BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90％,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P＜0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P＜0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P＜0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P＜0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P＜0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P＜0.05),the mitochondrial membrane potential decreased(P＜0.01),and the degree of damage in the high-fluoride group was more obvious(P＜0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.

ObjectiveTo investigate the effects of Astragali Radix polysaccharides （APS） on the polarization of BV2 microglial cells in an oxygen-glucose deprivation/reoxygenation （OGD/R） model through regulation of the Toll-like receptor 4 （TLR4）/nuclear factor-κB （NF-κB） signaling pathway. MethodsThe OGD/R injury model of BV2 microglia was established and divided into blank group， OGD/R group and APS group （0.4 g·L^-1 APS）. Neuroinflammatory injury was induced by lipopolysaccharide （LPS） and treated with APS. The cells were divided into blank group， LPS group （1 mg·L^-1 LPS） and APS group （0.4 g·L^-1 APS+1 mg·L^-1 LPS）. Cell viability was detected using the cell counting kit-8 （CCK-8） assay. Cell morphology was observed under an inverted microscope. Nitric oxide （NO） content in the cell supernatant was determined by the Griess assay. The secretion levels of tumor necrosis factor-α （TNF-α）， interleukin （IL）-6， IL-10， and IL-4 were measured by enzyme-linked immunosorbent assay （ELISA）. Immunofluorescence （IF） was used to detect the double-positive rates of ionized calcium-binding adapter molecule-1/inducible nitric oxide synthase （Iba-1⁺/iNOS⁺） and ionized calcium-binding adapter molecule-1/arginase 1 （Iba-1⁺/Arg1⁺）， as well as the nuclear translocation rate of nuclear factor-κB p65 （NF-κB p65）. Protein expression levels of Iba-1， iNOS， Arg1， TLR4， and NF-κB p65 were detected by Western blot. ResultsIn the OGD/R injury model， compared with the blank control group， BV2 microglial cells in the OGD/R group were activated and exhibited amoeboid morphological changes. The secretion levels of NO， TNF-α， and IL-6 were significantly increased （P<0.01）. The double-positive expression rate of Iba-1⁺/iNOS⁺ and the protein expression of Iba-1 and iNOS were significantly increased （P<0.01）. The nuclear translocation rate of NF-κB p65 and the protein expression levels of TLR4 and NF-κB p65 were significantly increased （P<0.01）. The levels of IL-10 and IL-4 were significantly decreased （P<0.01）， and the double-positive expression rate of Iba-1⁺/Arg1⁺ and Arg1 protein expression were significantly decreased （P<0.01）. Compared with the OGD/R group， the APS group （0.4 g·L^-1） showed reduced cell activation， significantly decreased secretion levels of NO， TNF-α， and IL-6 （P<0.01）， significantly decreased double-positive expression rate of Iba-1⁺/iNOS⁺ and relative protein expression of Iba-1 and iNOS （P<0.01）， significantly decreased nuclear translocation rate of NF-κB p65 and protein expression levels of TLR4 and NF-κB p65 （P<0.01）， significantly increased levels of IL-10 and IL-4 （P<0.01）， and significantly increased double-positive expression rate of Iba-1⁺/Arg1⁺ and Arg1 protein expression （P<0.01）. In the LPS-induced neuroinflammation model， compared with the blank control group， the LPS group showed increased cell activation， significantly increased levels of NO， TNF-α， and IL-6， significantly increased Iba-1⁺/iNOS⁺ double-positive expression rate， NF-κB p65 nuclear translocation rate， and protein expression levels of Iba-1， iNOS， TLR4， and NF-κB p65 （P<0.01）， while IL-10 and IL-4 levels， Iba-1⁺/Arg1⁺ double-positive expression rate， and Arg1 protein expression were significantly decreased （P<0.01）. Compared with the LPS group， the APS group showed reduced cell activation， significantly decreased levels of NO， TNF-α， and IL-6， Iba-1⁺/iNOS⁺ double-positive expression rate， NF-κB p65 nuclear translocation rate， and protein expression levels of Iba-1， iNOS， TLR4， and NF-κB p65 （P<0.01）， while IL-10 and IL-4 levels， Iba-1⁺/Arg1⁺ double-positive expression rate， and Arg1 protein expression were significantly increased （P<0.01）. ConclusionAPS may reduce microglial activation and promote their polarization toward the M2 phenotype by inhibiting activation of the TLR4/NF-κB signaling pathway， thereby alleviating the neuroinflammatory response induced by OGD/R.

Post-stroke cognitive impairment （PSCI） is a common and severe complication in stroke patients， significantly affecting their quality of life and social function. Despite increasing research on PSCI in recent years， effective therapeutic methods remain limited. The Wnt/β-catenin signaling pathway has emerged as a critical research focus in neuroscience due to its essential role in neuroprotection， neurorepair， and cognitive recovery. Dysregulation of the Wnt/β-catenin pathway is considered one of the key mechanisms in the onset and progression of PSCI. Traditional Chinese medicine （TCM）， with its multi-component， multi-target， and synergistic properties， has shown unique advantages in modulating the Wnt/β-catenin signaling pathway， providing a potential novel approach for PSCI treatment. TCM regulates the Wnt/β-catenin pathway through various mechanisms and exerts effects such as inhibiting cell apoptosis， maintaining blood-brain barrier integrity， reducing neuroinflammation， promoting neuroplasticity， and enhancing neurorepair， thereby improving post-stroke cognitive function. This review summarized the latest research progress on the regulation of the Wnt/β-catenin signaling pathway by TCM in intervening PSCI. It analyzed the mechanisms of action of various TCM components and compound formulas within this pathway， aiming to provide a theoretical basis for innovative strategies for PSCI treatment in the future and offer new research insights and practical guidance for the application of TCM in cerebrovascular diseases.

Post-stroke cognitive impairment （PSCI） is a common and severe complication in stroke patients， significantly affecting their quality of life and social function. Despite increasing research on PSCI in recent years， effective therapeutic methods remain limited. The Wnt/β-catenin signaling pathway has emerged as a critical research focus in neuroscience due to its essential role in neuroprotection， neurorepair， and cognitive recovery. Dysregulation of the Wnt/β-catenin pathway is considered one of the key mechanisms in the onset and progression of PSCI. Traditional Chinese medicine （TCM）， with its multi-component， multi-target， and synergistic properties， has shown unique advantages in modulating the Wnt/β-catenin signaling pathway， providing a potential novel approach for PSCI treatment. TCM regulates the Wnt/β-catenin pathway through various mechanisms and exerts effects such as inhibiting cell apoptosis， maintaining blood-brain barrier integrity， reducing neuroinflammation， promoting neuroplasticity， and enhancing neurorepair， thereby improving post-stroke cognitive function. This review summarized the latest research progress on the regulation of the Wnt/β-catenin signaling pathway by TCM in intervening PSCI. It analyzed the mechanisms of action of various TCM components and compound formulas within this pathway， aiming to provide a theoretical basis for innovative strategies for PSCI treatment in the future and offer new research insights and practical guidance for the application of TCM in cerebrovascular diseases.

BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90％,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P＜0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P＜0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P＜0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P＜0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P＜0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P＜0.05),the mitochondrial membrane potential decreased(P＜0.01),and the degree of damage in the high-fluoride group was more obvious(P＜0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.

Post-stroke cognitive impairment(PSCI) is one of the common complications in stroke patients, mainly manifested as impairments in memory, attention, and orientation. Adenosine monophosphate-activated protein kinase(AMPK), as a crucial intracellular energy sensor, has attracted extensive research attention in the field of PSCI in recent years. This paper provides a comprehensive review of the research progress on AMPK-related signaling pathways and their roles in PSCI, with a focus on exploring action mechanisms of AMPK in processes such as oxidative stress, neuroinflammation, autophagy, and apoptosis. Additionally, it summarizes the prospects and challenges of AMPK as a potential target for improving PSCI, aiming to offer new theoretical foundations for the clinical prevention and treatment of PSCI.

Objective:To explore the relationship between maternal rejection and internet addiction in college students, as well as the chain mediating role of rejection sensitivity and adaptability.Methods:From March to May 2024, a total of 1 119 college students were surveyed using the short-form Egna Minnen av Barndoms Uppforstran for Chinese(s-EMBU-C), internet addiction test(IAT), rejection sensitivity questionnaire(RSQ), and the China college student adjustment scale(CCSAS).SPSS 26.0 statistical software was used for independent sample t-test, one-way ANOVA, Pearson product moment correlation, and multiple linear regression analysis, and PROCESS 4.0 macro program was used for chain mediation analysis. Results:(1)Maternal rejection (11.19±2.97) was positively correlated with internet addiction (44.89±9.74)( r=0.60, P<0.01) and rejection sensitivity (102.93±55.63)( r=0.63, P<0.01), while negatively correlated with adaptability (200.19±14.18)( r=-0.56, P<0.01) among college students. Rejection sensitivity was positively correlated with internet addiction ( r=0.75, P<0.01) and negatively correlated with adaptability ( r=-0.76, P<0.01). Adaptability was negatively correlated with internet addiction ( r=-0.68, P<0.01). (2)Maternal rejection had a significant direct effect on internet addiction among college students (effect value=0.193, 95% CI=0.145-0.241), accounting for 32.06%(0.193/0.602) of the total effect. Rejection sensitivity mediated the relationship between maternal rejection and internet addiction (effect value=0.290, 95% CI=0.232-0.357), accounting for 48.17%(0.290/0.602) of the total effect. Adaptability also mediated this relationship (effect value=0.028, 95% CI=0.009-0.053), accounting for 4.65%(0.028/0.602) of the total effect. Additionally, there was a chain mediation effect of rejection sensitivity and adaptability on the relationship between maternal rejection and internet addiction (effect value=0.091, 95% CI=0.052-0.130), accounting for 15.12%(0.091/0.602) of the total effect. Conclusion:Maternal rejection can directly influence internet addiction in college students, and it can also indirectly influence internet addiction through the independent mediating effects of rejection sensitivity and adaptability, as well as through the chain mediating effects of both rejection sensitivity and adaptability.

Objective:To explore the effects of perceived stress on ego depletion of college students, as well as the mediating role of emotional eating and the moderating role of peer relationship.Methods:A cross-sectional survey of 1 088 college students was conducted using the perceived stress scale, the Dutch eating behavior questionnaire, the self-control resource depletion scale, and the peer relationship measurement from December 2023 to April 2024.PROCESS Macro program in SPSS 25.0 software was used to test the mediating effect of emotional eating and the moderating effect of peer relationship.Results:(1)The score of perceived stress, emotional eating, peer relationship and ego depletion were 39.26±8.35, 39.19±12.15, 2.00(1.00), and 18.19±7.15, respectively.(2)Perceived stress was positively correlated with emotional eating, ego depletion, and peer relationship( r=0.36, 0.61, 0.25, all P<0.01). Emotional eating was positively correlated with ego depletion and peer relationship( r=0.40, 0.19, both P<0.01). And ego depletion was positively correlated with peer relationship( r=0.23, P<0.01).(3)Emotional eating played a partial mediating role in the effect of perceived stress on ego depletion( β=0.077, 95% CI=0.053-0.104), and the mediating effect accounted for 12.38%(0.077/0.622) of the total effect.(4)Peer relationship played a moderating role between perceived stress and emotional eating. Under low peer relationship, perceived stress had a significant positive predictive effect on emotional eating( βsimple=0.46, P<0.01), and under high peer relationship, the predictive effect of perceived stress on emotional eating was significantly weaker( βsimple=0.26, P<0.01). Conclusions:Perceived stress not only directly affects ego depletion, but also indirectly affects ego depletion through emotional eating in college students.High levels of peer relationship can weaken the impact of perceived stress and high emotional eating on ego depletion.

Objective:To investigate the characteristics of m6A methylation modification patterns in mRNA of patients with major depressive disorder (MDD) and its effect in the pathogenesis of the disease.Methods:From March 2022 to May 2023, five untreated MDD patients were assigned to the MDD group, and five healthy individuals were enrolled as the healthy control group at the First Psychiatric Hospital of Harbin.Microarray analysis was performed to determine the m6A modification profiles and gene expression patterns of mRNA in MDD. Gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were conducted to elucidate the effect of m6A methylation in the development of depression. Finally, methylated RNA immunoprecipitation combined with quantitative PCR (MeRIP-qPCR) was used to validate the m6A methylation levels of key mRNAs (GRM4, CAMKK2). Data were analyzed using R software (version 4.2.0) with t-test and Fisher's exact test. Results:Significant differences in m6A-modified mRNAs were observed between MDD patients and healthy controls. A total of 513 mRNAs (180 hypermethylated and 333 hypomethylated) exhibited differential m6A modifications in MDD patients. GO and KEGG analysis revealed that hypermethylated mRNAs were primarily enriched in neuroactive ligand-receptor interactions, while hypomethylated mRNAs were associated with the AMP-activated protein kinase (AMPK) signaling pathway. Additionally, a total of 350 differentially expressed mRNAs were identified (171 upregulated and 179 downregulated), enriched in the cyclic adenosine monophosphate (cAMP) and tumor necrosis factor (TNF) signaling pathways. MeRIP-qPCR results demonstrated that the m6A methylation level of GRM4 in MDD patients (25.40±2.38) was significantly higher than that in healthy controls (9.40±1.00) ( t=13.88, P<0.05), whereas the methylation level of CAMKK2 (19.63±6.60) was significantly lower than that in healthy controls (30.51±7.20) ( t=2.48, P<0.05). Conclusion:The m6A modification expression profile is abnormal in patients with major depressive disorder, which may be involved in the pathogenesis and development of MDD, and the identification of key pathways may provide new clues and evidence for the development of more effective therapeutic targets for MDD.

Objective:To explore the role of retinoic acid-related orphan receptor α (RORα) in cognitive impairment induced by chronic alcohol consumption in mice.Methods:(1)The SPF grade RORαflox/flox transgenic mice aged 8 weeks were generated, and 22 transgenic mice were evenly divided into two groups by the method of matching body mass, which were the control group (Con group) and the alcohol group (EtOH group), with 11 mice in each group.(2)Emx1-Cre transgenic mice were used to selectively knock out the RORα gene in the forebrain of RORαflox/flox transgenic mice, producing conditional knockout mice (cKO mice) with the genotype RORαflox/flox-Emx1-Cre+ /+. Fourteen cKO mice were further split into two groups by the method of matching body mass, which were the conditional knockout group (cKO group) and the conditional knockout + alcohol group (cKO + EtOH group), with 7 mice in each group. A chronic alcohol use cognitive impairment model was developed in the EtOH group and cKO + EtOH group through the two-bottle free-choice method, while the Con group and cKO group were given two bottles of water for the same period. Cognitive abilities of mice in all groups were evaluated using behavioral novel object recognition test and Y-maze test.RORα mRNA and protein expression levels in the hippocampus of the Con group and EtOH group were assessed by RT-qPCR and Western blot, respectively.The GraphPad Prism 9.0 software was used for data analysis.One-way ANOVA was used for the comparison of multiple groups and Tukey test was used for further pairwise comparisons.Results:(1) Comparison between Con group and EtOH group: the relative levels of ROR α protein (0.63±0.04) and mRNA (0.78±0.03) in the hippocampus of mice in the EtOH group were significantly lower than those in the Con group((1.00±0.06), (1.00±0.05), both P<0.05). The duration of the EtOH group in the Y maze was significantly lower than that of the Con group ((212.30±32.05) s, (129.30±21.50) s, P<0.05), and the new object recognition index of the EtOH group was lower than that of the Con group ((14.73±25.49)% vs (-15.55±27.88)%, P=0.08). (2)Comparison between Con group and cKO group: the frequency and duration of entering the Y maze of mice in the cKO group ((7.43±2.30) times, (124.10±13.95) s) were lower than those in the Con group ((14.90±3.65) times, (212.30±32.05) s, both P<0.05). There was no statistically significant difference in the new object recognition index between the cKO group and the Con group ( P>0.05). (3) Comparison between the cKO+ EtOH group and the cKO group: the frequency ((2.71±1.11)times) and duration ((161.70±17.95) s) of entering the new heteroarm of Y maze in the KO+ EtOH group were lower than those in the cKO group ((7.43±2.30) times, (124.10±13.95) s, both P<0.05), and there was no statistically significant difference in the new object recognition index ( P>0.05). (4) Comparison between the cKO+ EtOH group and the EtOH group: the frequency of entering new heteroarm of the Y maze in the cKO+ EtOH group ((2.71±1.11)times) was significantly lower than that in the EtOH group (12.18±4.49) ( P<0.05), while there was no statistically significant difference in other behavioral results between the two groups ( P>0.05). Conclusion:Chronic alcohol consumption leads to cognitive impairment through the downregulation of RORα in the hippocampus of mice. Specific knockout of RORα in the forebrain exacerbates cognitive impairment induced by chronic alcohol use. RORα may represent a potential therapeutic target for cognitive impairment resulting from chronic alcohol consumption.