ObjectiveTo detect the proportion of splenic follicular helper T （Tfh） cells and their functionally related cytokine expression levels in the incomplete embryo implantation disorder （EID） model mice， and to explore the immunological mechanism of Tfh in infertility caused by embryo implantation disorder. MethodsSixteen female Kunming mice were randomly divided into two groups， with eight mice in each group. On day 4 of pregnancy， an incomplete EID mouse model was established by oral gavage of mifepristone suspension， while an equal volume of saline was administered to the control group. On day 8 of pregnancy， the mice were euthanized. Flow cytometry was used to detect the levels of Tfh cells in the spleen lymphocytes of both incomplete EID mice and normal control mice. qRT-PCR was performed to measure the mRNA levels of B-cell lymphoma 6 （Bcl-6） and C-X-C chemokine receptor type 5 protein （CXCR5） in the spleen lymphocytes of both groups. Western blot was employed to assess the protein expression levels of Bcl-6 and CXCR5 in the spleen lymphocytes of both groups. Serum levels of interleukin-4 （IL-4）， IL-6， and IL-21 were measured by ELISA. Immunohistochemistry （IHC） was used to observe the expression levels of progesterone receptor （PR）， Bcl-6， and CXCR5 proteins in the uterine endometrial tissue of mice in both groups. ResultsIncomplete-type EID mice had a reduced number of embryo implantation points and reduced endometrial PR expression. Flow assay results showed that the proportion of CD4⁺CXCR5⁺Tfh cells in splenic lymphocytes of incomplete-type EID mice was significantly higher than that of normal controls （P<0.05）. Compared with the normal control group， Bcl-6 and CXCR5 mRNA levels and protein levels were elevated in splenic lymphocytes of incomplete EID mice， with statistically significant differences （P<0.05）； serum IL-4， IL-6， and IL-21 levels were elevated in incomplete EID mice， and Bcl-6 and CXCR5 proteins in the endometrium were significantly elevated （P<0.05）. ConclusionThe increase of Tfh cells and their associated cytokines Bcl-6 and CXCR5 is associated with the development of incomplete EID， and may be involved in the development of female immune infertility.

The clinical diagnosis and treatment of urinary tract infection has long faced the challenges of insufficient standardization of diagnosis and treatment pathways,irrational use of antimicrobial drugs and high recurrence rate.How to optimize the hierarchical diagnosis and treatment pathway of urinary tract infection,standardize the use of antimicrobial drugs,and reduce the recurrence rate have always been the focus of clinical attention.There is significant heterogeneity in the existing diagnostic criteria for urinary tract infection,which seriously affects the comparability and evidence integration of clinical and research studies.In order to solve the above problems,a consensus on global multidisciplinary diagnostic criteria for urinary tract infection has been formed by international multidisciplinary experts after three rounds of Delphi method.Breaking through the traditional classification framework,the consensus innovatively established a four-dimensional quantitative scoring system including local symptoms and signs,systemic inflammatory response,quantitative analysis of pyuria and urine culture results,and established a hierarchical standard for stepwise urinary tract diagnosis according to the scoring threshold.Based on the key citations related to the consensus,this paper interprets in detail the basis for the selection of core indicators and the establishment of thresholds for the diagnosis of urinary tract infection in the consensus,and focuses on the key issues and implementation paths of the consensus in localization practice.This consensus provides a unified standard for standardizing the clinical diagnosis and treatment of urinary tract infection,improving the homogeneity of clinical research through standardized diagnostic processes,and promoting the standardization of UTI drug research and development and the rational use of antibiotics and precision.

AIM To observe the effects of Yiqi Jiedu Tongluo Formula(YQJDTL)on renal microvascular endothelial function and prevention of renal injury in a rat model of type 2 diabetes mellitus(T2DM).METHODS The SD rats were randomly divided into a normal group and a model group.The model group was administered with high-fat diet combined with a single intraperitoneal injection of STZ to establish the T2DM model.The successfully modeled rats were randomly divided into the model group,the canagliflozin group(9 mg/kg),and the low-dose and high-dose YQJDTL groups(4.77,9.45 g/kg).The corresponding doses of the drug were administered by gavage for a total of 12 weeks,during which the rats underwent observation of their general condition and blood glucose changes.After the end of administration,the rats had their levels of renal index,24-hour UP,serum SCr,BUN,TC,TG,HDL-C,LDL-C,ET-1 and NOS measured;their changes in renal microvasculature and the degree of renal fibrosis observed using HE staining,Masson staining,PAS staining,and PASM staining;their ultrastructure of the glomeruli observed using transmission electron microscopy;their renal protein expressions of TGF-β,SMAD2,SMAD3,Col-1,VEGFA and PKC detected by immunohistochemical staining and Western blot;and their renal mRNA expressions of VEGFA,TGF-β,SMAD2 determined by RT-qPCR.RESULTS Compared with the model group,the high-dose YQJDTL group showed decreased levels of renal index,blood glucose,TG,TC,HDL,24 h UP,BUN,SCr and ET-1(P＜0.05,P＜0.01);increased LDL and NOS levels(P＜0.05,P＜0.01);reduced renal inflammatory infiltration and fibrosis degree,inhibited fusion of foot processes and thickening of basement membrane;decreased renal protein expressions of TGF-β,SMAD2,SMAD3,VEGFA,PKC and Col-1(P＜0.05,P＜0.01);and decreased mRNA expressions of VEGFA,TGF-β and SMAD2(P＜0.01).CONCLUSION In the rat models of T2DM,YQJDTL can reduce their levels of blood glucose and lipids by improving the renal indices levels and the renal microvascular endothelial functions to alleviate renal fibrosis and microangiopathy as well,and the mechanism may be associated with the down-regulated expressions of TGF-β/SMAD and VEGF pathway-related proteins.

Objective:To analyze the differences in determination of fluoride level in drinking water by ion-selective electrode method and high-throughput rapid determination method.Methods:The precision test was carried out by using the two methods to measure two kinds of fluoride standard substances, water samples of external quality control assessment from 2021 to 2023 (two kinds each year) and the fluoride level in three drinking water samples (for 5 times/each sample). Accuracy testing was conducted by measuring the external quality control assessment water samples and the spiked recovery rates drinking water, and water samples were grouped (water fluoride ≤1.00, > 1.00 mg/L) and analyzed according to the "Hygienic Standards for Drinking Water" (GB 5749-85). SPSS 23.0 software was used for statistical analysis of the measurement results.Results:(1) The correlation coefficients ( r) of the working curves of the two methods were both > 0.990, meeting the quality control requirements. (2) In the precision test, when comparing the results of the two methods for detecting two kinds of fluoride standard substances, there was no statistically significant difference ( F = 0.36, 0.15, P = 0.564, 0.707), and the coefficients of variation ( CV) were all < 5%. The CV of the detection results of the external quality control assessment water samples and drinking water samples were < 5%. (3) In the accuracy test, when the fluoride concentration in water was ≤1.00 mg/L, there was no statistically significant difference in the spiked recovery rates between the two methods ( F = 0.49, P = 0.504). When the fluoride concentration in water was > 1.00 mg/L, there was a statistically significant difference in the spiked recovery rates between the two methods ( F = 24.75, P = 0.003). Conclusions:The ion-selective electrode method has the advantages of wide detection range and wide adaptability, while the high-throughput rapid determination method has high accuracy. Testing personnel can weigh and choose the appropriate determination method based on the actual laboratory conditions and sample concentration range.

The clinical diagnosis and treatment of urinary tract infection has long faced the challenges of insufficient standardization of diagnosis and treatment pathways,irrational use of antimicrobial drugs and high recurrence rate.How to optimize the hierarchical diagnosis and treatment pathway of urinary tract infection,standardize the use of antimicrobial drugs,and reduce the recurrence rate have always been the focus of clinical attention.There is significant heterogeneity in the existing diagnostic criteria for urinary tract infection,which seriously affects the comparability and evidence integration of clinical and research studies.In order to solve the above problems,a consensus on global multidisciplinary diagnostic criteria for urinary tract infection has been formed by international multidisciplinary experts after three rounds of Delphi method.Breaking through the traditional classification framework,the consensus innovatively established a four-dimensional quantitative scoring system including local symptoms and signs,systemic inflammatory response,quantitative analysis of pyuria and urine culture results,and established a hierarchical standard for stepwise urinary tract diagnosis according to the scoring threshold.Based on the key citations related to the consensus,this paper interprets in detail the basis for the selection of core indicators and the establishment of thresholds for the diagnosis of urinary tract infection in the consensus,and focuses on the key issues and implementation paths of the consensus in localization practice.This consensus provides a unified standard for standardizing the clinical diagnosis and treatment of urinary tract infection,improving the homogeneity of clinical research through standardized diagnostic processes,and promoting the standardization of UTI drug research and development and the rational use of antibiotics and precision.

Objective:To establish an oxidative stress injury model by using hydrogen peroxide (H 2O 2) to induce human ovarian granulosa cells COV434. Methods:Human ovarian granulosa cells line COV434 were randomly divided into 6 groups, control group was not treated, H 2O 2 groups were treated with H 2O 2 of 200 μmol/L, 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L for 0.5 h, 1 h, 2 h, 4 h and 6 h, respectively, and the cell viability was determined by CCK-8 method. The follow-up experiments were treated with different concentrations of H 2O 2 for 1 h. β-galactosidase staining was used to determine the degree of cell senescence. DCFH-DA fluorescence staining was determined by flow cytometry, and the level of reactive oxygen species (ROS) in cells was determined. JC-1 staining was used to determine the mitochondrial membrane potential of cells. Western blotting was used to determine the expression levels of apoptosis-related proteins Caspase-3 and Caspase-9. After the successful establishment of the model, in order to verify the usability of the cell model, the cells were pretreated with the antioxidant vitamin E for 12 h, followed by the addition of H 2O 2 for intervention, and the ROS level and mitochondrial membrane potential were measured. Results:The cell viability of the 200 μmol/L and 400 μmol/L groups decreased first and then increased compared with the control, and tended to be stable after 1 h of intervention, and there was no significant difference in cell viability at each time point (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, the cell viability gradually decreased with the treatment time and tended to stabilize after 1 h, and decreased significantly to nearly 50% ( P<0.001). When the concentration of H 2O 2 continued to increase to 800 μmol/L and 1 000 μmol/L, the cell viability gradually decreased with the treatment time and stabilized after 1 h, and decreased to less than 10% (all P<0.001). When the concentration of H 2O 2 was 200 μmol/L and 400 μmol/L, there was no significant difference in the ratio of β-galactosidase-positive cells and the relative ROS intensity after 1 h compared with the control (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, 800 μmol/L and 1 000 μmol/L, the ratio of β-galactosidase-positive cells and the relative ROS intensity increased significantly (β-galactosidase staining: P=0.011 at 600 μmol/L, P=0.003 at 800 μmol/L, P=0.005 at 1 000 μmol/L; the relative ROS intensity: P=0.002 at 600 μmol/L, P<0.001 at 800 μmol/L and 1 000 μmol/L). Compared with the control, the mitochondrial membrane potential of cells decreased gradually after intervention with different concentrations of H 2O 2, and was negatively correlated with H 2O 2 concentration (all P<0.001). There was no difference in the expression of Cleaved-Caspase-3 in the H 2O 2 group at 200 μmol/L compared with the control, and the expression was significantly increased at 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L (all P<0.001). The expressions of Caspase-3 and Caspase-9 were significantly increased in all H 2O 2 treated groups (all P<0.001). Compared with the model group, the relative ROS intensity of the vitamin E group was significantly reduced ( P=0.009), and the mitochondrial membrane potential was significantly increased ( P<0.001), but it could not be restored to the level of the control. Conclusion:Using 600 μmol/L H 2O 2 to continuously treat COV434 cells for 1 h can quickly establish a stable and effective oxidative stress injury model of human ovarian granulosa cells.

Objective:To establish an oxidative stress injury model by using hydrogen peroxide (H 2O 2) to induce human ovarian granulosa cells COV434. Methods:Human ovarian granulosa cells line COV434 were randomly divided into 6 groups, control group was not treated, H 2O 2 groups were treated with H 2O 2 of 200 μmol/L, 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L for 0.5 h, 1 h, 2 h, 4 h and 6 h, respectively, and the cell viability was determined by CCK-8 method. The follow-up experiments were treated with different concentrations of H 2O 2 for 1 h. β-galactosidase staining was used to determine the degree of cell senescence. DCFH-DA fluorescence staining was determined by flow cytometry, and the level of reactive oxygen species (ROS) in cells was determined. JC-1 staining was used to determine the mitochondrial membrane potential of cells. Western blotting was used to determine the expression levels of apoptosis-related proteins Caspase-3 and Caspase-9. After the successful establishment of the model, in order to verify the usability of the cell model, the cells were pretreated with the antioxidant vitamin E for 12 h, followed by the addition of H 2O 2 for intervention, and the ROS level and mitochondrial membrane potential were measured. Results:The cell viability of the 200 μmol/L and 400 μmol/L groups decreased first and then increased compared with the control, and tended to be stable after 1 h of intervention, and there was no significant difference in cell viability at each time point (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, the cell viability gradually decreased with the treatment time and tended to stabilize after 1 h, and decreased significantly to nearly 50% ( P<0.001). When the concentration of H 2O 2 continued to increase to 800 μmol/L and 1 000 μmol/L, the cell viability gradually decreased with the treatment time and stabilized after 1 h, and decreased to less than 10% (all P<0.001). When the concentration of H 2O 2 was 200 μmol/L and 400 μmol/L, there was no significant difference in the ratio of β-galactosidase-positive cells and the relative ROS intensity after 1 h compared with the control (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, 800 μmol/L and 1 000 μmol/L, the ratio of β-galactosidase-positive cells and the relative ROS intensity increased significantly (β-galactosidase staining: P=0.011 at 600 μmol/L, P=0.003 at 800 μmol/L, P=0.005 at 1 000 μmol/L; the relative ROS intensity: P=0.002 at 600 μmol/L, P<0.001 at 800 μmol/L and 1 000 μmol/L). Compared with the control, the mitochondrial membrane potential of cells decreased gradually after intervention with different concentrations of H 2O 2, and was negatively correlated with H 2O 2 concentration (all P<0.001). There was no difference in the expression of Cleaved-Caspase-3 in the H 2O 2 group at 200 μmol/L compared with the control, and the expression was significantly increased at 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L (all P<0.001). The expressions of Caspase-3 and Caspase-9 were significantly increased in all H 2O 2 treated groups (all P<0.001). Compared with the model group, the relative ROS intensity of the vitamin E group was significantly reduced ( P=0.009), and the mitochondrial membrane potential was significantly increased ( P<0.001), but it could not be restored to the level of the control. Conclusion:Using 600 μmol/L H 2O 2 to continuously treat COV434 cells for 1 h can quickly establish a stable and effective oxidative stress injury model of human ovarian granulosa cells.

Objective:To investigate the role of serotonergic (5-HT) neurons activation in the dorsal raphe nucleus (DRN) in heat-induced seizures and its influence on breathing patterns in Scn1a + /- mice. Methods:24-day-old male Scn1a + /- mice were used for experiments, and sudden unexpected death in epilepsy(SUDEP) model was established through heat induction for 5 consecutive days. (1) Fluoxetine intervention experiment: 20 male mice were randomly divided into model group( n=10) and fluoxetine group ( n=10) according the weight-matched method. The fluoxetine group received intraperitoneal injection of fluoxetine (10 mg/kg) 45 min before heat induction each day for 5 consecutive days, while the model group received equal volume of 0.9% NaCl solution. (2) Chemogenetic activation experiment: 20 male mice were randomly divided into vector control group( n=10) and chemogenetic activation group ( n=10) according the weight-matched method. Empty vector or rAAV-TPH2-hM3d(Gq)-EGFP-WPREs was stereotaxically injected into DRN 14 d prior to seizure induction, and deschloroclozapine (5 mg/kg) was intraperitoneally injected 30 min before heat induction. Seizure characteristics and survival were assessed through video monitoring, and respiratory parameters were monitored. Immunofluorescence was used to detect colocalization of tryptophan hydroxylase 2(TPH2) and c-Fos in DRN. Statistical analysis was performed using SPSS 24.0 and GraphPad Prism 9.0. The independent sample t-test or Mann-Whitney test was used for inter-group comparison. Results:(1) In the fluoxetine intervention experiment: the survival rates between the model group and fluoxetine group showed no statistically significant difference ( χ2=2.23, P>0.05). As for the frequency of grade Ⅳ seizures, the model group (1.50(1.25, 2.35)min) demonstrated higher frequency than the fluoxetine group (0.43(0.20, 0.67)min) ( t=-3.40, P<0.05).With respect to respiratory parameters, the model group demonstrated shorter expiratory time ((0.10±0.02) s) and inspiratory time ((0.15±0.02) s) compared to the fluoxetine group ((0.16±0.05) s, (0.19±0.04) s) ( t=-3.47, -3.73, both P<0.01). The respiratory rate in the model group ((269.96±44.84) times/min) was significantly higher compared to the fluoxetine group ((195.04±52.37) times/min) ( t=3.44, P<0.01). The tidal volume in the model group ((0.10±0.02) mL) was significantly lower than the fluoxetine group ((0.13±0.04) mL) ( t=-2.19, P<0.05).The number of TPH2+ /c-Fos+ co-expressing cells in the model group (11.00±4.00) was lower than that in the fluoxetine group (33.00±8.39)( t=-4.16, P<0.05). (2) In the chemogenetic activation experiment: compared to the vehicle group, the chemogenetic activation group demonstrated significantly enhanced survival rates ( χ2=5.83, P<0.05). As for the frequency of grade Ⅴ seizures, the vehicle group (2.11(1.62, 3.44) times/min) showed higher frequency compared to the chemogenetic activation group (0.81(0.00, 1.62) times/min) ( t=18.00, P<0.05). In terms of respiratory parameters, the vehicle group showed shorter expiratory time ((0.10±0.01) s) and inspiratory time ((0.14±0.01) s) compared to the chemogenetic activation group ((0.12±0.01) s, (0.15±0.01) s) ( t=-2.78, -2.50, both P<0.05). The respiratory rate in the vehicle group ((208.37±9.73) times/min) was significantly higher than the chemogenetic activation group ((191.85±8.83) times/min) ( t=3.98, P<0.01). The tidal volume in the vehicle group ((0.09±0.01) mL) was significantly lower than the chemogenetic activation group ((0.12±0.02) mL) ( t=-4.77, P<0.001).The number of TPH2+ /c-Fos+ co-expressing cells in the vehicle group (9.00±3.46) was lower than that in the chemogenetic activation group (43.00±11.02)( t=-5.20, P<0.01). Conclusion:Specific activation of serotonergic neurons in the DRN can ameliorate heat-induced epileptic symptoms, improve respiratory function, and prolong survival time in Scn1a + /- mice.

Objective To explore the correlation between the characteristics of the endometrial microbiome in patients with recurrent implantation failure(RIF)and the subsequent transplantation outcomes.Methods A total of 438 RIF patients underwent embryo transfer again in our hospital were retrospective selection.According to the pregnancy status of the patients 8 weeks after embryo transfer,patients were divided into the pregnancy group(n=85)and the non-pregnancy group(n=353).The clinical data,characteristics and composition diversity of the endometrial microbiome were compared between the two groups.Binary Logistic regression was used to analyze the influencing factors,and the receiver operating characteristic(ROC)was employed to predict the efficacy of the influencing factors.The interaction between the above-mentioned risk factors and Shannon index in the subsequent transplantation outcomes of RIF patients was further analyzed.Results The levels of basal estradiol(E2),fasting insulin(Fins),total cholesterol(TC)and triglycerides(TG)were significantly higher in the non-pregnant group than those in the pregnant group(P＜0.05).Meanwhile,there were significant differences in the composition of endometrial microorganisms between the two groups(P＜0.05).Among them,the abundance of Fusobacterium phylum,the abundance of Bacillus genus and the α-diversity(Chao1,Shannon,Simpson)index all showed significant differences(all P＜0.05).Binary Logistic regression multivariate analysis showed that the abundance of Fusobacterium phylum increased(OR=1.628,95%CI:1.416-1.841),the abundance of Bacillus genus decreased(OR=0.725,95%CI:0.557-0.934)and E2 increased(OR=1.654,95%CI:1.343-1.965).The elevated insulin(OR=1.691,95%CI:1.393-1.980)and decreased Shannon index(OR=0.388,95%CI:0.075-0.697)were independent risk factors for failure after subsequent transplantation.ROC curve analysis showed that the area under the curve(AUC)of the Shannon index was 0.836(95%CI:0.782-0.890),with the highest predictive efficacy.There was significant interaction among the subgroups of Fusobacteria,Bacillus genus,E2 and insulin in Shannon index on the subsequent transplantation outcomes of patients with RIF.Conclusion The independent risk factors for subsequent transplantation failure in RIF patients can be used as sensitive indicators to predict the subsequent transplantation outcomes of RIF patients,and Shannon index has a higher clinical predictive value.

AIM To observe the effects of Yiqi Jiedu Tongluo Formula(YQJDTL)on renal microvascular endothelial function and prevention of renal injury in a rat model of type 2 diabetes mellitus(T2DM).METHODS The SD rats were randomly divided into a normal group and a model group.The model group was administered with high-fat diet combined with a single intraperitoneal injection of STZ to establish the T2DM model.The successfully modeled rats were randomly divided into the model group,the canagliflozin group(9 mg/kg),and the low-dose and high-dose YQJDTL groups(4.77,9.45 g/kg).The corresponding doses of the drug were administered by gavage for a total of 12 weeks,during which the rats underwent observation of their general condition and blood glucose changes.After the end of administration,the rats had their levels of renal index,24-hour UP,serum SCr,BUN,TC,TG,HDL-C,LDL-C,ET-1 and NOS measured;their changes in renal microvasculature and the degree of renal fibrosis observed using HE staining,Masson staining,PAS staining,and PASM staining;their ultrastructure of the glomeruli observed using transmission electron microscopy;their renal protein expressions of TGF-β,SMAD2,SMAD3,Col-1,VEGFA and PKC detected by immunohistochemical staining and Western blot;and their renal mRNA expressions of VEGFA,TGF-β,SMAD2 determined by RT-qPCR.RESULTS Compared with the model group,the high-dose YQJDTL group showed decreased levels of renal index,blood glucose,TG,TC,HDL,24 h UP,BUN,SCr and ET-1(P＜0.05,P＜0.01);increased LDL and NOS levels(P＜0.05,P＜0.01);reduced renal inflammatory infiltration and fibrosis degree,inhibited fusion of foot processes and thickening of basement membrane;decreased renal protein expressions of TGF-β,SMAD2,SMAD3,VEGFA,PKC and Col-1(P＜0.05,P＜0.01);and decreased mRNA expressions of VEGFA,TGF-β and SMAD2(P＜0.01).CONCLUSION In the rat models of T2DM,YQJDTL can reduce their levels of blood glucose and lipids by improving the renal indices levels and the renal microvascular endothelial functions to alleviate renal fibrosis and microangiopathy as well,and the mechanism may be associated with the down-regulated expressions of TGF-β/SMAD and VEGF pathway-related proteins.