ObjectiveTo investigate the mechanism of action of Jiedu Huayu granules in improving liver injury in mice with acute liver failure （ALF） by observing its effect on a mouse model of ALF after prophylactic administration， and to provide a basis for clinical medication. MethodsA total of 60 specific pathogen-free male C57BL/6J mice were divided into normal group， model group， Jiedu Huayu granules group （JDHY group）， and farnesoid X receptor （FXR） agonist （GW4064） group using a random number table， with 15 mice in each group. The model of ALF was induced by a single intraperitoneal injection of D-galactosamine combined with lipopolysaccharide. The mice in the JDHY group were given prophylactic administration of 0.3 g/mL drug solution of Jiedu Huayu granules by gavage for 3 days before modeling， those in the normal group and the model group were given 0.9% NaCl solution by gavage， and those in the GW4064 group were given intraperitoneal injection of GW4064 for 3 consecutive days before modeling. The mice were sacrificed after modeling， and serum and liver tissue samples were collected. A veterinary automatic biochemical analyzer was used to measure the serum levels of total bilirubin （TBil）， total bile acids （TBA）， gamma-glutamyl transferase （GGT）， alanine aminotransferase （ALT）， and aspartate aminotransferase （AST） in mice from each group； HE staining was used to observe liver pathological changes； RT-PCR was used to measure the mRNA expression levels of FXR， fibroblast growth factor 15 （FGF15）， fibroblast growth factor receptor 4 （FGFR4）， small heterodimer partner （SHP）， and bile salt export pump （BSEP） in mice， and Western blot was used to measure the protein expression levels of FXR， FGF15， FGFR4， SHP， and BSEP. A one-way analysis of variance was used for comparison between groups， and the Dunett method was used for further comparison between two groups. ResultsCompared with the normal group， the model group had significant increases in the serum levels of TBil， ALT， AST， TBA， and GGT （all P<0.01）， and compared with the model group， the JDHY group and the GW4064 group had significant reductions in the serum levels of TBil， ALT， AST， TBA， and GGT （all P <0.01）. HE staining showed that compared with the model group， the JDHY group and the GW4064 group had milder pathological injury， a reduction in the area of hepatocyte necrosis， and alleviation of cellular swelling and edema. Compared with the normal group， the model group had significant reductions in the mRNA and protein expression levels of FXR， FGF15， FGFR4， SHP， and BSEP in liver tissue （all P <0.01）， and compared with the model group， the JDHY group and the GW4064 group had significant increases in the mRNA and protein expression levels of FXR， FGF15， FGFR4， SHP， and BSEP in liver tissue （all P <0.05）. ConclusionJiedu Huayu granules may alleviate liver injury in mice with ALF through the FXR/SHP axis.

BACKGROUND: The transcription factor POU2F1 regulates the expression levels of microRNAs in neoplasia. However, the miR-29b1/a cluster modulated by POU2F1 in gastric cancer (GC) remains unknown. METHODS: Gene expression in GC cells was evaluated using reverse-transcription polymerase chain reaction (PCR), western blotting, immunohistochemistry, and RNA in situ hybridization. Co-immunoprecipitation was performed to evaluate protein interactions. Transwell migration and invasion assays were performed to investigate the biological behavior of GC cells. MiR-29b1/a cluster promoter analysis and luciferase activity assay for the 3'-UTR study were performed in GC cells. In vivo tumor metastasis was evaluated in nude mice. RESULTS: POU2F1 is overexpressed in GC cell lines and binds to the miR-29b1/a cluster promoter. POU2F1 is upregulated, whereas mature miR-29b-3p and miR-29a-3p are downregulated in GC tissues. POU2F1 promotes GC metastasis by inhibiting miR-29b-3p or miR-29a-3p expression in vitro and in vivo . Furthermore, PIK3R1 and/or PIK3R3 are direct targets of miR-29b-3p and/or miR-29a-3p , and the ectopic expression of PIK3R1 or PIK3R3 reverses the suppressive effect of mature miR-29b-3p and/or miR-29a-3p on GC cell metastasis and invasion. Additionally, the interaction of PIK3R1 with PIK3R3 promotes migration and invasion, and miR-29b-3p , miR-29a-3p , PIK3R1 , and PIK3R3 regulate migration and invasion via the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway in GC cells. In addition, POU2F1 , PIK3R1 , and PIK3R3 expression levels negatively correlated with miR-29b-3p and miR-29a-3p expression levels in GC tissue samples. CONCLUSIONS The POU2F1 - miR-29b-3p / miR-29a-3p-PIK3R1 / PIK3R1 signaling axis regulates tumor progression and may be a promising therapeutic target for GC.
MicroRNAs/metabolism* ; Humans ; Stomach Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement/physiology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Animals ; Mice ; Octamer Transcription Factor-1/metabolism* ; Mice, Nude ; Class Ia Phosphatidylinositol 3-Kinase/metabolism* ; Neoplasm Invasiveness ; Gene Expression Regulation, Neoplastic/genetics* ; Male ; Immunohistochemistry ; Female

Objective:To explore the clinical phenotypes and genetic characteristics of a pedigree with Distal arthrogryposis type 5D (DA5D) caused by compound heterozygous variants in the ECEL1 gene. Methods:A child (proband) diagnosed with DA5D and his family members (proband′s parents and sister) who was admitted to the Department of Rehabilitation Medicine of Henan Children′s Hospital in July 2022 due to " multiplex distal arthrogryposis" were enrolled into this study. Clinical data of the proband were collected and peripheral blood samples were obtained from the proband and members of his family about 3 mL. Trio-whole genome sequencing (trio-WGS) was carried out to detected the genetic variations of the proband and his family members. The candidate′s pathogenic gene variants were screened and analyzed by Genome Aggregation Database (gnomAD) and other databases. The screened variants wer annotated for clinical phenotypes using databases like the Online Mendelian Inheritance in Man (OMIM). The pathogenicity of the candidate variants was predicted by bioinformatics tools such as Provean. Based on the guidelines of the American College of Medical Genetics and Genomics (ACMG), pathogenicity ratings were conducted for variant sites. The protein conservation and mutation structure prediction of ECEL1 protein among species were carried out though MEGA-X and PyMOL. The research protocol of this study was reviewed by the Ethics Committee of Henan Provincial Children′s Hospital (Approval No. 2023-H-H01), and informed consent for clinical research was obtained from the guardians of the probands.Results:The proband had multiplex distal arthrogryposis involving hands, feet, knees, and ankles, and had right ptosis, micrognathia, low auricular position, and upturned nose. The parents and sister both had normal phenotypes. Trio-WGS and Sanger sequencing revealed that the child had compound heterozygous variants of paternal c. 1742_c.1743insT and maternal c. 2314T>G, for which the father and sister were carriers of the c. 1742_c.1743insT heterozygous variant and the mother was carrier of c. 2314T>A. Neither mutation site has been reported. According to guidelines of ACMG, the c. 1742_c.1743insT variant was classified as likely pathogenic (PSV1+ PM2_Supporting), and c. 2314T>G was classified as uncertain (PM2_Supporting+ PM3+ PP3). The results of conserved analysis of amino acid residue sequences of ECEL1 protein showed that the missense mutation of the maternal c. 2314T>G(p.Cys772Gly) was highly conserved among humans and other seven species. The protein structure prediction revealed that the c.1742_c.1743insT frameshift mutation led to the protein truncation, and the c. 2314T>G missense mutation resulted in the failure of forming 1 disulfide bond.Conclusion:The compound heterozygous variants of ECEL1 gene were considered to be pathogenic for this DA5D patient, which have expanded the mutational spectrum of the ECEL1 gene and provided a reference for clinical diagnosis as well as genetic counseling for this family.

Objective:To investigate the effect and preliminary mechanism of hypervirulent Klebsiella pneumoniae (hvKP) on the immune response to sepsis induced by liver abscess in mice. Methods:C57BL/6 mice were intraperitoneally injected with hvKP strain NTUH-K2044 or classical Klebsiella pneumoniae (cKP) strain HS11286 suspension to prepare the model of sepsis. The survivals rates of mice within 24 h were recorded. HE staining was used to observed the inflammatory cell infiltration in mouse liver tissues. The levels of neutrophil marker lymphocyte antigen 6G (Ly6G) in mouse liver tissues were detected by immunohistochemistry. The activity of reactive oxygen species (ROS) in mouse liver macrophages and peripheral blood monocytes was measured by ROS assay kit. The activation of p105/p50 and p65 in NF-κB signaling pathway in mouse liver macrophages and peripheral blood monocytes was detected by Western blot. The expression of IL-1β, IL-6 and TNF-α at mRNA and protein levels in mouse liver macrophages and peripheral blood monocytes were detected by qRT-PCR and ELISA, respectively. One-way analysis of variance and t test were used for statistical analysis. Results:Compared with cKP, hvKP infection could induce C57BL/6 mice to develop obvious liver abscess with massive inflammatory cell infiltration. Moreover, the level of Ly6G in liver tissues was significantly higher in hvKP-infected mice than in cKP-infected mice ( P<0.000 1), but the survival rate of hvKP-infected mice was significantly lower than that of cKP-infected mice ( P<0.000 1). hvKP significantly promoted the ROS activity ( P<0.000 1) and enhanced the phosphorylation of p105/p50 and p65 in NF-κB signaling pathway in mouse liver macrophages and peripheral blood monocytes as compared with cKP ( P<0.001). The expression of IL-1β, IL-6 and TNF-α at mRNA and protein levels in mouse liver macrophages and peripheral blood monocytes were significantly higher in hvKP-infected mice than in cKP-infected mice ( P<0.001). Conclusion:hvKP can promote the development of liver abscess and induce sepsis in mice.

Objective:To investigate the role of TcpC, a virulence factor of uropathogenic Escherichia coli (UPEC), in inhibiting the formation of neutrophil extracellular traps (NETs) in mouse bone marrow neutrophils, and to analyze its pathogenic mechanism. Methods:C57BL/6 mice were injected with either wild-type (CFT073 wt) or tcpc gene-knockout UPEC CFT073(CFT073 Δ tcpc) to establish a mouse model of cystitis. Mice were sacrificed 3 d post-infection, and their bladders were collected to observe gross pathological changes. Hematoxylin-eosin (HE) staining was used to assess histopathological changes in bladder tissues, and immunohistochemistry was performed to localize TcpC in bladder tissues. Bacterial loads in urine samples were quantified using the ten-fold dilution method, and the presence of tcpc gene in genomic DNA from bladder or urine samples was confirmed by PCR. The expression of TcpC at mRNA and protein levels in mouse bone marrow nuetrophils infected with CFT073 wt was detected by qRT-PCR and Western blot. The effects of UPEC infection on expression of NETs-related proteins and the production of pro-inflammatory factors in mouse bone marrow neutrophils were analyzed by Western blot and ELISA, respectively. Reactive oxygen species(ROS) level and bacterial viability in mouse bone marrow nuetrophils were measured using ROS and bacterial viability detection kits. Results:Compared to the CFT073 Δ tcpc group, the bladder of CFT073 wt group mice exhibited significant enlargement, extensive inflammatory cell infiltration, and the presence of TcpC in bladder tissue. The bacterial load in the urine of CFT073 wt -infected mice was significantly higher than that in the CFT073 Δ tcpc group ( P<0.01). PCR confirmed the presence of the tcpc gene in bladder and urine samples from CFT073 wt-infected mice. Increased expression of TcpC at both mRNA and protein levels was observed in CFT073 wt-infected mouse bone marrow neutrophils. CFT073 wt infection inhibited the mRNA and protein expression of NETs-related proteins and reduced the production of pro-inflammatory factors in mouse bone marrow neutrophils. TcpC suppressed ROS level and promoted the survival of CFT073 wt in mouse bone marrow neutrophils. Conclusions:TcpC enhances the pathogenicity of UPEC CFT073 by inhibiting the formation and activation of NETs in mouse bone marrow neutrophils. This study provides new insights into the pathogenic mechanisms of UPEC and the immune evasion strategies of other pathogenic bacteria, as well as potential targets for clinical prevention and treatment of UPEC-induced urinary tract infections.

Hepatocellular carcinoma （HCC） is a malignant tumor with high incidence and mortality rates worldwide. Recent studies have shown that neutrophil extracellular traps （NETs） play an important role in the development， progression， and immune escape of HCC. NETs are released by neutrophils and mainly consist of DNA， histones， and antimicrobial molecules， and in addition to immune defense， they are also involved in the initiation， metastasis， and thrombosis of HCC. This article elaborates on the formation and regulatory mechanisms of NETs， explores their potential mechanisms in the initiation， metastasis， immune escape， and thrombosis of HCC， and discusses the prospect of NETs as a target for the diagnosis and treatment of HCC， in order to provide new ideas for the precise treatment of HCC in the future and promote the early diagnosis and effective treatment of HCC.

Hepatic encephalopathy(HE)is a common complication of severe liver disease in the end stage,and it is urgently needed to improve the rate of effective treatment and clarify the pathogenesis of HE.The liver is a crucial hub for immune regulation,and disruption of immune homeostasis is a key factor in the pathological mechanisms of HE.As the main metabolites of intestinal flora,short-chain fatty acids(SCFAs)play a vital role in the biological processes of both innate and adaptive immunity and can regulate the proliferation and differentiation of immune cells maintain the homeostasis of intestinal microenvironment and the integrity of barrier function.Studies have shown that SCFAs participate in bidirectional and dynamic interactions with the liver-gut-brain axis through immunomodulatory pathways,thereby playing an important role in the diagnosis,treatment,and prognostic evaluation of HE.Starting from the immunoregulatory effect of SCFAs,this article summarizes and analyzes the crosstalk relationship between SCFAs and the liver-gut-brain axis and the significance of SCFAs in the diagnosis and treatment of HE,in order to provide new ideas for optimizing clinical prevention and treatment strategies.

OBJECTIVE: To explore the clinical phenotypes and genetic characteristics of a pedigree with Distal arthrogryposis type 5D (DA5D) caused by compound heterozygous variants in the ECEL1 gene. METHODS: A child (proband) diagnosed with DA5D and his family members (proband's parents and sister) who was admitted to the Department of Rehabilitation Medicine of Henan Children's Hospital in July 2022 due to "multiplex distal arthrogryposis" were enrolled into this study. Clinical data of the proband were collected and peripheral blood samples were obtained from the proband and members of his family about 3 mL. Trio-whole genome sequencing (trio-WGS) was carried out to detected the genetic variations of the proband and his family members. The candidate's pathogenic gene variants were screened and analyzed by Genome Aggregation Database (gnomAD) and other databases. The screened variants were annotated for clinical phenotypes using databases like the Online Mendelian Inheritance in Man (OMIM). The pathogenicity of the candidate variants was predicted by bioinformatics tools such as Provean. Based on the guidelines of the American College of Medical Genetics and Genomics (ACMG), pathogenicity ratings were conducted for variant sites. The protein conservation and mutation structure prediction of ECEL1 protein among species were carried out though MEGA-X and PyMOL. The research protocol of this study was reviewed by the Ethics Committee of Henan Provincial Children's Hospital (Approval No. 2023-H-H01), and informed consent for clinical research was obtained from the guardians of the probands. RESULTS: The proband had multiplex distal arthrogryposis involving hands, feet, knees, and ankles, and had right ptosis, micrognathia, low auricular position, and upturned nose. The parents and sister both had normal phenotypes. Trio-WGS and Sanger sequencing revealed that the child had compound heterozygous variants of paternal c.1742_c.1743insT and maternal c.2314T>G, for which the father and sister were carriers of the c.1742_c.1743insT heterozygous variant and the mother was carrier of c.2314T>A. Neither mutation site has been reported. According to guidelines of ACMG, the c.1742_c.1743insT variant was classified as likely pathogenic (PSV1+PM2_Supporting), and c.2314T>G was classified as uncertain (PM2_Supporting+PM3+PP3). The results of conserved analysis of amino acid residue sequences of ECEL1 protein showed that the missense mutation of the maternal c.2314T>G (p.Cys772Gly) was highly conserved among humans and other seven species. The protein structure prediction revealed that the c.1742_c.1743insT frameshift mutation led to the protein truncation, and the c.2314T>G missense mutation resulted in the failure of forming 1 disulfide bond. CONCLUSION The compound heterozygous variants of ECEL1 gene were considered to be pathogenic for this DA5D patient, which have expanded the mutational spectrum of the ECEL1 gene and provided a reference for clinical diagnosis as well as genetic counseling for this family.
Humans ; Pedigree ; Arthrogryposis/genetics* ; Male ; Female ; Heterozygote ; Phenotype ; Mutation ; Child ; Metalloendopeptidases

OBJECTIVE Melatonin(MEL),a natural hormone with broad-spectrum anticancer effects,has been shown to potentiate the therapeutic outcomes of conventional chemotherapy.This study aims to investigate the combined effects of Doxorubicin(DOX)and MEL on head and neck squamous cell carcinoma(HNSCC)cell lines.METHODS The effects of MEL and DOX on cell proliferation in HNSCC cell lines TU686 and CAL-27 were assessed using the MTT assay.The effects of MEL and DOX on reactive oxygen species(ROS)expression levels in HNSCC cell lines were detected using DCFH-DA fluorescent probe.The effects of MEL and DOX on 8-hydroxy-2′-deoxyguanosine(8-oxodG)levels in HNSCC cell lines were measured by enzyme-linked immunosorbent assay(ELISA).The effects of MEL and DOX on γ-H2AX protein levels in HNSCC cell lines were analyzed via Western blot.The effects of MEL and DOX on antioxidant enzymes levels in HNSCC cell lines were evaluated based on spectrophotometry.The effects of MEL and DOX on cell apoptosis in HNSCC cell lines were detected using an ELISA cell death detection kit.RESULTS The MTT assay revealed that MEL significantly enhanced the cytotoxic effects of DOX in HNSCC cell lines(tTU686=13.51,tCAL-27=17.580,all P<0.05).The combination of MEL and DOX markedly increased intracellular ROS levels(FTU686=89.984,FCAL-27=102.853,all P<0.05),while the expression of antioxidant enzymes was significantly reduced(TU686:the F values of CAT,GPx,GR,GST and SOD are 176.035,34.662,20.260,120.105 and 184.254,all P<0.05;CAL-27:the F values are 96.801,177.398,97.849,102.750 and 186.608 respectively,all P<0.05).Furthermore,this combination treatment significantly elevated the expression levels of 8-oxodG(FTU686=200.078,FCAL-27=663.982,all P<0.05)and γ-H2AX(FTU686=192.500,FCAL-27=285.700,all P<0.01)in HNSCC cell lines.Additionally,compared to single-agent treatment,MEL significantly enhanced DOX-induced apoptosis in HNSCC cell lines(FTU686=2718.253,FCAL-27=5185.334,all P<0.01).CONCLUSION The combination of MEL and DOX can enhance cytotoxic effects on HNSCC cell lines,increase intracellular ROS levels,induce DNA oxidative damage,and impair cellular antioxidant defense capacity,thereby effectively promoting HNSCC cell apoptosis.This combination may represent a novel therapeutic approach to improve clinical outcomes in HNSCC.

Objective:To evaluate the clinical efficacy of Xiaoxuming Decoction combined with conventional Western medicine therapy in the treatment of patients with ischemic stroke in the recovery period.Methods:A randomized controlled clinical study was conducted. A total of the 118 patients with wind phlegm obstructing collaterlas syndrome during the recovery period of ischemic stroke in our hospital from September 2023 to July 2024 were selected as the observation subjects. They were divided into two groups using a random number table method, with 59 patients in each group. The control group was treated with conventional Western medicine therapy, while the TCM group was treated with modified Xiaoxuming Decoction on the basis of the control group. Both groups were treated for 2 months and followed up for 1 month. TCM syndrome scoring was performed before and after treatment, Barthel Index was used to evaluate daily living ability, and carotid artery ultrasound detector was used to evaluate the stability of carotid vascular plaques. Inter group comparisons were performed using t test, χ2 test, or repeated measures analysis of variance (RM-ANOVA). Results:RM-ANOVA showed that the time effect and inter group effect of TCM syndrome integration in the TCM group were significantly different from those in the control group ( Ftime=55.56, Ptime<0.001); Fbetween=18.94, Pbetween<0.001); there was no statistical significance in the interaction effect compared to the control group ( Finteraction=0.24, Pinteraction=0.866); the time effect, inter group effect, and interaction effect of Barthel Index in the TCM group were significantly different from those in the control group ( Ftime=44.57, Ptime<0.001); Fbetween=18.94, Pbetween<0.001; Finteraction=7.45, Pinteraction<0.001). The number of patients with unstable plaques in the TCM group after 3 months of treatment was lower than that in the control group ( χ2=4.52, P=0.033). Conclusion:The combination of modified Xiaoxuming Decoction and conventional Western medicine therapy can effectively improve the clinical symptoms and daily living ability of patients in the recovery period of ischemic stroke, improve the stability of cervical vascular plaques, and the clinical efficacy becomes more significant over time.