目的： 利用网络药理学和分子对接技术分析“茵陈-玉竹”药对治疗糖尿病肾病的机制。方法： 通过中药系统药理学数据库及分析平台(TCMSP)、中医药百科全书(ETCM)、中医药分子机制生物信息学注释数据库(BATMAN-TCM)检索“茵陈-玉竹”药对的主要化学成分，通过药物靶点数据库(TTD)、人类在线孟德尔遗传(OMIM)数据库、DisGeNET、GeneCards等数据库收集糖尿病肾病的疾病靶点，进而筛选茵陈、玉竹与糖尿病肾病的交集核心靶点。利用Cytoscape 3.7.2软件构建“化合物-靶点-疾病网络”，同时基于DAVID数据库对靶点进行GO功能富集和KEGG通路富集分析。结果： 获取“茵陈-玉竹”药对主要活性成分31个，作用靶点351个，糖尿病肾病相关基因2 911个，交集基因185个，其中起关键作用的活性成分为槲皮素、滨蒿内酯、莨菪亭、D-香芹酮和异鼠李素；构建可视化PPI网络得到185个节点，851条边，平均度值9.2，获得前列腺素内过氧化物酶2(PTGS2)、核因子κB1(NFKB1)、丝裂原活化蛋白激酶1(MAPK1)、MAPK3、MAPK14等“茵陈-玉竹”药对治疗糖尿病肾病的关键靶点。GO富集分析获得1 181条相关通路，KEGG通路富集获得180条信号通路。结论： “茵陈-玉竹”药对可能通过调控磷脂酰肌醇3激酶苏氨酸蛋白激酶(PI3K-Akt)、MAPK、糖基化终末产物糖基化终产物受体(AGE-RAGE)等信号通路，抑制各种炎症反应与氧化应激，调节异常代谢，从而对糖尿病肾病产生治疗作用。

       外泌体是含有来源细胞内容物的细胞外小囊泡，通过传递蛋白、核酸及脂类等生物活性分子介导细胞间通讯。肿瘤来源的外泌体与肿瘤的耐药性密切相关，环状RNA（circular RNA,circRNA）是一种环形稳定的内源性非编码RNA。近年来，关于外泌体转移circRNA在多种肿瘤耐药发展中的作用研究取得进展。因此，本文总结了外泌体circRNA参与肿瘤发病和耐药机制的研究内容。

 Objective： To explore the gene expression profiles of Henle-loop cell subset in renal tissue of the calcium oxalate kidney stone rat model. Methods： The rat model of calcium oxalate kidney stone was constructed by ethylene glycol and 1% ammonium chloride. The model was validated by Von Kossa′s staining. Henle-loop cells were identified using single-cell sequencing method. The gene expression profiles of Henle-loop cell subsets in renal tissue of calcium oxalate kidney stone rat model were performed via comparative analysis. The enrichment analysis of differential genes was performed through GO, KEGG, and GSEA bioinformatics tools. Results： Compared with the control group, black or brownish black staining of calcium salt deposition were observed in the kidney tissue of the experimental group rats, indicating the successful construction of the calcium oxalate kidney stone rat model. Single cell sequencing analysis revealed that there was a total of 3 510 differentially expressed genes in the Henle-loop cell subset of the experimental group, of which 880 were upregulated and 2 630 were downregulated. LOC499584, Il7, dual specificity phosphatase 1 (Dusp1), and matrix Gla protein (Mgp) were upregulated most significantly, while HORMA domain containing 2 (Hormad2), LOC361914, Jun dimerization protein 2 (Jdp2), and somatomedinB and thrombospondin type-1 domain-containing protein (Sbspon) were downregulated most significantly. KEGG analysis revealed significant changes in signaling pathways such as oxidative phosphorylation, metabolism pathways, propanoate metabolism, autophagy, lysosome and endocytosis in the experimental group of Henle-loop cells. Conclusion： This study reveals for the first time the gene expression profile characteristics of Henleloop cell subsets in renal tissue of the calcium oxalate kidney stone rat model, providing a molecular basis for the investigation of kidney stone formation and related kidney injury.

  Objective： To investigate the role of heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) in ferroptosis of pancreatic cancer cells and its potential mechanisms. Methods： Three pancreatic cancer cell lines PaTu8988, MIA-paca2 and PANC1 were treated with different concentrations of Erastin for 3 days. The cell viability was detected by CCK8 method. The half inhibitory concentrations (IC50) of Erastin in three kinds of pancreatic cancer cell lines were calculated and their resistance capabilities to Erastin were compared; qRT-PCR and Western blotting were used to detect the relative expression levels of mRNA and protein of hnRNPA2B1 in pancreatic cancer cells. The differential expression of the hnRNPA2B1 in pancreatic cancer tissues and normal tissues was analyzed by GEPIA, and the survival prognosis of patients with differential expression of hnRNPA2B1 was analyzed from clinical data sets from GEO database. PaTu8988 cells was transfected with sh-Control and sh-hnRNPA2B1 plasmid, respectively, to knock down hnRNPA2B1; PANC1 cells was transfected with Vector and Flag-hnRNPA2B1 plasmid, respectively, to overexpress hnRNPA2B1; and the transfection efficiency was verfied by qRT-PCR and Western blotting, respectively; the effects of knockdown or overexpression of hnRNPA2B1 on the migration and invasion were detected by Transwell assay, and the effects on the proliferation of pancreatic cancer cells were detected by CCK8 assay. The cells in sh-Control, sh-hnRNPA2B1, Vector and Flag-hnRNPA2B1 group were treated with control (0 μmol/L Erastin) and Erastin (IC50 concentration), respectively, then flow cytometry was used to determine the lipid peroxidation level, and colorimetry was used to determine the malindialdehyde (MDA) and tissue iron concentration; in addition to Erastin, the sh-hnRNPA2B1 and Flag-hnRNPA2B1 groups were added with Ferrostatin-1, Necrostatin-1, ZVAD-FMK, respectively, the cells activity was detected by trypan blue dye exclusion. The relative expression levels of RNA and protein of transferrin receptor (TFRC), ferritin heavy chain, glutathione peroxidase 4 and solute carrier family 7 member 11 weredetected by qRT-PCR and Western blotting, respectively. Results： The resistance of three types of pancreatic cancer cells to Erastin from high to low were PaTu8988, MIA-paca2 and PANC1, corresponding Erastin IC50 was 18.020, 15.760 and 2.947 μmol/L, respectively (P<0.05). The relative expression level of hnRNPA2B1 was the highest in PaTu8988 cells, followed by MIA-paca2 cells and the lowest in PANC1 cells (P<0.05), which was the same with Erastin IC50. The expression level of hnRNPA2B1 in pancreatic cancer tissues was significantly higher than that in normol tissues, and the prognosis of patients with higher  expression of hnRNPA2B1 was poor (P<0.05). Down-regulating the expression of hnRNPA2B1, the migration, invasion and proliferation ability of PaTu8988 cells were greatly reduced, while up-regulating was the opposite (P<0-05). Erastin-induced cell death was significantly restored by Ferrostatin-1 not by ZVAD-FMK or Necrostatin-1 (P<0.05); compared with sh-Control group, the level of lipid peroxidation, MDA and iron concentration in sh-hnRNPA2B1 group were significantly increased (P<0.05); compared with Vector group, Flag-hnRNPA2B1 group showed lower level of lipid peroxidation, MDA and ironconcentration (P<0.05). Upregulation of hnRNPA2B1 inhibited the expression of TFRC, while interference with hnRNPA2B1 produced the opposite effect (P<0.05). Conclusion： hnRNPA2B1 could inhibit t he intracelluar accumulation of iron by inhibiting the expression of TFRC, thus promoting the resistance of pancreatic cancer cells to ferroptosis.

Objective： To study the effects of di-(2-ethylhexyl) phthalate (DEHP) on embryonic development of zebrafish and its potential molecular mechanism. Methods： Zebrafish embryos were randomly divided into 5 groups: blank control group, dimethyl sulfoxide group, and 8, 40, 200 μg/L DEHP groups, which were exposed to 96 hours post-fertilization (hpf). The mortality rate, hatching rate, malformation rate, heart rate, body length and the number of spontaneous movements of embryos were recorded. The activity of superoxide dismutase (SOD), glutathione catalase (GPx) and malondialdehyde (MDA) content in larvae were measured by enzymatic analysis. Real-time quantitative PCR was used to determine the expression of mRNA related to cell apoptosis, glucose transport, and the synthesis of proteins, fatty acids, and glycogen. Results： (1) Compared with the control group, the embryo/larvae mortality of zebrafish in 8 μg/L DEHP group was significantly increased (P＜0.01), and the larvae body length was greatly decreased (P＜0.05); in 40 and 200 μg/L DEHP groups, the mortality and malformation rate of zebrafish embryo/larvae were significantly increased (P＜0.01), and the hatching rate, heart rate, body length and the number of embryonic autonomous movement were greatly decreased (P＜0.05 or P＜0.01). (2) Compared with the control group, the activity of GPx in zebrafish larvae in 8 μg/L DEHP group was significantly decreased (P＜0.05); the activities of SOD and GPx in zebrafish larvae were significantly decreased and the content of MDA was greatly increased in 40 and 200 μg/L DEHP groups (P＜0.01). (3) Compared with the control group, the expression of Pi3k mRNA in glucose transport signaling pathway in 8 μg/L DEHP group was significantly up-regulated (P＜0.01); the mRNA expressions of Bax/Bcl-2, Caspase-3 and Ras were greatly up-regulated (P＜0.01), and the mRNA expressions of Akt and Pi3k in glucose transport signaling pathway were significantly up-regulated (P＜0.01), the mRNA expression of Erk1/2 was greatly down-regulated (P＜0.05 or P＜0.01), and the mRNA expressions of Mtor, Gsk-3 and GSK-3β in protein, fatty acid and glycogen synthesis signaling pathways were significantly up-regulated (P＜0.05 or P＜0.01) in 40 and 200 μg/L DEHP groups. Conclusion： DEHP may induce apoptosis, disrupt the expression of metabolism-related mRNA, cause oxidative damage, and thus affect the growth and development of zebrafish embryos, resulting in developmental toxicity.
［Key words］di-(2-ethylhexyl) phthalate; zebrafish embryo; developmental toxicity; mechanism of toxicity

Objective: To investigate the effect of human umbilical cord mesenchymal stem cell derived small extracellular vesicles (hucMSC-sEVs) in attenuating the damage of diabetic kidney disease (DKD) in rats. Methods: HucMSCs were isolated and cultured, and hucMSC-sEVs were extracted from the culture media supernatant. The DKD rat model was established by high fat diet combined with injection of streptozocin, rats were randomly divided into control group, DKD group, and hucMSCsEVs group, with 6 rats in each group. The hucMSC-sEVs group received tail vein injection of hucMSC-sEVs 8 weeks after modeling, and kidney tissues were collected at week 24. Renal tissue pathology and fibrotic changes in rats were assessed using HE staining, PAS staining and Sirius Red staining. The expression of apoptosisrelated proteins Bcl-2 and Bax in kidney tissues was evaluated using Western bloting. Additionally, the expression levels of THBS1 and its receptors CD36 and CD47 in rat renal tissues were determined using histochemical staining, immunofluorescence staining, and Western bloting. Results: Compared with the control group, the DKD group exhibited significant pathological changes in the kidneys, such as thickening of the glomerular basement membrane and mesangial proliferation, dilation of renal tubules accompanied by vacuolar degeneration, and marked interstitial fibrosis with infiltration of inflammatory cells. The expression of the apoptosis marker Bax in kidney tissues showed a significant increase (P<0.05), while the expression of the anti-apoptotic marker Bcl-2 exhibited a significant decrease (P<0.01). The expression levels of THBS1 and its receptors CD36 and CD47 were also significantly elevated (P<0.001). In contrast, compared with the DKD group, the hucMSC-sEVs group demonstrated marked alleviation of renal tissue pathological damage and fibrosis. The expression of Bax significantly decreased (P<0.01), while the expression of Bcl-2 significantly increased (P<0.01). Additionally, the expression levels of THBS1 and its receptors CD36 and CD47 in the kidneys were significantly reduced (P<0.001). Conclusion: HucMSC-sEVs can significantly reduce the renal tissue injury in DKD, which may be related to the targeted inhibition of THBS1 expression.
［Key words］human umbilical cord mesenchymal stem cells; small extracellular vesicles; diabetic kidney disease; THBS1

Objective: To explore the effect of CXCL14 on pyroptosis of adipocytes and the formation of atherosclerosis in diabetic microenvironment. Methods: ① ApoE^-/- mice were intraperitoneally injected with streptozotocin to construct diabetes mellitus, and diabetic ApoE^-/- mice were fed with high fat diet for 20 weeks, and the diabetic atherosclerosis model (model group) was constructed, and control mice were only fed on a high-fat diet (AS group); ② Anti-CXCL14 short peptide was injected subcutaneously on the medial hind limb of diabetic mice (anti-CXCL14 group), and control diabetic mice were injected subcutaneously with normal saline only; ③ Adeno-associated virus (AAV) was injected in situ into posterior inguinal subcutaneous adipose tissue in diabetic mice to inhibit gastrointestinal dermatin (GSDMD)mediated pyroptosis, divided into: AAV-shscramble group, AAV-shGSDMD group, anti-CXCL14+AAV-shscramble group, anti-CXCL14+AAV-shGSDMDgroup. After 20 weeks of high-fat diet of the mice, the serum of the mice was collected under anesthesia, and the blood lipid levels (total cholesterol, triglycerides, LDL-C, and HDL-C) of the mice were analyzed by biochemical detection kit. The mice were sacrificed, and the epididymal adipose tissue was scanned by electron microscopy to observe the changes of fat cells. qRT-PCR was used to analyze the changes of pyroptosisrelated factors GSDMD, aspartate proteolytic enzyme-1 (Caspase-1), NLR family pyridine domain protein 3 (NLRP3) inflammasome, interleukin-1β (IL-1β) and inflammatory factor interleukin-6 (IL-6). The mouse aorta was isolated and extracted, and the relative area of atherosclerotic plaques was observed by HE staining and oil red O staining. Results: Compared with the AS group, the hypertrophy and number of adipocytes of adipose tissue in the epididymis in the model group decreased (P<0.01), the size of aorticplaque increased, the levels of total cholesterol, triacylglycerol and LDL-C were significantly increased, and HDL-C was significantly reduced. The levels of pyroptosisrelated factors and IL-6 in adipose tissue of epididymis were significantly increased (P<0.05), indicating that diabetes promoted pyroptosis of AS plaque and adipose tissue in mice. Injection of anti-CXCL14 short peptide could reduce the size of aortic plaque, improve blood lipid levels, and inhibit adipose tissue pyroptosis, which increases the number of fat cells. After GSDMD knockdown, the number of adipocytes increased and the area of aortic plaques decreased. However, after the injection of anti-CXCL14 immune peptide, there was no significant change in atherosclerosis in the AAV-shGSDMDgroup. Conclusion: Anti-CXCL14 can attenuate adipose tissue pyroptosis and alleviate the development of diabetic atherosclerosis.
［Key words］CXCL14; pyroptosis; adipose tissue; atherosclerosis; diabetic microenvironment

Objective： To explore the hub genes and potential mechanism of ferroptosis in the development of atherosclerosis (AS) in patients with type 2 diabetes mellitus (T2DM) based on bioinformatics. Methods： The datasets GSE20966 (T2DM) and GSE43292 (AS) were obtained from the GEO database. Differentially expressed genes (DEGs) were identified using the Limma R package. Heatmaps and volcano plots were drawn, and crossanalysis was performed to obtain DEGs associated with the two diseases. GO and KEGG enrichment analysis was performed to explore the biological functions of DEGs. Ferroptosis-related genes (FRGs) obtained from the FerrDb database were crosslinked, and hub genes were screened using LASSO regression and random forest analysis. ROC curves and validation sets GSE76895 (T2DM) and GSE28829 (AS) were used for verification. Finally, the gene-miRNA network was drawn. Results： A total of 606 DEGs were identified related to the T2DM and the AS datasets. Twenty potential genes were obtained by cross-analyzing with FRGs. Cyclin-depedent kinase inhibitors 1A (CDKN1A), poly ADPribose polymerase 8 (PARP8), phosphatidylethanolamine binding protein 1 (PEBP1), and progesterone receptor membrane component 1 (PGRMC1) were hub genes that affected AS in T2DM patients through ferroptosis. The area under the curve (AUC) of the ROC curves in the datasets GSE20966 and GSE43292 of the four genes was all greater than 0.7, which had diagnostic value. PEBP1 and PGRMC1 were significantly down-regulated in the validation sets GSE76895 and GSE28829. In addition, 13 miRNAs were closely associated with 4 hub genes. Conclusion： CDKN1A, PARP8, PEBP1 and PGRMC1 are involved in AS in T2DM patients through ferroptosis and may become new therapeutic targets.
［Key words］ferroptosis; type 2 diabetes mellitus; atherosclerosis; bioinformatics; machine learning

Objective： To explore intercellular communication-related signaling molecules in tumor microenvironment (TME) of castration-resistant prostate cancer (CRPC) and analyze their relationship with tumor occurrence and development. Methods： The main components of the TME in CRPC was analyzed using singlecell RNA sequencing (scRNA-seq) data (GSE137829) from CRPC patients found in the Gene Expression Omnibus (GEO) database. CellPhoneDB was used to examine interactions between different cells within the microenvironment, with a focus on revealing the connections between cancer-associated fibroblasts (CAFs) and malignant epithelial cells. CellChat was applied to analyze signaling molecules involved in communication between CAFs and malignant epithelial cells. Data from The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) were integrated to identify signaling molecules closely related to the prognosis of CRPC patients. siRNA was used to knock down the expression of the signaling molecule Delta-like canonical Notch ligand 3 (DLL3) in CRPC cells, and the knockdown efficiency was validated by real-time quantitative PCR and Western blotting. The effects of DLL3 on the biological behaviors of CRPC cells, including proliferation, migration, and invasion, were investigated using CCK8 assays, EdU assays, and Transwell assays with Matrigel, respectively. Singlegene differential analysis, overrepresentation analysis (ORA), and gene set enrichment analysis (GSEA) were also performed to identify pathways enriched with DLL3-related differential expression genes. Results： The results of single-cell transcriptomic sequencing indicated that the main cellular components of the TME in CRPC include malignant epithelial cells, CAFs and immune cells. Close communication between CAFs and malignant epithelial cells was mediated by interaction-related signaling molecules, with 63 signaling molecules identified as closely related to the communication between CAFs and malignant epithelial cells. Univariate Cox regression analysis identified key signaling molecules, including tumor necrosis factorrelated ligand superfamily member 12 (TNFSF12) and DLL3, whose transcriptional expression were significantly associated with the prognosis of CRPC patients. Notably, high expression of DLL3 was significantly associated with poor prognosis in CRPC patients. In vitro experimental results demonstrated that knockdown of DLL3 significantly inhibited the growth, proliferation, migration and invasion capabilities of CRPC cells. KEGG and GSEA enrichment analyses indicated that differentially expressed genes related to DLL3 were significantly enriched in metabolic pathways. Conclusion： In the TME, CAFs and malignant epithelial cells communicate closely through intercellular signaling molecules. Among these signaling molecules, the expression of DLL3 is negatively correlated with cancer prognosis and affects the biological behavior of CRPC cells, suggesting that DLL3 may be a key signaling molecule in the malignant progression of human CRPC.
［Key words］castration-resistant prostate cancer; scRNA-seq; tumor microenvironment; cell communication; DLL3