Objective·To investigate the role and mechanism of sirtuin 5(SIRT5)in pulmonary microvascular endothelial cell injury in sepsis.Methods·Wild-type(WT)and Sirt5 gene knockout C57BL/6 male mice underwent cecal ligation and puncture(CLP)surgery.Following euthanasia,lung tissues were collected.Pulmonary inflammation was assessed using hematoxylin and eosin(H-E)staining;vascular leakage was evaluated by Evans blue(EB)staining;coagulation function in mice was analyzed via immunofluorescence staining of lung tissues.Immunohistochemical staining was employed to detect vascular cell adhesion molecule-1(VCAM-1)protein expression,thereby assessing endothelial inflammation in CLP-treated mice.By using gene editing technology,SIRT5 was knocked down or overexpressed in human umbilical vein endothelial cells(HUVECs),and the cells were subsequently stimulated with lipopolysaccharide(LPS)to induce endothelial inflammation.Protein expression levels of VCAM-1,tissue factor(TF),and other endothelial injury markers were detected by Western blotting,and inflammatory cytokines such as interleukin-6(IL-6)and IL-1β,were detected by quantitative real-time PCR(qPCR).In addition,transcriptomic sequencing was performed on HUVECs overexpressing SIRT5,and key genes including F2R-like thrombin or trypsin receptor 3(F2RL3),serpin family A member 3(SERPINA3),and transforming growth factor β2/β3(TGF-β2/3)were validated by qPCR.Results·Sirt5 knockout significantly aggravated lung injury in CLP mice,reducing their survival rates(P<0.001).H-E staining showed increased inflammatory infiltration in the lung tissue of the mice,while EB staining indicated increased vascular leakage(P<0.001).Immunofluorescence revealed elevated fibrinogen deposition.In HUVECs with SIRT5 knockdown,the protein levels of VCAM-1 and TF,as well as the mRNA levels of inflammatory factors including IL-6,IL-1β,VCAM-1,and E-selectin,were significantly upregulated(all P<0.001),whereas overexpression of SIRT5 reversed these effects.Transcriptome sequencing analysis indicated that SIRT5 regulated endothelial inflammation and coagulation responses by inhibiting the F2RL3/SERPINA3/TGF-β pathway.Conclusion·SIRT5 negatively regulates the F2RL3/SERPINA3/TGF-β signaling axis,thereby alleviating endothelial inflammation and promoting coagulation responses,suggesting its potential protective role in sepsis-induced lung injury.

Objective·To investigate the role and mechanism of sirtuin 5(SIRT5)in pulmonary microvascular endothelial cell injury in sepsis.Methods·Wild-type(WT)and Sirt5 gene knockout C57BL/6 male mice underwent cecal ligation and puncture(CLP)surgery.Following euthanasia,lung tissues were collected.Pulmonary inflammation was assessed using hematoxylin and eosin(H-E)staining;vascular leakage was evaluated by Evans blue(EB)staining;coagulation function in mice was analyzed via immunofluorescence staining of lung tissues.Immunohistochemical staining was employed to detect vascular cell adhesion molecule-1(VCAM-1)protein expression,thereby assessing endothelial inflammation in CLP-treated mice.By using gene editing technology,SIRT5 was knocked down or overexpressed in human umbilical vein endothelial cells(HUVECs),and the cells were subsequently stimulated with lipopolysaccharide(LPS)to induce endothelial inflammation.Protein expression levels of VCAM-1,tissue factor(TF),and other endothelial injury markers were detected by Western blotting,and inflammatory cytokines such as interleukin-6(IL-6)and IL-1β,were detected by quantitative real-time PCR(qPCR).In addition,transcriptomic sequencing was performed on HUVECs overexpressing SIRT5,and key genes including F2R-like thrombin or trypsin receptor 3(F2RL3),serpin family A member 3(SERPINA3),and transforming growth factor β2/β3(TGF-β2/3)were validated by qPCR.Results·Sirt5 knockout significantly aggravated lung injury in CLP mice,reducing their survival rates(P<0.001).H-E staining showed increased inflammatory infiltration in the lung tissue of the mice,while EB staining indicated increased vascular leakage(P<0.001).Immunofluorescence revealed elevated fibrinogen deposition.In HUVECs with SIRT5 knockdown,the protein levels of VCAM-1 and TF,as well as the mRNA levels of inflammatory factors including IL-6,IL-1β,VCAM-1,and E-selectin,were significantly upregulated(all P<0.001),whereas overexpression of SIRT5 reversed these effects.Transcriptome sequencing analysis indicated that SIRT5 regulated endothelial inflammation and coagulation responses by inhibiting the F2RL3/SERPINA3/TGF-β pathway.Conclusion·SIRT5 negatively regulates the F2RL3/SERPINA3/TGF-β signaling axis,thereby alleviating endothelial inflammation and promoting coagulation responses,suggesting its potential protective role in sepsis-induced lung injury.

OBJECTIVES: To investigate the role and mechanism of circRNA-SR-related CTD associated factor 8 (SCAF8) in regulating endothelial cell pyroptosis in high glucose environment. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured and divided into six groups. The normal control group and high glucose control group were cultured in cell culture medium with 5 and 33 mmol/L glucose, respectively. The RNA control group, circRNA-SCAF8 inhibition group, miR-93-5p overexpression group and miR-93-5p inhibition group were added with non-functional siRNA, circRNA-SCAF8 inhibitor, miR-93-5p overexpression molecule and miR-93-5p inhibitor in high glucose environment, respectively. Cell viability and pyroptosis were detected by cell counting kit-8 (CCK-8) assay, flow cytometry and Hoechst 33342/propidium iodide fluorescence double staining. Western blotting and enzyme-linked immunosorbent assay were used to detect the expression of pyroptosis-related factors including apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartic acid specific protease-1 (caspase-1) and Gasdermin D (GSDMD), NOD like receptor protein 3 (NLRP-3), thioredoxin interacting proteins (TXNIP), IL-18 and IL-1β. The expression of circRNA-SCAF8, miR-93-5p and TXNIP was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Fluorescence in situ hybridization (FISH) was used to locate circRNA-SCAF8 and miR-93-5p. Dual luciferase assay was used to verify the targeted regulatory relationship between miR-93-5p and upstream and downstream molecules. RESULTS: Compared with the RNA control group, the cell survival rate of circRNA-SCAF8 inhibition group and miR-93-5p overexpression group increased (both P<0.01), the pyroptosis decreased (both P<0.01), and the expressions of pyroptosis-related factors such as TXNIP, NLRP-3, caspase-1, GSDMD, ASC, IL-18 and IL-1β were significantly decreased (all P<0.05). The expression of miR-93-5p was significantly increased after inhibition of circRNA-SCAF8 (P<0.01), and the expression of circRNA-SCAF8 tended to decrease after overexpression of miR-93-5p, but with no statistical significance (P>0.05). Dual luciferase assay showed that miR-93-5p downre-gulated circRNA-SCAF8 expression by binding to the 3 ´ UTR region of circRNA-SCAF8, and miR-93-5p downregulated TXNIP expression by binding to the 3 ´ UTR region of TXNIP. FISH showed that circRNA-SCAF8 and miR-93-5p were both located in the cytoplasm and were highly associated in the cells. qRT-PCR showed that the relative expression of TXNIP increased or decreased after overexpression or inhibition of miR-93-5p compared with the RNA control group, respectively (both P<0.05), suggesting that miR-93-5p could regulate TXNIP gene expression. CONCLUSIONS CircRNA-SCAF8/miR-93-5p/TXNIP axis is involved in the regulation of pyroptosis in HUVECs under high glucose.
Humans ; Factor VIII ; RNA, Circular ; Endothelial Cells ; Interleukin-18 ; Pyroptosis ; In Situ Hybridization, Fluorescence ; Caspase 1 ; MicroRNAs/genetics* ; Carrier Proteins/genetics* ; RNA-Binding Proteins

BACKGROUND: Bevacizumab combined with platinum-based chemotherapy has been recommended as the first-line agent in advanced nonsquamous non-small cell lung cancer (NSCLC) without driven gene, but this regimen is not common in the second-line or later-line treatment of non-squamous NSCLC. The aim of this study is to investigate the efficacy and safety of bevacizumab combined with chemotherapy as second-line or later-line treatment in advanced non-squamous NSCLC. METHODS: We retrospectively reviewed the clinical data of advanced nonsquamous NSCLC patients who were treated with bevacizumab after first-line treatment failure and they were hospitalized in the Affiliated Cancer Hospital of Zhengzhou University from January 2014 to June 2017, and Kaplan-Meier method, Log-rank test and Cox model were used for analysis. RESULTS: A total of 62 patients were included in the analysis. The total objective response rate (ORR) was 32.2%, and the disease control rate (DCR) was 96.8%. The median progression-free survival (PFS) was 6.4 months (95%CI: 6.05-6.83), and the median overall survival (OS) was 20.4 months (95%CI: 12.98-27.76). In the subgroup analysis, there was no significant difference in median PFS between patients with brain metastases and those without brain metastases (6.2 months vs 6.4 months, P=0.052). Cycles of bevacizumab (>6 or ≤6 cycles) was an independent influencing factor of PFS (P=0.004). The most common adverse events were leukopenia, fatigue, nausea, thrombocytopenia and hypertension. CONCLUSIONS In the second-line or later-line treatment, bevacizumab combined with chemotherapy is an effective and safe regimen for advanced non-squamous NSCLC.
Aged ; Bevacizumab ; adverse effects ; therapeutic use ; Brain Neoplasms ; secondary ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; pathology ; Disease-Free Survival ; Female ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Male ; Middle Aged ; Retrospective Studies ; Safety ; Treatment Outcome

Objective To investigate the clinical significance of compensatory angiogenesis and the angiosome-based revascularization strategy for endovascular procedures in treating infrapopliteal critical limb ischemia in elderly diabetic patients.Methods A retrospective study of 194 patients with diabetic feet (Fontaine Ⅳ) who had undergone endovascular procedures at our hospital from January 2005 to December 2014 was performed.Based on the angiosome concept,all subjects were classified into the direct revascularization group (DR group),the indirect revascularization through collaterals group (IR-tc group) and the indirect revascularization without collaterals group (IR-wc group).After one-year follow-up,the therapeutic effects were analyzed and compared between the three groups.Results During the one-year follow-up,the rate of unhealed ulcers,the re-intervention rate and the rate of major adverse limb events (MALE) were higher in the IR-wc group than in the DR and IR-tc groups (P=0.000 and 0.000,P=0.025 and 0.013,P=0.014 and 0.023,respectively).The IR-wc group presented a lower limb salvage rate as compared with those in the DR and IR-tc groups (P=0.011 and 0.027).Patients with a single recanalized branch had a higher rate of unhealed ulcers than those with multiple recanalized branches in the IR-wc group (93.9％ vs.71.0％,P=0.015).Conclusions The angiosome-based revascularization strategy has shown important value in guiding endovascular procedures for patients with diabetic foot disease and,in particular,the compensatory angiogenesis approach appears to be critical in generating favorable short-term clinical outcomes in the treatment of diabetic foot disease.

Objective To observe and analyze cupping therapy for chronic wound healing.Methods Thirty-nine patients with chronic wounds were collected and randomly divided into cupping therapy group (n =20) and control group (n =19).The control group was treated with route dressing change once every other day,while the cupping therapy group was added cupping therapy.Compared the two groups of patients in general view,positive ratio of wound germiculture,area percentage of wound healing and pain score (VAS score).Results The pus of wounds was mostly drained out and the fresh tissue fluid leakage when patients were treated with cupping therapy.After three days of treatment ,the positive ratio of wound germiculture of the cupping group (6 5 % )was lower than that of the control group(79%),but the difference was not significant.After one-week treatment,the positive ratio of wound germiculture of the cupping group(40%)was significantly lower than that of the control group(73%)(χ2 =4.496, P =0.034).And the VAS score of the cupping group (2.20 ±1.00)was significantly lower than that of the control group (4.16 ±0.96)(t =-12.929,P =0.001).After two-week treatment,the area percentage of wound of the cup-ping group (80.68%)was significantly lower than that of the control group (92.28%)(t =-13.675,P =0.000). And 4 cases of the cupping group cured,while no patient was cured in the control group.Conclusion Cupping thera-py based on route dressing change has positive therapeutic effect on chronic wounds.And the advantages of lower cost and easier operations would make it suitable for middle-and low-income individuals and primary hospitals.

Integrin alphavbeta3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of alphavbeta3 integrin, a stable CHO-677 cell line expressing the murine alphavbeta3 heterodimer (designated as "CHO-677-malphavbeta3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits alphav and beta3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-malphavbeta3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-malphavbeta3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable alphavbeta3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of alphavbeta3 integrin, and as a cell model for FMDV research.
Animals ; Animals, Suckling ; CHO Cells ; Cloning, Molecular ; Cricetulus ; DNA, Complementary/genetics/metabolism ; Disease Susceptibility/virology ; Foot-and-Mouth Disease/*genetics/virology ; Foot-and-Mouth Disease Virus/*physiology ; Integrin alphaVbeta3/*genetics/metabolism ; Mice

VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore,anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.

Objective To study the treatment effect of multilevel atherosclerotic occlusive disease of lower extremity. Methods From July 2004 to January 2008,intraoperative iliac balloon angioplasty and stenting combined with blood vassel prosthesis or autogenous reversed great saphenous vein bypass were performed on 32 patients suffering from lower extremity multilevel atheresclerotic occlusive disease. Results Surgical procedures were technically successful in all patients. The effect was good,intermittent claudication disappear, and rest pain improved. Preoperative vs postoperative ABI was 0.28±0.14 vs 0.65±0.18 (P<0.05 ).Thirty patients were followed up,the mean following period was 18 months (range of 3-36 months).Conclusions Simultaneous intravaseular interventional therapy combined with vascular bypass are effective in the treatment for patients with severe and multilevel atheroselerotie occlusive disease of lower extremity, the operation is less traumatic and the procedures are easy to do.The result is satisfactory.