ObjectiveTo observe the effect of earthworm protein on the expression of phosphatidylinositol 3-kinase/protein kinase B/nuclear factor E₂-related factor 2 （PI3K/Akt/Nrf2） pathway in the aorta of spontaneously hypertensive rats （SHR） and explore mechanism of earthworm protein in treating hypertensive vascular endothelial dysfunction （VED）. MethodTen 10-week-old Wistar Kyoto （WKY） rats and fifty SHR rats were selected for a week of adaptive feeding. WKY rats were selected as the normal group， and fifty SHR rats were randomized according to body weight into model， valsartan （8×10^-3 g·kg^-1·d^-1）， and high-， medium-， and low-dose （0.2， 0.1， 0.05 g·kg^-1·d^-1， respectively） earthworm protein groups. The normal and model groups were administrated with equal volume of double distilled water by gavage. During the drug intervention period， the general situations of rats in each group were observed and their blood pressure was monitored at specific time points every other week before and after administration. After 8 weeks of drug intervention， enzyme-linked immunosorbent assay was employed to measure the levels of angiotensin-Ⅱ （Ang-Ⅱ） and endothelin-1 （ET-1） in the serum of rats in each group. The corresponding kits were used to determine the levels of nitric oxide （NO）， malondialdehyde （MDA）， glutathione peroxidase （GPX）， superoxide dismutase （SOD）， and ferrous ion （Fe²⁺）. Hematoxylin-eosin （HE） staining was employed to observe the changes in the intima of the aorta. Fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to measure the mRNA levels of PI3K， Akt， Nrf2， heme oxygenase-1 （HO-1）， and glutathione peroxidase 4 （GPX4） in the aortic tissue. Western blotting was used to determine the protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the thoracic aorta. ResultCompared with the normal group， the model group had decreased body mass， increased irritability， severe endothelial damage， elevated blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe²⁺ （P<0.01）， lowered NO level （P<0.01）， and down-regulated mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the aortic tissue （P<0.01）. Compared with the model group， drug intervention caused no significant change in the body mass， calmed the rats， alleviated the endothelial damage， lowered blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe²⁺ （P<0.01）， elevated the NO level （P<0.05）， and up-regulated the mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 （P<0.05）. ConclusionThe earthworm protein can exert antihypertensive effects by ameliorating VED in SHR. Specifically， it may regulate the PI3K/Akt/Nrf2 signaling pathway to inhibit oxidative stress and ferroptosis.

Aim To establish an efficient and stable isolation method of primary mouse brain vascular smooth muscle cells and endothelial cells,and provide experimental materials for the investigation of pathogenesis and treatment of brain vascular diseases.Methods Brain vascular smooth muscle cells were isolated by dextran gradient centrifugation with enzymatic digestion,and endothelial cells were isolated by immunomagnetic beads sorting.Morphology and growth characteristics of two types of cells were observed with an inverted phase contrast microscope,their purity were identified by immunofluorescence,and their proliferation characteristics were observed by CCK-8 assay.At the level of cellular func-tion,angiogenic capacity of endothelial cell was assessed by angiogenesis assay and smooth muscle cell responsiveness to platelet-derived growth factor(PDGF)was assessed by migration assay.Results The two types of cells isolated using this method grew vigorously and were in good condition.Smooth muscle cells exhibited typical"peak valley"growth,and immunofluorescence results showed cytoplasmic specific smooth muscle α-SMA and SM22α expression was positive.En-dothelial cells exhibited typical"cobblestone like"growth,with positive expression of platelet endothelial cell adhesion molecule CD31 and atresia zone protein 1.Conclusion This study established a reliable and efficient method for sim-ultaneously isolating two types of cerebrovascular cells,the isolated cells have high purity,good activity,and stable charac-teristics after passage,which were sufficient to meet the needs of subsequent experiments.

Objective Prior studies have established a connection between albuminuria and various inflammatory reactions,highlighting that an increase in C-reactive protein by 1 mg/L increases the likelihood of albuminuria by 2%.Recent investigations indicate a positive correlation between the systemic immune-inflammation index(SII)and increased urinary protein excretion.In addition,elevated levels of the systemic inflammatory response index(SIRI)also correlate with a higher prevalence of albuminuria.The aggregate index of systemic inflammation(AISI)offers a more comprehensive indicator of inflammation,providing an extensive assessment of systemic inflammatory status compared to SII and SIRI.Yet,the specific relationship between AISI and albuminuria remains unclear.This study aims to explore this association in U.S.adults.Methods We analyzed data from the National Health and Nutrition Examination Survey(NHANES)for 2007-2018,excluding pregnant women and individuals under 18.Cases with missing data on AISI,urinary albumin concentration,and other covariates were also excluded.AISI was computed using the formula:AISI=(platelet count×neutrophil count×monocyte count)/lymphocyte count.Albuminuria was defined as the urinary albumin-to-creatinine ratio exceeding 30 mg/g.Continuous variables were presented in the form of the mean±standard error,and categorical variables in percentages.We utilized weighted t-tests and chi-square tests for baseline comparisons.We applied weighted multivariable logistic regression and generalized additive models(GAM)to explore the association between AISI and albuminuria and to assess potential nonlinear relationships.Results The study included 32 273 participants,with an average age of(46.75±0.24)years old.The cohort comprised 48.73% males and 51.27% females.The prevalence of albuminuria was 9.64%.The average logarithmic value of log2AISI was 7.95±0.01,and were categorized into tertiles as follows:Quartile 1(Q1)(4.94 to 7.49),Q2(7.49 to 8.29),and Q3(8.29 to 10.85).As log2AISI increased,so did the prevalence of hypertension,diabetes,congestive heart failure,and albuminuria,all showing statistically significant increases(P<0.001).Similarly,the use of antihypertensive,lipid-lowering,and hypoglycemic drugs was also more prevalent(P<0.001).Statistically significant differences were observed across the three groups concerning age,race and ethnicity,formal education,alcohol consumption,smoking status,systolic and diastolic blood pressures,body mass index,estimated glomerular filtration rate,HbA1c,alanine aminotransferase,aspartate aminotransferase,albumin,creatinine,uric acid,and high-density lipoprotein cholesterol(P<0.05).However,no significant differences were noted in the total cholesterol or the sex ratios among the groups.The association between log2AISI and albuminuria was assessed using weighted multivariable logistic regression,and the detailed results are presented in Table 2.In model 1,without adjusting for covariates,each unit increase in log2AISI was associated with a 32% increase in the risk of albuminuria(odds ratio[OR]=1.32,95% confidence interval[CI]:1.27-1.38,P<0.001).Model 2 was adjusted for age,gender,race,and education level,and showed a similar trend,with each unit increase in log2AISI associated with a 31% increased risk(OR=1.31,95% CI:1.26-1.37,P<0.001).Model 3,which was further adjusted for all covariates,revealed that each unit increase in log2AISI was associated with a 20% increase in the risk of albuminuria(OR=1.20,95% CI:1.15-1.26,P<0.001).The study also transformed log2AISI from a continuous to a categorical variable for analysis.Compared with Q1,the risk of albuminuria in Q3,after adjusting for all covariates,significantly increased(OR=1.37,95% CI:1.22-1.55,P<0.001).Q2 also demonstrated a higher risk compared with Q1(OR=1.13,95% CI:1.06-1.36,P=0.004).The trend test indicated a dose-effect relationship between increasing log2AISI and the rising risk of albuminuria.GAM revealed a nonlinear relationship between log2AISI and albuminuria,with distinct trends noted between sexes.Segmented regression based on turning points showed significant effects among women,although the slope difference between the segments was not significant.In men,a significant threshold effect was observed;below the log2AISI of 7.25,increases in log2AISI did not enhance the risk of albuminuria,but above this threshold,the risk significantly increased.As part of a sensitivity analysis,weighted multivariable logistic regression was performed by changing the outcome variable to macroalbuminuria and adjusting for all covariates.The analysis showed that for every unit increase in log2AISI,the risk of developing macroalbuminuria increased by 31% (OR=1.31,95% CI:1.15-1.49,P<0.001).Compared with Q1,the risk of albuminuria in Q3 increased by 69% (OR=1.69,95% CI:1.27-2.25,P<0.001),and in Q2,it increased by 40% (OR=1.40,95% CI:1.03-1.92,P=0.030).Subgroup analysis and interaction results showed that the positive association between AISI and proteinuria risk was stronger in men than in women.Similarly,the association was stronger in people with hypertension compared with those with normal blood pressure,and higher in overweight people compared with those of normal weight.Furthermore,smokers and drinkers showed a stronger positive association between AISI and the risk of proteinuria than non-smokers and non-drinkers do.These results suggest that sex,blood pressure,body mass index,smoking,and alcohol consumption interact with AISI to influence the risk of proteinuria.Conclusion There is a robust positive association between AISI and increased risks of albuminuria in US adults.As log2AISI increases,so does the risk of albuminuria.However,further validation of this conclusion through large-scale prospective studies is warranted.

Cardiovascular System/pathology* ; Constriction, Pathologic/genetics* ; Point Mutation ; Neurofibromin 1/genetics* ; Animals ; Mice

Objective:To analyze the development prospect of ChatGPT in the field of medical research management and the feasibility of application.Methods:The literature was reviewed and analyzed.Results:This paper describes the application of ChatGPT involving the orderly promotion of medical scientific research projects, the working mode of scientific research managers, and the development of scientific research management platform.Conclusions:The development of ChatGPT makes it have its unique advantages in medical scientific research management, but also has some shortcomings, which need to be reasonably understood and scientifically applied.

Objective To study the inhibitory effect of antimicrobial peptide LL-37 on Candida albicans through its ability to promote the secretion of immune factors by vaginal epithelial cells. Methods (1) LL-37 prokaryotic expression vector pET-Duet/LL-37 was constructed and its expression was induced in Escherichia coli M15. The expressed LL-37 fusion protein was purified and identified by western blot. Antifungal activity of the purified protein was initially identified by Kirby-Bauer (K-B) method. (2) Purified LL-37 protein was added to human vaginal epithelial cells co-cultured with Candida, and inhibitory effect on Candida growth was determined by the glucose consumption method. Interferon γ (IFN-γ), interleukin 10 (IL-10) concentration and IFN-γ/IL-10 ratio were measured by ELISA at different time points. Results (1) LL-37 fusion protein was purified to 96% purity at a concentration of 433.92 μg/ml, and was shown to possess anti-fungal activity confirmed by the K-B method. (2) A Candida-vaginal epithelial cells co-culture system was successfully constructed. LL-37 recombinant protein inhibited the growth of Candida with absorbance values significantly higher in the treatment group compared to the control group at all measured time points (12-hour:3.008±0.003 versus 2.967±0.003, 24-hour:2.941±0.003 versus 2.601±0.003, 48-hour:2.893 ± 0.004 versus 2.409 ± 0.003; all P<0.01). Furthermore, the rate of decrease was also much slower compared to the control group. In both control and experimental groups, IFN-γ and IL-10 secretion levels were observed to rise at first peaking at 24 hours and subsequently decrease. For each time period, IFN-γconcentration in the experimental group was significantly higher at 24 hours compared to the control group [(104.00 ± 1.07) versus (85.17 ± 0.28) pg/ml, P<0.01]. In contrast, IL-10 concentrations were significantly lower than the control group at all time points (P<0.01). IFN-γ/IL-10 ratio was also observed to be significantly higher than the control group at all measured time points (P<0.01). Conclusions (1) Recombinant protein LL-37 could significantly inhibit the growth of Candida. (2) By influencing the secretion of immune factors such as IFN-γ, IL-10, etc, recombinant protein LL-37 is able to adjust vaginal epithelial cells local immunity, and enhance resistance to Candida infection.

Objective To study the effects of antimicrobial peptide LL-37 expressed and purified from prokaryotes on candida albicans growth. Methods (1) Thirty female Kunming mice were treated with estrogen and white candida yeast suspension were poured into vagina to establish a vulvovaginal candidiasis (VVC) murine model. After successful establishing the VVC mouse model, mice were randomly sorted into test group (n=15) and control group (n=15) . Suspension(30μl, 100μg/ml)of recombinant peptide LL-37 expressed and purified in Prokaryotes was given by intravaginal administration to the test group for 5 days, while the same amount of phosphate buffered saline (PBS) was given to the control group. (2) Tweenty-four hours after treatment, the fungal burden and colony-forming unit (CFU) of vaginal fluids were evaluated. All mice were subsequently sacrificed and vaginal tissues were harvested for tissue homogenate preparation. ELISA was used to determine the levels of nterleukin-10(IL-10)and interferon-γ(IFN-γ)in the isolated vaginal tissues. Results (1) VVC mouse model was established successfully in this study. Vaginal mucosa congestion, edema, vaginal plica disappearing were obviously observed in the control group. After treatment with recombinant protein LL-37 vaginal mucosa has no obvious change in the test group. (2) Fungal burden and CFU of vaginal fluids were significantly lower in the test group [(4.8±1.0)×104 CFU/ml] than that in the control group [(8.5 ± 2.1) × 104 CFU/ml, P=0.017]. IFN-γlevel of the test group was increased [(257 ± 11) vs (197 ± 4) pg/ml, P=0.000], while the level of IL-10 was reduced [ (287 ± 15) vs (379 ± 17) pg/ml P=0.000] resulting in a the ratio of IFN-γ/IL-10 was in significantly higher in test group (0.892±0.008 vs 0.496±0.013, P=0.000). Conclusion Recombinant protein LL-37 expressed and purified from prokaryotes inhibits the growth candida albicans and improves vaginal immunity by adjusting IFN-γand IL-10 secretion in the VVC mouse model, highlighting the therapeutic potential of LL-37 for VVC.

Objective Tracking of the vaginal microflora recovery and the expression of immune factors from untreated and treated patients with bacterial vaginosis (BV) by using different administration regimens and studying the relationship of treatment results and regimen selections. Methods 25 healthy females were selected as a control group and 100 BV patients were randomly divided into 4 groups (n=25/group). Group A: Intravaginal administration of metronidazole (× 7 d), Group B:Continuous intravaginal administration of metronidazole (× 7 d) and then live Lactobacillus Capsule (× 7 d) , Group C: Intravaginal administration of nifuratel (× 7 d), Group D: Continuous intravaginal administration of nifuratel (× 7 d) and then live Lactobacillus Capsule (×7 d). The microecological assessment system and EILSA were used to compare the clinical efficacy, vaginal microflora recovery and the changes in IL-8, TLR2 and TNF-αof the vaginal lavage fluid in healthy women or patients with bacterial vaginosis before and after treatments by four treatment strategies. Results ① The vaginal microflora imbalance, flora disturbance, pH value increased were presented in BV group compared with the control group.②Compared to the median of IL-8, TLR2 and TNF-α in vaginal lavage fluids of control group, there was no significant difference in IL-8 level but both TLR2 and TNF-αwere significantly increased (P<0.05) in BV group. The immune factors had no significantly difference in all BV groups.③The therapeutic effect in each BV groups was compared after stopping treatment for 7 days. The cure rate and the vaginal microflora recovery rate were significant higher in group B and D than group A and C (P<0.05). ④ After treatment there was no significant change in IL-8 level but there was an obviouslydecrease in TLR2 and TNF-α(P<0.05). The decreased levels are more significant in groups B and D than groups A and C (P<0.05). Conclusion By combining with the microecological assessment system to evaluate the therapeutic effect of BV, our research suggests that the sequence schemes of nifuratel plus live Lactobacillus Capsule is more effective in therapy effect, restoring normal vaginal micro-ecological environment and vaginal local immunity than metronidazole used alone.

Objective To construct the prokaryotic expression vector for HD-5 and purify the recombinant HD-5 protein then analyze its antifungal activity. Methods The HD-5 gene was cloned by PCR, then was inserted into prokary-otic expression plasmid pQE-30Xa to construct pQE-30Xa/HD-5. After sequencing, pQE-30Xa/HD-5 was transformed in-to E.coli M15 cells. Its expression was induced by IPTG and confirmed by SDS-PAGE. The recombinant protein was purified through Ni-NTA affinity purification system. The antifungal activity was tested by disk diffusion method. Results HD-5 gene and pQE-30Xa/HD-5 vector were obtained successfully. E.coli M15 strains was used to express HD-5 fusion protein. After purification, the fusion protein was confirmed by Western blot. The disk diffusion test confirmed that the fusion pro-tein can inhibit Candida albicans. Conclusion Expression vector pQE-30Xa/HD-5 was successfully constructed. The HD-5 fusion protein was expressed in E.coli successfully, which showed a certain degree of antifungal activity.

BACKGROUND:The functions of homo heterogeneous ribonucleoprotein E1 are very wide. It can participate in the expression of skeleton proteins in the nervous system. OBJECTIVE:To construct the recombinant plasmid of homo heterogeneous ribonucleoprotein E1 and observe its expression in nerve cells for further studying the functions of it in neurocytes. METHODS:Using pcDNATM4/His C, the homo heterogeneous ribonucleoprotein E1 was subcloned into recombinant plasmid E1-pcDNATM 4/His C, fol owed by enzyming and sequencing. After SH-SY5Y cells were transfected with the recombinant plasmid, western blot analysis and real time RT-PCR were used to detect the expression of homo heterogeneous ribonucleoprotein E1 in SH-SY5Y cells. And the growth of SH-SY5Y cells was observed. RESULTS AND CONCLUSION:We successful y constructed the eukaryotic expressed vector of homo heterogeneous ribonucleoprotein E1. The recombinant plasmids were verified to express in SH-SY5Y cells correctly at mRNA and protein levels. And SH-SY5Y cells generated quickly after homo heterogeneous ribonucleoprotein E1 was over-expressed. The homo heterogeneous ribonucleoprotein E1 is an important protein in neural development. And this vector offers the premise for further studying its function in nervous system.