1.Establishment and Evaluation of New Mouse Model of Rheumatoid Arthritis Combined with Interstitial Lung Disease
Liting XU ; Qingyu ZHAO ; Chao YANG ; Lianhua HE ; Congcong SUN ; Shuangrong GAO ; Lili WANG ; Chunfang LIU ; Na LIN
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(6):81-90
ObjectiveTo establish a mouse model of rheumatoid arthritis with interstitial lung disease (RA-ILD) in DBA/1 mice using Porphyromonas gingivalis (Pg) infection combined with collagen-induced arthritis (CIA), and to comprehensively evaluate pathological characteristics in joints, lungs, and serum. MethodsForty DBA/1 mice were randomly divided into four groups, i.e., Control, Pg infection (Pg), CIA, and Pg infection combined with CIA (Pg+CIA), with 10 mice in each group. Arthritis clinical symptoms were evaluated by recording arthritis incidence and clinical scores. Micro-CT scanning was used to assess knee joint pathology. Histopathological changes and collagen deposition in knee joints and lung tissues were analyzed using hematoxylin-eosin (HE) and Masson staining. Immunohistochemistry was performed to detect protein expression of α-smooth muscle actin (α-SMA), typeⅠ collagen (ColⅠ), and fibronectin (FN) in lung tissues. Real-time quantitative polymerase chain reaction(Real-time PCR)was used to measure mRNA expression levels of α-SMA, ColⅠ, FN, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β in lung tissues. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of Pg, cyclic citrullinated peptide (CCP), and immunoglobulin G (IgG). ResultsJoint lesions: The CIA and Pg+CIA groups showed 100% arthritis incidence, with evident joint redness, swelling, and deformity. The number of affected limbs was 27 and 28, and clinical scores were 68 and 70, respectively. No obvious clinical symptoms were observed in the Pg group. Histopathological and imaging analyses showed severe joint lesions in the CIA and Pg+CIA groups, with significantly increased histopathological scores, bone mineral density, bone volume fraction, trabecular thickness, and trabecular number compared to the Control group (P<0.01). No obvious joint pathology was observed in the Pg group. Lung lesions: The Pg+CIA group exhibited marked alveolar inflammation, interstitial inflammatory cell infiltration, and alveolar wall thickening, with pronounced blue staining of collagen fibers. Histopathological scores and collagen area ratios were significantly higher than those of the Control, Pg, and CIA groups (P<0.05). Lung protein and mRNA expression levels of α-SMA, ColⅠ, and FN were markedly increased, and mRNA levels of IL-6, TNF-α, and IL-1β were significantly elevated compared to the Control group (P<0.05). Serology: The Pg+CIA group showed significantly higher levels of CCP, Pg, and IgG compared with the Control, Pg, and CIA groups (P<0.05). ConclusionDBA/1 mice subjected to Pg infection combined with CIA exhibited pronounced symptoms and pathological features of RA-ILD, along with elevated serum anti-CCP antibody levels. This model represents a novel RA-ILD mouse model, providing a valuable experimental tool for investigating RA-ILD pathogenesis and developing new therapeutics, and serves as a basis for establishing anti-cyclic citrullinated peptide antibody (ACPA)-positive RA-ILD animal models.
2.Indoor environment management and CO 2 volume concentration of primary and secondary school classrooms in winter across three provinces and municipalities of China
Chinese Journal of School Health 2026;47(2):163-167
Objective:
To evaluate the classroom environmental management and CO 2 volume concentration in primary and secondary schools from Liaoning, Tianjin, and Shanghai, thereby providing a scientific basis for developing targeted strategies to improve classroom air quality.
Methods:
From December 16 to 26, 2024, by using stratified random cluster sampling method, the questionnaire survey was conducted in 72 primary and secondary schools (24 each of primary, junior high, and regular high schools) across Liaoning, Tianjin and Shanghai. Information on heating, ventilation and other classroom environmental management was collected. Additionally, 108 classrooms were selected for on site microclimate measurements, including temperature, humidity, wind speed and CO 2 volume concentration. Univariate analysis and multiple linear regression models were employed to explore related factors of classroom CO 2 volume concentration.
Results:
Among the three provinces/municipalities, 20.8% of schools regularly monitored the microclimate. The overall compliance rate for classroom CO 2 volume concentration was 17.6%. Multiple linear regression analysis showed that CO 2 volume concentration in regular and junior high school classrooms were higher than in primary school classrooms ( β=0.067, 0.046, 95%CI =0.036-0.099, 0.013-0.080); classrooms ventilated regularly in the morning and afternoon had higher CO 2 volume concentration than those ventilated during every break between classes ( β=0.043, 95%CI = 0.007- 0.080); both temperature ( β=0.010, 95%CI =0.004-0.016) and humidity ( β=0.003, 95%CI =0.002-0.004) were positively correlated with CO 2 volume concentration (all P <0.05).
Conclusions
Excessive CO 2 volume concentration in primary and secondary school classrooms is a prominent issue, and ventilation frequency is a key intervenable factor for controlling CO 2 levels. It is recommended to promote ventilation during every break between classes as a core management measure and to emphasize air quality supervision in regular high school classrooms.
3.Risk factors for suicidal ideation among unemployed callers to the Beijing psychological assistance hotline
Xingxue LI ; Zikang LIU ; Liting ZHAO ; Hong LIANG
Chinese Mental Health Journal 2025;39(10):843-848
Objective:To explore the risk factors of suicidal ideation among callers with unemployment issues to the Beijing psychological assistance hotline.Methods:Totally 802 callers from the Beijing psychological assis-tance hotline from 2020 to 2023,who identified unemployment as their primary concern.Suicidal ideation was as-sessed by directly asking callers if they had experienced suicidal thoughts within the past two weeks.General demo-graphic information was collected,along with factors related to suicidal ideation,including distress,hope,depres-sion,substance abuse,physical problems,history of previous suicide attempts,chronic life events,acute life events,history of abuse,fear of being attacked,and family or friends' history of suicide.Results:A total of 517 individuals(64.4%)reported experiencing suicidal ideation within the two weeks prior to their call.Regression analysis indi-cated that high levels of hope(OR=0.32,95%CI:0.23-0.44)were a protective factor against suicidal ideation among unemployed callers.In contrast,severe depressive symptoms(OR=2.47,95%CI:1.77-3.45),a history of previous suicide attempts(OR=1.64,95%CI:1.05-2.56),and chronic life events(OR=1.55,95%CI:1.11-2.16)were identified as significant risk factors for suicidal ideation in this group.Conclusion:A history of prior suicide attempts,chronic life events,and high levels of depression are significant risk factors for suicidal idea-tion among unemployed callers,whereas a high level of hope serves as a protective factor.
4.Experimental animal models for rheumatoid arthritis-associated interstitial lung disease
Qianqian YAN ; Lianhua HE ; Lili WANG ; Liting XU ; Aimin ZHOU ; Chunfang LIU ; Na LIN
Science of Traditional Chinese Medicine 2025;3(2):124-136
Background: Rheumatoid arthritis (RA) is a systemic inflammatory disease primarily affecting the joints of the limbs. As the disease progresses, it can involve multiple organ systems. Interstitial lung disease (ILD) is the most common pulmonary manifestation of RA. Reported animal models of RA-ILD include adjuvant-induced arthritis (AA), collagen-induced arthritis (CIA), and transgenic mouse arthritis. However, the establishment criteria and evaluation methods for these models lack uniform standards, and they fail to fully replicate the clinicopathological characteristics of RA-ILD. This limitation significantly hinders research into the pathogenesis and development of therapeutic drugs for RA-ILD. Objective: The aim of the study was to review literature in China and abroad on RA-ILD animal models, analyze current research progress, identify existing issues, and propose research recommendations. Methods: Literature searches were conducted using the English keywords “rheumatoid arthritis, interstitial lung disease, model” and the Chinese keywords “(rheumatoid arthritis), (interstitial lung disease), (model)” or “(rheumatoid arthritis), (lung interstitial lesions), (model).” The search was performed in PubMed, Web of Science, CNKI, Wanfang Data, and VIP (China Science and Technology Journal Database) for articles published before November 2024. A total of 41 articles were included. Results and conclusions: The CIA model and the CIA model combined with bleomycin are commonly used due to their similarities to the histopathology and disease manifestations of human RA-ILD. Additionally, these models have appropriate cost and modeling duration, along with a high success rate, making them preferable choices. Transgenic animal models exhibit pathological features similar to the nonspecific interstitial pneumonia subtype of human RA-ILD and are useful for studying the genetic effects on RA-ILD. However, they have drawbacks such as high economic costs, long modeling durations, and a low success rate in some cases. The AA model is easy to establish, requires a short modeling period, and has low experimental costs. However, it lacks the chronic pathological development characteristic of human RA and exhibits a degree of self-limitation in lesion progression. Among other models, the comprehensive HLA-DQ8 transgenic mouse model can be used to study the combined effects of genetic and environmental factors on RA-ILD. The collagen autoantibody-induced arthritis model combined with bleomycin has a short modeling period, but it does not align well with the disease course of RA-ILD. These established animal models provide valuable insights into the pathogenesis of RA-ILD, the identification of novel biomarkers, and the development of new therapeutic approaches. Future research should focus on identifying an animal model that better replicates the physiological and pathological changes of clinical RA-ILD while being more convenient, cost-effective, and comprehensive in reflecting disease progression.
5.Oxylipidomics Combined with Transcriptomics Reveals Mechanism of Jianpi Huogu Prescription in Treating Steroid-induced Osteonecrosis of Femoral Head in Rats
Lili WANG ; Qun LI ; Zhixing HU ; Qianqian YAN ; Liting XU ; Xiaoxiao WANG ; Chunyan ZHU ; Yanqiong ZHANG ; Weiheng CHEN ; Haijun HE ; Chunfang LIU ; Na LIN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(11):190-199
ObjectiveTo unveil the mechanism of Jianpi Huogu prescription (JPHGP) in ameliorating the dyslipidemia of steroid-induced osteonecrosis of the femur head (SONFH) by oxylipidomics combined with transcriptomics. MethodsSixty SD rats were assigned into normal, model, low-, medium-, and high-dose (2.5, 5, 10 g·kg-1, respectively) JPHGP, and Jiangushengwan (1.53 g·kg-1) groups. Lipopolysaccharide was injected into the tail vein at a dose of 20 μg·kg-1 on days 1 and 2, and methylprednisolone sodium succinate was injected at a dose of 40 mg·kg-1 into the buttock muscle on days 3 to 5. The normal group received an equal volume of normal saline. Drug administration by gavage began 4 weeks after the last injection, and samples were taken after administration for 8 weeks. Hematoxylin-eosin staining was conducted to reveal the histopathological changes of the femoral head, and the number of adipocytes, the rate of empty bone lacunae, and the trabecular area were calculated. Micro-computed tomography was used for revealing the histological and histomorphometrical changes of the femoral head. Enzyme-linked immunosorbent assay was employed to measure the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), apolipoprotein A1 (ApoA1), and apolipoprotein B (ApoB). At the same time, the femoral head was collected for oxylipidomic and transcriptomic detection. The differential metabolites and differential genes were enriched and analyzed, and the target genes regulating lipid metabolism were predicted. The predicted target proteins were further verified by molecular docking, immunohistochemistry, and Western blot. ResultsCompared with the normal group, the model group showcased thinning of the femoral head, trabecular fracture, karyopyknosis, subchondral cystic degeneration, increases in the number of adipocytes and the rate of empty bone lacunae (P<0.01), a reduction in the trabecular area (P<0.01), decreases in BMD, Tb.Th, Tb.N, and BV/TV, and increases in Tb.Sp and BS/BV (P<0.01). Compared with the model group, the JPHGP groups showed no obvious thinning of the femoral head or subchondroidal cystic degeneration. The high- and medium-dose JPHGP groups presented declines in the number of adipocytes and the rate of empty bone lacunae, an increase in the trabecular area (P<0.05, P<0.01), rises in BMD, Tb.Th, Tb.N, and BV/TV, and decreases in Tb.Sp and BS/BV (P<0.05, P<0.01). Compared with the normal group, the model group showcased raised serum levels of TG, TC, LDL, and ApoB and lowered serum levels of HDL and ApoA1 (P<0.01). Compared with the model group, the JPHGP groups had lowered serum levels of TG, TC, LDL, and ApoB (P<0.05, P<0.01) and a risen serum level of ApoA1 (P<0.05, P<0.01). Moreover, the serum level of HDL in the high-dose JPHGP group increased (P<0.01). A total of 19 different metabolites of disease set and drug set were screened out by oxylipidomics of the femoral head, and 119 core genes with restored expression were detected by transcriptomics. The enriched pathways were mainly concentrated in inflammation, lipids, apoptosis, and osteoclast differentiation. Molecular docking, immunohistochemistry, and Western blot results showed that compared with the normal group, the model group displayed increased content of 5-lipoxygenase (5-LO) and peroxisome proliferator-activated receptor γ (PPARγ) in the femoral head (P<0.01). Compared with the model group, medium- and high-dose JPHGP reduced the content of 5-LO and PPARγ (P<0.05, P<0.01). ConclusionJPHGP can restore the levels of oxidized lipid metabolites by regulating the 5-LO-PPARγ axis to treat SONFH in rats. Relevant studies provide experimental evidence for the efficacy mechanism of JPHGP in the treatment of SONFH.
6.Oxylipidomics Combined with Transcriptomics Reveals Mechanism of Jianpi Huogu Prescription in Treating Steroid-induced Osteonecrosis of Femoral Head in Rats
Lili WANG ; Qun LI ; Zhixing HU ; Qianqian YAN ; Liting XU ; Xiaoxiao WANG ; Chunyan ZHU ; Yanqiong ZHANG ; Weiheng CHEN ; Haijun HE ; Chunfang LIU ; Na LIN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(11):190-199
ObjectiveTo unveil the mechanism of Jianpi Huogu prescription (JPHGP) in ameliorating the dyslipidemia of steroid-induced osteonecrosis of the femur head (SONFH) by oxylipidomics combined with transcriptomics. MethodsSixty SD rats were assigned into normal, model, low-, medium-, and high-dose (2.5, 5, 10 g·kg-1, respectively) JPHGP, and Jiangushengwan (1.53 g·kg-1) groups. Lipopolysaccharide was injected into the tail vein at a dose of 20 μg·kg-1 on days 1 and 2, and methylprednisolone sodium succinate was injected at a dose of 40 mg·kg-1 into the buttock muscle on days 3 to 5. The normal group received an equal volume of normal saline. Drug administration by gavage began 4 weeks after the last injection, and samples were taken after administration for 8 weeks. Hematoxylin-eosin staining was conducted to reveal the histopathological changes of the femoral head, and the number of adipocytes, the rate of empty bone lacunae, and the trabecular area were calculated. Micro-computed tomography was used for revealing the histological and histomorphometrical changes of the femoral head. Enzyme-linked immunosorbent assay was employed to measure the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), apolipoprotein A1 (ApoA1), and apolipoprotein B (ApoB). At the same time, the femoral head was collected for oxylipidomic and transcriptomic detection. The differential metabolites and differential genes were enriched and analyzed, and the target genes regulating lipid metabolism were predicted. The predicted target proteins were further verified by molecular docking, immunohistochemistry, and Western blot. ResultsCompared with the normal group, the model group showcased thinning of the femoral head, trabecular fracture, karyopyknosis, subchondral cystic degeneration, increases in the number of adipocytes and the rate of empty bone lacunae (P<0.01), a reduction in the trabecular area (P<0.01), decreases in BMD, Tb.Th, Tb.N, and BV/TV, and increases in Tb.Sp and BS/BV (P<0.01). Compared with the model group, the JPHGP groups showed no obvious thinning of the femoral head or subchondroidal cystic degeneration. The high- and medium-dose JPHGP groups presented declines in the number of adipocytes and the rate of empty bone lacunae, an increase in the trabecular area (P<0.05, P<0.01), rises in BMD, Tb.Th, Tb.N, and BV/TV, and decreases in Tb.Sp and BS/BV (P<0.05, P<0.01). Compared with the normal group, the model group showcased raised serum levels of TG, TC, LDL, and ApoB and lowered serum levels of HDL and ApoA1 (P<0.01). Compared with the model group, the JPHGP groups had lowered serum levels of TG, TC, LDL, and ApoB (P<0.05, P<0.01) and a risen serum level of ApoA1 (P<0.05, P<0.01). Moreover, the serum level of HDL in the high-dose JPHGP group increased (P<0.01). A total of 19 different metabolites of disease set and drug set were screened out by oxylipidomics of the femoral head, and 119 core genes with restored expression were detected by transcriptomics. The enriched pathways were mainly concentrated in inflammation, lipids, apoptosis, and osteoclast differentiation. Molecular docking, immunohistochemistry, and Western blot results showed that compared with the normal group, the model group displayed increased content of 5-lipoxygenase (5-LO) and peroxisome proliferator-activated receptor γ (PPARγ) in the femoral head (P<0.01). Compared with the model group, medium- and high-dose JPHGP reduced the content of 5-LO and PPARγ (P<0.05, P<0.01). ConclusionJPHGP can restore the levels of oxidized lipid metabolites by regulating the 5-LO-PPARγ axis to treat SONFH in rats. Relevant studies provide experimental evidence for the efficacy mechanism of JPHGP in the treatment of SONFH.
7.Resveratrol protects against oxidative stress damage during sperm cryopreservation by mediating the NRF2 signaling pathway
Lei XIN ; Yutong ZHANG ; Liting LIU ; Jing TAN ; Jingjing ZHANG ; Zongliu DUAN ; Yang GAO ; Jun YANG ; Biao YU ; Qunshan SHEN ; Xiaojin HE
Chinese Journal of Reproduction and Contraception 2025;45(10):1038-1046
Objective:To investigate the protective effects of resveratrol (RSV) on human sperm cryopreservation and explore its underlying protective mechanisms.Methods:A total of 165 normal fresh semen samples were collected from the Reproductive Medicine Center, Department of Obstetrics and Gynecology and Human Sperm Bank of the First Affiliated Hospital of Anhui Medical University between December 2022 and December 2024. Among them, 65 samples were used to obtain semen parameters before and after conventional freezing. Each sample of the other 104 samples was mixed at a 2∶1 volume ratio with cryoprotectant containing 0, 10 -?, 10 -?, or 10 -? mol/L RSV, followed by cryopreservation in liquid nitrogen for 24 h. Post-thaw assessments included routine sperm parameters, sperm DNA fragmentation index (DFI) evaluated by sperm chromatin dispersion assay, reactive oxygen species (ROS) levels measured via flow cytometry, RSV and nuclear factor erythroid 2-related factor 2 (NRF2) interactions examined by molecular docking and cellular thermal shift assay (CETSA), NRF2 protein contents analyzed by immunofluorescence and Western blotting, mRNA levels of NRF2 and downstream antioxidant proteins Heme Oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) quantified by qRT-PCR and effects of NRF2 inhibitor ML385 on sperm parameters. Results:Compared with fresh samples, conventional cryopreservation significantly reduced sperm motility (all P<0.001). The addition of 10 -? mol/L RSV significantly improved the percentage of forward motile sperm after freezing (26.98%±8.98% vs. 19.61%±8.03%, P<0.001) while reducing DFI (9.84%±3.81% vs. 15.06%±4.22%, P<0.001) and ROS levels ( P<0.001) compared with the post-freezing group without the addition of RSV. Both molecular docking analysis and CETSA confirmed that RSV interacted with NRF2. Notably, sperm cryopreserved with 10 -? mol/L RSV exhibited significantly higher contents of NRF2 and its downstream effectors HO-1 and NQO1 compared with the post-freezing group without the addition of RSV (all P<0.001). This protective effect was markedly attenuated by co-treatment with the NRF2 inhibitor ML385, as evidenced by a significant decline in sperm motility ( P<0.001). Conclusion:RSV exerts cryoprotective effects likely through NRF2-mediated antioxidant pathways, reducing oxidative stress and enhancing post-thaw sperm quality.
8.Revealing potential mechanism of Danggui Buxue decoction in regulating immuno-suppression via PI3K-AKT signaling pathway using network pharmacology and in vivo experiment
Huan HUANG ; Pingrui YANG ; Xifeng LI ; Fuxing GUI ; Yutao LI ; Li ZHANG ; Shuo LIU ; Yufei XIE ; Hongxu DU ; Shicheng BI ; Liting CAO
Chinese Journal of Veterinary Science 2025;45(2):350-361
Danggui Buxue decoction(DBD)is a classic prescription with immunomodulatory and hematopoietic effects.Previous studies have shown the DBD has potential to be used as an oral im-mune booster.However,its immunomodulatory effects and mechanism of action have not been thoroughly studied,especially the protective mechanism of immunomodulatory regulation in the state of immunosuppressive is still unclear.The aim of this study was to explore the protective mechanism of DBD in the immunosuppressive state using network pharmacology combined with animal experiments verification.The active components,core targets and signaling pathways of DBD in treating immunosuppression were obtained using network pharmacology tools.On this ba-sis,the active components of DBD were identified using HPLC-MS,and in vivo studies were con-ducted at the same time.The key active components of DBD obtained using network pharmacology included quercetin,kaempferol and formononetin.The core targets included TP53,RELA,TNF,AKT1,and IL-6.KEGG pathway enrichment analysis showed that phosphoinositide 3-kinase(PI3K)-protein kinase B(AKT)may play an important role in the treatment of immunosuppres-sive diseases using DBD.Molecular docking confirmed that each core target had good binding activ-ity with its corresponding compounds.Animal experiments showed that after DBD intervention,the mRNA gene and protein expression of RELA,TNF,and IL-6 in the serum was significantly down-regulated.The mRNA expression of PI3K and AKT in the ileum and PI3K protein expression were also downregulated.In conclusion,DBD exerts its role in treating immunosuppressive diseases by regulating the PI3K-AKT signaling pathway.
9.Epidemiological study on traditional Chinese medicine treatment for inflammatory bowel disease in Jiangsu Province from 2019 to 2023
Chujun NI ; Zexing LIN ; Haiyang JIANG ; Jie WU ; Peizhao LIU ; Jiaqi KANG ; Chengliang QIAN ; Haiqing LIU ; Liting DENG ; Huan YANG ; Chenling WU ; Yun ZHAO
Chinese Journal of Inflammatory Bowel Diseases 2025;09(4):318-325
Objective:To explore patterns of traditional Chinese medicine (TCM) use among patients with inflammatory bowel disease (IBD) in Jiangsu Province, China from 2019 to 2023.Methods:Using data from the IBD health data platform of the National Healthcare Big Data (Eastern) Center, a retrospective cohort study was conducted. We performed descriptive analyses on hospitalised patients diagnosed with IBD between 2019 and 2023, who received TCM treatment.Results:The study included 11 095 case records from 4 760 patients, with TCM diagnoses primarily indicating diarrhoea and abdominal pain. Ulcerative colitis (UC) accounted for 4 782 hospitalizations (3 103 patients), while Crohn's disease (CD) accounted for 6 313 hospitalizations (1 657 patients). Patient demographics showed a trend towards younger age and a higher proportion of males. Treatment utilisation was highest in southern Jiangsu compared with the central and northern regions. In terms of disease burden, all treatment costs showed a downward trend. In terms of external TCM therapies, UC patients tend to prefer plasters and enemas, while CD patients are more inclined to use acupuncture. Regarding herbal medicine, licorice, white atractylodes, and white peony root are commonly used single herbs for IBD patients.Conclusions:The number of IBD patients treated with TCM in Jiangsu Province has steadily increased from 2019 to 2023. It is important to identify effective TCM treatment methods to reduce the burden of patients.
10.Resveratrol protects against oxidative stress damage during sperm cryopreservation by mediating the NRF2 signaling pathway
Lei XIN ; Yutong ZHANG ; Liting LIU ; Jing TAN ; Jingjing ZHANG ; Zongliu DUAN ; Yang GAO ; Jun YANG ; Biao YU ; Qunshan SHEN ; Xiaojin HE
Chinese Journal of Reproduction and Contraception 2025;45(10):1038-1046
Objective:To investigate the protective effects of resveratrol (RSV) on human sperm cryopreservation and explore its underlying protective mechanisms.Methods:A total of 165 normal fresh semen samples were collected from the Reproductive Medicine Center, Department of Obstetrics and Gynecology and Human Sperm Bank of the First Affiliated Hospital of Anhui Medical University between December 2022 and December 2024. Among them, 65 samples were used to obtain semen parameters before and after conventional freezing. Each sample of the other 104 samples was mixed at a 2∶1 volume ratio with cryoprotectant containing 0, 10 -?, 10 -?, or 10 -? mol/L RSV, followed by cryopreservation in liquid nitrogen for 24 h. Post-thaw assessments included routine sperm parameters, sperm DNA fragmentation index (DFI) evaluated by sperm chromatin dispersion assay, reactive oxygen species (ROS) levels measured via flow cytometry, RSV and nuclear factor erythroid 2-related factor 2 (NRF2) interactions examined by molecular docking and cellular thermal shift assay (CETSA), NRF2 protein contents analyzed by immunofluorescence and Western blotting, mRNA levels of NRF2 and downstream antioxidant proteins Heme Oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) quantified by qRT-PCR and effects of NRF2 inhibitor ML385 on sperm parameters. Results:Compared with fresh samples, conventional cryopreservation significantly reduced sperm motility (all P<0.001). The addition of 10 -? mol/L RSV significantly improved the percentage of forward motile sperm after freezing (26.98%±8.98% vs. 19.61%±8.03%, P<0.001) while reducing DFI (9.84%±3.81% vs. 15.06%±4.22%, P<0.001) and ROS levels ( P<0.001) compared with the post-freezing group without the addition of RSV. Both molecular docking analysis and CETSA confirmed that RSV interacted with NRF2. Notably, sperm cryopreserved with 10 -? mol/L RSV exhibited significantly higher contents of NRF2 and its downstream effectors HO-1 and NQO1 compared with the post-freezing group without the addition of RSV (all P<0.001). This protective effect was markedly attenuated by co-treatment with the NRF2 inhibitor ML385, as evidenced by a significant decline in sperm motility ( P<0.001). Conclusion:RSV exerts cryoprotective effects likely through NRF2-mediated antioxidant pathways, reducing oxidative stress and enhancing post-thaw sperm quality.


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