BACKGROUND: Temozolomide (TMZ) resistance is a significant challenge in treating glioblastoma (GBM). Collagen remodeling has been shown to be a critical factor for therapy resistance in other cancers. This study aimed to investigate the mechanism of TMZ chemoresistance by GBM cells reprogramming collagens. METHODS: Key extracellular matrix components, including collagens, were examined in paired primary and recurrent GBM samples as well as in TMZ-treated spontaneous and grafted GBM murine models. Human GBM cell lines (U251, TS667) and mouse primary GBM cells were used for in vitro studies. RNA-sequencing analysis, chromatin immunoprecipitation, immunoprecipitation-mass spectrometry, and co-immunoprecipitation assays were conducted to explore the mechanisms involved in collagen accumulation. A series of in vitro and in vivo experiments were designed to assess the role of the collagen regulators prolyl 4-hydroxylase subunit alpha 1 (P4HA1) and yes-associated protein (YAP) in sensitizing GBM cells to TMZ. RESULTS: This study revealed that TMZ exposure significantly elevated collagen type I (COL I) expression in both GBM patients and murine models. Collagen accumulation sustained GBM cell survival under TMZ-induced stress, contributing to enhanced TMZ resistance. Mechanistically, P4HA1 directly binded to and hydroxylated YAP, preventing ubiquitination-mediated YAP degradation. Stabilized YAP robustly drove collagen type I alpha 1 ( COL1A1) transcription, leading to increased collagen deposition. Disruption of the P4HA1-YAP axis effectively reduced COL I deposition, sensitized GBM cells to TMZ, and significantly improved mouse survival. CONCLUSION P4HA1 maintained YAP-mediated COL1A1 transcription, leading to collagen accumulation and promoting chemoresistance in GBM.
Temozolomide ; Humans ; Glioblastoma/drug therapy* ; Animals ; Mice ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics* ; YAP-Signaling Proteins ; Hydroxylation ; Dacarbazine/pharmacology* ; Adaptor Proteins, Signal Transducing/metabolism* ; Transcription Factors/metabolism* ; Collagen/biosynthesis* ; Collagen Type I/metabolism* ; Prolyl Hydroxylases/metabolism* ; Antineoplastic Agents, Alkylating/therapeutic use*

OBJECTIVES: To explore the underlying pharmacological mechanisms and its potential effects of Chinese medicine herbal formula Sini Powder (SNP) on hepatocellular carcinoma (HCC). METHODS: The active components of SNP and their in vivo distribution were identified using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Construction of component-target-disease networks, protein-protein interaction network, Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and molecular docking were employed to analyze the active components and anti-HCC mechanisms of SNP. Cell viability assay and wound healing assay were utilized to confirm the effect of SNP-containing serum (2.5%, 5.0%, 10%, 20%, and 40%), isoprenaline or propranolol (both 10, 100, and 1,000 µ mol/L) on proliferation and migration of HepG 2 or Huh7 cells. Meanwhile, the effect of isoprenaline or propranolol on the β 2 adrenergic receptor (ADRB2) mRNA expression on HepG2 cells were measured by real-time quantitative reverse transcription (RT-qPCR). Mice with subcutaneous tumors were either subjected to chronic restraint stress (CRS) followed by SNP administration (364 mg/mL) or directly treated with SNP (364 mg/mL). These two parallel experiments were performed to validate the effects of SNP on stress responses. Stress-related proteins and hormones were quantified using RT-qPCR, enzyme-linked immunosorbent assay, and immunohistochemistry. Metagenomic sequencing was performed to confirm the influence of SNP on the gut microbiota in the tumor-bearing CRS mice. RESULTS: The distribution of the 12 active components of SNP was confirmed in various tissues and feces. Network pharmacology analysis confirmed the anti-HCC effects of the 5 active components. The potential anti-HCC mechanisms of SNP may involve the epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein kinase Src (SRC) and signal transducer and activator of transcription 3 (STAT3) pathways. SNP-containing serum inhibited the proliferation of HepG2 and Huh7 cells at concentrations of 2.5% and 5.0%, respectively, after 24 h of treatment. Furthermore, SNP suppressed tumor progression in tumor-bearing mice exposed to CRS. SNP treatment also downregulated the expressions of stress-related proteins and pro-inflammatory cytokines, primarily by modulating the gut microbiota. Specifically, the abundance of Alistipes and Prevotella, which belong to the phylum Bacteroidetes, increased in the SNP-treated group, whereas Lachnospira, in the phylum Firmicutes, decreased. CONCLUSION SNP can combat HCC by alleviating stress responses through the regulation of gut microbiota.
Animals ; Gastrointestinal Microbiome/drug effects* ; Liver Neoplasms/microbiology* ; Carcinoma, Hepatocellular/microbiology* ; Humans ; Drugs, Chinese Herbal/therapeutic use* ; Powders ; Cell Proliferation/drug effects* ; Mice ; Molecular Docking Simulation ; Cell Line, Tumor ; Hep G2 Cells ; Receptors, Adrenergic, beta-2/genetics* ; Stress, Physiological/drug effects* ; Cell Movement/drug effects* ; Male ; Protein Interaction Maps/drug effects* ; Cell Survival/drug effects* ; Proto-Oncogene Mas

ObjectiveTriple-negative breast cancer (TNBC) is the breast cancer subtype with the worst prognosis, and lacks effective therapeutic targets. Colony stimulating factors (CSFs) are cytokines that can regulate the production of blood cells and stimulate the growth and development of immune cells, playing an important role in the malignant progression of TNBC. This article aims to construct a novel prognostic model based on the expression of colony stimulating factors-related genes (CRGs), and analyze the sensitivity of TNBC patients to immunotherapy and drug therapy. MethodsWe downloaded CRGs from public databases and screened for differentially expressed CRGs between normal and TNBC tissues in the TCGA-BRCA database. Through LASSO Cox regression analysis, we constructed a prognostic model and stratified TNBC patients into high-risk and low-risk groups based on the colony stimulating factors-related genes risk score (CRRS). We further analyzed the correlation between CRRS and patient prognosis, clinical features, tumor microenvironment (TME) in both high-risk and low-risk groups, and evaluated the relationship between CRRS and sensitivity to immunotherapy and drug therapy. ResultsWe identified 842 differentially expressed CRGs in breast cancer tissues of TNBC patients and selected 13 CRGs for constructing the prognostic model. Kaplan-Meier survival curves, time-dependent receiver operating characteristic curves, and other analyses confirmed that TNBC patients with high CRRS had shorter overall survival, and the predictive ability of CRRS prognostic model was further validated using the GEO dataset. Nomogram combining clinical features confirmed that CRRS was an independent factor for the prognosis of TNBC patients. Moreover, patients in the high-risk group had lower levels of immune infiltration in the TME and were sensitive to chemotherapeutic drugs such as 5-fluorouracil, ipatasertib, and paclitaxel. ConclusionWe have developed a CRRS-based prognostic model composed of 13 differentially expressed CRGs, which may serve as a useful tool for predicting the prognosis of TNBC patients and guiding clinical treatment. Moreover, the key genes within this model may represent potential molecular targets for future therapies of TNBC.

Background and Objectives: Osteoblasts are derived from bone marrow mesenchymal stem cells (BMMSCs) and playimportant role in bone remodeling. While our previous studies have investigated the cell subtypes and heterogeneity in osteoblasts and BMMSCs separately, cell-to-cell communications between osteoblasts and BMMSCs in vivo in humans have not been characterized. The aim of this study was to investigate the cellular communication between human primary osteoblasts and bone marrow mesenchymal stem cells. Methods: and Results: To investigate the cell-to-cell communications between osteoblasts and BMMSCs and identifynew cell subtypes, we performed a systematic integration analysis with our single-cell RNA sequencing (scRNA-seq) transcriptomes data from BMMSCs and osteoblasts. We successfully identified a novel preosteoblasts subtype which highly expressed ATF3, CCL2, CXCL2 and IRF1. Biological functional annotations of the transcriptomes suggested that the novel preosteoblasts subtype may inhibit osteoblasts differentiation, maintain cells to a less differentiated status and recruit osteoclasts. Ligand-receptor interaction analysis showed strong interaction between mature osteoblasts and BMMSCs. Meanwhile, we found FZD1 was highly expressed in BMMSCs of osteogenic differentiation direction. WIF1 and SFRP4, which were highly expressed in mature osteoblasts were reported to inhibit osteogenic differentiation. We speculated that WIF1 and sFRP4 expressed in mature osteoblasts inhibited the binding of FZD1 to Wnt ligand in BMMSCs, thereby further inhibiting osteogenic differentiation of BMMSCs. Conclusions Our study provided a more systematic and comprehensive understanding of the heterogeneity of osteogenic cells. At the single cell level, this study provided insights into the cell-to-cell communications between BMMSCs and osteoblasts and mature osteoblasts may mediate negative feedback regulation of osteogenesis process.

Bungarus Parvus, a precious animal Chinese medicinal material used in clinical practice, is believed to be first recorded in Ying Pian Xin Can published in 1936. This study was carried out to analyze the names, geographical distribution, morphological characteristics, ecological habits, poisonousness, and medicinal parts by consulting ancient Chinese medical books and local chronicles, Chinese Pharmacopeia, different processing standards of trditional Chinese medicine(TCM) decoction pieces, and modern literatures. The results showed that the earliest medicinal record of Bungarus Parvus was traced to 1894. In 1930, this medicinal material was used in the formulation of Annao Pills. The original animal, Bungarus multicinctus, was recorded by the name of "Bojijia" in 1521. The morphological characteristics, ecological habits, and poisonousness of the original animal are the same in ancient and modern records. The geographical distribution is similar between the ancient records and modern documents such as China Medicinal Animal Fauna. The dried body of young B. multicinctus is used as Bungarus Parvus, which lack detailed references. As a matter of fact, it is still inconclusive whether there are differences between young snakes and adult snakes in terms of active ingredients, pharmacological effects, and clinical applications. This study clarified the medicinal history and present situation of Bungarus Parvus. On the basis of the results, it is suggested that systematic comparison on young and adult B. multicinctus should be carried out to provide references for revising the medicinal parts of B. multicinctus.
Animals ; Bungarus ; Snakes ; China ; Medicine, Chinese Traditional ; Drugs, Chinese Herbal

OBJECTIVE: To study the mechanism of Shengmai Injection (SMI, ) on anti-sepsis and protective activities of intestinal mucosal barrier. METHODS: The contents of 11 active components of SMI including ginsenoside Rb1, Rb2, Rb3, Rd, Re, Rf, Rg1, Rg2, ophioposide D, schisandrol A and schisantherin A were determined using ultra-performance liquid chromatography. Fifty mice were randomly divided into the blank, the model, the low-, medium- and high-dose SMI groups (0.375, 0.75, 1.5 mL/kg, respectively) by random number table, 10 mice in each group. In SMI group, SMI was administrated to mice daily via tail vein injection for 3 consecutive days, while the mice in the blank and model groups were given 0.1 mL of normal saline. One hour after the last SMI administration, except the blank group, the mice in other groups were intraperitoneally injected with lipopolysaccharide (LPS) saline solution (2 mL/kg) at a dosage of 5 mL/kg for development of endotoxemia mice model. The mice in the blank group were given the same volume of normal saline. Inflammatory factors including interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), interleukin (IL)-2 and IL-10 were measured by flow cytometry. Myosin light-chain kinase (MLCK), nuclear factor κB (NF-κB) levels, and change of Occludin proteins in jejunum samples were analyzed by Western blot. RESULTS: The decreasing trends of INF-γ, TNF-α and IL-2 were found in serum of SMI treatment groups. In SMI-treated mice, the content of Occludin increased and MLCK protein decreased compared with the model group (P<0.05 or P<0.01). The content of cellular and nuclear NF-κB did not change significantly (P>0.05). CONCLUSION SMI may exert its anti-sepsis activity mainly through NF-κB-pro-inflammatory factor-MLCK-TJ cascade.
Animals ; Drug Combinations ; Drugs, Chinese Herbal ; Mice ; NF-kappa B/metabolism* ; Occludin ; Saline Solution ; Sepsis/drug therapy* ; Tumor Necrosis Factor-alpha/metabolism*

Objective:To analyze the effect of transtracheal pressure (TTP) on the application of a speaking valve in critically ill patients after tracheostomy.Methods:A retrospective analysis was conducted of 50 patients wearing a speaking valve after tracheostomy. Patients who had been wearing a speaking valve for 30min or more were the tolerance group, while those with less than 30min were the intolerance group. Transtracheal pressure was monitored during resting breathing, forced expiration and speaking. Linear regression models were evaluated to isolate the factors best predicting tolerance. The changes in respiratory muscle contraction before and after wearing a speaking valvs were evaluated using ultrasound. The patients′ satisfaction with wearing a speaking valve was also recorded.Results:TTP during speaking significantly predicted tolerance. The baseline values of diaphragmatic thickening fraction and physical functioning also positively predicted tolerance. Acute physiology and chronic health (APACHE II) score was a significant negative predictor. After wearing the speaking valve, the average contraction of the rectus abdominis, external oblique, internal oblique and transverse abdominis muscles increased significantly. Both groups expressed high satisfaction with the speaking valves.Conclusions:Transtracheal pressure during speaking can help predict the tolerance for wearing a speaking valve among critically ill patients after a tracheostomy. Baseline diaphragmatic thickening fraction, physical functioning and APACHE II score can predict the duration of speaking valve tolerance.

ObjectiveTo observe the effect of Linggui Zhugantang （LG） on the blood-brain barrier （BBB） model of Alzheimer's disease （AD） in vitro and to explore the mechanism of LG in repairing the BBB injury in AD. MethodA total of 50 male SPF rats were randomized into five groups： high-dose （4.8 g·kg^-1）， medium-dose （2.4 g·kg^-1）， and low-dose （1.2 g·kg^-1） LG groups， western medicine （0.5 g·kg^-1 donepezil hydrochloride） group， and normal group （normal saline of equivalent volume）. They received （ig） corresponding drugs twice a day for 7 d. Drug-containing serum was respectively collected from the abdominal aorta 1 h after the last administration. The BBB injury of AD in vitro was induced with the cell co-culture method， and 6 groups were designed： normal group， model group， high-， medium-， and low-dose LG groups， and western medicine group. The model group was added with 100 μL amyloid β_1-42 （Aβ_1-42， final concentration： 5 μmol·L^-1）， and high-dose， medium-dose， and low-dose LG groups and the western medicine group were added with corresponding 10% drug-containing serum in addition to the 100 μL Aβ_1-42 （final concentration： 5 μmol·L^-1）. Cell survival rate was detected by methyl thiazolyl tetrazolium （MTT） assay， expression of BBB-related skeleton proteins （claudin-5， ZO-1， occludin）， matrix metalloproteinase-2 （MMP-2）， and matrix metalloproteinase-9 （MMP-9） by Western blot， and content of inflammatory factors interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） by enzyme-linked immunosorbent assay （ELISA）. BBB Aβ transporter low-density lipoprotein receptor-related protein 1 （LRP-1） and advanced glycation end product receptor （RAGE） at different time points in high-dose， medium-dose， and low-dose LG groups were determined by Real-time PCR and Western blot. ResultCell survival rate of the model group was lower than that of the normal group （P<0.05） and the survival rates of the western medicine group and high-dose LG group was higher than that in the model group （P<0.05）. The skeleton proteins were down-regulated and MMP-2 and MMP-9 were up-regulated in the model group compared with those in the normal group （P<0.05）. The expression of skeleton proteins was higher （P<0.05） and that of MMP-2 and MMP-9 was lower （P<0.05） in the western medicine group and high-dose LG group than in the model group. Compared with the model group， only the medium-dose LG group showed the up-regulation （P<0.05） of claudin-5 （P<0.05） and the decrease （P<0.05） of MMP-2. IL-1β， IL-6， and TNF-α in the model group were up-regulated （P<0.05） compared with those in the normal group， and those inflammatory factors in the western medicine group and high-dose and medium-dose LG groups were lower （P<0.05） than those in the model group. LRP-1 expression was up-regulated and RAGE expression was down-regulated at 3 h compared with those at 0 h （P<0.05）， while the expression of the two became stable at 6， 12， 24， 36 h. At 3 h， LRP-1 expression was down-regulated and RAGE expression was up-regulated in model group compared with those in the normal group at 3 h （P<0.05）. Moreover， the LRP-1 content was higher and RAGE content was lower in the western medicine group and high-dose LG group than in the model group. ConclusionLG can repair the BBB injury in vitro by inhibiting the expression of inflammatory factors and MMP-2， MMP-9， promoting the expression of skeletal proteins， and regulating the balance of transporters.

Activation and inflammation of microglial correlate with progressive neuronal apoptosis in neurodegenerative disorders such as Parkinson’ s disease (PD). γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, has recently been shown to play an inhibitory role in the immune system, but the mechanism is unclear. In this study, the results showed that LPS promoted the release of inflammatory factors in a dose-dependent manner compared with the control group (P<0. 01). Meanwhile, cell viability and cytotoxicity assays showed that the released inflammatory factors could induce the decline of SH-SY5Y cell viability. BV2 microglia cells were pretreated with GABA and Muscimol, a GABA

Objective To investigate the prevalence and influencing factors of intestinal protozoan infections among rural children in Henan Province. Methods A total of 104 survey sites were sampled from 35 counties (cities) in Henan Province using the stratified cluster sampling method to investigate the prevalence of intestinal protozoan infections among rural children from 2014 to 2015. The trophozoites and cysts of intestinal protozoa were identified using the iodine staining method and the physiological saline direct smear method (one detection for one stool sample). The prevalence of intestinal protozoan infections was compared among rural children with different characteristics, and the factors affecting intestinal protozoan infections among rural children were identified. Results The overall prevalence of intestinal protozoan infections was 0.60% (40/6 771) among rural children in Henan Province from 2014 to 2015. There were 7 species of intestinal protozoa identified, and there was no species-specific prevalence (χ² = 37.732, P = 0.000). No significant differences were found in prevalence of intestinal protozoan infections among rural children in terms of gender (χ² = 1.793, P = 0.181), age (χ² = 1.443, P = 0.486), occupation (χ² = 0.219, P = 0.896) or ecological region (χ² = 1.700, P = 0.637). In addition, terrain (χ² = 2.311, P = 0.510), economic level (χ² = 4.322, P = 0.229), source of drinking water (χ² = 0.731, P = 0.393), eating raw vegetables (χ² = 1.134, P = 0.287) and deworming (χ² = 1.089, P = 0.297) had no remarkable effects on the prevalence of intestinal protozoan infections among rural children in Henan Province; however, the prevalence of intestinal protozoan infections varied significantly among rural children living in regions with different coverage of non-harmless toilets (χ² = 10.050, P = 0.018). Conclusion The prevalence of intestinal protozoan infections is low among rural children in Henan Province.