简介:AbstractObjective:The role of Vitamin D-binding protein (DBP) in preeclampsia (PE) pathogenesis is unknown. In this study, we compared the expression of DBP in the placentas of PE patients with the placentas of normotensive pregnant women with placenta previa controls, and aimed to explore the effect of DBP on endothelial cells (ECs) and the underlying mechanism.Methods:DBP expression in placental tissues collected from PE patients and controls was evaluated by immunohistochemistry. The downregulation and upregulation of DBP expression in HTR-8/SVneo cells were examined using DBP-targeting small interfering RNA (siRNA) and DBP-expression vector, respectively. The conditioned media of these DBP-overexpressing and DBP-siRNA HTR-8/SVneo cells were collected and added to human umbilical vein EC (HUVEC) cultures. Angiogenic effects on HUVECs were assessed by tube formation assays, and the proliferation and migration of HUVECs were examined using the Real-Time Cell Analyzer. The expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR)-2, as well as the phosphorylation of different residues of VEGFR-2 in HUVECs, were determined by western blotting.Results:DBP expression was significantly increased in the placental tissues collected from PE patients. The conditioned medium of DBP-overexpressing HTR-8/SVneo cells potently inhibited tube formation by HUVECs, in addition to their proliferation and migration. Furthermore, treatment of HUVECs with the conditioned medium of DBP-overexpressing HTR-8/SVneo cells decreased the phosphorylation of VEGFR-2 at tyrosine 996, whereas the treatment of these cells with the conditioned medium of DBP-siRNA HTR-8/SVneo cells increased the phosphorylation of VEGFR-2 at tyrosine 951, 996, and 1,175.Conclusions:The expression of DBP is increased in the placentas of PE patients. DBP plays potential roles in endothelial dysfunction, which contributes to PE development, by inhibiting tyrosine phosphorylation of VEGFR-2 in ECs.
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简介:Objective:Toinvestigatethebiologicalfunctionofplatelet-derivedgrowthfactorB(PDGF-B)onthesurvivalandproliferationofcatcornealendothelialcellssoastoprovidebasesforfurtherstudiesofitsroleinwoundrepairanditsclinicalapplication.Methods:TotalRNAwasextractedfromtheplacentatissuesofhealthypregnantwomenundergoinghysterotokotomyandPDGFeDNAwasobtainedwithre-versetranscription-polymerasechainreaction(RT-PCR).TheprokaryoticexpressionvectorpET-PDGF-BwasconstructedandexpressedtherecombinantPDGF-BinEscherichiacoli(E.coli)BL21(DE3).AfterpurificationandrefoldingonNi2+-chelationaffinitychromatography(NTA)column,itwasusedtoculturecatcornealendothelialcells.Cellproliferationwastestedbymodifiedtertrazoliumsalt(MTT)andflowcytometer.Andthemorphologicchangeandtheultrastructurewereob-servedunderaninvertedphasecontrastmicroscope,ascan-ningelectronmicroscopeandatransmissionelectonmicroscope,respectively.Results:PDGF-Bchainpeptide(PDGF-BB)genewassuccessfullyinsertedintotheprokaryoticexpressionvector,pET-28a(+).ThepurifiedrecombinedproteinpET-PDGF-Bshowedasinglebandonsodiumdodecylsulfatepolyacry-lamidegelelectropheresis(SDS-PAGE)withthemolecularweightofabout27u,whichwasinagreementwiththede-ducedvalue.MTTandflowcytometryshowedthatPDGF-BBpromotedthesurvivalandproliferationofcatcornealen-dothelialcells.Conclusions:Theconstructionofrecombinantprokary-oticexpressionvectorpET-PDGF-BandthepreparationofPDGF-BBproteinprovideafoundationforfurtherstudyofthefunctionofPDGF-BBandproducingbiologicalPDGF-BBprotein.TheexpressedPDGF-BBpromotestheprolif-erationofculturedcatcornealendothelialcells.
简介:瞄准:在微容器密度(MVD)和脉管的内皮生长的表情调查差别,并且在MVD之中探索关联在前列腺癌症(PCa)之间的因素(VEGF),VEGF-C和VEGFreceptor-3(VEGFR-3)纸巾和邻近的良性的纸巾,Jewett-Whitmore阶段,格利森分数和在PCa的前进的VEGF,VEGF-C和VEGFR-3的表情。方法:免疫组织化学的途径被采用在癌症区域和71个主要职业人员静电干扰腺癌标本的外部良性的区域检测CD34,VEGF,VEGF-C和VEGFR-3的表情。统计分析然后被执行根据试验性并且诊所数据。结果:都显著地与邻近的良性的上皮(P<0.01)相比在恶意的上皮/癌症房间在VEGF,VEGF-C和VEGFR-3的调整表情上面被发现。当在肿瘤区域(P<0.01)比较VEGF-C或VEGFR-3的表示时,在阶段D的病人在阶段A,B或C比病人有一个显著地更高的分数。另外,重要关联在Jewett-Whitmore阶段和VEGF-C之间被观察(r=0.738,P<0.01),临床的阶段和VEGFR-3(r=0.410,P<0.01),VEGF-C和格利森分数(r=0.401,P<0.01),VEGFR-3和格利森分数(r=0.581,P<0.001)并且MVD和VEGF(r=0.492,P<0.001)。结论:VEGF和VEGF-C的增加的表情仔细与PCa的前进被联系。为PCa前进的增加的VEGF表示的主要贡献到过起来调整MVD,它维持了肿瘤织物的生长优点。然而,VEGF-C和VEGFR-3的增加的表情的主要角色是提高lymphangiogenesis并且提供一条主要小径让癌症房间传播。
简介:Toclonethegenecodingtheimmunodominantregioninthechlamydialprotease-likeactivityfactor(CPAF)fromChlamydophilapneumoniae,toanalyzeimmunocompetenceoftheexpressedprotein,andtoevaluateitsvalueinserodiagnosis,theCPAFimmunodominantregiongenewasamplified,ligatedintoapGEX6p-2vector,andthentheexpressedrecombinantproteinwaspurifiedwithglutathioneS-transferase(GST)agarosegelFFafterrenaturation,thenidentifiedbySDS-PAGEandWesternblot.AnewindirectELISAwasdevelopedwiththepurifiedproteinascoatingantigen.TheimmunogenicityoftherecombinantproteinwasevaluatedbyimmunizationtoNewZealandrabbits,anditsimmunoreactivitywasanalyzedbyreactingwithanti-C,pneumoniaeantibody.300clinicalserasampleswererespectivelyde-tectedbymicroimmunofluorescence(MIF)asreferencemethodandtheindirectELISA,andthediffer-encebetweenthetwomethodswasanalyzed.Cross-reactivityagainstChlamydiatrachomatiswasinvesti-gatedwiththeindirectELISAtodetectanti-C,trachomatispositiveantisera.Theresultsindicatedthata51.3kDarecombinantproteinwasobtained.Westernblotassayprovedthattherecombinantproteincouldmerelyspecificallyreactwithhumananti-C.pneumoniaeantisera.ThetitersofthespecificIgGan-tibodiesintheimmunizedNewZealandrabbitswereabove1:16000.Anti-C.pneumoniaeIgGpositiveandnegativereferencesereweredetectedwiththeindirectELISA,andtheconcordancerateofnegativeandpositiveresultswereboth100%(40/40).ThesensitivityandspecificityoftheindirectELISAincomparisonwithMIFwere93.8%(45/48)and100%(252/252)separatelybydetecting300clinicalserasamples,andtheconcordanceratebetweenthetwomethodswas99.0%.NocrossreactionagainstC.trachomatiswasfoundwiththeindirectELISAtodetectanti-C,trachomatispositiveantisera.Incon-clusion,thepreparedrecombinantproteinoftheCPAFimmunodominantregionshowsexcellentimmuno-competenceandcanbeusedtodevelopanewindirect
简介:AbstractBackground:Vascular endothelial dysfunction is considered a key pathophysiologic process for the development of acute lung injury. In this study, we aimed at investigating the effects of unfractionated heparin (UFH) on the lipopolysaccharide (LPS)-induced changes of vascular endothelial-cadherin (VE-cadherin) and the potential underlying mechanisms.Methods:Male C57BL/6 J mice were randomized into three groups: vehicle, LPS, and LPS + UFH groups. Intraperitoneal injection of 30 mg/kg LPS was used to induce sepsis. Mice in the LPS + UFH group received subcutaneous injection of 8 U UFH 0.5 h before LPS injection. The lung tissue of the mice was collected for assessing lung injury by measuring the lung wet/dry (W/D) weight ratio and observing histological changes. Human pulmonary microvascular endothelial cells (HPMECs) were cultured and used to analyze the effects of UFH on LPS- or tumor necrosis factor-alpha (TNF-α)-induced vascular hyperpermeability, membrane expression of VE-cadherin, p120-catenin, and phosphorylated myosin light chain (p-MLC), and F-actin remodeling, and on the LPS-induced activation of the phosphatidylinositol-3 kinase (PI3K)/serine/threonine kinase (Akt)/nuclear factor kappa-B (NF-κB) signaling pathway.Results:In vivo, UFH pretreatment significantly attenuated LPS-induced pulmonary histopathological changes (neutrophil infiltration and erythrocyte effusion, alveolus pulmonis collapse, and thicker septum), decreased the lung W/D, and increased protein concentration (LPS vs. LPS + UFH: 0.57 ± 0.04 vs. 0.32 ± 0.04 mg/mL, P = 0.0092), total cell count (LPS vs. LPS + UFH: 9.57 ± 1.23 vs. 3.65 ± 0.78 × 105/mL, P= 0.0155), polymorphonuclear neutrophil percentage (LPS vs. LPS+ UFH: 88.05% ± 2.88% vs. 22.20% ± 3.92%, P = 0.0002), and TNF-α (460.33 ± 23.48 vs. 189.33 ± 14.19 pg/mL, P = 0.0006) in the bronchoalveolar lavage fluid. In vitro, UFH pre-treatment prevented the LPS-induced decrease in the membrane expression of VE-cadherin (LPS vs. LPS + UFH: 0.368 ± 0.044 vs. 0.716 ± 0.064, P = 0.0114) and p120-catenin (LPS vs. LPS + UFH: 0.208 ± 0.018 vs. 0.924 ± 0.092, P = 0.0016), and the LPS-induced increase in the expression of p-MLC (LPS vs. LPS + UFH: 0.972 ± 0.092 vs. 0.293 ± 0.025, P = 0.0021). Furthermore, UFH attenuated LPS- and TNF-α-induced hyperpermeability of HPMECs (LPS vs. LPS + UFH: 8.90 ± 0.66 vs. 15.84 ± 1.09 Ω·cm2, P = 0.0056; TNF-α vs. TNF-α + UFH: 11.28 ± 0.64 vs. 18.15 ± 0.98 Ω·cm2, P = 0.0042) and F-actin remodeling (LPS vs. LPS + UFH: 56.25 ± 1.51 vs. 39.70 ± 1.98, P = 0.0027; TNF-α vs. TNF-α + UFH: 55.42 ± 1.42 vs. 36.51 ± 1.20, P = 0.0005) in vitro. Additionally, UFH decreased the phosphorylation of Akt (LPS vs. LPS + UFH: 0.977 ± 0.081 vs. 0.466 ± 0.035, P = 0.0045) and I kappa B Kinase (IKK) (LPS vs. LPS + UFH: 1.023 ± 0.070 vs. 0.578 ± 0.044, P = 0.0060), and the nuclear translocation of NF-κB (LPS vs. LPS + UFH: 1.003 ± 0.077 vs. 0.503 ± 0.065, P = 0.0078) in HPMECs, which was similar to the effect of the PI3K inhibitor, wortmannin.Conclusions:The protective effect of UFH against LPS-induced pulmonary endothelial barrier dysfunction involves VE-cadherin stabilization and PI3K/Akt/NF-κB signaling.
简介:AbstractBackground:Both bone marrow mesenchymal stem cell (BM-MSC) and transforming growth factor-β1 (TGF-β1) have a strong anti-inflammatory capacity in stroke. But their relationship has not been well addressed. In this study, we investigated how intravenous BM-MSC transplantation in rats effected the expression of TGF-β1 48 h post cerebral ischemia, and we analyzed the main cells that produce TGF-β1.Methods:We used a distal middle cerebral artery occlusion (dMCAO) model in twenty Sprague-Dawley (SD) rats. The rats were randomly divided into two groups: the ischemic control group and the postischemic BM-MSC transplantation group. One hour after the dMCAO model was established, the rats were injected in the tail vein with either 1 ml saline or 1 × 106 BM-MSCs suspended in 1 ml saline. ELISAs were used to detect TGF-β1 content in the brain infarct core area, striatum and the plasma at 48 h after cerebral infarction. Immunofluorescent staining of brain tissue sections for TGF-β1, Iba-1, CD68 and NeuN was performed to determine the number and the proportion of double stained cells and to detect possible TGF-β1 producing cells in the brain tissue.Results:Forty-eight hours after ischemia, the TGF-β1 content in the infarcted area of the BM-MSC transplantation group (23.94 ± 4.48 pg/ml) was significantly lower than it was in the ischemic control group (34.18 ± 4.32 pg/ml) (F = 13.534, P = 0.006). The TGF-β1 content in the rat plasma in the BM-MSC transplantation group (75.91 ± 12.53 pg/ml) was significantly lower than it was in the ischemic control group (131.18 ± 16.07 pg/ml) (F = 36.779, P = 0.0002), suggesting that after transplantation of BM-MSCs, TGF-β1 levels in the plasma decreased, but there was no significant change in the striatum area. Immunofluorescence staining showed that the total number of nucleated cells (1037.67 ± 222.16 cells/mm2) in the infarcted area after transplantation was significantly higher than that in the ischemic control group (391.67 ± 69.50 cells/mm2) (F = 92.421, P < 0.01); the number of TGF-β1+ cells after transplantation (35.00 ± 13.66 cells/mm2) was significantly reduced in comparison to that in the ischemic control group (72.33 ± 32.08 cells/mm2) (F = 37.680, P < 0.01). The number of TGF-β1+/Iba-1+ microglia cells in the transplantation group (3.67 ± 3.17 cells/mm2) was significantly reduced in comparison to that of the ischemic control group (13.67 ± 5.52 cells/mm2) (F = 29.641, P < 0.01). The proportion of TGF-β1+/Iba-1+ microglia cells out of all Iba-1+ microglia cells after transplantation (4.38 ± 3.18%) was significantly decreased compared with that in the ischemic control group (12.81 ± 4.86%) (F = 28.125, P < 0.01).Conclusions:Iba-1+ microglia is one of the main cell types that express TGF-β1. Intravenous transplantation of BM-MSCs does not cooperate with TGF-β1+ cells in immune-regulation, but reduces the TGF-β1 content in the infarcted area and in the plasma at 48 h after cerebral infarction.
简介:AbstractBackground:The transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) has been proven associated with the pathogenesis of asthmatic airway remodeling, in which the Wnt/β-catenin pathway plays an important role, notably with regard to TGF-β1. Recent studies have shown that 1α, 25-dihydroxyvitamin D3(1α, 25(OH)2D3) inhibits TGF-β1-induced EMT, although the underlying mechanism have not yet been fully elucidated.Methods:Alveolar epithelial cells were exposed to 1α, 25(OH)2D3, ICG-001, or a combination of both, followed by stimulation with TGF-β1. The protein expression of E-cadherin, α-smooth muscle actin, fibronectin, and β-catenin was analyzed by western blotting and immunofluorescence analysis. The mRNA transcript of Snail was analyzed using RT-qPCR, and matrix metalloproteinase 9 (MMP-9) activity was analyzed by gelatin zymogram. The activity of the Wnt/β-catenin signaling pathway was analyzed using the Top/Fop flash reporters.Results:Both 1α, 25(OH)2D3 and ICG-001 blocked TGF-β1-induced EMT in alveolar epithelial cells. In addition, the Top/Fop Flash reporters showed that 1α, 25(OH)2D3 suppressed the activity of the Wnt/β-catenin pathway and reduced the expression of target genes, including MMP-9 and Snail, in synergy with ICG-001.Conclusion:1α, 25(OH)2D3 synergizes with ICG-001 and inhibits TGF-β1-induced EMT in alveolar epithelial cells by negatively regulating the Wnt/β-catenin signaling pathway.
简介:AbstractObjective:Pulmonary Mycobacterium tuberculosis infection can trigger cellular and humoral innate immune responses, which may cause death of the pathogen and or host cells/tissue. We aimed to determine the cytotoxic response of phagocytes in patients with pulmonary Mycobacterium tuberculosis infection based on plasma tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), and superoxide dismutase (SOD) levels.Methods:In this observational study, patients newly infected with pulmonary Mycobacterium tuberculosis (n=31; age 37-62 years) and age-matched uninfected volunteers (n=50) were recruited as test and control volunteers, respectively in Owo, Nigeria. The study protocol was reviewed and approved by the Research and Ethics Committee of the Department of Medical Laboratory Science, Achievers University, Owo, Nigeria (AUO/MLS/VII/2009/212). Anti-hepatitis C virus, human immunodeficiency virus antigen/antibody, hepatitis B virus surface antigen, and plasma TNF-α were determined by enzyme-linked immunosorbent assay, SOD, and MDA were determined by colorimetry, Plasmodium by Giemsa thick blood film staining, and acid-fast bacilli in sputum were detected by Ziehl-Neelsen staining.Results:All participants had normal blood glucose levels and tested negative for human immunodeficiency virus antigen/antibody, anti-hepatitis C virus, hepatitis B virus surface antigen, and Plasmodium spp., and had no medical history of cancer. Infected patients had significantly higher plasma MDA and TNF-α levels and significantly lower SOD levels compared with control subjects (all P<0.05).Conclusion:Mycobacterium tuberculosis infection elicited a cytotoxic response by phagocytes, evidenced by significant increases in MDA and TNF-α and a significant decrease in SOD levels.
简介:AbstractPurpose:Severe damage to the femoral head in patients with osteonecrosis has a high impact on morbidity. Despite early diagnosis, the treatment outcome is still unsatisfactory. This study aimed to explore the expression of vascular endothelial growth factor (VEGF) and cyclic guanine monophosphate (cGMP) serum level as the risk factors of femoral head osteonecrosis in alcohol-exposed Wistar rats.Methods:This was an experimental study using randomized post-test only control group design, with samples using 10-14 weeks Wistar male rats. Rats were then divided into 6 groups: 3 groups without intervention, and 3 groups with intervention using 40% alcohol given perorally. Each one group from intervention and control group was euthanized by the end of the week for 3 consecutive weeks. Proximal femurs were examined under microscope for osteonecrosis, immunohistochemically for VEGF, and blood serum for cGMP levels.Results:VEGF expression in the femoral head of alcohol-exposed Wistar rats was lower than those not exposed to alcohol (p < 0.005). Blood serum cGMP levels of alcohol-exposed Wistar rats were higher than those not exposed to alcohol (p < 0.005). The number of necrotic osteocytes in the femoral head of Wistar rats exposed to alcohol was greater than those not exposed to alcohol (p < 0.005). There are significant differences between VEGF, cGMP levels, and number of necrotic osteocytes in the control group and treatment at 1st, 2nd, and 3rd week (p < 0.005).Conclusions:Based on the result of this study, VEGF and cGMP may be considered as diagnostic biomarkers for alcohol-induced femoral head osteonecrosis.
简介:Objective:Toinvestigatetheexpressionandpatternofvascularendothelialgrowthfactor(VEGF)anditsfetalliverkinase-1(Flk-1)receptorinspinalcordanddorsalrootgangliaafterneurotomyofsciaticnerveinrats.Methods:Forty-fiveadultmaleWistarratsweredividedrandomlyintoacontrolgroup(n=5)andanexperimentalgroup(n=40).ThebilateralsciaticnervesoftheratsintheexperimentalgroupunderwentneurotomyandtheL4-L6spinalcordandthecorrespondingdorsalrootgangliawereharvestedrespectivelyat8hours,and1,3,5,7,10,14and21days(8subgroupswith5ratseach)afteroperation.Theratsinthecontrolgrouponlyunderwentanexposureofsciaticnervewithoutneurotomy.ImmunohistochemistryandimageanalysiswereusedtostudytheexpressionofVEGFanditsFlk-1receptor.Results:BothVEGFandFlk-1receptorexpressedinthenormalratspinalcordanddorsalrootganglia.Inresponsetoneurotomy,theirexpressionreachedahigherlevelandpersistedforashorttimethendeclinedtothenormallevelrapidly.Besides,positivestainingofFlk-1wasobservedinbothglialcellsandnervefibers,whichlocatedinthewhitematterofthespinalcord.Conclusions:VEGFcanpromotetheregenerationofperipheralnervesfromtheangleofcentralneurons,whichestablishestheexperimentalandtheoreticalfoundationforVEGFtreatingperipheralnerveinjuries.
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简介:摘要BackgroundDelayed encephalopathy (DE) is the most severe complication after acute carbon monoxide (CO) poisoning, which seriously affects the outcome of patients and leads to a high disability rate. Prior studies have shown that hyperbaric oxygen (HBO2) therapy is therapeutic for DE due to reducing immune-mediated neuropathology and thus improving cognitive performance.MethodsIn our present perspective study, five DE patients were treated regularly with HBO2 therapy. The mini-mental state examination (MMSE) and Barthel index (BI) were intermittently collected during their hospitalization for mental and physical status evaluation, the peripheral bloods were serially sampled to determine the concentration changes of circulating stem cells, as well as corresponding BDNF and neural markers.ResultsMMSE and BI showed series of improvements after multiple HBO2 therapies. The CD34+/CD90+ and CD34+/CD133+ dual positive cells, which were categorized as circulating stem cells, were observed an overall up-regulation since the beginning of the DE onset upon the application of HBO2 therapy. Characteristic neurotrophin BDNF, neural markers such as nestin and synaptophysin (SYP) were also up-regulated after exposure of HBO2. Conclusion The application of HBO2 therapy is of significance in improving the cognition of DE patients, along with mobilized circulating stem cells.ConclusionWe primarily infer that the CD34+/CD90+ and CD34+/CD133+ cells were mobilized by HBO2 exposure and have played a positive role in cognition improvement on DE patients by up-regulation of BDNF, nestin and SYP. The altering amount of circulating stem cells mobilized in peripheral blood could be a potential marker on predicting the outcome of DE.
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简介:AbstractBackground:Head and neck cancers (HNCs) are a heterogeneous group of tumors that progress owing to varied enviromental and genetic risk factors. Viral infections are threatening and adept at altering the expression of cellular transcription factors such as nuclear factor kappa B (NF-κB) and deregulation of other cellular proteins like NF kappa B inhibitor alpha (IκBα). The present study was conducted to detect high-risk genotypes of human papillomavirus (HPV) and protein expression of NF-κB signaling pathway in HNC patients with HPV infection.Methods:For HPV detection, genomic DNA from 152 HNC tumors was extracted formalin-fixed paraffin-embedded tissue DNA kit. For genotyping, polymerase chain reaction (PCR) using a general primer, HPV type-specific primers and agarose gel electrophoresis were performed. Immunohistochemistry (IHC) was also performed on 4-μm thick tissue sections using HPV E6 monoclonal antibody. Protein expression analysis of NF-κB signaling pathway including p50, p65, and IκBα was performed using IHC.Results:PCR analysis showed that 24.3% (37/152) of HNC cases were HPV positive. Among HPV positive, 86.5% (32/37) were tobacco users, while among HPV negative, 66.9% (77/115) were tobacco users. A significant association of HPV positivity and tobacco user was observed by univariate analysis [P < 0.01; odds ratio (OR): 0.310, 95% confidence interval (CI): 0.110 to 0.870]. More HPV positive patients were with poor oral hygiene (78.3%) when compared with patients with good oral hygiene (21.6%) [P < 0.03, OR: 2.440, 95% CI: 1.650 to 3.600]. The results of the logistic regression analysis showed that age, tobacco use and oral hygiene are significant predictors (P < 0.02). PCR and IHC staining results confirmed that HPV16 was predominant among HNC cases (64.8%) when compared with HPV18 (35.2%). Expression of NF-κB proteins (p50, p65, and IκBα inhibitor) were also observed in HPV and non-HPV infected HNC tissues. IHC expression of p50, and p65 showed nuclear staining, while IκBα inhibitor showed cytoplasmic staining. Protein expression in HPV cases was higher as compared to HPV naive cases (P < 0.05).Conclusions:From the study, it can be established that the use of tobacco, oral hygiene, and HPV infection may be synergistically involved in modulating the expression of NF-κB signaling pathway for the development and progression of HNC in the Pakistani population.