Hydrogen sulfide (H₂S) plays a crucial neuroprotective role in Parkinson's disease (PD). Cystathionine-β-synthase (CBS), a key enzyme involved in H₂S biosynthesis, exhibits expression deficiencies that are closely linked to PD progression. This suggests that enhancing the CBS-H₂S signaling axis to restore H₂S homeostasis may be a critical approach for preventing and treating PD. In the present study, the H₂S donor sulforaphane (SFN) was investigated to elucidate its neuroprotective mechanisms through activation of the CBS-H₂S axis. siRNA-mediated silencing of Nrf2 and CBS was employed to clarify the role of each in SFN's effects. Our findings demonstrate that SFN promotes CBS expression and H₂S synthesis, activates mitophagy to clear damaged mitochondria, reduces mitochondrial-derived reactive oxygen species (mtROS) levels, and inhibits the activation of NLRP3 inflammasomes and caspase-1. In the MPTP-induced PD mouse model, SFN improved motor performance, increased the survival rate of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra, and restored dopamine metabolism in the striatum, normalizing the DOPAC/DA and 5-HIAA/5-HT ratios. Electron microscopy revealed that SFN facilitated the clearance of damaged mitochondria through autophagosomes and blocked mtROS-mediated NLRP3 inflammasome activation. In the MPP+-induced BV-2 microglial cell model, SFN upregulated CBS expression, enhanced H₂S synthesis, increased the LC3-II/I ratio, and inhibited p62 degradation, thereby promoting the recovery of mitochondrial membrane potential and reducing ROS release. These effects were still observed under Nrf2 silencing conditions, indicating that SFN's neuroprotective effects are mediated through the CBS-H₂S axis independently of Nrf2 signaling. Collectively, these findings indicate that SFN reshapes the CBS-H₂S signaling axis, activates mitochondrial autophagy, and suppresses inflammation, offering novel insights into multi-target therapeutic approaches for PD and underscoring the essential role of H₂S in neuroprotection.

Sex disparity is a hepatocellular carcinoma (HCC) hallmark, demonstrating aggressiveness and mortality more frequently in men than in women. However, the components of its basis remain largely unknown. It was identified in HCC frequent loss of heterozygosity of general transcript factor IIH subunit 2 (GTF2H2), a potential estrogen pathway gene. GTF2H2 functions in nucleotide excision repair (NER) and basal transcription, but the function of GTF2H2 in cancer has not been described in depth. Here, it was identified that GTF2H2 inhibited growth and invasive mobility and induced apoptosis of HCC cells, which was up-regulated by estrogen-dependent estrogen receptor alpha (ERα) signaling. Mechanistically, in vitro estrogen-treated HCC models with GTF2H2 knockdown and over-expression showed estrogen-regulated GTF2H2 promoted NER of HCC genomic DNA and inhibited cell cycle progression, and down-regulated PAM/NF-κB pathway. Xenografted HCC mice models showed higher tumor progression of HCC with low GTF2H2 expression in ovariectomized female mice or male mice, but could be rescued by GTF2H2 over-expression, which was also observed in spontaneous tumor mice models. Clinical association analysis of HCC cases showed GTF2H2 expression was higher in female HCC, with correlation positively with ERα expression. Taken together, the estrogen-regulated GTF2H2 may be involved in the development and sex disparity of HCC by maintaining NER-related genomic stability and affecting PAM/NF-κB signaling pathway.

Background and Aims:Despite advancements in diagnostic and therapeutic strategies, hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality. Antioxidant-1 (ATOX1) has been implicated in oncogenic processes across various cancer types; however, its specific role in HCC remains unclear. This study aimed to investigate the function of ATOX1 and its underlying molecular mechanisms in HCC. Methods:Immunohistochemical analysis was conducted to assess ATOX1 expression in HCC tissues. Cell Counting Kit-8, colony formation, Transwell migration, flow cytometry, and reactive oxygen species (ROS) assays were employed to evaluate the malignant behaviors of tumor cells. A xenograft mouse model was employed to assess the effects of ATOX1 knockdown on tumor growth in vivo. DCAC50 treatment was performed to inhibit the copper transport function of ATOX1. RNA sequencing was conducted to explore the potential molecular mechanisms of ATOX1 in HCC. Results:ATOX1 expression was significantly elevated in HCC tumor tissues. ATOX1 promoted cell proliferation, colony formation, and migration. Knockdown of ATOX1 suppressed tumor growth in vivo. Mechanistically, ATOX1 activated c-Myb, and thus enhanced the malignant phenotype of HCC cells via activation of the PI3K/AKT signaling pathway. Additionally, ATOX1 reduced intracellular copper accumulation and inhibited ROS production and apoptosis. Inhibition of ATOX1 by DCAC50 decreased cell proliferation while increasing ROS levels and apoptosis in HCC cells. Notably, acetylcysteine reversed the reduction in c-Myb expression induced by ATOX1 knockdown. Conclusions:ATOX1 may promote HCC carcinogenesis through the activation of the c-Myb/PI3K/AKT pathway and the inhibition of copper accumulation and oxidative stress.

Objective:To explore the perinatal management of patients with WD during pregnancy, and to determine its genetic etiology and the possibility of fetal morbidity using the genetic detection of amniotic fluid and umbilical cord blood.Method:In terms of fine management during the perinatal period, a case of K-F ring was found in the Ophthalmology Department of Beijing Friendship Hospital, Capital Medical University in March 2019 due to eye astringency and eye swelling, and the hepatology department further diagnosed WD for one artificial abortion. After the second pregnancy in October 2020, multidisciplinary consultation and standardized treatment during pregnancy including gynecology and obstetrics, liver disease center, anesthesiology department, gastroenterology department and nutrition department were carried out. The genomes of patients' venous blood, amniotic fluid and umbilical cord blood were extracted and analyzed for ATP7B gene variation by Sanger sequencing.Result:Through multi-disciplinary collaborative management, the patient gave birth successfully in the case of pregnancy complicated with liver cirrhosis, portal hypertension, splenomegaly with hyperfunction, thrombocytopenia, anemia, esophageal and gastric varices and other complications. The phenotype of the newborn was normal, and the Apgar score was 10-10-10. Sequencing results showed that the patient had ATP7B p.Arg778Leu and p.Val890Met, which were missense heterozygous variants reported in the mutation database, and ACMG was classified as pathogenic variants. The results of amniotic fluid and umbilical cord blood showed that the fetus had only p.Arg778Leu single heterozygous variation, and it was predicted that there would be no clinical phenotype of WD.Conclusion:Perinatal multidisciplinary collaborative management has important protective significance for the successful pregnancy of patients with WD. Genetic screening of amniotic fluid and umbilical cord blood is conducive to early detection of fetal WD.

为快速准确、低成本、高通量地检测我国人群常见的遗传性胆红素代谢障碍及胆汁淤积综合征,选择了10个易感基因的全部外显子及内含子剪切区的SNP/CNV,建立了基于二代测序技术(next generation sequencing,NGS)的靶向捕获测序方法.通过6例已知突变位点的样本对该方法的准确性进行验证,准确率为100％.收集首都医科大学附属北京友谊医院遗传性胆红素代谢障碍及胆汁淤积综合征患者39例进行检测,共检测到58种突变.检测结果与HGMD、ClinVar、OMIM突变数据库比较,未报道的突变通过千人基因组数据集对比并按照哈温平衡检验(HWE_P>0.05)和χ2检验确定新突变19种.检测到的不同突变类型有效地揭示了该类疾病的遗传多样性.NGS方法的建立及应用为临床诊断提供了新的技术手段.

The mutations in modifier genes may contribute to some inherited diseases including Wilson disease (WD). This study was designed to identify potential modifier genes that contribute to WD. A total of 10 WD patients with single or no heterozygous ATP7B mutations were recruited for whole-exome sequencing (WES). Five hundred and thirteen candidate genes, of which the genetic variants present in at least two patients, were identified. In order to clarify which proteins might be involved in copper transfer or metabolism processes, the isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify the differentially expressed proteins between normal and CuSO4-treated cell lines. Thirteen genes/proteins were identified by both WES and iTRAQ, indicating that disease-causing variants of these genes may actually contribute to the aberrant copper ion accumulation. Additionally, the c.86C > T (p.S29L) mutation in the SLC31A2 gene (coding CTR2) has a relative higher frequency in our cohort of WD patients (6/191) than reported (0.0024 in gnomAD database) in our healthy donors (0/109), and CTR2(S29L) leads to increased intracellular Cu concentration and Cu-induced apoptosis in cultured cell lines. In conclusion, the WES and iTRAQ approaches successfully identified several disease-causing variants in potential modifier genes that may be involved in the WD phenotype.

Wilson disease (WD) is an autosomal recessive copper metabolic disorder caused by mutations in ATP7B. Sanger sequencing is currently used for ATP7B variant identification. However, the ATP7B gene contains 21 exons, which makes sequencing of the entire gene both complex and time‐consuming. Therefore, a simpler assay is urgently needed.

Colorectal cancer (CRC) is common worldwide. Aldehyde dehydrogenase 1B1 (ALDH1B1), a member of the ALDH1 family, serves as a biomarker for cancer stem cells. We hypothesized that ALDH1B1 expression is associated with colorectal tumors. Immunohistochemistry was used to detect ALDH1B1 expression across a commercial colorectal tissue microarray. The signal intensities of the positively stained tissues were expressed using the mean integrated optical density (mean IOD). We also analyzed ALDH1B1 mRNA expression in the Oncomine database. The associations between ALDH1B1 expression and CRC stage and prognosis were then evaluated using the web-based tools, GEPIA and UALCAN. Analysis of the tissue microarray revealed that the expression of ALDH1B1 was significantly higher in colorectal adenomas and colorectal adenocarcinoma (IOD/area values=0.117±0.070 and 0.168±0.0168, respectively) compared with normal and cancer-adjacent tissues (IOD/area values=0.051±0.028 and 0.068±0.053). For samples collected in the hospital, ALDH1B1 was highly expressed in the adenoma (IOD/area=0.103±0.054) and CRC (IOD/area=0.116±0.059) tissues compared with the cancer-adjacent tissues (IOD/area=0.066±0.024, p<0.05). The expression of ALDH1B1 in tissues from two resources was not found to be significantly associated with CRC stage. In Oncomine, ALDH1B1 mRNA expression was increased in the colorectal tumor tissues compared with the normal colorectal tissues (p=0.024) and its expression was independent of CRC stage and prognosis (p<0.05). Thus, while the protein and mRNA expression of ALDH1B1 suggests that it is a potential marker of colorectal tumors, its expression is independent of CRC stage and prognosis.

MarvelD3, a recently identified tight junction membrane protein, could be associated with hepatocellular carcinoma (HCC). We aimed to investigate the role of marvelD3 in Epithelial–Mesenchymal Transition (EMT) and migration of HCC and explore the underlying molecular mechanisms. First, we assessed marvlD3 expression in HCC and normal liver tissues and found loss of marvelD3 expression was significantly correlated with the occurrence and TNM stage of HCC. Second, we detected that marvelD3 was downregulated in HCC cells with transforming growth factor β1 and snail/slug-induced EMT. Finally, we analyzed expression of marvelD3 protein was significantly associated with EMT and the NF-κB signaling pathway. Our study demonstrated that MarvelD3 inhibited EMT and migration of HCC cells along with inhibiting NF-κB signaling pathway. Abbreviations: HCC, Hepatocellular carcinoma; TJ, Tight junction; MARVEL, MAL and related proteins for vesicle trafficking and membrane link; EMT, Epithelial–mesenchymal transition; NF-κB, Nuclear factor kappa B; TAMPs, Tight junction-associated marvel proteins; TGF-β1, Transforming growth factor-β1; MMP9, matrix metallopeptidase 9; RT-PCR, Real-time PCR; IHC, Immunohistochemistry; IF, Immunofluorescence.

Background: Colorectal cancer (CRC) comprises a large proportion of malignant tumors, and early detection of CRC is critical for effective treatment and optimal prognosis. We aimed to discover and validate serum autoantibodies for early detection of CRC. Methods: Combined with CRC-associated autoantibodies discovered by serological proteome and multiplex analyses, 26 predefined autoantibodies were evaluated in 315 samples (130 CRCs, 75 advanced adenomas, and 110 healthy controls) by protein microarray analysis. Autoantibodies with potential detection value were verified by enzyme-linked immunosorbent assays (ELISAs). Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the accuracy of the biomarkers. Results: Four serum autoantibodies (ALDH1B1, UQCRC1, CTAG1, and CENPF) showed statistically different levels between patients with advanced neoplasm (CRC or advanced adenoma) and controls in microarray analysis, which were validated by ELISAs. Among the four biomarkers, the ALDH1B1 autoantibody showed the highest detection value with area under the curve (AUC) values of 0.70 and 0.74 to detect CRC and advanced adenoma with sensitivities of 75.68 and 62.31% and specificities of 63.06 and 73.87%, respectively. By combining the four biomarkers, the performance was improved with an AUC of 0.79 to detect CRC and advanced adenomas. Conclusion: The ALDH1B1 autoantibody has a good potential for early detection of CRC and advanced adenoma, and measuring serum autoantibodies against tumor-associated antigens may improve detection of early CRC.

Background: Dubin-Johnson syndrome (DJS) is a rare autosomal recessive disorder characterized by predominantly conjugated hyperbilirubinemia that is caused by pathogenic mutations in the adenosine triphosphate-binding cassette subfamily C member 2 ( ABCC2 ) gene, which encodes multidrug resistance-associated protein 2 (MRP2). However, little is known about the causative mutation of DJS in China. Recently, we have reported ABCC2 p.G693R mutation in two unrelated cases. In the present study, we investigated the pathogenicity of the ABCC2 p.G693R mutation in DJS in China. Methods: Clinical and genetic analysis was conducted for the two patients with the ABCC2 p.G693R mutation. Whole exome sequencing for mutations in other known hyperbilirubinemia-related genes was conducted for the cases with ABCC2 p.G693R. Expression and cellular localization of the mutant MRP2 p.G693R were analyzed by Western blotting and immunofluorescence assay, respectively. Organic anion transport activity was evaluated by the analysis of glutathione-conjugated-monochlorobimane. Results: The two DJS patients with ABCC2 p.G693R mutation, which was conserved among different species, showed typical hyperbilirubinemia phenotype. No pathogenic mutation was identified in the other known hyperbilirubinemia related genes. Functional studies in three cell lines showed that the expression, localization and the organic anion transport activity were significantly compromised by MRP2 p.G693R mutation compared with wild-type MRP2. Conclusions: The recurrent ABCC2 p.G693R mutation is associated with loss of function of the MRP2 protein and may result in hyperbilirubinemia in DJS in China.

PHP14 inhibition via adeno-associated virus (AAV)-mediated gene silencing could potently attenuate carbon tetrachloride (CCl4)-induced liver fibrosis. PHP14 could regulate the migration of macrophages to the site of injury in vivo. PHP14 knockdown in vivo influenced the environment of fibrogenesis and relevant signaling pathways, subsequently affecting myofibroblast activation.

目的 探讨通用转录因子ⅡH亚基2(GTF2H2)在肝癌细胞中的核苷酸切除修复(NER)功能及对肝癌细胞增殖和凋亡的影响.方法 使用siRNA构建GTF2H2敲低的肝癌细胞模型,采用细胞计数试剂盒(CCK8)实验检测肝癌细胞的增殖能力;流式细胞术检测肝癌细胞株的凋亡情况.定量实时聚合酶链式反应(q-RT-PCR)检测增殖细胞核抗原(PCNA)和凋亡蛋白半胱天冬酶(Caspase-3)的表达情况.进一步构建肝癌细胞的紫外线(UV)照射模型,分析GTF2H2敲低后UV照射引起的NER的标志物环丁烷嘧啶二聚体(CPD)和嘧啶(6-4)嘧啶酮光产物(6-4PP)的变化.结果 GTF2H2敲低的肝癌细胞增殖能力相对明显增强,增殖蛋白PCNA的表达相对增多.相反,凋亡能力相对减弱,凋亡蛋白Caspase-3的表达相对降低.而GTF2H2敲低后,UV照射所致的NER标志物CPD和6-4PP含量相对增加.结论 GTF2H2在肝癌细胞中发挥了NER功能,抑制了肝癌细胞的增殖,促进其凋亡,是肝癌发生的一个潜在抑制因子.

Rotor syndrome, a rare autosomal-recessive genetic disorder characterized by conjugated hyperbilirubinemia, is caused by biallelic pathogenic variants in both SLCO1B1 and SLCO1B3 genes. Long interspersed nuclear elements (LINEs) make up about 17% of the human genome and insertion of LINE-1 in genes can result in genetic diseases. In the current study, we examined SLCO1B1 and SLCO1B3 genes in two Chinese patients diagnosed with Rotor syndrome based on laboratory tests. In one patient, a novel exon 4 inversion variant was identified. This variant may have been induced by LINE-1 retrotransposon insertion into SLCO1B3 intron 3, and was identified using genome walking. Splicing assay results indicated that the exon inversion, resulting in SLCO1B3 exon 4 (122 bp) exclusion in the mature mRNA, might generate a premature termination codon. Here, we describe an exon inversion contributing to the molecular etiology of Rotor syndrome. Our results may inform future diagnoses and guide drug prescriptions and genetic counseling.

目的 探讨雌激素对HepG2和BEL7402两种肝癌细胞增殖及迁移能力的抑制和诱导凋亡的作用及其相应机制.方法 利用MTS试验检测不同浓度的雌激素在不同时间点对HepG2和BEL7402两种肝癌细胞增殖能力的影响.Annexin V/PI流式细胞术检测不同浓度的雌激素诱导HepG2和BEL7402细胞凋亡的情况.Transwell迁移实验检测雌激素对HepG2和BEL7402细胞迁移能力的影响.采用Western blotting法检测不同浓度下雌激素对肝癌细胞中NF-κB信号通路的活性及其下游相关基因c-Myc、CyclinD1、基质金属蛋白酶(MMP-9)、血管内皮生长因子(VEGF)和Bcl-2表达的影响.结果 MTS试验结果显示,在不同时间点雌激素有效抑制了HepG2和BEL7402细胞的增殖,并随着雌激素浓度的升高其抑制肝癌细胞增殖的效果逐渐增强,与此同时,雌激素也降低了c-Myc、CyclinD1等增殖相关基因的表达.Annexin V/PI流式细胞术数据显示雌激素诱导HepG2和BEL7402细胞的凋亡并呈明显剂量依赖性,而抗凋亡蛋白Bcl-2的表达出现下降.Transwell迁移实验证明雌激素明显抑制了HepG2和BEL7402细胞的迁移能力并且降低了MMP-9和VEGF的表达.验证了雌激素可有效抑制HepG2和BEL7402肝癌细胞中NF-κB信号通路的活性.结论 雌激素通过下调N F-κB信号通路的活性及下游相关分子的表达,而抑制肝癌细胞的增殖与迁移并诱导其凋亡.

Hepatocellular carcinoma (HCC) is more frequently observed and aggressive in men compared with women. Increasing evidence demonstrates that the sex disparity appears to be mediated by the stimulatory effects of androgens and the protective effects of estrogen in the development and progression of HCC. In the past few decades, studies on the sex difference of HCC mainly focused on the effect of sex hormones on the transactivation of hepatitis B virus X protein and the release of inflammatory cytokines, and these studies have further intensified in recent years. Sex hormones are also involved in genetic alterations and DNA damage repair in hepatocytes through binding to their specific cellular receptors and affecting the corresponding signaling pathways. Furthermore, the theory of sex chromosomes participating in HCC has been considered. The present review discussed the recent advances in the molecular mechanisms of sex disparity in HCC, with the aim of improving the understanding of the underlying critical factors and exploring more effective methods for the prevention and treatment of HCC.

Wilson disease (WD) is a rare autosomal recessive genetic disorder that is associated with various mutations in the ATP7B gene. Although ATP7B variants are frequently identified, the exact mutation patterns remain unknown because of the absence of pedigree studies, and the functional consequences of individual ATP7B variants remain to be clarified. In this study, we recruited 65 clinically diagnosed WD patients from 60 unrelated families. Pedigree analysis showed that besides several ATP7B homozygous variants (8/65, 12.3%), compound heterozygous variants (43/65, 66.2%) were present in the majority of WD patients. There were 20% of the patients had one (12/65, 18.5%) or multiple (1/65, 1.5%) variants in only a single allele, characterized by a high ratio of splicing or frameshift variants. Nine ATP7B variants were cloned into the pAG426GPD yeast expression vector to evaluate their functional consequences, and the results suggested different degrees of functional disruption from mild or uncertain to severe, consistent with the corresponding phenotypes. Our study revealed the complex ATP7B mutation patterns in WD patients and the applicability of a yeast model system to the evaluation of the functional consequences of ATP7B variants, which is essential for WD cases that are difficult to interpret.

Dubin-Johnson syndrome (DJS) is a rare, autosomal recessive disorder characterized by predominantly conjugated hyperbilirubinemia, caused by a mutation in the adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2) gene coding the multidrug resistance-associated protein 2 (MRP2) protein. ABCC2 mutations have been identified in patients with DJS worldwide; however, the mutation pattern of ABCC2 in China is not well studied. In the present study, the mutation pattern of the ABCC2 gene in Chinese patients with DJS was investigated. A total of 7 clinically confirmed patients with DJS were enrolled, and mutation analysis of the ABCC2 gene was performed by Sanger sequencing of genomic DNA extracted from whole blood. All 32 exons and the adjacent splice junction areas were sequenced. All cases were identified to harbor at least one non-synonymous variant in the ABCC2 gene, including three known mutations in 3 cases and three novel variants (p.G693R, p.G808V and p.E647X) in the other 4 cases, with the known p.R393W and the novel p.G693R and p.E647X variants identified in 2 of the 7 cases (28.6%), respectively. All the identified mutations were heterozygous, and 1 case presented with a compound heterozygous mutation, namely p.G693R/p.G808V, while the other cases carried only one single mutation. The loss of membrane expression of MRP2 caused by the novel nonsense variant, p.E647X, was confirmed by immunohistochemical analysis of liver biopsy. The present study provided the first report on the mutation patterns of the ABCC2 gene in Chinese patients with DJS, and the clinical association of these mutations with the syndrome.