OBJECTIVES: To study the effect of CDC20 knockdown on proliferation, migration and invasion of cervical cancer cells and its underlying mechanism. METHODS: CDC20 expression in cervical cancer tissues was analyzed using the TCGA database, and the protein expressions of CDC20 and β-Catenin in clinical specimens of cervical cancer and adjacent tissues were detected using immunohistochemistry. A dual target sgRNA2&7 sequence for CDC20 gene was designed for CDC20 gene knockdown in cervical cancer C33A cells using CRISPR/Cas9 technology, and CDC20 mRNA and protein expression levels in the transfected cells were detected using qRT-PCR and Western blotting. The changes in proliferation, cell cycle, apoptosis, migration and invasiveness of the transfected cells were evaluated using colony-forming assay, fluorescence activated cell sorting (FACS) and Transwell assay. In the animal experiment, naïve C33A cells and the cells with CDC20 knockdown were injected subcutaneously into the left and right axillae of nude mice (n=5) to observe tumor growth. The expressions of CDC20 and β-Catenin proteins in transfected cells and the xenograft were analyzed using Western blotting, and their interaction was confirmed by co-immunoprecipitation (CoIP) and immunofluorescence co-localization assays. RESULTS: Cervical cancer tissues expressed significantly higher CDC20 and β‑Catenin levels than the adjacent tissues. C33A cells with CDC20 knockdown showed reduced proliferation, increased apoptosis, and lowered migration and invasion abilities. CDC20 knockdown significantly suppressed the growth of C33A cell xenograft in nude mice, and the tumor-bearing mice did not exhibit obvious body mass changes. CDC20 and β-Catenin levels were both significantly lowered in C33A cells with CDC20 knockdown. Co-immunoprecipitation and co-localization assays confirmed the interaction between CDC20 and β‑Catenin. CONCLUSIONS CDC20 is highly expressed in cervical cancer tissues, and CDC20 knockdown can suppress proliferation, invasion, and metastasis while enhancing apoptosis of C33A cells, which is closely related with the regulation of the Wnt/β-Catenin signaling pathway.
Humans ; Uterine Cervical Neoplasms/metabolism* ; Female ; Cdc20 Proteins/genetics* ; Cell Proliferation ; Animals ; Cell Movement ; Neoplasm Invasiveness ; Apoptosis ; Mice, Nude ; beta Catenin/metabolism* ; CRISPR-Cas Systems ; Mice ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis

OBJECTIVE: To investigate the role of the SIRT1/NF-κB pathway in mediating the effect of puerarin against lipopolysaccharide (LPS)-induced acute kidney injury (AKI). METHODS: Fifteen BALB/C mice were randomized into control group, LPS group and puerarin treatment group, and in the latter two groups, the mice were given an intraperitoneal injection of LPS (5 mg/kg), followed by daily injection of normal saline for 3 days or injection of puerarin (25 mg/kg) given 1 h later and then on a daily basis for 3 days. On day 5 after modeling, the kidney tissues were taken for histological observation and detection of cell apoptosis. The renal function indexes including urea nitrogen (BUN), serum creatinine (Scr) and kidney injury molecule 1 (KIM-1) and the levels of tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β) were measured, and the expressions of SIRT1 and NF-κB-p65(acetyl K310) in the renal tissues were detected. RESULTS: Intraperitoneal injection of LPS caused obvious glomerular capillary dilatation, hyperemia, renal interstitial edema, and renal tubular epithelial cell swelling and deformation in the mice. The mouse models of LPS-induced AKI also showed significantly increased renal tubular injury score and renal cell apoptosis (P < 0.01) with increased serum levels of BUN, Scr, KIM-1, TNF-α and IL-1β (P < 0.01), enhanced renal expressions of TNF-α, IL-1β and NF-κB p65(acetyl K310) (P < 0.01) and lowered renal expression of SIRT1 (P < 0.05). Treatment with puerarin effectively alleviated LPS-induced renal interstitial edema and renal tubular epithelial cell shedding, lowered renal tubular injury score (P < 0.01) and renal cell apoptosis rate (P < 0.01), and decreased serum levels of BUN, Scr, KIM, TNF-α and IL-1β (P < 0.01). Puerarin treatment significantly reduced TNF-α, IL-1β and NF-κB p65 (acetyl K310) expression in the renal tissue (P < 0.05) and increased SIRT1 expression by 17% (P < 0.05) in the mouse models. CONCLUSION Puerarin can effectively alleviate LPS-induced AKI in mice possibly by modulating the SIRT1/NF-κB signaling pathway.
Animals ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; Lipopolysaccharides ; Sirtuin 1 ; Tumor Necrosis Factor-alpha ; Acute Kidney Injury ; Disease Models, Animal ; Edema

AIM: To explore the role of phosphatidylinositiol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathways in the inhibitory effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-induced tissue factor (TF) expression in vascular endothelial cells.METHODS: The mRNA expression of TF was detected by real-time fluorescent quantitative PCR.The protein levels of TF and Akt was determined by Western blot.The content of the nitric oxide (NO) was measured by nitrate reduction method.RESULTS: Compared with control group, incubating endothelial cells with ox-LDL significantly induced TF expression at mRNA and protein levels and the dephosphorylation of Akt protein, and decreased NO production.Incubation of the endothelial cells with puerarin for 1 h and then treatment of the cells with ox-LDL decreased the TF expression at mRNA and protein levels, increased Akt protein phosphorylation and intracellular NO content.Co-incubation of the endothelial cells with PI3K inhibitor LY294002 and puerarin for 1 h and then treatment of the cells with ox-LDL augmented the TF expression at mRNA and protein levels and the Akt protein dephosphorylation, and decreased NO production.Co-incubation of the endothelial cells with eNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and puerarin significantly decreased the inhibitory effect of puerarin on ox-LDL-induced TF expression at mRNA and protein levels in the endothelial cells, and reduced Akt protein phosphorylation and NO production.CONCLUSION: Puerarin inhibits ox-LDL-induced TF expression at mRNA and protein levels in the human umbilical vein endothelial cells via activation of PI3K/Akt/eNOS signaling pathway.

Objective To investigate the effects of puerarin on the expression of human umbilical vein endothelial cells (HUVECs) tissue factor (TF) and tissue factor pathway inhibitor (TFPI) induced by oxidized low-density lipoprotein (ox-LDL).Methods After HUVECs were incubated with different concentrations of puerarin and 50 mg/L ox-LDL,the expression of TF and TFPI mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blot respectively.Results Compared with control,treatment with ox-LDL caused the augment of TF mRNA and protein expression (P<0.01),and the decrease of TFPI mRNA and protein expression.However,50,100,and 200 μmol/L puerarin blunted the augment of TF mRNA and protein expression and weakened the inhibition of TFPI mRNA and protein expression induced by ox-LDL(P<0.01).Conclusions Puerarin reduces HUVECs TF and TFPI mRNA and protein induced by ox-LDL.

OBJECTIVETo evaluate the protective effect of quercetin against lipopolysaccharide (LPS)-induced cardiac injury in mice.

METHODSC57BL/6J mice were randomized into 4 groups to receive intraperitoneal injection of saline (negative control) or LPS (20 mg/kg), or fed with quercetin (100 mg/kg for 7 days) with or without subsequent LPS injection (quercetin+LPS group and quercetin control group, respectively). Six hour after LPS injection, the mice were tested for cardiac function with an echocardiograph, and the protein expressions of Bax, Bcl-2, iNOS, and eNOS in the myocardium were evaluated with Western blotting; serum NO concentration was also measured. The survival of the mice within 5 days after LPS injection was recorded to draw the survival curve.

RESULTSQuercetin pretreatment significantly improved the cardiac function of LPS-challenged mice (P<0.05), and attenuated LPS-induced increment in myocardial iNOS expression and decrement in eNOS level. LPS significantly increased the myocardial Bax expression and slightly decreased Bcl-2 expression; quercetin pretreatment decreased Bax expression to the control level and significantly lowered Bax/Bcl-2 ratio as compared with the LPS group. Serum NO level was significantly increased by nearly 2.5 folds in LPS-challenged mice, but was markedly decreased with quercetin pretreatment (P<0.05). The 5-day survival rate of LPS-treated mice was 10%, which was increased to 45% in quercetin- pretreated mice (P<0.05).

CONCLUSIONQuercetin can alleviate LPS-induced cardiac dysfunctions in mice to increase their survival rate following LPS challenge.

Animals ; Cardiotonic Agents ; pharmacology ; Heart ; drug effects ; Lipopolysaccharides ; adverse effects ; Mice ; Mice, Inbred C57BL ; Myocardium ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Quercetin ; pharmacology

Educational reform of pathophysiology oriented to clinical application is to pass the physician qualifica -tion examination .One of essential approach is to implement pathophysiology teaching with the translational medical philosophy and promote the harmonious development of physician -patient relationship with the utilization of the de-velopment and changes of disease in the teaching process .In that way, the pathophysiology in basic and clinical medicine is worthy of the name of “bridge”, and ultimately achieves the goal of “the transformation and develop-ment of the cultivation of clinical application talents”.

Objective To evaluate the protective effect of quercetin against lipopolysaccharide (LPS)-induced cardiac injury in mice. Methods C57BL/6J mice were randomized into 4 groups to receive intraperitoneal injection of saline (negative control) or LPS (20 mg/kg), or fed with quercetin (100 mg/kg for 7 days) with or without subsequent LPS injection (quercetin+LPS group and quercetin control group, respectively). Six hour after LPS injection, the mice were tested for cardiac function with an echocardiograph, and the protein expressions of Bax, Bcl-2, iNOS, and eNOS in the myocardium were evaluated with Western blotting;serum NO concentration was also measured. The survival of the mice within 5 days after LPS injection was recorded to draw the survival curve. Results Quercetin pretreatment significantly improved the cardiac function of LPS-challenged mice (P<0.05), and attenuated LPS-induced increment in myocardial iNOS expression and decrement in eNOS level. LPS significantly increased the myocardial Bax expression and slightly decreased Bcl-2 expression; quercetin pretreatment decreased Bax expression to the control level and significantly lowered Bax/Bcl-2 ratio as compared with the LPS group. Serum NO level was significantly increased by nearly 2.5 folds in LPS-challenged mice, but was markedly decreased with quercetin pretreatment (P<0.05). The 5-day survival rate of LPS-treated mice was 10%, which was increased to 45% in quercetin-pretreated mice (P<0.05). Conclusion Quercetin can alleviate LPS-induced cardiac dysfunctions in mice to increase their survival rate following LPS challenge.

Objective To evaluate the protective effect of quercetin against lipopolysaccharide (LPS)-induced cardiac injury in mice. Methods C57BL/6J mice were randomized into 4 groups to receive intraperitoneal injection of saline (negative control) or LPS (20 mg/kg), or fed with quercetin (100 mg/kg for 7 days) with or without subsequent LPS injection (quercetin+LPS group and quercetin control group, respectively). Six hour after LPS injection, the mice were tested for cardiac function with an echocardiograph, and the protein expressions of Bax, Bcl-2, iNOS, and eNOS in the myocardium were evaluated with Western blotting;serum NO concentration was also measured. The survival of the mice within 5 days after LPS injection was recorded to draw the survival curve. Results Quercetin pretreatment significantly improved the cardiac function of LPS-challenged mice (P<0.05), and attenuated LPS-induced increment in myocardial iNOS expression and decrement in eNOS level. LPS significantly increased the myocardial Bax expression and slightly decreased Bcl-2 expression; quercetin pretreatment decreased Bax expression to the control level and significantly lowered Bax/Bcl-2 ratio as compared with the LPS group. Serum NO level was significantly increased by nearly 2.5 folds in LPS-challenged mice, but was markedly decreased with quercetin pretreatment (P<0.05). The 5-day survival rate of LPS-treated mice was 10%, which was increased to 45% in quercetin-pretreated mice (P<0.05). Conclusion Quercetin can alleviate LPS-induced cardiac dysfunctions in mice to increase their survival rate following LPS challenge.

Aim To investigate the mechanism for the inhibitory effect of hydrogen sulfide on the expression of tissue factor(TF)induced by oxidative low-density lipoprotein(ox-LDL)in endothelial cells.Methods Human umbilical vein endothelial cells (HUVECs ) were treated with 50 mg·L-1 ox-LDL in the absence or presence of different concentrations of NaHS (25 , 50,100 and 200 μmol·L-1 )for 24 h.The mRNA expression and protein content of TF in HUVECs were determined by reverse transcription PCR and ELISA, respectively.The content of intracellular reactive oxy-gen species (ROS)was determined by DCFH,an oxi-dative sensitive fluorescent indicator.The activation of nuclear factor-kappaB (NF-κB)was estimated by its expression in nuclear extracts analyzed by Western blot.Results Ox-LDL induced TF mRNA expression and increased TF protein content in HUVECs.The in-crease in intracellular ROS production and the activa-tion of NF-κB were observed in HUVECs treated with ox-LDL.However,NaHS could markedly inhibit the increases in TF mRNA and protein levels induced by ox-LDL.Also the elevation of intracellular ROS pro-duction and the activation of NF-κB elicited by ox-LDL were significantly suppressed by pretreatment with NaHS.In addition,pretreatment with BAY 1 1-7082 (10 μmol·L-1 ),the inhibitor of NF-κB or N-acetyl-L-cysteine(1 mmol·L-1 ),an antioxidant,could also decrease the TF mRNA and protein level as well as ROS production and NF-κB activation induced by ox-LDL in HUVECs,similar to the effects of 200 μmol· L-1 NaHS.Conclusion The mechanism for the in-hibitory effect of H2 S on the ox-LDL- induced TF ex-pression in endothelial cells may be related to inhibi-ting intracellular ROS production and subsequently NF-κB activation.

Objective To explore the solution to the secure problem of EMR based upon No.1 Military Medical Project.Methods The existing EMR system provides medical records service and the EMR are stored and managed in ordinary or established file format in the database server.Based upon above database characters related to EMR and the requirement of EMR security,the MRBACKUP system was developed to resolve EMR backup,recovery and dump.Results The MRBACKUP system safeguarded the security of EMR in the lowest cost,the least system resources and the highest efficiency.Conclusion The new secure solution to EMR can better resolve the security problems in the widely-used EMR in hospitals.