Patients suffering from nerve injury often experience exacerbated pain responses and complain of memory deficits. The dorsal hippocampus (dHPC), a well-defined region responsible for learning and memory, displays maladaptive plasticity upon injury, which is assumed to underlie pain hypersensitivity and cognitive deficits. However, much attention has thus far been paid to intracellular mechanisms of plasticity rather than extracellular alterations that might trigger and facilitate intracellular changes. Emerging evidence has shown that nerve injury alters the microarchitecture of the extracellular matrix (ECM) and decreases ECM rigidity in the dHPC. Despite this, it remains elusive which element of the ECM in the dHPC is affected and how it contributes to neuropathic pain and comorbid cognitive deficits. Laminin, a key element of the ECM, consists of α-, β-, and γ-chains and has been implicated in several pathophysiological processes. Here, we showed that peripheral nerve injury downregulates laminin β1 (LAMB1) in the dHPC. Silencing of hippocampal LAMB1 exacerbates pain sensitivity and induces cognitive dysfunction. Further mechanistic analysis revealed that loss of hippocampal LAMB1 causes dysregulated Src/NR2A signaling cascades via interaction with integrin β1, leading to decreased Ca²⁺ levels in pyramidal neurons, which in turn orchestrates structural and functional plasticity and eventually results in exaggerated pain responses and cognitive deficits. In this study, we shed new light on the functional capability of hippocampal ECM LAMB1 in the modulation of neuropathic pain and comorbid cognitive deficits, and reveal a mechanism that conveys extracellular alterations to intracellular plasticity. Moreover, we identified hippocampal LAMB1/integrin β1 signaling as a potential therapeutic target for the treatment of neuropathic pain and related memory loss.
Animals ; Laminin/genetics* ; Hippocampus/metabolism* ; Neuralgia/metabolism* ; Cognitive Dysfunction/etiology* ; Male ; Peripheral Nerve Injuries/metabolism* ; Extracellular Matrix/metabolism* ; Integrin beta1/metabolism* ; Pyramidal Cells/metabolism* ; Signal Transduction

Objective:To analyze and confirm the ambiguous results of HLA-DRB1 genotyping in one case.Methods:HLA genotyping was performed on a sample of hematopoietic stem cell donor using Illumina MiSeq-based next-generation sequencing(NGS).The ambiguous results of HLA-DRB1 locus were further analyzed and confirmed through PacBio SMRT third-generation sequencing.Results:The Illumina MiSeq-based NGS typing results suggested the presence of a new HLA-DRB1*11 allele(DRB1*11:NEW,12:01)in the specimen,with a mismatch of G＞A located in the 40th residue of exon 1 compared with the nearest allele DRB1*11:01:01:03.However,due to the long sequence of intron 1,this observed mutation site was so far away from the near heterozygous sites that no reads could cover this gap.Therefore,it was impossible to determine which consensus the mutation site was located in,and the NGS-based genotyping results were obtained from the random allocation by the software,which was ambiguous and unreliable.In order to confirm the results,the long-read third generation sequencing technology based on PacBio was applied to genotype the DRB1 locus.The results showed that the DRB1 typing was HLA-DRB1*11:01,12:10.E1-40A was actually located in the allele HLA-DRB1*12:XX,which was exactly matched with HLA-DRB1*12:10.Conclusion:For some new alleles suggested by NGS,especially the ambiguous ones that are far away from other heterozygous sites,it is necessary to analyze and confirm them by other methods such as the third-generation long-read sequencing technology to obtain reliable results.

Objective To explore the clinical characteristics of myelodysplastic syndrome(MDS)with U2 small nuclear RNA cofactor 1(U2AF1)mutation in different age groups,as well as the efficacy and prognosis of an arsenic-containing traditional Chinese medicine(TCM)compound prescription(Qinghuang Capsules combined with Bushen Yijing Formula).Methods A retrospective analysis was conducted on the clinical data of patients with MDS who were hospitalized in the Hematology Department Ward of Xiyuan Hospital,China Academy of Chinese Medical Sciences,and received arsenic-containing TCM compound treatment from November 30,2020,to September 30,2023.Stratified by age,the U2AF1 mutation and wild-type groups aged＜65 years and≥65 years were compared in terms of sex,TCM syndrome,World Health Organization classification,MDS Revised International Prognostic Score System(IPSS-R)score,blood routine indicators,serum lactate dehydrogenase content,nephroblastoma 1(WT1)expression level,bone marrow puncture and biopsy indicators,and chromosomal prognostic grades,et al.Furthermore,the efficacy of arsenic-containing TCM compound were compared in the U2AF1 mutation and wild-type groups among different age groups,as well as the influence of age on the survival prognosis of MDS patients with U2AF1 mutation.Results A total of 201 patients with MDS were included.104 patients were under 65 years old,among whom 20 had U2AF1 mutation,and 84 had wild-type.Ninety-seven patients were aged 65 years or older,among whom 19 patients had the U2AF1 mutation and 78 had the wild-type.Among patients aged＜65 years,the U2AF1 mutation group had a higher proportion of male patients and very low-risk/low-risk patients with an IPSS-R score≤3(P＜0.05),a lower mean corpuscular volume(MCV)(P＜0.05),and a relatively higher proportion of peripheral blood cell line 1 reduction than the wild-type group(P＜0.05).Among patients aged≥65 years,the MCV in the U2AF1 mutation group was lower(P＜0.05),and the expression level of the bone marrow WT1 gene and the proportion of patients with reticular fiber grade 4 were relatively higher than in the wild-type group(P＜0.05).The total effective rate of the arsenic-containing TCM compound for patients with U2AF1 mutation was 61.5％(24/39),and the total response rate was 30.8％(12/39).The total effective rate for the wild-type patients was 67.9％(110/162),and the total response rate was 29.6％(48/162).No significant difference was observed in the total effective and response rates.Kaplan-Meier survival analysis of 39 patients with U2AF1 mutation revealed that the median overall survival(mOS)of patients older than 65 years had not been reached.The 1-,2-,and 3-year survival rates were 93.8％,84.4％,and 84.4％,respectively.The mOS of the patients aged≥65 years was 35 months(95％confidence interval[CI]:7.559-62.441),and the 1-,2-,and 3-year survival rates were 66.2％,58.9％,and 29.4％,respectively.The mOS of patients in the aged≥65 years group was significantly lower than that in the aged＜65 years group(P＜0.05),and no significant difference was observed in median progression-free survival between the two groups.Conclusion The U2AF1 mutation is closely associated with the clinical characteristics of MDS.However,age and the presence of U2AF1 mutation have no significant effect on the total effective and response rates of arsenic-containing TCM compound.Age is a significant factor influencing the prognosis of patients with MDS with U2AF1 mutation.Patients aged 65 years or older have a shorter survival time than those younger than 65 years.

Objective To analyze the characteristics of methadone-related poisoning cases and provide a reference for forensic identification.Methods A total of 71 cases of methadone-related poisoning re-ported from 1998 to 2023 in China and 26 cases of methadone-related deaths reported from 2013 to 2018 in Italy were retrieved from databases including PubMed,Wanfang and CNKI.The general infor-mation,forensic pathological and toxicological characteristics were analyzed.Results Among the 71 methadone-related poisoning cases in China,there were 54 cases(76.06%)of poisoning without death and 17 cases(23.94%)of death from poisoning.There were 54 male cases(76.06%),and 51 cases(71.83%)aged 19 to 39 years old.There were 35 cases(49.30%)of poisoning caused by methadone alone,and 32 cases(45.07%)were poisoning caused by methadone combined with other substances or drugs including heroin and benzodiazepines.Most of the poisoned showed coma,respiratory depres-sion and miosis.Signs of asphyxia were often found by autopsy.The mass concentration of methadone detected in the blood of 6 deceased ranged from 0.112 to 3.000 mg/L.Among the 26 methadone-related deaths in Italy,22 cases were male(84.62%).There were 6 cases(23.08%)caused by methadone alone,and 20 cases(76.92%)died from methadone combined with other substances or drugs.The mass concentration of methadone in blood ranged from 0.181 to 4.059 mg/L.Conclusion The propor-tions of poisoning cases caused by methadone alone and methadone combined with other substances or drugs are comparable in China.The majority of deceased caused by methadone poisoning shows typi-cal triad of coma,respiratory depression and miosis,which helps forensic experts determine the cause of death related to methadone.Additionally,it is necessary to increase the routine testing of the con-centration of methadone and its combined substances or drugs in deceased,and collect data for the in-terpretation of the results of related cases.

Objective To explore the clinical characteristics of myelodysplastic syndrome(MDS)with U2 small nuclear RNA cofactor 1(U2AF1)mutation in different age groups,as well as the efficacy and prognosis of an arsenic-containing traditional Chinese medicine(TCM)compound prescription(Qinghuang Capsules combined with Bushen Yijing Formula).Methods A retrospective analysis was conducted on the clinical data of patients with MDS who were hospitalized in the Hematology Department Ward of Xiyuan Hospital,China Academy of Chinese Medical Sciences,and received arsenic-containing TCM compound treatment from November 30,2020,to September 30,2023.Stratified by age,the U2AF1 mutation and wild-type groups aged＜65 years and≥65 years were compared in terms of sex,TCM syndrome,World Health Organization classification,MDS Revised International Prognostic Score System(IPSS-R)score,blood routine indicators,serum lactate dehydrogenase content,nephroblastoma 1(WT1)expression level,bone marrow puncture and biopsy indicators,and chromosomal prognostic grades,et al.Furthermore,the efficacy of arsenic-containing TCM compound were compared in the U2AF1 mutation and wild-type groups among different age groups,as well as the influence of age on the survival prognosis of MDS patients with U2AF1 mutation.Results A total of 201 patients with MDS were included.104 patients were under 65 years old,among whom 20 had U2AF1 mutation,and 84 had wild-type.Ninety-seven patients were aged 65 years or older,among whom 19 patients had the U2AF1 mutation and 78 had the wild-type.Among patients aged＜65 years,the U2AF1 mutation group had a higher proportion of male patients and very low-risk/low-risk patients with an IPSS-R score≤3(P＜0.05),a lower mean corpuscular volume(MCV)(P＜0.05),and a relatively higher proportion of peripheral blood cell line 1 reduction than the wild-type group(P＜0.05).Among patients aged≥65 years,the MCV in the U2AF1 mutation group was lower(P＜0.05),and the expression level of the bone marrow WT1 gene and the proportion of patients with reticular fiber grade 4 were relatively higher than in the wild-type group(P＜0.05).The total effective rate of the arsenic-containing TCM compound for patients with U2AF1 mutation was 61.5％(24/39),and the total response rate was 30.8％(12/39).The total effective rate for the wild-type patients was 67.9％(110/162),and the total response rate was 29.6％(48/162).No significant difference was observed in the total effective and response rates.Kaplan-Meier survival analysis of 39 patients with U2AF1 mutation revealed that the median overall survival(mOS)of patients older than 65 years had not been reached.The 1-,2-,and 3-year survival rates were 93.8％,84.4％,and 84.4％,respectively.The mOS of the patients aged≥65 years was 35 months(95％confidence interval[CI]:7.559-62.441),and the 1-,2-,and 3-year survival rates were 66.2％,58.9％,and 29.4％,respectively.The mOS of patients in the aged≥65 years group was significantly lower than that in the aged＜65 years group(P＜0.05),and no significant difference was observed in median progression-free survival between the two groups.Conclusion The U2AF1 mutation is closely associated with the clinical characteristics of MDS.However,age and the presence of U2AF1 mutation have no significant effect on the total effective and response rates of arsenic-containing TCM compound.Age is a significant factor influencing the prognosis of patients with MDS with U2AF1 mutation.Patients aged 65 years or older have a shorter survival time than those younger than 65 years.

Objective:To analyze and confirm the ambiguous results of HLA-DRB1 genotyping in one case.Methods:HLA genotyping was performed on a sample of hematopoietic stem cell donor using Illumina MiSeq-based next-generation sequencing(NGS).The ambiguous results of HLA-DRB1 locus were further analyzed and confirmed through PacBio SMRT third-generation sequencing.Results:The Illumina MiSeq-based NGS typing results suggested the presence of a new HLA-DRB1*11 allele(DRB1*11:NEW,12:01)in the specimen,with a mismatch of G＞A located in the 40th residue of exon 1 compared with the nearest allele DRB1*11:01:01:03.However,due to the long sequence of intron 1,this observed mutation site was so far away from the near heterozygous sites that no reads could cover this gap.Therefore,it was impossible to determine which consensus the mutation site was located in,and the NGS-based genotyping results were obtained from the random allocation by the software,which was ambiguous and unreliable.In order to confirm the results,the long-read third generation sequencing technology based on PacBio was applied to genotype the DRB1 locus.The results showed that the DRB1 typing was HLA-DRB1*11:01,12:10.E1-40A was actually located in the allele HLA-DRB1*12:XX,which was exactly matched with HLA-DRB1*12:10.Conclusion:For some new alleles suggested by NGS,especially the ambiguous ones that are far away from other heterozygous sites,it is necessary to analyze and confirm them by other methods such as the third-generation long-read sequencing technology to obtain reliable results.

Objective:To confirm the sequence of a null allele HLA-C*08:127N produced by a base insertion.Methods:PCR sequence-specific oligonucleotide probe(SSOP)and PCR sequence-based typing(SBT)were used for HLA routine detection,which discovered abnormal sequence maps of HLA-C in one acute myeloid leukemia patient.The sequence of the above loci was confirmed by next generation sequencing(NGS)technology.Results:The SSOP typing result showed that HLA-C locus was C*03:04,C*08:01,while the sequence was suspected to be inserted or deleted in exon 3 by SBT,and finally confirmed by NGS as C*03:04,C*08:127N.Conclusion:When base insertion produces HLA null alleles,SBT analysis software cannot provide correct results,but NGS technology can more intuitively obtain accurate HLA typing results.

OBJECTIVES: To identify cuproptosis- and ferroptosis-related genes involved in nonalcoholic fatty liver disease and to determine the diagnostic value of hub genes. METHODS: The gene expression dataset GSE89632 was retrieved from the Gene Expression Omnibus database to identify differentially expressed genes (DEGs) between the non-alcoholic steatohepatitis (NASH) group and the healthy group using the 'limma' package in R software and weighted gene co-expression network analysis. Gene ontology, kyoto encyclopedia of genes and genomes pathway, and single-sample gene set enrichment analyses were performed to identify functional enrichment of DEGs. Ferroptosis- and cuproptosis-related genes were obtained from the FerrDb V2 database and available literatures, respectively. A combined signature for cuproptosis- and ferroptosis-related genes, called CRF, was constructed using the STRING database. Hub genes were identified by overlapping DEGs, WGCNA-derived key genes, and combined signature CRF genes, and validated using the GSE109836 and GSE227714 datasets and real-time quantitative polymerase chain reaction. A nomogram of NASH diagnostic model was established utilizing the 'rms' package in R software based on the hub genes, and the diagnostic value of hub genes was assessed using receiver operating characteristic curve analysis. In addition, immune cell infiltration in NASH versus healthy controls was examined using the CIBERSORT algorithm. The relationships among various infiltrated immune cells were explored with Spearman's correlation analysis. RESULTS: Analysis of GSE89632 identified 236 DEGs between the NASH group and the healthy group. WGCNA highlighted 8 significant modules and 11,095 pivotal genes, of which 330 genes constituted CRF. Intersection analysis identified IL6, IL1B, JUN, NR4A1, and PTGS2 as hub genes. The hub genes were all downregulated in the NASH group, and this result was further verified by the NASH validation dataset and real-time quantitative polymerase chain reaction. Receiver operating characteristic curve analysis confirmed the diagnostic efficacy of these hub genes with areas under the curve of 0.985, 0.941, 1.000, 0.967, and 0.985, respectively. Immune infiltration assessment revealed that gamma delta T cells, M1 macrophages, M2 macrophages, and resting mast cells were predominantly implicated. CONCLUSIONS Our investigation underscores the significant association of cuproptosis- and ferroptosis-related genes, specifically IL6, IL1B, JUN, NR4A1, and PTGS2, with NASH. These findings offer novel insights into the pathogenesis of NASH, potentially guiding future diagnostic and therapeutic strategies.
Non-alcoholic Fatty Liver Disease/pathology* ; Humans ; Ferroptosis/genetics* ; Copper/metabolism* ; Gene Ontology ; Gene Expression Profiling

ObjectiveTo explore the protective mechanism of paeoniflorin on mice with ulcerative colitis （UC） through the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） autophagy pathway. MethodUC mouse model was established by allowing mice freely drink 4% DSS， and 56 BALB/c male mice were randomly divided into model group， AMPK inhibitor group （20 mg·kg^-1）， paeoniflorin （50 mg·kg^-1） + inhibitor （20 mg·kg^-1） group， and high dose （50 mg·kg^-1）， medium dose （25 mg·kg^-1）， and low dose （12.5 mg·kg^-1） paeoniflorin groups. After seven days of drug intervention， the protective effect of paeoniflorin on mice with UC was determined by comparing the body weight， disease activity index （DAI） changes， and Hematoxylin-eosin （HE） staining results. Enzyme linked immunosorbent assay （ELISA） was used to detect the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the serum of mice in each group， and immunofluorescence was utilized to detect microtubule-associated protein 1 light chain 3 （LC3） content in the colon， AMPK， mTOR proteins， and their phosphorylated proteins including p-AMPK and p-mTOR in the colon tissue were detected by Western blot， and the mRNA expression levels of AMPK， mTOR， Beclin1， LC3， and p62 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group showed a decrease in body mass， an increase in DAI score， and severe pathological damage to the colon. The levels of inflammatory factors including TNF-α and IL-6 increased in serum （P<0.01）， while the protein levels of LC3 and p-AMPK/AMPK were down-regulated in colon tissue， and those of p-mTOR/mTOR were up-regulated （P<0.01）. The mRNA expression levels of AMPK and LC3 were down-regulated， while the mRNA expression levels of mTOR and p62 were up-regulated （P<0.01）. Compared with the model group and the paeoniflorin + inhibitor group， the mice treated with paeoniflorin showed an increase in body mass， a decrease in DAI score， a reduction in pathological damage to colon tissue， and a reduction in the levels of inflammatory factors of TNF-α and IL-6 in serum （P<0.05）. The protein levels of LC3 and p-AMPK/AMPK in colon tissue were up-regulated， while the protein levels of p-mTOR/mTOR were down-regulated （P<0.01）. The mRNA expression levels of AMPK， Beclin1， and LC3 were up-regulated， while the mRNA expression of mTOR and p62 were down-regulated （P<0.01）. The colon tissue of the inhibitor group was severely damaged， and the trend of various indicators was completely opposite to that of the high dose paeoniflorin group. ConclusionPaeoniflorin can enhance autophagy and reduce inflammatory damage in mice with UC by activating the AMPK/mTOR signaling pathway and thus play a protective role.

Objective:To discuss the protective effect of sodium butyrate(NaB)on acute liver injury in the mice induced by lipopolysaccharide(LPS)combined with D-galactosamine(D-Gal),and to clarify its mechanism.Methods:Thirty male Kunming mice were randomly divided into control group,model group,and NaB group,and there were 10 mice in each group.The mice in NaB group were given 200 mg·kg-1·d-1 NaB,while the mice in control group and model group were given an equal volume of sterile water.The mice in model group and NaB group were intraperitoneally injected with 20 μg·kg-1 LPS and 600 mg·kg-1 D-Gal to induce the acute liver injury models.The body weights and liver weights of the mice in various groups were detcted,and the liver index was calculated.HE staining was used to observe the pathomorphology of liver tissue of the mice in various groups;kits were used to detect the activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum,and the activities of total superoxide dismutase(T-SOD)and catalase(CAT),and the levels of malondialdehyde(MDA)in liver tissue of the mice in various groups;Western blotting method was used to detect the expression levels of nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)proteins in liver tissue of the mice in various groups.Results:There were no significant differences in body weights of the mice among various groups(P>0.05).Compared with control group,the liver index of the mice in model group was significantly increased(P<0.01).Compared with model group,the liver index of the mice in NaB group was significantly decreased(P<0.01).The HE staining results showed that the liver tissue of the mice in control group exhibited normal structure,with clear boundaries of hepatocytes,consistent size,radially arranged around the central vein,and the nucleus located in the center of the cells;in model group,the arrangement of hepatocytes was disordered,the cells were swollen,there were multiple foci of hepatocellular necrosis,inflammatory cell infiltration,and hemorrhage;compared with model group,the cells in NaB group showed improved hepatocellular structure and reduced inflammatory infiltration.Compared with control group,the activities of ALT and AST in serum of the mice in model group were significantly increased(P<0.01);compared with model group,the activities of ALT and AST in serum of the mice in NaB group were significantly decreased(P<0.05 or P<0.01).Compared with control group,the activities of T-SOD and CAT in liver tissue of the mice in model group were significantly decreased(P<0.01),and the level of MDA was significantly increased(P<0.01);compared with model group,the activities of T-SOD and CAT in liver tissue of the mice in NaB group were significantly increased(P<0.05 or P<0.01),and the level of MDA was significantly decreased(P<0.01).The Western blotting results showed that compared with control group,the expression levels of Nrf2 and HO-1 proteins in liver tissue of the mice in model group were significantly decreased(P<0.05);compared with model group,the expression levels of Nrf2 and HO-1 proteins in liver tissue of the mice in NaB group were significantly increased(P<0.01).Conclusion:NaB has a protective effect on LPS/D-Gal induced acute liver injury in the mice,and its mechanism may be related to the upregulation of the expressions of Nrf2 and HO-1 proteins and the increas of the activity of oxidant enzyme in liver tissue by NaB,thereby reduces the liver oxidative stress level of liver.