Study On The Role Of Lactic Acid And TDAG8 In HPH

Objective:By observing the changes in the expression of lactic acid,cytokines,TDAG8 and HIF-1α in clinical patients and rats,we can understand the role of lactic acid and TDAG8 in HPH.Methods:1.Clinical part:Select 10 patients with hypoxic pulmonary hypertension who were diagnosed in the Department of Respiratory and Critical Care Medicine,the Third Clinical College of Inner Mongolia Medical University from April 2019 to December 2019 as the HPH group,and 10 patients with healthy physical examination during the same period as the NC group.PCR was performed to detect the expression of TDAG8,HIF-1α,PDK1,NDRG3,and CD206 m RNA in peripheral blood,and the expression of PDGF and VEGF was detected by Elisa method.2.The animal part:SD rats were randomly divided into normal group(NC group),hypoxia group(HPH group),and TDAG8 gene interference group(HPH+TDAG8-group)with 6 each.In HPH group,hypoxic pulmonary arteries were established by normal pressure and hypoxia for 21 days High-pressure rat model,the HPH+TDAG8-group was injected with TDAG8 sh RNA lentivirus into the tail vein of rats 1 day before HPH modeling,7days,and 14 days to achieve gene interference.After 21 days of successful modeling,keep them Blood and heart and lung tissue specimens.Evaluation of model establishment and lentivirus interference effect at organ level(1)Evaluation of right heart hypertrophy index and pathological section to evaluate pulmonary artery remodeling;(2)In vivo fluorescence tracing method to verify rat tail vein TDAG8 sh RNA lentivirus injection into rats The situation in the body.At the cell level,the expression effect of lentivirus-mediated sh RNA interference on TDAG8 rat alveolar macrophages was detected.Real-time PCR was used to detect the expression of TDAG8,HIF-1α,PDK1,NDRG3 and CD206 m RNA in the peripheral blood of model rats.Western Blot method was used to detect the protein expression of TDAG8,HIF-1α,PDK1,NDRG3 and CD206 in the peripheral blood of model rats.ELisa method was used to detect the expression of PDGF and VEGF in rat peripheral blood serum and the content of lactic acid in rat lung tissue homogenate.The cell level was used to stimulate pulmonary vascular smooth muscle cells through the serum of model rats,and the cell proliferation experiment was carried out by MTT method to reflect the proliferation of PASMCs by measuring the OD value.Results :1.Clinical part:(1)Comparison of general data of patients with hypoxic pulmonary hypertension and healthy physical examinations.There is no difference in age between HPH group and NC group(t=2.01,P>0.05);FEV1% of HPH group is lower than that of NC group(t=6.92,P< 0.0001);FEV1%FVC in HPH group was lower than that in NC group(t=7.32,P<0.0001);PASP in HPH group was lower than that in NC group(t=7.1,P<0.0001).(2)The expression of TDAG8,HIF-1α,PDK1,NDRG3,CD206,PDGF,and VEGF m RNA in peripheral blood detected by PCR was significantly higher in the HPH group than in the NC group(t=6.253,6.687,4.945,5.452,6.098 respectively,P <0.001);The expression of PDGF and VEGF in the peripheral blood detected by Elisa method was significantly higher in the HPH group than in the NC group(t=6.5,5.3,P<0.0001),and the difference was statistically significant.(3)In the HPH group,TDAG8 was positively correlated with HIF-1α,PDK1,NDRG3,CD206,PDGF,and VEGF(r values were 0.764,0.747,0.763,0.703,respectively,P values were all <0.05);HIF-1α and PDK1 NDRG3,CD206 are positively correlated(r values are 0.694,0.734,0.762,respectively,P values are less than 0.05);NDRG3 is positively correlated with CD206,PDGF,and VEGF(r values are 0.753,0.763,0.741,respectively,P values are less than 0.05).(4)ROC curve: The AUC of TDAG8,HIF-1α,PDK1,NDRG3,CD206,PDGF,and VEGF are 0.96,0.97,0.95,0.93,0.96,0.98 and 0.97,respectively.The above factors predict hypoxic pulmonary hypertension.Value,PDGF has the largest AUC,so PDGF has better diagnostic value.2.The animal part(1)The hypoxic pulmonary hypertension rat model was successfully established:(1)The average weight of the rats after 21 days: the HPH group was 212±1.45,which was significantly higher than the NC group(t=6.435,P<0.0001);the HPH+TDAG8-group was 191.9 ±5.938,which was significantly higher than that of the NC group(t=8.996,P=0.0001);the HPH+TDAG8-group was significantly less than that of the HPH group(t=6.52,P=0.0001),both of which were different;(2)Right ventricular hypertrophy index(RVHI): The HPH group was0.440±0.0178,which was significantly higher than the NC group(t=7.051,P<0.0001);the HPH+TDAG8-group was 0.409±0.0150,which was significantly higher than the NC group(t=4.265,P<0.01);HPH The +TDAG8-group was significantly reduced compared with the HPH group(t=3.184,P<0.05);(3)In vivo fluorescence tracing method verified the success of TDAG8-specific sh RNA lentivirus injection in the tail vein of rats.(2)PCR detection of TDAG8,HIF-1α,PDK1,NDRG3,and CD206 m RNA in the peripheral blood of rats was significantly higher in the HPH group than in the NC group(t=10.1,11.4,9.25,10.68,12.96,respectively,P<0.0001);Compared with the NC group,the HPH+TDAG8-group was significantly higher(t=10.1,11.4,9.25,10.68,12.96,respectively,P<0.05);in the HPH+TDAG8-group,it was significantly lower than the HPH group(t=4,2.58,3.13,3.477,5.181,P <0.05);and there are significant differences between the three groups(F are 30.7,31.1,23.4,31.29,59.09,P <0.0001).The PDGF and VEGF in the peripheral blood of rats detected by ELisa method were significantly higher in the HPH group than in the NC group(t=7.49,11.8,P<0.0001);in the HPH+TDAG8-group than the NC group was significantly higher(t=5.22,5.9,P<0.01);in the HPH+TDAG8-group,it was significantly lower than the HPH group(t=3.9,6.53,P<0.01),and there were differences among the three groups(F were 37.1,74.4,respectively,P<0.0001).TDAG8,HIF-1α,PDK1,NDRG3,and CD206 proteins in the peripheral blood of rats by Western Blot method were significantly higher in the HPH group than in the NC group(t=9.04,15.1,7.76,7.22,9.76,respectively,P<0.01);in HPH The +TDAG8-group was significantly higher than the NC group(t=8.77,10.9,14.3,9.25,7.6,all P<0.001);the HPH+TDAG8-group was significantly lower than the HPH group(t=2.47,5.98,2.5,4.86,3.48,P <0.05),there are differences among the three groups(F are 43.2,114,41.6,39.4,47.5,respectively,P <0.001).The lactic acid content of rat lung homogenate detected by Elisa method was significantly higher in the HPH group than in the NC group(t=7.86,P<0.0001),and was significantly higher in the HPH+TDAG8-group than in the NC group(t=6,P< 0.001),the HPH+TDAG8-group was significantly lower than the HPH group(t=4.5,P<0.01),and there was a difference(F=41.5,P<0.0001).MTT method detected rat serum to stimulate rat pulmonary vascular smooth muscle cell proliferation in the HPH group was significantly higher than that in the NC group(t=6.13,P<0.01),and in the HPH+TDAG8-group was significantly higher than that in the NC group(t=2.46,P<0.05),the HPH+TDAG8-group was significantly lower than the HPH group(t=4.23,P<0.01),and there was a difference between the three groups(F=23.6,P=0.0005).Correlation analysis showed that in the HPH group,TDAG8 was positively correlated with HIF-1α,PDK1,NDRG3,and CD206(r values were 0.899,0.915,0.899,0.890,all P<0.05),and HIF-1α was correlated with PDK1,NDRG3,and CD206 There was a positive correlation(r values were 0.916,0.899,0.891,respectively,P<0.05),and NDRG3 was positively correlated with CD206,PDGF,and VEGF(r values were0.847,0.852,0.830,respectively,P<0.05).Conclusion:1.Clinical studies have shown that the levels of TDAG8,HIF-1α,PDK1,NDRG3,CD206,PDGF,and VEGF in HPH patients are significantly increased,and TDAG8 is positively correlated with HIF-1α,PDK1,NDRG3,and CD206,and HIF-1α is positively correlated with PDK1,NDRG3,and CD206 It is positively correlated,and NDRG3 is positively correlated with CD206,PDGF,and VEGF.ROC analysis showed that PDGF has the most diagnostic value for hypoxic pulmonary hypertension.2.In HPH model rats,TDAG8,HIF-1α,PDK1,NDRG3,CD206 gene and protein expression levels are also significantly increased,and molecular level PDGF and VEGF expression levels are also significantly increased.Correlation analysis shows that TDAG8 and HIF-1α,PDK1,NDRG3,CD206 are positively correlated,HIF-1α is positively correlated with PDK1,NDRG3,CD206,NDRG3 is positively correlated with CD206,PDGF,VEGF,and CD206 is positively correlated with PDGF,VEGF,which is consistent with the results of HPH patients.Using lentiviral gene interference technology to interfere with the expression of TDAG8 in HPH model rats can partially reduce the expression of HIF-1α,PDK1,NDRG3,CD206,PDGF,and VEGF.The correlation analysis did not change significantly.4.Lactic acid and TDAG8 have a regulatory effect in HPH,and the mechanism may involve the activation of TDAG8/HIF-1α/PDK1/lactic acid signaling pathway,which ultimately leads to enhanced glycolysis,increased lactic acid production,and lactation of NDRG3,independent of HIF-1α Stable expression promotes the polarization of macrophages to M2 type,increases the secretion of cytokines such as PDGF and VEGF,induces the proliferation of pulmonary vascular smooth muscle cells,and promotes the process of pulmonary vascular remodeling.