1.Association between exposure to heatwave and sudden death among residents in Jiangsu Province,China
Changkui OU ; Yanling ZHONG ; Rui LI ; Yi LIN ; Ruijun XU ; Tingting LIU ; Tingting WANG ; Hong SUN ; Yuewei LIU
Journal of Public Health and Preventive Medicine 2026;37(1):22-28
Objective To quantitatively assess the exposure-response association between exposure to heatwave and sudden death, estimate the attributable excess deaths, and identify potential vulnerable subgroups. Methods A time-stratified case-crossover study was conducted among residents who died from sudden death in Jiangsu Province, China between 2015 and 2021. Heatwave events in Jiangsu Province, defined using varying relative temperature thresholds and durations, were identified using temperature data from the China Meteorological Administration Land Data Assimilation System (CLDAS V2.0). Individual heatwave exposure was assessed based on each subject's residential address. The exposure-response association between heatwave and sudden death was evaluated using conditional logistic regression model combined with a Distributed Lag Nonlinear Model(DLNM). Heatwave-attributable excess deaths were estimated. Stratified analyses by sex and age were performed to assess potential effect modifications. Results Under all definitions, exposure to heatwave was significantly associated with an increased risk of sudden death, and the risk increased with the intensity of heatwave. Using the P95_3d definition (temperature exceeding the 95th percentile for ≥3 consecutive days), heatwave was significantlyassociated with a 56% increased risk of sudden death (95% CI: 31%, 86%). The population-attributable fraction of sudden death due to heatwave exposure was 1.45% (95% CI: 0.97%, 1.90%). Stratified analyses indicated no statistically significant differences in the association between heatwave exposure and sudden death across age or sex subgroups. Conclusion Heatwave exposure was associated with an increased risk of sudden death. Reducing heatwave exposure during summer may help lower the occurrence of sudden death.
2.Comparative Study on Effect of Jingui Shenqiwan and Liuwei Dihuangwan on Reproductive Ability and Brain Function of Normal Mice
Hong SUN ; Fan LEI ; Chenggong LI ; Rui LUO ; Shixian HU ; Bin REN ; Juan HAO ; Yi DING ; Lijun DU
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):1-14
ObjectiveTo explore the effects of Jingui Shenqiwan (JSW) and Liuwei Dihuangwan (LDW) on the reproductive ability and brain function of normal mice and compare the actions of the two medications. MethodsSeven groups of female and male mice were divided at a ratio of 2∶1. Except for the control group, the other six groups were as follows: a group of both males and females receiving JSW (3.0 g·kg-1), a group of both males and females receiving LDW (4.5 g·kg-1), a group of males receiving water and females receiving JSW, a group of males receiving water while females receiving LDW, a group of females receiving water while males receiving JSW, and a group of females receiving water while males receiving LDW. Each group was administered the drug for 14 days and then caged together at a 2∶1 (female∶male) ratio to detect the number of pregnant mice and calculate the pregnancy rate. Pregnant mice continued receiving the drug until they naturally gave birth, which was followed by the observation of newborn mice, calculation of their average number, and the measurement of the offspring's preference for sugar water and neonatal recognition index. At the end of the experiment, the weights of the thymus and spleen were measured to calculate the organ coefficients, and mRNA or protein expression was analyzed in the brain and testes or ovaries. A 1% sucrose solution was used to examine the euphoria of their brain reward systems, while novel object recognition test (NOR) was applied to assess their memory capabilities. mRNA expression was detected using real-time quantitative polymerase chain reaction (Real-time PCR) assay, and protein expression was analyzed with Western blot. ResultsCompared with the control group, oral administration of JSW to both male and female mice for 14 days significantly increased the pregnancy rate of female mice on day 2 after being caged together (P<0.05), while LDW showed a trend but no statistical significance. Additionally, compared with the control group, JSW could upregulate the gene expression of gonadotropin-releasing hormone (GnRH) in the thalamus, as well as reproductive stem cell factor (SCF) and tyrosine kinase receptor (c-Kit) in the testes and reproductive stem cell marker mouse vasa homologue (MVH) in the ovaries, upregulate the expression of proteins influencing neuronal functional activity, such as brain-derived neurotrophic factor (BDNF), in hippocampal neurons (P<0.05), and enhance sucrose preference in male mice (P<0.05). Compared with the control group, JSW significantly increased sucrose preference and novel object recognition index in offspring mice (P<0.05), which was related to the upregulation of hippocampal dopamine D1 receptor (D1R) and N-methyl-D-aspartate receptor (Nmdar) gene expression. Compared with the control group, both JSW and LDW could upregulate the protein expression of glucocorticoid receptor (GR), BDNF, and tyrosine kinase receptor B (TrkB) in the hippocampus of offspring mice (P<0.05). ConclusionJSW significantly enhances the reproductive ability of normal mice, which is not only related to the release of gonadotropin but also associated with its regulation of brain function. Additionally, JSW has a certain regulatory effect on the brain function of the offspring mice.
3.Comparison between ultrafiltration and dextran gel method in the purification of Tfn/PCL micelles
Lingbo YU ; Yadong ZHANG ; Rui XU ; Yuyu SUN ; Huiyun WANG ; Jinjin YANG ; Yanan CUI
Acta Universitatis Medicinalis Anhui 2026;61(2):258-263
ObjectiveTo compare the differences between the ultrafiltration method and the dextran gel filtration method during the purification of Tfn-modified PCL micelles by using purification efficiency and micelle purity as indicators. MethodsCoumarin-6 (C6) was used as a fluorescent probe and was loaded into HOOC-PEG-PCL to form PCL micelles by the film-dispersion method. Tfn was then conjugated to the surface of PCL micelles via an amidation reaction, resulting in two types of micelles: Tfn/PCLH and Tfn/PCLL. The pharmaceutical properties of the two types of micelles were characterized. The micelles were then purified through ultrafiltration method and dextran gel method respectively, and the efficiency of the two methods, along with the purity of the final micelles, was compared. The density of Tfn on the surface of PCL micelles was also calculated. ResultsThe hydrated diameter of PCL micelles was approximately 73 nm, and the C6 loading efficiency was around 0.046%. The size increased to 134 nm and 158 nm for Tfn/PCLL and Tfn/PCLH, respectively. The micelle population was monodisperse. The purification results showed that, for the ultrafiltration method, after two and one rounds of purification, the Tfn/C6 ratio stabilized at 23.6 and 3.4 for Tfn/PCLH and Tfn/PCLL, respectively. For the dextran gel filtration method, the Tfn/C6 ratio reached 23.7 for the Tfn/PCLH group after two rounds of purification. However, for the Tfn/PCLL group, the Tfn/C6 ratio increased during four rounds of dextran gel purification, and a significant difference (P = 0.042 4) was observed between the first and last filtrations. The density of Tfn in the final micelles were calculated. For the ultrafiltration method, the Tfn density of Tfn/PCLH and Tfn/PCLL were 94.9% and 13.8%, respectively. For the dextran gel filtration method, the density of the two micelles were 95.6% and 14.4%, respectively. For Tfn/PCLL group, the density results revealing a statistically significant difference (P=0.000 2). ConclusionThe purification efficiency of the two methods is comparable. However, the purity of the final micelles shows a significant difference, with the dextran gel filtration method resulting in higher purity, particularly for the Tfn/PCLL micelles.
4.Successful treatment of extracorporeal membrane oxygenation bridging to lung transplantation in a patient with rapidly progressive interstitial lung disease
Yi GONG ; Xinyu LING ; Rui YAN ; Bo SUN ; Ke MA ; Guifang WANG ; Chang CHEN
Chinese Journal of Clinical Medicine 2026;33(1):154-159
A 42-year-old male with chest tightness and dyspnea was admitted to the hospital. Chest CT indicated diffuse interstitial lung infiltration. Despite receiving anti-infective therapy, glucocorticoid therapy, and immunosuppressive agents, the patient developed refractory hypoxaemia. Endotracheal intubation and invasive mechanical ventilation failed to improve oxygenation. Therefore the patient was diagnosed with rapidly progressive interstitial lung disease (RP-ILD) accompanied by type Ⅰ respiratory failure. Veno-venous (VV) extracorporeal membrane oxygenation (ECMO) was initiated, and oxygenation improved in this patient. The patient subsequently underwent bilateral lung transplantation with veno-arterio-venous (VAV) ECMO support. ECMO machine was withdrawn on day 1, and extubation was achieved on day 9 after surgery. Histopathology revealed fibrotic nonspecific interstitial pneumonia (NSIP) with hyaline membrane formation. The patient developed ICU-acquired myasthenia and received early rehabilitation, with gradual recovery of muscle strength. During follow-up, graft lung function remained stable. This case demonstrates that ECMO can serve as a bridge to lung transplantation in RP-ILD patients.
5.Neuroprotective Effects of Transcranial Magneto-acoustic Stimulation on Parkinson’s Disease Model Mice by Regulating Mitophagy and Mitochondrial Homeostasis
Shuai ZHANG ; Yan-Bin WANG ; Yi-Hao XU ; Jin-Rui MI ; Xiao-Chao LU ; Yu-Chen AN ; Ji-Zhou LIU ; Jia-Qi SUN
Progress in Biochemistry and Biophysics 2026;53(5):1457-1470
ObjectiveTranscranial magneto-acoustic stimulation (TMAS) is an emerging non-invasive neuromodulation technique that may provide a novel non-pharmacological intervention strategy for Parkinson's disease (PD). PD is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), leading to motor impairments such as bradykinesia, tremor, and rigidity. Increasing evidence indicates that mitochondrial dysfunction and impaired mitochondrial quality control are central mechanisms underlying dopaminergic neuronal loss. In particular, abnormalities in mitophagy and mitochondrial fission-fusion balance contribute substantially to oxidative stress, energy metabolic failure, and neuronal injury. At present, most clinical treatments for PD mainly alleviate symptoms but do not effectively halt disease progression. Therefore, exploring new interventions targeting the core pathological mechanisms is of considerable significance. This study aims to investigate whether TMAS can improve neural damage and motor dysfunction in PD mice by regulating mitophagy and the fission/fusion dynamic balance, thereby providing theoretical and experimental support for its application in PD treatment. MethodsMale C57BL/6 mice were used in this study. A PD model was established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 consecutive days. After model induction, mice in the intervention group received TMAS once daily for 14 consecutive days, whereas the corresponding control group received sham stimulation. The stimulation target was positioned over the primary motor cortex (M1). Motor performance was evaluated using the pole test and the open-field test. To verify the activation effect of TMAS on the target cortical region, c-Fos immunohistochemistry was performed in the M1. To assess nigral dopaminergic neuronal injury, tyrosine hydroxylase (TH) immunohistochemistry was used to quantify TH-positive neurons in the SNc. Mitochondrial function was evaluated by measuring reactive oxygen species (ROS) levels and adenosine triphosphate (ATP) content in the SNc. Western blot was further performed to determine the expression of mitophagy-related proteins, including PINK1, Parkin, LC3-II, and p62, as well as mitochondrial dynamics-related proteins, including Drp1 and Opa1. ResultsTMAS significantly increased the number of c-Fos-positive cells in M1 (P<0.000 1), indicating effective activation of neurons in the targeted cortical region. Compared with the control group, MPTP-treated mice exhibited marked motor dysfunction, including a significant reduction in total distance traveled in the open-field test (P<0.000 1) and mean speed (P=0.000 1), as well as significant prolongation of turn time and total climbing time in the pole test (P<0.000 1). These behavioral impairments were accompanied by a substantial loss of TH-positive dopaminergic neurons in the SNc, whereas TMAS significantly increased TH-positive neuron survival (P<0.000 1). In parallel, MPTP induced a pronounced increase in ROS levels and a significant reduction in ATP content, indicating severe mitochondrial dysfunction and energy metabolism impairment (P<0.01). TMAS treatment significantly improved motor performance, as reflected by the reversal of MPTP-induced impairment in the open-field and pole tests, and significantly reduced ROS accumulation (P<0.01) while restoring ATP production (P<0.001). At the molecular level, MPTP markedly downregulated PINK1 and Parkin, decreased p62 expression, increased LC3-II accumulation, elevated Drp1 expression, and reduced Opa1 expression, whereas TMAS significantly reversed these abnormalities, suggesting restoration of mitophagy-related mitochondrial quality control and re-establishment of mitochondrial fission-fusion balance. Collectively, these findings indicate that TMAS ameliorates MPTP-induced neurotoxicity and restores mitochondrial homeostasis and energy metabolism. ConclusionTMAS effectively attenuates neural damage and improves motor dysfunction in MPTP-induced PD mice. Its neuroprotective effects are closely associated with multidimensional regulation of the mitochondrial quality control system, including restoration of PINK1/Parkin-mediated mitophagy and rebalancing of Drp1/Opa1-related mitochondrial dynamics. Rather than acting only as a symptomatic neuromodulatory intervention, TMAS may influence a key pathological axis of PD by improving mitochondrial homeostasis in SNc and protecting nigral dopaminergic neurons. These findings provide experimental evidence supporting TMAS as a promising non-invasive physical intervention for PD.
6.Neuroprotective Effects of Transcranial Magneto-acoustic Stimulation on Parkinson’s Disease Model Mice by Regulating Mitophagy and Mitochondrial Homeostasis
Shuai ZHANG ; Yan-Bin WANG ; Yi-Hao XU ; Jin-Rui MI ; Xiao-Chao LU ; Yu-Chen AN ; Ji-Zhou LIU ; Jia-Qi SUN
Progress in Biochemistry and Biophysics 2026;53(5):1457-1470
ObjectiveTranscranial magneto-acoustic stimulation (TMAS) is an emerging non-invasive neuromodulation technique that may provide a novel non-pharmacological intervention strategy for Parkinson's disease (PD). PD is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), leading to motor impairments such as bradykinesia, tremor, and rigidity. Increasing evidence indicates that mitochondrial dysfunction and impaired mitochondrial quality control are central mechanisms underlying dopaminergic neuronal loss. In particular, abnormalities in mitophagy and mitochondrial fission-fusion balance contribute substantially to oxidative stress, energy metabolic failure, and neuronal injury. At present, most clinical treatments for PD mainly alleviate symptoms but do not effectively halt disease progression. Therefore, exploring new interventions targeting the core pathological mechanisms is of considerable significance. This study aims to investigate whether TMAS can improve neural damage and motor dysfunction in PD mice by regulating mitophagy and the fission/fusion dynamic balance, thereby providing theoretical and experimental support for its application in PD treatment. MethodsMale C57BL/6 mice were used in this study. A PD model was established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 consecutive days. After model induction, mice in the intervention group received TMAS once daily for 14 consecutive days, whereas the corresponding control group received sham stimulation. The stimulation target was positioned over the primary motor cortex (M1). Motor performance was evaluated using the pole test and the open-field test. To verify the activation effect of TMAS on the target cortical region, c-Fos immunohistochemistry was performed in the M1. To assess nigral dopaminergic neuronal injury, tyrosine hydroxylase (TH) immunohistochemistry was used to quantify TH-positive neurons in the SNc. Mitochondrial function was evaluated by measuring reactive oxygen species (ROS) levels and adenosine triphosphate (ATP) content in the SNc. Western blot was further performed to determine the expression of mitophagy-related proteins, including PINK1, Parkin, LC3-II, and p62, as well as mitochondrial dynamics-related proteins, including Drp1 and Opa1. ResultsTMAS significantly increased the number of c-Fos-positive cells in M1 (P<0.000 1), indicating effective activation of neurons in the targeted cortical region. Compared with the control group, MPTP-treated mice exhibited marked motor dysfunction, including a significant reduction in total distance traveled in the open-field test (P<0.000 1) and mean speed (P=0.000 1), as well as significant prolongation of turn time and total climbing time in the pole test (P<0.000 1). These behavioral impairments were accompanied by a substantial loss of TH-positive dopaminergic neurons in the SNc, whereas TMAS significantly increased TH-positive neuron survival (P<0.000 1). In parallel, MPTP induced a pronounced increase in ROS levels and a significant reduction in ATP content, indicating severe mitochondrial dysfunction and energy metabolism impairment (P<0.01). TMAS treatment significantly improved motor performance, as reflected by the reversal of MPTP-induced impairment in the open-field and pole tests, and significantly reduced ROS accumulation (P<0.01) while restoring ATP production (P<0.001). At the molecular level, MPTP markedly downregulated PINK1 and Parkin, decreased p62 expression, increased LC3-II accumulation, elevated Drp1 expression, and reduced Opa1 expression, whereas TMAS significantly reversed these abnormalities, suggesting restoration of mitophagy-related mitochondrial quality control and re-establishment of mitochondrial fission-fusion balance. Collectively, these findings indicate that TMAS ameliorates MPTP-induced neurotoxicity and restores mitochondrial homeostasis and energy metabolism. ConclusionTMAS effectively attenuates neural damage and improves motor dysfunction in MPTP-induced PD mice. Its neuroprotective effects are closely associated with multidimensional regulation of the mitochondrial quality control system, including restoration of PINK1/Parkin-mediated mitophagy and rebalancing of Drp1/Opa1-related mitochondrial dynamics. Rather than acting only as a symptomatic neuromodulatory intervention, TMAS may influence a key pathological axis of PD by improving mitochondrial homeostasis in SNc and protecting nigral dopaminergic neurons. These findings provide experimental evidence supporting TMAS as a promising non-invasive physical intervention for PD.
7.Effect of Shenge Bushen Capsules and Its Polysaccharides and Flavonoids on Precocious Puberty in Young Mice
Hong SUN ; Fan LEI ; Chenggong LI ; Shixian HU ; Weihua WANG ; Bin REN ; Juan HAO ; Rui LUO ; Lijun DU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(1):95-103
ObjectiveTo explore the effect of Shenge Bushen Capsules (SBC) on sexual development in normal 3-week-old mice. MethodsThe experiment consisted of two parts. In the first part, mice were divided into four groups: The control group and the low, medium, and high-dose SBC groups (234.7, 469.4, 938.7 mg·kg-1, respectively). In the second part, mice were divided into four groups: Control group, Pseudostellariae Radix polysaccharide (PRP) group, total flavonoids group, and SBC group, all receiving a dose of 469.4 mg·kg-1. After 7 days of administration, the vaginal opening of female mice and the descent of testes and scrotum in male mice, as well as the ovarian and testicular organ indices, were observed. After 4 weeks of administration, female and male mice were housed together for 2 days, and the pregnancy rate of females was monitored. After delivery, the pregnant female mice continued receiving the treatment for 4 weeks, and the sexual development of their offspring, including vaginal opening, testicular descent, and organ indices of ovaries and testes, was observed. Serum sex hormones were measured by enzyme-linked immunosorbent assay (ELISA), and the expression of gonadotropin-releasing hormone (GnRH) and growth hormone (GH) proteins in the hypothalamus was assessed by Western blot. ResultsCompared with the control group, there was no significant effect on the vaginal opening of female mice or the descent of testes in male mice after 7 days of SBC administration. After 4 weeks of administration, the pregnancy rate in the low-dose group was significantly reduced (P<0.05), but no significant effects were observed in the other groups. The three doses of SBC did not significantly affect the ovarian or testicular organ indices, and there was no significant upregulation in the expression of GnRH or GH in the hypothalamus. The primary component of SBC, Pseudostellariae Radix polysaccharide, significantly reduced the vaginal opening in female mice after 7 days of administration (P<0.05). After 4 weeks, the serum estradiol levels of non-pregnant female mice were decreased (P<0.05), but there was no significant effect on the expression of GnRH or GH proteins in the hypothalamus of either male or female mice. Additionally, there were no significant effects on precocious puberty indicators, such as vaginal opening and testicular descent, in the offspring mice. ConclusionSBC does not significantly promote precocious puberty in young mice, and it does not have any noticeable effects on the pregnancy rate of adult mice or the sexual development of their offspring.
8.Effect of Rhei Radix et Rhizoma Before and After Steaming with Wine on Intestinal Flora and Immune Environment in Constipation Model Mice
Yaya BAI ; Rui TIAN ; Yajun SHI ; Chongbo ZHAO ; Jing SUN ; Li ZHANG ; Yonggang YAN ; Yuping TANG ; Qiao ZHANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(2):192-199
ObjectiveTo study on the different therapeutic effects and potential mechanisms of Rhei Radix et Rhizoma(RH) before and after steaming with wine on constipation model mice. MethodsFifty-four male ICR mice were randomly divided into control group, model group, lactulose group(1.5 mg·kg-1), high, medium and low dose groups of RH and RH steaming with wine(PRH)(8, 4, 1 g·kg-1). Except for the control group, the constipation model was replicated by gavage of loperamide hydrochloride(6 mg·kg-1) in the other groups. After 2 weeks of modeling, each administration group was gavaged with the corresponding dose of drug solution, and the control and model groups were given an equal volume of normal saline, 1 time/d for 2 consecutive weeks. After administration, the feces were collected for 16S rRNA sequencing, the levels of gastrin(GAS), motilin(MTL), interleukin-6(IL-6), γ-interferon(IFN-γ) in the colonic tissue were detected by enzyme-linked immunosorbent assay(ELISA), the histopathological changes of colon were observed by hematoxylin-eosin(HE) staining, flow cytometry was used to detect the proportion changes of CD4+, CD8+ and regulatory T cell(Treg) in peripheral blood. ResultsCompared with the control group, the model group showed significantly decrease in fecal number in 24 h, fecal quality and fecal water rate(P<0.01), the colon was seen to have necrotic shedding of mucosal epithelium, localized intestinal glands in the lamina propria were degenerated, necrotic and atrophied, a few lymphocytes were seen to infiltrate in the necrotic area in a scattered manner, the contents of GAS and MTL, the proportions of CD4+, CD8+ and Treg were significantly reduced(P<0.01), the contents of IL-6 and IFN-γ were significantly elevated(P<0.05, P<0.01). Compared with the model group, the fecal number in 24 h, fecal quality and fecal water rate of high-dose groups of RH and PRH were significantly increased(P<0.05, P<0.01), the pathological damage of the colon was alleviated to varying degrees, the contents of GAS, MTL, IL-6 and IFN-γ were significantly regressed(P<0.05, P<0.01), and the proportions of CD4+ and CD8+ were significantly increased(P<0.01), although the proportion of Treg showed an upward trend, there was no significant difference. In addition, the results of intestinal flora showed that the number of amplicon sequence variant(ASV) and Alpha diversity were decreased in the model group compared with the control group, and there was a significant difference in Beta diversity, with a decrease in the relative abundance of Lactobacillus and an increase in the relative abundances of Bacillus and Helicobacter. Compared with the model group, the ASV number and Alpha diversity were increased in the high-dose groups of RH and PRH, and there was a trend of regression of Beta diversity to the control group, the relative abundance of Lactobacillus increased, and the relative abundances of Bacillus and Helicobacter decreased. ConclusionRH and PRH can improve dysbacteriosis, promote immune system activation, inhibit the release of inflammatory factors for enhancing the gastrointestinal function, which may be one of the potential mechanisms of their therapeutic effect on constipation.
9.Peripubertal DEHP exposure impairs reproductive function in adult male mice by inducing damage to Sertoli cells and disrupting the blood-testis barrier
Guiyong XU ; Lu ZHANG ; Fengqiong SUN ; Rui YANG ; Ziyuan ZHOU ; Guanghong YANG
Journal of Army Medical University 2025;47(15):1695-1707
Objective To investigate the mechanism by which peripubertal exposure to di-2-ethylhexyl phthalate(DEHP)induces reproductive injury in adult male mice through impairment of Sertoli cell function and the blood-testis barrier.Methods A total of 180 male C57BL/6J mice(3 weeks old)were randomly divided into corn oil group(control group),and 5,25,125,250 and 500 mg/kg DEHP exposure groups.All groups received daily oral gavage for 5 consecutive weeks.Ten mice from each group were sacrificed for first material collection when they were 9 weeks old.The remaining F0 generation mice were mated with unexposed adult female mice at a male-to-female ratio of 1∶2 at 14 weeks of age for a period of 2 weeks.The second material collection was completed at 16 weeks of age,with 20 mice taken from each group.A computer-assisted analysis system was used to detect sperm density and motility in 9-week-old and 16-week-old male mice.The average birth body weight of F1 generation pups,survival rate of pups at 4 d after birth per litter,number of pups per litter,and male-to-female sex ratio within the litter were recorded.ELISA was used to detect hormone levels in the serum.Sperm abnormalities were observed using eosin staining.HE staining was used to observe pathological changes in testicular tissue.Immunofluorescence assay was utilized to detect the protein expression of tight junction protein 1(zonula occludens-1,ZO-1)and gap junction protein 43(connexin43,Cx43)related to the blood-testis barrier in testicular tissue,as well as the protein expression of apoptosis-related molecule Cleaved-Caspase3.Immunohistochemistry was used to detect the expression of Sertoli cell marker SOX9 protein in testicular tissue.TUNEL was used to detect apoptosis in testicular tissue.Comparative toxicogenomics database(CTD)was used to screen for mono-2-ethylhexyl phthalate(MEHP)induced reproductive damage-associated genes and combined with GO/KEGG and Reactome enrichment analysis to predict possible signaling pathways.An in vitro exposure model was established by treating TM4 cells with MEHP at concentrations of 0,50,100,200 and 400 μmol/L,respectively.CCK-8 assay,flow cytometry and Western blotting were applied to detect the cell viability,apoptosis,and protein expression of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)and Bcl-2-associated X protein(Bax),and blood-testis barrier-related proteins ZO-1 and Cx43.Results Compared with the control group,the pregnancy rate was decreased from 80.0%to 52.5%,and the total number of pups was decreased from 212 to 125 in the female mice mating with the male from the 500 mg/kg group.Compared with the control group,the sperm density and motility were significantly reduced,and the total rate of sperm abnormalities was obviously increased in the 9-week-old DEHP-exposed mice(P<0.05),and even in the 16-week-old mice,the sperm motility was still in a dose-dependent downward trend(P<0.05).In the 25,125,250 and 500 mg/kg DEHP exposure groups,the thickness of the seminiferous epithelium was obviously decreased,with more cellular vacuolization and notable epithelial atrophy and degeneration.Cell apoptosis in the testicular tissue was enhanced,while the expression of ZO-1 and Cx43 were decreased in the 250 and 500 mg/kg exposure groups.The levels of androgen-binding protein,gonadotropin-releasing hormone,and testosterone in the serum were decreased in a dose-dependent manner(P<0.05),while those of luteinizing hormone and follicle-stimulating hormone showed a dose-dependent increase(P<0.05).Bioinformatics analysis showed that 153 genes related to male system diseases were screened out by CTD for MEHP.GO enrichment,KEGG pathway,and Reactome enrichment analyses displayed that the apoptosis signaling pathway was significantly enriched.CCK-8 assay showed that TM4 cell viability was decreased in a dose-dependent manner after MEHP treatment when compared with the control group(P<0.05).Western blotting indicated that MEHP treatment reduced the expression levels of Bcl-2,ZO-1 and Cx43(P<0.05),and enhanced that of Bax in comparison with the control group(P<0.05).Conclusion Peripubertal DEHP exposure may lead to reduced sperm quality and reproductive damage in adult male mice by disrupting the blood-testis barrier and activating Sertoli cell apoptosis.
10.Determination of 71 Kinds of Per-and Polyfluoroalkyl Substances in Eckloniae Thallus by QuEChERS-Liquid Chromatography-Tandem Mass Spectrometry
Dan-Rui TANG ; Jing SUN ; Ling CAO ; Dan-Yi JIANG ; Hai-Wei SHI ; Lu-Lu WU
Chinese Journal of Analytical Chemistry 2025;53(7):1177-1185,中插9-中插25
A QuEChERS cleanup-ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method was established for simultaneous determination of traditional and emerging 71 kinds of per-and polyfluoroalkyl substances(PFAS)in Chinese herbal medicine Eckloniae Thallus.The extraction solvent employed here was 0.2%formic acid-acetonitrile with sodium chloride-anhydrous sodium sulfate(3:1,m/m)as salting-out agent,and the purification was carried out using graphitized carbon black and octadecyl-bonded silica as adsorbents.The sample separation was carried out on a Horizon C18 chromatographic column(150 mm×2.1 mm,1.6 μm)by gradient elution with 2.5 mmol/L ammonium acetate aqueous solution(containing 2.5 mmol/L acetic acid)and acetonitrile as the mobile phases.The flow rate was set at 0.4 mL/min,with an injection volume of 5 μL.The chromatographic separation of each target compound and internal standard substance could be achieved within 15 min.An electrospray ion source was utilized for simultaneous scanning of positive and negative ions under the scheduled multiple reaction monitoring(sMRM)mode,and the internal standard method was used for quantitative analysis.The results of method validations showed that the 71 kinds of PFAS had good linearity,with a correlation coefficient(r)greater than 0.997,spiked recoveries of 76.2%?122.0%and relative standard deviations(RSDs)of 5.5%?14.8%.The limits of detection were in the range of 0.3-60 ng/kg and the limits of quantification were in the range of 0.8-199 ng/kg.The developed method was used for detection of PFAS in 14 batches of Eckloniae Thallus samples,and a total of 19 kinds of PFAS were detected.The findings indicated that PFAS residues were commonly present in Eckloniae Thallus,with perfluorocarboxylic acids(PFCAs)exhibiting the highest concentrations.The method was simple,robust and efficient in preparation,sensitive in detection with high throughput,and suitable for monitoring residual PFAS compounds,including both ionic and neutral in food-medicine homology samples such as Eckloniae Thallus.


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