs) related to gastric inflammation-cancer transformation were identified from the Gene Expression Omnibus (GEO) database. Then, we obtained the potential therapeutic targets of JQH in treating gastric inflammation-cancer transformation by intersecting drugs and disease targets. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) analyses of the potential therapeutic targets were conducted using R software. Next, we conducted molecular docking and in vitro experiments to validate our results. RESULTS: We obtained 214 potential therapeutic targets of JQH by intersecting drugs and disease targets. We found that the potential mechanisms of JQH in treating gastric inflammation-cancer transformation might be related to JAK-STAT, Wnt, p53 and VEGF signaling pathways. The molecular docking indicated that quercetin, as the main active ingredient of JQH, might inhibit gastric inflammation-cancer transformation by binding with specific receptors. Our experimental results showed that quercetin inhibited cells proliferation (P < 0.001), promoted cell apoptosis (P < 0.001), reduced the secretion of pro-inflammatory cytokines (P < 0.001) and promoted the secretion of anti-inflammatory cytokines (P < 0.001) in MNNG-induced GES-1 cells. Furthermore, quercetin inhibited cells proliferation (P < 0.001) and reduced mRNA and protein level of markers of PLGC (P < 0.001) in CDCA-induced GES-1 cells. CONCLUSION: These results provide the material basis and regulatory mechanisms of JQH in treating gastric inflammation-cancer transformation.

Astragaloside IV ameliorates experimental autoimmune myasthenia gravis by regulating CD4 + T cells and altering gut microbiota.

BACKGROUND: Myasthenia gravis (MG) is an antibody-mediated autoimmune disease and its pathogenesis is closely related to CD4 + T cells. In recent years, gut microbiota is considered to play an important role in the pathogenesis of MG. Astragaloside IV (AS-IV) is one of the main active components extracted from Astragalus membranaceus and has immunomodulatory effects. To study the immunomodulatory effect of AS-IV and the changes of gut microbiota on experimental autoimmune myasthenia gravis (EAMG) mice, we explore the possible mechanism of AS-IV in improving MG. METHODS: In this study, network pharmacology was utilized to screen the crucial targets of AS-IV in the treatment of MG. Subsequently, a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to identify potential pathways through which AS-IV acts against MG. Furthermore, experimental investigations were conducted to validate the underlying mechanism of AS-IV in MG treatment. Before modeling, 5 mice were randomly selected as the control group (CFA group), and the other 10 were induced to EAMG model. These mice were randomly divided into EAMG group and EAMG + AS-IV group, n = 5/group. In EAMG + AS-IV group, AS-IV was administered by gavage. CFA and EAMG groups were given the same volume of PBS. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. At the last administration, the feces were collected for 16S RNA microbiota analysis. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected by flow cytometry. The levels of IFN-γ, IL-17 and TGF-ß in serum were measured by ELISA. Furthermore, fecal microbial transplantation (FMT) experiments were performed for exploring the influence of changed intestinal flora on EAMG. After EAMG model was induced, the mice were treated with antibiotics daily for 4 weeks to germ-free. Then germ-free EAMG mice were randomly divided into two groups: FMT EAMG group, FMT AS-IV group, n = 3/group. Fecal extractions from EAMG and EAMG + AS-IV groups as gathered above were used to administered daily to the respective groups for 4 weeks. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected at the last administration. The levels of IFN-γ, IL-17 and TGF-ß in serum were measured by ELISA. RESULTS: The network pharmacology and KEGG pathway analysis revealed that AS-IV regulates T cell pathways, including T cell receptor signaling pathway and Th17 cell differentiation, suggesting its potential in improving MG. Further experimental verification demonstrated that AS-IV administration improved muscle strength and body weight, reduced the level of Th1 and Th17 cells, enhanced the level of Treg cells, and resulted in alterations of the gut microbiota, including changes in beta diversity, the Firmicutes/Bacteroidetes (F/B) ratio, and the abundance of Clostridia in EAMG mice. We further conducted FMT tests and demonstrated that the EAMG Abx-treated mice which were transplanted the feces of mice treated with AS-IV significantly alleviated myasthenia symptoms, reduced Th1 and Th17 cells levels, and increased Treg cell levels. CONCLUSION: This study speculated that AS-IV ameliorates EAMG by regulating CD4 + T cells and altering the structure and species of gut microbiota of EAMG.

Paris polyphylla extract attenuates colitis in mice by regulating PPAR-γ mediated Treg/Th17 balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Paris polyphylla Sm. (P.P), is a widely-used traditional Chinese medicine (TCM) in the treatment of wound, throat sores and snakebites. Furthermore, P.P was recorded as an anti-inflammatory drug by the Chinese Pharmacopoeia. AIM OF THE STUDY: We sought to decipher the anti-inflammatory effect of P.P on ulcerative colitis (UC); specifically, to explore whether P.P attenuates colitis by restoring the regulatory T cells (Tregs) and T helper 17 (Th17) cells balance and its mechanism. MATERIAL AND METHODS: We treated experimental colitis mice with extracts of Paris polyphylla (EPP). The percentage of Tregs and Th17 cells were measured using flow cytometry, and their secreted cytokines levels were evaluated employing ELISA. The expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) in colon tissues was detected using immunofluorescence. Furthermore, GW9662, a PPAR-γ antagonist, was used to validate the mechanism of EPP in restoring the Treg/Th17 balance. RESULTS: The EPP effectively alleviated the clinical symptoms and inflammatory cytokine levels in mice with colitis. EPP treatment also restored the impaired Treg/Th17 balance in mice. Furthermore, EPP treatment promoted PPAR-γ expression and reduced HIF-1α and p-STAT3 expression in colon tissues, whereas PPAR-γ inhibition blocked the effects of EPP in mice models. CONCLUSION: Our study indicates that EPP exhibit excellent anti-inflammatory properties via restoring PPAR-γ/STAT3/HIF-1α axis-mediated Treg/Th17 balance in colitis mice. Hence, P. polyphylla is a promising medicinal plant-based alternative for managing colitis that requires further clinical validation.

In silico and in vivo demonstration of the regulatory mechanism of Qi-Ge decoction in treating NAFLD.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD), a chronic and progressive liver disease, often causes steatosis and steatohepatitis. Qi-Ge decoction (QGD) shows a good effect against NAFLD in the clinic. But the molecular mechanism for QGD in improving NAFLD is unknown. PURPOSE: This study explored the molecular mechanism of QGD in NAFLD model rats using comprehensive network pharmacology, molecular docking and in vivo verification strategies. METHODS: Active components and targets of QGD were obtained from public database. The overlapped genes between QGD and NAFLD targets were analyzed by enrichment analysis. Active components and targets were used to predict molecular docking analysis. Finally, seven key targets were screened out and the gene expression were verified in the NAFLD rat's liver tissues after QGD treatment. RESULTS: Fifty-eight common QGD therapeutic targets were associated with NAFLD. Molecular docking demonstrated that seven targets had strong binding ability for the corresponding active ingredients. GO analysis identified 18 biological process entries, which were mainly related to regulation of lipid storage, lipid localization and peptide transport. KEGG analysis identified multiple signaling pathways, which were mainly associated with tumor necrosis factor signaling and NAFLD. In vivo data confirmed that the effect of QGD in the treatment of NAFLD was mainly exerted through improving liver steatosis and inflammatory cell infiltration. Additionally, QGD upregulated the expression of MAPK8 and ESR1 and downregulated the transcriptional expression of IL6, VEGFA, CASP3, EGFR and MYC. These targets may affect lipid metabolism by regulating lipid storage and inflammation. CONCLUSION: The integration of results obtained in silico and in vivo indicated that QGD regulates multiple targets, biological processes and signaling pathways in NAFLD, which may represent a complex molecular mechanism by which QGD improves NAFLD.Key messagesQGD intervention is related to multiple biological processes such as inflammation, oxidation and cell apoptosis in NAFLD.Lipid and atherosclerosis, TNF signaling pathway, IL-17 signaling pathway, non-alcoholic fatty liver disease and AGE-RAGE signaling pathway in diabetic complications are the main pathways for QGD intervention NAFLD.The active components of QGD can form good binding with relevant target proteins through intermolecular forces, exhibiting excellent docking activity.

Examining the Mechanisms of Huachansu Injection on Liver Cancer through Integrated Bioinformatics Analysis.

OBJECTIVE: The objective of this study is to explore the potential anti-liver cancer mechanism of Huachansu injection through integrated bioinformatics analysis. METHODS: Active ingredients of Huachansu injection (extraction of toad skin) were obtained, and their potential drug targets were predicted via SwissTargetPrediction database. Liver cancer disease targets were identified from the GEO (Gene Expression Omnibus) dataset and four public databases. Then Protein-Protein Interaction (PPI) network of toad skin was constructed. GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis were performed subsequently. Finally, molecular docking was performed using Auto Dock Vina. RESULTS: In the search for therapeutic targets, twenty active components of toad skin were screened for further study, five hundred and sixty-eight targets of components were identified. In the search for disease targets, three thousand two hundred and twenty-seven genes were identified after removal of duplicated genes, one hundred and fifty-nine genes were up-regulated in liver cancer samples while two hundred and seventy-eight were down-regulated in liver cancer patients. After predicting the therapeutic targets of the components, the results were cross-checked with the disease targets, thirteen up-regulated targets and ten down-regulated targets were obtained. Finally, in the results of molecular docking, seven targets (CDK1, AKR1B1, MMP12, AURKB, CHEK1, AURKA, TTK) were potential up-regulated targets, three targets (SHBG, SRD5A2, NR1I2) were potential down-regulated targets, all of which have the best binding energy and molecular interactions. CONCLUSION: CDK1, AKR1B1, MMP12, AURKB, CHEK1, AURKA, and TTK could be potential upregulated target proteins of Huachansu injection for treating liver cancer. The mechanism of Huachansu injection in the treatment of liver cancer through these up-regulated targets is related to cell cycle, cellular senescence, viral carcinogenesis, p53 signaling pathway. SHBG, SRD5A2, and NR1I2 could be potential down-regulated target proteins of Huachansu injection in treating liver cancer.

miR-196a-5p Correlates with Chronic Atrophic Gastritis Progression to Gastric Cancer and Induces Malignant Biological Behaviors of Gastric Cancer Cells by Targeting ACER2.

BACKGROUND: As the prognosis of early gastric cancer (EGC) is significantly better than that of advanced gastric cancer (AGC), the development of biomarkers to monitor the progression of chronic atrophic gastritis (CAG) to gastric cancer (GC) is essential. METHODS: Stomach tissue miRNA and mRNA sequences from patients with chronic non-atrophic gastritis (CNAG), CAG, precancerous lesions of gastric cancer (PLGC), and GC were analyzed. A publicly available GC-related miRNA microarray dataset was obtained from the Gene Expression Omnibus database. Spearman's correlation and differential gene analyses, and clinical validation were used to identify novel miRNAs correlating with CAG progression to GC. miRNA targets were predicted using weighted gene co-expression analysis and databases. A dual-luciferase reporter assay was performed to check for direct interaction between miR-196a-5p and ACER2. The CCK-8 and wound healing assays, and flow cytometry were performed to evaluate cell proliferation, migration, and apoptosis. RESULTS: miR-196a-5p was correlated with CAG progression to GC. Overexpression of miR-196a-5p promoted GC cell proliferation and migration and inhibited apoptosis, whereas suppression of miR-196a-5p exerted the opposite effect. Based on the prediction and luciferase assays, ACER2 was identified as the target of miR-196a-5p. ACER2 was downregulated in GC cell lines. Knockdown of ACER2 increased GC cell proliferation rates and migration ability and inhibited apoptosis, while ACER2 overexpression led to the opposite effect. CONCLUSIONS: miR-196a-5p correlated with CAG progression to GC and induced malignant biological behaviors of GC cells by targeting ACER2, providing a novel monitoring biomarker and target for GC prevention.

N6-methylandenosine-related immune genes correlate with prognosis and immune landscapes in gastric cancer.

Objectives: This study aimed to probe into the significance of N6-methyladenosine (m6A)-related immune genes (m6AIGs) in predicting prognoses and immune landscapes of patients with gastric cancer (GC). Methods: The clinical data and transcriptomic matrix of GC patients were acquired from The Cancer Genome Atlas database. The clinically meaningful m6AIGs were acquired by univariate Cox regression analysis. GC patients were stratified into different clusters via consensus clustering analysis and different risk subgroups via m6AIGs prognostic signature. The clinicopathological features and tumor microenvironment (TME) in the different clusters and different risk subgroups were explored. The predictive performance was evaluated using the KM method, ROC curves, and univariate and multivariate regression analyses. Moreover, we fabricated a nomogram based on risk scores and clinical risk characteristics. Biological functional analysis was performed based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. The connectivity map was used to screen out potential small molecule drugs for GC patients. Results: A total of 14 prognostic m6AIGs and two clusters based on 14 prognostic m6AIGs were identified. A prognostic signature based on 4 m6AIGs and a nomogram based on independent prognostic factors was constructed and validated. Different clusters and different risk subgroups were significantly correlated with TME scores, the distribution of immune cells, and the expression of immune checkpoint genes. Some malignant and immune biological processes and pathways were correlated with the patients with poor prognosis. Ten small molecular drugs with potential therapeutic effect were screened out. Conclusions: This study revealed the prognostic role and significant values of m6AIGs in GC, which enhanced the understanding of m6AIGs and paved the way for developing predictive biomarkers and therapeutic targets for GC.

Translation and evaluation of the simplified Chinese version of the rating form of IBD patient concerns.

BACKGROUND: Inflammatory bowel disease (IBD) has become a global public health problem. The prevalence of IBD in China increased annually in past two decades. METHODS: This study was to translate and validate the rating form of IBD patients' concerns (RFIPC), and to describe disease-related worries and concerns of patients with IBD. The simplified Chinese version of the RFIPC was developed according to translation and back-translation procedure. Patients with IBD were consecutively enrolled from the First Affiliated Hospital of Guangzhou University of Chinese Medicine. The participants were assessed using the RFIPC and the Short Inflammatory Bowel Disease Questionnaire (SIBDQ). Internal consistency, test-retest reliability, measurement error, confirmatory factor analysis (CFA) and correlation of the RFIPC with the SIBDQ were performed to evaluate the psychometric characteristics of the RFIPC. RESULTS: A total of 116 patients with IBD, 73 with ulcerative colitis (UC) and 43 with Crohn's disease (CD), were enrolled in this study. Thirty-seven of them recompleted the questionnaires for the second time between 7 and 14 days after the first interview. The results of CFA indicated the original structure of the RFIPC was reasonable. Cronbach's alpha value of the RFIPC were 0.97. The intraclass correlation coefficients of four domains ranged from 0.85 to 0.92. The standard error of measurement was 7.10. The correlation coefficients between total score of the RFIPC and the SIBDQ score ranged from - 0.54 to - 0.70. Median total score of the RFIPC was 39.4 (IQR 24.0-59.3). Patients with severe symptoms reported higher scores of the RFIPC. The uncertain nature of disease, having surgery, having an ostomy bag, developing cancer, feeling out of control, being a burden on others and financial difficulties were highest concerns of patients with IBD. Comparing with patients with UC, patients with CD had more concerns of the ability to have children and being treated as different (P < 0.05). CONCLUSIONS: The simplified Chinese version of RFIPC is a valid and reliable tool. It could be used for assessing disease-related worries and concerns of patients with IBD in China. Specific concerns of patients with UC and CD are different, therefore, health workers should consider the specific needs of UC and CD patients.

Analysis of the Efficacy of Acupuncture Combined with Rehabilitation Training in the Treatment of Upper Limb Spasm after Stroke: A Systematic Review and Meta-Analysis.

Objective: Systematic evaluation of the efficacy of acupuncture combined with cognitive rehabilitation training in the treatment of upper limb spasm after cerebral apoplexy. Methods: The data of CNKI, CBM, CQVIP, Wanfang, and the libraries of Pubmed and Cochrane were searched by computer, and the related literatures about acupuncture combined with cognitive rehabilitation training in the treatment of cognitive dysfunction after stroke were searched. The search time is from January 1, 1995 to January 1, 2022. All data segments were independently analyzed and extracted by two evaluators. After evaluating the quality of the methodology, meta-analysis was carried out by using the RevMan5.4 software. Results: Finally, 11 studies were included, with a total of 789 subjects. The results of meta-analysis indicated that acupuncture combined with cognitive rehabilitation training was superior to simple cognitive rehabilitation training or drugs in the following aspects, the difference exhibited statistically significant, the total effective rate (RR = 1.58, 95% CI), latency of P300 (MD = -18.46, 95% CI), amplitude of P300 (MD = 1.23, 95% CI (0.82), P < 0.00001, 95% CI (0.31)), and activity of daily living (ADL), respectively, were significantly higher compared to the control group (P < 0.00001), and the difference was statistically significant (P < 0.05). Based on the results of systematic evaluation, the GRADE system recommendation classification method is used to evaluate the quality of evidence. The results show that the level of evidence is low and the intensity of recommendation is weak. Conclusion: The results of this meta-analysis suggest that the curative effect of acupuncture combined with cognitive rehabilitation training is better compared to simple cognitive rehabilitation training or drugs. However, due to the low quality of the original literature, it needs to be confirmed by multicenter, high-quality, large-sample randomized blind controlled trials in the future.

Mass Cytometry and Single-Cell Transcriptome Analyses Reveal the Immune Cell Characteristics of Ulcerative Colitis.

Background: The pathogenesis of ulcerative colitis (UC) is closely related to immunity. The immune characteristic differences between active UC (UCa) and inactive UC (UCin) have not been completely explained. Mass cytometry (CyTOF) and single-cell RNA sequencing (scRNA-seq) were used to analyze the immune cells of UCa, UCin and healthy control (HC) subjects to determine the specific immune characteristics. Methods: The immune cell subsets among UCa, UCin, HC were distinguished using CyTOF analysis. scRNA-seq analysis was used to validate the results of CyTOF. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to understand the roles of differential immune cell subsets. Results: After CyTOF analysis and validation of scRNA-seq analysis, differential immune cell subsets mainly contained TNF+IL-17A++ effector memory (EM) Tregs, CXCR3+CTLA4+ EM Tregs, CXCR3++CCR7+ B cells, HLA-DR+CCR7+ dendritic cells (DCs) and CTLA-4+ natural killer (NK) cells. In comparison to HC, CCR6+TNF+CD161+ EM T cells were highly enriched in UCa and UCin. Besides, UCa was characterized by an increase in CD38+TNF+ EM Tregs, CXCR3+CCR4+ naïve B cells, HLA-DR+CD14+IL21+ macrophages/monocytes, HLA-DR+CCR7+ DCs, AHR+CD14+ cytotoxic NK (cNK) cells and CD8A+IFNG+ cNK cells. Decreases in CD38+CD27+ plasmablasts, CXCR3+CD38+ regulatory NK cells, and CXCR3+CCR7+ tolerant NK cells in UCa were discovered. Conclusions: Novel immune cell subsets which was used to distinguish UCa, UCin and HC were identified. This information might be utilized to distinguish the patients with UCa and UCin.

Interpretation of a Quantitative Diagnosis Model of Traditional Chinese Medicine Syndromes Based on Computer Adaptive Testing.

Objectives: The aim of this study is to interpret a quantitative diagnosis model of traditional Chinese medicine (TCM) syndromes based on computer adaptive testing (CAT), from the perspective of both patients and clinicians. Methods: In this cross-sectional study, patients with postprandial distress syndrome completed the CAT model of TCM syndromes and the Chinese version of the Quality of Life Questionnaire for Functional Digestive Disorders (Chin-FDDQL); the clinicians' diagnosis was concurrently recorded. The patients completed this questionnaire again after 14 ± 2 days. The kappa test and paired chi-square test were used to evaluate the consistency between the CAT model and clinical diagnosis. Minimal clinically important differences (MCID) of the Chin-FDDQL scores were used to assess clinical efficacy from the patients' perspective. Logistic regression was used to examine the association between changes in the CAT model syndrome domain scores and changes in clinical outcomes. Results: Changes in the CAT model syndrome domain scores may affect the clinical outcomes of patients with the total scores of Chin-FDDQL (all P < 0.05). There was a correlation between changes in the CAT model syndrome domain scores and the patients' clinical outcomes. Different syndrome elements had different effects on various Chin-FDDQL domains, which was consistent with the theory of TCM. Conclusions: This study proposes a method for the clinical interpretation of the CAT model of TCM syndromes, including evidence derived from the application. It may provide a reference for future interpretation of other CAT models.

Effectiveness of modified Buzhong Yiqi decoction in treating myasthenia gravis: study protocol for a series of N-of-1 trials.

BACKGROUND: Myasthenia gravis (MG) is an acquired autoimmune disease with high heterogeneity. The disease is chronic, relapsing repeatedly and progressive with acute exacerbation occasionally. Although the treatment of MG has developed, it is still unsatisfactory and has some unexpected side effects. Traditional Chinese medicine (TCM) has shown great potential in MG treatment, including relief of muscle weakness syndrome, improvement of patient's quality of life, and reduction of side effects of western medicine. The purpose of this study is to evaluate the effectiveness of modified Buzhong Yiqi decoction (MBYD) as an add-on therapy for MG through a small series of N-of-1 trials. METHODS: Single-centre, randomized, double-blind, 3 crossover N-of-1 trials will be conducted to enroll patients with MG diagnosed as spleen-stomach deficiency syndrome or spleen-kidney deficiency syndrome in TCM. Each N-of-1 trial has 3 cycles of two 4-week periods containing the MBYD period and placebo period. The wash-out interval of 1 week is prior to switching each period. PRIMARY OUTCOME: quantitative myasthenia gravis (QMG). SECONDARY OUTCOMES: the following scales: myasthenia gravis composite (MGC), myasthenia gravis activities of daily living profile (MG-ADL), myasthenia gravis quality of life (MG-QOL); the level of CD4+FoxP3+Treg cells and cytokines (IL-4, IL-17A, INF-γ, TGF-ß) in the peripheral blood; the alterations of the composition of gut microbiota; reduction of the side effects of western medicine. DISCUSSION: Used by WinBUGS software, we will conduct a hierarchical Bayesian statistical method to analyze the efficacy of MBYD in treating MG in individuals and populations. Some confounding variables such as TCM syndrome type and potential carryover effect of TCM will be introduced into the hierarchical Bayesian statistical method to improve the sensitivity and applicability of the trials, and the use of prior available information within the analysis may improve the sensitivity of the results of a series of N-of-1 trials, from both the individual and population level to study the efficacy of TCM syndrome differentiation. We assumed that this study would reveal that MBYD is effective for MG and provide robust evidence of the efficacy of TCM to treat MG. TRIAL REGISTRATION: Chinese Clinical Trial Register, ID: ChiCTR2000040477 , registration on 29 November 2020.

Mitochondrial dynamics and biogenesis indicators may serve as potential biomarkers for diagnosis of myasthenia gravis.

Due to challenges in diagnosing myasthenia gravis (MG), identifying novel diagnostic biomarkers for this disease is essential. Mitochondria are key organelles that regulate multiple physiological functions, such as energy production, cell proliferation and cell death. In the present study, Mfn1/2, Opa1, Drp1, Fis1, AMPK, PGC-1α, NRF-1 and TFAM were compared between patients with MG and healthy subjects to identify potential diagnostic biomarkers for MG. Blood samples were collected from 50 patients with MG and 50 healthy subjects. The participants' demographic information and routine blood test results were recorded. Mitochondrial dynamics were evaluated and levels of Mfn1/2, Opa1, Drp1, Fis1, AMPK, PGC-1α, NRF-1 and TFAM were determined in peripheral blood mononuclear cells using western blotting and reverse transcription-quantitative PCR, respectively. Receiver operating characteristic curve analysis was used to evaluate the diagnostic accuracy of these indicators. The areas under the curve values of Mfn1/2, Opa1, Drp1, Fis1,AMPK, PGC-1α, NRF-1 and TFAM were 0.5408-0.8696. Compared with control subjects, mRNA expression levels of Mfn1/2, Opa1, AMPK, PGC-1α, NRF-1 and TFAM were lower, while those of Drp1 and Fis1 were higher in patients with MG. The protein expression levels of all these molecules were lower in patients with MG than in control subjects. These results suggested that mitochondrial dynamics and biogenesis indicators may be diagnostic biomarkers for MG.

Exploration of Potential Roles of m5C-Related Regulators in Colon Adenocarcinoma Prognosis.

Objectives: The purpose of this study was to investigate the role of 13 m5C-related regulators in colon adenocarcinoma (COAD) and determine their prognostic value. Methods: Gene expression and clinicopathological data were obtained from The Cancer Genome Atlas (TCGA) datasets. The expression of m5C-related regulators was analyzed with clinicopathological characteristics and alterations within m5C-related regulators. Subsequently, different subtypes of patients with COAD were identified. Then, the prognostic value of m5C-related regulators in COAD was confirmed via univariate Cox regression and least absolute shrinkage and selection operator (LASSO) Cox regression analyses. The prognostic value of risk scores was evaluated using the Kaplan-Meier method, receiver operating characteristic (ROC) curve. The correlation between the two m5C-related regulators, risk score, and clinicopathological characteristics were explored. Additionally, Gene Set Enrichment Analysis (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and Gene Ontology (GO) analysis were performed for biological functional analysis. Finally, the expression level of two m5C-related regulators in clinical samples and cell lines was detected by quantitative reverse transcription-polymerase chain reaction and through the Human Protein Atlas database. Results: m5C-related regulators were found to be differentially expressed in COAD with different clinicopathological features. We observed a high alteration frequency in these genes, which were significantly correlated with their mRNA expression levels. Two clusters with different prognostic features were identified. Based on two independent prognostic m5C-related regulators (NSUN6 and ALYREF), a risk signature with good predictive significance was constructed. Univariate and multivariate Cox regression analyses suggested that the risk score was an independent prognostic factor. Furthermore, this risk signature could serve as a prognostic indicator for overall survival in subgroups of patients with different clinical characteristics. Biological processes and pathways associated with cancer, immune response, and RNA processing were identified. Conclusion: We revealed the genetic signatures and prognostic values of m5C-related regulators in COAD. Together, this has improved our understanding of m5C RNA modification and provided novel insights to identify predictive biomarkers and develop molecular targeted therapy for COAD.