Objective To investigate the impact of MDR1-targeting small interfering RNA (siRNA) on diffuse large B-cell lymphoma cell OCI-LY1 proliferation.Methods A20 gene was silenced using RNA interference.An optimal concentration and treatment duration of vincristine were selected using MTT.Before and after siRNA transfection, proliferation of OCI-LY1 cells was assayed using MTT assay, and cellular apoptosis was detected using FCM before or after the treatment of the cells with VCR.Detection of A20, NF-κB (p65) and Pgp proteins were conducted using Western blot whereas mRNA of the A20 and MDR1 genes were examined using real time PCR.Results1)Proliferation of OCI-LY1 cells was enhanced (P<0.001) after the transfection with siRNA-2,(P<0.05).In addition, cell proliferation curve was declined after VCR stimulation, but the decrease was slower in siRNA-transfected cells than the untransfected counterparts.2)Apoptostic rate was lower in siRNA-transfected cells than theuntransfected counterparts, and the rate was higher in the cells after treatment with the drug for 24 h (P<0.05).Increased apoptosis was more obvious in control OCI-LY1 cells than in siRNA-transfected cells after treatment with VCR(P<0.05).3)The expression of MDR1 mRNA and Pgp (P<0.001) was significantly increased after transfection, but the expression of MDR1 mRNA and Pgp were significantly decreased (P<0.05).The expression in VCR group was significantly lower than that in siRNA-transfected cells+VCR group (P<0.01).ConclusionsA20 siRNA could effectively enhance NF-kappa B expression in OCI-LY1 cells.NF-kappa B may up regulate the expression of its downstream genes such as MDR1 and cause apoptosis, in turn enhancing the inhibition of cell proliferation.VCR can reduce MDR1 mRNA and Pgp expression in OCI-LY1 cells and the effect of VCR could be attenuated by A20 siRNA.

Objective To detect the A20 gene deletion,investigate the impacts of A20 gene deletion on clinicopathological features and prognosis of DLBCL,and relationship between activation of NF-κB pathway and relative molecular pathogenesis.Methods A20 gene deletion was detected by fluorescence in situ hybridization (FISH).The expression of A20,Survivin,P65 and Ki-67 were detected by immunohistochemistry stain.Apoptosis was assayed by TUNEL.Follow-up and statistical analysis were done.Results The deletion rate of A20 gene was 21.7％.The deletion rate of A20 gene was obviously higher in ABC-like DLBCL than that in GCB-like DLBCL (30.6％ vs.8.3％,P＜0.05).It was observed that there was a negative correlation between A20 protein expression and A20 gene deletion (r=-0.259,P =0.023).The expression of P65 and Survivin protein was positively correlated with the A20 gene deletion (r=0.280,P =0.015;r =0.313,P =0.007).Apoptosis rate was significantly reduced in DLBCL patients with A20 gene deletion.The apoptosis rate was higher in cases with positive expression of A20 protein,while that was lower in cases with positive expression of p65 and Survivin protein than those with negative expression of corresponding protein.There was no statistically significant difference in apoptosis rate between ABC-like and GCB-like DLBCL patients (P＞0.05).COX regression analysis indicated that age,A20 gene deletion,types of DLBCL and Ki67 expression were independent factors associated with survival status.Log-rank test showed that there was a statistical difference in survival status between the cases with and without A20 gene deletion (P=0.015).Conclusion A20 gene deletion may associate with the attenuation of A20 protein expression.The latter weakens negative feedback regulation of A20 protein for NF-κB pathway.An up-regulated expression of Survivin and abnormal proliferation and apoptosis may be result from the abnormal activation of NF-κB.A20 gene deletion brings certain influence on clinical course and prognosis of DLBCL.

Objective To observe the expression of serine/threonine kinase 31 (STK31) in osteosarcoma and its effect on the malignant biological behavior of osteosarcoma.Methods Fifteen cases of osteosarcoma specimens and adjacent normal tissue were collected.The expression of STK31 in tumor tissues and normal tissue were detected by immunohistochemistry,real-time quantitative PCR and Western blot.The STK31 knockout plasmids PGenesil-STK31-shRNA or control plasmid pGenesil-1 were transfected into osteosarcoma cell line MG63 cells.The effect of STK31 on the proliferation of MG63 cells was detected by CCK8 cell activity assay.Tanswell experiment was used to observed the effect of STK31 on the migration ability of osteosarcoma cells.Results Immunohistochemical showed that STK31 expressed in the tumor tissue,and it was significantly higher than the adjacent normal tissues;Real time quantitative PCR[(3.65±0.83)vs.(1.05±0.14),P＜0.05] and Western blot also revealed that STK31 expression in tumor tissue were significantly higher than adjacent normal tissues(P＜0.05);CCK8 experiments showed that knockdown STK31 inhibited proliferation of MG63 cell when compared with the control group after 36 h[(1.71±0.17)vs.(1.39±0.11),P＜0.05],72 h[(2.15±0.21)vs.(1.54±0.14),P＜0.05];Tansewell experiments showed that transfection of pGenesil-STK31-shRNA could suppress MG63 cell's migration[(13±4)vs.(55±8),P＜0.05].Conclusion STK31 is overexpression in osteosarcoma with increased biological activity of osteosarcoma cells.

Objective To evaluate the preoperative nutritional status of patients with gastric carcinoma by using the European Nutritional Risk Screening 2002(NRS 2002)and its prediction for postoperative nutrition-related complications.Methods We prospectively evaluated the nutritional risk of 314 gastric cancer patients admitted in one center from 2004 to 2007 with NRS 2002 with China's normal body mass index(BMI),in terms of postoperative complications,mortality and hospital stay.Results NRS 2002 scoring system was applicable in 93.1% cases.Preoperatively 125 patients were of score≥3,accounting for 39.8% of this group.The postoperative complication rate(26.2%)was higher than 13.8% in those with normal preoperative nutritional scores(NRS 2002 score＜3)(P＜0.05);The odds ratio to develop a complication was 0.642 in patients with preoperative nutritional risk score(P＜0.05),and 1.596 in patients with clinicopathological stage of gastric cancer(P＜0.01).The correlation between length of hospital stay and nutritional risk was also assessed by Pearson correlation analysis.The Pearson coefficient was 0.177(P=0.002).Conclusion Preoperative nutrition score(NRS 2002)≥3 predicts higher postoperative complications and longer hospital stay.Preoperative nutritional support is necessary in patients with preoperative nutrition score(NRS 2002)≥3.

Objective To investigate the clinical value of nutritional risk screening 2002(NRS2002)and malnutritional universal screening tools(MUST)in the preoperative nutrition risk evaluation of patients with gastric cancer.Methods The preoperative nutritional risk of 3 14 patients who had been admitted to the Third Affiliated Hospital of Sun Yat-sen University from January 2004 to December 2007 was assessed by subjective global assessment(SGA),NRS 2002 and M UST,and the influence of nutritional risk on the incidence of postoperative complications and hospital stay was investigated.All data were analyzed by Wilcoxon test,Kappa test and Logistics regression analysis.Results Compared with SGA,the sensitivity,specificity,positive predicting value and negative predicting value were 86.7%,74.2%,86.9% and 73.8% for NRS2002,and were 73.1%,70.6%,74.8% and 68.7% for MUST.Compared with MUST,NRS2002 had a higher consistency with SGA(K_(NRS2002)=0.601,K_(MUST)=0.436).Logistic regression analysis revealed that patients with higher MUST or NRS2002 score had higher incidence of postoperative complications and longer hospital stay.In the aspect of hospital stay,the relative risk of MUST was 2.517,which was lower than 3.426 of NRS2002.The relative risk of MUST was 0.529,which was lower than 0.642 of NRS2002 in the aspect of incidence of postoperative complications.Conclusions NRS2002 and MUST are suitable for preoperative nutritional risk screening of patients with gastric cancer,and the score of NRS2002 or MUST is associated with the incidence of postoperative complications and length of hospital stay.However,NRS2002 is more accurate than MUST in the reflection of nutritional risk of patients with gastric cancer.

Objective:To investigate the expression and significance of endoplasmic reticulum stress apoptosis pathway related proteins in renal cortex of rats with chronic fluorosis.Methods:Twenty four healthy SD rats were divided into 4 groups (6 rats/group, half male and half female) according to their body mass (100 - 120 g) by random number table method, rats in control group drank tap water (fluoride content < 0.5 mg/L), and in low, medium and high fluoride groups drank tap water with fluoride content (sodium fluoride) of 10, 50 and 100 mg/L, respectively. After 180 days of feeding, dental fluorosis was examined, 24-hour urine sample was collected and the content of fluoride in urine was detected by fluoride ion selective electrode method. Renal tissue was taken after anesthesia, and the pathological changes of renal cortex were observed by hematoxylin-eosin (HE) staining. The expressions of endoplasmic reticulum stress apoptosis pathway related proteins [inositol-requiring enzyme 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), C-Jun N-terminal kinase (JNK) and phosphorylated JNK (P-JNK)] were determined by immunohistochemical staining in rat renal cortex.Results:No dental fluorosis was found in control group. The incidence of dental fluorosis in low, medium and high fluoride groups were 2/6, 5/6 and 6/6, respectively. Compared with control group [(5.707 ± 1.190) mg/L], the urinary fluoride in low, medium and high fluoride groups [(17.028 ± 3.006), (34.378 ± 12.045), (94.759 ± 31.773) mg/L] was significantly higher ( P < 0.05), and the urinary fluoride in high fluoride group was higher than that in low and medium fluoride groups ( P < 0.05). HE staining showed that, compared with control group, the cell volume of renal tubules and glomeruli in medium and high fluoride groups increased, the cells arranged closely, and the eosinophilia of the cytoplasm increased. The immunohistochemical staining results showed that there was no significant difference in the expression of JNK protein in rat renal cortex between control group and low, medium and high fluoride groups ( F = 0.07, P > 0.05). The expressions of IRE1α, ASK1 and P-JNK proteins in rat renal cortex in high fluoride group were higher than those of control, low and medium fluoride groups ( P < 0.05), and the expressions of IRE1α and ASK1 proteins in medium fluoride group were significantly higher than those in control and low fluoride groups ( P < 0.05). Conclusion:Long-term excessive fluoride intake can lead to renal cortex injury in rats, and the mechanism of injury may be related to the activation of IRE1α-ASK1-JNK endoplasmic reticulum stress apoptosis pathway.

BACKGROUND:Previous studies have shown that N-methyl-D-aspartic acid receptor(NMDA)receptors are associated with fluorine,but the role in fluoride-induced endoplasmic reticulum stress remains unclear. OBJECTIVE:To observe the changes of excitatory neurotransmitter NMDA receptor and endoplasmic reticulum stress IRE1α-ASK1-JNK pathway protein expression in brain tissue of rats with experimental fluorosis,and to investigate the pathogenesis of neurological injury in fluorosis by giving NMDA receptor inhibitor to SH-SY5Y cells. METHODS:(1)Animal model:18 1-month-old SD rats were randomly divided into control group(drinking water fluoride content<0.5 mg/L),low fluoride group(drinking water fluoride content 10.0 mg/L)and high fluoride group(drinking water fluoride content 100.0 mg/L),with 6 rats in each group,half of each sex.After 6 months of fluoride intake,the rats were observed for the occurrence of dental fluorosis,and the 24-hour urinary fluoride content was measured.After anesthesia and euthanasia,the brain tissue of rats was taken to observe the pathological changes.Western blot assay was used to detect NMDA receptors and IRE1α,ASK1 and JNK protein expression in the brain tissue.(2)Cell model:SH-SY5Y cells were cultured in vitro and treated with sodium fluoride at final concentrations of 0.3 mmol/L and 3 mmol/L.The fluoride-stained cells were interfered with 10 μmol/L NMDA receptor antagonists Ifenprodil and MK-801 to observe the relevant protein changes. RESULTS AND CONCLUSION:(1)The incidence of dental fluorosis and urinary fluoride level in rats in the high fluoride group were significantly higher than that in the control and low fluoride groups(P<0.05).(2)Compared with the control group,the cytoplasm of neuronal cells in the CA3 area of the hippocampus in the low fluoride group was slightly more basophilic,while the neuronal cells in the CA3 area of the high fluoride group were disorganized,with increased basophilicity and some of the nuclei solidified.(3)In rat brain tissue,the expressions of NR2A in the high fluoride group and NR2B in the low fluoride group were significantly higher compared with the control group(P<0.05),and NR2B,IRE1,ASK1,and p-JNK protein expression levels were increased in the high fluoride group compared with the control and low fluoride groups(P<0.05).(4)In SH-SY5Y cells,NR1,NR2A and NR2B protein expressions were significantly increased in the high fluoride group compared to the control group(P<0.05).The protein levels of NR1 and NR2A were significantly reduced in the high fluorine + Ifenprodil group and high fluorine + MK-801 group compared with the high fluorine group(P<0.05).NR2B protein expression was significantly lower in the high fluorine + Ifenprodil group than that in the high fluorine group(P<0.05).(5)In SH-SY5Y cells,IRE1,ASK1,and p-JNK protein expression was significantly higher in the high fluoride group compared with the control group(P<0.05),while ASK1 and p-JNK protein expressions were significantly decreased in the high fluorine + Ifenprodil group and high fluorine + MK-801 group compared with the high fluorine group(P<0.05).IRE1 protein level was significantly lower in the high fluorine + Ifenprodil group than that in the high fluorine group(P<0.05).(6)It is concluded that excessive fluorine intake activates NMDA receptors in the central nervous system,causing increased expression of endoplasmic reticulum stress IRE1α,ASK1,and p-JNK proteins,and the use of NMDA receptor inhibitors has a mitigating effect on endoplasmic reticulum stress caused by fluorosis.