Semaphorin 7A impairs barrier function in cultured human corneal epithelial cells in a manner dependent on nuclear factor-kappa B

●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0,125,250,or 500 ng/mL for 24,48,or 72h in vitro.Transepithelial electrical resistance(TEER)as well as Dextran-fluorescein isothiocyanate(FITC)permeability assays were conducted to assess barrier function.To quantify tight junctions(TJs)such as occludin and zonula occludens-1(ZO-1)at the mRNA level,reverse transcriptionpolymerase chain reaction(RT-PCR)analysis was performed.Immunoblotting was used to examine the activity of the nuclear factor-kappa B(NF-κB)signaling pathway and the production of TJs proteins.Immunofluorescence analyses were employed to localize the TJs.Enzyme-linked immunosorbent assay(ELISA)and RT-PCR were utilized to observe changes in interleukin(IL)-1βlevels.To investigate the role of NF-κB signaling activation and IL^(-1)βin Sema7A’s anti-barrier mechanism,we employed 0.1μmol/L IκB kinase 2(IKK2)inhibitor IV or 500 ng/mL IL^(-1)receptor(IL-1R)antagonist.●RESULTS:Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time-and dose-dependent manner,as well as altering the localization of TJs.Furthermore,Sema7A stimulated the activation of inhibitor of kappa B alpha(IκBα)and expression of IL-1β.The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists.●CONCLUSION:Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins,as well as the expression of IL-1β.These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.

Delayed hepatocarcinogenesis through antiangiogenic intervention in the nuclear factor-kappa B activation pathway in rats

BACKGROUND: The active form of nuclear factor-kappa B (NF-kappa B) is involved in the initiation, generation, and development of hepatocellular carcinoma (HCC), and is up-regulated in inflammation-associated malignancies. We investigated the dynamic expression of NF-kappa B and its influences on the occurrence of HCC through antiangiogenic (thalidomide) intervention in NF-kappa B activation. METHODS : Hepatoma models were induced with 2-fluorenylacetamide (2-FAA, 0.05%) in male Sprague-Dawley rats, and thalidomide (100 mg/kg body weight) was administered intragastrically to intervene in NF-kappa B activation. The pathological changes in the liver of sacrificed rats were assessed after hematoxylin and eosin staining. NF-kappa B mRNA was amplified by RT-nested PCR. The alterations of NF-kappa B and vascular endothelial growth factor (VEGF) expression were analyzed by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blotting. RESULTS: Rat hepatocytes showed denatured, precancerous, and cancerous stages in hepatocarcinogenesis, with an increasing tendency of hepatic NF-kappa B, NF-kappa B mRNA, and VEGF expression, and their values in the HCC group were higher than those in controls (P<0.001). In the thalidomide-treated group, the morphologic changes generated only punctiform denaturation and necrosis at the early or middle stages, and nodular hyperplasia or a little atypical hyperplasia at the final stages, with the expression of NF-kappa B (chi(2)=9.93, P<0.001) and VEGF (chi(2)=8.024, P<0.001) lower than that in the 2-FAA group. CONCLUSION: NF-kappa B is overexpressed in hepatocarcinogenesis and antiangiogenic treatment down-regulates the expression of NF-kappa B and VEGF, and delays the occurrence of HCC. (Hepatobiliary Pancreat Dis Int 2010; 9: 169-174)

Urinary trypsin inhibitor attenuates hepatic ischemia-reperfusion injury by reducing nuclear factor-kappa B activation

BACKGROUND: Urinary trypsin inhibitor (UTI) inhibits the inflammatory response and protects against ischemia-reperfusion (I/R) injury. The inflammatory response is mediated by nuclear factor-kappa B (NF-kappa B) and its related target genes and products such as vascular endothelial cell adhesion molecule and CXC chemokines. We aimed to assess the roles of those mediators in a UTI-treated mouse model of hepatic I/R injury. METHODS: Treatment group 1 (UTI given 5 minutes prior to liver ischemia), treatment group 2 (UTI given 5 minutes after the anhepatic phase) and a control group were investigated. Blood and liver samples were obtained and compared at 1, 3, 6 and 24 hours after reperfusion. RESULTS: Attenuation of pathological hepatocellular damage was greater in the treatment groups than in the control group (P < 0.05). Compared with the control group, the UTI treatment groups showed significantly lower serum alanine aminotransferase and aspartate aminotransferase levels, decreased myeloperoxidase activity, and reduced NF-kappa B activation. Also downregulated was the expression of tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein-2 at the mRNA level. P-selectin protein and intercellular adhesion molecule-1 protein expression were also downregulated. In addition, the treatment group I showed a better protective effect against I/R injury than the treatment group 2. CONCLUSIONS: UTI reduces NF-kappa B activation and downregulates the expression of its related mediators, followed by the inhibition of neutrophil aggregation and infiltration in hepatic I/R injury. The protective role of UTI is more effective in prevention than in treatment.

Characteristics of hepatic nuclear-transcription factor-kappa B expression and quantitative analysis in rat hepatocarcinogenesis

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. We analyzed the expression of miclear-transcription factor-kappa B (NF-kappa B) during hepatocarcinogenesis in order to evaluate its dynamic expression and its clinical value in the development and diagnosis of HCC. METHODS: Hepatoma models were induced by oral administration of 2-acetamidoflurene (2-FAA) to male Sprague-Dawley rats. Morphological changes were observed after hematoxylin and eosin staining. The cellular distribution of NF-kappa B expression during different stages of cancer development was investigated by immunohistochemistry, and the level of NF-kappa B expression in liver tissues was quantitatively analyzed by ELISA. The gene fragments of hepatic NF-kappa B were amplified by nested-polymerase chain reaction assay. RESULTS: Hepatocytes showed vacuole-like degeneration during the early stages, then had a hyperplastic nodal appearance during the middle stages, and finally progressed to tubercles of cancerous nests with high differentiation. The NF-kappa B-positive material was buff-colored, fine particles localized in the nucleus, and the incidence of NF-kappa B-positive cells was 81.8% in degeneration, 83.3% in precancerous lesions, and 100% in cancerous tissues. All of these values were higher than those in controls (P<0.01). Hepatic NF-kappa B expression and hepatic NF-kappa B-mRNA were also higher during the course of HCC development (P<0.01). CONCLUSION: The NF-kappa B signal transduction pathway is activated during the early stages of HCC development, and its abnormal expression may be associated with the occurrence of HCC.

Dietary saturated fatty acid and polyunsaturated fatty acid oppositely affect hepatic NOD-like receptor protein 3 inflammasome through regulating nuclear factor-kappa B activation

AIM: To investigate the effect of different dietary fatty acids on hepatic inflammasome activation.METHODS: Wild-type C57BL/6 mice were fed either a high-fat diet or polyunsaturated fatty acid (PUFA)-enriched diet. Primary hepatocytes were treated with either saturated fatty acids (SFAs) or PUFAs as well as combined with lipopolysaccharide (LPS). The expression of NOD-like receptor protein 3 (NLRP3) inflammasome, peroxisome proliferator-activated receptor-&#x003b3; and nuclear factor-kappa B (NF-&#x003ba;B) was determined by real-time PCR and Western blot. The activity of Caspase-1 and interleukine-1&#x003b2; production were measured.RESULTS: High-fat diet-induced hepatic steatosis was sufficient to induce and activate hepatic NLRP3 inflammasome. SFA palmitic acid (PA) directly activated NLRP3 inflammasome and increased sensitization to LPS-induced inflammasome activation in hepatocytes. In contrast, PUFA docosahexaenoic acid (DHA) had the potential to inhibit NLRP3 inflammasome expression in hepatocytes and partly abolished LPS-induced NLRP3 inflammasome activation. Furthermore, a high-fat diet increased but PUFA-enriched diet decreased sensitization to LPS-induced hepatic NLRP3 inflammasome activation in vivo. Moreover, PA increased but DHA decreased phosphorylated NF-&#x003ba;B p65 protein expression in hepatocytes.CONCLUSION: Hepatic NLRP3 inflammasome activation played an important role in the development of non-alcoholic fatty liver disease. Dietary SFAs and PUFAs oppositely regulated the activity of NLRP3 inflammasome through direct activation or inhibition of NF-&#x003ba;B.

Testosterone alleviates tumor necrosis factor-alpha-mediated tissue factor pathway inhibitor downregulation via suppression of nuclear factor-kappa B in endothelial cells

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor(TFPI)gene expression through the androgen receptor in endothelial cells.This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha(TNF-α).Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-α.TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction.To study the cellular mechanism of testosterone’s action,nuclear factor-kappa B(NF-κB)translocation was confirmed by electrophoretic mobility shift assays.We found that after NF-κB was activated by TNF-α,TFPI protein levels declined significantly by 37.3%compared with controls(P<0.001),and the mRNA levels of TFPI also decreased greatly(P<0.001).A concentration of 30 nmol L-1 testosterone increased the secretion of TFPI compared with the TNF-α-treated group.NF-κB DNA-binding activity was significantly suppressed by testosterone(P<0.05).This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-κB activity.

Inhibitory effects of Shuanghuanglian injection on nuclear factor-kappa B expression in mice with viral encephalitis in a time-and dose-dependent manner

Previous studies have confirmed that the anti-virus effects of Shuanghuanglian injection may be associated with nuclear factor-kappa B activity. This study observed nuclear factor-kappa B expression in mice with viral encephalitis, and showed significant decreases in nuclear factor-kappa B protein and mRNA levels following Shuanghuanglian injection. The inhibitory effect was more significant with prolonged intervention duration and increased treatment dose. These findings verify that Shuanghuanglian injection plays a therapeutic role in viral encephalitis by reducing expression of nuclear factor-kappa B in a time- and dose-dependent manner.

Shuanghuanglian injection downregulates nuclear factor-kappa B expression in mice with viral encephalitis

A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension. Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and protein expression of intelectin-2 and nuclear factor-kappa B in the viral encephalitis and control groups. Nuclear factor-kappa B and intelectin-2 mRNA and protein expression were significantly increased in mice with viral encephalitis. After intraperitoneal injection of Shuanghuanglian at a dose of 1.5 mg/kg for 5 successive days, intelectin-2 and nuclear factor-kappa B protein and mRNA expression were significantly decreased. To elucidate the relationship between intelectin-2 and nuclear factor-kappa B, mice with viral encephalitis were administered an intracerebral injection of 107 pfu recombinant lentivirus expressing intelectin shRNA. Both protein and mRNA levels of intelectin and nuclear factor-kappa B in brain tissue of mice were significantly decreased. Experimental findings suggest that Shuanghuanglian injection may downregulate nuclear factor-kappa B production via suppression of intelectin production, thus inhibiting inflammation associated with viral encephalitis.

Effects of Five Chitosan Oligosaccharides on Nuclear Factor-kappa B Signaling Pathway

The effects of five chito-oligomers, from dimer to hexamer （chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose） separated from chitosan oligosaccharides, on nuclear factor -kappaB （NF-rd3） signaling pathway were investigated by using luciferase assay and laser scanning microscopy. The expression of NF-rd3 downstream genes （cyclin DI, TNFa and IL-6） were tested by real time PCR. We found that all five chitosan oligosaccharides increased NF-KB-dependent luciferase gene expression and NF-KB downstream genes transcription, and the most significant were chitotetraose and chitohexaose. In addition, laser scanning microscopy experiments showed that chitotetraose and chitohexaose also activated the p65 subunite of NF-kB translocating from cytoplasm to nucleus, which suggested that they were the most potent activators of NF-kB signaling pathway.

Effects of transplantation of microencapsulated rabbit sciatic nerve on nuclear factor-kappa B expression after spinal cord injury in rats

BACKGROUND： It has been reported that nuclear factor-kappa B （NF- κB）, activated after spinal cord injury in rats, plays a key role in inflammatory responses in the central nervous system. OBJECTIVE： To investigate the effects of transplantation of microencapsulated rabbit sciatic nerve on NF- κB expression and motor function after spinal cord injury in rats, and to compare the results with the transplantation of rabbit sciatic nerve alone. DESIGN, TIME AND SETTING： This completely randomized, controlled study was performed at the Department of Neurobiology, Medical College of Nanchang University between December 2007 and July 2008. MATERIALS： A rabbit anti-NF- κB P65 monoclonal antibody was made by the Santa Cruz Company, USA and a streptavidin peroxidase immunohistochemical kit was provided by the Sequoia Company, China. METHODS： Eight rabbits were used to prepare a sciatic nerve cell suspension that was divided into two parts： one stored for transplantation, and the other mixed with a 1.5% sodium alginate solution. One hundred and twenty adult Sprague Dawley rats weighing 220-250 g were randomly divided into four groups： the microencapsulated cell group （n = 36）, the non-encapsulated cell group （n = 36）, the saline group （n = 36） and the sham operation group （n = 12）. The first three groups underwent a right hemisection injury of the spinal cord at the T10 level, into which was transplanted a gelatin sponge soaked with 10 μL of a microencapsulated nerve tissue/cell suspension （microencapsulated cell group）, a tissue/cell suspension （non-encapsulated cell group） or physiological saline （saline group）. In the sham operation group the vertebrae were exposed, but the spinal cord was not injured, and no implantation was given. MAIN OUTCOME MEASURES： Pathological changes were detected using hematoxylin-eosin staining; NF- κB expression was quantified using immunohistochemical staining; motor function was assessed using the Basso, Beattie and Bresnahan （BBB） scale. RESULTS： Spinal cord injuries, such as neuronal death and inflammatory cell infiltration, were found in the microencapsulated cell group, the non-encapsulated cell group and the saline group. However, the damage in the microencapsulated cell group was milder than in the non-encapsulated cell or saline groups. NF- κB expression in the microencapsulated cell group, the non-encapsulated cell group and the saline group was increased after spinal cord injury; it reached a peak after 24 hours, gradually decreased after 3 days, and was close to normal levels after 7 days. NF- κB expression in the microencapsulated cell group was significantly lower than in the saline group and the non-encapsulated cell group （P 〈 0.05）. With time, the motor function of the animals in each group improved to a certain extent, but did not reach normal levels. There were no significant differences in BBB scores between the different groups on post-operative day 3; however, the BBB scores for the microencapsulated cell group and the non-encapsulated cell group were significantly higher than the saline group on post-operative day 7 （P 〈 0.05）. In addition, the motor function recovered better in the microencapsulated cell group than in the non-encapsulated cell group （P 〈 0.05）. CONCLUSION： The transplantation of microencapsulated rabbit sciatic nerve can inhibit NF- κB expression and inflammatory reactions and promote recovery of motor function after spinal cord injury in rats. The effects of microencapsulated cell transplantation are superior to those of transplantation of cells alone.

Effect of Nuclear Factor-kappa B on Vascular Endothelial Growth Factor mRNA Expression of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia

In order to investigate the effect of nuclear factor kappa B (NF κB) on vascular endothelial growth factor (VEGF) mRNA expression of human pulmonary artery smooth muscle cells (HPASMCs) in hypoxia, the cultured HPASMCs in vitro were stimulated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF κB. The NF κB p65 nuclei positive expression was detected by immunocytochemical technique. The IκBα protein expression was measured by Western blot. RT PCR was used to detect the VEGF mRNA expression of HPASMCs. The results showed that no significant change was observed in the NF κB p65 nuclei positive expression of cultured HPASMCs during 6 h-24 h in normoxia, but the levels of NF κB p65 nuclei positive expression of cultured HPASMCs were significantly increased in hypoxia groups as compared with those in all normoxia groups ( P <0.05). The IκBα protein expression of cultured HPASMCs showed no significant change during 6 h-24 h in normoxia, but significantly decreased in hypoxia as comapred with that in normoxia groups ( P <0.05). PDTC (1 to 100 μmol/L) could inhibit the VEGF mRNA expression of HPASMCs in a concentration dependent manner in hypoxia. In conclusion, NF κB can be partly translocation activated from cytoplasm into nuclei in the cultured HPASMCs under hypoxia. The inhibition of NF κB activation can decrease the VEGF mRNA expression. It is suggested that the activation of NF κB is involved in the VEGF mRNA expression of HPASMCs under hypoxia.

Treatment of COVID-19 by Controlling the Activity of the Nuclear Factor-Kappa B

Heavy infection of the virus leads to overproduction of cytokines. The overproduction of cytokine (cytokines storms) is responsible for the critical cases and deaths of COVID-19. The nuclear factor kappa-B stimulates the expression of the genes, which is responsible for cytokines storm and RNA transcription. The COVID-19 virus can be controlled by inhibition of nuclear factor kappa-B. Nuclear factor kappa-B is controlled by inhibition of hydrogen peroxide and inhibitor kappa-B kinase enzyme.

基于骨免疫学论中医药抑制类风湿关节炎骨破坏的研究进展

类风湿关节炎(RA)是一种以滑膜炎、软骨与骨破坏为主要病理表现的自身免疫性疾病,致残率较高。RA免疫及炎症反应与骨细胞代谢互为影响,其核心环节为破坏机体核因子κB受体活化因子配体/核因子κB受体活化因子/骨保护素(RANKL/RANK/OPG)信号通路的平衡,导致成骨细胞减少,以及破骨细胞凋亡减退及异常活化。西药目前以抑制炎症反应及相关细胞因子分泌,减缓疾病进展,但长期使用其不良反应难以忽视。中医药在防治骨破坏中研究逐步深入,但在基础及临床研究方面仍存在一定局限性。

野菊花水提物对RAW264.7炎症细胞模型的抗炎作用及其机制

目的建立以脂多糖(LPS)诱导的RAW 264.7巨噬细胞为模型,探讨野菊花提取液(CID)通过核转录因子(NF-κB)信号通路发挥抗炎活性的作用及其分子机制。方法以噻唑蓝(MTT)法检测不同浓度CID对RAW 264.7巨噬细胞活性的影响以筛选适宜的实验浓度;分别采用Griess法和酶联免疫吸附试验(ELISA)测定50、100、200μg·mL^(-1) CID干预后各组细胞中一氧化氮(NO)和肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的释放量;实时荧光定量聚合酶链式反应(RT-PCR)分析各组中环氧合酶-2(COX-2)和诱导型一氧化氮合酶(iNOS)mRNA的相对表达水平;免疫印迹实验(WB)观察各组中nuclear factor-kappa B p65(NF-κB p65)、inhibitor kappa B(IκB-α)和磷酸化IκB-α(p-IκB-α)的蛋白表达。结果50~200μg·mL^(-1)的CID可显著降低LPS诱导RAW264.7巨噬细胞中NO、TNF-α和IL-6的生成量(P<0.01),并能下调COX-2和iNOX mRNA的相对表达(P<0.01)、下调p-IκB-α、总的NF-κB p65、细胞核NF-κB p65的蛋白相对含量(P<0.01),并上调IκB-α、细胞质NF-κB p65的相对含量(P<0.01)。结论CID可有效降低LPS诱导RAW 264.7巨噬细胞的炎症因子释放,其机制可能与通过减少TNF-α等关键蛋白表达以及通过抑制NF-κB等炎症信号通路激活来抑制炎症发生有关。

Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

PTHrP促进RANKL诱导巨噬细胞分化为破骨细胞参与中耳胆脂瘤骨破坏

目的:骨质进行性吸收破坏是中耳胆脂瘤最重要的临床特征之一,可导致一系列颅内外并发症,而目前中耳胆脂瘤骨破坏的机制尚未明确。本研究旨在探究甲状旁腺激素相关蛋白(parathyroid hormone-related protein,PTHrP)参与中耳胆脂瘤骨破坏的机制。方法:收集后天性中耳胆脂瘤患者的25例胆脂瘤标本和13例外耳道正常皮肤组织标本。采用免疫组织化学染色方法检测PTHrP、核因子κB受体活化因子配体(receptor activator for nuclear factor-kappa B ligand,RANKL)和骨保护素(osteoprotegerin,OPG)在中耳胆脂瘤和外耳道正常皮肤组织中的表达,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法检测中耳胆脂瘤和外耳道正常皮肤组织中是否存在TRAP阳性多核巨噬细胞。选取小鼠单核巨噬细胞RAW264.7细胞进行干预,分为RANKL干预组和PTHrP+RANKL共同干预组,采用TRAP染色法检测2组破骨细胞的生成情况,实时聚合酶链反应(real-time polymerase chain reaction,real-time PCR)检测干预后2组破骨细胞相关基因TRAP、组织蛋白酶K(cathepsin K,CTSK)和活化T细胞核因子1(nuclear factor of activated T cell cytoplasmic 1,NFATc1)的mRNA表达水平,骨吸收陷窝实验检测2组破骨细胞的骨吸收功能。结果:免疫组织化学染色结果显示,PTHrP和RANKL在中耳胆脂瘤组织中的表达均显著增高,OPG表达降低(均P<0.05),且PTHrP的表达与RANKL、RANKL/OPG比值均呈显著正相关,与OPG表达呈显著负相关(分别r=0.385、r=0.417、r=-0.316,均P<0.05)。同时,PTHrP、RANKL的表达水平与中耳胆脂瘤的骨破坏程度均呈显著正相关(分别r=0.413、r=0.505,均P<0.05)。TRAP染色结果显示中耳胆脂瘤上皮周围基质中有大量TRAP阳性细胞,并存在细胞核数量为3个或3个以上的TRAP阳性破骨细胞。RANKL或PTHrP+RANKL联合干预5 d后,与RANKL干预组相比,PTHrP+RANKL联合干预组的破骨细胞数量显著增加(P<0.05),且破骨细胞相关基因TRAP、CTSK和NFATc1的mRNA表达水平均升高(均P<0.05)。骨吸收陷窝扫描电镜结果显示RANKL干预组、PTHrP+RANKL联合干预组的骨片表面均形成骨吸收陷窝;与RANKL干预组相比,PTHrP+RANKL联合干预组的骨片表面骨吸收陷窝数量显著增加(P<0.05),面积也更大。结论:PTHrP可能通过促进RANKL诱导胆脂瘤组织周围基质中的巨噬细胞分化为破骨细胞,参与中耳胆脂瘤骨破坏。

柚皮素对口腔鳞状细胞癌细胞的干预作用研究

目的:探究柚皮素(NRG)对口腔鳞状细胞癌(OSCC)细胞增殖、凋亡、迁移与侵袭的影响。方法:采用浓度(μmol/L)为0、5、10、15、20、25、30的NRG处理OSCC CAL-27细胞,CCK-8法检测细胞活力;将CAL-27细胞分为低、中、高剂量NRG组(NRG-L、NRG-M组、NRG-H组)、Compound C(AMPK抑制剂)组、NRG-H+Compound C组、对照组(NC组,正常培养),CCK-8与EdU染色、流式细胞术、划痕实验、Transwell分别检测细胞增殖、凋亡、迁移、侵袭;Western blot检测天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)、增殖细胞核抗原(PCNA)、基质金属蛋白酶(MMP)-2、MMP-9、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、沉默信息调节蛋白1(SIRT1)、乙酰化核因子κB p65(Ac-NF-κB p65)蛋白表达。结果:选取5、10、20μmol/L NRG分别作为后续处理CAL-27细胞的低、中、高剂量;与NC组比较,NRG-L组、NRG-M组、NRG-H组EdU阳性率、划痕愈合率、A 450值、侵袭细胞数及MMP-2、PCNA、MMP-9、Ac-NF-κB p65蛋白下调,细胞凋亡率及p-AMPK、Caspase-3、SIRT1蛋白上调(P<0.05);与NC组相比,Compound C组EdU阳性率、划痕愈合率、A 450值、侵袭细胞数及MMP-2、PCNA、MMP-9、Ac-NF-κB p65蛋白升高,细胞凋亡率及p-AMPK、Caspase-3、SIRT1蛋白表达下调(P<0.05);Compound C逆转了高剂量NRG对CAL-27细胞增殖、迁移、凋亡及侵袭的影响。结论:NRG抑制CAL-27细胞增殖、迁移与侵袭,并诱导细胞凋亡,其机制可能与激活AMPK进而抑制NF-κB通路有关。

β-紫罗兰酮通过NF-κB途径抑制乳腺癌细胞增殖

目的探讨β-紫罗兰酮(β-Ionone,BI)通过调节核因子-κB(Nuclear factor kappa-B,NF-κB)对乳腺癌细胞增殖过程的抑制作用及其可能的机制。方法采用亚甲基蓝(Methylene blue,MB)法和MTT法测定人乳腺癌BT549细胞和MCF-7细胞活性,孔雀石绿磷酸盐法检测蛋白磷酸酶2A(Protein phosphatase 2A,PP2A)活性、免疫印迹法检测磷酸化P65(p-P65)(s536和s311)、PP2A(A、B和C)和磷酸化共济失调毛细血管扩张突变基因(Phosphorylation-ataxia telangiectasia-mutated gene,p-ATM)(s1981)蛋白水平。结果BI可明显抑制人乳腺癌BT549细胞和MCF-7细胞的增殖,且呈时间和剂量依赖性,差异具有统计学意义(P<0.01)。MCF-7细胞经BI处理后,NF-κB活性被显著抑制,表现为磷酸化P65(s536和s311)的蛋白水平显著降低,PP2A的蛋白水平升高,差异具有统计学意义(P<0.05)。此外,BI还显著地降低PP2A抑制剂冈田酸(Okadaic acid,OA)对MCF-7细胞中P65蛋白和ATM蛋白的磷酸化作用。结论该研究表明BI通过抑制NF-κB活性来抑制乳腺癌细胞的增殖,其机制可能是BI通过增加PP2A活性调节NF-κB通路。

创伤性脑损伤患者血清AQP4和NF-κB p65表达与神经功能缺损程度及预后的关系

目的探讨创伤性脑损伤(TBI)患者血清水通道蛋白4(AQP4)和核因子κB(NF-κB p65)表达与神经功能缺损程度及预后的关系。方法选取2021年3月至2023年3月于河南科技大学第一附属医院开元急诊科收治的128例TBI患者作为研究对象(观察组),根据美国国立卫生院神经缺损评估量表(NIHSS)将患者分为重度组31例、中度组45例和轻度组52例;根据格拉斯哥预后量表(GOS)评分将患者分为预后不良组37例和预后良好组91例。另选取同期于本院体检的128例健康志愿者作为对照组。比较各组受检者的一般资料及血清AQP4和NF-κB p65水平,采用Spearman法分析血清AQP4、NF-κB p65水平与NIHSS评分的相关性,采用受试者工作特征曲线(ROC)分析血清AQP4、NF-κB p65对TBI患者预后不良的预测价值。结果观察组患者的血清AQP4、NF-κB p65水平分别为(27.37±6.34)μg/L、(2.27±0.24)ng/mL,明显高于对照组的(12.65±3.21)μg/L、(0.36±0.11)ng/mL,差异均具有统计学意义(P<0.05)。Spearman相关性分析结果显示,TBI患者血清AQP4、NF-κB p65水平与NIHSS评分均呈正相关(r=0.605、0.612,P<0.05)。入院24 h血清AQP4、NF-κB p65水平比较,重度组>中度组>轻度组,48 h、72 h有同样的趋势,差异均具有统计学意义(P<0.05)。预后不良组患者的血清AQP4、NF-κB p65水平分别为(34.65±7.51)μg/L、(2.71±0.40)ng/mL,明显高于预后良好组的(24.41±6.48)μg/L、(2.09±0.22)ng/mL,差异均有统计学意义(P<0.05)。血清AQP4、NF-κB p65两者联合预测TBI患者预后不良的曲线下面积(AUC)为0.938,高于各单一指标的0.873、0.830,联合预测的敏感度为91.89%,特异度为85.71%,两者联合优于血清AQP4、NF-κB p65各自单独预测(Z两者联合-AQP4=2.564、Z两者联合-NF-κB p65=2.555,P=0.010、0.011)。结论TBI患者血清AQP4、NF-κB p65水平上升与神经功能缺损程度和不良预后有关,可作为预测预后的潜在标志物。

室旁核H_(2)S通过TLR4/MyD88/NF-κB信号通路对盐敏感性高血压大鼠的影响

目的研究室旁核(PVN)硫化氢(H_(2)S)对高盐诱导的高血压大鼠Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核转录因子κB(NF-κB)信号通路的影响,探讨H_(2)S降压的作用机制。方法选择40只成年雄性Dahl盐敏感性大鼠,鼠龄8周,体质量260~280 g。随机把大鼠分成正常对照组(Control组)、正常给药组(Control+GYY组)、高盐模型组(Model组)、高盐给药组(Model+GYY组),每组10只。于第4周末,利用微型渗透泵向Control+GYY组和Model+GYY组大鼠双侧PVN区域持续注射H_(2)S的缓释供体GYY4137,其余组大鼠全部注射等量人工脑脊液。连续注射6周后进行取材,通过酶联免疫吸附分析(ELISA)法来检测PVN H_(2)S及血浆去甲肾上腺素(NE)的水平,免疫荧光法检测TLR4、MyD88的表达,免疫组化法检测磷酸化核转录因子κB(p-NF-κB p65)、磷酸化κB抑制蛋白激酶β(p-IKKβ)的表达,通过Western blot法检测TLR4、MyD88、p-NF-κB p65/NF-κB p65的蛋白表达。结果Model组平均动脉压(MAP)、血浆NE较Control组升高(t=10.204、24.155、23.967、25.657、22.069、31.036、23.054、28.189,P<0.05),PVN中H_(2)S水平降低(t=14.348,P<0.05),Model+GYY组与Model组相比MAP、NE水平降低(t=8.295、12.931、10.992、16.064、12.228、16.017,P<0.05),PVN中H_(2)S水平升高(t=7.889,P<0.05)。免疫荧光或免疫组化结果显示,Model组PVN中TLR4、MyD88、pNF-κB p65、p-IKKβ因子的表达水平较Control组均升高(t=8.844、10.344、6.813、3.909,P<0.05),Model+GYY组与Model组相比PVN中TLR4、MyD88、p-NF-κB p65、p-IKKβ因子的表达水平均降低(t=4.719、4.313、3.234、4.955,P<0.05)。Western blot结果表明,与Control组相比,Model组大鼠PVN中TLR4、MyD88、p-NF-κB p65/NF-κB p65蛋白表达水平升高(t=15.951、6.537、7.394,P<0.05);与Model组大鼠相比,Model+GYY组TLR4、MyD88、p-NF-κB p65/NF-κB p65蛋白表达水平下降(t=9.415、4.901、8.997,P<0.05)。结论室旁核H_(2)S可明显降低高盐诱导的高血压大鼠的血压、降低外周交感神经活动,其机制可能与抑制TLR4/MyD88/NF-κB信号通路有关。