This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

Insulin-like growth factor 2 (IGF2) is a critical endocrine mediator implicated in male reproductive physiology. To investigate the correlation between IGF2 protein levels and various aspects of male infertility, specifically focusing on sperm quality, inflammation, and DNA damage, a cohort of 320 male participants was recruited from the Women's Hospital, Zhejiang University School of Medicine (Hangzhou, China) between 1 st January 2024 and 1 st March 2024. The relationship between IGF2 protein concentrations and sperm parameters was assessed, and Spearman correlation and linear regression analysis were employed to evaluate the independent associations between IGF2 protein levels and risk factors for infertility. Enzyme-linked immunosorbent assay (ELISA) was used to measure IGF2 protein levels in seminal plasma, alongside markers of inflammation (tumor necrosis factor-alpha [TNF-α] and interleukin-1β [IL-1β]). The relationship between seminal plasma IGF2 protein levels and DNA damage marker phosphorylated histone H2AX (γ-H2AX) was also explored. Our findings reveal that IGF2 protein expression decreased notably in patients with asthenospermia and teratospermia. Correlation analysis revealed nuanced associations between IGF2 protein levels and specific sperm parameters, and low IGF2 protein concentrations correlated with increased inflammation and DNA damage in sperm. The observed correlations between IGF2 protein levels and specific sperm parameters, along with its connection to inflammation and DNA damage, underscore the importance of IGF2 in the broader context of male reproductive health. These findings lay the groundwork for future research and potential therapeutic interventions targeting IGF2-related pathways to enhance male fertility.
Humans ; Male ; Insulin-Like Growth Factor II/metabolism* ; Infertility, Male/genetics* ; DNA Damage ; Adult ; Inflammation/metabolism* ; Spermatozoa/metabolism* ; Semen Analysis ; Semen/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Histones/metabolism* ; Interleukin-1beta/metabolism*

An assay was established for detection of toxigenic Clostridioides difficile by combining microfluidic chip analysis with immunochromatography,and its performance was evaluated and compared with those of the Xpert C.difficile/Epi and VIDAS CD AB tests.Primer pairs were designed according to the tcdB and tpi genes in C.difficile.The specificity,limit of detection,reproducibility,and stability were evaluated.A total of 215 stool samples from patients with diarrhea were collected and tested in parallel with the Xpert C.difficile/Epi,VIDAS CDAB,and our assay.C.difficile was isolated from samples,and the tcdB gene was identified when discrepant results were obtained from the three above assays.Our assay showed no cross-reaction with other diarrhea-associated pathogens.Its reproducibility was 100％in testing of two standard plasmids containing tcdB and tpi genes at two concentrations(105 and 102 copies/μL).Two standard plasmids were detected after the PCR and immunochromatography reagents had been stored for 3,6,9,and 12 months,and all the results were posi-tive.The limit of detection was 10 copies/μL for toxigenic C.difficile.Testing of 33 samples positive for C.difficile with our assay(33/215,15.3％)yielded findings statistically coherent with those of the Xpert C.difficile/Epi test(kappa value=0.965).The sensitivity,specificity,positive predictive value,and negative predictive value of our assay,with respect to Xpert C.difficile/Epi as the standard,were 94.3％,100.0％,100.0％,and 98.9％;these values were significantly higher than those of VIDAS CDAB(60.0％,98.9％,91.3％,and 92.7％)(Kappa=0.714,OR=157.50,95％CI:62.03-847.28,P=0.013).In conclusion,our newly developed assay is specific,stable,and reproducible,and may be used for rapid and accu-rate detection of toxigenic C.difficile.The assay could be used for C.difficile infection screening in outpatient and emergen-cy,community medical service center,and epidemiological settings.

An assay was established for detection of toxigenic Clostridioides difficile by combining microfluidic chip analysis with immunochromatography,and its performance was evaluated and compared with those of the Xpert C.difficile/Epi and VIDAS CD AB tests.Primer pairs were designed according to the tcdB and tpi genes in C.difficile.The specificity,limit of detection,reproducibility,and stability were evaluated.A total of 215 stool samples from patients with diarrhea were collected and tested in parallel with the Xpert C.difficile/Epi,VIDAS CDAB,and our assay.C.difficile was isolated from samples,and the tcdB gene was identified when discrepant results were obtained from the three above assays.Our assay showed no cross-reaction with other diarrhea-associated pathogens.Its reproducibility was 100％in testing of two standard plasmids containing tcdB and tpi genes at two concentrations(105 and 102 copies/μL).Two standard plasmids were detected after the PCR and immunochromatography reagents had been stored for 3,6,9,and 12 months,and all the results were posi-tive.The limit of detection was 10 copies/μL for toxigenic C.difficile.Testing of 33 samples positive for C.difficile with our assay(33/215,15.3％)yielded findings statistically coherent with those of the Xpert C.difficile/Epi test(kappa value=0.965).The sensitivity,specificity,positive predictive value,and negative predictive value of our assay,with respect to Xpert C.difficile/Epi as the standard,were 94.3％,100.0％,100.0％,and 98.9％;these values were significantly higher than those of VIDAS CDAB(60.0％,98.9％,91.3％,and 92.7％)(Kappa=0.714,OR=157.50,95％CI:62.03-847.28,P=0.013).In conclusion,our newly developed assay is specific,stable,and reproducible,and may be used for rapid and accu-rate detection of toxigenic C.difficile.The assay could be used for C.difficile infection screening in outpatient and emergen-cy,community medical service center,and epidemiological settings.

Aim To explore the effects of Lycium berry seed oil on Nrf2/ARE pathway and oxidative damage in testis of subacute aging rats. Methods Fifty out of 60 male SD rats, aged 8 weeks, were subcutaneously injected with 125 mg • kg"D-galactosidase in the neck for 8 weeks to establish a subacute senescent rat model. The presence of senescent cells was observed using P-galactosidase ((3-gal), while testicular morphology was examined using HE staining. Serum levels of testosterone (testosterone, T), follicle-stimulating hormone ( follicle stimulating hormone, FSH ) , luteinizing hormone ( luteinizing hormone, LH ) , superoxide dis-mutase ( superoxide dismutase, SOD ) , glutathione ( glutathione, GSH) and malondialdehyde ( malondial-dehyde, MDA) were measured through ELISA, and the expressions of factors related to aging, oxidative damage, and the Nrf2/ARE pathway were assessed via immunohistochemical analysis and Western blotting. Results After successfully identifying the model, the morphology of the testis was improved and the intervention of Lycium seed oil led to a down-regulation in the expression of [3-gal and -yH2AX. The serum levels of SOD, GSH, T, and FSH increased while MDA and LH decreased (P 0. 05) . Additionally, there was an up-regulated expression of Nrf2, GCLC, NQOl, and SOD2 proteins in testicular tissue ( P 0. 05 ) and nuclear expression of Nrf2 in sertoli cells. Conclusion Lycium barbarum seed oil may reduce oxidative damage in testes of subacute senescent rats by activating the Nrf2/ARE signaling pathway.

Objective To investigate the distribution patterns of traditional Chinese medicine(TCM)constitution in 959 patients with endometriosis(EMs).Methods From January 2019 to November 2019,959 EMs patients were selected from Guang'anmen Hospital of China Academy of Chinese Medical Sciences,Beijing Obstetrics and Gynecology Hospital Affiliated to Capital Medical University,Beijing Hospital,Dongfang Hospital of Beijing University of Chinese Medicine,Beijing Friendship Hospital Affiliated to Capital Medical University,and Fuxing Hospital Affiliated to Capital Medical University.The general clinical information of the patients was recorded and then the TCM constitution was identified.After that,the correlation of TCM constitution distribution with concurrent constitution and the relationship of TCM constitution distribution with age and the complication of dysmenorrhea were analyzed.Results(1)The constitution types of EMs patients listed in descending order of the proportion were yang deficiency constitution(65.1％,624/959),qi stagnation constitution(58.4％,560/959),qi deficiency constitution(52.8％,506/959),blood stasis constitution(44.2％,424/959),phlegm-damp constitution(42.5％,408/959),damp-heat constitution(41.9％,402/959),yin deficiency constitution(39.6％,380/959),balanced constitution(26.8％,257/959),and inherited special constitution(16.6％,159/959).Among the patients,there were fewer patients with single constitution,accounting for 20.2％(194/959),and most of them had concurrent constitution types,accounting for 79.8％(765/959).(2)The association rule mining based on Apriori algorithm obtained 33 related rules.The concurrent constitution types of qi deficiency-yang deficiency,blood stasis-yang deficiency,and blood stasis-qi stagnation were the association rules with high confidence.(3)Compared with patients aged 35 years and below,the patients over 35 years old were predominated by high proportion of blood stasis constitution(P＜0.05).Compared with patients without dysmenorrhea,the patients with dysmenorrhea had the increased proportion of biased constitutions and the decreased proportion of balanced constitution(P＜0.05 or P＜0.01).Conclusion Yang deficiency constitution,qi stagnation constitution,qi deficiency constitution and blood stasis constitution are the high-risk constitution types of EMs patients.The concurrent constitution types are commonly seen in EMs patients,which are more common than single biased constitution.Management of EMs patients with the methods of warming yang,relieving stagnation,benefiting qi and activating blood will be helpful for correcting the biased constitutions in time and preventing disease progression,which will achieve the preventive treatment efficacy through TCM constitution correction.

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.

This study aimed to provide scientific evidence for predicting quality markers(Q-markers) of Elephantopus scaber by establishing UPLC fingerprint of E. scaber from different geographical origins and determining the content of 13 major components, as well as conducting in vitro anti-cancer activity investigation of the main components. The chromatographic column used was Waters CORTECS UPLC C_(18)(2.1 mm×150 mm, 1.6 μm), and the mobile phase consisted of acetonitrile and 0.1% formic acid solution(gradient elution). The column temperature was set at 30 ℃, and the flow rate was 0.2 mL·min~(-1). The injection volume was 1 μL, and the detection wavelength was 240 nm. The UPLC fingerprint of E. scaber was fitted using the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 edition) to determine common peaks, evaluate similarity, identify and determine the content of major components. The CCK-8 assay was used to explore the inhibitory effect of the main components on the proliferation of lung cancer cells. The results showed that in the established UPLC fingerprint of E. scaber, 35 common peaks were identified. Thirteen major components, including neochlorogenic acid(peak 1), chlorogenic acid(peak 2), cryptochlorogenic acid(peak 3), caffeic acid(peak 4), schaftoside(peak 6), galuteolin(peak 9), isochlorogenic acid B(peak 10), isochlorogenic acid A(peak 12), isochlorogenic acid C(peak 18), deoxyelephantopin(peak 28), isodeoxyelephantopin(peak 29), isoscabertopin(peak 31), and scabertopin(peak 32) were identified and quantified, and a quantitative analysis method was established. The results of the in vitro anti-cancer activity study showed that deoxyelephantopin, isodeoxyelephantopin, isoscabertopin, and scabertopin in E. scaber exhibited inhibition rates of lung cancer cell proliferation exceeding 80% at a concentration of 10 μmol·L~(-1), higher than the positive drug paclitaxel. These results indicate that the fingerprint of E. scaber is highly characteristic, and the quantitative analysis method is accurate and stable, providing references for the research on quality standards of E. scaber. Four sesquiterpene lactones in E. scaber show significant anti-cancer activity and can serve as Q-markers for E. scaber.
Humans ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/chemistry* ; Asteraceae/chemistry* ; Lung Neoplasms/drug therapy*

OBJECTIVE: To analyze the factors related to pregnancy of endometriosis and whether Chinese herbal medicines (CHMs) can improve pregnancy outcomes in patients with endometriosis in long-term management. METHODS: This multicenter cohort study retrospectively analyzed the clinical data of endometriosis patients with fertility needs from January 2019 to November 2019. A total of 252 patients with endometriosis from 5 level-III Grade A hospitals in Beijing were included in this study. Univariate and multivariate logistic regression analysis were performed for the relevant factors. The propensity score matching (PSM) function of SPSS software was used to match the CHMs group with the non-CHMs group. The pregnancy rate and live birth rate were analyzed. RESULTS: The results of univariate analysis showed that age, disease course, presence of infertility, presence of adenomyosis, time after surgery or use of gonadotropin-releasing hormone agonist (GnRH-a), use of CHMs and follow-up time were influencing factors of pregnancy in endometriosis patients (P<0.05). The results of multivariate analysis showed that age, presence of adenomyosis, time after surgery or use of GnRH-a, use of CHMs and follow-up time were independent factors affecting pregnancy in endometriosis patients, among which, age ⩾35 years old, presence of adenomyosis and follow-up time >6 months were independent risk factors (OR=0.445, 0.348, 0.140, respectively, P<0.05), time after surgery or use of GnRH-a ⩽6 months and use of CHMs were independent protective factors (OR=3.839, 3.842, respectively, P<0.05). After PSM, 99 pairs of two groups were matched successfully. The pregnancy rate of the CHMs group was higher than that of the non-CHMs group [55.56% (55/99) vs. 36.36% (36/99), P<0.05]. The live birth rate of the CHMs group was higher than that of the non-CHMs group [49.49% (49/99) vs. 35.35% (35/99), P<0.05]. CONCLUSION CHMs can effectively improve clinical pregnancy rate and live birth rate of patients with endometriosis in the chronic disease management.
Pregnancy ; Female ; Humans ; Adult ; Pregnancy Outcome ; Endometriosis/surgery* ; Retrospective Studies ; Cohort Studies ; Adenomyosis ; Gonadotropin-Releasing Hormone ; Plant Extracts ; Fertilization in Vitro

OBJECTIVES: To observe the effects on the glucose-lipid metabolism and the expression of zinc-α2-glycoprotein (ZAG) and glucose transporter 4 (GLUT4) in the femoral quadriceps and adipose tissue after electroacupuncture (EA) at "Pishu" (BL 20), "Weiwanxiashu" (EX-B 3), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) in the rats with diabetes mellitus type 2 (T2DM), so as to explore the effect mechanism of EA in treatment of T2DM. METHODS: Twelve ZDF male rats were fed with high-sugar and high-fat fodder, Purina #5008 for 4 weeks to induce T2DM model. After successfully modeled, the rats were randomly divided into a model group and an EA group, with 6 rats in each one. Additionally, 6 ZL male rats of the same months age were collected as the blank group. The rats in the EA group were treated with EA at bilateral "Pishu" (BL 20), "Weiwanxiashu" (EX-B 3), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), with continuous wave, 15 Hz in frequency, and 2 mA in intensity. The electric stimulation lasted 20 min each time. EA was delivered once daily, 6 times a week for 4 weeks. Separately, the levels of fasting blood glucose (FBG) was measured before modeling, before and after intervention, and the body mass of each rat was weighted before and after intervention. After intervention, the levels of the total cholesterol (TC), triacylglycerol (TG) and free fatty acid (FFA) in serum were detected using enzyme colorimetric method; and the levels of the serum insulin (INS) and ZAG were detected by ELISA. Besides, the insulin sensitivity index (HOMA-ISI) was calculated. With Western blot technique adopted, the protein expressions of ZAG and GLUT4 in the femoral quadriceps and adipose tissue were determined. RESULTS: After intervention, compared with the blank group, the levels of FBG and body mass, and the levels of serum TC, TG, FFA and INS increased (P<0.01), while HOMA-ISI decreased (P<0.01); the level of ZAG in the serum and the protein expressions of ZAG and GLUT4 in the femoral quadriceps and adipose tissue dropped (P<0.01) in the model group. In the EA group, compared with the model group, the levels of FBG and body mass, and the levels of serum TC, TG, FFA and INS were reduced (P<0.01), and HOMA-ISI increased (P<0.01); the level of ZAG in the serum and the protein expressions of ZAG and GLUT4 in the femoral quadriceps and adipose tissue increased (P<0.01, P<0.05). CONCLUSIONS Electroacupuncture can effectively regulate glucose-lipid metabolism, improve insulin resistance and sensitivity in the rats with T2DM, which is associated with the modulation of ZAG and GLUT4 expression in the skeletal muscle and adipose tissue.
Rats ; Male ; Animals ; Glucose/metabolism* ; Electroacupuncture ; Rats, Sprague-Dawley ; Diabetes Mellitus, Type 2/therapy* ; Lipid Metabolism ; Triglycerides ; Adipose Tissue/metabolism* ; Acupuncture Points