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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.
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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.
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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.