Objective:To investigate the association between glutathione S-transferase pi (GSTP1) gene polymorphism and toxici-ties related to high-dose methotrexate (HD-MTX) in children with acute lymphoblastic leukemia (ALL). Methods:GSTP1 genotypes and allelic frequencies in 51 children with ALL were determined by Nest PCR, denaturing gel gradient electrophoresis (DGGE), and DNA sequencing. HD-MTX adverse reactions were analyzed using the National Cancer Institute Common Toxicity Criteria (NCICTC). Results:We identified three SNPs of GSTP1, including rs1695 (A313G), rs1138272 (G439T), and rs4891 (T555C). The wild types, het-erozygous types, and homozygous types of GSTP1 rs1695/rs4891 polymorphisms were detected in 32 cases (62.7%), 16 cases (31.4%), and 3 cases (5.9%), respectively. GSTP1 rs1695/rs4891 polymorphisms included only one heterozygous type and one homozygous type. The allele frequencies of the three SNPs were 21.6%, 2.9%, and 21.6%. The AG+GG/TC+CC genotype of GSTP1 rs1695/rs4891 was associated with decrease in the odds of peripheral hemoglobin (OR=0.25, 95%CI=0.06-1.00, P=0.049). The AG+GG/TC+CC genotype of GSTP1 rs1695/rs4891 in standard and intermediate-risk ALL children was significantly correlated with higher odds of gastrointesti-nal toxicity (OR=0.125, 95%CI=0.02-0.78, P=0.026). Conclusion:GSTP1 rs1695 (A313G)/rs4891 (T555C) gene polymorphism is as-sociated with the reduction of peripheral hemoglobin in ALL children and with the odds of gastrointestinal toxicity in standard and inter-mediate-risk ALL children who receive high-dose methotrexate.

AIM:Atherosclerotic calcification is highly linked with plaque instability and cardiovascular events .Adenosine monophosphate-activated protein kinase ( AMPK) has been involved in the pathogenesis of various cardiovascular disease .The contributions of AMPKαsubunits to the development of atherosclerotic calcification in vivo remained unknown .We hypothesized that AMPKαsubunits may play a role in the development of atherosclerotic calcification .METHODS: Atherosclerotic calcification was generated by 24-week fed of western diet in ApoE-/-background mice .Calcification was evaluated in aortic roots and innominate arteries of ApoE-/-mice or in mice with dual deficiencies of ApoE and AMPKαsubunits globally ( AMPKα1 and AMPKα2 ) , or vascular smooth muscle cell ( VSMC)-specific or macrophage-specific knockout of AMPKα1 with atherosclerotic calcification pone diet . The mechanism of AMPKα1 in regulating Runx2 was further explored in human aortic VSMC .RESULTS: Ablation of AMPKα1 but not AMPKα2 in ApoE-/-background promoted atherosclerotic calcification with increased Runt -related transcription factor ( Runx2 ) expression in VSMC compared with ApoE-/-mice.Conversely, chronic administration of metformin, which activated AMPK, markedly reduced ath-erosclerotic calcification and Runx2 expression in ApoE-/-mice but had less effects in ApoE-/-/AMPKα1 -/-mice.Furthermore, VSMC-but not macrophage-specific deficiency of AMPKα1 in ApoE-/-background promoted atherosclerotic calcification in vivo com-pared with the controls .AMPKα1 silencing in human aortic VSMC prevented Runx 2 from proteasome degradation to trigger osteoblastic differentiation of VSMC .Conversely , activation of AMPK led to Runx 2 instability by inducing its small ubiquitin-like modifier modifi-cation (SUMOylation).Protein inhibitor of activated STAT-1 (PIAS1), the SUMO E3-ligase of Runx2, was directly phosphorylated by
AMPKα1 at serine 510, to enhance its SUMO E3-ligase activity.Ablation of PIAS1 serine 510 phosphorylation inhibited metformin-in-duced Runx2 SUMOylation, and subsequently prevented the effect of metformin on reducing oxLDL-triggered Runx2 expression in hu-man aortic VSMC.CONCLUSION:Deficiency of AMPKα1 in VSMC increases Runx2 expression and promotes atherosclerotic calcifi-cation in vivo.AMPKα1 phosphorylates PIAS1 to enhance Runx2 SUMOyalation and subsequent degradation .

Objective To investigate the diagnosis and treatment of functional pancreatic endocrine tumors (PETs). Methods The clinical data of 45 patients with functional PETs who were admitted to the Southwest Hospital from January 1998 to December 2008 were retrospectively analyzed. Etiologic and localization diagnosis were made preoperatively according to the manifestation and the results of color doppler ultrasound and computed tomography, respectively. Eight patients received resection of the body and tail of the pancreas and spleen, 32 received tumor enucleation, one received resection of the pancreaticobiliary junction and four received pancreaticoduodenectomy. All patients received chemotherapy after the operation. Results Thirty-four cases of the PETs were benign and the rest eleven cases were malignant. Eight cases of the PETs were in the head of the pancreas,26 in the tail of the pancreas, seven in the body of the pancreas and four cases were with multiple PETs. The diameters of the PETs were 0. 3-5.0 cm, and the diameters of the PETs in 19 cases were above 2.0 cm. Eight patients were complicated with pancreatic leakage, two with incision infection and one with abdominal infection. Of the 33 patients with insulinoma, the blood glucose of five patients with multiple PETs was still abnormal after the operation, three patients underwent reoperation and the other two were treated by diazoxide to control the blood glucose in the normal range. The clinical symptoms of the seven patients with gastrinoma disappeared after the operation, and the gastric ulcer was healed, the 12-hour gastric juice volume and the level of the gastric acid were in the normal range after a continuous treatment with proton pump inhibition agents for 6 months. Necrotizing or migratory rash and diabetes of the four patients with glucagonoma were cured three weeks later, and the level of the amino acid was back to normal. Diarrhea and electronic disturbance of one patient with vasoactive intestinal peptide tumor were alleviated after the operation. Thirty-nine patients were followed up for 20-120 months. Of the 32 patients with benign PETs, two patients had tumor recurrence, and three patients died of other diseases. Of the seven patients with malignant PETs, two patients survived, and three patients died of hepatic metastasis or tumor recurrence, two patients died of other diseases. Conclusions Surgical treatment is effective for the treatment of functional PETs. Palliative resection of the tumor also can obviously improve the life quality of patients with fuctional PETs.

Objective:To prepare a novel vascular endothelial growth inhibitor-soluble chimeric protein VEGI~+,so as to lay a basis for studying its biological activity.Methods:Chimeric molecule VEGI~+ was constructed by grafting oligopeptide CTTH- WGFTLC to extraeellular region of VEGI(VEGI_(23-174)).Before ligation into pET30a(+)expression vector,PCR product of the recombinant gene was cloned into pGEM-T vector and verified by restriction enzyme digestion and DNA sequencing,then pET30a-VEGI was used to transfect BL21(modified E.coli strain).The chimeric protein was purified by metal affinity chro- matography.Western blotting and coomassie blue staining were used for protein identification.Results:The chimeric molecule VEGI~+ was confirmed by restriction enzyme digestion and DNA sequencing.The constructed pET30a-VEGI was confirmed by enzymatic digestion.The expression was mainly in the form of inclusion body.SDS-PAGE electrophoresis and Western blotting revealed a chimeric protein about 23000,with a purity of about 90%.Conclusion:We have successfully constructed the recom- binant plasmid pET30a-VEGI~+ and expressed it in E.coll.And we have obtained high purity of soluble chimeric protein VEGI~+ through affinity chromatography.

Abstract: Objective　To analyze the laboratory microscopic re-examination results of malaria cases in Nantong of the National Notifiable Disease Report System from 2014 to 2021 by Nantong Malaria Diagnostic Reference Laboratory, so as to evaluate the malaria diagnosis ability of Nantong Malaria Diagnostic Reference Laboratory. Methods　The blood smear and blood samples of malaria cases in Nantong from 2014 to 2021 of the National Notifiable Disease Report System were collected. Nantong Malaria Diagnostic Reference Laboratory and Jiangsu Institute of Parasitic Diseases carried out the re-examination of municipal and provincial laboratories, taking the results of provincial laboratory as the standard to compare and analyze the re-examination results of Nantong Malaria Diagnostic Reference Laboratory. Results　From 2014 to 2021, the two-level laboratories in Nantong city and Jiangsu Province re-examined the blood samples of 297 malaria cases. The microscopic examination and PCR re-examination results at the provincial level were the same：292 positive cases and 5 negative cases. The qualitative coincidence rate between Nantong microscopic re-examination results and the provincial re-examination results was 100% (297/297), without misjudgment and omission. The coincidence rate of Plasmodium typing was 96.23% (281/292). The coincidence rate of P. falciparum, P. vivax, P. ovale and P. malaria were 99.57% (234/235), 62.50% (5/8), 89.47% (34/38) and 72.73% (8/11) respectively. The consistency test results showed that the Kappa value of Plasmodium typing results between municipal and provincial laboratories was 0.89. The Kappa values of P. falciparum, P. vivax， P. ovale and P. malaria were 0.98, 0.58, 0.87 and 0.79 respectively. Conclusion　The malaria diagnosis ability of Nantong Malaria Diagnostic Reference Laboratory is generally good, and it is necessary to improve the ability of Plasmodium typing.

OBJECTIVETo investigate the mechanism of rhubarb in regulating aquaporin-4 in rats with blood-brain barrier damage after acute cerebral hemorrhage (CH).

METHODSCH model was induced by stereospecific injection of auto-blood into caudate nucleus of rats, and the brain water content and neurological defect were detected to evaluate cerebral edema and neurological defect level. Also, the blood-brain barrier damage was observed by Evan's blue staining; the changes of blood-brain barrier tight junction and astrocyte end feet at different time points were observed with electron microscope; and the AQP-4 mRNA and protein expression were measured with RT-PCR and Western blot.

RESULTSRhubarb showed effects in reducing cerebral edema. Evan's blue result indicated the blood-brain barrier was evidently damaged at the 12th hour after CH, with blood-brain barrier tight junction damaged and astrocyte end feet process swelled obviously, but these changes could be relieved by rhubarb. The AQP-4 mRNA and protein expression in rats increased significantly 24 hrs after modeling (P < 0.05) and reached the peak value at 72 hrs, and decreased gradually after then. Rhubarb also showed inhibiting transcription and translation of AQP-4 gene.

CONCLUSIONRhubarb could alleviate cerebral edema via reducing blood-brain barrier tight junction damage and astrocyte end feet process swelling, which might be realized by the inhibition on transcription and translation of AQP-4 gene.

Animals ; Aquaporin 4 ; biosynthesis ; genetics ; Blood-Brain Barrier ; drug effects ; Brain Edema ; drug therapy ; etiology ; Cerebral Hemorrhage ; drug therapy ; metabolism ; physiopathology ; Drugs, Chinese Herbal ; therapeutic use ; Male ; Phytotherapy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rheum

OBJECTIVESTo construct the expression vector pGenesil-3-shRNA that can express the short hairpin RNAs (shRNA) silencing Kir6.2 gene and to study its influence on the proliferation and invasion of HepG2 cells.

METHODSTwo shRNA silencing Kir6.2 genes were transcript synthesized intracellularly by expressed templates of plasmid vector pSilence-3, and the target sequence of Kir6.2 gene was inserted into the upstream of the reporter gene in order to construct the recombinant plasmid vector pGenesil-3. Plasmids containing 2 different sequences of human Kir6.2 mRNA coding region were constructed and transfected into HepG2 cells by using lipofectamine 2000 methods. The experiment was divided into 4 groups: SK (normal), SK-HK (negative control), SK-K1 (transfected with the interfering sequence 1) and SK-K2 (transfected with the interfering sequence 2) groups. A selected single clone was cultured after screening by G418. The expression of Kir6.2 protein was detected by Western blot. MTT assay and Transwell system were used to observe the proliferation and invasion of HepG2 cells.

RESULTSThe recombinant expression plasmid pGenesil-3 was successfully constructed and underexpression of Kir6.2 gene in HepG2 cells was detected by Western blot. Underexpression of Kir6.2 gene significantly decreased the proliferation and invasion of the HepG2 cells.

CONCLUSIONshRNA can inhibit the expression of Kir6.2 gene in the HepG2 cells, and underexpression of Kir6.2 gene decreased the proliferation and invasion of the HepG2 cells.

Cell Proliferation ; Genetic Vectors ; Hep G2 Cells ; Humans ; Neoplasm Invasiveness ; Plasmids ; Potassium Channels, Inwardly Rectifying ; genetics ; RNA, Small Interfering ; genetics

OBJECTIVEVasoactive intestinal peptide (VIP) is a neuro-peptide that can modulate immunity in several aspects. Previous reports showed that VIP attenuates the deleterious consequences of severe infection and septic shock by regulating production of inflammatory cytokines in immune activated cells. Intestine is one of the major organ of immune system and it may trigger multiple organ dysfunction syndrome in sepsis. The present study was planned to study the change of serum TNF-alpha, IL-1beta, IL-10 level and histopathological alteration of intestinal tract, and protective effects of VIP on endotoxic shock in rat.

METHODSTwenty eight SD rats were randomly divided into 3 groups, including control group (8 rats), LPS shock group (10 rats), and LPS + VIP group (10 rats). Endotoxic shock model was established by administration of a single dose of 10 mg/kg LPS in LPS shock group, a bolus of 5 nmol VIP intravenous injection following LPS in LPS + VIP group. The rats in the control group were given the same volume of normal saline injection. Blood samples were taken at time points of 1, 2, 4, and 6 hours after intervention from each group for measuring the level of TNF-alpha, IL-1beta and IL-10 by ELISA. Pathological changes of the intestine were observed by light microscope and electron microscope at the animals death or at the end of the experiment.

RESULTSSerum TNF-alpha, IL-1beta and IL-10 levels elevated at each time point in LPS shock group and LPS + VIP group (P < 0.05 or P < 0.01). TNF-alpha concentration reached the peak level 2 h after LPS injection; IL-1beta and IL-10 increased continuously till the end of the experiment. In LPS + VIP group, TNF-alpha and IL-1beta elevated slightly and IL-10 increased significantly as compared with LPS shock group (P < 0.01). Leukocyte infiltration, ischemia, segmental hemorrhage or necrosis appeared in intestine under light microscope and cell swelling, cytoplasmic vacuoles and organelle damage were observed under electron microscope. However, pathological changes in LPS + VIP group were milder than those in LPS group.

CONCLUSIONSVIP improved endotoxic shock-associated histopathological alteration of intestine, down-regulated pro-inflammatory cytokines production and up-regulated anti-inflammatory cytokines. These effects may suggest a protective mechanism of VIP in septic shock. VIP is a potential immunoregulatory substance in treatment of septic shock.

Animals ; Biomarkers ; blood ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Female ; Interleukin-10 ; blood ; Interleukin-1beta ; blood ; Intestines ; drug effects ; pathology ; ultrastructure ; Lipopolysaccharides ; toxicity ; Male ; Rats ; Rats, Sprague-Dawley ; Shock, Septic ; blood ; drug therapy ; immunology ; Tumor Necrosis Factor-alpha ; blood ; Vasoactive Intestinal Peptide ; administration & dosage ; immunology ; pharmacology

OBJECTIVETo observe the effect of rhubarb in treating secondary damage of central nerve system (CNS) in rats with acute hemorrhagic stroke (AHS) and to explore the possible mechanism.

METHODSThe rat's AHS model was established by autologous blood injection, the effect of rhubarb on the secondary damage of CNS, plasminogen (PLG) in brain and tissue type plasminogen activator (t-PA) were observed.

RESULTS(1) The nerve function deficit signs reappeared in about 70% model rats 4 - 6 days after modeling and reached the peak at day 6 - 8, scored as 1.63 +/- 0.72 on day 4 and as 2.32 +/- 1.12 on day 7; (2) Rhubarb could effectively improve the secondary nerve function damage, with the nerve deficit scores kept to 1.24 +/- 0.19 from day 4 on, and no sign of secondary CNS damage was shown. The nerve deficit score was 1.22 +/- 0.15 on day 7 in the rhubarb treated group, showing significant difference as compared with that in the model group (P<0.05); (3) The specific amplified products of t-PA mRNA on day 3 and that of PLG mRNA on day 7 in CNS of model group were significantly higher than those in the sham operated group and the rhubarb treated group.

CONCLUSIONRhubarb could effectively reduce the secondary CNS damage in rats with AHS, it might be related with the suppressive effect of rhubarb on tPA mRNA and PLG mRNA expression in CNS.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Intracranial Hemorrhages ; blood ; pathology ; physiopathology ; Male ; Plasminogen ; biosynthesis ; genetics ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Rheum ; chemistry ; Stroke ; blood ; pathology ; physiopathology

OBJECTIVETo investigate the renal protective activity of Hsian-tsao Mesona procumbens Hemsl. water extracts in diabetic rats.

METHODSThirty Sprague-dawley female rats were randomly divided into three groups (n = 10 each), "control group" with intraperitoneal saline injection, "diabetic group" with 60 mg of intraperitoneal streptozotocin injection per kg of body weight and "Hsian-tsao group" with intragastric administration of Hsian-tsao extraction everyday for 4 weeks after intraperitoneal streptozotocin injection. The body weight and blood sugar were measured before and after model induction in the three groups. Thrombospondin-1 (TSP-1) expressions in the kidney were monitored by immunohistochemistry. Kidney ultrastructural changes were also analyzed by using transmission electron microscopy.

RESULTSBefore diabetic model induction, there were no significant differences among the three groups in body weight and blood sugar. Four weeks after the induction of diabetes, the differences became statistically significant. Electron microscopy also revealed disruption of the foot processes of the podocytes and other damages in diabetic group. These damages were significantly less severe in Hsian-tsao group when compared with the diabetic group. TSP-1 expressions in the kidney were significantly increased in both the diabetic group and Hsian-tsao group, but it was relatively lower in Hsian-tsao group than in diabetic group.

CONCLUSIONOur results showed that Hsian-tsao treatment in the diabetic rats effectively prevented the pathological alterations in the kidney and decreased the TSP-1 expression. It was suggested that Hsian-tsao had protective effect on the kidneys of the diabetic rats.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; pathology ; Diabetic Nephropathies ; metabolism ; pathology ; prevention & control ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Kidney ; drug effects ; metabolism ; pathology ; ultrastructure ; Lamiaceae ; chemistry ; Rats ; Rats, Sprague-Dawley ; Thrombospondin 1 ; metabolism