| Objective: The hepatopulmonary syndrome (HPS) is characterized by a defect in arterial oxygenation induced by pulmonary vascular dilatation in the setting of liver diseased or portal hypertension, accompanying with respiratory dysfunction.The basal pathological alteration is intrapulmonary vasodilatation, which is often related to the nitric oxide (NO) overproduction.NO is an important mediator for intrapulmonary vasodilatation. It could stimulate the hyperplasia endothelialis and peripherad growth of micrangium, which lead to widened alveolar septum and diminished alveolar volume.The end-results are descending of partial pressure of oxygen in artery (PaO2) and widened of alveolar-arterial PO2 difference (P(A-a)O2). NO is produced by NO synthases (NOS) with the substrate of L-arginine. Now, it is considered that endothelin (ET) could bind to ETB receptor in endodermis, which leading to endothelial NOS (eNOS) activation, and following NO production. Unlike eNOS, inducible NOS (iNOS) activation dues to pulmonary macrophage activation or aggregation may increase NO production.Phosphatidylinositol 3 kinase (PI3K) family is characterized by catalyzing the phosphorylation of phosphatidylinositol (PI) at the 3-OH position of the inositol phospholipids, and producing phosphatidylinositoltriphosphate (PIP3) that is the intracellular second messenger. This enzyme is composed of two subunits: an 85-kilodalton regulatory subunit and a 110-kilodalton catalytic subunit.The p85 subunit includes two SH2 domains and one SH3 domain. Having been activated by growth factor receptors, SH2 domains would directly bind to phosphotyrosine residues of growth factor receptors or adaptors, and then,the p85-p110 complex is brought to the membrane for next message transduction. Akt, also known as protein kinase B (PKB), which is identified as the human homologues for the viral oncogene v-akt, is a serine/threonine kinase downstream target of PI3K. To date, three members of this PKB family have been isolated, named Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ. Although they are products of different genes, they are closely related to each other, with up to 80% of amino acid homology. Nevertheless, their expression is different in distinct tissues. Activation of three isoforms is in a similar way that phosphorylation of two sites, one in the activation domain and another in the COOH-terminal hydrophobic motif, and both of them are necessary for full activity. Activated Akt could activate or restrain a series of substrates through phosphorylation, for example, NF-κB, Bad, caspase9 and GSK-3 and so on, to adjust cellular proliferation, differentiate, apoptosis and migration.Bacterium translocation (BT) leads to overproduction of TNF-α, the latter is an important factor in the development of HPS. It has been identified that the monocytes in circulating could stick to and accumulate in pulmonary vascular endodermis.Where they can differentiate into pulmonary intravascular cmacrophages (PIM), which could be activated by LPS. Activation of PIM could release a great of mediators of inflammation, for example, TNF-α, NO, IL-1, IL-6 and IL-8 etc. Especially, TNF-αcould induce local release of monocyte chemotactic factors and expression of macrophage and endothelium adhesion molecules. In addition, TNF-αcould activate NF-κB through PI3K/Akt signal pathway. NF-κB participates in the transcribing process of numerous inflammatory factors, including iNOS and TNF-αexpression by macrophage. So that iNOS induces NO overproduction and TNF-αand NF-κB that could activate each other in circles. Then the cascade reaction is triggered, which promotes the development of HPS.The prevalence of HPS in the setting of cirrhosis ranges between 10% - 20%, with prognosis mala. Owing to unknown pathogeny of HPS, there is still no effective medical therapy to reverse this condition. Liver transplantation is the only effective method among choices of treatment though mortality is comparatively high. So it is important to define its pathogeny, and look for effective therapeutic approach.For the past years, TNF-αMcAb has been used for treatment of Crohn's disease, rheumatoid arthritis and ankylosing spondylitis etc, and shows well effect. So we conclude that TNF-αMcAb should be effective on HPS through combining to TNF-α. Up to now, there is few people have done the similar study. Therefore, we carried out this study. The aim of the present study was to evaluate if TNF-αMcAb influences experimental HPS when administered after CBDL and to define the mechanisms about the development of HPS.Metheds: Male Sprague-Dawley rats, weighing 250±25g underwent sham operation (6 rats), CBDL (30 rats, divided into 5 subgroups: 1week,2week,3week,4week and 5week after ligation), CBDL+TNF-αMcAb (24 rats, divided into 4 subgroups: treating 1week, 2week, 3week, and 4week after 1 week of ligation). CBDL+TNF-αMcAb ( 0.1g/kg/2d ) was given by intraperitoneal injection for 1 week, 2week, 3week and 4 week , respectively, beginning 1 week after CBDL. At the same time, Rats in CBDL group were given NS for the same dose. Rat samples were gotten at different timepoints, as follow 1 wk, 2 wk, 3 wk, 4 wk, 5 wk after CBDL (Sham group were gotten at the fifth week). Hepatopulmonary syndrome was assessed by measurements of alveolar-arterial PO2 difference (P(A-a)O2). The level of NO and TNF-αwas measured. Liver histopathological changes were evaluated by hematoxylin and eosin staining and Masson's trichrome methods. Lung histopathological changes were evaluated by hematoxylin and eosin staining. Immunohistochemistry, Western Blot and RT-PCR were employed to investigate the expression and the change of PI3K, Akt and p-Akt in rats'lung.Results:1. Histopathological changes of liver and lung:1.1 Liver:①Control group: Hematoxylin and eosin staining showed that there were normal hepatic lobules, and hepatic plate to line in order. Masson showed that there was a little connective tissue in portal area.②CBDL group: Hematoxylin and eosin staining and Masson's trichrome methods showed that the rats of CBDL group were with extensive biliary ducts accrementition and dilation, even complete cirrhotic nodule;③TNF-αMcAb treatment group: Hematoxylin and eosin staining showed that there was also biliary ducts accrementition, but not significant difference compared with the sham group in liver. Masson showed that there was increasing collagen fiber in portal areas, but not complete cirrhotic nodule.1.2 Lung:①Control group: micrangiums of lung were eumorphism, alveolus wall and alveolus space were regular;②CBDL group: Hematoxylin and eosin staining showed that there was widened interalveolar septum, and obviously inflammatory reaction in lung interstitium. Macrophages were found. Capillary density increased, which showed that there was vasodilatation in lung tissue;③TNF-αMcAb treament group: Hematoxylin and eosin staining showed that there was a gentle vasodilatation and widened interalveolar septum.2. Hepatic function check:①CBDL group: ALT in plasma obviously increased after CBDL for 1 week(177.00±56.32 U/L). Compared with the control group(56.33±9.29 U/L), which showed that there existed obvious difference(P<0.05). Following, the level of ALT in plasma fell off, but it was notablely elevated compared with that of control group.②TNF-αMcAb treatment group: Having treated with TNF-αMcAb for 2week, the level of ALT in plasma decreased obviously compared with that of CBDL group (P<0.05). Following, the downtrend continued, up to 4 week (63.00±7.55), it was still higher than that of control group, but the difference was not significant (P>0.05) .3. Measurement of plasma TNF-α:①CBDL group: 1 week(1.60±0.07 ng/ml) after CBDL, the level of TNF-αin plasma increased significantly. Then, it kept the rising tendency. At the third week(3.18±0.54 ng/ml)after CBDL, it reached the peak. Between the third week and the fifth week after CBDL, it had a transient drop. Following, it returned to increase. But, it was at a higher level compared with that of control group (P<0.05).②TNF-αMcAb treament group: Compared with the CBDL group, the level of TNF-αin plasma decreased obviously after one week treatment. Then, the level of TNF-αkept downturn. Until the fourth week(1.21±0.13 ng/ml) after the CBDL, the level of TNF-αin plasma was also much lower than the CBDL group(2.43±0.43)(P<0.05),but compared with that of control group, there was no significant difference (P>0.05).4. The blood gas analysis:①CBDL group: two weeks(17.65±2.67 mmHg) after the CBDL, P(A-a)O2 began to increase. Compared with that of control group(6.70±3.20)mmHg, which showed that there existed obvious difference(P<0.05). Later, it kept increasing.②TNF-αMcAb treatment group: The P(A-a)O2 increasing of the treatment group was restrained after 2 weeks treatment, then it kept increasing. After treatment for 4 week(10.5±8.51 mmHg),to contrast that of CBDL group(51.8±8.49 mmHg),there was significant difference(P<0.05). Compared with that of control group, the level of the P(A-a)O2 was also higher , but no significant difference (P>0.05).5. Measurement of plasma NO:①CBDL group: One week after CBDL, the level of NO in plasma increased significantly. Then, it remained elevated tendency. It reached peak at the third week(95.67±15.41 umol/l) after CBDL. Then it had a transient drop. But, it was at a higher level compared with that of control group(36.19±1.59 umol/l)(P<0.05).②TNF-αMcAb: Compared with that of CBDL group, the level of TNF-αin plasma decreased obviously after one week treatment. Then, the level of TNF-αkept decreasing. Until the third week(45.47±9.58)and 4 week(44.42±9.27)after the CBDL, the level of NO in plasma was obviously lower than CBDL that of group at the same time( 72.22±6.11 umol/l , 64.31±1.75 umol/l ), and there was statistical significance. The level of NO in plasma was also higher than that of control group, but the difference was not significant (P>0.05).6. Measurement of the expression of PI3K in lung6.1 RT-PCR showed as follow:①CBDL group:The expression of PI3K mRNA peaked at the second week(1.75±0.15)after CBDL,and there was a transient descent at the third week(1.07±0.19),then a ascensus at fourth week (1.26±0.16)and another descent at the fifth week(0.98±0.11).Compared with the expression of PI3K mRNA of sham group(0.28±0.08),there was significant difference.②TNF-αMcAb treatment group: Compaerd with CBDL group, the expression of PI3K mRNA was obviously depressed at the second week (0.34±0.14).Then, the level of the PI3K mRNA kept ascending, but it's aways lower than that of sham group, and the difference was significant(P<0.05).6.2 Immunohistochemistry showed as follow:①CBDL group: The positive protein expression of PI3K was higher than that in the control group, the cell kytoplasm is brown, and the positive expression localized in the kytoplasm of phagocyte.The second week(0.31±0.10), expression of PI3K protein was up to peak,which was higher than that of control group(0.06±0.01)(P<0.05). Next, it had a temporary descent.Then, it returned to increase. Compared with that of control group, there was significant difference (P<0.05).②TNF-αMcAb treatment group:After 1 week treament ( 0.15±0.03, the protein expression of PI3K was obviously restrained, compared with that of CBDL group, there was significant difference (P<0.05). Following, it kept on this level. Compared with that of control group, there was significant difference (P<0.05).6.2 Western blot showed as follow: The result was similar with IHC.7 Measurement of the expression of Akt and p-Akt in lung7.1 RT-PCR of Akt showed as follow:①CBDL group: Expression of Akt mRNA showed gradually elevated tendency. At the third week, it reached peak (2.07±0.18). Next, it kept descending. But compared with sham group (0.58±0.06), the expression of Akt mRNA was higher. And the difference was significant.②TNF-αMcAb treatment group: The tendency of the expression of Akt mRNA was similar with that of CBDL group, but level of the expression of Akt mRNA was obviously depressed, and there was significant difference (P<0.05). At the third week, the difference was not significant (P>0.05).But compared with that of sham group, there was significant difference (P<0.05).7.2 Immunohistochemistry of p-Akt showed as follow:①CBDL group: The positive protein expression of p-Akt was higher than that in the control group, the cell kytoplasm is brown, and the positive expression localized in the kytoplasm of phagocyte. After CBDL, protein expression of p-Akt was uo to peak on the fourth weeek(0.31±0.03. Compared with that of control group(0.09±0.03, there was significant difference (P<0.05).②TNF-αMcAb treatment group: compared with that CBDL group, the expression of p-Akt decreased. After treatment for 4 weeks(0.20±0.03, it was much lower than CBDL group(P<0.05), but still higher than that of control group(P<0.05).7.3 Western blot of Akt showed as follow:①CBDL group: After CBDL, the expression of Akt kept increasing. At the third week(2.45±0.23, it was up to peak. Following, its expression was significantly restrained, but there was significant difference between it and the control group(1.08±0.20)(P<0.05).②TNF-αMcAb treatment group: For 1 week treatment, compared with that of CBDL group, the expression of Akt was visiblely inhibited. Following, it kept on this level. Compared with that of control group, there was significant difference (P<0.05).7.4 Western blot of p-Akt showed as follow: The result was similar with IHC.8 Analysis of correlation of TNF-αand NO in plasmaCoefficient correlation was 0.719, and probability value of coefficient correlation was 0.005<0.05, there was statistical significance, which showed correlativity between TNF-αand NO in plasma of rats with HPS. The direct correlation indicated that NO would increase along with TNF-α.Conclusions:1 Liver function changes were the foundation of the lung function changes in the forming process of HPS;2 TNF-αmay probably be an important essential cause of the development of HPS;3 Development of HPS accompanies by increasing expression of PI3K and Akt protein. Activation of Akt increasing showes activation of PI3K/Akt signalling pathway, which suggested that PI3K/Akt was concerned with HPS happenning; 4 TNF-αMcAb may combine with TNF-αin the plasma, and decrease the expression of TNF-α, partly inhibited expression and activation of PI3K and Akt protein, and the release of NO, then improve HPS.
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