ObjectiveTo investigate the association of serum exosomal microRNAs （miRNAs） with hepatic inflammatory injury and histological outcomes in patients with chronic hepatitis B （CHB）. MethodsPeripheral serum samples were collected from six healthy adults and six patients with CHB， and size exclusion chromatography was used to extract exosomes. Small RNA sequencing and transcriptomic analysis were used to identify the serum exosomal miRNAs associated with liver inflammatory injury and fibrosis， and quantitative real-time PCR was used for validation in a mouse model of acute liver injury induced by lipopolysaccharide/D-galactosamine， a rat model of liver fibrosis induced by carbon tetrachloride， and 84 CHB patients undergoing liver biopsy twice before and after treatment. The independent-samples t test was used for comparison of normally distributed continuous data between two groups； an analysis of variance was used for comparison between multiple groups， and the Tukey test was used for further comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups； the Kruskal-Wallis H test was used for comparison between multiple groups， and the Dunn test was used for further comparison between two groups. The chi-square test or the Fisher’s exact test was used for comparison of categorical data between groups. The univariate and multivariate Logistic regression analyses were used to investigate influencing factors. ResultsAbnormal expression of serum exosomal miR-122-5p was observed in patients with CHB， and it was downregulated in patients with confluent necrosis and advanced fibrosis. In the mouse model of acute liver injury and the rat model of liver fibrosis， compared with the control group， the model group had a significant reduction in the expression level of miR-122-5p in the liver （P=0.048 and 0.014）， and compared with the patients with mild liver injury， the patients with severe confluent necrosis and advanced fibrosis showed a significant reduction in the expression level of miR-122-5p in liver tissue （P<0.05）. Among the 84 CHB patients， the patients with severe hepatic confluent necrosis or advanced liver fibrosis had a significantly lower expression level of serum exosomal miR-122-5p than those with mild liver injury （P<0.001 and P=0.003）. The multivariate Logistic regression analysis showed that the expression level of miR-122-5p was an independent influencing factor for confluent necrosis （odds ratio ［OR］=0.001， 95% confidence interval ［CI］： 0.000‍ ‍—‍ ‍0.037， P=0.005） and liver fibrosis degree （OR=0.568， 95%CI： 0.331‍ ‍—‍ ‍0.856， P=0.019）. In addition， compared with the patients with low expression of miR-122-5p， the patients with high expression of miR-122-5p before treatment had a significantly higher reversal rate of liver fibrosis after 72 weeks of antiviral therapy （64.3% vs 38.1%， P=0.029）. ConclusionSerum exosomal miR-122-5p in CHB patients is closely associated with the progression of hepatic confluent necrosis and fibrosis， and the reduction in the expression level of miR-122-5p may aggravate hepatic confluent necrosis， promote the progression of fibrosis， and affect the histological outcome of CHB patients after antiviral therapy.

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy with a poor prognosis, despite advancements in treatment. Many patients struggle with relapse or refractory disease. Investigating the role of the super-enhancer (SE) regulated gene ubiquitin-specific protease 20 (USP20) in T-ALL could enhance targeted therapies and improve clinical outcomes. Analysis of histone H3 lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) data from six T-ALL cell lines and seven pediatric samples identified USP20 as an SE-regulated driver gene. Utilizing the Cancer Cell Line Encyclopedia (CCLE) and BloodSpot databases, it was found that USP20 is specifically highly expressed in T-ALL. Knocking down USP20 with short hairpin RNA (shRNA) increased apoptosis and inhibited proliferation in T-ALL cells. In vivo studies showed that USP20 knockdown reduced tumor growth and improved survival. The USP20 inhibitor GSK2643943A demonstrated similar anti-tumor effects. Mass spectrometry, RNA-Seq, and immunoprecipitation revealed that USP20 interacted with hypoxia-inducible factor 1 subunit alpha (HIF1A) and stabilized it by deubiquitination. Cleavage under targets and tagmentation (CUT&Tag) results indicated that USP20 co-localized with HIF1A, jointly modulating target genes in T-ALL. This study identifies USP20 as a therapeutic target in T-ALL and suggests GSK2643943A as a potential treatment strategy.

OBJECTIVES: To investigate the active components and possible mechanisms of n-butanol fraction of Periploca forrestii Schltr. ethanol extract for treating Alzheimer's disease (AD). METHODS: The active components of n-butanol fraction of Periploca forrestii Schltr. ethanol extract were analyzed using UPLC-QE-MS technique. In a SD rat model of AD induced by treatment with AlCl₃ and D-gal, the therapeutic effects of low, moderate and high doses of the n-butanol fraction, saline, and donepezil hydrochloride were evaluated using ELISA, HE and Nissl staining, immunohistochemistry and Western blotting. The therapeutic mechanisms of the n-butanol fraction were explored using network pharmacology and molecular docking. RESULTS: Seventeen active components were identified from the n-butanol fraction of Periploca forrestii Schltr. ethanol extract, including phenylpropanoids, flavonoids, anthraquinones, triterpenoids, steroids, and volatile oils. In the rat models of AD, treatment with the n-butanol fraction significantly lowed AChE content in the hippocampus, increased the contents of ACh, SOD, CAT, and GSH-Px, enhanced the expressions of neuronal apoptotic factors Bcl-2, PI3K, Akt, p-PI3K, and p-Akt, and reduced the expressions of Bax and caspase-3 proteins. The treatment also dose-dependently up-regulated hippocampal expressions of Nrf-2, HO-1 and BDNF and down-regulated Keap-1, Aβ and Tau expressions. Bioinformatics analysis identified 14 key intersected targets (including TNF, AKT1 and ESR1) between the n-butanol fraction and AD. CONCLUSIONS The therapeutic effect of n-butanol fraction of Periploca forrestii Schltr. ethanol extract in AD mice is mediated by its multiple active components that regulate multiple targets and pathways.
Animals ; Rats, Sprague-Dawley ; Rats ; 1-Butanol/chemistry* ; Plant Extracts/pharmacology* ; Periploca/chemistry* ; Ethanol/chemistry* ; Alzheimer Disease/drug therapy* ; Male ; Molecular Docking Simulation ; Apoptosis/drug effects*

Ischemic stroke (IS) is a prevalent neurological disorder often resulting in significant disability or mortality. Resveratrol, extracted from Polygonum cuspidatum Sieb. et Zucc. (commonly known as Japanese knotweed), has been recognized for its potent neuroprotective properties. However, the neuroprotective efficacy of its derivative, (E)-4-(3,5-dimethoxystyryl) quinoline (RV02), against ischemic stroke remains inadequately explored. This study aimed to evaluate the protective effects of RV02 on neuronal ischemia-reperfusion injury both in vitro and in vivo. The research utilized an animal model of middle cerebral artery occlusion/reperfusion and SH-SY5Y cells subjected to oxygen-glucose deprivation and reperfusion to simulate ischemic conditions. The findings demonstrate that RV02 attenuates neuronal mitochondrial damage and scavenges reactive oxygen species (ROS) through mitophagy activation. Furthermore, Parkin knockdown was found to abolish RV02's ability to activate mitophagy and neuroprotection in vitro. These results suggest that RV02 shows promise as a neuroprotective agent, with the activation of Parkin-mediated mitophagy potentially serving as the primary mechanism underlying its neuroprotective effects.
Animals ; Ubiquitin-Protein Ligases/genetics* ; Mitophagy/drug effects* ; Resveratrol/analogs & derivatives* ; Neuroprotective Agents/pharmacology* ; Humans ; Neurons/metabolism* ; Reactive Oxygen Species/metabolism* ; Ischemic Stroke/genetics* ; Male ; Quinolines/pharmacology* ; Mice ; Fallopia japonica/chemistry* ; Mitochondria/metabolism* ; Reperfusion Injury/metabolism* ; Rats ; Mice, Inbred C57BL ; Disease Models, Animal

In order to provide basic information for the utilization and development of famous classical formulas containing Bletillae Rhizoma， this article systematically analyzes the historical evolution of the name， origin， harvesting and processing of Bletillae Rhizoma by reviewing the ancient materia medica， prescription books， medical books and modern literature. The research results showed that Baiji（白及） was the main name， some scholars took Baiji（白芨） as its main name， and there were many other names such as Baiji（白给）， Baigen（白根）， Baiji（白苙）. The mainstream source of Bletillae Rhizoma was the tubers of Bletilla striata， and drying， large， white， solid， root-free and skin removed completely were the good quality standards. With the promotion of wild to cultivated medicinal materials， there were certain differences between their traits， and the quality evaluation indexes should be adjusted accordingly. The origin of records in the past dynasties was widely distributed， with Guizhou and Sichuan having high production and good quality in modern times. The harvesting period is mostly in spring and autumn， and harvested in autumn was better. The processing and processing technology is relatively simple， and it was used fresh or powdered in past dynasties， while it is mainly sliced for raw use in modern times. Based on the results， it is suggested that the tubers of Bletilla striata of Orchidaceae should be used in the famous classical formulas， and it should be uniformly written as Baiji（白及）. And if the original formula indicates the requirement of processing， it should be operated according to the requirement， if the requirement of processing is not indicated， it can be used in raw form as medicine.

ObjectiveTo investigate the effect of transplantation of bone marrow mesenchymal stem cells （BMSCs） co-cultured with bone marrow-derived M2 macrophages （M2-BMDMs）， named as BMSC^M2， on a rat model of liver cirrhosis induced by carbon tetrachloride （CCl₄）/2-acetaminofluorene （2-AAF）. MethodsRat BMDMs were isolated and polarized into M2 phenotype， and rat BMSCs were isolated and co-cultured with M2-BMDMs at the third generation to obtain BMSC^M2. The rats were given subcutaneous injection of CCl₄ for 6 weeks to establish a model of liver cirrhosis， and then they were randomly divided into model group （M group）， BMSC group， and BMSC^M2 group， with 6 rats in each group. A normal group （N group） with 6 rats was also established. Since week 7， the model rats were given 2-AAF by gavage in addition to the subcutaneous injection of CCl₄. Samples were collected at the end of week 10 to observe liver function， liver histopathology， and hydroxyproline （Hyp） content in liver tissue， as well as changes in the markers for hepatic stellate cells， hepatic progenitor cells， cholangiocytes， and hepatocytes. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the N group， the M group had significant increases in the activities of serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） （P<0.01）； compared with the M group， the BMSC and BMSC^M2 groups had significant reductions in ALT and AST （P<0.01）， and the BMSC^M2 group had significantly better activities than the BMSC group （P<0.05）. Compared with the N group， the M group had significant increases in Hyp content and the mRNA and protein expression levels of alpha-smooth muscle actin （α-SMA） in the liver （P<0.01）； compared with the M group， the BMSC and BMSC^M2 groups had significant reductions in Hyp content and the expression of α-SMA （P<0.05）， and the BMSC^M2 group had a significantly lower level of α-SMA than the BMSC group （P<0.01）. Compared with the N group， the M group had significant increases in the mRNA expression levels of the hepatic progenitor cell markers EpCam and Sox9 and the cholangiocyte markers CK7 and CK19 （P<0.01） and significant reductions in the expression levels of the hepatocyte markers HNF-4α and Alb （P<0.01）； compared with the M group， the BMSC and BMSC^M2 groups had significant reductions in the mRNA expression levels of EpCam， Sox9， CK7， and CK19 （P<0.05） and significant increases in the mRNA expression levels of HNF-4α and Alb （P<0.05）， and compared with the BMSC group， the BMSC^M2 group had significant reductions in the mRNA expression levels of EpCam and CK19 （P<0.05） and significant increase in the expression level of HNF-4α （P<0.05）. ConclusionM2-BMDMs can enhance the therapeutic effect of BMSCs on CCl₄/2-AAF-induced liver cirrhosis in rats， which provides new ideas for further improving the therapeutic effect of BMSCs on liver cirrhosis.

ObjectiveTo investigate whether anti-PD-1 monoclonal antibody can improve the efficacy and safety of cryoablation combined with lenvatinib in the treatment of unresectable hepatocellular carcinoma （HCC）. MethodsA retrospective analysis was performed for 232 patients with unresectable HCC who were treated at The Fifth Medical Center of Chinese PLA General Hospital from January 2018 to December 2022， among whom 128 received cryoablation combined with lenvatinib （double combination） and 104 received cryoablation combined with lenvatinib and anti-PD-1 monoclonal antibody （triple combination）. Propensity score matching was performed at a ratio of 1∶1， and finally there were 86 patients in each group. The two groups were evaluated in terms of objective response rate （ORR）， disease control rate （DCR）， overall survival （OS）， progression-free survival （PFS）， and adverse events （AEs）. The independent-samples t test was used for comparison of normally distributed continuous data between two groups， and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups； the chi-square test was used for comparison of categorical data between two groups. Survival curves were plotted， and the Kaplan-Meier method was used to calculate the survival rate of patients in both groups， while the log-rank test was used for comparison between the two groups. The Cox regression model was used to calculate hazard ratio （HR） and 95% confidence interval （CI） and perform the univariate and multivariate analyses of influencing factors for prognosis. ResultsThe median follow-up time was 28 months， and there were 33 deaths （38.0%） in the triple combination group and 40 deaths （46.0%） in the double combination group. Compared with the double combination group， the triple combination group had significantly higher ORR （35.6% vs 14.5%， P=0.008） and DCR （86.1% vs 64.1%， P=0.003）. OS and PFS in the triple combination group were significantly higher than those in the double combination group （P=0.045 and 0.026）. The univariate and multivariate Cox proportional-hazards regression model analyses showed that treatment regimen （HR=0.60， P=0.038） and alpha-fetoprotein level （HR=2.37， P=0.001） were independent risk factors for OS， and treatment regimen （HR=0.65， P=0.025）， diabetes mellitus （HR=1.94， P=0.005）， whether or not to have received local treatment （HR=0.63， P=0.014）， and distant metastasis （HR=0.58， P=0.009） were independent risk factors for PFS. There was no significant difference in the incidence rate of AEs between the two groups （P>0.05）. ConclusionFor patients with unresectable HCC， the triple combination of cryoablation， lenvatinib， and anti-PD-1 monoclonal antibody significantly improves the treatment outcome and survival of patients compared with the double combination of cryoablation and lenvatinib， without increasing AEs， which provides a clinical basis for optimizing the treatment regimen for unresectable HCC.

The advent of chimeric antigen receptor (CAR)-T cell immunotherapies has led to breakthroughs in the treatment of hematological malignancies. However, their success in treating solid tumors has been limited. CAR-natural killer (NK) cells have several advantages over CAR-T cells because NK cells can be made from pre-existing cell lines or allogeneic NK cells with a mismatched major histocompatibility complex (MHC), which means they are more likely to become an "off-the-shelf" product. Moreover, they can kill cancer cells via CAR-dependent/independent pathways and have limited toxicity. Macrophages are the most malleable immune cells in the body. These cells can efficiently infiltrate into tumors and are present in large numbers in tumor microenvironments (TMEs). Importantly, CAR-macrophages (CAR-Ms) have recently yielded exciting preclinical results in several solid tumors. Nevertheless, CAR-T, CAR-NK, and CAR-M all have their own advantages and limitations. In this review, we systematically discuss the current status, progress, and the major hurdles of CAR-T cells, CAR-NK cells, and CAR-M as they relate to five aspects: CAR structure, therapeutic mechanisms, the latest research progress, current challenges and solutions, and comparison according to the existing research in order to provide a reasonable option for treating solid tumors in the future.

Hepatocellular carcinoma （HCC） is the sixth most common cancer in the world. In recent years， the clinical early diagnosis and treatment protocols of HCC have been improved， whereas the prognosis of patients is still not satisfactory， which is due to the fact that the mechanism of HCC development has not been fully elucidated. Therefore， it is of great significance to explore the molecular mechanisms and key regulatory links of hepatocellular carcinoma development to further improve the diagnosis and treatment of HCC in China. Epigenetics has become a research hotspot because of its reversibility and easy regulation. According to relevant studies， HCC involves the accumulation of multiple genetic and epigenetic changes during the initiation， promotion， and progression stages. HCC is categorized as infantile malnutrition with accumulation， hypochondriac pain， tympan ites， and abdominal mass in traditional Chinese medicine （TCM）. In the treatment of HCC， TCM with low toxicity， multi-targets， and multi-mechanisms can inhibit tumor growth， alleviate the clinical symptoms， and enhance the quality of life of the patients. Chinese medicines and their active ingredients exert anti-HCC effects through epigenetic regulation of DNA methylation， histone modification， and non-coding RNA. Abnormal gene expression due to epigenetic regulation disorders is involved in all stages of HCC development. There are few studies on epigenetic regulation in TCM treatment of HCC， and there is still much room for development in basic and clinical trials. This paper reviews the mechanism of epigenetic regulation in HCC and summarizes the experimental results of TCM research on the related mechanism， with a view to providing a theoretical basis for future research on the mechanism of HCC development and clinical diagnosis and treatment of hepatocellular carcinoma with TCM.