Objective To evaluate whether Vibrio vulnificus secreted exotoxin-hemolysin (VVH) can activate platelet, an important blood immune cell, and to explore the possible molecular mechanism of platelet activation by VVH. Methods Transcriptomics and immunohistochemistry were used to analyze whether Vibrio vulnificus infection caused platelet activation in mice. Then, flow cytometry was used to identify whether VVH was the main stimulator of platelet activation. Naturally expressed VVH toxin was purified and prepared. The effects of extracellular and intracellular Ca²⁺ signal inhibitors on VVH activated platelets were evaluated by flow cytometry and Western blotting. The immune activation effect of VVH in the early stage of Vibrio vulnificus infection was analyzed in vivo. Results VVH was the main stimulator of platelet activation in Vibrio vulnificus culture supernatant. Natural VVH can induce the increase of P-selectin (CD62P) on platelet surface, the formation of platelet-neutrophil complex (PNC), and the release of platelet microvesicles. The activation mechanism may be related to the VVH pore-dependent Ca²⁺-calmodulin (CaM) -myosin light chain kinase (MLCK) signaling pathway, which led to the release of platelet alpha particles and cascade activation of platelets. In a mouse model of ALD infected by Vibrio vulnificus gavage, VVH was strongly associated with platelet activation. Conclusion This study shows that VVH is an important platelet activating molecule in the early stage of Vibrio vulnificus infection, and its induction of platelet activation may be related to the pathogenic process.
Animals ; Platelet Activation/drug effects* ; Hemolysin Proteins/pharmacology* ; Vibrio vulnificus/metabolism* ; Mice ; Blood Platelets/drug effects* ; Vibrio Infections/immunology* ; P-Selectin/metabolism* ; Bacterial Proteins ; Female

Objective To explore the mechanism of TPT1-AS1 targeting miR-324/TWIST1 axis to regulate the proliferation, invasion, migration and angiogenesis of epithelial ovarian cancer (EOC) cells, thereby affecting ovarian cancer (OC) progression. Methods RT-qPCR was used to detect the expression of TPT1-AS1 and miR-324 in 29 OC lesions and adjacent tissue samples. The two OC cell models of TPT1-AS1 overexpression and miRNA324 knockdown were constructed, and the cell proliferation, invasion and migration abilities were detected by CCK-8, Transwell^TM and scratch test. Western blot analysis was used to detect the protein expression levels of TWIST1, epithelial cadherin (E-cadherin), Vimentin, and vascular endothelial growth factor A (VEGF-A) in OC cells. Fluorescence in situ hybridization (FISH) and RNA pull-down experiments were used to verify the interaction between TPT1-AS1 and miR-324. Immunohistochemistry and Targetscan bioinformatics analysis were used to verify the negative regulatory role of miR-324 in the epithelial-mesenchymal transition (EMT) process. Results The TPT1-AS1 expression was significantly higher in OC tissues than that in para-cancerous tissues, while the miR-324 expression was significantly lower. In SKOV3 cells with TPT1-AS1 overexpression, the miR-324 expression decreased significantly, and TPT1-AS1 was negatively correlated with miR-324. It was also found that TPT1-AS1 and miR-324 were co-expressed in OC cells, and there was a direct binding relationship between them. Down-regulation of miR-324 significantly promoted the proliferation, invasion and migration of SKOV3 cells. Further studies revealed that miR-324 had a binding site at the 3'-UTR end of the TWIST1, a key transcription factor for EMT. Inhibiting miR-324 expression increased the transcription level of TWIST1, leading to a decrease in E-cadherin protein expression and an increase in Vimentin protein expression. Additionally, the downregulation of miR-324 resulted in an increased expression level of VEGF-A protein, which in turn enhanced angiogenesis of OC. Conclusion TPT1-AS1 promotes EOC cell proliferation, invasion, migration and angiogenesis by negatively regulating the miR-324/TWIST1 axis, thus promoting the development of OC. These findings provide new potential targets for the diagnosis and treatment of OC.
Humans ; MicroRNAs/metabolism* ; Female ; Cell Movement/genetics* ; Ovarian Neoplasms/blood supply* ; Twist-Related Protein 1/metabolism* ; Cell Line, Tumor ; Neovascularization, Pathologic/genetics* ; Neoplasm Invasiveness ; Carcinoma, Ovarian Epithelial/metabolism* ; Nuclear Proteins/metabolism* ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic ; RNA, Long Noncoding/metabolism* ; Cadherins/genetics* ; Vascular Endothelial Growth Factor A/genetics* ; Vimentin/genetics* ; Angiogenesis

Objective To investigate the effect and mechanism of baicalin on blood lipid metabolism and immune function in rats with gestational diabetes mellitus (GDM). Methods Female rats fed with high-fat and high-sugar diet and male rats fed with ordinary diet were caged together to prepare pregnant rats, and the GDM rat model was established by intraperitoneal injection of streptozotocin (35 mg/kg). GDM rats were randomly divided into a model group, a fasudil (FA) (RhoA/RocK inhibitor) group (10 mg/kg), low-dose (100 mg/kg) and high-dose (200 mg/kg) baicalin groups, and a high-dose baicalin combined with LPA (RhoA/RocK activator) group (200 mg/kg baicalin+1 mg/kg LPA ), with 12 rats in each group. Another 12 pregnant rats fed with high-fat and high-sugar diet were selected as the control group. After 2 weeks of corresponding drug intervention in each group, the level of fasting blood glucose (FBG) was detected by blood glucose meter. The level of fasting insulin (FINS) in serum was detected by ELISA, and the insulin resistance index (HOMA-IR) was calculated. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) in serum, and the levels of immunomodulator tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-10 in peripheral blood were detected by the kit. The histopathological changes of liver were observed by HE staining. The proportion of T lymphocyte subsets in peripheral blood was detected by flow cytometry. The mRNA and protein expressions of Ras homolog gene family member A (RhoA), Rho associated coiled-coil forming protein kinase 1 (ROCK1), and ROCK2 in liver tissue were detected by real-time quantitative PCR and Western blot. Results Compared with the control group, the levels of FBG, FINS, HOMA-IR, ALT, AST, TG, TC, and LDL-C in serum, the levels of TNF-α, IL-6, the percentage of CD8⁺T cell in peripheral blood, and the mRNA and protein expression of RhoA, ROCK1, and ROCK2 in liver tissue in the model group were higher; the level of HDL-C in serum, the percentage of IL-10 levels, CD3⁺T cells, CD4⁺T cell, and CD4⁺T/CD8⁺T ratio in peripheral blood were lower. Compared with the model group, the levels of FBG, FINS, HOMA-IR, ALT, AST, TG, TC, and LDL-C in serum, the levels of TNF-α, IL-6, the percentage of CD8⁺T cell in peripheral blood, and the mRNA and protein expression of RhoA, ROCK1, and ROCK2 in liver tissue in the the FA group and low-dose and high-dose baicalin groups were lower; the level of HDL-C in serum, IL-10 level, the percentage of CD3⁺T cells, CD4⁺T cell, and CD4⁺T/CD8⁺T ratio in peripheral blood were higher. LPA could obviously weaken the improvement effects of baicalin on blood lipid metabolism and immune function in GDM rats. Conclusion Baicalin may improve blood lipid metabolism and immune function in GDM rats by inhibiting the RhoA/ROCK pathway.
Animals ; Female ; Diabetes, Gestational/metabolism* ; Pregnancy ; rho-Associated Kinases/genetics* ; Flavonoids/pharmacology* ; Rats ; rhoA GTP-Binding Protein/genetics* ; Lipid Metabolism/drug effects* ; Male ; Signal Transduction/drug effects* ; Rats, Sprague-Dawley ; Blood Glucose/metabolism* ; Lipids/blood* ; Tumor Necrosis Factor-alpha/blood* ; rho GTP-Binding Proteins

Pseudorabies (PR) is an infectious disease caused by the pseudorabies virus (PRV), affecting various domesticated and wild animals. Since pigs are the only natural hosts of PRV, PR poses a serious threat to the pig farming industry. Currently, PR is primarily prevented through vaccination with inactivated vaccines or genetically modified attenuated live vaccines. Developing safe and effective genetically engineered vaccines would facilitate the eradication and control of PR. In this study, the PRV vaccine strain Bartha-K61 was used as the reference strain. The gB protein was expressed via the baculovirus-insect cell expression system. Non-denaturing gel electrophoresis confirmed that the gB protein could form a trimeric structure. The purified gB protein was used to immunize mice, and the immune effect was evaluated by a challenge test. The results showed that the gB antigen induced a strong immune response in mice, with the serum-neutralizing antibody titer above 1:70. The lymphocyte stimulation index reached more than 1.29, and the level of (interferon gamma, IFN-γ) release was higher than 100 pg/mL. After immunization, mice were challenged with the virus at a dose of 10⁴ TCID₅₀/mL, 200 μL per mouse, and the clinical protection rate was 100%. Immunohistochemistry, histopathological section, and tissue viral load results showed that the pathological damage and viral load in the gB-immunized group were significantly lower than those in the PBS group. In summary, the gB protein obtained in this study induced strong humoral and cellular immune responses in mice, laying a foundation for developing a recombinant gB protein subunit vaccine.
Animals ; Mice ; Baculoviridae/metabolism* ; Viral Envelope Proteins/biosynthesis* ; Herpesvirus 1, Suid/genetics* ; Pseudorabies/immunology* ; Swine ; Pseudorabies Vaccines/genetics* ; Antibodies, Viral/blood* ; Insecta/cytology* ; Mice, Inbred BALB C ; Female ; Viral Vaccines/immunology*

In this study, we constructed a series of recombinant Fc variants of immunoglobulin G4 (IgG4), screened the fragment crystallizable (Fc) variants with significantly prolonged serum half-lives, and analyzed the relationship between mutation site and half-life, aiming to provide a theoretical basis for the development of IgG4 antibodies and Fc fusion protein-based drugs. Nine gene sites were selected for mutation, and different mutation sites were combined. The variant expression plasmids pET24b-Fc were constructed by molecular cloning and point mutation. The plasmids were transformed into Escherichia coli BL21(DE3) for the expression of different recombinant proteins of Fc. Fc2 and Fc3 variants had slightly lower recombinant protein yields, and the expression of other variants was not affected. The toxicity of different Fc variants was determined by cell counting kit-8 (CCK-8) and calcein acetoxymethyl ester/ propidium iodide (calcein AM/PI) in vitro and enzyme-linked immuno sorbent assay (ELISA) in vivo. The results showed that the recombinant Fc variants had good biocompatibility and safety. Finally, the Fc variants were labeled with fluorescent markers, and the effects of different mutations on their serum half-lives were investigated by in vivo experiments. The Fc5 variant with prolonged serum half-life was successfully screened out, which provided a theoretical and practical basis for the optimal design of IgG4 subtype antibody and Fc fusion protein drugs.
Immunoglobulin G/blood* ; Immunoglobulin Fc Fragments/biosynthesis* ; Half-Life ; Animals ; Escherichia coli/metabolism* ; Humans ; Recombinant Fusion Proteins/biosynthesis* ; Recombinant Proteins/biosynthesis* ; Mice

Objective To explore the effect of rehmanniae radix extract(RRE)on chondrocyte apoptosis in the rabbit model of knee osteoarthritis(KOA)by regulating the miR-485-5p/heat shock protein 90 beta family member 1(Hsp90b1)axis.Methods New Zealand rabbits were randomly assigned into control,KOA,low-dose RRE,medium-dose RRE,high-dose RRE,celecoxib,high-dose RRE+antagonist control,and high-dose RRE+miR-485-5p antagonist groups,with 12 rabbits in each group.Rabbits in other groups except the control group were modeled for KOA with the improved Hulth method.After modeling for 8 weeks,the rabbits were administrated with corresponding agents for 4 weeks.The changes in the activity rating of rabbits were recorded.ELISA was employed to measure the levels of tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 in the serum.Safranine O-fast green staining was conducted to reveal the pathological changes in the cartilage tissue and Mankin scoring was performed.TUNEL was employed to detect chondrocyte apoptosis.Real-time fluorescence quantitative PCR was performed to determine the expression of miR-485-5p in the cartilage tissue.Western blot was employed to determine the protein levels of Hsp90b1,cleaved cysteinyl aspartate-specific proteinase-3(Caspase-3),and Bcl2-associated-X(Bax)in the cartilage tissue.The dual-luciferase reporter assay was employed to examine the relationship between miR-485-5p and Hsp90b1.Results Compared with the control group,the KOA group showed down-regulated expression of miR-485-5p,elevated levels of TNF-α and IL-6 in the serum,cartilage erosion and losses,and increases in activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.001).Compared with the KOA group,RRE at low,medium,and high doses,and celecoxib up-regulated the expression of miR-485-5p,lowered the levels of TNF-α and IL-6 in the serum,alleviated the pathological damage to the cartilage tissue,and decreased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).Compared with the high-dose RRE group and the high-dose RRE+antagonist control group,high-dose RRE+miR-485-5p antagonist down-regulated the expression of miR-485-5p,elevated the levels of TNF-α and IL-6 in the serum,exacerbated the pathological damage to the cartilage tissue,and increased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).The results indicated that there was a targeted regulatory relationship between miR-485-5p and Hsp90b1.Conclusion RRE may inhibit the expression of Hsp90b1 by up-regulating miR-485-5p,thereby inhibiting chondrocyte apoptosis in the rabbit model of KOA.
Animals ; Rabbits ; Apoptosis/drug effects* ; Chondrocytes/pathology* ; Osteoarthritis, Knee/drug therapy* ; MicroRNAs/metabolism* ; Rehmannia/chemistry* ; Disease Models, Animal ; Tumor Necrosis Factor-alpha/blood* ; Plant Extracts/pharmacology* ; Interleukin-6/blood* ; HSP90 Heat-Shock Proteins/metabolism* ; Male ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVES: To investigate the correlation between bone mineral density (BMD) and bone metabolic markers in preschool children and the influencing factors for BMD, and to provide a clinical basis for promoting bone health in children. METHODS: A retrospective analysis was performed for the data of 127 preschool children who underwent physical examination in the Department of Child Health Care of the First Affiliated Hospital of Xinjiang Medical University, from June to December 2024. BMD and bone metabolic markers were measured, and physical examination was performed. A multiple linear regression analysis was used to investigate the effect of general information on BMD Z-score in preschool children. Spearman's rank correlation test was used to investigate the correlation of BMD Z-score with 25-hydroxyvitamin D (25-OHD), serum bone Gla protein (BGP), and parathyroid hormone (PTH). RESULTS: BMD Z-score significantly differed by ethnicity, weight category, and height category (all P<0.05). The multiple linear regression analysis indicated that weight and height significantly influenced BMD Z-score (P<0.05), whereas sex, age, ethnicity, and parental education level did not (P>0.05). In children, BMD Z-score was positively correlated with 25-OHD level (r_s=0.260, P<0.001) and BGP level (r_s=0.075, P=0.025) and was negatively correlated with PTH level (r_s=-0.043, P=0.032). CONCLUSIONS Weight, height, 25-OHD, BGP, and PTH are influencing factors for BMD in preschool children. In clinical practice, combined measurement of bone metabolic markers may provide a scientific basis for early identification of children with abnormal BMD and prevention of osteoporosis and osteomalacia.
Humans ; Bone Density ; Child, Preschool ; Female ; Male ; Retrospective Studies ; Vitamin D/blood* ; Parathyroid Hormone/blood* ; Biomarkers/blood* ; Osteocalcin/blood* ; Bone and Bones/metabolism* ; Calcium-Binding Proteins/blood* ; Linear Models ; Matrix Gla Protein ; Extracellular Matrix Proteins/blood* ; Body Weight ; Infant

OBJECTIVES: To investigate the role of apelin in regulating proliferation, migration and angiogenesis of bladder cancer cells and the possible regulatory mechanism. METHODS: GEO database was used to screen the differentially expressed genes in bladder cancer tissues and cells. Bladder cancer and paired adjacent tissues were collected from 60 patients for analysis of apelin expressions in relation to clinicopathological parameters. In cultured bladder cancer J82 cells and human umbilical vein endothelial cells (HUVECs), the effects of transfection with an apelin-overexpressing plasmid or specific siRNAs targeting apelin, fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 1 (FGFR1) on proliferation and migration of J82 cells and tube formation in HUVECs were examined using plate cloning assay, Transwell assay, and angiogenesis assay; the changes in FGF2 expression and FGFR1 phosphorylation were detected using Western blotting. RESULTS: The expression level of apelin was significantly higher in bladder cancer tissues than adjacent tissues, and bladder cancer cell lines (T24 and J82) also expressed higher mRNA and protein levels of apelin than SV-HUC-1 cells. Apelin expression level in bladder cancer tissues was correlated with tumor invasion, distant metastasis and advanced TNM stages. Apelin knockdown significantly suppressed proliferation and migration of J82 cells and decreased the total angiogenic length of HUVECs. In contrast, apelin overexpression significantly promoted proliferation and migration and enhanced FGFR1 phosphorylation in J82 cells, and increased the total angiogenesis length in HUVECs, but this effects were effectively mitigated by transfection of the cells with FGF2 siRNA or FGFR1 siRNA. CONCLUSIONS High expression of apelin promotes J82 cell proliferation and migration and HUVEC angiogenesis by promoting activation of the FGF2/FGFR1 pathway.
Humans ; Urinary Bladder Neoplasms/blood supply* ; Receptor, Fibroblast Growth Factor, Type 1/metabolism* ; Cell Proliferation ; Cell Movement ; Fibroblast Growth Factor 2/metabolism* ; Neovascularization, Pathologic ; Human Umbilical Vein Endothelial Cells ; Cell Line, Tumor ; Signal Transduction ; Apelin ; Intercellular Signaling Peptides and Proteins/genetics* ; Female ; Male ; Angiogenesis

OBJECTIVES: To examine the changes in serum levels of endoplasmic reticulum stress (ERS) proteins GRP78/CHOP in patients with lupus nephritis (LN) and analyze their diagnostic value and association with renal pathological features. METHODS: From a sample bank established based on a multicenter cohort study of systemic lupus erythematosus (SLE), 60 LN patients and 35 SLE patients without renal involvement were randomly selected. ELISA was used to detect serum levels of GRP78 and CHOP in the patients to analyze their correlation with clinical features and their diagnostic ability for LN and active LN. MRL/lpr mice were used as an animal model of LN to examine their serum levels of GRP78 and CHOP expression and renal expressions of endoplasmic reticulum apoptosis-related proteins. RESULTS: Serum GRP78 and CHOP levels were significantly higher in LN patients than in SLE patients without renal involvement (P<0.05), and were also higher in active LN patients than in patients in the stable phase (P<0.05). Correlation analysis indicated that serum GRP78 and CHOP levels were positively correlated with SLEDAI scores and 24-h urinary protein. ROC analysis showed that CHOP had a high diagnostic ability for LN (AUC=0.762) and active LN (AUC=0.933). Consistent with the clinical findings, serum GRP78 and CHOP levels were elevated in LN mice, and the expressions of PERK and IRE1α pathway proteins were also increased in the kidneys of the mice. TUNEL staining showed increased renal cell apoptosis and elevated renal expressions of apoptosis-related proteins in LN mice. CONCLUSIONS Serum levels of GRP78/CHOP are increased in LN patients possibly in association with ERS-induced apoptosis mediated by the PERK/IRE1α dual pathway.
Endoplasmic Reticulum Chaperone BiP ; Lupus Nephritis/blood* ; Transcription Factor CHOP/blood* ; Heat-Shock Proteins/blood* ; Animals ; Apoptosis ; Humans ; Mice ; Mice, Inbred MRL lpr ; Female ; Adult ; Endoribonucleases/metabolism* ; Male ; eIF-2 Kinase/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Young Adult ; Endoplasmic Reticulum Stress ; Kidney/metabolism* ; Middle Aged ; Signal Transduction

OBJECTIVES: To investigate the therapeutic mechanism of Danzhi Jiangtang Capsule (DZJTC) for repairing renal vascular endothelial injury in rats with diabetic nephropathy (DN). METHODS: Fifty male SD rat models of DN, established by left nephrectomy, high-sugar and high-fat diet and streptozotocin injection, were randomized into DN model group, low-, medium-, and high-dose DZJTC treatment groups, and DAPT (a γ-secretase inhibitor) treatment group, with 10 rats with normal feeding as the control group. DZJTC was administered by daily gavage at 0.315, 0.63, or 1.26 g/kg, and DAPT (20 mg/kg, dissolved in 50% CMC-Na solution) was given by gavage every other day for 4 weeks; normal saline was given in the control and model groups. After treatment, the levels of creatinine (CRE), blood urea nitrogen (BUN), and microalbuminuria (mALB) were detected with ELISA, and renal pathologies were observed by transmission electron microscopy. Renal expressions of vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) were measured by immunohistochemistry, and the protein expressions of CD31 and Notch signaling pathway components were detected using Western blotting. RESULTS: The rat models of DN showed significantly increased CRE, BUN, and mALB levels, obvious renal pathologies under electron microscopy, increased renal VEGF, ET-1 and CD31 expressions, and upregulated Notch1, NICD, and MAML1 protein levels. Treatment with DZJTC at the 3 doses and DAPT significantly reduced CRE, BUN, and mALB levels, improved renal pathology, decreased VEGF, ET-1 and CD31 expressions, and lowered Notch1, NICD and MAML1 levels, and the effects were the most pronounced with high-dose DZJTC. CONCLUSIONS DZJTC ameliorates hyperproliferation and dysfunction of renal vascular endothelium in DN rats possibly by regulating renal VEGF and ET-1 levels via inhibiting NICD- and MAML1-mediated Notch signaling pathway.
Animals ; Male ; Drugs, Chinese Herbal/therapeutic use* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Diabetic Nephropathies/drug therapy* ; Receptor, Notch1/metabolism* ; Kidney/blood supply* ; Diabetes Mellitus, Experimental ; Down-Regulation ; Endothelium, Vascular/metabolism* ; Nuclear Proteins/metabolism*