Expressions of TNF-α and NF-κB and their relation in diabetic rat spleen
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Abstract:
Objective To observe the changes of pathology and the expression of tumour necrosis factor-α(TNF-α) and nuclear factor-κB(NF-κB) in diabetic rat spleen.Methods A total of 26 streptozotocins-induced diabetic rats were included in this study, immunohistochemistry detection of TNF-α and NF-κB were taken at the 1st,3rd,6th month after establishment of model respectively.Results The expressions of TNF-α and NF-κB were found in diabetic rat spleen and increased along with the development of diabetes.Conclusion The expression of TNF-α and NF-κB could be related with complication of spleen (pathological and functional changes) in the diabetic rats.Cite
Objective
To observe the dynamic changes of TNF-α, IL-1β, IL-6, IL-10, and expressions of Notch-1, NF-κB mRNA and their protein levels in the brain tissue of septic mice and intervention effects of intrathecal injection of lentiviral vector of miR-34a gene for regulating Notch-1/NF-κB signaling pathway.
Methods
A total of 54 mice of clean grade were divided randomly(random number)into four groups, namely sham group(n=9), in which sham-operated laparotomy was performed; CLP group(n=15), in which the cecum ligation operation(CLP) was performed; NC group(n=15), in which intrathecal injection of lentivirus 5 μL(concentration 5×108 TU/mL), one time per day, for 3 days, then CLP was performed on the seventh day; intervention group(n=15), in wihch intrathecal injection of miR-34a lentivirus 5 μL(concentration 5×108 TU/mL), one time per day, for 3 days, then CLP was performed on the seventh day. The mice of four groups were sacrificed 24 h after modeling or operation. The changes of behavior of mice was observed and the neurological scores were assessed 24 h after CLP. The levels of TNF-α, IL-1β, IL-6 and IL-10 in the brain were measured by ELISA method. The mRNA expression and protein levels of Notch-1 and NF-κB in the brain tissue were measured by real-time PCR and Western blot method, respectively. Pathological changes of brain tissue were observed under light microscope.
Results
The neurological scores, the cerebral TNF-α, IL-6 levels, the mRNA expression and protein level of NF-κB(P 0.05). The IL-10 level (P 0.05). The degree of cerebral damage found under light microscope was also ameliorated in intervention group compared with CLP group 24 h after modeling.
Conclusions
The effects of miR-34a via regulating Notch-1/NF-κB signaling pathway on brain function exerts cerebral protective effects in septic mice.
Key words:
miR-34a; Notch-1; NF-κB; Lentiviral vector; Mice
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Objective: To explore the mechanism of Xuebijing injection in the treatment of acute paraquat poisoning by means of studying the expression of TNF-alpha, NF-kappa B, Caspase-3 and the changes of cell apoptosis rate detected by TUNEL in the lung tissue of acute paraquat-induced rats. Methods: On the base of random number table, 126 Wister rats weighing 220 g to 270 g were divided into 3 groups: (1) Control group: 42 rats, (2) Poisoned group: 42 rats, (3) Treatment group: 42 rats. On 1(st)、3(rd)、7(th)、14(th)、21(st)、28(th)、and 35(th) day, six rats from each group were anaesthetized by intraperitoneal injection of chloral hydrate. To cut the chest and take the lung tissue samples. The expression levels of Tumor Necrosis Factor-alpha, Nuclear Factor-kappa B and Caspase-3 protein in lung tissue were detected by immunohistochemical staining, as well as apoptotic cell rate was detected by TUNEL staining. Results: The expression levels of Tumor Necrosis Factor-alpha, Nuclear Factor-kappa B, Caspase-3 protein and TUNEL staining in the lung tissue of the poisoned group was significantly higher than that of the control group (P<0.05) . Compared with the poisoned group, the expression of TNF-alpha, NF-kappa B, Caspase-3 and TUNEL in treatment group decreased significantly (P<0.05) , but they were still higher than those of the control group, and the difference was statistically significant compared with the control group (P<0.05) . Conclusion: Apoptosis and TNF-alpha, NF-kappa B and Caspase-3 play an important role in lung injury of paraquat-induced rats. Xuebijing injection can inhibit the expression of TNF-alpha, NF-kappa B, Caspase-3 in lung tissue, reduce the apoptosis rate and alleviate the damage of lung tissue in paraquat-poisoning rats.目的： 通过研究血必净对急性百草枯中毒大鼠肺组织中炎性因子的影响及细胞凋亡率，探索血必净治疗急性百草枯中毒的机制。 方法： 将126只Wister大鼠采用随机数字表法分成对照组（42只）、染毒组（42只）、治疗组（42只）。在不同处理的第1、3、7、14、21、28、35天，每组各取6只，留取右下肺组织标本，行免疫组化检测肺组织肿瘤坏死因子-α（TNF-α）、核因子-κB（NF-κB）、Caspase-3蛋白的表达水平，TUNEL染色检测凋亡细胞率。 结果： 染毒组大鼠肺组织中TNF-α、NF-κB、Caspase-3及TUNEL凋亡细胞率明显高于对照组，差异有统计学意义（P<0.05）。经血必净治疗后，与染毒组比较，TNF-α、NF-κB、Caspase-3及TUNEL凋亡细胞率明显降低，差异有统计学意义（P<0.05），但仍高于对照组，与对照组比较有统计学意义（P<0.05）。 结论： 细胞凋亡和细胞因子TNF-α、NF-κB、Caspase-3在百草枯所致大鼠肺损伤中起重要作用；血必净能抑制肺组织细胞TNF-α、NF-κB及Caspase-3的表达，降低其调控的细胞凋亡率。.
Intraperitoneal injection
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Objective
To determine the effect of Xinjikang on signaling pathway and apoptosis of Toll-like receptors 4 (TLR4) -nuclear factor-κB (TLR4-NF-κB) in rats with viral myocarditis (VMC) .
Methods
Eighty 3-month-old male rats were randomly divided into the model group (n=30) , Xinjikang group (n=30) and normal control group (n=20) . The model group receieved intraperitoneal injection of Coxsackievirus B3 (CVB3) . In addition, the Xinjikang group was injected with Xinjikang decoction (15 g/kg) . The normal group was inoculated intraperitoneally with 0.2 ml virus-free Eagle’s solution. The cardiac index (body weight/cardiac weight) in each group was statistically analyzed. The pathological changes of heart tissues were examined by HE staining. The expression levels of TLR-4 and NF-κB in myocardial tissues were determined by immunohistochemistry. The protein expression levels of TLR-4 and NF-κB in myocardial tissues were determined by Western Blotting. The myocardial cell apoptosis index (AI) was examined by in situ end labeling (TUNEL) method.
Results
(1) The mortality in the Xinjikang group was significantly lower than that in the model group, whereas the median survival time was significantly longer than that in the model group (P Xinjikang group>normal control group (P Xinjikang group>normal control group (P<0.05) .
Conclusion
Xinjikang may reduce the mortality of VMC rats, prolong the median survival time, inhibit the expression of TLR4 and NF-κB, and improve the myocardial cell apoptosis.
Key words:
Myocarditis; TLR4; NF-κB; Apoptosis
Viral Myocarditis
Intraperitoneal injection
Coxsackievirus
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Objective To investigate the variation and significance of the expressions of NF-κB, inducible nitric oxide synthase(iNOS) and cyclooxygenase-2(COX-2) in the renal tissue of insulin-resistant rat. Methods Thirty healthy male Wistar rats were bred since 2 months old, and they were randomly divided into normal control(NC) group(n=15) and insulin-resistant(IR) group(n=15). Insulin resistance rat model was reproduced by feeding with high fat and sucrose diet. Hyperinsulinemic-euglycemic clamp test was used to verify the reproduction of the model. The kidneys of the rats were obtained after the successful reproduction of the model. The change in renal histology was observed by HE staining, and the expressions of iNOS and COX-2 in the kidneys were detected by immunohistochemistry staining. The mRNA expressions of NF-κB, iNOS and COX-2 in the kidneys were assessed with RT-PCR. DNA binding activity of NF-κB in the rat's kidney was assessed with electrophoretic mobility shift assay(EMSA). Results HE staining showed that, compared with NC group, the early lesions of the renal tissue, such as glomerular enlargement and mesangial region broadening, could be seen in IR group. Immunohistochemical staining showed that the positive expressions of iNOS and COX-2 were up-regulated significantly in IR group than in NC group(P0.05). RT-PCR revealed that the expressions of NF-κB mRNA, iNOS mRNA and COX-2 mRNA in renal tissue were significantly higher in IR group than in NC group(P0.05). EMSA showed that the binding activity of NF-κB in renal tissue increased significantly in IR group than in NC group(P0.05). Conclusion NF-κB activation is present in the kidney tissue in the insulin resistance rat, which may upregulate the expression of downstream target gene iNOS and COX-2, resulting in damage to kidney tissue. The activation of NF-κB may be one of the initiative factors that lead to the kidney lesion of the insulin resistance rat.
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To evaluate the effects of etanercept on the expression of Fas, tumor necrosis factor-alpha (TNF-α) and caspase-8 in the early stage of the apoptotic pathway in diabetic rats, and to explore the therapeutic effect of etanercept on diabetic retinopathy.A total of 60 Sprague-Dawley (SD) rats were randomly and evenly divided into 3 groups with 20 rats each, including control group, and diabetic groups with or without treatment. Streptozotocin (STZ)-induced diabetic rats were established for diabetic groups. Blood glucose and body weight were measured weekly. All the rats were sacrificed at the 12wk after treatment. The expressions of Fas, TNF-α and caspase-8 in rat retina were quantitatively detected by PCR and Western blot. The leakage of Evan blue was adopted to measure the retinal vascular leakage quantitatively, and to compare it among different groups. TUNEL method was used to compare the amount of apoptotic bodies quantitatively in rat retina ganglion cells under electron microscope.The expressions of Fas, TNF-α and caspase-8 in each group were compared via PCR and Western blot, in which the diabetic group with treatment was lower than those without treatment (P<0.01), but all the diabetic groups were higher than the control group (P<0.01). Evans blue leakage in the diabetic treatment group was lower than those without treatment (P<0.01), but those in the control group was the lowest compared with the other two groups (P<0.01). TUNEL method showed that the apoptotic bodies of retina in the diabetic treatment group was lower than those without treatment (P<0.01), while those in the control group was the lowest compared with the other two groups (P<0.01).Etanercept can effectively reduce the expression of Fas, TNF-α and caspase-8, as well as the retinal leakage and retinal cell apoptosis in diabetic rats.
Evans Blue
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To investigate the relationship between NF-kappa B activity and IFN-gamma gene expression, as well as the histopathological changes following liver transplants, both with and without cyclosporin A (CsA) treatment.Sixty male Wistar and Thirty male SD rats were subjected to orthotopic liver transplants. Fourty-five of the Wistar rats were used as recipients, and were divided into 3 groups: group I, syngeneic control (Wistar-to-Wistar); group II, acute rejection (SD-to-Wistar); and group III: acute rejection treated with cyclosporin A by intramuscular injection (SD-to-Wistar + CSA). After the liver transplants, electrophoretic gel mobility shift assay was used to analyze NF-kappa B activity in the splenocytes of recipient rats, and RT-PCR was used to measure IFN-gamma gene expression in grafted liver specimens. In addition, histopathological examinations were performed to assess the severity of acute liver rejection.In group I, low levels of NF-kappa B activity were only detectable on day 5 and 7 post-transplant, and only weak IFN-gamma mRNA expression was observed at all time points. By contrast, both high NF-kappa B activity and high expression levels of IFN-gamma mRNA were detected at all time points in group II. In group III, NF-kappa B activity and IFN-gamma mRNA expression were significantly inhibited, as compared to group II (P < 0.05). A good correlation was found between NF-kappa B activity and IFN-gamma mRNA expression (r = 0.815). In addition, NF-kappa B activity and IFN-gamma mRNA expression mirrored histopathological changes in all three experimental groups.Changes in IFN-gamma mRNA expression levels after liver transplantation are at least partially due to changes in levels of NF-kappa B activity. CsA appears to downregulate NF-kappa B activity, thus inhibiting IFN-gamma gene transcription. Blocking the NF-kappa B mediated transcription pathway may be of benefit in preventing liver transplant rejection.
Kappa
Splenocyte
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The aim of the present study was to explore the role of catechin on the expression of heat shock protein 27 (HSP27), nuclear factor-kappa beta (NF-κB) and inflammatory factors IL-1β, IL-6 and TNF-α in streptozotocin (STZ)-induced diabetic retinopathy (DR) rats.A total of 150 rats were selected and randomly assigned to five groups: control group, STZ group, and three groups with different concentrations of catechin (low, middle and high concentrations). After STZ induction, DR rats were treated with different concentrations of catechin (50 mg/kg/day, low catechin group; 100 mg/kg/ day, middle catechin group; 200 mg/kg/day, high catechin group) by intravitreal injection for eight weeks. Hematoxylin and eosin (H&E) staining was used to observe the pathological changes in retinal tissues. The mRNA and protein levels of HSP27 in the retina were determined by RT-PCR and western blotting. Furthermore, the expression of NF-κB p65 and p-NF-κB p65 were determined by western blotting, while the expression of IL-1β, IL-6, and TNF-α were determined by ELISA.HSP27 levels increased in STZ-induced DR rats, and became further upregulated after catechin treatment. Furthermore, IL-1β, IL-6, and TNF-α levels were upregulated in the retinas of STZ-induced DR rats, but these changes were partially inhibited after treatment with catechin. Moreover, the application of catechin inhibited the activation of NF-κB, which was upregulated in STZ-induced DR. A negative correlation was observed between the concentrations of catechin and the expression of inflammatory factors.Catechin can weaken DR induced by STZ by increasing HSP27 levels and decreasing the production of associated inflammatory factors. This could help to treat DR in clinic.
Hsp27
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The aim of this study was to investigate the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in juvenile rats with nephrotic syndrome, and to explore its effects on inflammatory changes and renal injury.24 Sprague-Dawley (SD) rats were randomly divided into the normal group (n=12) and model group (n=12). Rats in the normal group were intraperitoneally injected with normal saline. Meanwhile, rats in the model group were given azithromycin hydrochloride injection to establish the model of nephrotic syndrome. After 24 h of modeling, the samples were collected. The expression of NF-κB was detected via immunohistochemistry. Moreover, the protein expression of NF-κB was determined through Western blotting. Quantitative Polymerase Chain Reaction (qPCR) was used to measure the messenger ribonucleic acid (mRNA) expression levels of interleukin-1 (IL-1) and IL-6. Meanwhile, the content of IL-1 and IL-6 was detected by enzyme-linked immunosorbent assay (ELISA). The serum levels of urea nitrogen and serum creatinine were measured by an automatic biochemical analyzer. Furthermore, the correlation between NF-κB protein with IL-1 and IL-6 were studied via Pearson analysis.Compared with the normal group, rats in the model group exhibited significantly increased expression and protein expression of NF-κB (p<0.05). Meanwhile, the mRNA expression levels and content of IL-1 and IL-6 (p<0.05), as well as the serum levels of urea nitrogen and creatinine (p<0.05) of the model group were markedly higher than those of the normal group. Furthermore, NF-κB protein was positively correlated with IL-1 and IL-6 contents.NF-κB is highly expressed in juvenile rats with nephrotic syndrome, which promotes the expressions of inflammatory factors (IL-1 and IL-6) and aggravates the renal injury.
Blood urea nitrogen
Group A
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Objective To evaluate the changes in expression of NF-κB, IL-6 and TNF-α in spinal cord in a rat model of bone cancer. Methods Seventy-two female SD rats weighing 150-180 g were randomly divided into 3 groups (n = 24 each): control group (group C);sham operation group (group S) and bone cancer pain group (group BP). Bone cancer was induced by intra-tibial inoculation of 1 × 105 Walker 256 breast cancer cells. Paw withdrawal threshold to mechanical stimulation was measured with yon Frey filaments. The expression of NF-κB p65, IL-6 and TNF-α mRNA in the spinal cord was determined by RT-PCR and the expression of NF-κB p65 by immuno-histochemistry and NF-κB p65 positive cell count was determined. Results The paw withdrawal threshold was significantly lower and the expression of NF-κB p65, NF-κB p65 mRNA, IL-6 mRNA, TNF-α mRNA and NF-κB p65 positive cell count in the spinal cord were significantly higher in group BP than in group C and S ( P ＜0.05 or 0.01 ). Conclusion Intra-tibial inoculation of Walker 256 breast cancer cells activates NF-κB in the spinal cord, leading to the increased release of IL-6 and TNF-α and mechanical hyperalgesia.
Key words:
Bone neoplasms; Pain; NF-kappa B; Interleukin-6; Tumor necrosis factor-alpha
Bone cancer
Group B
Group A
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Melatonin (MT) is an indoleamine hormone that can counteract ischemia‑induced organ injury through its antioxidant effects. The aim of the present study was to investigate the protective effects of exogenous MT against hemorrhagic shock (HS)‑induced hepatic ischemic injury in rats, and the role of the nuclear factor (NF)‑κB signaling pathway in this process. A rat model of HS‑induced hepatic ischemic injury was established. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH), tumor necrosis factor (TNF)‑α, interferon (IFN)‑γ, interleukin (IL)‑6 and IL‑1β were measured every 6 h, and the 24‑h survival rate of the rats was analyzed. All surviving rats were sacrificed after 24 h. Pathological changes in the liver and the hepatocyte apoptosis rate were observed by hematoxylin and eosin staining and TUNEL assay, respectively, and the expression levels of NF‑κB p65 and NF‑κB inhibitor α (IκBα) were analyzed by reverse transcription‑quantitative PCR analysis and western blotting. The results demonstrated that the serum levels of ALT, AST, LDH, GDH, TNF‑α, IFN‑γ, IL‑6 and IL‑1β gradually increased after HS compared with those in rats subjected to a sham procedure, but this increase was attenuated by MT. Furthermore, the survival rate of the MT group was significantly higher compared with that of the HS group. The degree of pathological hepatic injury, the hepatocyte apoptosis rate, and the hepatic levels of TNF‑α, IFN‑γ, IL‑6 and IL‑1β were significantly decreased in the MT group compared with the HS group. In addition, the mRNA expression of NF‑κB p65 was significantly decreased and the mRNA expression of IκBα was significantly increased in the MT group compared with the sham group. Furthermore, the NF‑κB p65 protein levels in the MT group were significantly increased in the cytosol but decreased in the nucleus, and the IκBα protein levels were increased while those of phosphorylated IκBα were decreased compared with those in the HS group. Therefore, it may be inferred that exogenous MT alleviates HS‑induced hepatic ischemic injury in rats via the inhibition of NF‑κB activation and IκBα phosphorylation.
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