Identification of SULF1 as a Shared Gene in Idiopathic Pulmonary Fibrosis and Lung Adenocarcinoma / 中国肺癌杂志

BACKGROUND@#Idiopathic pulmonary fibrosis (IPF) is an idiopathic chronic, progressive interstitial lung disease with a diagnosed median survival of 3-5 years. IPF is associated with an increased risk of lung cancer. Therefore, exploring the shared pathogenic genes and molecular pathways between IPF and lung adenocarcinoma (LUAD) holds significant importance for the development of novel therapeutic approaches and personalized precision treatment strategies for IPF combined with lung cancer.@*METHODS@#Bioinformatics analysis was conducted using publicly available gene expression datasets of IPF and LUAD from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis was employed to identify common genes involved in the progression of both diseases, followed by functional enrichment analysis. Subsequently, additional datasets were used to pinpoint the core shared genes between the two diseases. The relationship between core shared genes and prognosis, as well as their expression patterns, clinical relevance, genetic characteristics, and immune-related functions in LUAD, were analyzed using The Cancer Genome Atlas (TCGA) database and single-cell RNA sequencing datasets. Finally, potential therapeutic drugs related to the identified genes were screened through drug databases.@*RESULTS@#A total of 529 shared genes between IPF and LUAD were identified. Among them, SULF1 emerged as a core shared gene associated with poor prognosis. It exhibited significantly elevated expression levels in LUAD tissues, concomitant with high mutation rates, genomic heterogeneity, and an immunosuppressive microenvironment. Subsequent single-cell RNA-seq analysis revealed that the high expression of SULF1 primarily originated from tumor-associated fibroblasts. This study further demonstrated an association between SULF1 expression and tumor drug sensitivity, and it identified potential small-molecule drugs targeting SULF1 highly expressed fibroblasts.@*CONCLUSIONS@#This study identified a set of shared molecular pathways and core genes between IPF and LUAD. Notably, SULF1 may serve as a potential immune-related biomarker and therapeutic target for both diseases.

Imputation method for dropout in single-cell transcriptome data / 生物医学工程学杂志

Single-cell transcriptome sequencing (scRNA-seq) can resolve the expression characteristics of cells in tissues with single-cell precision, enabling researchers to quantify cellular heterogeneity within populations with higher resolution, revealing potentially heterogeneous cell populations and the dynamics of complex tissues. However, the presence of a large number of technical zeros in scRNA-seq data will have an impact on downstream analysis of cell clustering, differential genes, cell annotation, and pseudotime, hindering the discovery of meaningful biological signals. The main idea to solve this problem is to make use of the potential correlation between cells and genes, and to impute the technical zeros through the observed data. Based on this, this paper reviewed the basic methods of imputing technical zeros in the scRNA-seq data and discussed the advantages and disadvantages of the existing methods. Finally, recommendations and perspectives on the use and development of the method were provided.

Liver macrophages show an immunotolerance phenotype in nonalcoholic fatty liver combined with Porphyromonas gingivalis-lipopolysaccharide infection / 华西口腔医学杂志

OBJECTIVES@#This study aimed to explore the functions and potential regulatory targets of local macrophages in nonalcoholic fatty liver combined with Porphyromonas gingivalis (P. gingivalis)infection.@*METHODS@#Single-cell RNA sequencing was used to analyze the phenotypes and functional changes in various cells in the liver tissue of nonalcoholic steatohepatitis (NASH) mice fed with P. gingivalis. Real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, and immunofluorescence staining were applied to observe the inflammation and expression levels of macrophage antigen presenting functional markers in the NASH liver. Oil red staining was performed to observe the accumulation of local adipose tissue in the NASH liver. Results were verified through RT-PCRand RNA sequencing using P. gingivalis-lipopolysaccharide treated mouse peritoneal macrophages.@*RESULTS@#In comparison with healthy livers with Kupffer cells, the NASH liver combined with P. gingivalis infection-related macrophages showed significant heterogeneity. C1qb, C1qc, Mafb, Apoe, and Cd14 were highly expressed, but Cd209a, H2-Aa, H2-Ab1, and H2-DMb1, which are related to the antigen presentation function, were weakly expressed. Further in vivo and in vitro investigations indicated that the activation and infiltration of these macrophages may be due to local P. gingivalis-lipopolysaccharide accumulation.@*CONCLUSIONS@#P. gingivalis-lipopolysaccharide induces a local macrophage immunotolerance phenotype in nonalcoholic fatty liver, which may be the key mechanism of periodontitis pathogen infection that promotes NASH inflammation and pathogenesis. This study further clarifies the dysfunction and regulatory mechanisms of macrophages in the pathogenesis of P. gingivalis-infected NASH, thereby providing potential therapeutic targets for its clinical treatment.

Circulating Exosomal LncRNAs as Novel Diagnostic Predictors of Severity and Sites of White Matter Hyperintensities / 生物医学与环境科学（英文）

OBJECTIVE@#Exosomal long noncoding RNAs (lncRNAs) are the key to diagnosing and treating various diseases. This study aimed to investigate the diagnostic value of plasma exosomal lncRNAs in white matter hyperintensities (WMH).@*METHODS@#We used high-throughput sequencing to determine the differential expression (DE) profiles of lncRNAs in plasma exosomes from WMH patients and controls. The sequencing results were verified in a validation cohort using qRT-PCR. The diagnostic potential of candidate exosomal lncRNAs was proven by binary logistic analysis and receiver operating characteristic (ROC) curves. The diagnostic value of DE exo-lncRNAs was determined by the area under the curve (AUC). The WMH group was then divided into subgroups according to the Fazekas scale and white matter lesion site, and the correlation of DE exo-lncRNAs in the subgroup was evaluated.@*RESULTS@#In our results, four DE exo-lncRNAs were identified, and ROC curve analysis revealed that exo-lnc_011797 and exo-lnc_004326 exhibited diagnostic efficacy for WMH. Furthermore, WMH subgroup analysis showed exo-lnc_011797 expression was significantly increased in Fazekas 3 patients and was significantly elevated in patients with paraventricular matter hyperintensities.@*CONCLUSION@#Plasma exosomal lncRNAs have potential diagnostic value in WMH. Moreover, exo-lnc_011797 is considered to be a predictor of the severity and location of WMH.

Preliminary Results of Single-cell Transcriptome Sequencing in Renal Arterial Lesions of Takayasu Arteritis / 中国医学科学院学报

Objective To explore the preliminary application of single-cell RNA sequencing (scRNA-seq) in the renal arterial lesions in Takayasu arteritis (TA) patients. Methods This study included 2 TA patients with renal artery stenosis treated by bypass surgery in the Department of Vascular Surgery,Beijing Hospital.The obtained 2 renal artery samples were digested with two different protocols (GEXSCOPE kit and self-made digestion liquid) before scRNA-seq and bioinformatics analysis. Results A total of 2920 cells were obtained for further analysis.After unbiased cluster analysis,2 endothelial cell subsets,2 smooth muscle cell subsets,1 fibroblast subset,2 mononuclear macrophage subsets,1 T cell subset,and 1 undefined cell subset were identified.Among them,the two subsets of smooth muscle cells were contractile and secretory,respectively.The results of scRNA-seq indicated that enzymatic hydrolysis with GEXSCOPE kit produced a large number of endothelial cells (57.46%) and a small number of immune cells (13.21%).However,immune cells (34.64%) were dominant in the cells obtained by enzymatic hydrolysis with self-made digestive liquid. Conclusion scRNA-seq can be employed to explore the cellular heterogeneity of diseased vessels in TA patients.Different enzymatic digestion protocols may impact the proportion of different cells.

Single-cell profiling reveals a potent role of quercetin in promoting hair regeneration

Hair loss affects millions of people at some time in their life, and safe and efficient treatments for hair loss are a significant unmet medical need. We report that topical delivery of quercetin (Que) stimulates resting hair follicles to grow with rapid follicular keratinocyte proliferation and replenishes perifollicular microvasculature in mice. We construct dynamic single-cell transcriptome landscape over the course of hair regrowth and find that Que treatment stimulates the differentiation trajectory in the hair follicles and induces an angiogenic signature in dermal endothelial cells by activating HIF-1α in endothelial cells. Skin administration of a HIF-1α agonist partially recapitulates the pro-angiogenesis and hair-growing effects of Que. Together, these findings provide a molecular understanding for the efficacy of Que in hair regrowth, which underscores the translational potential of targeting the hair follicle niche as a strategy for regenerative medicine, and suggest a route of pharmacological intervention that may promote hair regrowth.

Single-nucleus transcriptomics reveals a gatekeeper role for FOXP1 in primate cardiac aging

Aging poses a major risk factor for cardiovascular diseases, the leading cause of death in the aged population. However, the cell type-specific changes underlying cardiac aging are far from being clear. Here, we performed single-nucleus RNA-sequencing analysis of left ventricles from young and aged cynomolgus monkeys to define cell composition changes and transcriptomic alterations across different cell types associated with age. We found that aged cardiomyocytes underwent a dramatic loss in cell numbers and profound fluctuations in transcriptional profiles. Via transcription regulatory network analysis, we identified FOXP1, a core transcription factor in organ development, as a key downregulated factor in aged cardiomyocytes, concomitant with the dysregulation of FOXP1 target genes associated with heart function and cardiac diseases. Consistently, the deficiency of FOXP1 led to hypertrophic and senescent phenotypes in human embryonic stem cell-derived cardiomyocytes. Altogether, our findings depict the cellular and molecular landscape of ventricular aging at the single-cell resolution, and identify drivers for primate cardiac aging and potential targets for intervention against cardiac aging and associated diseases.

Single-nucleus profiling unveils a geroprotective role of the FOXO3 in primate skeletal muscle aging

Age-dependent loss of skeletal muscle mass and function is a feature of sarcopenia, and increases the risk of many aging-related metabolic diseases. Here, we report phenotypic and single-nucleus transcriptomic analyses of non-human primate skeletal muscle aging. A higher transcriptional fluctuation was observed in myonuclei relative to other interstitial cell types, indicating a higher susceptibility of skeletal muscle fiber to aging. We found a downregulation of FOXO3 in aged primate skeletal muscle, and identified FOXO3 as a hub transcription factor maintaining skeletal muscle homeostasis. Through the establishment of a complementary experimental pipeline based on a human pluripotent stem cell-derived myotube model, we revealed that silence of FOXO3 accelerates human myotube senescence, whereas genetic activation of endogenous FOXO3 alleviates human myotube aging. Altogether, based on a combination of monkey skeletal muscle and human myotube aging research models, we unraveled the pivotal role of the FOXO3 in safeguarding primate skeletal muscle from aging, providing a comprehensive resource for the development of clinical diagnosis and targeted therapeutic interventions against human skeletal muscle aging and the onset of sarcopenia along with aging-related disorders.

Acute Developmental Toxicity of Panax notoginseng in Zebrafish Larvae / 中国结合医学杂志

OBJECTIVE@#To evaluate toxicity of raw extract of Panax notoginseng (rPN) and decocted extract of PN (dPN) by a toxicological assay using zebrafish larvae, and explore the mechanism by RNA sequencing assay.@*METHODS@#Zebrafish larvae was used to evaluate acute toxicity of PN in two forms: rPN and dPN. Three doses (0.5, 1.5, and 5.0 µ g/mL) of dPN were used to treat zebrafishes for evaluating the developmental toxicity. Behavior abnormalities, body weight, body length and number of vertebral roots were used as specific phenotypic endpoints. RNA sequencing (RNA-seq) assay was applied to clarify the mechanism of acute toxicity, followed by real time PCR (qPCR) for verification. High performance liquid chromatography analysis was performed to determine the chemoprofile of this herb.@*RESULTS@#The acute toxicity result showed that rPN exerted higher acute toxicity than dPN in inducing death of larval zebrafishes (P<0.01). After daily oral intake for 21 days, dPN at doses of 0.5, 1.5 and 5.0 µ g/mL decreased the body weight, body length, and vertebral number of larval zebrafishes, indicating developmental toxicity of dPN. No other adverse outcome was observed during the experimental period. RNA-seq data revealed 38 genes differentially expressed in dPN-treated zebrafishes, of which carboxypeptidase A1 (cpa1) and opioid growth factor receptor-like 2 (ogfrl2) were identified as functional genes in regulating body development of zebrafishes. qPCR data showed that dPN significantly down-regulated the mRNA expressions of cpa1 and ogfrl2 (both P<0.01), verifying cpa1 and ogfrl2 as target genes for dPN.@*CONCLUSION@#This report uncovers the developmental toxicity of dPN, suggesting potential risk of its clinical application in children.

Evaluation of peripheral blood T-lymphocyte subpopulations features in patients with hepatitis B virus-related acute-on-chronic liver failure based on single-cell sequencing technology / 中华肝脏病杂志

Objective: T lymphocyte exhaustion is an important component of immune dysfunction. Therefore, exploring peripheral blood-exhausted T lymphocyte features in patients with hepatitis B virus-related acute-on-chronic liver failure may provide potential therapeutic target molecules for ACLF immune dysfunction. Methods: Six cases with HBV-ACLF and three healthy controls were selected for T-cell heterogeneity detection using the single-cell RNA sequencing method. In addition, exhausted T lymphocyte subpopulations were screened to analyze their gene expression features, and their developmental trajectories quasi-timing. An independent sample t-test was used to compare the samples between the two groups. Results: Peripheral blood T lymphocytes in HBV-ACLF patients had different differentiation trajectories with different features distinct into eight subpopulations. Among them, the CD4(+)TIGIT(+) subsets (P = 0.007) and CD8(+)LAG3(+) (P = 0.010) subsets with highly exhausted genes were significantly higher than those in healthy controls. Quasi-time analysis showed that CD4(+)TIGIT(+) and CD8(+)LAG3(+) subsets appeared in the late stage of T lymphocyte differentiation, suggesting the transition of T lymphocyte from naïve-effector-exhausted during ACLF pathogenesis. Conclusion: There is heterogeneity in peripheral blood T lymphocyte differentiation in patients with HBV-ACLF, and the number of exhausted T cells featured by CD4(+)TIGIT(+)T cell and CD8(+)LAG3(+) T cell subsets increases significantly, suggesting that T lymphocyte immune exhaustion is involved in the immune dysfunction of HBV-ACLF, thereby identifying potential effective target molecules for improving ACLF patients' immune function.

Single cell RNA sequencing technology applicated for drug discovery / 药学学报

Single cell RNA sequencing (scRNA-seq) is an advanced technology to study the transcriptome information at the single cell level. The application of this technology can attribute to analyze the heterogeneous map of cells in the process of disease development, and precisely identify the specific cell subsets that are responsive to pharmacological therapy. Currently, scRNA-seq technology has been widely applied in the field of drug research, including studies on therapeutic targets, drug-induced adverse reactions, drug resistance and vaccine. This work reviews the application of scRNA-seq technology in drug discovery, which offers a scientific basis for personalized and accurate medication therapy.

Advances in single-cell RNA sequencing in the retina / 中华眼底病杂志

Retina is composed of a heterogeneous population of cell types, each with a unique biological function. Even if the same type of cells, due to genetic heterogeneity will lead to cell function differences. In the past, traditional molecular biological methods cannot resolve variations in their functional roles that arise from these differences, and some cells are difficult to define due to the lack of specific molecular markers or the scarcity of numbers, which hindered the understanding and research of these cells. With the development of biotechnology, single-cell RNA sequencing can analyze and resolve differences in single-cell transcriptome expression profiles, characterize intracellular population heterogeneity, identify new and rare cell subtypes, and more definitely define the characteristics of each cell type. It clarifies the origin, function, and variations in cell phenotypes. Other attributes include pinpointing both disease-related characteristics of cell subtypes and specific differential gene expression patterns, to deepen our understanding of the causes and progression of diseases, as well as to aid clinical diagnosis and targeted therapy.

Changes in metabolic capacity and gene expression of brown adipose tissue in the first and third trimester of pregnancy / 中华内分泌代谢杂志

Objective:To explore gene expression and metabolic capacity changes of brown adipose tissue(BAT)during different gestation periods.Methods:A normal pregnancy model was established using C57BL/6J mice, while infertile mice of the same age were served as the control group. The morphological alteration of BAT during pregnancy as well as the gene expression of uncoupling protein 1(UCP1) and other fat browning and mitochondrial marker genes were detected. Moreover, BATs from early and late gestation were selected to screen differentially expressed genes in relation to pregnancy progressing by RNA sequencing(RNA-seq), and gene ontology(GO) and Kyoto gene and gene sequencing(KEGG)were performed.Results:With pregnancy progressing, the size of BAT lipid droplets was substantially enlarged, UCP1 protein expression was decreased( P<0.01), and the fat browning marker genes(Ucp1, Dio2, and Pgc1α)and the mitochondrial marker gene CytC were downregulated( P<0.001). Additionally, a total of 1 298 distinct genes were identified by RNA-seq, 906 of which were upregulated and 392 were downregulated at later stage of pregnancy. GO and KEGG analyses revealed that the differentially expressed genes were mainly enriched in bioregulatory functional pathways such as lipid metabolism, sex steroid hormones, and inflammatory factors. Conclusion:BAT in mice showed larger lipid droplets and reduced thermogenic and metabolic capacity during late gestation, and BAT gene expression was significantly different in different periods of gestation, so reduced metabolic capacity of BAT may contribute to metabolic abnormality during pregnancy.

Application of single cell RNA sequencing in the treatment of diabetes / 中华内分泌代谢杂志

Single-cell RNA sequencing (scRNA-seq) is used for transcriptome profiling at the individual cell level, which is capable of screening in differentially gene expression that results from genetic mutation. Islet-based developmental atlas and heterogeneity characterization are currently the main applications of scRNA-seq in diabetes. scRNA-seq also can be used to mark and purify the functional β cells from resident adult stem cells in the pancreatic islets, which is expected to improve the outcome of islet β cells transplantation in type 1 diabetic patients. In addition, the technique can aid in learning diabetic β cell dedifferentiation and immunomodulatory functions. Although the study of scRNA-seq in diabetic retinopathy, nephropathy, atherosclerosis, and peripheral neuropathy is still at a nascent stage, scRNA-seq has great potential in a wide range of biomedical and clinical applications.

High-throughput transcriptional profiling of perturbations by Panax ginseng saponins and Panax notoginseng saponins using TCM-seq / 药物分析学报

Panax ginseng(PG)and Panax notoginseng(PN)are highly valuable Chinese medicines(CM).Although both CMs have similar active constituents,their clinical applications are clearly different.Over the past decade,RNA sequencing(RNA-seq)analysis has been employed to investigate the molecular mechanisms of extracts or monomers.However,owing to the limited number of samples in standard RNA-seq,few studies have systematically compared the effects of PG and PN spanning multiple conditions at the transcriptomic level.Here,we developed an approach that simultaneously profiles transcriptome changes for multiplexed samples using RNA-seq(TCM-seq),a high-throughput,low-cost workflow to molecularly evaluate CM perturbations.A species-mixing experiment was conducted to illustrate the accuracy of sample multiplexing in TCM-seq.Transcriptomes from repeated samples were used to verify the robustness of TCM-seq.We then focused on the primary active components,Panax notoginseng sa-ponins(PNS)and Panax ginseng saponins(PGS)extracted from PN and PG,respectively.We also char-acterized the transcriptome changes of 10 cell lines,treated with four different doses of PNS and PGS,using TCM-seq to compare the differences in their perturbing effects on genes,functional pathways,gene modules,and molecular networks.The results of transcriptional data analysis showed that the tran-scriptional patterns of various cell lines were significantly distinct.PGS exhibited a stronger regulatory effect on genes involved in cardiovascular disease,whereas PNS resulted in a greater coagulation effect on vascular endothelial cells.This study proposes a paradigm to comprehensively explore the differences in mechanisms of action between CMs based on transcriptome readouts.

Investigation of endothelial cell pathological characteristics in murine choroidal neovascularization model based on single-cell RNA sequencing / 中华实验眼科杂志

Objective:To investigate the molecular expression and pathological features of endothelial cell (EC) in a murine model of choroidal neovascularization (CNV) based on single-cell RNA sequencing (scRNA-seq).Methods:Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid, respectively.Single-cell suspension was prepared by continuous digestion with trypsin/type Ⅰ collagenase at 37 ℃, and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group, with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um (10x Genomics) instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC, revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1, mitochondrial outer membrane proteins Tomm20 and mt-Co1, and capillary markers Kdr and Plvap were observed by immunofluorescence staining, and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University (No.20200181).Results:The cell viability of the single-cell suspension prepared from choroidal-scleral fragments and choroidal scrapings was 99.4% and 99.1%, respectively, both of which met the sequencing requirements.The percentage of EC detected by flow cytometry was approximately 1.58%.The scRNA-seq result revealed that both the normal control and CNV groups contained 13 choroidal cell clusters.Compared with the normal control group, the proportions of rod/cone photoreceptor cells, EC and hematopoietic cells all increased, while the retinal pigment epithelium (RPE) and Schwan cells reduced in the CNV group.Among all clusters, EC constituted 18.4%.The pseudo-time analysis demonstrated that EC could be further divided into 4 states.The percentage of state 2 EC was 29.1% in the CNV group, which was significantly higher than 9.5% in the normal control group.Differentially expressed gene analysis showed that the expression of mitochondrion-related genes, including mt-Nd4 and mt-Atp6, were upregulated in state 2 EC, while capillary-related genes, including Kdr and Esm1, were downregulated.Immunofluorescent staining revealed that the area of Tomm20 and mt-Co1 in Pecam1-positive EC in the CNV area was (19.50±4.68)% and (4.64±2.82)%, respectively, which were both higher than (3.00±2.09)% and (0.18±0.34)% in normal area ( t=7.88, 3.84; both at P<0.01). The area of Kdr and Plvap in Pecam1-positive EC in the CNV area was (1.50±0.29)% and (0.79±0.97)%, respectively, which were both lower than (31.30±5.44)% and (10.43±2.28)% in the normal area ( t=13.40, 9.48; both at P<0.01). The vascular diameter in the CNV area was (5.52±1.85)μm, which was larger than (4.21±1.84)μm in the normal area ( t=9.57, P<0.001). Conclusions:When CNV occurs, the proportion of EC in choroid increases, and CNV-EC shows pathologic features of mitochondrial metabolic activation and loss of capillary properties, suggesting the mitochondrial activation of EC may play a role in the formation of CNV.

Single-cell RNA sequencing deciphers transcriptional profiles of hepatocytes in mouse with hepatic alveolar echinococcosis / 中国血吸虫病防治杂志

Objective To investigate the cell composition and the transcriptional characteristics in microenvironments of hepatic tissues in mice at late stage of Echinococcus multilocularis infection at a single-cell level. Methods Peri-lesion and paired distal hepatic specimens were collected from two BALB/c mice (6 to 8 weeks old) infected with E. multilocularis for single-cell RNA sequencing. The Seurat package in the R software was employed for quality control of data, multi-sample integration and correction of batch effects, and uniform manifold approximation and projection (UMAP) algorithm was used for cell clustering. Cell types were annotated using classical marker genes. Differentially expressed genes were screened in each cell type through differential gene expression analysis, and the biological roles of cells were predicted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Results A total of 43 710 cells from peri-lesion and distal hepatic tissues of E. multilocularis-infected mice were analyzed, and were classified into 11 cell types, including neutrophils, T cells, macrophages, granulocyte-monocyte progenitor cells, B cells, plasma cells, basophils, hepatic stellate cells, endothelial cells, hepatocytes, and platelets. T cells were the largest population of immune cells in the microenvironment of hepatic tissues, including five CD4+ T cell subsets, two CD8+ T cell subsets and phosphoantigen-reactive γδT cells. The proportions of CD4+ helper T cells and cytotoxic CD4+ T cells decreased and the proportion of T helper 2 (Th2) cells increased in peri-lesion tissues relative to distal hepatic tissues. In addition, the differentially expressed genes in Th2 cells were associated with negative regulation of the immune system, and the highly expressed genes in cytotoxic CD4+ T cells correlated with activation of the immune system. Conclusions Single-cell RNA sequencing deciphers the cell composition and distribution in microenvironments of hepatic tissues from mice infected with E. multilocularis, and the increased proportion of Th2 cells in peri-lesion hepatic tissues may be associated with formation of immunosuppressive microenvironments.

Mechanism of gambogenic acid inhibiting the proliferation of melanoma based on RNA-seq sequencing technology / 中国临床药理学与治疗学

AIM: By analyzing the effect of gambogenic acid (GNA) on the mRNA expression profile of melanoma xenograft model mice, the possible mechanism of GNA in the treatment of melanoma was explored. METHODS: The inhibitory effect of GNA on melanoma cells was studied by measuring the cell survival rate by MTT method in vitro and observing the cell morphology under an inverted microscope. In the in vivo experiment, the effect of GNA on the growth of xenografted tumors in melanoma mice was observed by comparing the results of HE (hematoxylin-eosin) staining and immunohistochemistry (Ki-67), and the tumor weight and tumor weight ratio were recorded. RNA-seq sequencing technology was used to sequence the GNA medium-dose group and the model group, and the screened mRNAs were analyzed by GO and KEGG, and finally the screening results of differentially expressed genes were verified by real-time quantitative fluorescent PCR. RESULTS: After different doses of GNA acted on the melanoma mouse model, a large area of necrosis occurred in the tumor tissue of the model mouse, and the tumor growth was significantly inhibited. A total of 36 differentially expressed mRNAs were identified by mRNA sequencing, of which 30 were up-regulated and 6 were down-regulated. The possible functions of the mRNAs were predicted according to the genomic adjacency analyzed by GO and KEGG. The expression of the selected differential mRNAs was further verified by real-time quantitative PCR technology. The results showed that the mRNA expressions of Cidec, Ces1d, Mylk4, and Igkv9-123 were up-regulated, and the mRNA expressions of Ryr3 and Hapln1 were down-regulated. CONCLUSION: GNA can inhibit the proliferation of melanoma cells in vitro and in vivo, and its mechanism is related to the regulation of cytokine-cytokine receptor interaction, NF-κB, MAPK, and other pathways of mRNA expression.

Gene expression in rhesus macaques prefrontal cortex after multiple sevoflurane exposures / 中国临床药理学与治疗学

AIM: To analyze differentially expressed genes after multiple sevoflurane anesthesia in rhesus macaques was studied. METHODS: Rhesus macaques received three anesthetic exposures to sevoflurane on postnatal day (P) 7 and then on P21 and P35. RNA sequencing analysis was used in the studies. RESULTS: Transcriptomic analysis of differentially expressed genes (DEGs) revealed that up-regulated genes were mainly enriched in the regulation of positive regulation of gastrulation and odontogenesis, while down-regulated genes were primarily enriched in the regulation of transcriptional activator activity, RNA polymerase II transcription regulatory region sequence-specific binding, and MAPK signaling pathway. CONCLUSION: Differentially expressed genes (DEGs) were analyzed in the prefrontal cortex of rhesus macaques after multiple sevoflurane exposures by RNA sequencing to screen essential genes for subsequent studies on neurotoxicity induced by sevoflurane, providing a solid basis for the study of the mechanism of general anesthesia.

Sensitizing effect of d-borneol on cisplatin-resistant NSCLC based on transcriptomics and its mechanism / 中国药理学通报

Aim To explore the key targets of d-borneol combined with eisplatin for sensitization of cisplatin-resistant NCSLC cells by RNA-Seq and verify its mechanism. Methods Cisplatin-resistant human large cell lung cancer cells (H460/CDDP) were inoculated into the right armpit of male BALB/c nude mice (4 weeks old) to construct a xenograft tumor model. Then they were randomly divided into control group, vehicle group, eisplatin group, and combination group (d-borneol + eisplatin) with 6 nude mice and treated for 14 d. After last administration of 24 h, the tumor tissue was taken for RNA-Seq. And then real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were used to verify the expression of cell cycle-related molecules. Results RNA-seq analysis showed that there were significant differences in gene expression between the eisplatin group and combined group, and they were significantly enriched in cell cycle. RT-PCR and IHC results showed that d-borneol combined with eisplatin could significantly inhibit the expressions of cyclins (cyclin A2, cyclin D3) and cyclin-dependent kinases (CDK2, CDK6) and promote the expression of its upstream molecular cyclin-dependent kinase inhibitor CD-KI (P21, P27) (P<0. 05, P<0.01). Conclusions d-Borneol increases the sensitivity of eisplatin by increasing the expression of P21 and P27 and inhibiting the expression of cyclinA2/D3 and CDK2/6 to induce cell cycle arrest and inhibit the malignant proliferation of H460/CDDP cells, thereby achieving the effect of anti-drug sensitization.