BACKGROUND:Dapagliflozin,an inhibitor of sodium-glucose cotransporter 2,can delay the progression of atherosclerosis by regulating glucose metabolism,inhibiting inflammation and improving endothelial cell function. OBJECTIVE:To study the effect of dapagliflozin on cell pyroptosis and endothelial dysfunction induced by oxidized low-density lipoprotein. METHODS:Human umbilical vein endothelial cells were divided into a control group(no intervention),a model group(treated with oxidized low-density lipoprotein for 24 hours),and a dapagliflozin group(treated with oxidized low-density lipoprotein + dapagliflozin for 24 hours).Endothelial cell proliferation activity was measured by cell counting kit-8 assay.The levels of intercellular adhesion molecule 1,vascular cell adhesion molecule 1,and monocyte chemotactic protein-1 in cell supernatant were detected using ELISA.Nitric oxide level in the cells was detected by nitrate reductase assay.The pyroptosis rate and characteristics of endothelial cells were detected by Hoechst 33342/PI fluorescence co-staining and lactate dehydrogenase release assay.The protein expression levels of NLRP3,caspase-1,GSDMD,interleukin-1β,and interleukin-18 were detected by western blot assay. RESULTS AND CONCLUSION:(1)Oxidized low-density lipoprotein could cause pyroptosis and dysfunction of endothelial cells.(2)Compared with the control group,the level of nitric oxide and cell activity were decreased(P<0.05),while lactate dehydrogenase,intercellular adhesion molecule 1,vascular cell adhesion molecule 1,and monocyte chemotactic protein-1 levels were significantly increased in the model group(P<0.05).Compared with the model group,cell activity and nitric oxide levels significantly increased(P<0.05),but lactate dehydrogenase,intercellular adhesion molecule 1,vascular cell adhesion molecule 1,and monocyte chemotactic protein-1 levels were significantly diminished in the dapagliflozin group(P<0.05).(3)Compared with the model group,cell pyroptosis rate and the protein expression of pyroptosis factor NLRP3,caspase-1,GSDMD,interleukin-18 and interleukin-1β significantly reduced in the dapagliflozin group(P<0.05).(4)The results indicate that dapagliflozin inhibits oxidized low-density lipoprotein-induced endothelial pyroptosis and ameliorates endothelial cell dysfunction.

Objective : To investigate the role of dapagliflozin ( DAPA) in ox⁃LDL⁃induced pyroptosis of human myeloid leukemia monocytes (THP⁃1) derived foam cells . Methods : THP⁃1 ⁃derived foam cell pyroptosis model was constructed by ox⁃LDL⁃induced THP⁃1derived macrophages . The experimental groups were set as follows : the blank control group(NC) , the ox⁃LDL group(ox⁃LDL) , and the drug intervention group(ox⁃LDL + DAPA) . Oil Red Ostaining was used to detect the foam cell levels of macrophages . The cell proliferation and toxicity assay kit was used to detect the effect of DAPA on foam cell viability . Hoechst 33342/propidium iodide(PI) double staining was used to detect THP⁃1 derived foam cell pyroptosis . Cell immunofluorescence double staining was used to detect the effect of DAPA on the expression of pyroptosis key factor Caspase⁃1 in foam cells . The activity of lactate dehydrogenase (LDH) in the cell culture medium was detected using a microplate enzyme⁃linked immunosorbent assay. qRT⁃PCR and Western blot were used to detect the mRNA and protein expression levels of Nod⁃like receptor pyrindomain containing 3 (NLRP3) , cystein⁃containing aspartate⁃specific protease⁃1( Caspase⁃1 ) , apoptosis⁃associated⁃speck⁃like protein containing CARD(ASC) ,gasdermin⁃D (GSDMD) , interleukin(IL) Ⅳ18 and IL⁃1β , respectively . Results : The CCK⁃8 assay indicated that the optimal intervention concentration of DAPA was 10 μmol/L. Oil Red O staining confirmed the successful construction of the THP⁃1 ⁃derived foam cell pyroptosis model . Compared with the blank control group , the expression levels of NLRP3 , Caspase⁃1 , ASC , GSDMD , IL⁃18 , IL⁃1β mRNA and protein significantly increased in ox⁃LDL group(P < 0. 05) , as well as the number of PI⁃positive cells and LDH activity(P < 0. 05) , the fluorescence intensity of Caspase⁃1 and the number of redlipid droplets in the cytoplasm of the cells . However , these effects were significantly reversed after DAPA intervention in the ox⁃LDL + DAPA group(P < 0. 05) . Conclusion DAPA inhibits ox⁃LDL⁃induced pyroptosis in THP⁃1 ⁃derived foam cells .

The European Association for the Study of Liver Diseases issued the "Clinical Practice Guidelines for the Management of Hepatic Encephalopathy" in 2022, which included recommendations for clinical diagnosis, assessment, treatment, management, and prevention. The Society’s "Hepatic Encephalopathy Clinical Practice Guidelines in Chronic Liver Disease," which was last published in 2014, and the "Guidelines for the Diagnosis and Treatment of Hepatic Encephalopathy in Cirrhosis," which the Chinese Society of Hepatology, Chinese Medical Association, released in 2018, have certain differences and updates in terms of comparison to terminology, grading and classification, diagnosis, clinical evaluation and treatment, management, and prevention. Herein, the updated points of this guideline and the differences between it and our nation’s guidelines are summarized in order to refine and understand the guiding role of the new version of the guideline for the clinical treatment of hepatic encephalopathy and provide aid for standardizing clinical diagnosis and treatment.

Objective To study the effect of microalbuminuria (MAU) on artery elasticity in middle-aged and elderly essential hypertension (EH) patients with CHD.Methods Two hundred and forty-six EH patients were divided into middle-aged EH group (n=114) and elderly EH group (n=132) with 45 EH-free subjects undergoing physical examination served as a control group.The CF-PWV,Ba-PWV and CSP in patients included in this study were measured with an arteriosclerosis detector.Results The volume of ACR and incidence of CHD were significantly higher in middle-aged and elderly EH groups than in control group (19.30％ and 16.67％ vs 0％,P＜0.05).The number of 1-,2-and 3-branch lesions was significantly greater,the ba-PWV and ACR volume were significantly higher in elderly EH group than in middle-aged EH group (P＜0.05).The number of 3-branch lesions was significantly greater,the CSP,CF-PWV,ba-PWV and ACR volume were significantly higher in middle-aged and elderly EH groups with 3-branch lesions than in those with 1-and 2-branch lesions (P＜0.05).Multivariate linear regression analysis showed that age,CF-PWV,CSP,MAU were positively associated with the number of coronary artery lesions (P＜0.05,P＜0.01).Conclusion CF-PWV and CSP can show the severity of CHD and renal damage.The renal vascular damage is exacerbated in elderly EH patients with CHD.

Objective To investigate the mechanisms of metformin (Met) for protecting H9C2 myocardial cells damage induced by saturated fatty acids (palmitic acid,PA) in rats.Methods Rat H9C2 myocardial cell lines in blank control group,PA group and three different concentrations of metformin and PA combination groups were cultured for 24 h.Then Western blot was adopted to detect the protein expression of nuclear factor κB p65 (NFκB p65),intercellular cell adhesion molecule-1 (ICAM1),phosphorylated inhibitor of nuclear factor κB (p-IκBα) and phosphorylated adenosine monophosphate activated protein kinase(p-AMPK);the quantitative real-time PCR(qRT-PCR) was used to detect the mRNA expression of nuclear factor of nuclear factor κB(NFκB),chemokine (C-C motif) ligand 2 (CCL2) andintercellular cell adhesion molecule-1(ICAM1).Results Compared with the control groups,the protein expression of NFκB p65,p-IκBα and ICAM1 in the PA group was increased (P<0.05);compared with the PA group,the protein expression of NFκB p65,IκBα and ICAM1 in the Met+PA combination groups was decreased in various degrees with the Met concentration increase (all P<0.05),while the protein expression of p-AMPK was significantly risen with the Met concentration increase (all P<0.05).Compared with the control group,the mRNA expression of CCL2 and ICAM1 in the PA group was increased (P<0.05);compared with the PA group,the mRNA expression of CCL2 and ICAM1 in the Met+PA combination groups was decreased (P<0.05).Conclusion Met can alleviate H9C2 myocardial cellular damage caused by saturated fatty acid inducing increase of CCL2 and ICAM1 expression.

Objective To construct a eukaryotic expression plasmid of pcDNA 3.1-Ag85A-CD226, and to use it as DNA vaccine then further evaluate its immunogenicity through oral administration in a mouse model.Methods The CD226-PCR2.1-ToPo plasmid was used as the template to clone CD 226 gene by PCR.The CD226 gene was then inserted into pcDNA 3.1-Ag85A plasmid to construct the recombinant plas-mid of pcDNA3.1-Ag85A-CD226.After identified by restriction enzyme analysis and sequencing , the re-combinant plasmid was transfected into HEK 293 cells by using lipofection .The expression of Ag85A-CD226 gene in HEK293 cells was detected by RT-PCR, Western blot and indirect immunofluorescence assay .The purified recombinant plasmid was used to prepare the Ag 85A-CD226 DNA vaccine by liposomal encapsula-tion.The vaccine was administered intragastrically to mice .The activities of NK cells , the cytokine levels in the supernatants of spleen cell cultures and the mRNA level of cytokines in the intestines were evaluated to analyze the immunogenicity of Ag85A-CD226 DNA vaccine.Results The Ag85A-CD226 DNA vaccine was prepared successfully .The expression of Ag85A-CD226 fusion protein was detected in HEK293 cells.The activities of NK cells from mice vaccinated with Ag 85A-CD226 DNA vaccine were higher than those from other control groups (P<0.01).The level of TNF-α, IFN-γand IL-2 in the supernatants of spleen cell cul-tures and in the intestines were significantly up-regulated in comparison with other control groups ( P <0.01).The level of IL-4 in the supernatants of spleen cell cultures was down-regulated in the experimental group (P<0.01), but the level of IL-4 in intestines showed no significant difference among the five groups (P>0.05).Conclusion The Ag85A-CD226 DNA vaccine could significantly enhance Th1 type immune responses systemically and in the intestine as in comparison with those vaccinated with single dose of Ag 85A DNA vaccine or CD226 DNA vaccine.

Objective: To study expressions of small ubiquitin-related modifier protein（SUMO）4 (SUMO4), nuclear factor (NF)- κB and inhibitory factor of NF-κB (IκB) in kidneys of rats with type 2 diabetes mellitus (T2DM). Methods: A total of ten 40-week-old male Goto-Kakizaki (GK) rats （with spontaneous diabetes mellitus）of specific-pathogen free (SPF) grade, and ten 40-week-old male Wistar rats of SPF grade were selected. The lesion of renal tissue was observed by hematoxylin eosin (HE) staining. Expressions of SUMO4, NF-κB and IκB in renal tissue were observed by immunohistochemistry methods. Results: In the GK rats, glomerular capillary ball hypertrophy, basilar membrane slightly thickening; glomerular mesangial cells hyperplasia, hypertrophy and renal tubular epithelial cells hypertrophy were observed. Compared with normal Wistar rats, expression levels of NF-κB [(0.232±0.034) vs. (0.634±0.058)], IκB [(0.242±0.027) vs. (0.712±0.078)] and SUMO4 [(0.160±0.031) vs. (0.545±0.045)] significantly increased in renal tissue of GK rats (P<0.01 all). Conclusion: Compared with Wistar rats, expressions of NF-κB, IκB and SUMO4 significantly increase in renal tissue of GK rats, suggesting that SUMO inhibiting transcriptional activity of NF-κB may exist in kidneys of T2DM rats. Therefore, sumoylation may be a new therapeutic target for inhibit renal microvascular lesion of diabetic disease.

The aim of this study was to investigate the influences of heme oxygenase-1, carbon monoxide and nitricoxide synthase, nitrogen monoxide systems on vascular remodeling of injured balloon carotid artery in rabbits and the intercorrelations among the two systems after balloon angioplasty. Seventy rabbits were randomly divided into seven groups, i. e., control group, SH group, Chol group, Arg group, L-NAME group, Hem group, and Znpp group. The control group received normal chow, while all the rabbits the rest six groups received 1.5% cholesterol diet. Among the six test groups, to those in Chol group and SH group nothing else was added except the 1.5% cholesterol. L-arginine or L-nitro-arginine methylester was added to those in the Arg group and in the L-NAME group with drinking water. Hemin or zincprotoporphyrin IX was added to those in Hem group and in Znpp group by injecting the medicine into the abdominal cavity. After two weeks, the experimental groups underwent balloon injury at one side common carotid artery. Compared to Chol group, the HO-1 activity and CO production increased significantly. The intima area was reduced distinctly in Hem group, while there were opposite results in Znpp group. Compared with that in Chol group, the NF-kappaB activity of Arg group and Hem group were lower significantly. That of L-NAME group and Znpp group were higher significantly. Compared with that in the Chol group, the cNOS activity and NO production were eleveated markedly in Arg group while they were decreased markedly in L-NAME group. The intima area was reduced significantly in Arg group, while in L-NAME group they were not different from those in Chol group. These results suggested that the reciprocal relationship between HO-1/CO and NOS/NO system in restenosis may play the inhibitory role against neointimal proliferation and vascular wall remodeling after balloon angioplasty.
Animals ; Carbon Monoxide ; metabolism ; Carotid Arteries ; metabolism ; pathology ; physiopathology ; Catheterization ; adverse effects ; Heme Oxygenase-1 ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Rabbits ; Random Allocation

Objective To investigate the protective effect of heme oxygenase-1 (HO-1)/carbon monoxide (CO) system on restenosis after balloon angioplasty and relative mechanism. Methods Fifty rabbits were randomly divided into 5 groups. Control group received normal chow (Control group), the other rabbits received 1.5% cholesterol diet (Chol group and SH group) or 1.5%cholesterol diet plus hemin (Hem group) or zinc protoporphyrin Ⅸ (Znpp group) for 10 weeks. At the third week of experiment, the three experimental groups underwent balloon injury at one side common carotid artery. Results Compared with control group, arterial nitric oxide production and nitricoxide synthase activity were significantly decreased, while HO-1 expression and CO production were significantly increased (all P＜0. 01 ) in Chol group. The intima thickness and ratio of intima/media (I/M) were lower in Hem group than in Chol group [(281.47± 21.10) μm vs. (442.17 ±59.14) μm, 2.49 ± 0. 17 vs. 3.99 ± 0. 52, respectively]. While arterial HO-1 expression and CO production were increased markedly, endothelin-1 expression was distinctly reduced in Hem group group than in Chol group. Compared with Chol group, arterial HO-1 expression and CO production were decreased obviously, while endothelin-1 expression and intima thickness and ratio of intima/media [(698.71±58. 37) μm, 6.17±0. 52]were significantly increased in Znpp group (all P＜0. 01).Conclusions The HO-1/CO system plays a protective role on improving endothelium function and restraining neointimal proliferation by compensating and regulating nitricoxide synthase/nitric oxide system and lowering endothelin -1 expression so as to inhibit restenosis after balloon injury.

OBJECTIVETo study the membrane mobility of aquaporin 7 (AQP7) by cloning stably transfected CHO cells with expression of pEGFP-C1-AQP7, in which AQP7 cDNA was fused downstream and in frame to pEGFP-C1 gene.

METHODSThe full sequence of AQP7 was amplified by RT-PCR and then recombined in the downstream of the green fluorescent protein gene in the pEGFP-C1 vector. The recombinant vector pEGFP-C1-AQP7 was stably transfected into CHO cells. With fluorescent microscopy, immunocytochemical stain and Western blot, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization.

RESULTS(1) The sequence of AQP7 cDNA of the Wistar rat was logged into the GenBank (access number: AY157737). (2) Identification demonstrated that pEGFP-C1-AQP7 fusion protein stably expressed in CHO cells. (3) With fluorescence microscopy, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization.

CONCLUSIONThe CHO cell line with stable pEGFP-C1-AQP7 expression was set up successfully for advanced research.

Animals ; Aquaporins ; biosynthesis ; genetics ; CHO Cells ; Cricetinae ; Cricetulus ; DNA, Complementary ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Male ; Molecular Sequence Data ; Rats ; Rats, Wistar ; Recombinant Fusion Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Testis ; metabolism ; Transfection