Effect Of HMGB-1 On The Expression Of TDAG8 In Hypoxic Pulmonary Hypertension And Its Mechanism

Objective:The effects of HMGB-1 on the expression of TDAG8 and the regulation of inflammatory response in hypoxic pulmonary hypertension(HPH)mice were studied by lentivirus interference technique,and the related signaling pathways were elucidated.Methods:Eighteen SD mice were randomly divided into normal group(NC group),hypoxic group(HPH group),HMGB-1 gene interference group(HPH+HMGB-1~-group).The hypoxic pulmonary hypertension mice model was established by hypobaric hypoxia in HPH group.The specific shRNA lentivirus in tail vein of mice in HPH+HMGB-1~-group was interfered by hypoxic model D10 and d20,resPectively.After 28 days of successful modeling,blood and tissue sAMPles were collected.Establishment of organ-level evaluation model and the effect of lentivirus interference:1.Assessment of right heart hypertrophy index and pathological section to evaluate pulmonary artery remodeling;2.In vivo fluorescence tracing and tissue frozen section to verify the injection of HMGB-1 specific shRNA lentivirus into tail vein of mice.Cell level:To detect the effect of lentivirus-mediated shRNA interference on the expression of HMGB-1 in mouse peritoneal macrophages RAW264.7.Gene level:1.RT-PCR was used to detect the expression of HMGB-1 in lung tissue of model mice;2.Western-blot was used to detect the expression of HMGB-1,TDAG8 and ERK phosphorylation.Molecular level:ELISA was used to detect the expression of HMGB-1,TLR4,NF-KB,IL-6,IL-1beta,HIF-1 in alveolar lavage fluid and the cumulative expression of cAMP.Variance analysis was used to test the differences between groups,and pearson analysis was used to analyze the correlation.Results:1.The establishment of hypoxic pulmonary hypertension model in mice was successful:(1)After 28 days,the average weight of mice in HPH group was significantly lower than that in NC group,P=0.0001;the HPH+HMGB-1~-group was lower than that in NC group,P=0.001;the HPH+HMGB-1~-group was lower than that in HPH group,P=0.01;the right ventricular hypertrophy index(RVHI)had statistical significance among the three groups,P<0.05;and(2)pathological section showed that the pulmonary artery lumen in NC group was larger than that in HPH group.The pulmonary artery wall of rats was normal,the pulmonary artery wall thickening lumen of HPH group was severely narrowed comPared with NC group,P=0.0001;the lumen of HPH+HMGB-1-group was narrower than that of NC group,P=0.001;the pulmonary artery wall thickening and lumen of HPH group was significantly narrower than that of HPH+HMGB-1-group,P=0.01.2.The exPression of HPH inflammatory factors was up-regulated and HMGB-1 signal transduction pathway was activated:HMGB-1 gene and HMGB-1 protein in lung tissue and HMGB-1 expression in alveolar lavage fluid were higher in HPH group than in NC group,P<0.05,indicating that HMGB-1 expression in HPH group was higher than in NC group;TLR4,NF-KB,IL-1b,IL-6expression in alveolar lavage fluid was higher in HPH group than in NC group,P<0.05;Pearson correlation analysis indicated that HMGB-1 expression in HPH group was higher than in NC group,P<0.05.Molecular level was positively correlated with gene and protein level,P<0.05;pearson correlation analysis showed that HMGB-1 was positively correlated with TLR4,NF-KB,IL-1beta and IL-6,indicating that the expression of HMGB-1 was increased in HPH,the expression of inflammatory factors IL-1beta and IL-6 was up-regulated,and TLR4/NF-kB signaling pathway was activated.3.Intravenous injection of HMGB-1specific shRNA lentivirus significantly inhibited the expression of HMGB-1 in lung tissue,down-regulated inflammatory factors and alleviated inflammation:1.Macrophage RAW264.7verified the interference effect of HMGB-1 shRNA lentivirus:After transfection of HMGB-1shRNA lentivirus by PCR,the expression of HMGB-1 gene was significantly decreased,P<0.05.(2)Both in vivo fluorescence tracing and tissue frozen section can verify the success of tail vein injection of HMGB-1 specific shRNA lentivirus in mice.(3)The expression of HMGB-1 in lung tissue,HMGB-1 protein and alveolar lavage fluid was lower in HPH+HMGB-1~-group than in HPH group(P<0.05).pearson correlation analysis showed that the expression level of HMGB-1 in HPH+HMGB-1-group was positively correlated with gene and protein levels,P<0.05,with statistical significance.The expression of TLR4,NF-KB,IL-1b and IL-6 in alveolar lavage fluid in HPH+HMGB-1~-group was lower than that in HPH group(P<0.05).4.The expression of HIF-1 in HPH was increased:the expression of HIF-1 in alveolar lavage fluid in HPH group was significantly higher than that in NC group(t=6.53,P=0.0001);the expression of HIF-1 in HPH group was significantly higher than that in HPH+HMGB-1~-group(t=3.77,P=0.001).Linear correlation analysis indicated that there was a positive correlation between HIF and HMGB-1 expression.5.Increased expression of HIF-1 in HPH leads to microacid environment and activation of TDAG8/cAMP expression:1.The expression of TDAG8 protein in lung tissue of mice in HPH group was higher than that in NC group,P<0.05;2.The accumulation of cAMP in alveolar lavage fluid was increased by ELISA method:the expression of TDAG8 protein in HPH group was significantly higher than that in NC group(t=8.54,P=0.0001),with statistical significance.Pearson correlation analysis showed that TDAG8 was positively correlated with cAMP,and HIF-1 was positively correlated with TDAG8 and cAMP ex Pression.6.In HMGB-1-mediated HPH inflammation,the expression of HIF-1 increased,lactic acid accumulated and acidic environment formed.The activation of TDAG8/cAMP Pathway Predicted that HMGB-1 could Positively regulate the expression of TDAG8 in HPH.The expression of TDAG8 and cAMP in HPH grouP was higher than that in NC group,P<0.05,with significant difference;the expression of TDAG8and cAMP in HPH+HMGB-1-group was lower than that in HPH group,P<0.05,with significant difference.pearson correlation analysis showed that HMGB-1 was Positively correlated with the expession of TDAG8 and cAMP.Conclusion:1.The establishment of hypoxic pulmonary hypertension model in mice was successful;2.The expression of HMGB-1 in HPH was significantly increased,and inflammatory factors(IL-1,IL-6)were up-regulated,activating TLR4/NF-kB signaling pathway.HMGB-1 was positively correlated with TLR4,NF-kB,IL-1 and IL-6,indicating that HMGB-1 mediated inflammatory response in HPH;3.The expression of HIF-1 in HPH was increased,and the expression of TDAG8 and cAMP was positively correlated with TDAG8 and cAMP.The results showed that HIF-1 activated TDAG8/cAMP signaling pathway;4.After HMGB-1 gene interference,the expression of inflammatory factors was down-regulated.pathological results showed that the degree of pulmonary artery stenosis in HPH+HMGB-1~-group was significantly reduced in hypoxic group,indicating that the inflammatory reaction of HPH was alleviated;5.The expression of TDAG8 and cAMP was increased in HPH group,and the role of TDAG8 in negative regulation of inflammatory factors was enhanced.The expression of TDAG8 and cAMP decreased in HPH+HMGB-1-1group,and the signal transduction of HMGB-1 group.This study predicted that HMGB-1could regulate the expression of TDAG8 by positive feedback.