Functional constipation （FC） is a common functional disorder of the intestines， mainly characterized by reduced bowel movement frequency， difficulty in defecation， a sensation of incomplete evacuation， and hard stools， which severely affect patients’ quality of life. Research indicates that the pathogenesis of FC is closely related to gut microbiota dysbiosis and abnormal bile acid secretion. Bile acids， as endogenous natural laxatives， promote bowel movements by enhancing colonic secretion and regulating intestinal motility； meanwhile， gut microbiota influence colonic transit function by regulating the enteric nervous system， immune system， and their metabolic products. Based on an overview of the relationship between gut microbiota and bile acid metabolism， this article systematically reviews the current research status on the mechanisms of traditional Chinese medicine （TCM） in treating FC by regulating the balance of the gut microbiota-bile acid axis. It is found that single Chinese medicinal herbs （such as Atractylodes macrocephala）， isolated compounds （such as Platycodon grandiflorum polysaccharides）， herbal formulas （such as Shanger huang pill）， acupuncture， and moxibustion can up-regulate the abundance of beneficial bacteria， reshape the microbial structure， correct bile acid metabolism， and activate the Takeda G-protein receptor 5/farnesoid X receptor pathway to treat FC.

ObjectiveTo construct a stable monoclonal human embryonic kidney 293 （HEK293） cell line expressing recombinant human coagulation factor Ⅶ （rhFⅦ） and evaluate the expression level and procoagulant bioactivity of rhFⅦ. MethodsThe plasmid pCDNA3.1-EGFP-FⅦ was transfected into HEK293 cells to verify the effectiveness of the transfection system. The plasmid pCDNA3.1-FⅦ was transfected into HEK293 cells， and monoclonal stable transfected cell lines were selected using geneticin （G418）. The transcription of the FⅦ gene was identified by reverse transcription polymerase chain reaction （RT-PCR）. The expression level of rhFⅦ in the supernatant of the monoclonal stable transfected cell line was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The concentration of rhFⅦ was determined by enzyme-linked immunosorbent assay （ELISA）， and the procoagulant activity of rhFⅦ was measured by human coagulation factor Ⅶ potency assay. ResultsHEK293 cells transfected with pcDNA3.1-EGFP-FⅦ showed green fluorescence， indicating that rhFⅦ was successfully expressed in the supernatant of HEK293 cells after transient transfection with pcDNA3.1-FⅦ. The monoclonal stable transfected cell line was obtained by G418 screening. RT-PCR identified that the FⅦ gene was integrated into the genome of the monoclonal stable transfected cell line. The cell viability was good as detected by Cell Counting Kit-8， and a single band of rhFⅦ was obtained by purification of the cell supernatant. The highest rhFⅦ expression was （1.27±0.09） mg/L， and the highest procoagulant activity was （380.29±13.80）%. ConclusionThe monoclonal HEK293 cell lines which can express rhFⅦ protein efficiently and stably with excellent procoagulant bioactivity is successfully screened.

Background 900 MHz radiofrequency radiation (RF) is a commonly used frequency in modern wireless communication devices, and its potential health effects have drawn much attention, especially its impact on bone metabolism, which has not been fully clarified. Objective To investigate the effects of 900 MHz RF on the bone tissue and osteoblast senescence of mice, as well as the dose-effect relationship. Methods In vivo, 3-month-old female C57BL/6 mice were divided into five groups (n=10): sham exposure, low-dose RF (50 μW·cm⁻²), medium-dose RF (150 μW·cm⁻²), high-dose RF (450 μW·cm⁻²), and D-galactose positive control (D-gal). Treatments were administered for 4 h per day for 28 d. Bone mineral density (BMD) and microstructure, including bone volume (BV), tissue volume (TV), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th), were assessed by Micro-CT; bone morphology was examined after hematoxylin and eosin (HE) staining; osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-κΒ ligand (RANKL) expression was detected by immunohistochemistry; serum OPG, tartrate-resistant acid phosphatase 5b (TRACP-5b), plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6), and C-X-C motif chemokine ligand 15 (CXCL15) levels were measured by enzyme-linked immunosorbent assay (ELISA); mRNA expression of Tp53, Cdkn1a, and Cdkn2a in bone tissue was analyzed by reverse transcription polymerase chain reaction (RT-PCR). In vitro, MC3T3-E1 pre-osteoblasts were grouped into sham, low-dose RF (50 μW·cm⁻²), medium-dose RF (150 μW·cm⁻²), high-dose RF (450 μW·cm⁻²), and H₂O₂ control, groups, and were exposed for 4 h per day for 5 d. Cell morphology was observed by microscopy; viability was tested by cell counting kit-8 (CCK-8); senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining; P53 and P21 protein expression was detected by Western blot; Tp53 and Cdkn1a mRNA levels were measured by RT-PCR. Results In vivo, RF at each dose significantly reduced the BMD of the mice's femurs and the bone microstructure parameters, such as BV/TV, Tb.N, and Tb.Th (P<0.05). Among them, Tb.Sp only increased in the 150 μW·cm⁻² RF group (P<0.05), with a looser bone network; fewer, sparser trabeculae and increased marrow fat were observed after HE staining; down-regulated OPG and up-regulated RANKL expression levels were observed by immunohistochemistry; the ELISA test revealed that the serum OPG levels in the 150 μW·cm⁻² RF group and the 450 μW·cm⁻² RF group of mice were significantly decreased (P<0.05), while the indicator in the 50 μW·cm⁻² RF group showed a decreasing trend but the difference was not statistically significant (P>0.05), TRACP-5b rose, and PAI-1, IL-6, and CXCL15 levels increased (P<0.05); the RT-PCR results showed thatTp53, Cdkn1a, and Cdkn2a mRNA expression was upregulated (P<0.05). In vitro, radiofrequency radiation induced cell flattening, reduced viability (P<0.05), increased SA-β-gal-positive cells (P<0.05), and upregulated P53, P21, Tp53, and Cdkn1a expression (P<0.05). Conclusion 900 MHz RF disrupts bone metabolism in mice by inhibiting bone formation, promoting resorption, and inducing osteoblast senescence, accelerating bone aging. The 150 μW·cm⁻² RF dose exhibits the most pronounced effect, reflecting a nonlinear “window effect,” highlighting potential health risks.

Objective: To determine the association between exposure to entertainment screen content on mobile phones and symptoms of anxiety-depression co-morbidity among college students,so as to provide evidence for mental health interventions. Methods: A baseline survey was conducted from April to May 2019. A total of 1 135 college students were selected from one university each in Shangrao City, Jiangxi Province and Hefei City, Anhui Province using cluster random sampling method. A follow up study was conducted in November 2019, resulting in 1 110 matched valid responses. Self rating questionnaires were used to assess the exposure of entertainment screen content. The Depression Anxiety Stress Scale-21(DASS-21) and the Patient Health Questionnaire-9 (PHQ-9) were used to evaluate the anxiety symptoms, depressive symptoms, and symptoms of anxiety-depression co-morbidity among college students. A multivariate binary Logistic regression model was constructed following initial intergroup comparisons with Chi-square test to determine the association between baseline exposure to mobile entertainment screen content and the risk of symptoms of anxiety depression co-morbidity at baseline and the 6 month follow up. Results: The prevalence rates of symptoms of anxiety-depression co-morbidity among college students were 25.4% and 20.6% at baseline and follow up, respectively.After adjusting for confounding factors such as gender, self rated family economic status and self rated health status, the results of multivariate binary Logistic regression analysis showed that compared with the appropriate exposure level group, the exposure of entertainment screen content on mobile phones at baseline, including frequent exposure to reading( OR =1.65,95% CI =1.14-2.39), occasional exposure to other entertainment screen content ( OR =1.46,95% CI =1.01-2.10)and frequent exposure to other entertainment screen content( OR =1.76,95% CI =1.20-2.60), increased the co-occurrence risk of symptoms of anxiety-depression co-morbidity among college students during the follow up period (all P <0.05). Conclusion Occasional or frequert exposure to mobile entertainment screen content can increase the risk of symptoms of anxiety depression co-morbidity among college students.

BACKGROUND:Choline kinase alpha is a key enzyme in phospholipid metabolism,involved in the synthesis of phosphatidylcholine,and plays an important role in maintaining cell membrane integrity and signal transduction.Research has shown that choline kinase alpha is highly expressed in various tumors and is closely related to cell proliferation,metabolic reprogramming,and tumor progression.As a potential therapeutic target,the role of choline kinase alpha in tumor metabolism and mitochondrial function still needs further exploration.OBJECTIVE:To evaluate the effects and the underlying mechanisms of choline kinase alpha on the proliferation and apoptosis of glioma U87MG and U251 cells.METHODS:Short hairpin RNA of choline kinase alpha and its empty vector control were transfected into U87MG and U251 glioma cells.Mitochondrial morphology was observed by transmission electron microscopy.Mitochondrial structure and functional protein levels were assessed by western blot assay.Reactive oxygen species levels in cells were measured using a reactive oxygen species fluorescent probe.Mitochondrial membrane potential was assessed with a JC-1 assay.Intracellular adenosine triphosphate levels were measured by chemiluminescence.Cell proliferation was evaluated using a CCK-8 assay.Apoptosis levels were analyzed by flow cytometry.The mitochondrial fission inhibitor Mdivi-1 was used to protect the mitochondrial function of the choline kinase α-silenced lentiviral cells.Finally,U87MG cells were subcutaneously injected to construct a subcutaneous tumor model in nude mice.The tumor growth in nude mice was observed before and after choline kinase alpha silencing and after the use of the mitochondrial fission inhibitor Mdivi-1.RESULTS AND CONCLUSION:(1)Compared with the empty control group,the mitochondria of U87MG and U251 cells in the choline kinase alpha silencing lentivirus group exhibited significant structural abnormalities in mitochondria,such as vacuolization and cristae disruption.The expressions of mitochondrial structure and function-related proteins TOM20,ACO2,and ATP5A were significantly decreased(P＜0.01,P＜0.001),the expression of SOD2 was significantly increased(P＜0.01,P＜0.000 1),the fluorescence intensity of reactive oxygen species was significantly increased(P＜0.01),the mitochondrial membrane potential and adenosine triphosphate level were significantly decreased(P＜0.01,P＜0.001),the cell proliferation ability was reduced(P＜0.01),and the apoptosis level was increased(P＜0.001).(2)Following Mdivi-1 treatment,the fluorescence intensity of reactive oxygen species in U87MG and U251 cells decreased(P＜0.05,P＜0.01),mitochondrial membrane potential and adenosine triphosphate levels were significantly restored(P＜0.05,P＜0.01,P＜0.001),cell proliferation ability was improved(P＜0.05,P＜0.01),and apoptosis level was decreased(P＜0.05).(3)In addition,the in vitro subcutaneous tumor formation experiment of nude mice showed that compared with the empty control group,the mass and growth rate of subcutaneous tumors formed by U87MG cells in the choline kinase alpha silencing lentivirus group were significantly reduced(P＜0.000 1).After Mdivi-1 treatment,the mass and growth rate of tumors were significantly increased(P＜0.000 1).(4)The results show that choline kinase alpha silencing affects the proliferation and apoptosis of glioma cells by inducing mitochondrial dysfunction.

BACKGROUND:Choline kinase alpha is a key enzyme in phospholipid metabolism,involved in the synthesis of phosphatidylcholine,and plays an important role in maintaining cell membrane integrity and signal transduction.Research has shown that choline kinase alpha is highly expressed in various tumors and is closely related to cell proliferation,metabolic reprogramming,and tumor progression.As a potential therapeutic target,the role of choline kinase alpha in tumor metabolism and mitochondrial function still needs further exploration.OBJECTIVE:To evaluate the effects and the underlying mechanisms of choline kinase alpha on the proliferation and apoptosis of glioma U87MG and U251 cells.METHODS:Short hairpin RNA of choline kinase alpha and its empty vector control were transfected into U87MG and U251 glioma cells.Mitochondrial morphology was observed by transmission electron microscopy.Mitochondrial structure and functional protein levels were assessed by western blot assay.Reactive oxygen species levels in cells were measured using a reactive oxygen species fluorescent probe.Mitochondrial membrane potential was assessed with a JC-1 assay.Intracellular adenosine triphosphate levels were measured by chemiluminescence.Cell proliferation was evaluated using a CCK-8 assay.Apoptosis levels were analyzed by flow cytometry.The mitochondrial fission inhibitor Mdivi-1 was used to protect the mitochondrial function of the choline kinase α-silenced lentiviral cells.Finally,U87MG cells were subcutaneously injected to construct a subcutaneous tumor model in nude mice.The tumor growth in nude mice was observed before and after choline kinase alpha silencing and after the use of the mitochondrial fission inhibitor Mdivi-1.RESULTS AND CONCLUSION:(1)Compared with the empty control group,the mitochondria of U87MG and U251 cells in the choline kinase alpha silencing lentivirus group exhibited significant structural abnormalities in mitochondria,such as vacuolization and cristae disruption.The expressions of mitochondrial structure and function-related proteins TOM20,ACO2,and ATP5A were significantly decreased(P＜0.01,P＜0.001),the expression of SOD2 was significantly increased(P＜0.01,P＜0.000 1),the fluorescence intensity of reactive oxygen species was significantly increased(P＜0.01),the mitochondrial membrane potential and adenosine triphosphate level were significantly decreased(P＜0.01,P＜0.001),the cell proliferation ability was reduced(P＜0.01),and the apoptosis level was increased(P＜0.001).(2)Following Mdivi-1 treatment,the fluorescence intensity of reactive oxygen species in U87MG and U251 cells decreased(P＜0.05,P＜0.01),mitochondrial membrane potential and adenosine triphosphate levels were significantly restored(P＜0.05,P＜0.01,P＜0.001),cell proliferation ability was improved(P＜0.05,P＜0.01),and apoptosis level was decreased(P＜0.05).(3)In addition,the in vitro subcutaneous tumor formation experiment of nude mice showed that compared with the empty control group,the mass and growth rate of subcutaneous tumors formed by U87MG cells in the choline kinase alpha silencing lentivirus group were significantly reduced(P＜0.000 1).After Mdivi-1 treatment,the mass and growth rate of tumors were significantly increased(P＜0.000 1).(4)The results show that choline kinase alpha silencing affects the proliferation and apoptosis of glioma cells by inducing mitochondrial dysfunction.

ObjectiveTo explore the feasibility of constructing a programmed death receptor-1（PD-1） molecular probe for non-invasive micro-positron emission tomography/computed tomography （Micro-PET/CT） imaging of PD-1 protein in mouse S180 sarcoma. MethodsA transgenic PD-1 C57 S180 sarcoma mouse model was established using the S180 sarcoma cell injection. Furthermore， ¹²⁴I-anti-PD-1 monoclonal antibody probe was synthesized. 18.5 MBq of the ¹²⁴I-anti-PD-1 probe was injected into the tail vein of transgenic PD-1 C57 mice. Subsequently， S180 sarcoma was imaged using Micro-PET/CT. ResultsStudy successfully established a transgenic PD-1 C57 S180 sarcoma mouse model. Immunohistochemical （IHC） results showed PD-1 protein expression in S180 sarcoma. Micro-PET/CT imaging successfully visualized the PD-1 protein receptor in S180 sarcoma at different time points （20， 48， 72， and 120 h） after probe injection. ConclusionThe ¹²⁴I-anti-PD-1 monoclonal antibody molecular probe successfully targets the PD-1 receptor in S180 sarcoma of transgenic PD-1 C57 mice， and presents clear Micro-PET/CT immunoassay results， thus it potentially enables the non-invasive screening of patients with PD-1 positive malignant tumors.

low transit constipation （STC） is a common functional intestinal disorder caused by impaired colonic transit function， characterized by reduced bowel movement frequency， hard stools， and difficulty in defecation. The mitogen-activated protein kinase （MAPK） signaling pathway， which mainly includes extracellular signal-regulated kinase （ERK）， c-Jun N-terminal kinase （JNK）， and p38 subtypes， plays a critical regulatory role in the occurrence and development of STC. This paper systematically reviews the multiple pathogenic mechanisms of the MAPK signaling pathway in STC and the research progress of traditional Chinese medicine （TCM） intervention.At the mechanistic level， the MAPK signaling pathway promotes the progression of STC through the following links：（1） Activation of p38 upregulates the expression of aquaporin 3 （AQP3）/AQP4 in the colon， leading to excessive reabsorption of water in the intestinal lumen； （2） It forms a positive feedback loop with nuclear factor-κB （NF-κB） to maintain low-grade intestinal inflammation， releases inflammatory factors such as tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β）， and inhibits smooth muscle contraction； （3） Overactivation of p38 downregulates the expression of occludin and mucin 2 while upregulates the expression of claudin-2， thereby disrupting the mucosal barrier； （4） The JNK/p38 signaling pathway activates the caspase cascade to induce apoptosis of intestinal epithelial cells， neurons， and interstitial cells of Cajal； （5） Abnormal ERK signaling and excessive activation of p38/JNK inhibit intestinal smooth muscle contraction and reduce 5-hydroxytryptamine secretion， ultimately resulting in impaired colonic transit function.At the intervention level， TCM compound formulas and single herbs have been proven to improve STC by regulating the MAPK signaling pathway. Their effects are syndrome type-dependent：yin-nourishing formulas （Zengye Chengqi Tang， Tongbian Tang） mainly regulate the ERK/AQP axis； yang-warming formulas （Jichuan Jian） target both ERK/JNK and anti-apoptosis； heat-clearing formulas （Sanren Tang） focus on p38/NF-κB anti-inflammation. A single drug can simultaneously cover multiple aspects including water metabolism， inflammation， barrier function， apoptosis， and intestinal motility.Current relevant studies still have limitations such as mechanisms mostly remaining at the correlational level and a lack of disease-syndrome integrated research models. Future studies should combine specific inhibitors or gene knockout to identify core targets， establish disease-syndrome integrated STC models， and use network pharmacology and molecular docking techniques to deeply analyze the fine mechanism of “component-target-phenotype”， so as to provide high-quality evidence for the precise regulation of the MAPK signaling pathway by TCM in the intervention of STC.

[Objective] To explore the temperature parameters affecting the polybrene test and determine the optimal temperature conditions for detecting unexpected antibodies of the Rh system. [Methods] The reaction of IgG human anti-D antibody with different dilutions (undiluted, 1∶2, 1∶4, 1∶8, 1∶16, 1∶32，1∶64) with D antigen-positive red blood cells was detected by manual polybrene test (MPT). Different temperatures (25℃ and 37℃) were set, and the reaction time with low ionic medium was 4 minutes. The agglutination integral value of anti-D and red cell depolymerization time were compared to observe the effect of enhanced agglutination reaction, thereby establishing the test temperature reaction conditions for enhancing the MPT. The same reaction condition was applied to 36 blood samples containing unexpected antibodies of the Rh system, and the effect of enhanced MPT was observed in comparison with the polybrene method and the antiglobulin test (column agglutination). [Results] With all other conditions held constant, when low ionic medium was added, the incubation temperature of 25℃ and 37℃ resulted in different total agglutination integral values for anti-D (20.9±2.025 vs 25.5±2.635), and the comparison showed a significant difference (P<0.05). When the antibody dilution was 1∶16, the incubation temperature of 25℃ and 37℃ resulted in different agglutination integral values (3.9±0.738 vs 5.8±0.632), and the comparison showed a significant difference (P<0.05). Erythrocyte depolymerization time (62.8±8.149 vs 90.1±10.713) was significantly different (P<0.05). At a dilution of 1∶32, the incubation temperatures of 25℃ and 37℃ resulted in different agglutination integral values (2.5±0.527 vs 4.3±0.675), as well as different red blood cell dissociation times (35.4±7.792 vs 57.4±10.885)(P<0.05), and the comparison showed a significant difference (P<0.05), with no differences observed in the other groups. In the detection of 36 Rh system unexpected antibody samples, when the antibody titer was ≤2, the enhanced polybrene method had a higher positive rate, and when the antibody titer was ≥4, the detection rates of the three methods were consistent. [Conclusion] The reference temperature condition for the modified MPT is incubation at 37℃ for 4 min after the addition of low ionic medium. The application of this temperature condition to unexpected antibody samples of Rh system could achieve a significant enhancement effect, thereby increasing transfusion safety for the treatment of emergency patients, and is worth popularizing.

ObjectiveTo investigate the role of paraventricular nucleus (PVN) corticotropin releasing hormone (CRH) neurons in chronic restraint stress (CRS)-induced anxiety-like behavior. And whether exercise relieves chronic restraint stress-induced anxiety through PVN CRH neurons. MethodsTwenty 8-week-old male C57BL/6J mice were randomly divided into control (Ctrl) group and chronic restraint stress (CRS) group. The open field test (OFT) and elevated plus maze (EPM) were used to evaluate anxiety-like behavior of the mice. Food intake was recorded after CRS. Immunofluorescence staining was used to label the expression of c-Fos expression in PVN and calculate the co-expression of c-Fos and CRH neurons. We used chemogenetic activation of PVN CRH neurons to observed the anxiety-like behavior. 8-week treadmill training (10-16 m/min, 60 min/d, 6 d/week) were used to explore the role of exercise in ameliorating CRS-induced anxiety behavior and how PVN CRH neurons involved in it. ResultsCompared with Ctrl group, CRS group exhibited significant anxiety-like behavior. In OFT, the mice in CRS groups spent less time in center area (P<0.001). In EPM, the time in open arm in CRS group were significantly decreased (P<0.001). Besides, food intake was also suppressed in CRS group compared with Ctrl group (P<0.05). Compared with Ctrl group, CRS significantly increase c-Fos expression in PVN and most of CRH neurons co-express c-Fos (P<0.001). Chemogenetic activation of PVN CRH neurons induced anxiety-like behavior (P<0.05) and inhibited feeding behavior (P<0.01). Exercise relieves chronic restraint stress-induced anxiety (P<0.001) and relieved the anorexia caused by chronic restraint stress (P<0.05). Aerobic exercise inhibited the CRS labeled c-Fos in PVN CRH neurons (P<0.001). Furthermore, ablation of PVN CRH neurons attenuated CRS induced anxiety-like behavior. ConclusionCRS activated PVN CRH neurons, induced anxiety-like behavior and reduced food intake. 8-week exercise attenuated CRS-induced anxiety-like behavior through inhibiting PVN CRH neuron. Ablation of CRH PVN neurons ameliorated CRS-induced anxiety-like behavior. These finding reveals a potential neural mechanism of exercise-relieving CRS-induced anxiety-like behavior. This provides a new idea and theoretical basis for the treatment of anxiety and related mental disorders.