Objective To construct a Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)long-fragment fusion protein plasmid;investigate the expression,extraction,and purification of CaMK Ⅱ;and identify its binding to the Cav1.2 channel.Methods The extracted pGEX-6p-1/CaMK Ⅱ long-fragment plasmid was transformed into Escherichia coli BL21 receptor cells and cultured in a shaking incubator for 12 h.Isopropyl β-D-thiogalactoside was added to promote GST fusion protein expression.Next,the GST-CaMK Ⅱ long frag-ment was isolated and purified with GS-4B using dithiothreitol(DTT)combined with ultrasonic crushing.After treatment with the PreScis-sion protease,the GST label was removed to obtain the CaMK Ⅱ long-fragment protein.The molecular weight and relative purity of the CaMKⅡ long-fragment protein were determined using 15％sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The concentration of the purified protein was determined using the Bradford method.The binding ability of the CaMK Ⅱ long-fragment pro-tein to the Cav1.2 channel protein was evaluated using the pull-down method combined with Western blotting.Results The sequencing results showed that the CaMK Ⅱ long fragment was successfully constructed.A CaMK Ⅱ long-fragment protein with high purity and con-centration was obtained using DTT combined with ultrasonic crushing.This protein can bind to the CT1 protein of cardiac Cav1.2 calcium channel.Conclusion In this study,we successfully constructed a CaMKⅡ long-fragment plasmid.The CaMKⅡ long-fragment protein was extracted and purified,and was determined to bind to Cav1.2 channel proteins and exhibit biological activity.Collectively,this study provides a basis for further study of the function of CaMK Ⅱ.

Objective To investigate the effect of the Shenghua Sanjia decoction on endometrial damage after induced abortion in rats by regulating the Fas/Fas ligand(FasL)signaling pathway.Methods Fifty pregnant rats were randomly assigned to five groups:normal con-trol,model,low-and high-dose Shenghua Sanjia decoction(12.6 g/kg and 25.2 g/kg,respectively),and dydrogesterone(3 mg/kg)groups,with 10 rats in each group.In all groups except the normal control group,rats were administered 16.6 mg/kg mifepristone combined with 100 μg/kg misoprostol to establish an artificial abortion model,and the endometrial injury model was established by a mechanical injury method.After administering the respective drug intervention,enzyme-linked immunosorbent assay(ELISA)was used to measure serum levels of estrogen(E2),progesterone(P),and prolactin(PRL).Histopathological changes of the endometrium were evaluated using HE staining.Masson staining was employed to assess endometrial fibrosis,whereas TdT-mediated dUTP nick-end labeling(TUNEL)was uti-lized to detect apoptosis in the endometrial tissue.Additionally,the mRNA expression levels of the estrogen receptor(ER),progesterone receptor(PR),prolactin receptor(PR LR),vascular endothelial growth factor(VEGF),Fas,and FasL in the endometrial tissue were measu-red using real-time quantitative PCR.Finally,Western blotting was performed to analyze the protein expression levels of transforming growth factor-β(TGF-β),matrix metalloproteinase-9(MMP-9),Fas,and FasL in the endometrial tissue.Results Compared to the normal control group,the model group showed a decreased E2,P,and PRL serum levels,a reduction in the mRNA expressions of ER,PR,PRLR,and VEGF as well as decreased protein expression of MMP-9 in the endometrial tissue.However,the apoptosis rate,mRNA expression of Fas and FasL,and protein expression of TGF-β,Fas,and FasL in the endometrial tissue were all increased(P＜0.05).Lastly,the endo-metrial tissue was found to be severely damaged with the collagen fibers increased.Compared to the model group,the low-and high-dose Shenghua Sanjia decoction and dydrogesterone groups showed an increase in the serum levels of E2,P,and PRL,as well as an elevation in the mRNA expression of ER,PR,PRLR,and VEGF;similarly the protein expression of MMP-9 in the endometrial tissue was increased.However,the apoptosis rate,mRNA expression of Fas and FasL,and protein expression of TGF-β,Fas,and FasL in the endometrial tissue were all decreased(P＜0.05).Conversely,the histopathological damage of the endometrium was found to be alleviated with the collagen fibers decreased.Conclusion The Shenghua Sanjia decoction can reduce endometrial fibrosis and apoptosis,while promoting endome-trial repair after the induction of abortion in rats.The underlying mechanism may be associated with the inhibition of the Fas/FasL signa-ling pathway activity.

Objective To explore the role of ferroptosis in DIC through bioinformatics analysis of hub genes involved in ferroptosis in doxorubicin-induced cardiomyopathy(DIC),combined with in vitro experimental validation.Methods Divalent iron fluorescence staining confirms the occurrence of ferroptosis in myocardial cells of DIC.The GSE207737 dataset was retrieved from the Gene Expression Comprehensive Database(GEO)and intersected with the FerrDb database to identify ferroptosis-related genes.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses of the intersected genes and intersecting the genes obtained from LASSO regression analysis and SVM-SFR machine learning methods were used to obtain ferroptosis hub genes for DIC.Real-time PCR was used to validate H9C2 cells in the control and DIC model groups,and Western blotting was used to further validate those whose bioinformatics and real-time PCR results that did not match.Results Thirty-eight ferroptosis-related genes in DIC were identified,and GO and KEGG analyses showed that these genes mainly participate in cell metabolism.Five hub genes for ferroptosis in DIC were obtained using machine learning methods:Mpc1,Prdx1,Kdm4a,Alox 12b,and Tfrc.Through in vitro experiments,the mRNA expression levels of Mpc1,Prdx1,and Kdm4a were downregulated in the DIC model group compared to those in the control group(P＜0.001),whereas the mRNA expression level of Alox12b was upregulated(P＜0.001).There were no significant differences in the mRNA or protein expression levels of Tfrc(P＞0.05).Conclusion Mpc1,Prdx1,Kdm4a,and Alox12b are key genes involved in ferroptosis in doxorubicin-induced cardiomyopathy and potential targets for the prevention and treatment of doxorubicin-induced cardiomyopathy in ferroptosis.

Objective To investigate the effect of macrophage polarization induced by colorectal cancer exosomes on the anti-tumor activity of CD8+T cells.Methods M0 macrophages were co-incubated with PBS,HT-29 cells,and LoVo exosomes(HT-29 and LoVo exo)for 48 h,and termed the PBS,HT-29 exo,and LoVo exo groups.Real-time quantitative polymerase chain reaction was performed to detect M2 macrophage biomarker CD206,Arginase-1,IL-10,and CD163 mRNA expressions as well as M1 macrophage biomarker iNOS and IL-1β mRNA expressions.CD8+T-cells were co-incubated with the macrophages of the aforementioned groups(PBS+M0,HT-29 exo+M0,and LoVo exo+M0 groups)for 48 h.Flow cytometry was performed to detect CD8+T PD-1 expression.Next,the PBS+M0,HT-29 exo+M0,and LoVo exo+M0 groups of CD8+T-cells were incubated with HT-29 or LoVo cells for 24 h,respectively(PBS+M0/CD8+T,HT-29 exo+M0/CD8+T,and LoVo exo+M0/CD8+T groups).Subsequently,the enzyme-linked immunosorbent assay(ELISA)was used to detect the concentration of IFN-γ,perforin,and granzyme B in the cell supernatant.The cell lysis rates of HT-29 and LoVo cells were detected through cytotoxicity experiments.Results Compared with the PBS group,CD206,Arginase-1,IL-10,and CD 163 mRNA expressions of macrophages in the HT-29 exo and LoVo exo groups were significantly upregulated(P＜0.05),whereas iNOS and IL-1βmRNA expressions were significantly downregulated(P＜0.05).Compared with the PBS+M0 group,the HT-29 exo+M0 and LoVo exo+M0 groups exhibited significantly increased PD-1 expression(P＜0.05).Compared with the PBS+M0/CD8+T group,the IFN-γ,perforin,and granzyme B levels in the cell culture supernatant of the HT-29 exo+M0/CD8+T and LoVo exo+M0/CD8+T groups were significantly reduced(P＜0.05),and the cell lysis rates of HT-29 and LoVo were significantly reduced(P＜0.001).Conclusion M2 macrophage induced by HT-29 and LoVo exo can inhibit the tumor-killing function of CD8+T cells.

Objective To investigate the correlation between seminal plasma oxidation-reduction potential(ORP)and normalized ORP(nORP)with sperm DNA fragmentation index(DFI),high DNA staining(HDS),and motility parameters,and to explore whether these parameters can be used as biomarkers for evaluating male fertility potential.Methods A total of 309 men who attended the andrology clinic at the Department of Reproductive Medicine,General Hospital of Northern Theater Command,between October 2023 and December 2023,were selected in this study.We analyzed the correlation between seminal plasma ORP and nORP and parameters such as DFI using the electrode method,HDS,age,days of abstinence,semen volume,total sperm count,sperm concentration,sperm motility(forward motile sperm count,forward motile sperm percentage,non-forward sperm count,non-forward sperm percentage,immotile sperm count,immotile sperm percentage,and sperm motility rate),and motility indices(curvilinear velocity[VCL],straight-line velocity[VSL],average velocity[VAP],linear index[LIN],straightness index[STR],and wobble coefficient[WOB]).The nORP results were divided into three groups and analyzed in relation to each of the parameters mentioned above.A generalized linear regression model was used to assess the relationship between nORP and HDS,with smooth curve fitting performed to visualize this relationship.Results Pearson and Spearman correlation analyses revealed that both ORP and nORP were positively correlated with semen volume,percentage of immotile sperm,and DFI,but negatively correlated with forward motile sperm count,percentage of forward motile sperm,sperm motility rate,VSL,and VAP(P＜0.05).Additionally,ORP was positively correlated with abstinence days and immotile sperm count(P＜0.05)and negatively correlated with LIN,STR,and WOB(P＜0.05).However,nORP was positively correlated with HDS(P＜0.05),and negatively correlated with total sperm count,sperm concentration,non-forward sperm count,non-forward sperm percentage,immotile sperm count,and VCL(P＜0.05).The nORP group showed significant differences in semen volume,total sperm count,sperm concentration,forward motile sperm count,forward motile sperm percentage,non-forward sperm count,non-forward sperm percentage,immotile sperm count,immotile sperm percentage,sperm motility rate,VCL,VSL,VAP,DFI,and HDS(P＜0.05).A smooth curve fitting of nORP and HDS,adjusted for age and abstinence days,demonstrated a linear upward trend in nORP as HDS increased.Conclusion Both ORP and nORP are associated with sperm DFI and key motility parameters.Moreover,nORP is closely related to HDS,which reflects sperm maturity,offering a more comprehensive indicator of semen quality.Thus,nORP can be utilized as a biomarker for assessing male fertility potential.

Microbiota can be recognized by the human immune system,trigger specific immune responses,and play a significant role in the onset and progression of diseases through intricate regulatory mechanisms.The tumor microenvironment comprises the peritumoral extracellular matrix,immune cells,tumor-associated fibroblasts,and endothelial cells,among others.In this context,the tumor immune microenvironment,characterized by immune cells,significantly influences cancer progression and treatment.Notably,the tumor immune microenvironment is closely associated with the tumor microbiota.The presence of respiratory microbiota is correlated with the typing and staging of non-small cell lung cancer.This study investigated the mechanisms by which respiratory microbiota affect innate and adaptive immune cells within the tumor immune microenvironment and explored the role of respiratory microbiota in the immunotherapy of non-small cell lung cancer.The aim was to identify potential biomarkers that could enhance the diagnosis and treatment of non-small cell lung cancer.

Objective To establish a cellular model of Parkinson disease by treating retinoic acid(RA)-differentiated SH-SY5Y cells with α-synuclein preformed fibrils(α-Syn PFF).Methods SH-SY5Y cells were divided into undifferentiated and RA-differentiated groups.The expression levels of tyrosine hydroxylase(TH),dopamine transporter(DAT),lymphocyte activation gene 3(LAG3),and Nestin proteins in the cells were detected using Western blotting,whereas those of microtubule-associated protein 2(MAP2)and neu-ronal nuclei(NeuN)were detected using immunofluorescence staining.Furthermore,RA-treated SH-SY5Y cells were divided into control and α-Syn PFF groups,and their levels of chromatin condensation were detected using Hoechst 33342 staining.Nitric oxide(NO)levels were measured using a NO assay kit.Additionally,the protein levels of TH,poly(ADP-ribose)(PAR),and poly(ADP-ribose)poly-merase(PARP)in these cells were detected using Western blotting,whereas their expression levels of phosphorylated α-Syn(pS129-α-Syn)and phosphorylated histone H2AX(γH2AX)were detected using immunofluorescence staining.Results Treatment with RA resulted in a reduction in cell body size and the elongation of protrusions in SH-SY5Y cells.The results of Western blotting showed that RA treatment could increase the TH,DAT,and LAG3 levels and decrease the Nestin level in SH-SY5Y cells(P＜0.05).α-Syn PFF treat-ment decreased the TH protein level and increased the PAR,PARP-l,and cleaved PARP-1 levels in differentiated SH-SY5Y cells(P＜0.05).According to the immunofluorescence results,RA treatment increased the expression levels of MAP2 and NeuN in SH-SY5Y cells(P＜0.001).The α-Syn PFF treatment increased the expression levels of γH2AX and pS129-α-Syn in RA-differentiated SH-SY5Y cells(P＜0.01).The Hoechst 33342 staining results showed that α-Syn PFF treatment led to chromatin condensation in the differentiated SH-SY5Y cells(P＜0.001)and increased the NO levels(P＜0.01).Conclusion A cellular model of Parkinson disease can be established by treating RA-differentiated SH-SY5Y cells with α-Syn PFF.

Objective To explore the role of icariin on diabetic osteoporosis(DOP)in rats by regulating estrogen reoeptor 1(ER1).Methods Fifty SD rats were randomly divided into five groups:control,diabetes(STZ),low-dose icariin[four weeks after successful establishment of diabetic model rats,50 mg/(kg·d)icariin was administered by gavage for eight weeks],high-dose icariin[100 mg/(kg·d)icariin administered by gavage for eight weeks],and high-dose icariin+ER1-siRNA group[icariin 100 mg/(kg·d)administered by gavage for eight weeks.A total of 10 μL ER1-siRNA was injected via the tail vein once every four weeks,two times in total],with 10 rats in each group.HE staining was used to detect the pathological morphology of the femoral tissue.Enzyme-linked immunosorbent assay(ELISA)was used to measure serum ALP,TRAP,and blood calcium concentrations.Protein-protein interaction(PPI)network analysis predicted icariin core targets and validated molecular docking.ER1,PDE5A,Wnt,and β-cantenin expression levels in rat femoral tissue were detected using Western blotting.Results In the STZ group,the number of empty lacunae in the subchondral region increased,ALP expression and blood calcium concentration decreased(P＜0.01),and TRAP expression increased(P＜0.01),compared with the control group.In the low-and high-dose icariin groups,blood glucose levels decreased(P＜0.01),the number of empty bone lacunae in the subchondral region decreased,ALP expression and blood calcium concentration increased(P＜0.01),and TRAP expression decreased(P＜0.01),compared with the STZ group.PPI network analysis and molecular docking showed that icariin stably bound to the ER1 protein structure.Com-pared with the control group,expression of ER1,Wnt,and β-cantenin decreased in STZ group(P＜0.01).The expression of ER1,Wnt,and β-cantenin in the high-dose icariin group increased compared with the STZ group(P＜0.01).Moreover,expression of ER 1,Wnt,andβ-cantenin decreased in the high-dose icariin+ER1-siRNA group,compared with the high-dose icariin group(P＜0.01).The protective effects of icariin were reversed by ER1-siRNA administration.Conclusion Icariin could significantly improve osteoporosis in diabetes rats,which may be relevant to the activation of the ER 1-mediated Wnt/β-cantenin pathway.

Objective To discuss the risk factors for impaired fasting glucose(IFG)and construct and validate a predictive model based on column charts of the risk of IFG occurrence.Methods This retrospective study included 3 037 individuals who underwent routine physical examinations at a hospital in Shenyang between August and December 2022.The population was randomly divided into a training group(n=2 126)and a validation group(n=911)in a 7∶3 ratio,and physical examination data were collected.LASSO regression analy-sis was used to screen predictive variables and logistic regression analysis was used to further screen and construct a column chart pre-dictive model.The validation group was used to conduct an internal validation of the feasibility of the model,and the area under the curve(AUC)of receiver operator characteristic(ROC)and goodness of fit tests were used to evaluate the model effectiveness.Results Among the 3 037 included individuals,2 880 did not experience IFG and 157 did.The results showed that age(OR=1.04,95％CI:1.02-1.05),body mass index(BMI,OR=1.10,95％CI:1.05-1.17),systolic blood pressure(SBP,OR=1.01,95％CI:1.00-1.03),triglycerides(TG,OR=1.20,95％CI:0.99-1.51),and a history of hypertension(OR=1.28,95％CI:1.04-1.59)were independent risk factors for IFG occurrence in this population.Based on these variables,a column chart prediction model was constructed.In the training group,the model predicted an AUC of 0.722(95％CI:0.68-0.77)for IFG occurrence,while in the validation group,it predicted an AUC of 0.907(95％CI:0.87-0.94)for IFG occurrence.The results of the Hosmer-Lemeshow goodness of fit test showed that the models of the training and validation groups were not significantly different(P＞0.05);that is,the actual probability was consistent with the prediction probability of the model,and the models calibration was good.Conclusion A risk prediction model for IFG occurrence that included five variables:age,BMI,SBP,TG,and history of hypertension could be construted.This model might help to identify high-risk groups for IFG early and allow for inter-vention in a timely manner.

Objective To investigate the role of reactive oxygen species(ROS)and the extracellular signal-regulated kinase(ERK)/cAMP response element binding protein(CREB)signaling pathway in arsenic-induced apoptosis in HT-22 cells.Methods HT-22 cells were cultured in vitro and exposed to NaAsO2.The cells were divided into the following groups:control group,4,6,8,and 10 μmol/L NaAsO2 groups,NAC(5 mmol/L)group,and NAC(5 mmol/L)+NaAsO2(10 μmol/L)group.ROS levels were measured using a DCFH-DA assay.The expression of ERK/CREB signaling pathway-related and apoptosis-related proteins were analyzed by Western blotting.Apop-tosis rates were evaluated using flow cytometry.Results Compared with the control group,ROS levels in HT-22 cells significantly increased,while p-ERK protein level in the nucleus and p-CREB and Bcl-2 protein levels in the cells significantly decreased in the NaAsO2 only exposed groups(P＜0.05).Additionally,the apoptosis rate significantly increased in the 8 and 10 μmol/L NaAsO2 groups compared with the control group(P＜0.05).In the NAC+NaAsO2 group,ROS levels and the apoptosis rate significantly decreased while p-ERK protein level in the nucleus and p-CREB and Bcl-2 protein levels in the cells significantly increased compared with the 10 μmol/L NaAsO2 group(P＜0.05).Conclusion Arsenic exposure induces oxidative stress,inhibits the nuclear translocation of p-ERK,and dis-rupts the CREB signaling pathway,leading to apoptosis.