ObjectiveTo elucidate the underlying mechanism through which Zhuluan decoction suppresses excessive autophagy in human ovarian granulosa cells （KGN） and ameliorates premature ovarian insufficiency （POI） via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodsThe optimal concentration of cyclophosphamide for inducing a POI model in KGN cells was identified via the cell counting kit-8 （CCK-8） assay. Subsequently， the impacts of varying concentrations of Zhuluan decoction-containing serum on the viability of the KGN cell model were assessed. After the optimal drug concentration was determined， KGN cells were categorized into the following groups： blank control （20% blank serum）， model （20% blank serum + 5 μmol·L^-1 cyclophosphamide）， Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide）， autophagy inhibitor （20% blank serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， autophagy inhibitor + Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， and estradiol valerate （20% estradiol valerate-containing serum + 5 μmol·L^-1 cyclophosphamide）. Following 48 hours of incubation， flow cytometry was utilized to measure the apoptosis rate of KGN cells in each group. Western blotting was employed to quantify the protein levels of PI3K， phosphorylated （p）-Akt， Akt， p-mTOR， and mTOR， along with the expression levels of autophagy-related proteins such as Beclin1， autophagy-related 5 homolog （ATG5）， and microtubule-associated protein 1 light chain 3 （LC3）， in each group. Additionally， monodansylcadaverine （MDC） staining was performed to evaluate the extent of autophagy in each group. ResultsIncubation of KGN cells with 5 μmol·L^-1 cyclophosphamide for 48 h successfully established a POI model， marked by a significant inhibition of KGN cell proliferation. Notably， the inhibitory effect of cyclophosphamide on KGN cell proliferation exhibited a positive correlation with its concentration. Zhuluan decoction-containing serum at 20% and 30% promoted cell proliferation and mitigated the inhibitory effect of cyclophosphamide on KGN cell proliferation， with comparable therapeutic efficacy observed at both concentrations. Compared with the blank control group， the model group displayed an elevated apoptosis rate （P<0.01）， reduced protein levels of PI3K， p-Akt， and p-mTOR （P<0.01）， increased protein levels of Beclin1， LC3， and ATG5 （P<0.01）， no significant alterations in the protein levels of Akt and mTOR， and an enhanced MDC autophagy fluorescence intensity （P<0.01）. In comparison to that the model group， the apoptosis rates in the blank control group， model group， Zhuluan decoction-containing serum group， autophagy inhibitor group， autophagy inhibitor + Zhuluan decoction-containing serum group， and estradiol valerate group all reduced （P<0.05， P<0.01）， with the most pronounced reduction observed in the autophagy inhibitor + Zhuluan decoction-containing serum group. The protein levels of PI3K， p-Akt， and p-mTOR were higher in other groups than in the model group （P<0.05， P<0.01）， being the highest in the autophagy inhibitor + Zhuluan decoctio-containing serum group （P<0.01）. The protein levels of Beclin1 and ATG5 were lower in other groups than in the model group （P<0.05， P<0.01）. The expression level of LC3 declined in the Zhuluan decoction-containing serum group and the estradiol valerate group （P<0.05， P<0.01）， while it decreased without statistical significance in the autophagy inhibitor group and the autophagy inhibitor + Zhuluan decoction-containing serum group. ConclusionZhuluan decoction may activate the PI3K/Akt/mTOR pathway to inhibit excessive autophagy and counteract the detrimental effects of cyclophosphamide on the KGN cell model， thus managing POI.

ObjectiveTo elucidate the underlying mechanism through which Zhuluan decoction suppresses excessive autophagy in human ovarian granulosa cells （KGN） and ameliorates premature ovarian insufficiency （POI） via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodsThe optimal concentration of cyclophosphamide for inducing a POI model in KGN cells was identified via the cell counting kit-8 （CCK-8） assay. Subsequently， the impacts of varying concentrations of Zhuluan decoction-containing serum on the viability of the KGN cell model were assessed. After the optimal drug concentration was determined， KGN cells were categorized into the following groups： blank control （20% blank serum）， model （20% blank serum + 5 μmol·L^-1 cyclophosphamide）， Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide）， autophagy inhibitor （20% blank serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， autophagy inhibitor + Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， and estradiol valerate （20% estradiol valerate-containing serum + 5 μmol·L^-1 cyclophosphamide）. Following 48 hours of incubation， flow cytometry was utilized to measure the apoptosis rate of KGN cells in each group. Western blotting was employed to quantify the protein levels of PI3K， phosphorylated （p）-Akt， Akt， p-mTOR， and mTOR， along with the expression levels of autophagy-related proteins such as Beclin1， autophagy-related 5 homolog （ATG5）， and microtubule-associated protein 1 light chain 3 （LC3）， in each group. Additionally， monodansylcadaverine （MDC） staining was performed to evaluate the extent of autophagy in each group. ResultsIncubation of KGN cells with 5 μmol·L^-1 cyclophosphamide for 48 h successfully established a POI model， marked by a significant inhibition of KGN cell proliferation. Notably， the inhibitory effect of cyclophosphamide on KGN cell proliferation exhibited a positive correlation with its concentration. Zhuluan decoction-containing serum at 20% and 30% promoted cell proliferation and mitigated the inhibitory effect of cyclophosphamide on KGN cell proliferation， with comparable therapeutic efficacy observed at both concentrations. Compared with the blank control group， the model group displayed an elevated apoptosis rate （P<0.01）， reduced protein levels of PI3K， p-Akt， and p-mTOR （P<0.01）， increased protein levels of Beclin1， LC3， and ATG5 （P<0.01）， no significant alterations in the protein levels of Akt and mTOR， and an enhanced MDC autophagy fluorescence intensity （P<0.01）. In comparison to that the model group， the apoptosis rates in the blank control group， model group， Zhuluan decoction-containing serum group， autophagy inhibitor group， autophagy inhibitor + Zhuluan decoction-containing serum group， and estradiol valerate group all reduced （P<0.05， P<0.01）， with the most pronounced reduction observed in the autophagy inhibitor + Zhuluan decoction-containing serum group. The protein levels of PI3K， p-Akt， and p-mTOR were higher in other groups than in the model group （P<0.05， P<0.01）， being the highest in the autophagy inhibitor + Zhuluan decoctio-containing serum group （P<0.01）. The protein levels of Beclin1 and ATG5 were lower in other groups than in the model group （P<0.05， P<0.01）. The expression level of LC3 declined in the Zhuluan decoction-containing serum group and the estradiol valerate group （P<0.05， P<0.01）， while it decreased without statistical significance in the autophagy inhibitor group and the autophagy inhibitor + Zhuluan decoction-containing serum group. ConclusionZhuluan decoction may activate the PI3K/Akt/mTOR pathway to inhibit excessive autophagy and counteract the detrimental effects of cyclophosphamide on the KGN cell model， thus managing POI.

"Sinew prescription correspondence" is the principle of selecting prescriptions for channel sinew diseases. On the basis of the theory of syndrome differentiation and treatment， the pulse manifestation corresponds to the channel sinew syndrome， which can improve the flexibility and standardization of clinical prescriptions. From the perspective of systematic dialectical sphygmology， this paper explains the dialectical relationship between channel sinew theory and pulse body elements， pulse wall elements， pulse elements and blood flow elements， and clarifies the internal relationship between pulse manifestation and prescriptions at the level of channel sinew disease. The prescription is derived from the method， while the method is established with the syndrome， and the prescription is unified by the method. According to the theory of "sinew prescription correspondence"， the treatment ideas of channel sinew diseases were analyzed from the perspective of channel sinew distribution， functional characteristics and structural changes. On this basis， the diagnosis of channel sinew disease and the application of prescriptions are expanded， and the research on the internal treatment and diagnosis mode of "pulse manifestation-channel sinew-zang fu （脏腑）" is prospected， so as to expand the differentiation and treatment methods of channel sinew theory.

Objective To study the spatial distribution of the incidence of pulmonary tuberculosis in Hubei Province and its influencing factors, so as to improve the theoretical basis for scientific development of tuberculosis prevention and control measures in the future. Methods The data of reported incidence of tuberculosis and related influencing factors in various counties and districts of Hubei Province in 2020 were collected. Global Moran's I index, hotspot analysis and geographically weighted regression (GWR) model analysis were used to calculate the spatial autocorrelation of the incidence of tuberculosis, and to analyze the influencing factors affecting the incidence rate of tuberculosis. Results There were obvious regional differences in the space distribution of the incidence rate of tuberculosis. Hot spot analysis showed positive spatial correlation and obvious clustering. The GWR model (AICc=784.251) in this study had higher AICc value compared to the ordinary least squares regression (OLS) model (AICc=804.2585). The GWR model showed that the increase in the proportion of the population aged 65 and above and the proportion of the ethnic minority population had a significant promoting effect on the increase of the incidence rate of tuberculosis, and there was significant spatial heterogeneity. The effect of PM_2.5 concentration on the incidence rate of pulmonary tuberculosis varied in different regions, and the degree of effect was also different. Conclusion The proportion of people aged 65 and above and the proportion of ethnic minorities may significantly influence the incidence of pulmonary tuberculosis. The effect of PM_2.5 concentration varies in different regions, so targeted measures should be formulated according to the situation in different regions.

BACKGROUND:Growth factor is the key effect molecule that plays a role in platelet-rich plasma in clinical treatment.There are differences in the concentration of growth factor after different activators activate platelet-rich plasma,which is an important factor affecting clinical efficacy. OBJECTIVE:To analyze the influence of different activators on the mass concentration of growth factors in platelet-rich plasma. METHODS:Totally 12 healthy volunteers were recruited to collect EDTA-K2 anticoagulant venous blood.Secondary centrifugation was used to prepare platelet-rich plasma.The difference in mass concentrations of growth factors was compared between venous blood and platelet-rich plasma.The platelet-rich plasma was mixed with four activators(normal saline,thrombin,calcium gluconate,calcium gluconate+thrombin)according to the volume ratio of 10:1,and incubated in a constant temperature water bath at 37 °C for 30 minutes.After centrifugation,the supernatant was extracted and the mass concentration of growth factor was detected.The bacterial growth in supernatant was measured by blood agar plate.Pearson correlation was used to analyze the correlation between different activators and the mass concentration of growth factor in platelet-rich plasma,and the correlation between the value of thrombocytometer and the mass concentration of growth factors in platelet-rich plasma. RESULTS AND CONCLUSION:(1)The mass concentrations of platelet-derived growth factor-BB,platelet-derived growth factor-AB,vascular endothelial growth factor,and epidermal growth factor in platelet-rich plasma were 8.7,22.2,2.3,and 2.8 times of those in venous blood,respectively(P<0.05).(2)Compared with normal saline group,the mass concentrations of platelet-derived growth factor BB,platelet-derived growth factor AB,vascular endothelial growth factor,and epidermal growth factor were increased in the thrombin group,calcium gluconate group,and calcium gluconate+thrombin group(P<0.05).The mass concentration of platelet-derived growth factor BB in the thrombin group and calcium gluconate group was higher than that in the calcium gluconate+thrombin group(P<0.05),and the mass concentration of platelet-derived growth factor AB in the thrombin group was higher than that in the calcium gluconate group and calcium gluconate+thrombin group(P<0.05).Epidermal growth factor mass concentration in the thrombin group was lower than that in the calcium gluconate group and calcium gluconate+thrombin group(P<0.05).(3)The results of blood agar plate test showed no bacterial growth in the supernatant of the four groups.(4)Pearson correlation analysis showed that the mass concentration of platelet-derived growth factor BB in platelet-rich plasma was strongly positively correlated with thrombin(r=0.683,P<0.05),and the mass concentration of vascular endothelial growth factor was strongly positively correlated with thrombin,calcium gluconate,calcium gluconate+thrombin stimulant(r=0.730,0.789,0.686,P<0.05).There was no correlation between the value of thrombocytometer and the mass concentration of four kinds of growth factors(P>0.05).(5)The results suggest that different activators have an impact on the concentration of growth factors in platelet-rich plasma.It is suggested to choose different activators to improve clinical efficacy according to different growth factor mass concentrations and treatment needs.

Extensive epigenetic reprogramming involves in memory CD8+ T-cell differentiation. The elaborate epigenetic rewiring underlying the heterogeneous functional states of CD8+ T cells remains hidden. Here, we profile single-cell chromatin accessibility and map enhancer-promoter interactomes to characterize the differentiation trajectory of memory CD8+ T cells. We reveal that under distinct epigenetic regulations, the early activated CD8+ T cells divergently originated for short-lived effector and memory precursor effector cells. We also uncover a defined epigenetic rewiring leading to the conversion from effector memory to central memory cells during memory formation. Additionally, we illustrate chromatin regulatory mechanisms underlying long-lasting versus transient transcription regulation during memory differentiation. Finally, we confirm the essential roles of Sox4 and Nrf2 in developing memory precursor effector and effector memory cells, respectively, and validate cell state-specific enhancers in regulating Il7r using CRISPR-Cas9. Our data pave the way for understanding the mechanism underlying epigenetic memory formation in CD8+ T-cell differentiation.
CD8-Positive T-Lymphocytes/metabolism* ; Cell Differentiation ; Chromatin/immunology* ; Animals ; Mice ; Immunologic Memory ; Epigenesis, Genetic ; SOXC Transcription Factors/immunology* ; NF-E2-Related Factor 2/immunology* ; Mice, Inbred C57BL ; Gene Regulatory Networks ; Enhancer Elements, Genetic

Objective:To investigate the impact of early invasive arterial blood pressure (IBP) monitoring on survival and neurological outcomes in out-of-hospital cardiac arrest (OHCA) patients undergoing extracorporeal cardiopulmonary resuscitation (ECPR).Methods:This retrospective cohort study analyzed 44 OHCA patients receiving ECPR between January 2021 and January 2023. Patients were divided into: Early intervention group : IBP established within 3 min of ECMO initiation; Late intervention group : IBP established after ICU admission. Baseline characteristics, ECMO parameters, and clinical outcomes were compared. Multivariable logistic regression (adjusted for age, initial rhythm, etc.) and Spearman's correlation were used.Results:This study included a total of 44 patients treated with OHCA and ECPR, divided into an early intervention group of 23 cases and a late intervention group of 21 cases. The early intervention group showed significantly higher: Survival to discharge (43.5% vs. 9.5%, P<0.05), Good neurological recovery (CPC 1-2: 34.8% vs. 9.5%, P<0.05).Early intervention independently predicted survival (adjusted OR=18.84, 95% CI:1.97-179.98, P=0.01). Stratified analysis by pH (cutoff 7.0) demonstrated consistent benefits in both pH>7.0 ( aOR=0.392, 95% CI:0.106-0.678) and pH≤7.0 subgroups ( aOR=0.385, 95% CI: 0.075-0.695; interaction P=0.183). Early IBP positively correlated with CPC scores ( ρ=0.40, P=0.007). Conclusions:Early IBP monitoring significantly improves survival and neurological outcomes in OHCA-ECPR patients, supporting its integration into standardized protocols.

Objective To discuss the preliminary application of CRISPR-Cas9 gene editing technology in repair of antithrombin gene(SERPINC1)c.318_319insT mutation.Methods The single guide RNA(sgRNA)was designed by CRISPR online design website,and AT c.318_319 insT mutant and CRISPR-Cas9 repairsome were constructed.The gene fragments from the wild-type gene,AT c.318_319 insT mutant and CRISPR-Cas9 repairsome were transferred into lentiviral expression vectors,and then PCR sequencing was performed for verification.The successfully constructed lentiviral recombinant plasmids were transfected into the human embryonic kid-ney cells(HEK293T).After cell culture,HEK293T cells were lysed.The AT:Ag levels in the cell lysing reagents from wild-type gene,CRISPR-Cas9 repairsome and mutant were compared by ELISA and Western blot,respectively.The recombinant AT protein was characterized in vitro by cellular immunofluorescence assay.Results Both the AT c.318_319insT mutant and CRISPR-Cas9 repair-some were successfully constructed.The results of experiments with HEK293T cells in vitro showed that the wild-type AT:Ag in the cell lysing reagents was set as 100％,the AT:Ag of CRISPR-Cas9 repairsome was 47％,and the AT:Ag of AT c.318_319insT was 22％,which were consistent with the results of western blot and cellular immunofluorescence assay.Conclusion The cellular experiments in vitro verified that CRISPR-Cas9 gene editing technology could effectively repair the SERPINC1 c.318_319 insT mutation in situ,which might provide the experimental support for the application of CRISPR-Cas9 gene editing technology in the gene therapy of hereditary thrombotic diseases.

Objective To investigate the relationship between serum trimethylamine oxide(TMAO),endo-thelial cell specific molecule 1(Endocan)and cardiac function and pregnancy outcome in patients with hyper-tensive disorders of pregnancy(HDP).Methods A total of 182 patients with HDP admitted to Handan Ma-ternal and Child Health Hospital from January 2021 to June 2023(HDP group)and 98 healthy pregnant women admitted to this hospital during the same period(control group)were selected as research subjects.Serum TMAO,Endocan and left ventricular cardiac function indexes[left ventricular ejection fraction(LVEF),left ventricular end-diastolic volume(LVEDV)and left ventricular end-systolic volume(LVESV)]were compared between the two groups.According to pregnancy outcome,HDP patients were divided into poor outcome group(78 cases)and good outcome group(104 cases).Spearman correlation analysis was used to analyze the correlation between serum TMAO and Endocan and cardiac function indexes in HDP patients,and multi-factor Logistic regression was used to analyze the influencing factors of adverse pregnancy outcomes in HDP patients.The predictive value of serum TMAO and Endocan for adverse pregnancy outcomes in HDP patients was analyzed by receiver operating characteristic(ROC)curve.Results Compared with control group,serum TMAO,Endocan,LVEDV and LVESV were increased in HDP group,and LVEF was decreased(P＜0.05).Serum TMAO and Endocan in HDP patients were negatively correlated with LVEF(P＜0.05),and positively correlated with LVEDV and LVESV(P＜0.05).The incidence of adverse pregnancy outcomes in 182 HDP patients was 42.86％(78/182).Preeclampsia(PE),severe preeclampsia(SPE),24 h urine protein increase,LVEDV increase,LVESV increase,TMAO increase,Endocan increase were independent risk factors for adverse pregnancy outcomes in HDP patients,and LVEF increase was protective factor(P＜0.05).The area under the curve of serum TMAO combined with Endocan in predicting adverse pregnancy outcomes in HDP patients was 0.880,which was greater than 0.793 and 0.788 predicted by serum TMAO and Endocan a-lone.Conclusion The increase of serum TMAO and Endocan levels in HDP patients are relate to the decrease of cardiac function and adverse pregnancy outcomes,and the combined detection of the two has high predictive value for adverse pregnancy outcomes in HDP patients.

Objective To explore the relationship between serum pregnancy-specific protein 1(PSG1),stress-induced protein 2(Sestrin 2),growth arrest-specific protein 6(Gas6)and uterine artery blood flow pa-rameters and fetal growth restriction(FGR)in patients with gestational hypertension(GH).Methods A to-tal of 485 GH patients admitted to Handan Maternal and Child Health Hospital from January 2020 to October 2023 were selected as the research objects and divided into the occurrence group(81 cases)and the non-occur-rence group according to whether FGR occurred.The correlations between serum PSG1,Sestrin 2,Gas6 and uterine artery blood flow parameters[pulse index(PI),resistance index(RI),ratio of peak systolic velocity to end diastolic velocity(S/D)]was analysed,as well as the related influencing factors of FGR in GH patients.In addition,a Nomogram model based on the influencing factors was constructed to analyze the predictive value.Results The serum PSG1 level in occurrence group was significantly lower than that in non-occurrence group,and the serum Sestrin 2,Gas6 levels and PI,RI,S/D values were significantly higher than those in non-occurrence group(P＜0.05).Pearson correlation results showed that serum PSG1 was negatively correlated with the uterine artery blood flow parameters PI,RI,and S/D,and the levels of serum Sestrin 2 and Gas6 were positively correlated with the uterine artery blood flow parameters PI,RI,and S/D(P＜0.05).Gestational di-abetes mellitus,umbilical cord abnormalities,high Sestrin 2,high Gas6,high PI,high RI,and high S/D were independent risk factors for the occurrence of FGR in GH patients(P＜0.05),and increased PSG1 level was protective factor for the occurrence of FGR in GH patients(P＜0.05).Receiver operating characteristic(ROC)curve analysis showed that the area under the curve(AUC)of the Nomogram prediction model con-structed based on the influencing factors for predicting the occurrence of FGR in GH patients was 0.982,and the sensitivity and specificity were 0.943 and 0.938,respectively.The internal verification of the Bootstrap method shows that the Bias-corrected prediction curve basically coincides with the Ideal line,and the consis-tency index(C-index)was 0.964,indicating that the model was relatively stable.The decision curve shows that the threshold probability of this model was 0.01-1.00 and the net return rate was above 0.Conclusion Ser-um PSG1,Sestrin 2 and Gas6 in GH patients are closely related to uterine artery blood flow parameters and FGR,and the three are the influencing factors for the occurrence of FGR in GH patients.The constructed No-mogram model has a good predictive efficacy for FGR.