ObjectiveThis paper aims to investigate and evaluate the staged efficacy and safety of the representative empirical prescription of the “Zhu Ke Jiao” theory， Qijia Rougan prescription， combined with entecavir in the treatment of hepatic fibrosis in chronic hepatitis B. MethodsA multicenter randomized controlled clinical study was conducted， and 101 patients diagnosed with chronic hepatitis B-related hepatic fibrosis （CHB-HF） who met the diagnosis and inclusion criteria were randomly assigned to an observation group （Qijia Rougan prescription + entecavir） and a control group （entecavir）. The treatment duration was 24 weeks. Liver stiffness measurement （LSM）， fibrosis-4 index （FIB-4）， portal vein diameter， hepatitis B serology， biochemical indicators， hepatic fibrosis markers in serum ［hyaluronic acid （HA）， laminin （LN）， procollagen Ⅲ peptide （PⅢP）， and type Ⅳ collagen （Ⅳ-C）］， and traditional Chinese medicine syndrome scores were used as efficacy evaluation indicators. Efficacy assessments and explorations of different staged subgroups of Qijia Rougan prescription were conducted according to LSM values based on the Metavir pathological staging standard. ResultsA total of 98 cases were included for statistical analysis， with 49 cases in the observation group and 49 in the control group. The general data of the patients in both groups were comparable. Compared with the same group before treatment， the observation group showed a significant reduction in LSM and FIB-4 （P<0.01）， as well as notable improvements in LN， Ⅳ-C， and various TCM syndrome scores （P<0.05， P<0.01）. When compared to the control group after treatment， the observation group demonstrated significant improvements in LSM， FIB-4， and various TCM syndrome score indicators （P<0.05， P<0.01）， indicating that the observation group performed better than the control group. Subgroup analysis of the regression of hepatic fibrosis stages showed that compared to the same group before treatment， the observation group had better improvement in regression of stages F2 and F3 （P<0.05）. When compared to the control group after treatment， the observation group exhibited superior improvement in regression of stage F3 （P<0.05）. No adverse events occurred in either group during the treatment period. ConclusionCompared with entecavir alone， the combination of Qijia Rougan prescription and entecavir significantly improves the degree of hepatic fibrosis and clinical TCM symptoms in patients. The optimal intervention period is primarily during stage F3， which is a potential “interception” point of the “Zhu Ke Jiao” theory.

[摘 要] 目的：探讨异位表达血红蛋白亚基（HBA/HBB）对缺氧条件下嵌合抗原受体T细胞（CAR-T细胞）功能障碍的改善作用及其对肿瘤细胞的杀伤效应。方法：全基因合成技术合成靶向HER2的CAR序列，构建共表达HBA或HBB的CAR慢病毒载体，包装慢病毒后感染人原代T淋巴细胞，制备异位表达HBA/HBB的CAR-T细胞，命名为HBA CAR-T和HBB CAR-T。采用缺氧探针检测小鼠实体瘤缺氧状态。通过流式细胞术检测瘤内CAR-T细胞占比、异位表达血红蛋白亚基的CAR-T细胞阳性率及CAR-T细胞的活性氧、凋亡水平。WB法检测HBA CAR-T和HBB CAR-T内相关血红蛋白亚基表达情况，采用细胞计数板计数检测细胞增殖水平，通过萤光素酶报告基因法检测CAR-T细胞对肿瘤细胞的杀伤能力，qPCR检测CAR-T细胞中缺氧诱导因子-1α（HIF-1α）表达水平，利用MitoXpress Intra试剂盒检测CAR-T细胞内氧气含量。结果：不同细胞构建的实体瘤模型均存在明显缺氧情况，且CAR-T细胞浸润水平与缺氧程度呈显著负相关（P < 0.000 1）。HBA CAR-T与HBB CAR-T构建成功（阳性率 > 60%），相应血红蛋白亚基可稳定表达。缺氧环境下HBA CAR-T和HBB CAR-T的ROS水平、凋亡水平显著下降，增殖、对肿瘤细胞的体外杀伤能力显著强于传统CAR-T细胞（均P < 0.05）。HBA CAR-T与HBB CAR-T内HIF-1α表达降低（均P < 0.001），且缺氧程度显著降低（均P < 0.001）。结论：异位表达血红蛋白亚基可改善缺氧条件下CAR-T细胞功能障碍并增强其对肿瘤细胞的体外杀伤作用。

Metabolic associated fatty liver disease （MAFLD） has become one of the most prevalent chronic liver diseases worldwide， with the core pathological feature of pathological accumulation of triglycerides in hepatocytes， and as a key rate-limiting enzyme for triglyceride synthesis in the liver， 2-acylglycerol O-acyltransferase 2 （DGAT2） plays a central role in this process. Cell and animal model studies have confirmed that inhibition of DGAT2 can effectively alleviate hepatic steatosis and delay disease progression， and subsequent clinical trials have also shown the potential clinical application value in the treatment of MAFLD. This article systematically reviews the mechanism of action of DGAT2 in the development and progression of MAFLD and comprehensively summarizes the therapeutic strategies targeting DGAT2， including the advances in both monotherapy and drug combinations， in order to provide new ideas for individualized treatment of MAFLD patients.

Objective: To explore the differential lipid metabolites in the plasma of mice with idiopathic pulmonary fibrosis(IPF). Methods : Thirty SPF C57BL/6 male mice were randomly divided into 2 groups with 15 mice in each group. The experimental groups were divided into control group and bleomycin(BLM) group. The model of idiopathic pulmonary fibrosis was induced by one-time intratracheal infusion of BLM(1 mg/kg). Hematoxylin-eosin(HE) staining was used to observe the lung histopathology. The collagen fiber deposition in lung tissue was observed by Sirius red staining. The differential lipid metabolites in plasma of IPF mice were screened and enriched by lipidomics. Results : HE staining showed that the pulmonary tissue structure was disordered, alveolar septum was broken and alveolar wall was destroyed in BLM group. Sirius red staining showed a large amount of collagen fiber deposition in the lung interstitium of BLM group. The results of lipidomics analysis showed that the lipid metabolism profile of BLM group changed, 15 differential lipid metabolites were screened out, of which 11 differential lipid metabolites were up-regulated, and 4 differential lipid metabolites were down-regulated, mainly concentrated in glycerophosphoglycerophosphates, glycerophosphocholines, steroid lactones, etc. Conclusion The lipid metabolism profile of BLM group mice changes, differential lipid metabolites such as phosphoglycolate phosphatase(PGP)(18:0/18:0), PGP(i-12:0/i-24:0), PGP(i-13:0/a-25:0), and phosphatidylcholine(PC)(18:0/14:0), PC(18:3/16:0), lysophosphatidylcholine(LPC)(16:1), and LPC(18:3) may play an important role in the progression of IPF. These findings provide a new reference for further study of the molecular mechanism of IPF, and also provide a potential new target for clinical treatment.

Objective:To discuss the construction of a NOD-signal regulatory protein α(SIRPA)gene knock-in mouse model based on clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)9 technology,and to provide the reference for the establishment of more advanced humanized mouse models.Methods:Based on CRISPR/Cas9 technology,the NOD-background SIRPA gene was knocked into the BALB/c mouse fertilized eggs,and homozygous mice with stable genotypes(SIRPA-KI mice)were obtained through expansion and breeding for experiments;PCR primers were designed,and mouse genotypes were identified by nucleic acid electrophoresis.The mice were divided into C57BL/6 group,BALB/c group,and SIRPA-KI group according to their strains,and 3 mice with similar ages were randomly selected from each group for experiments.Mature bone marrow-derived macrophages were co-incubated with human CD47-Fc fusion protein,stained with Streptavidin PE/Cy7,and the mean fluorescence intensity(MFI)of PE/Cy7 was detected by flow cytometry to compare the ability of SIRPA in the mice from various groups to bind human CD47 Fc.Each mouse was intravenously injected with 2×10? carboxyfluorescein diacetate,succinimidyl ester(CFSE)-labeled human red blood cells,and the phagocytosis of human red blood cells by macrophages in various groups was detected by in vivo phagocytosis assay.One BALB/c mouse and one SIRPA-KI mouse were randomly selected to induce mature bone marrow-derived macrophages,and the phagocytosis of human red blood cells by macrophages in various groups was detected by in vitro phagocytosis assay.Results:BLAB/c SIRPANOD/NOD homozygous mice were successfully obtained.The flow cytometry results showed that compared with C57BL/6 group,the MFI of the mice in BALB/c group was significantly increased(P<0.01);compared with C57BL/6 group and BALB/c group,the MFI of the mice in SIRPA-KI group was significantly increased(P<0.01).The in vivo phagocytosis assay results showed that the macrophages in C57BL/6 group exhibited the fastest overall clearance rate of human red blood cells;at 6 h of macrophage phagocytosis,compared with SIRPA-KI group,the residual percentage of the human red blood cells in C57BL/6 group was significantly decreased(P<0.01)and was closed to 0%;compared with SIRPA-KI group,the residual percentage of the human red blood cells in BALB/c group was significantly decreased(P<0.05).The in vitro phagocytosis assay results showed that the macrophages in BALB/c group significantly phagocytosed the human red blood cells,with a high phagocytic index of 30.7%;compared with BALB/c group,the phagocytic index of the macrophages in SIRPA-KI group was significantly decreased(P<0.01).Conclusion:The study successfully constructs a mouse model with enhanced affinity for human CD47 and significantly inhibited phagocytosis of human red blood cells by knocking the NOD-background SIRPA gene into the BALB/c strain using CRISPR/Cas9 technology,providing a superior human cell xenotransplantation efficiency.

Background and purpose:Epithelial ovarian cancer(EOC)has a poor prognosis and is prone to developing resistance to platinum-based chemotherapy.Exosomes are currently believed to be involved in platinum resistance in ovarian cancer.This study explored the role and mechanism of exosomes on platinum resistance of ovarian cancer.Methods:Through ultracentrifugation,exosomes were isolated and analyzed using electron microscopy,particle size studies,and Western blot for detailed exosome analysis.We detected the expression levels of exosomal proteins and related signaling pathways.Exosomal protein expression profile was analyzed by proteomics.Key differential proteins were screened by intersecting with existing drug resistance datasets.Furthermore,patients diagnosed with EOC via pathological analysis at the Fudan University Shanghai Cancer Center from August 2023 to August 2024,who underwent surgery and fulfilled the eligibility requirements,were gathered to examine the link between the expression of interferon-stimulated gene 15(ISG15)in serum exosomes and resistance to platinum medication.The expression levels of related proteins in serum exosomes were quantitatively detected by enzyme-linked immunosorbent assay(ELISA).Receiver operating characteristic(ROC)curve was plotted to explore the predictive value of serum exosomal protein in ovarian cancer resistance.Results:Exosomes derived from platinum-sensitive and drug-resistant ovarian cancer cells were extracted and proteomic analysis was further performed.We identified 9 differential proteins associated with platinum-resistance and found the key molecule interferon-stimulated gene 15(ISG15).Compared with sensitive cells,the expression of ISG15 in exosomes of drug-resistant ovarian cancer cells was significantly upregulated.Exosome tracer tests showed that exosomes were successfully taken up by ovarian cancer receptor cells.After coculture with drug-resistant exosomes,the expression level of ISG15 in ovarian cancer receptor cells was increased.Knockdown ISG15 in EOC cells decreased the expression of ISG15 in exosomes,and incubation of ISG15-knockdown exosomes decreased the cell viability on the condition of platinum drugs,indicating that ISG15 in exosomes regulated platinum resistance of ovarian cancer cells.Moreover,ISG15 could regulate the expression of multidrug resistance protein 1(MDR1)through phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)/nuclear factor-kappa-light-chain-enchancer of activated B cells(NF-κB)signaling pathway.This study included a total of 87 patients.Clinical serum samples also showed that the expression of ISG15 in exosomes was higher in platinum-resistant ovarian cancer patients than in sensitive ovarian cancer patients.Using serum exosomal ISG15 as an indicator to distinguish between sensitivity and resistance,the area under ROC curve was 0.779(P＜0.05),cutoffvalue was 27.35ng/mL,sensitivity was 70.2%,and specificity was 76.4%.Conclusion:ISG15 in exosomes can regulate the expression of MDR1 in ovarian cancer cells through PI3K/AKT/NF-κB signaling pathway,and promote platinum resistance in ovarian cancer cells.

Volumes 9 and 10 of Sun Simiao's Supplement to Important Formulas Worth a Thousand Gold Pieces record part of Trea-tise on Cold Damage,which contain Sun Simiao's unique thoughts on six-meridian treatment.Sun Simiao's treatment of Taiyang me-ridian emphasizes the classification of prescriptions and syndromes,advocates the correct treatment methods,and establishes three guidelines;Yangming meridian emphasizes the differentiation of cold and heat syndromes;except Taiyang meridian,all meridians em-phasize the differentiation of outline syndromes.It has made important contributions to the inheritance and development of the six-me-ridian thought of Treatise on Cold Damage in later generations.

Objective:To explore the correlation between skeletal muscle mass and strength with metabolic syndrome in children.Methods:This cross-sectional study was conducted involving 383 children aged 10 to 15 years who visited the Department of Child Health Care, Children′s Hospital of Nanjing Medical University from June 2021 to December 2022. Their height, weight, waist circumference, body composition, grip strength and blood pressure were measured. Relative skeletal muscle mass, muscle-to-fat ratio, and grip strength-to-body weight index were calculated. The levels of fasting blood glucose, lipids and insulin were tested. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Children were divided into the metabolic syndrome group and the non-metabolic syndrome group. Independent t test or Mann-Whitney U test etc. was used to compare the difference between groups. Spearman correlation analysis and binary Logistic regression were used to investigate the correlation between skeletal muscle mass and strength and metabolic syndrome. The area under the curve (AUC) of receiver operating characteristic (ROC) curve was used to compare the accuracy of the index of skeletal muscle in predicting metabolic syndrome in children. Results:Among the 383 children, 282 (73.6%) were male, at the age of 11.4 (10.6, 12.5) years. There were 216 children (56.4%) diagnosed with obesity and 90 children (23.5%) diagnosed with metabolic syndrome. Relative skeletal muscle mass, muscle-to-fat ratio, and grip strength-to-body weight index of the metabolic syndrome group were all lower than those in the non-metabolic syndrome group (all P<0.001). After adjusting for sex and age, relative skeletal muscle mass, muscle-to-fat ratio, and grip strength-to-body weight index were all negatively correlated with body mass index ( r=-0.84, -0.38, -0.63), waist circumference ( r=-0.76, -0.36, -0.70), systolic blood pressure ( r=-0.42, -0.21, -0.38), diastolic blood pressure ( r=-0.33, -0.18, -0.24), triglycerides ( r=-0.29, -0.13, -0.23), fasting insulin ( r=-0.28, -0.20, -0.29), and HOMA-IR ( r=-0.26, -0.18, -0.26) (all P<0.05), and positively correlated with high-density lipoprotein cholesterol ( r=0.38, 0.13, 0.31, all P<0.01). After adjusting for sex and age, high relative skeletal muscle mass, high muscle-to-fat ratio, and high grip strength-to-body weight index all decreased the risks of metabolic syndrome ( OR=0.80, 0.55, 0.90), obesity ( OR=0.53, 0.64, 0.82), hypertension ( OR=0.86, 0.58, 0.92), low high-density lipoprotein cholesterol ( OR=0.83, 0.62, 0.92), hypertriglyceridemia ( OR=0.88, 0.78, 0.96). After adjusting for sex and age, high relative skeletal muscle mass and high grip strength-to-body weight index all decreased the risks of hyperglycemia ( OR=0.93 and 0.95, all P<0.05). ROC curve analysis showed that the relative skeletal muscle mass, muscle-to-fat ratio, and grip strength-to-body weight index all had good predictive accuracy of metabolic syndrome in children (AUC=0.79, 0.71, 0.76), with optimal cutoff values of 40%, 1.2, and 35%, respectively. Conclusions:High relative skeletal muscle mass, high muscle-to-fat ratio, and high grip strength-to-body weight index are all protective factors for metabolic syndrome in children. Regular measurement of skeletal muscle mass and grip strength can aid in the early identification and prevention of obesity and metabolic syndrome during childhood .

Background and purpose:Epithelial ovarian cancer(EOC)has a poor prognosis and is prone to developing resistance to platinum-based chemotherapy.Exosomes are currently believed to be involved in platinum resistance in ovarian cancer.This study explored the role and mechanism of exosomes on platinum resistance of ovarian cancer.Methods:Through ultracentrifugation,exosomes were isolated and analyzed using electron microscopy,particle size studies,and Western blot for detailed exosome analysis.We detected the expression levels of exosomal proteins and related signaling pathways.Exosomal protein expression profile was analyzed by proteomics.Key differential proteins were screened by intersecting with existing drug resistance datasets.Furthermore,patients diagnosed with EOC via pathological analysis at the Fudan University Shanghai Cancer Center from August 2023 to August 2024,who underwent surgery and fulfilled the eligibility requirements,were gathered to examine the link between the expression of interferon-stimulated gene 15(ISG15)in serum exosomes and resistance to platinum medication.The expression levels of related proteins in serum exosomes were quantitatively detected by enzyme-linked immunosorbent assay(ELISA).Receiver operating characteristic(ROC)curve was plotted to explore the predictive value of serum exosomal protein in ovarian cancer resistance.Results:Exosomes derived from platinum-sensitive and drug-resistant ovarian cancer cells were extracted and proteomic analysis was further performed.We identified 9 differential proteins associated with platinum-resistance and found the key molecule interferon-stimulated gene 15(ISG15).Compared with sensitive cells,the expression of ISG15 in exosomes of drug-resistant ovarian cancer cells was significantly upregulated.Exosome tracer tests showed that exosomes were successfully taken up by ovarian cancer receptor cells.After coculture with drug-resistant exosomes,the expression level of ISG15 in ovarian cancer receptor cells was increased.Knockdown ISG15 in EOC cells decreased the expression of ISG15 in exosomes,and incubation of ISG15-knockdown exosomes decreased the cell viability on the condition of platinum drugs,indicating that ISG15 in exosomes regulated platinum resistance of ovarian cancer cells.Moreover,ISG15 could regulate the expression of multidrug resistance protein 1(MDR1)through phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)/nuclear factor-kappa-light-chain-enchancer of activated B cells(NF-κB)signaling pathway.This study included a total of 87 patients.Clinical serum samples also showed that the expression of ISG15 in exosomes was higher in platinum-resistant ovarian cancer patients than in sensitive ovarian cancer patients.Using serum exosomal ISG15 as an indicator to distinguish between sensitivity and resistance,the area under ROC curve was 0.779(P＜0.05),cutoffvalue was 27.35ng/mL,sensitivity was 70.2%,and specificity was 76.4%.Conclusion:ISG15 in exosomes can regulate the expression of MDR1 in ovarian cancer cells through PI3K/AKT/NF-κB signaling pathway,and promote platinum resistance in ovarian cancer cells.

Objective Exploring the therapeutic mechanism of Yishen Jiangzhuo Granules(YSJZG)on chronic kidney disease(CKD)based on mitochondrial dynamics and apoptosis of renal tubular epithelial cells.Methods The CKD model of rats with 5/6 nephrectomy was adopted and divided into 6 groups according to random number table:sham operation control group,model group,emodin group(500 mg/kg/d),Yishen Jiangzhuo granule low,middle and high dose groups.After 8 weeks of treatment with YSJZG,serum creatinine(SCR)and urea nitrogen(BUN),pathological changes of renal cortex,mitochondrial morphology and ultrastructure were detected,and mitochondrial kinetic protein in renal tubular epithelial cells was detected by immunohistochemistry(Drp1,Fis1)and fusion proteins(Opa1,Mfn1)were detected by Western blot,and apoptotic proteins(CytC,Bax)in cytoplasm and mitochondria were detected by real-time PCR.Results Renal injury:Compared with the model group,YSJZG groups significantly reduced the levels of SCR and BUN,renal tubular degeneration and necrosis,and mitochondrial structural damage in rats.Renal tubule mitochondrial dynamic protein:Compared with the model group,the expression of division proteins Drp1 and Fis1 was downregulated,the expression of fusion proteins Opa1 and Mfn1 was upregulated,and transmission electron microscopy observed that the mitochondrial fragmentation changes were relatively mild.Apoptosis related indicators and mtDNA copy number of renal tubular cells:Compared with the model group,the content of Bax protein in renal tubular epithelial cells of YSJZG groups increased significantly in cytoplasm(P<0.05)and decreased significantly in mitochondria(P<0.05).The content of CytC protein decreased significantly in cytoplasm(P<0.05)and increased significantly in mitochondria(P<0.05).The copy number of mtDNA increased significantly(P<0.05),and the total levels of SMAC,CytC and Bax mRNA decreased significantly(P<0.05).Correlation between mitochondrial dynamic protein and apoptosis in renal tubular cells:Pearson correlation analysis showed that the expression of Drp1 and Fis1 was negatively correlated with the expression of CytC in mitochondria,and positively correlated with the expression of CytC in cytoplasm.The expression levels of fusion proteins Opa1 and Mfn1 showed a significant positive correlation with CytC expression in mitochondria,and a significant negative correlation with CytC expression in cytoplasm.Conclusion YSJZG can significantly delay the progression of CKD,and its mechanism may be achieved by regulating mitochondrial dynamics of renal tubular epithelial cells,thereby inhibiting endogenous cell apoptosis pathway.