OBJECTIVE To investigate the effects and mechanism of Wenpi tongluo kaiqiao formula （WPTL） against neuronal necroptosis in Alzheimer’s disease （AD） mice based on the Z-DNA binding protein 1 （ZBP1）/mixed lineage kinase domain-like protein （MLKL） signaling pathway. METHODS Forty APP/PS1 transgenic AD mice were randomly divided into model group， WPTL low-dose （WPTL-L） group （10.4 g/kg， calculated by the raw medicine）， WPTL high-dose （WPTL-H） group （20.8 g/kg， calculated by the raw medicine） and donepezil hydrochloride group （3 mg/kg）， with 10 mice in each group； another 10 C57BL/6J mice were selected as normal control group. Intragastric administration， once a day， for 30 consecutive days. Twenty-four hours after the last administration， Morris water maze test was performed to evaluate learning and memory abilities； the pathological morphology of hippocampal tissues was observed； the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-4 （IL-4） were determined； the expressions of amyloid precursor protein （APP）， Tau protein， and ZBP1/MLKL signaling pathway-related proteins in hippocampal tissues were detected； the positive expression of phosphorylated receptor-interacting protein kinase 3 （p-RIPK3） in the neurons of hippocampal tissues and mRNA expression of ZBP1 were measured in hippocampal tissues. RESULTS Compared with normal control group， the escape latency of mice in model group was prolonged significantly on day 3 to 5 （P＜0.05）， the times of crossing platform reduced significantly （P＜0.05）， and obvious pathological changes were observed in the hippocampal tissue. The level of TNF- α， the expressions of APP， p-Tau and ZBP1， the phosphorylation levels of RIPK1， RIPK3 and MLKL， the fluorescence intensity of p-RIPK3 as well as the mRNA expression of ZBP1 were significantly increased （P＜0.05）， while the serum level of IL-4 was decreased significantly （P＜0.05）. Compared with model group， above indexes were reversed significantly in administration groups （P＜0.05）， and pathological damage of hippocampal tissue was alleviated. CONCLUSIONS WPTL can inhibit the ZBP1/MLKL signaling pathway， reduce neuronal necroptosis in AD mice， and inhibit inflammatory responses， thereby improving learning and spatial memory abilities in AD mice.

OBJECTIVE To investigate the intervention effect and its potential mechanism of Yifei xuanfei jiangzhuo formula by inhibiting mitochondrial fission in a vascular dementia （VaD） model rats. METHODS VaD rat model was established by bilateral common carotid artery ligation. The experimental animals were randomly divided into sham operation group （SHAM）， model group （MOD），Yifei xuanfei jiangzhuo formula low-dose group （YFXF-L）， Yifei xuanfei jiangzhuo formula high-dose group （YFXF-H）， and Donepezil hydrochloride group （positive control）， with 9 animals in each group. After 30 days of intervention， the spatial learning memory ability was assessed by Morris water maze experiment； HE staining was used to observe histopathological changes in CA1 area of hippocampus； ELISA was used to detect the levels of serum inflammatory factors [interleukin-1β （IL-1β） and IL-4]； Western blot was used to detect the expressions of heat shock protein 90 （HSP90）/mixed lineage kinase domain-like protein （MLKL）/dynamin-related protein 1 （Drp1） pathway-related proteins， mitochondrial fusion proteins （MFN1， MFN2）， and adenosine triphosphate synthase 5A （ATP5A） in hippocampal tissues. The immunohistochemistry was used to detect the level of phosphorylated MLKL （p-MLKL）； real-time fluorescence quantitative PCR was adopted to detect mRNA expressions ofHSP90， MFN1， MFN2 and ATP5A. RESULTS Compared with SHAM group， the escape latency of rats in the MOD group was significantly prolonged， the number of crossing the platform was significantly reduced， and the hippocampal tissues showed typical neuronal damage characteristics， the positive expression level of p-MLKL and the serum level of IL-1β significantly increased， while the serum level of IL-4 significantly decreased， the protein and mRNA expression of HSP90， as well as the protein expressions of p-MLKL/MLKL and p-Drp1（Ser616）/Drp1 were all significantly increased in hippocampal tissue， the protein and mRNA expressions of MFN1， MFN2 and ATP5A， and protein expression of p-Drp1（Ser637）/Drp1 were all significantly decreased （P＜0.05）. After the intervention of Yifei xuanfei jiangzhuo formula， above indicators in each treatment group were all significantly reversed （P＜0.05）. CONCLUSIONS Yifei xuanfei jiangzhuo formula may alleviate neuronal damage and neuroinflammatory responses in VaD rats by regulating the HSP90/MLKL/Drp1 signaling pathway， inhibiting mitochondrial fission， thereby maintaining mitochondrial dynamic balance and improving mitochondrial function.

OBJECTIVES: To explore the mechanisms of Qingre Lidan Jiedu Recipe (QLJR) for improving cognitive dysfunction in rats with high copper load. METHODS: Seventy-five male SD rats were randomized into normal control group, model group, QLJR group, penicillamine (PCA) group, and QLJR+ PCA group. Except for those in the control group, all the rats were fed a high-copper diet for 12 weeks. The effects of the treatments on cognitive function of the rats were assessed using the Barnes maze and passive avoidance tests. Hippocampal expressions of NIX, FUNDC1 and LC3 of the rats were detected using Western blotting and immunofluorescence staining, and changes in mitochondrial morphology were observed with transmission electron microscopy. RESULTS: Behavioral tests showed prolonged target hole latency, shortened latency to enter the dark chamber, and increased error counts of the rats in the model group, which were significantly improved in QLJR+PCA group; the error counts were significantly lower in QLJR+PCA group than in either QLJR or PCA group. Among all the groups, the hippocampal expressions of NIX and FUNDC1 were the lowest and LC3 I/II expression the highest in the model group; NIX and FUNDC1 expressions were significantly higher and LC3 I expression was lower in QLJR+PCA group than in QLJR group and PCA group. Immunofluorescence staining revealed weakened NIX and FUNDC1 expressions and enhanced LC3 expression in the hippocampus of the rats in the model group as compared with those in the normal control and QLJR+PCA groups, but their expressions did not differ significantly between QLJR and PCA groups. The rats in the model group showed obvious structural disarray of the mitochondria, which were improved in all the treatment groups. CONCLUSIONS QLJR improves cognitive dysfunction in rats with high copper load possibly by regulating mitophagy.
Animals ; Male ; Rats, Sprague-Dawley ; Rats ; Drugs, Chinese Herbal/therapeutic use* ; Copper/toxicity* ; Mitophagy/drug effects* ; Hippocampus/drug effects* ; Cognition Disorders/drug therapy* ; Cognitive Dysfunction/chemically induced*

Objective To investigate the impact of overwork on vascular endothelial barrier function in mice.Methods Thirty KM mice were randomized equally into control,overwork for 2 weeks(W2)group and 4 weeks(W4)group.In the latter two groups,the mice were subjected to continuous standing in water for 8 h followed by restraint for 3 h to simulate overwork on a daily basis for 2 and 4 weeks.After modeling,4 mice from each group were intraperitoneally injected with Evans blue dye to assess vascular permeability.In the other 6 mice,serum IL-1β levels were measured using ELISA,and arterial tissues were collected for histological examination and detection of mRNA expressions of occludin,claudin-5,ZO-1,JAM-A and VE-cadherin;immunofluorescence assay was used to detect the protein expressions of claudin-5,ZO-1,VE-cadherin,and Syndecan-1.Results The mice in W2 and W4 groups exhibited slower weight gain,hair loss,reduced activity,and significantly increased serum IL-1β levels.Vascular permeability was significantly increased in W4 group.In W2 group,the endothelial cells were swollen and dissociated,and the intima was rough and irregular;arterial intimal rupture was observed in W4 group.The mRNA expressions of occludin,claudin-5,ZO-1 and JAM-A in the arterial tissues were significantly increased in W2 group but decreased in W4 group,while VE-cadherin mRNA expression were reduced in both groups(P<0.05).The protein expressions of claudin-5,ZO-1,VE-cadherin,and Syndecan-1 were all significantly reduced in W4 group.Conclusion Prolonged overwork can cause damage of the intercellular junction complexes in arterial endothelial cells and the endothelial glycocalyx to result in impaired barrier function and increased vascular permeability in mice.

Objective:To explore potential predictors of refractory Mycoplasma pneumoniae pneumonia (RMPP) in early stage. Methods:The prospective multicenter study was conducted in Zhejiang, China from May 1 st, 2019 to January 31 st, 2020. A total of 1 428 patients with fever >48 hours to <120 hours were studied. Their clinical data and oral pharyngeal swab samples were collected; Mycoplasma pneumoniae DNA in pharyngeal swab specimens was detected. Patients with positive Mycoplasma pneumoniae DNA results underwent a series of tests, including chest X-ray, complete blood count, C-reactive protein, lactate dehydrogenase (LDH), and procalcitonin. According to the occurrence of RMPP, the patients were divided into two groups, RMPP group and general Mycoplasma pneumoniae pneumonia (GMPP) group. Measurement data between the 2 groups were compared using Mann-Whitney U test. Logistic regression analyses were used to examine the associations between clinical data and RMPP. Receiver operating characteristic (ROC) curves were used to analyse the power of the markers for predicting RMPP. Results:A total of 1 428 patients finished the study, with 801 boys and 627 girls, aged 4.3 (2.7, 6.3) years. Mycoplasma pneumoniae DNA was positive in 534 cases (37.4%), of whom 446 cases (83.5%) were diagnosed with Mycoplasma pneumoniae pneumonia, including 251 boys and 195 girls, aged 5.2 (3.3, 6.9) years. Macrolides-resistant variation was positive in 410 cases (91.9%). Fifty-five cases were with RMPP, 391 cases with GMPP. The peak body temperature before the first visit and LDH levels in RMPP patients were higher than that in GMPP patients (39.6 (39.1, 40.0) vs. 39.2 (38.9, 39.7) ℃, 333 (279, 392) vs. 311 (259, 359) U/L, both P<0.05). Logistic regression showed the prediction probability π=exp (-29.7+0.667×Peak body temperature (℃)+0.004×LDH (U/L))/(1+exp (-29.7+0.667×Peak body temperature (℃)+0.004 × LDH (U/L))), the cut-off value to predict RMPP was 0.12, with a consensus of probability forecast of 0.89, sensitivity of 0.89, and specificity of 0.67; and the area under ROC curve was 0.682 (95% CI 0.593-0.771, P<0.01). Conclusion:In MPP patients with fever over 48 to <120 hours, a prediction probability π of RMPP can be calculated based on the peak body temperature and LDH level before the first visit, which can facilitate early identification of RMPP.

Objective To investigate the effect of TRPC5 on intermittent hypoxia-induced myocardial pyroptosis.Methods TRPC5 knock out(TRPC5-/-)and SD rats were each randomly divided into chronic intermit-tent hypoxia group(CIH group)and normoxia group(Control group)(n=6 for each group).The myocardial fibrosis in rats was visualized by Masson staining,the levels of serum inflammatory factors were measured by ELISA and the relative expression levels of TRPC 5 and pyroptosis-related proteins were determined by Western blotting.Results Masson staining showed that the collagen volume fraction in the TRPC5-/--CIH group was higher than that in the TRPC5-/--Control group and lower than that in the WT-CIH group.The ELISA results showed that the serum levels of IL-1,IL-6,and TGF-β in the TRPC5-/--CIH group were higher than those in the TRPC5-/--Control group and lower in the WT-CIH group.Western blotting results showed that the relative expression levels of pyroptosis related proteins caspase-1,NLRP3,GSDMD,and GSDMD-N in the TRPC5-/--CIH group were higher than those in the TRPC5-/--Control group and lower than those in the WT-CIH group.Conclusion TRPC5 deficiency alleviates myocardial pyroptosis and myocardial fibrosis induced by hypoxia.

Objective:To explore the relationship between the hypertensive snowbirds′ length of migratory stay and their blood pressure control and blood pressure levels.Methods:This study was a cross-sectional study. A population of snowbirds with hypertension was recruited between October and November 2022, and a structured questionnaire was used to collect their self-measured blood pressure and length of stay in Hainan Province. The blood pressure control status is determined based on self-measured blood pressure. According to the self-measured blood pressure to determine whether the blood pressure was well controlled. The associations between snowbirds′ length of stay and their blood pressure control as well as their self-measured blood pressure were analyzed using restricted cubic splines.Results:A total of 362 research subjects were included, 169(46.7%) of whom were male, and their age was (69.7±7.0) years old. The participants′ self-measured systolic blood pressure and diastolic blood pressure were (129.1±16.2) mmHg (1 mmHg=0.133 kPa) and (78.9±10.1) mmHg, respectively. Overall, 174 (48.1%) participants attained adequate blood pressure control. The median length of stay in Wuzhishan City was 7(6, 7) months. There was an inverted U-shaped association between snowbirds′ length of stay and blood pressure control (overall: P=0.023; nonlinearity: P=0.014), where participants with a length of stay of 7 months had the highest rate of blood pressure control. There is a U-shaped curve relationship between length of stay and systolic blood pressure (overall: P=0.001; nonlinearity: P=0.033), and a linear negative correlation with diastolic blood pressure ( β=-1.19, P=0.003). Conclusions:Compared with hypertensive snowbirds with too long or too short lengths of stay, snowbirds who stayed in Wuzhishan City for seven months have better blood pressure control, and systolic blood pressure is also lower.

Objective To investigate the impact of overwork on vascular endothelial barrier function in mice.Methods Thirty KM mice were randomized equally into control,overwork for 2 weeks(W2)group and 4 weeks(W4)group.In the latter two groups,the mice were subjected to continuous standing in water for 8 h followed by restraint for 3 h to simulate overwork on a daily basis for 2 and 4 weeks.After modeling,4 mice from each group were intraperitoneally injected with Evans blue dye to assess vascular permeability.In the other 6 mice,serum IL-1β levels were measured using ELISA,and arterial tissues were collected for histological examination and detection of mRNA expressions of occludin,claudin-5,ZO-1,JAM-A and VE-cadherin;immunofluorescence assay was used to detect the protein expressions of claudin-5,ZO-1,VE-cadherin,and Syndecan-1.Results The mice in W2 and W4 groups exhibited slower weight gain,hair loss,reduced activity,and significantly increased serum IL-1β levels.Vascular permeability was significantly increased in W4 group.In W2 group,the endothelial cells were swollen and dissociated,and the intima was rough and irregular;arterial intimal rupture was observed in W4 group.The mRNA expressions of occludin,claudin-5,ZO-1 and JAM-A in the arterial tissues were significantly increased in W2 group but decreased in W4 group,while VE-cadherin mRNA expression were reduced in both groups(P<0.05).The protein expressions of claudin-5,ZO-1,VE-cadherin,and Syndecan-1 were all significantly reduced in W4 group.Conclusion Prolonged overwork can cause damage of the intercellular junction complexes in arterial endothelial cells and the endothelial glycocalyx to result in impaired barrier function and increased vascular permeability in mice.

[Objective]To investigate the association between screen content and the frequency of screen exposure at the age of one and a half years and hyperactive behavior in preschool,and to explore how the association is affected by the interaction between outdoor activities and screen behaviors,which could provide theoretical basis and feasible solutions for the prevention and intervention of behavioral problems in childhoood.[Methods]The survey was conducted from June 2022 to June 2023 in Huicheng District,Huizhou (China) stratified by whole cluster sampling methods. Parents and teachers of 5648 children in 61 kindergartens were sampled for questionnaire surveys. The Conners Teacher Rating Scale (TRS) was used to investigate hyperactive behavior. A self-administered questionnaire was used to investigate basic demographic information of children,screen content,frequency of screen exposure and outdoor activities at the age of one and a half years. Multivariate logistic regression was used to explore the association between video screen behavior and hyperactive behavior and its interaction with outdoor activities by controlling for covariates such as children's age,gender,and parental education.[Results]Result showed the overall prevalence of 3.2％ for hyperactive behavior,2.1％ for conduct problems,2.1％ for hyperactivity problems,1.3％ for inattention-passivity problems,and 0.9％ for hyperactivity index. After adjusting for confounding factors,multiple logistic regression analysis showed that screen exposure of "two to four times a week" at one and a half years old was associated with an increased detection rate of hyperactive behaviors in preschool children,with an estimated ORs (95％ CI) of 1.682 (1.141,2.480). Daily screen exposure was associated with increased detection rates of hyperactive behavior,conduct problems,hyperactivity issues,inattention-passivity problems,and hyperactivity index in pre-school age. The estimated ORs (95％ CI) were 2.136 (1.218,3.746),2.321 (1.185,4.546),2.300 (1.208,4.380),2.776 (1.267,6.085) and 3.640 (1.525,8.687),respectively. But the above associations were not found in children who were engaged in daily outdoor activities at the age of one and a half years (P value for interaction<0.001). No association was found between screen content and hyperactive behavior (P>0.05).[Conclusions]Frequency of screen exposure in early childhood is significantly associated with hyperactive behavior problems in preschool,and outdoor activities could weaken the correlation between high-frequency screen exposure and hyperactive behavior,suggesting that parents and schools should prioritize scientifically guiding children's video viewing behavior and outdoor activities,ensuring a well-arranged daily life,to lay a good foundation for the healthy development of children's behavior.