| BackgroundThe pathogenesis of PAH is not yet completely clear,however,pulmonary vascular remodeling is the basic pathophysiology of the occurrence and development of PAH,which causing PVR increased,right ventricular overload and right heart failure.According to the previous works,many factors were involved in the pathogenesis of PAH,including apoptosis,immune factors and inflammatory factors,in which inflammation captured people's attention in recent years.Infiltration of inflammatory cell,including T lymphocytes,B lymphocytes and macrophages,was detected firstly around the plexiform lesion of arterial wall in patients with PAH by Tuder et al,thus opening the door of correlation between PAH and inflammation for people.Afterwards,Humbert argued that serum levels of antinuclear antibody,IL-1 and IL-6 increased in patients with idiopathic PAH,and IL-1 receptor antagonists can reduce the symptoms of PAH rats established by monocrotaline(MCT).Studies have shown that high mobility group box protein 1(HMGB1)is a potent proinflammatory cytokines,significantly rised in serum of PAH patients,and administration of HMGB1 inhibitors can lower the mPAP of PAH rats,reduce inflammation,which indicating the relationship between inflammation and PAH.At present,many scholars turn their attention to mitigation of vascular remodeling and promot of angiogenesis in terms of pathophysiology of PAH,HGF was acclaimed as an obvious cytokine with pro-angiogenesis effect.The application of gene transfection and other molecular biology techniques confirmed that HGF indeed improved blood supply and function of ischemic necrosis of tissue in limb ischemia and animal models of myocardial infarction.Previous study demonstrated that transfection of HGF gene to MCT-PAH rats can reduce the PVR of PAH rats,decline mPAP,improve the PAH hemodynamics and right heart function.Study about the role of HGF in the regulation of inflammation of bone marrow derived macrophages,done by Coudriet et al,manifested that HGF can decrease the level of IL-6,up-regulate level of IL-10 known as a anti-inflammatory cytokine and increase phosphorylation and inactivation of glycogen synthase kinase 3-?(GSK3?),which is an important regulator of inflammation by increasing the release of inflammatory cytokines,besieds,IL-6 is one of the important factors associated with PAH closely.Giving an inhibitor of inflammatory cytokines(interleukin or HMGB1 inhibitor)to rat model of PAH can reduce serum levels of inflammatory factors,decline mPAP and right ventricular hypertrophy,which impling the feasibility of intervening PAH by anti-inflammation.Heat shock protein(HSP),mainly in lung,is a kind of stress protein with effect of self-protection generated under heat stress and regulating the synthesis of other proteins through anti-oxidation and anti-inflammatory,moreover,HSP70 is the main protein with protective effect in HSP family.Works confirmed that HSP70 was highly expressed in pulmonary artery smooth muscle cells with acute lung injury or hypoxic pulmonary hypertension,with effect of inhibiting a variety of inflammatory cytokines including IL-1?,IL-1,IL-6,TNF-?,ICAM-1,and so on,besides,it can also reduce the generation of oxygen free radicals,playing a important role in the protection of the body.Studies have demonstrated that the anti-inflammatory mechanism of HSP70 is related to the down-regulation of the expression of nuclear factor kappa B(NF-?B),which is is an important signaling pathways of inflammatory response,involving in the transcriptional regulation of many cytokines,including adhesion molecules and chemokines.Under normal conditions,NF-?B stably present a trimer in the cytoplasm binding to I?B?(inhibition protein of NF-?B),alternatively,I?B? inactivation or the amount decline caused by external factors can resulte in NF-?B transfer from the cytosol to the nucleus,exerting biological effects by activating multiple signaling pathways.Studies have shown that HSP70 can reduce phosphorylation of I?B?,thereby improving I?B? protein level,down regulating NF-?B level to extenuate the inflammation.Previous studies have emphasized that HGF may play a protective role in the apoptosis of hepatocytes(induced by TNF-a)by increasing the expression of HSP70,and whether a similar mechanism involved in the PAH is not reported.In this study,we observed the effect of HGF on PAH through the establishment of PAH rat model,futhermore,the effect of HSP70/NF-?B pathway on the mechanism of HGF intervening PAH by anti-inflammatory was investigated.Objective1.The effect of HGF on intervening PAH rats,and the changes of hemodynamics and right ventricular function of rats.2.Mechanism and signaling pathway of HGF to improve PAH by anti-inflammation.MethodsFourty male wistar rats were randomly divided into five groups(eight for each grop):1)Control group injected with normal saline(1mg/kg);2)PAH group PAH ras model was established by injecting monocrotaline(60mg/kg)intraperitoneally;3)HGF group HGF(1mg/kg)was injected by intravenous when PAH rat model was successfully established two weeks;4)HGF-Double Group A double dose(2mg/kg)of HGF was injected by intravenous after PAH rat model was successfully established two weeks.5)HGF+Quercetin group:HSP70 inhibitors(Quercetin,5mg/100g)was given to PAH rats 6 hours before HGF administration..1.General changes were observed in each group of rats,such as weight,shortness of breath,eating and death and so on.2.MPAP and PVR were measured in each group.Rats were anesthetized with chloral hydrate when observing to four weeks,A endotracheal tube was inserted to exposed trachea to maintain breathing with a ventilator,then,a pressure transducer connected to the tee and the right 3Frliller cardiac catheterization,which was filled with a certain concentration of heparin saline.The right cardiac catheter,which is connected to the pressure transducer,is placed to the pulmonary artery.The right ventricle and pulmonary artery pressure waveforms were recorded by the multi-hannel physiological recorder.3.RVHI were measured in each groupThoracotomy heart was removed and brine with ice saline,moreover,bilateral atrium and aorta root were cut off along the atrioventricular groove,while the right ventricular free walls were cut down along with ventricular septal,lastly,after water-absorbing filter paper,weighed the right ventricular free wall(RV)and left ventricle plus septum(LV+S),and calculated the index of right heart ventricular hypertrophy[RVHI=RV/(LV + S)].4.Right heart function was measured by EchocardiographyThe right heart function was observed at the forth week,and the rat was placed left 30 degrees,on the left sternal Die probe,and 10-30 angle of midline sternum.Tricuspid annular plane systolic excursion(TAPSE),isovolumic contraction peak velocity at the tricuspid annulus(IVCv),right ventricular fractional area change(RVFAC),right ventricular and left ventricular diameter were measured with section of apical four chamber and right ventricular tricuspid annulus longitudinal direction of the side walls for each group.5.Routine pathological observation of lung tissue and the measurement of lung perivascular inflammation score.The lung was taken with clipping size 4mm massive specimens after the rat was anesthetized,and fixed on more than 24 hours to prepare a wax block sectioned and stained.Thirty pulmonary arteries with diameter 50-150um were selected from HE staining of lung pathology,to investegating the inflammation around the pulmonary artery,then the score were recorded 04 point according to the degree of inflammatory cell infiltration around pulmonary vessels.6.The serum levels of IL-6,TNF-a,ICAM-1 and HMGB1.After anesthetized by chloral hydrate,the rat was fixed in shelves supine for surgery The blood sample was aspirated with a syringe about 2ml after exposing the abdominal aorta clearly,-20 ? for storage after centrifugation,and ELISA will be done after the collection was completed in all groups.7.The expression of HSP70 protein in lung tissue was detected by immunohistochemistry.8.The level of HMGB1mRNA and HSP70mRNA in lung tissue was detected by RT-PCR9.The expression of I?B? and NF-?Bp65 Protein in lung tissue was detected by Western blot10.Statistical analysisStatistical analysis was performed by using the SPSS 15.0 statistical software package.Measured data were assessed for normality by using the Kolmogorov-Smirnov test.Normally distributed data were represented as the mean ± standard deviation.T test was used to comparison between the two groups,while multi-group comparison using SNK test and Waller-Duncan test for inconsistent sample size.Comparison of non-normal distribution datausing Mann-Whitney Test,while chi-squared test was used for count data.Differences with P<0.05 were considered statistically significant..Results1.PAH rats began to appear shun fur and slow performance after MCT injection for second weeks with lower body weight,and the symptom gradually worsened with the extension of time.Some rats appeared snout cyanosis,besides,two PAH rats died at the forth week,which had action of obvious shortness of breath,purple cyanosis,and poor eating performance before a few days of death.Autopsy of lung tissue of the died rats showed a little flaky bleeding at the surface and ascites in lung.The symptom of HGF group was reduced significantly,no rat death,just slight cyanosis.Weight of rats in HGF group increased obviously comparing PAH group,but still reduced compared with control group.The symptoms of HGF+Q group get worse than HGF roup,including shortness of breath,decreased feeding,weighing less than HGF group,and a rat died before the forth week,but still improved comparing to the PAH group.The performance and weight of the HGF-D group rats was the same to HGF group.2.The level of mPAP and PVR in HGF group was significantly lower than PAH group,which was still higher campared with control group,the level of mPAP group and PVR in HGF+Q group was higher than HGF group,but still lower than the PAH group.There was no significant difference between HGF-D group and HGF group.3.The level of RVHI in HGF group decreased significantly compared with PAH group,but still higher than the control group,there was no significant difference between HGF-D group and HGF group.The RVHI level of HGF+Q group rats was significantly higher than that of HGF group,but still lower than that of group PAH.4.The level of TAPSE,IVCv and RVFAC in HGF group and HGF-D group were both higher than PAH group,however,still lower than the control group;No statistical significance was showed between HGF group and HGF-D group;For RVETD/LVETD,HGF group and HGF-D group were both lower than the PAH group and higher than the control.Right heart function of rats in HGF+Q group decreased comparing with HGF group,the level of TAPSE,IVCv and RVFAC were lower than HGF group,but still higher than that of PAH group,the level of RVETD/LVETD is higher than that of HGF group,lower than PAH group,suggesting that the right heart function still improved comparing with the PAH group..5.The pulmonary artery wall of PAH group was significantly thickened,with significantly hyperplasia and hypertrophy or stenosis of smooth muscle,small acinar pulmonary artery muscularization,perivascular mononuclear cell infiltration,the number of alveolar macrophages was significantly increased in PAH group;There was no obvious thickening of the pulmonary artery wall in HGF group,otherwise,the inflammatory response was reduced for a small amount of mononuclear cell infiltration was seen around the pulmonary artery;Compared with the low dose group,the inflammatory response in group HGF-D was decreased and no significant difference.Changes including alveolar structural damage,inflammatory cell infiltration and pulmonary artery thickening in HGF+Quercetin group get worse than significantly the HGF group,but slightly improved compared with PAH group.The structure of lung tissue and alveolus in control appeared normal,and infiltration of inflammatory cells of perivascular was hardly observed.HE staining around the small pulmonary arteries of lung tissue in PAH group show significant mononuclear cell infiltration,perivascular inflammation points,which was significantly higher than control group;The extent of vascular inflammation reduced in HGF group with inflammation score,which was lower than PAH group;There was no significant difference between HGF group and HGF-D group.Inflammation score of HGF+Q group was significantly higher than HGF group,however,still was improved compareing with PAH group.6.The level of IL-6,TNF-a,ICAM-1 and HMGB1 in HGF group were significantly lower than the PAH group,but still higher than the control group;There was no statistically significant between HGF group and HGF-D group,the level of IL-6,TNF-a,ICAM-1 and HMGB1 of HGF-D group was lower than PAH group,still higher than the control.7.The relative expression of HMGB1mRNA in PAH group was significantly higher than control group.The level of HMGB1 mRNA in HGF group was lower than PAH group,and the difference was statistically significant.The level of HMGB1mRNA in HGF+Q group was higher than HGF group,which was still lower than that PAH group.8.Immunohistochemical revealed that HSP70 expression in HGF group was obvious,mainly cytoplasmic staining and expressed in the bronchial epithelium,alveolar and vascular endometrial epithelial cells,while a strong expression with tan or brown.HSP70 expression in PAH group weakened compared with HGF group with brown or pale yellow bule.Control group and HGF + Q group expressed negative cytoplasm uncolored or pale yellow.In the HGF group,the positive rate of HSP70 and strong positive expression rate were significantly higher than group PAH.The positive expression rate of HSP70 in PAH groupwas higher than control group.The negative expression rateor positive expression rate of HGF+Q group had no significantly different compared with control group.9.The level of relative expression of HSP70mRNA in HGF group was significantly higher than control and PAH group;the relative expression of HSP70mRNA in lung tissue of HGF and PAH group was significantly higher than HGF+Q group.There was no statistically significant difference between HSP70 inhibitor group(HGF + Q)and control group,suggesting that quercetin sufficiently suppressed the expression of HSP70.10.Expression of I?Ba in HGF group was significantly higher than control group and PAH group.I?Ba protein levels of PAH group was lower than the control group.I?Ba protein levels of HGF+Q group was higher than PAH group,significantly lower than HGF groupand no statistically significant compared with control group.11.NF-?Bp65 protein levels of HGF group was lower than PAH group(P<0.05)and HGF +Q group,still higher than control group.The level of NF-?Bp65 protein in HGF + Q group was still lower than PAH group.Conclusions1.HGF alleviate inflammation in lung tissue of PAH rats by reduce levels of inflammatory factors and anti-inflammatory,decline mPAP,PVR and RVHI to improve right heart function and symptoms of PAH rats.2.The high expression of HSP70 in lung tissue was induced after HGF administration in PAH rats,moreover,which can inhibite NF-?B signaling pathway,suggesting that the HSP70/NF-?B pathway is one of the important mechanisms for HGF improving PAH.BackgroundPulmonary hypertension(PH)is a group of diseases characterized by an increase in pulmonary vascular resistance,which can lead to right ventricular failure.The European Society of Cardiology(ESC)issued a new version of "pulmonary hypertension diagnosis and treatment guidelines" in August 29,2015.PAH is a hemodynamic and pathophysiological state,which is defined that mean pulmonary arterial pressure(mPAP)should be greater than or equal to 25 mmHg,measured by right heart catheterization(RHC)at sea level conditions.Early diagnosis of PH is more difficult,because the symptoms of PH including shortness of breath,chest distress,chest pain,fatigue and so on are also common in other cardiovascular diseases.RHC as a gold standard for PH diagnosis have reached a consensus,however,it is difficult to promote as early indicators for PH screening as an invasive examination.Echocardiography is easy to be accepted as a noninvasive examination,following some limitations that pulmonary artery systolic pressure(PASP)measurement may not be entirely in line with the actual mPAP.For that,the combination with biomarkers to improve the accuracy of prediction is the focus and direction of the research,which may provide an opportunity to improve the suivival and quality of life for PH patients with early detection and intervention.Hepatocyte growth factor(HGF)is a multifunctional cytokine derived from mesenchymal with the activities of mitogenic,motogenic and morphogenic in various cell types.Furthermore,HGF was widely distributed in the small intestine,brain,thymus,bronchial,alveolar epithelial cells,syncytial trophoblast cells and vascular smooth muscle cells et al,which can be detected in normal human plasma also.Studies have damonstrated that HGF levels in the early stage of acute lung injury or pulmonary embolism(PE)was elevated,especially showing a time-varying curve with PE,which suggesting the correlation between HGF and PE,however,the level of serum HGF in patients with PH has not been reported.The present study observes the serum levels of HGF in patients with varying degrees of PH,and the relationship between HGF and mPAP,PVR,NT-proBNP and CRP et al,to investigate the level and significance of HGF for PH patients.Objective1.Serum HGF level and significance of patients with pulmonary hypertension.2.The correlation between HGF and mPAP,PVR and Cardiac index(CI)in patients with pulmonary hypertension.MethodsTwenty-five healthy people were enrolled to control group,while seventy-three patients in PH group enrolled with standard by "Suggestion of diagnosis and treatment of pulmonary hypertension" drafted by the World PH Forum in 2013.PH patients were divided into three groups according to mPAP.Patients with mPAP between 25 and 30 mmHg were diagnosed with mild PH(L group).Patients with mPAP greater than 30 mmHg but less than 50 mmHg were diagnosed with moderate PH(M group).Patients with mPAP of 50 mmHg or greater were diagnosed with severe PH(H group).In addition,patients with pulmonary hypertension were divided into two groups:idiopathic pulmonary hypertension(IPAH)group and secondary pulmonary hypertension(PPH)group.All patients gave their informed consent for inclusion in this study.Demographic information,including age,sex,and comorbidities(e.g,systemic hypertension,diabetes,smoking),was collected from all participants.MPAP,PVR and CI data and other indicators were checked by RHC in PAH patients,Peripheral blood samples were obtained from all participants.Serum was isolated from the blood and stored at-80 ? until analysis.Serum levels of HGF were measured by sandwich enzyme-linked immunosorbent assays(ELISAs).The O.D value of the sample was measured,and the light absorption value concentration curve was drawn..The HGF concentration values were determined from the curve of absorbance vs.concentration.Besides,NT-proBNP,C-reactionprotein(CRP)and alanine aminotransferase(ALT)were detected by automatic biochemical analyzer.Statistical analysisMeasured data were assessed for normality by using the Kolmogorov-Smirnov test.Normally distributed data were represented as the mean ± standard deviation,otherwise show with M(P25,P75).Data were compared between two groups using the t test,while SNK test was used for multi-group comparison.Mann-Whitney Test was used to analysis unnormally sistuibuted data.Linear correlation analysis was used to determine the relation between two indicators,and Multivariate stepwise regression analysis for the correlation between mPAP and HGF.For count data,the chi-squared test was used.Statistical analysis was performed by using the SPSS 15.0 statistical software package.Differences with P<0.05 were considered statistically significant.Results1 There was no statistically significant difference for age,gender(female population)and the rate of obesity in four groups.The proportion of hypertension,diabetes and smoking in the three PH groups(L,M and H)showed no statistically differences also.The proportion of PH patients receiving drug therapy,including prostaglandins,endothelin antagonists,phosphodiesterase inhibitors,diuretics and combination therapy(greater than or equal two therapeutic drugs),increased along with elevation of pulmonary artery pressure.2 PVR level in H group and M group were significantly higher than group L,there was statistical significance difference between M group and L groups;CI levels were decreased with the elevation of mPAP,CI of H group and M group lower than L group,H group is lower in comparison with M group;3 Significantly higher levels of plasma HGF were observed in the PH group(2.220±1.670ng/ml)compared to healthy controls(0.152 ±0.038ng/ml).The HGF level of the group with mild PHwas significantly higher than that of the healthy control group.HGF level of H group was higher than the group with moderateand mild PH for M or L vs.H group,additionally,there was significantly statisticallydifference betweensecondary PH group(2.649 ±1.875 ng/mL)and idiopathic PH group(1.632 ± 1.192ng/mL).4 Serum level of NT-proBNP and CRP in PH group was significantly higher than control group.The level of H group was higher compared with M group and L group,and M group was higher than L group,in addition,the level of NT-proBNP and CRP of PPH group was significantly higher than IPAH group..5 HGF levels of PH was positively related with mPAP,PVR,NT-proBNP and CRP(r=0.835,0.769,0.523 and 0.804,P<0.05),The HGF levels had a negative correlation with CI(r=-0.701).For the IPAH group,the level of HGF was positively correlated with mPAP,PVR,NT-proBNP and CRP(r=0.877,0.792,0.352 and 0.761,P<0.05),with a negative correlation with CI similarly(r=-0.765,P<0.05).The level of HGF was also positively correlated with mPAP,PVR,NT-proBNP and CRP in PPH group(r=0.735,0.711,0.652 and 0.821,P<0.05),with a negative correlation with CI(r=-0.614,P<0.05).The level of NT-proBNP in PH patients was positively correlated with mPAP,PVR and CRP(r=0.62,0.58 and 0.598,P<0.05),with a negative correlation with CI(r=-0.614,P<0.05).For the IPAH group,the level of NT-proBNP was positively correlated with mPAP PVR and CRP(r=0.525,0.439 and 0.468,P<0.05),with a negative correlation with CI similarly(r=-0.427,P<0.05).The level of NT-proBNP was also positively correlated with mPAP PVR and CRP in PPH group(r=0.852,0.72 and 0.755,P<0.05),with a negative correlation with CI(r=-0.631,P<0.05).Serum levels ofCRPalso showed positively correlation withmPAP and PVR(r=0.856,0.831,P<0.05),and a negative correlation with CI(r=-0.641,P<0.05).For the IPAH group,the level of CRPwas positively correlated with mPAPand PVR(r=0.748,0.722,P<0.05),with a negative correlation with CI similarly(r=-0.535,P<0.05).The level of CRP was also positively correlated with mPAPand PVR in PPH group(r=0.834,0.802,P<0.05),with a negative correlation with CI(r=-0.776,P<0.05).6.ROC curve prediction(1)NT-proBNP:area under the curve was 0.819,cut point sensitivity and specificity were 0.531 and 0.757 respectively.(2)CRP:area under the curve was 0.745,cut point sensitivity and specificity were 0.678 and 0.586 respectively.(3)HGF:area under the curve was 0.777,cut point sensitivity and specificity were 0.690 and 0.495 respectively.(4)Combined with NT-proBNP,CRP and HGF:area under the curve was 0.893,cut point sensitivity and specificity were 0.832 and 0.783 respectively.7.The correlation between mPAP(Y)andHGF(X1),NT-proBNP(X2),CRP(X3),PVR(X4)and CI(X5)in multivariate stepwise regression showed that HGF and PVR were correlated with mPAP(R=0.935,R2=0.874,P<0.05),which suggesting that there is a correlation between HGF and mPAP.Conclusions1.The serum HGF levels in patients with PH increased significantly and was positively correlated with mPAP,suggesting a relation between HGF and PH.2.The level of CRP of PH patients was obviously higher and was positively correlated with mPAP and PVR,indicating a relation between inflammatory factors and PH.3.NT-proBNP,CRP and HGF were associated with PH,and combined with HGF could increase the predictive value of pulmonary hypertension. |
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