The Mechanism Of Hmgbl In Nsclc And Ali

The incidence and mortality of Lung cancer continued to increase; some cytokines play a very important role in lung cancer. Studies have shown that, HMGB1has a very close relationship with non-small cell lung cancer characteristic such as development and metastasis.The high mobility group protein B1(high-mobility group box-Bl of HMGB1) is widely present in eukaryotic cells and it plays important role in biological processes. When HMGBl's expression levels increased, it can promote cell differentiation, migration and promote cell proliferation. HMGB1can also promote cell growth and gene transcription, the high mobility roup protein have very close relationship with tumor biological characteristics, such as tumorigenesis, invasion,1metastasis.High mobility group proteins has variety of biological activity corresponding receptor,the advanced glycation end products (RAGE) receptor is the most important receptor of HMGB1. HMGB1binding to RAGE, two major downstream signal transduction pathways are activated:the first pathway is that the guanosine triphosphatase (of Rac) and CDC42pathway is activated, resulting in corresponding biological effects, including the cell remodeling, motility, migration and tumor's growth, migration and proliferation; the second pathway is that the mitogen original activated protein kinase pathway is activated, nuclear factor-kappa B (of NFκB) is activated in this process, producing a large number of cytokines and chemokine, prompting immune cells to mature, also plays a very important role in immune receptor expression; addition, the third signal transduction pathway of the MAPK, P38, JNK and P42/P44may also be activated. This pathway can activate MMP-2and-9, they are downstream target in plasmin activation cascade.By degradation of extracellular matrix, it can promote tumor invasion.PurposeThis study shows that the expression of HMGB1in the non-small cell lung cancer is high, and the level of HMGB1has very close relationship with tumor clinical biological characteristics, to further explore and study the role of HMGB1in non-small cell lung cancer and pathogenesis.MethodApplication reverse transcriptase-polymerase chain reaction (RT-PCR) to test HMGBl mRNA expression in non-small cell lung cancer, application Western blot to test HMGB1protein level in non-small cell lung cancer, comparing HMGB1protein level in different stages of non-small cell lung cancer and different sizes, also comparing HMGBl protein levels in patients before and after surgery.Result1. Expression of HMGB1mRNA in human lung cancer A549cell line and bronchial epithelial cell line HBEBronchial epithelial cell line HBE as control cells, according to the formula2-averageAACT×100%of the target gene relative expression levels. The results show that HMGB1mRNA expression in lung adenocarcinoma A549cell line was significantly higher than that in bronchial epithelial cell line HBE.2. Expression of HMGB1protein level in human lung cancer A549cell line and bronchial epithelial cell line HBEHMGB1protein expression level in lung cancer cell lines A549were significantly higher than that in the bronchial epithelial cell line HBE.3. Serum HMGB1levels in patients with lung cancer and benign patientsThe mean value of serum HMGB1levels in145patients with lung cancer was76.1₃7.0ng/ml and was significantly higher than those in77COPD patients (39.8±10.8ng/ml), and49healthy control (7.7±6.1ng/ml, p<0.0001, respectively,) and the difference was also found between COPD patients and control (p<0.001).4. Comparison the serum HMGB1levels in patients with different tumor stage of NSCLCThe tumor stage was defined by the international staging system (TNM stage) for NSCLC to evaluate whether HMGB1was associated with lung cancer metastases to lymph nodes, distal organs, and tumor characteristics. The serum HMGB1levels were 30.2±5.9ng/ml,60.9±22.5ng/ml,99.0±23.1ng/ml and133.4±18.9ng/ml in patients with NSCLC of TNM stage â… ,â…¡, â…¢, and IV. There were significant differences among four groups (p<0.0001) and between two groupsMoreover, the serum HMGB1levels were notably increased in patients with distant metastasis (133.4±18.9ng/ml) than none distant metastasis (68.46±31.9ng/ml, p<0.0001).5. Comparison the serum HMGB1levels in patients with different size of lung cancerWe divided the tumors into three groups by their maximum size:<4cm and>4cm to compare the serum HMGB1levels in patients with different size of lung cancer. For the patients with<4cm and>4cm lung tumor lesions, the serum HMGB1levels were52.3±23.9ng/ml and105.3±28.3ng/ml (p<0.0001), and the significant positive correlation between the levels of serum HMGB1and the size of tumor (r=0.799,p<0.001).6. The serum HMGB1levels in patients with lung cancer before operation and one month after operationTo determine whether the serum HMGB1levels of preoperation patients were different from post-operation, we measured the serum HMGB1levels of the same operated patients before operation and one month after operation. The serum HMGB1levels were57.2±28.8ng/ml in patients with NSCLC before operation, and26.5±14.7ng/ml one month after operation (p<0.0001).ConclusionThis study shows that HMGBlplays a very important role in non-small cell lung cancer, it also shows that HMGB1expression level has very close relationship with tumor clinical and biological characteristics. HMGB1can also be tumor clinical marker. Acute lung injury (ALI) is the systemic inflammatory response which causes the damage in the lung. The symptoms of ALI are very severe, and can rapidly develop into ARDS and multiple organ failure. The mortality rate of ALI is very high, and its clinical manifestations are refractory hypoxemia and respiratory distress.The causes and mechanism of ALI is very complex, on the one hand, the balance between pro-inflammatory factors and anti-inflammatory factors plays a very important role in ALI, and on the other hand it also has relationship with environmental factors. In recent years, studies show that ALI has very close relationship with HMGB1, HMGB1is a late inflammatory mediator, plays an important role in the process of inflammation. HMGB1and inflammatory factors interact each other, inflammatory factors release HMGB1by stimulation factors, and HMGB1can also stimulate inflammatory factors, prompting inflammatory factors to release many inflammatory mediators.Ethyl pyruvate, as a chemical raw material, is an important food additive. Ethyl pyruvate is not only time-dependent anti-inflammatory agents but also dose-dependent anti-inflammatory agents, that means at different times and different doses will produce different results. Ethyl pyruvate can inhibit LPS-stimulated macrophages to release HMGB1.Ethyl pyruvate inhibit the inflammatory signal transduction pathway of NF-KB.PurposeThe purpose of this study is to explore the role and mechanism of HMGB1in acute lung injury which caused by lipopolysaccharide, and further study the efficacy of ethyl pyruvate at different times and different doses in acute lung injury, which may provide a new idea and method in treating acute lung injury.MethodTesting lung tissue HMGB1mRNA expression levels by reverse transcriptase-polymerase chain reaction (RT PCR); Testing HMGB1protein levels in lavage fluid by Western blotting; Testing tumor necrosis factor alpha (TNF-alpha),interleukin-1β (IL-1β) and white interleukin-6(IL-6) level by ELISA.Result1. Pathological changes in acute lung injury and EP treatment groupALI lung tissue has more inflammatory cells, some alveolar cavity with bloody exudate; EP treatment lung tissue in which the inflammatory cells in the alveolar space is significantly reduced.2. HMGB1protein level in mice bronchoalveolar lavage fluid in acute lung injury and EP treatment groupTo determine the levels of pro-inflammatory cytokines in BAL fluids, we flushed the airways with1.0ml PBS and collected the BAL fluids12h after ALI induction. As shown in Table1, levels of the cytokines HMGB1, TNF-IL-6and IL-1were substantially elevated in BAL fluids from LPS-induced ALI mice (versus controls; P <0.001). Treatment with EP (100mg/kg, i.p., immediately before intratracheal LPS instillation) significantly inhibited the release of HMGB1, TNF-, IL-6and IL-1into the BAL fluids of ALI mice (Table1), indicating that EP prevented LPS-induced ALI by attenuating the release of early (TNF-, IL-6, and IL-1) and late (HMGB1) systemic pro-inflammatory cytokines associated with lethality. EP reduced the permeability indices of the injured lungs.3. Different doses of ethyl pyruvate on mortalityTo evaluate the protective effect of EP against lethality in LPS-induced ALI mice, the mice received three different doses of EP (100,50and10mg/kg/d. i.p.) starting immediately before intratracheal LPS instillation. The results showed that the high EP dose (100mg/kg/d) protected against ALI lethality (survival in100mg/kg/d and50mg/kg/d of EP versus survival in vehicle-treated controls; P<0.0001and P=0.02respectively), but the effect was dose dependent; the low dose failed to protect significantly against death (survival in10mg/kg/d of EP versus survival in vehicle-treated controls; P=0.81)(Fig.2). Also, there was a dose-dependent reduction in the permeability index (100mg/kg/d and50mg/kg/d EP versus vehicle-treated control, P<0.0001;10mg/kg/d versus vehicle-treated control, P=1.00;4. Different times of ethyl pyruvate on mortalityTo assess the therapeutic efficacy of early EP treatment against ALI, EP was administered at different time points (0,12,24and48h after induction of ALI) at the same single dose of100mg/kg/d, i.p. The results showed that early administration of high dose EP (0,12or24h) significantly increased survival rate in the mice (survival after administration EP at0,12and24h versus survival in vehicle-treated controls; P <0.0001, P<0.0001and P=0.01, respectively)(Fig.4). However, no survival advantage occurred when EP was initiated at48h (survival after EP administration at48h versus survival in vehicletreated controls; P=0.75)(Fig.4). Moreover, the animals treated with EP beginning at0,12, and24h were significantly more active and alert and fed more rapidly than either the animals with treatment beginning at48h or controls treated with vehicle.ConclusionThis study shows that ethyl pyruvate can significantly inhibit the early cytokines such as TNF-alpha, IL-1β, IL-6and late cytokine HMGB1to be released, decrease animal mortality and systemic response syndrome in ALI, early high-dose application of ethyl pyruvate is more effective in the treatment of ALI.