ObjectiveTo evaluate the effects of drinking chlorinated water on the intestinal flora of rats and mice and to explore differences in the intestinal microecological responses of model animals to chlorine stimulation in chlorinated drinking water systems. MethodsSix 8-week-old male SD rats and six 8-week-old male C57BL/6J mice were acclimated for 5 days. Subsequently, the drinking water for both rats and mice was changed from pure water to water containing free chlorine at a concentration of 25.4–32.4 nmol/L. The intervention lasted for 8 weeks, during which all animals were fed the same diet. Fecal samples were collected before the intervention (week 0, as the control group) and on the last day of week 3 and week 8 (as the chlorinated water group), and microbial composition was analyzed by 16S rRNA sequencing, including α diversity (Chao1 and Shannon indices), β diversity [principal component analysis (PCA)], and linear discriminant analysis effect size (LEfSe) analysis. ResultsAfter 3 weeks of intervention, α diversity of the intestinal flora in both rats and mice in the chlorinated water group was significantly lower than that in the control group (P < 0.01). After 8 weeks of intervention, no significant difference in α diversity was observed between rats in the chlorinated water group and those in the control group, whereas α diversity in mice remained significantly lower than that in the control group (P < 0.01). β diversity analysis showed significant alterations in microbial structure in the chlorinated water group. LEfSe analysis indicated that, compared with the control group, the abundances of Bacteroides and Ruminococcus were significantly reduced in the chlorinated water group, and microbial disturbance was more pronounced in mice [linear discriminant analysis (LDA) > 4.0], with a greater decrease in microbial diversity and a larger number of differentially abundant genera. ConclusionConsumption of chlorinated water can alter the diversity and structure of the intestinal flora in both rats and mice, with mice being more sensitive to this exposure. These findings suggest that the potential effects of chlorine on gut microecology should be considered in drinking water management and model selection for animal experiments.

Objective To assess the clinical and safety outcomes of endovascular therapy(EVT)administered beyond 24 h after the onset of symptoms in patients with acute ischemic stroke(AIS)resulting from middle cerebral artery atherosclerotic occlusion.Methods We enrolled consecutive AIS patients with middle cerebral artery atherosclerotic occlusion who received EVT in our hospital between January 2020 and June 2023 and divided them into two groups based on the time from AIS onset to EVT:EVT≤24 h group and EVT＞24 h group.The National Institutes of Health Stroke Scale(NIHSS)scores after surgery,90-day functional independence(defined as mRS 0-2),symptomatic ICH(sICH),postoperative acute thrombosis,as well as mortality,were analyzed in the two groups of patients.Results A total of 161 patients were recruited,with 106 cases in the EVT≤24 h group and 55 cases in the EVT＞24 h group.The NIHSS scores declined after EVT in both groups(P＜0.01).The NIHSS scores after surgery were lower in the EVT≤24 h group(median:4.5 vs.7.0,P＜0.05).90-day functional independence(45.3％vs.30.9％,95％CI:0.93-3.68),sICH(5.7％vs.9.1％,95％CI:0.18-2.06),postoperative acute thrombosis(6.6％vs.3.6％,95％CI:0.37-9.34),and mortality(3.8％vs.1.8％,95％CI:0.23-19.42)did not differ significantly between the groups(P＞0.05).Conclusion Our findings support EVT feasibility in selected patients with middle cerebral artery atherosclerotic occlusion beyond 24 h.Prospective studies are warranted for confirmation.

OBJECTIVE To investigate the impact of dapagliflozin on contrast-induced acute kidney injury(CIAKI)and prognosis in patients with diabetes mellitus type 2(T2DM)and acute coronary syndrome(ACS)who underwent percutaneous coronary intervention(PCI).METHODS Retrospective selection of data on T2DM patients with ACS who underwent PCI treatment in the Cardiology Department of Tianjin Chest Hospital from January 1st 2021 to December 31st 2022.The patients were divided into dapagliflozin group(96 cases)and control group(148 cases)based on whether they received dapagliflozin or not.Renal function indicators were measured for all enrolled patients before PCI and at 48 h and 1 week after PCI,including blood urea nitrogen(BUN),serum creatinine(Scr),estimated glomerular filtration rate(eGFR),cystatin-C(Cys-C),kidney injury molecule-1(KIM-1)and β2-microglobulin(β2-MG).All patients were followed up for at least 1 year.The incidence of CIAKI and major adverse cardiac event(MACE)during follow-up were recorded for both groups.Logistic regression was used to analyze the impact of dapagliflozin on the occurrence of CIAKI,while the Log-rank test was applied to compare the incidence of MACE between the two groups.Cox regression was employed to analyze the impact of dapagliflozin on prognosis.RESULTS At 48 h and 1 week after PCI,serum levels of Cys-C,KIM-1 and β2-MG were significantly lower in the dapagliflozin group compared to the control group(P＜0.05).The incidence of CIAKI was lower in the dapagliflozin group compared to the control group(6.25%vs.14.86%,P=0.042).Logistic regression analysis revealed that dapagliflozin was an independent protective factor against CIAKI(OR=0.280,95%CI 0.101-0.780,P=0.015).During the follow-up period,the incidence of MACE was lower in the dapagliflozin group compared to the control group(7.29%vs.17.57%,P=0.049).Cox regression analysis indicated that dapagliflozin reduced the occurrence of MACE after PCI(HR=0.374,95%CI 0.161-0.866,P=0.022).CONCLUSIONS With adequate hydration,the use of dapagliflozin does not increase the risk of CIAKI following PCI in T2DM patients with ACS.

Objective:To establish a rapid and effective melanin-bleaching method to standardize and improve the molecular pathological analyses of melanoma.Methods:Fifteen cases of melanoma with melanin content exceeding 50% collected at the Precision Pathology Diagnosis Center, Weifang People′s Hospital, Weifang, China between 2023 and 2024 were included in the study. These tissue samples were subject to melanin-bleaching treatments using H 2O 2, potassium permanganate (KMnO 4), Tris-HCl or PBS method. The bleaching effects in each group were evaluated using hematoxylin and eosin (HE) staining. Thereafter, the total amount, purity, and integrity of DNA extracted from bleached tissues were analyzed using UV spectrophotometry and Qsep1 Bio-fragment analyzer. Finally, in order to compare the efficiency of DNA amplification, C-KIT and PDGFRA were examined using Sanger sequencing, and BRAF was detected using real-time quantitative PCR. Results:Among the 15 cases, there were seven males and eight females whose median age was 68 (range, 63 to 71) years. Bleached tissue in Tris-HCl group had the highest HE score, which was significantly higher than those in the groups of PBS, H 2O 2, KMnO 4, and control ( F=113.3, P<0.05). The total DNA amount in the control and Tris-HCl groups was significantly higher than those in the groups of PBS, H 2O 2, and KMnO 4, respectively ( F=275, P<0.05). Meanwhile, the mean A260/ A280 values of DNA obtained from bleached tissue in Tris-HCl and KMnO 4 were better than that of control, and the mean A260/ A230 values of Tris-HCl and KMnO 4 were higher than that of control, PBS, and H 2O 2. Meanwhile, the proportion of >5 000 bp,>3 000 bp, and >1 000 bp DNA fragments in Tris-HCl was all significantly higher than those in other groups ( F=147.9, F=127.9 and F=61.9, respectively, P<0.05). C-KIT and PDGFRA genes were both successfully amplified based on DNA obtained from bleached tissue in Tris-HCl group, and the sequencing peaks were clear and free of noticeable noises. Real-time quantitative PCR results showed that Tris-HCl method had lower cycle threshold values than other methods in detecting BRAF gene ( F=30.36, P<0.05). Conclusions:Tris-HCl method is a fast and effective melanin-bleaching method for analyzing melanoma, which could remove melanin effectively, confirm high quality of DNA and good integrity of DNA, and improve the amplification efficiency of sequencing for melanoma related genes. This new method may help pathologists with reliable molecular typing and identifying therapeutic targets for melanoma. It may greatly improve clinical management of melanoma patients.

It was a retrospective study. The propensity score matching was applied to divide the type 2 diabetes mellitus (T2DM) patients who have underwent percutaneous coronary intervention (PCI) into two groups: short-term (<4 weeks) sodium-glucose cotransporter 2 inhibitor (SGLT2i) group (213 patients) and control group (213 patients). The occurrence of contrast-induced acute kidney injury (CIAKI) after PCI was compared between the two groups. The results showed that the incidence of CIAKI in the SGLT2i group was not significantly different from the control group [10.8% (23/213) vs. 7.5% (16/213), χ2=1.383, P=0.313]. The incidence of CIAKI in patients with SGLT2i application time <1 week was higher than that in control patients, but the difference was not statistically significant [13.00% (16/123) vs. 7.5% (16/213), χ2=2.734, P=0.122]. Multivariate logistic regression analysis showed that short-term (<4 weeks) use of SGLT2i would not increase the risk of CIAKI after PCI in T2DM patients ( OR=0.507, 95% CI 0.238-1.077, P=0.077). Short-term application of SGLT2i before PCI may not increase the risk of CIAKI, but it is advisable to avoid initiating the application of SGLT2i before PCI as much as possible.

Objective:To investigate the effects of phillyrin(PHN)on the proliferation,invasion,and epithelial-mesenchymal transition(EMT)of glioma U251 cells by adjusting the high mobility group protein B1(HMGB1)/receptor of advanced glycation endproduct(RAGE)signaling pathway.Methods:Human glioma U251cells were assigned into the PHN-0 group(treated with 0 μmol/L PHN),the low,medium,and high-dose PHN groups(PHN-50、PHN-100、PHN-200 groups,treated with 50,100,and 200 μmol/L PHN respectively),the PHN+pcDNA-NC group(treated with 200 μmol/L PHN after transfection of pcDNA-NC plasmid),and the PHN+HMGB1 group(treated with 200 μmol/L PHN after transfection of overexpressed HMGB1 plasmid).The proliferation ability of cells in each group was detected by the CCK-8 method and the clone formation assay.The apoptosis level of cells in each group was detected by flow cytometry.The migration and invasion abilities of cells in each group were detected by the Transwell assay.ELISA was used to detect the IL-8 secretion level of cells in each group.Immunofluorescence was used to detect the positive rates of N-cadherin and E-cadherin in cells of each group.WB assay was performed to detect the expression levels of Toll like receptor 4(TLR4),nuclear factor-kappa B(NF-κ B),HMGB1,RAGE,N-cadherin,E-cadherin,cell cycle protein D1(cyclin D1),cyclin dependent kinase 2(CDK2),B-lymphoblastoma-2(Bcl-2),Bcl-2 associated X protein(BAX)proteins in cells of each group.Results:Compared with those in the PHN-0 group,the proliferation activity,the number of clone formation,the numbers of invasion and migration,IL-8 secretion levels,the positive rate and protein expression of N-cadherin,and the expressions of TLR4,NF-κB,HMGB1,RAGE,cyclin D1 and CDK2 protein in the PHN-50,PHN-100,and PHN-200 groups decreased significantly(all P<0.05);and the apoptosis rate,the positivity rate and protein expression of E-cadherin,and the BAX/Bcl-2 ratio increased significantly(all P<0.05).At the same time,overexpression of HMGB1 could reverse the inhibitory effects of PHN on the proliferation,migration,invasion and EMT of U251 cells,as well as its promoting effect on the apoptosis(all P<0.05).Conclusion:PHN inhibits the proliferation,invasion and EMT progression of glioma U251 cells through the HMGB1/RAGE signaling pathway.

Objective To investigate the regulatory role and mechanism of lobetyolin(LBT,a poly-acetylene glycoside isolated from the roots of Codonopsis pilosula)in the proliferation and apop-tosis of brain glioma cells based on the Akt/GSK-3β/Snail signaling pathway.Methods Human brain glioma cell line U-373MG was randomly divided into normal,SC79(Akt activator),LBT,and LBT+SC79 groups.After corresponding interventions,CCK-8 assay,colony formation assay,and flow cytometry were used to detect the proliferation and apoptosis of the cells.Western blot-ting was employed to measure the protein expression levels of the molecules related to prolifera-tion,apoptosis,and Akt/GSK-3β/Snail signaling pathway.After tumor xenograft nude mouse model of U-373MG cells was established,followed by grouping and interventions as above cell experiments,the tumor weight and volume were measured.Immunohistochemical assay and TUNEL assay were performed to detect the proliferation and apoptosis of tumor cells.Western blotting was applied to detect Akt/GSK-3β/Snail signaling pathway related proteins in the nude mouse groups.Results In the LBT+SC79 group,cell viability,number of formed colonies,pro-tein levels of cyclin D1,Bcl-2 and Snail,p-Akt/Akt and p-GSK-3β/GSK-3β,tumor weight and vol-ume,and positive ratios of Ki67,cyclin D1 and Bcl-2 in transplanted tumors were increased(P＜0.05),and cell apoptotic rate[(3.20±1.14)％vs(46.15±1.52)％,P＜0.05],Bax protein level(0.51±0.07 vs 0.89±0.06,P＜0.05),and positive ratios of TUNEL[(51.56±7.13)％vs(74.95±8.61)％,P＜0.05]and Bax[(32.71±5.43)％vs(41.86±4.90),P＜0.05]in transplanted tumors were declined when compared with the LBT group.Conclusion LBT can induce apoptosis and inhibit proliferation of brain glioma cells in vitro and in vivo by blocking activation of the Akt/GSK-3β/Snail signaling pathway.

Objective: To investigate whether zerumbone ( ZER) has the effect of preventing cisplatin ( Cis) -induced acute kidney injury (Cis-AKI) . Methods: The MTT method was used to detect the effect of different concentrations of ZER on the cell viability of Cis-AKI. The in vivo and in vitro models of Cis-AKI mice were estab- lished by dividing into control group , separate administration group , model group , and dose group. Western blot and immunofluorescence experiments were used to detect the expression changes of kidney injury marker-1 ( KIM- 1) , phosphorylated NF-κB p65 ( P-p65 ) , Cleaved casepase3 , receptor interacting protein kinase 1 ( RIPK1) , RIPK3 , and tumor necrosis factor-α (TNF-α) . Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expression of KIM-1 , TNF-α , interleukin-6 ( IL-6) , and monocyte chemoattractant protein-1(MCP-1) . Periodic acid-Schiff (PAS) staining confirmed the therapeutic effect of ZER on Cis-AKI. RNA-seq and cell thermal shift (CETSA) were used to explore possible target proteins. Results : MTT results showed that ZER could alleviate the decrease in cell viability of Cis-AKI ; in vivo and in vitro studies showed that compared with the model group , after treatment with ZER , its KIM-1 , P-p65 , Cleaved casepased3 , RIPK1 , RIPK3 , TNF -α expres- sion decreased significantly , and the mRNA expression of KIM-1 , TNF-α , IL-6 mRNA , and MCP-1 decreased. PAS staining showed that ZER had a therapeutic effect on Cis-AKI. RNA-seq and CETSA analysis showed that ZER might prevent and treat Cis-AKI by targeting the PIM1 protein. Conclusion ZER may alleviate Cis-AKI and im- prove inflammatory response and necroptosis by regulating PIM1 protein. ZER is expected to be a potential drug for the prevention and treatment of Cis-AKI.

Objective To investigate the regulatory role and mechanism of lobetyolin(LBT,a poly-acetylene glycoside isolated from the roots of Codonopsis pilosula)in the proliferation and apop-tosis of brain glioma cells based on the Akt/GSK-3β/Snail signaling pathway.Methods Human brain glioma cell line U-373MG was randomly divided into normal,SC79(Akt activator),LBT,and LBT+SC79 groups.After corresponding interventions,CCK-8 assay,colony formation assay,and flow cytometry were used to detect the proliferation and apoptosis of the cells.Western blot-ting was employed to measure the protein expression levels of the molecules related to prolifera-tion,apoptosis,and Akt/GSK-3β/Snail signaling pathway.After tumor xenograft nude mouse model of U-373MG cells was established,followed by grouping and interventions as above cell experiments,the tumor weight and volume were measured.Immunohistochemical assay and TUNEL assay were performed to detect the proliferation and apoptosis of tumor cells.Western blotting was applied to detect Akt/GSK-3β/Snail signaling pathway related proteins in the nude mouse groups.Results In the LBT+SC79 group,cell viability,number of formed colonies,pro-tein levels of cyclin D1,Bcl-2 and Snail,p-Akt/Akt and p-GSK-3β/GSK-3β,tumor weight and vol-ume,and positive ratios of Ki67,cyclin D1 and Bcl-2 in transplanted tumors were increased(P＜0.05),and cell apoptotic rate[(3.20±1.14)％vs(46.15±1.52)％,P＜0.05],Bax protein level(0.51±0.07 vs 0.89±0.06,P＜0.05),and positive ratios of TUNEL[(51.56±7.13)％vs(74.95±8.61)％,P＜0.05]and Bax[(32.71±5.43)％vs(41.86±4.90),P＜0.05]in transplanted tumors were declined when compared with the LBT group.Conclusion LBT can induce apoptosis and inhibit proliferation of brain glioma cells in vitro and in vivo by blocking activation of the Akt/GSK-3β/Snail signaling pathway.

Objective:To establish a rapid and effective melanin-bleaching method to standardize and improve the molecular pathological analyses of melanoma.Methods:Fifteen cases of melanoma with melanin content exceeding 50% collected at the Precision Pathology Diagnosis Center, Weifang People′s Hospital, Weifang, China between 2023 and 2024 were included in the study. These tissue samples were subject to melanin-bleaching treatments using H 2O 2, potassium permanganate (KMnO 4), Tris-HCl or PBS method. The bleaching effects in each group were evaluated using hematoxylin and eosin (HE) staining. Thereafter, the total amount, purity, and integrity of DNA extracted from bleached tissues were analyzed using UV spectrophotometry and Qsep1 Bio-fragment analyzer. Finally, in order to compare the efficiency of DNA amplification, C-KIT and PDGFRA were examined using Sanger sequencing, and BRAF was detected using real-time quantitative PCR. Results:Among the 15 cases, there were seven males and eight females whose median age was 68 (range, 63 to 71) years. Bleached tissue in Tris-HCl group had the highest HE score, which was significantly higher than those in the groups of PBS, H 2O 2, KMnO 4, and control ( F=113.3, P<0.05). The total DNA amount in the control and Tris-HCl groups was significantly higher than those in the groups of PBS, H 2O 2, and KMnO 4, respectively ( F=275, P<0.05). Meanwhile, the mean A260/ A280 values of DNA obtained from bleached tissue in Tris-HCl and KMnO 4 were better than that of control, and the mean A260/ A230 values of Tris-HCl and KMnO 4 were higher than that of control, PBS, and H 2O 2. Meanwhile, the proportion of >5 000 bp,>3 000 bp, and >1 000 bp DNA fragments in Tris-HCl was all significantly higher than those in other groups ( F=147.9, F=127.9 and F=61.9, respectively, P<0.05). C-KIT and PDGFRA genes were both successfully amplified based on DNA obtained from bleached tissue in Tris-HCl group, and the sequencing peaks were clear and free of noticeable noises. Real-time quantitative PCR results showed that Tris-HCl method had lower cycle threshold values than other methods in detecting BRAF gene ( F=30.36, P<0.05). Conclusions:Tris-HCl method is a fast and effective melanin-bleaching method for analyzing melanoma, which could remove melanin effectively, confirm high quality of DNA and good integrity of DNA, and improve the amplification efficiency of sequencing for melanoma related genes. This new method may help pathologists with reliable molecular typing and identifying therapeutic targets for melanoma. It may greatly improve clinical management of melanoma patients.