Background: Aristolochic acid II (AAII), a major nephrotoxic and carcinogenic component of aristolochic acids (AAs), has been less studied compared with its well-characterized analog, aristolochic acid I (AAI). Although AAs are known to induce carcinogenesis via DNA adduct formation, the toxicity mechanisms, environmental prevalence, and long-term health impacts of AAII remain poorly understood. Objective: This study aimed to systematically evaluate AAII’s acute and chronic toxicity, carcinogenic mechanisms, and environmental exposure patterns using integrated murine models and phytochemical analyses to clarify its toxicological profile and associated health risks. Methods: C57BL/6J mice were used in the following experiments: (1) determination of AAII content in 3 commonly used Aristolochia medicinal materials via liquid chromatography-mass spectrometry/mass spectrometry; (2) acute toxicity testing with single doses of 10, 20, or 40 mg/kg; and (3) chronic exposure with 1 or 10 mg/kg administered every other day for 24 weeks, followed by 21 to 40 weeks of postexposure monitoring. Histopathological examination, whole-exome sequencing, biochemical assays, and micronucleus tests were performed to assess multi-organ damage, tumorigenesis, genomic mutation signatures, and direct clastogenicity. Phytochemical analyses were used to evaluate environmental distribution. Results: (1) A single 40 mg/kg dose of AAII induced dose-dependent renal tubular degeneration without hepatotoxicity; (2) the 10 mg/kg group showed significant mortality (20%), tumor incidence (33.3%, primarily forestomach and bladder transitional cell carcinomas), persistent renal interstitial fibrosis, and subclinical hepatic injury. Chronic exposure to 1 mg/kg still induced 13.3% mortality and 15.5% tumor incidence over a 64-week period; (3) whole-exome sequencing revealed a predominance of C>T mutations and pathway enrichment in chemical carcinogenesis and cytochrome P450-mediated metabolism, indicating reactive metabolite-driven mechanisms distinct from classical AA-DNA adducts; and (4) no histopathological changes were observed in nontarget organs (brain, heart, and testes), and micronucleus assays confirmed the absence of direct clastogenicity. Conclusion: This study highlights the delayed carcinogenic risks of low-dose chronic AAII exposure and emphasizes the need to update regulatory frameworks to ensure the safe use of aristolochiaceae-containing herbal products.

Objective:To explore the differences between the Phoenix sepsis scoring system including Phoenix sepsis score (PSS) and Phoenix-8 organ dysfunction score (hereinafter referred to as Phoenix-8) and the commonly used pediatric sepsis scores in evaluating clinical characteristics and prognostic analysis of pediatric patients with severe sepsis diagnosed under traditional standards, namely the diagnostic criteria from the 2005 International Pediatric Sepsis Consensus Conference.Methods:This study was a retrospective observational study. From December 2020 to March 2023, 202 pediatric patients with severe sepsis meeting the inclusion criteria were admitted to the Children's Hospital of Nanjing Medical University. Based on the sepsis diagnostic criteria outlined in the International Consensus Criteria for Pediatric Sepsis and Septic Shock (2024), the pediatric patients were categorized into a sepsis group and a non-sepsis group. Sepsis group was further subdivided into a death subgroup and a survival subgroup based on the outcomes. The age, hospitalization costs, disease outcome indicators (e.g., mortality rate and incidence of septic shock), major organ (e.g., heart, liver, lungs, and kidneys) damage and their correlations, as well as PSS, Phoenix-8 and commonly used pediatric sepsis scores (e.g., pediatric sequential organ failure assessment (pSOFA), pediatric risk of mortality score Ⅲ (PRISM Ⅲ), pediatric logistic organ dysfunction-2 score (PELOD-2), pediatric multiple organ dysfunction score (P-MODS), pediatric critical illness score (PCIS), and pediatric early warning score (PEWS)) were collected and compared. Receiver operating characteristic (ROC) curve and precision-recall curve were plotted to evaluate the predictive ability of PSS, Phoenix-8, and commonly used pediatric sepsis scores for mortality risk in pediatric patients with severe sepsis under traditional standards. Predictive performance was quantified using the area under the ROC curve (AUROC). Univariate logistic regression analysis was employed to quantify the odds ratios of PSS and Phoenix-8 for predicting mortality risk. Patients with severe sepsis under traditional standards were further stratified into subgroups based on complications and comorbidities, including central nervous system (CNS) diseases, multiple infections, cardiovascular system diseases, shock, and malignancies. The Hosmer-Lemeshow goodness-of-fit test was used to assess calibration of PSS and Phoenix-8, and the DeLong test was used to compare whether there were statistically significant differences in the AUROC of PSS and Phoenix-8 for predicting mortality risk among different subgroups of pediatric patients. Results:Compared with those in non-sepsis group, pediatric patients in sepsis group were significantly older ( Z=-2.92, P<0.05) with higher incidences of septic shock and mortality, hospitalization costs, PRISM Ⅲ, PEWS, pSOFA, PELOD-2, PSS, and Phoenix-8 (with χ2 values of 21.28 and 13.64, respectively, Z values of -1.99, -5.33, -5.10, -8.55, -6.91, -10.98, and -9.93, respectively, P<0.05), and lower PCIS ( Z=-3.34, P<0.05). Compared with those in survival subgroup, hospitalization costs, PSS, Phoenix-8, PRISM Ⅲ, PEWS, pSOFA, PELOD-2, and P-MODS of pediatric patients in death subgroup was significantly higher (with Z values of -2.50, -3.50, -2.47, -5.11, -3.84, -2.94, -3.61, and -3.04, respectively, P<0.05). Compared with those in survival subgroup, the incidences of lung damage and liver damage of pediatric patients in death subgroup were also significantly higher (with χ2 values of 6.20 and 10.94, respectively, P<0.05), and 64.7% (97/150) of patients exhibited two or more concurrent organ damage. For predicting mortality risk in pediatric patients with severe sepsis under traditional standards, the AUROC values for PRISM Ⅲ, PCIS, PEWS, pSOFA, PELOD-2, P-MODS, PSS, and Phoenix-8 were approximately 0.70, with optimal cutoff values of 17.5, 91.0, 5.5, 4.5, 2.5, 4.5, 3.5, and 4.5, respectively; PELOD-2 demonstrated the highest sensitivity (0.83); while PRISM Ⅲ, PSS, and Phoenix-8 showed high specificity (>0.80). Univariate logistic regression analysis showed that for every 1-point increase in the PSS within 24 hours of pediatric intensive care unit admission, the relative risk of mortality increased by 63.7% (with odds ratio of 1.64, 95% confidence interval of 1.34-1.99, P<0.05). Similarly, for every 1-point increase in the Phoenix-8, the relative risk of mortality increased by 37.5% (with odds ratio of 1.38, 95% confidence interval of 1.18-1.60, P<0.05). The AUROC values (around 0.80) of PSS and Phoenix-8 for predicting mortality risk in pediatric patients with severe sepsis combined with CNS diseases, multiple infections, and cardiovascular system diseases were relatively high. In contrast, the AUROC values (0.60-0.80) for predicting mortality risk in pediatric patients with severe sepsis combined with shock or malignant tumors were moderate. All models passed the Hosmer-Lemeshow goodness-of-fit test ( P>0.05). The DeLong test indicated no statistically significant differences in predictive ability between PSS and Phoenix-8 across subgroups of pediatric patients ( P>0.05). Conclusions:PSS and Phoenix-8 exhibited higher specificity than most of the commonly used pediatric sepsis scores in predicting mortality risk under traditional standards. Both scores performed much better in predicting the mortality risk in pediatric patients with severe sepsis combined with CNS diseases, multiple infections, and cardiovascular system diseases.

Cervi Cornu Pantotrichum is a precious animal-derived Chinese medicinal material, while there are often adulterants derived from animals not specified in the Chinese Pharmacopeia in the market, which disturbs the safety of medication. This study was conducted with the aim of strengthening the quality control of Cervi Cornu Pantotrichum and standardizing the medication. To achieve digital identification of Cervi Cornu Pantotrichum from different sources, a digital identification model was constructed based on ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UHPLC-QTOF-MS~E) combined with an adaptive boosting algorithm(Adaboost). The young furred antlers of sika deer, red deer, elk, and reindeer were processed and then subjected to polypeptide analysis by UHPLC-QTOF-MS~E. Then, the mass spectral data reflecting the polypeptide information were obtained by digital quantification. Next, the key data were obtained by feature screening based on Gini index, and the digital identification model was constructed by Adaboost. The model was evaluated based on the recall rate, F_1 composite score, and accuracy. Finally, the results of identification based on the constructed digital identification model were validated. The results showed that when the Gini index was used to screen the data of top 100 characteristic polypeptides, the digital identification model based on Adaboost had the best performance, with the recall rate, F_1 composite score, and accuracy not less than 0.953. The validation analysis showed that the accuracy of the identification of the 10 batches of samples was as high as 100.0%. Therefore, based on UHPLC-QTOF-MS~E and Adaboost algorithm, the digital identification of Cervi Cornu Pantotrichum can be realized efficiently and accurately, which can provide reference for the quality control and original animal identification of Cervi Cornu Pantotrichum.
Animals ; Algorithms ; Antlers/chemistry* ; Boosting Machine Learning Algorithms ; Chromatography, High Pressure Liquid/methods* ; Deer ; Drugs, Chinese Herbal/chemistry* ; Mass Spectrometry/methods* ; Quality Control ; Reindeer ; Tandem Mass Spectrometry/methods* ; Tissue Extracts/analysis*

Emodin is a hydroxyanthraquinone compound that is widely distributed and has multiple pharmacological activities, including anti-diarrheal, anti-inflammatory, and liver-protective effects. Research indicates that emodin may be one of the main components responsible for inducing hepatotoxicity. However, studies on the mechanisms of liver injury are relatively limited, particularly those related to bile acids(BAs) metabolism. This study aims to systematically investigate the effects of different dosages of emodin on BAs metabolism, providing a basis for the safe clinical use of traditional Chinese medicine(TCM)containing emodin. First, this study evaluated the safety of repeated administration of different dosages of emodin over a 5-week period, with a particular focus on its impact on the liver. Next, the composition and content of BAs in serum and liver were analyzed. Subsequently, qRT-PCR was used to detect the mRNA expression of nuclear receptors and transporters related to BAs metabolism. The results showed that 1 g·kg~(-1) emodin induced hepatic damage, with bile duct hyperplasia as the primary pathological manifestation. It significantly increased the levels of various BAs in the serum and primary BAs(including taurine-conjugated and free BAs) in the liver. Additionally, it downregulated the mRNA expression of farnesoid X receptor(FXR), retinoid X receptor(RXR), and sodium taurocholate cotransporting polypeptide(NTCP), and upregulated the mRNA expression of cholesterol 7α-hydroxylase(CYP7A1) in the liver. Although 0.01 g·kg~(-1) and 0.03 g·kg~(-1) emodin did not induce obvious liver injury, they significantly increased the level of taurine-conjugated BAs in the liver, suggesting a potential interference with BAs homeostasis. In conclusion, 1 g·kg~(-1) emodin may promote the production of primary BAs in the liver by affecting the FXR-RXR-CYP7A1 pathway, inhibit NTCP expression, and reduce BA reabsorption in the liver, resulting in BA accumulation in the peripheral blood. This disruption of BA homeostasis leads to liver injury. Even doses of emodin close to the clinical dose can also have a certain effect on the homeostasis of BAs. Therefore, when using traditional Chinese medicine or formulas containing emodin in clinical practice, it is necessary to regularly monitor liver function indicators and closely monitor the risk of drug-induced liver injury.
Emodin/administration & dosage* ; Bile Acids and Salts/metabolism* ; Animals ; Male ; Liver/injuries* ; Chemical and Drug Induced Liver Injury/genetics* ; Drugs, Chinese Herbal/adverse effects* ; Humans ; Rats, Sprague-Dawley ; Mice ; Rats

OBJECTIVE: Huachansu injection (HCSI), a promising anti-cancer Chinese medicine injection, has been reported to have the potential for reducing the toxicity of chemotherapy and improving the quality of life for colorectal cancer (CRC) patients. The objective of this study is to explore the synergistic and detoxifying effects of HCSI when used in combination with irinotecan (CPT-11). METHODS: To investigate the effect of HCSI on anti-CRC efficacy and intestinal toxicity of CPT-11, we measured changes in the biological behavior of LoVo cells in vitro, and anti-tumor effects in LoVo cell xenograft nude mice models in vivo. Meanwhile, the effect of HCSI on intestinal toxicity and the uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) expression was investigated in the CPT-11-induced colitis mouse model. Subsequently, we measured the effect of HCSI and its 13 constituent bufadienolides on the expression of UGT1A1 and organic anion transporting polypeptides 1B3 (OATP1B3) in HepG2 cells. RESULTS: The combination index (CI) results showed that the combination of HCSI and CPT-11 exhibited a synergistic effect (CI < 1), which significantly suppressing the LoVo cell migration, enhancing G2/M and S phase arrest, and inhibiting tumor growth in vivo. Additionally, the damage to intestinal tissues was attenuated by HCSI in CPT-11-induced colitis model, while the increased expression of UGT1A1 in HepG2 cells and in mouse was observed. CONCLUSION The co-therapy with HCSI alleviated the intestinal toxicity induced by CPT-11 and exerted an enhanced anti-CRC effect. The detoxifying mechanism may be related to the increased expression of UGT1A1 and OATP1B3 by HCSI and its bufadienolides components. The findings of this study may serve as a theoretical insights and strategies to improve CRC patient outcomes. Please cite this article as: Jiang B, Meng ZY, Hu YJ, Chen JJ, Zong L, Xu LY, Zhang XQ, Zhang JX, Han YL. Huachansu injection enhances anti-colorectal cancer efficacy of irinotecan and alleviates its induced intestinal toxicity through upregulating UGT1A1-OATP1B3 expression in vitro and in vivo. J Integr Med. 2025; 23(5):576-590.
Irinotecan/therapeutic use* ; Animals ; Glucuronosyltransferase/genetics* ; Humans ; Colorectal Neoplasms/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Mice, Nude ; Mice ; Up-Regulation/drug effects* ; Male ; Xenograft Model Antitumor Assays ; Mice, Inbred BALB C ; Hep G2 Cells ; Cell Line, Tumor ; Intestines/drug effects* ; Amphibian Venoms

Objective:To explore the differences between the Phoenix sepsis scoring system including Phoenix sepsis score (PSS) and Phoenix-8 organ dysfunction score (hereinafter referred to as Phoenix-8) and the commonly used pediatric sepsis scores in evaluating clinical characteristics and prognostic analysis of pediatric patients with severe sepsis diagnosed under traditional standards, namely the diagnostic criteria from the 2005 International Pediatric Sepsis Consensus Conference.Methods:This study was a retrospective observational study. From December 2020 to March 2023, 202 pediatric patients with severe sepsis meeting the inclusion criteria were admitted to the Children's Hospital of Nanjing Medical University. Based on the sepsis diagnostic criteria outlined in the International Consensus Criteria for Pediatric Sepsis and Septic Shock (2024), the pediatric patients were categorized into a sepsis group and a non-sepsis group. Sepsis group was further subdivided into a death subgroup and a survival subgroup based on the outcomes. The age, hospitalization costs, disease outcome indicators (e.g., mortality rate and incidence of septic shock), major organ (e.g., heart, liver, lungs, and kidneys) damage and their correlations, as well as PSS, Phoenix-8 and commonly used pediatric sepsis scores (e.g., pediatric sequential organ failure assessment (pSOFA), pediatric risk of mortality score Ⅲ (PRISM Ⅲ), pediatric logistic organ dysfunction-2 score (PELOD-2), pediatric multiple organ dysfunction score (P-MODS), pediatric critical illness score (PCIS), and pediatric early warning score (PEWS)) were collected and compared. Receiver operating characteristic (ROC) curve and precision-recall curve were plotted to evaluate the predictive ability of PSS, Phoenix-8, and commonly used pediatric sepsis scores for mortality risk in pediatric patients with severe sepsis under traditional standards. Predictive performance was quantified using the area under the ROC curve (AUROC). Univariate logistic regression analysis was employed to quantify the odds ratios of PSS and Phoenix-8 for predicting mortality risk. Patients with severe sepsis under traditional standards were further stratified into subgroups based on complications and comorbidities, including central nervous system (CNS) diseases, multiple infections, cardiovascular system diseases, shock, and malignancies. The Hosmer-Lemeshow goodness-of-fit test was used to assess calibration of PSS and Phoenix-8, and the DeLong test was used to compare whether there were statistically significant differences in the AUROC of PSS and Phoenix-8 for predicting mortality risk among different subgroups of pediatric patients. Results:Compared with those in non-sepsis group, pediatric patients in sepsis group were significantly older ( Z=-2.92, P<0.05) with higher incidences of septic shock and mortality, hospitalization costs, PRISM Ⅲ, PEWS, pSOFA, PELOD-2, PSS, and Phoenix-8 (with χ2 values of 21.28 and 13.64, respectively, Z values of -1.99, -5.33, -5.10, -8.55, -6.91, -10.98, and -9.93, respectively, P<0.05), and lower PCIS ( Z=-3.34, P<0.05). Compared with those in survival subgroup, hospitalization costs, PSS, Phoenix-8, PRISM Ⅲ, PEWS, pSOFA, PELOD-2, and P-MODS of pediatric patients in death subgroup was significantly higher (with Z values of -2.50, -3.50, -2.47, -5.11, -3.84, -2.94, -3.61, and -3.04, respectively, P<0.05). Compared with those in survival subgroup, the incidences of lung damage and liver damage of pediatric patients in death subgroup were also significantly higher (with χ2 values of 6.20 and 10.94, respectively, P<0.05), and 64.7% (97/150) of patients exhibited two or more concurrent organ damage. For predicting mortality risk in pediatric patients with severe sepsis under traditional standards, the AUROC values for PRISM Ⅲ, PCIS, PEWS, pSOFA, PELOD-2, P-MODS, PSS, and Phoenix-8 were approximately 0.70, with optimal cutoff values of 17.5, 91.0, 5.5, 4.5, 2.5, 4.5, 3.5, and 4.5, respectively; PELOD-2 demonstrated the highest sensitivity (0.83); while PRISM Ⅲ, PSS, and Phoenix-8 showed high specificity (>0.80). Univariate logistic regression analysis showed that for every 1-point increase in the PSS within 24 hours of pediatric intensive care unit admission, the relative risk of mortality increased by 63.7% (with odds ratio of 1.64, 95% confidence interval of 1.34-1.99, P<0.05). Similarly, for every 1-point increase in the Phoenix-8, the relative risk of mortality increased by 37.5% (with odds ratio of 1.38, 95% confidence interval of 1.18-1.60, P<0.05). The AUROC values (around 0.80) of PSS and Phoenix-8 for predicting mortality risk in pediatric patients with severe sepsis combined with CNS diseases, multiple infections, and cardiovascular system diseases were relatively high. In contrast, the AUROC values (0.60-0.80) for predicting mortality risk in pediatric patients with severe sepsis combined with shock or malignant tumors were moderate. All models passed the Hosmer-Lemeshow goodness-of-fit test ( P>0.05). The DeLong test indicated no statistically significant differences in predictive ability between PSS and Phoenix-8 across subgroups of pediatric patients ( P>0.05). Conclusions:PSS and Phoenix-8 exhibited higher specificity than most of the commonly used pediatric sepsis scores in predicting mortality risk under traditional standards. Both scores performed much better in predicting the mortality risk in pediatric patients with severe sepsis combined with CNS diseases, multiple infections, and cardiovascular system diseases.

ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS^E） technique， we identified qualitatively the metabolites of aristolochic acid（AAs） in rat in order to analyze the metabolic differences between water extract of Aristolochiae fructus（AFE） and Aristolochic acid Ⅰ（AAⅠ）. MethodSD rats were selected and administered AFE（110 g·kg^-1·d^-1） or AAⅠ（5 mg·kg^-1·d^-1） by oral for 5 days， respectively. Serum， urine and feces were collected after administration. Through sample pretreatment， ACQUITY UPLC BEH C₁₈ column（2.1 mm×100 mm， 1.7 μm） was used with the mobile phase of 0.01% formic acid methanol（A）-0.01% formic acid water（B， containing 5 mmol·L^-1 ammonium acetate） for gradient elution（0-1 min， 10%B； 1-7 min， 10%-75%B； 7-7.2 min， 75%-95%B； 7.2-10.2 min， 95%B； 10.2-10.3 min， 95%-10%B； 10.3-12 min， 10%B） at a flow rate of 0.3 mL·min^-1. Positive ion mode of electrospray ionization（ESI⁺） was performed in the scanning range of m/z 100-1 200. In combination with UNIFI 1.9.4.053 system， the Pathway-MS^E was used to qualitatively analyze and identify the AAs prototype and related metabolites in biological samples（serum， urine and feces）， and to compare the similarities and differences of metabolites in rats in the subacute toxicity test between AFE group and AAⅠ group. ResultCompared with AAⅠ group， 6， 10， 13 common metabolites and 14， 20， 30 unique metabolites were identified in biological samples（serum， urine and feces） of AFE group， respectively. Moreover， the main AAs components always followed the metabolic processes of demethylation， nitrate reduction and conjugation. Compared with common metabolites in AAⅠ group， prototype components of AAⅠ in serum and most metabolic derivatives of AAⅠ［AAⅠa， aristolochic lactam Ⅰ（ALⅠ）a， 7-OHALⅠ and its conjugated derivatives］ in biological samples were significantly increased in AFE group（P<0.05， P<0.01）， except that the metabolic amount of ALⅠ in feces of AFE group was remarkably lowed than that of AAⅠ group（P<0.01）. In addition， a variety of special ALⅠ efflux derivatives were also identified in the urine and feces of the AFE group. ConclusionAlthough major AAs components in AFE all show similar metabolic rules as AAⅠ components in vivo， the coexistence of multiple AAs components in Aristolochiae Fructus may affect the metabolism of AAⅠ， and achieve the attenuating effect by increasing the metabolic effection of AAⅠ and ALⅠ.

Objective: To analyze the mortality trend and characteristics of chronic obstructive pulmonary disease (COPD) among residents in China from 2004 to 2020. Methods: From the area, gender, region, and age dimensions, the Joinpoint regression model was used to analyze the trend of COPD mortality rate from 2004 to 2020, extracted from the China Death Surveillance Dataset. Results: From 2004 to 2020, the mortality rate and age-adjusted mortality rate of COPD showed a downward trend (AAPC=-3.68%, P<0.001; AAPC=-7.27%, P<0.001), which were consistent with urban and rural subpopulations (mortality rate: AAPC=-3.62%, P=0.009, AAPC=-3.23%, P=0.014; age-adjusted mortality rate: AAPC=-7.26%, P<0.001, AAPC=-6.78%, P<0.001). The mortality rate of COPD in rural was higher than that of urban subpopulations (P<0.001). Also, the mortality rate and age-adjusted mortality rate of COPD showed a downward trend in males and females (mortality rate: AAPC=-3.00%, P<0.001, AAPC=-4.37%, P<0.001; age-adjusted mortality rate: AAPC=-6.73%, P<0.001, AAPC=-8.11%, P<0.001), and the COPD mortality rate for male was generally higher than female (P<0.001). Meanwhile, the mortality rate of COPD in eastern, central and western regions also showed a downward trend (AAPC=-3.87%, P<0.001; AAPC=-3.12%, P<0.001; AAPC=-1.37%, P=0.001), and western regions were significantly higher than that in central (P<0.001) and eastern (P<0.001) regions. The mortality rate of COPD in the age group of Chinese people showed a downward trend in<45, 45-59, and≥60 years groups (AAPC=-9.48%, P<0.001; AAPC=-9.03%, P<0.001; AAPC=-5.91%, P<0.001). Among them,≥60 years groups was significantly higher than that in<45 (P<0.001) and 45-59 (P<0.001) years groups, and the decline rate was slowest. Conclusion: In China, the mortality rate of COPD decreases from 2004 to 2020, and more efforts are needed to reduce COPD mortality, especially in western regions, rural populations, males and the elderly.
Aged ; Female ; Humans ; Male ; Middle Aged ; Asian People ; China/epidemiology* ; Mortality ; Pulmonary Disease, Chronic Obstructive ; Rural Population ; Urban Population ; Adult

MicroRNAs (miRNAs) are mediators of the aging process. The purpose of this work was to analyze the miRNA expression profiles of spermatozoa from men of different ages with normal fertility. Twenty-seven donors were divided into three groups by age (Group A, n = 8, age: 20-30 years; Group B, n = 10, age: 31-40 years; and Group C, n = 9, age: 41-55 years) for high-throughput sequencing analysis. Samples from 65 individuals (22, 22, and 21 in Groups A, B, and C, respectively) were used for validation by quantitative real-time polymerase chain reaction (qRT-PCR). A total of 2160 miRNAs were detected: 1223 were known, 937 were newly discovered and unnamed, of which 191 were expressed in all donors. A total of 7, 5, and 17 differentially expressed microRNAs (DEMs) were found in Group A vs B, Group B vs C, and Group A vs C comparisons, respectively. Twenty-two miRNAs were statistically correlated with age. Twelve miRNAs were identified as age-associated miRNAs, including hsa-miR-127-3p, mmu-miR-5100_L+2R-1, efu-miR-9226_L-2_1ss22GA, cgr-miR-1260_L+1, hsa-miR-652-3p_R+1, pal-miR-9993a-3p_L+2R-1, hsa-miR-7977_1ss6AG, hsa-miR-106b-3p_R-1, hsa-miR-186-5p, PC-3p-59611_111, hsa-miR-93-3p_R+1, and aeca-mir-8986a-p5_1ss1GA. There were 9165 target genes of age-associated miRNAs. Gene Ontology (GO) analysis of the target genes identified revealed enrichment of protein binding, membrane, cell cycle, and so on. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of age-related miRNAs for target genes revealed 139 enriched pathways, such as signaling pathways regulating stem cell pluripotency, metabolic pathways, and the Hippo signaling pathway. This suggests that miRNAs play a key role in male fertility changes with increasing age and provides new evidence for the study of the mechanism of age-related male fertility decline.
Humans ; Male ; Young Adult ; Adult ; Middle Aged ; MicroRNAs/genetics* ; Signal Transduction/genetics* ; Spermatozoa/metabolism* ; Gene Expression Profiling

Humans ; Ependymoma/secondary* ; Brain Neoplasms/pathology* ; Skin Neoplasms ; Melanoma ; Spinal Cord Neoplasms/pathology*