Research On The Mechanism Of Ma Xing Xiong Ting He Ji(Admixture) To Hypoxic Pulmonary Hypertension(HPH),Targeting On Rho/Rock Signal Pathway

Objective: through the establishment of hypoxic pulmonary hypertension (HPH) rat model, to explore the HPH pathological mechanism, observe the intervention effects for Ma Xing Xiong Ting He Ji (admixture) in releasing lung, removing blood stasis and dissolving phlegm pattern on pulmonary artery pressure increase and vascular structure reconstruction of intervention effects of HPH rats. And through the observation the expression level of rock-1gene and protein in HPH rats lung tissue, explore the control function of Rho/rock signaling pathways on hypoxic pulmonary hypertension and the intervention of molecular mechanism of hypoxic pulmonary hypertension by Ma Xing Xiong Ting He Ji (admixture) in releasing lung, removing blood stasis and dissolving phlegm pattern.Methods:50Sprague wrote-Dawley rats, half male and half female, randomly divided into normal group, the HPH model group and fasduil control group, medium dosage Chinese medicine group, high dosage Chinese medicine group. Atmospheric intermittent hypoxia method:(10±0.5)%for02, intermittent hypoxia for six to eight hours a day, anoxic for6days a week,4weeks, to establish HPH rats model. Put them in a low oxygen tank before medicine delivery, For fasudil group, intraperitoneal injection,10mg/kg, with hydroxyfasudil infection; For medium dosage Chinese medicine group,10g/kg of Ma Xing Xiong Ting He Ji(mixture) gavage; For high dosage Chinese medicine group,20g/kg of Ma Xing Xiong Ting He Ji(mixture) gavage; For normal group and the HPH model group,1ml saline lavage for every100g of body weigh. At21th day determine their mean pulmonary artery pressure (mPAP), average carotid artery pressure (mean cervical arterial pressure, mCAP), right heart hypertrophy index (right ventricular hypertrophy index, RVHI); Observe morphology changes of pulmonary arterioles under light microscopy, calculate the percentage of the thickness of vessel wall over the external diameter (WT%), the percentage of the wall area over the total area of blood vessel (WA%), the percentage of the lumen area over total area of the tube (LA%) by picture analysis. Determine the percentage of muscular arteries,partial muscular artery non muscular arteries,and muscular pulmonary arterioles and density of membrane nucleus,observe the reconstruction of pulmonary vascular structure.After confirming the successful modeling of HPH, apply Real time PCR,Westen bolt and other techniques,to determine the amount of the expression of Rho kinase gene and protein of lung tissue of the rats in each group by molecular biology method and determine Rho kinase substrate of lung tissue of the rats in each group the phosphorylated expression of MBS Thr-697.Results:(1)After21days of hypoxia,mPAP, RVHI,WT%,WA%,LA%and the portion of muscular arteries,partial muscular arteries,non-musclar arteries,and density of membrane nucleus in muscular pulmonary arterioles of rats in HPH model group are as follows respectively:(30.27±1.47mmHg,,(41.41±3.71)％,(55.23±3.07)％,(83.12±5.90)％,(16.88±1.74)％,(57.26±0.26)％,(17.46±2.78)％,(25.28±3.07)％,(9.41±1.90),and rormal group(16.14±0.38)mmHg,(24.65±1.85)％,(33.41±3.65)％,(54.23±4.61)％,(45.76±3.23)％,(7.88±0.83)％,(25.76±2.05)％,(66.36±3.65)％,(7.05±1.37).The comparison exists a dofference.(P<0.05).(2) After21days of hypoxia,mPAP, RVHI,WT%,WA%,LA%and the portion of muscular arteries,partial muscular arteries,non-muscular arteries,and density of membrane nucleus in muscular pulmonary arterioles of rats in Fasudi group are as follows respectively:(18.85±0.65)mmHg,(30.24±1.82)％,(38.15±2.96)％,(68.49±4.16)％,(31,50±3.53)％,(12.49±0.65)％,(36.58±2.55)％,(50.94±2.96)％,(13.13±2.61)；For medium dosage Chinese medicine group are:(21.61±0.38)mmHg,(28.19±1.67)％,(43.12±3.61)％,(65.49±4.15)％,(34.51±3.38)％,(20.71±0.21)％,(30.86±2.03)％,(48.43±3.61)％,(11.28±1.08)；For high dosage Chinese medicine group are(23.25±1.19)mmHg,(27.64±1.55)％,(44.53±3.76)％,(69.90±4.22)％,(30.10±3.37)％,(20.77±0.19)％,(29.08±2.57)％,(50.15±3.76)％,(7.26±1.68)；The three groups above all have comparison difference(P<0.05) respectively with HPH model group(30.27±1.47mmHg),(41.41±3.71)％,(55.23±3.07)％,(83.12±5.90)％,(16.88±1.74)％,(57.26±0.26)％,(17.46±2.78)％,(25.28±3.07)％,(9.41±1.90).(3)mCAP has no significant difference among groups(P>0.05).(4)It is obvious that the vessel wall of rats’ pulmonary arterioles in HPH model group is thicker; and there are significant remodeling of pulmonary arterioles: continuous damage on endothelial cells and vascular endothelial is generating cuboidally or columnarly to the lumen, even has necrosis and falls off, smooth muscle cells on nunica media has hypertrophy, hyperplasia and the lumen decreases. Compared with HPH model group, Fasudi intervention group, medium and high dosage Traditional Chinese medicine (TCM) group, rats’ pulmonary arterioles are with thinner walls and lumen with wider and the degree of hypertrophy and hyperplasia of vascular muscle cells and thickening of the tunica intima is reduced obviously.(5)After three weeks of hypoxia (21days) intervention, rats’ Rho kinase ROCK-1mRNA (2.680±0.061) and normal group (1.094±0.051) are increased in HPH model group, the difference was statistically significant (P<0.05). Method of shu in Bristol, in traditional Chinese medicine (TCM) and high dose group rats’ Rho kinase (ROCK-1mRNA (1.661±0.0.90),(2.035±0.078),(1.962±0.066)(1.094±0.051) in Fasudi group, medium and high dosage TCM group are higher than normal group, the difference was statistically significant (P<0.05), compared with the HPH model group (2.680±0.061),they are lower, and the difference has statistical significance (P<0.05).(6) By applying Western imprinting detection method, ROCK-1protein expression in HPH model group is increased than the normal group (P<0.05), the ROCK-1protein expression in Fasudi group, medium and high dosage TCM group, after the intervention of Ma Xing Xiong Ting He Ji(mixture),is increased than the normal group (P<0.05), compared with HPH model group there is a reduction in different degrees (P<0.05).(7) According to the results of western blot, P-MYPT1protein expression in HPH model group is increased than the normal group (P<0.05), P-MYPT1protein expression in Fasudi group, medium and high dosage TCM group, after the intervention of Ma Xing Xiong Ting He Ji(mixture), is increased than the normal group (P<0.05), while decreased compared the HPH model group (P<0.05).Conclusion:(1) Ma Xing Xiong Ting He Ji(mixture), with its lung-releasing-and-blood stasis-removing-phlegm-dissolving effect can reduce the increase of pulmonary artery pressure caused by hypoxia, improve the right ventricular hypertrophy, reconstruct pulmonary vascular structure, has specific prevention and intervention for hypoxic pulmonary hypertension.(2) the Rho/ROCK signaling pathways are closely associated with the occurrence and development of HPH.The intervention effect of Ma Xing Xiong Ting He Ji(mixture), with its lung-releasing-and-blood stasis-removing-phlegm-dissolving effect,for hypoxic pulmonary hypertension, is related to its ability to regulate the Rho/ROCK signaling pathways, inhibit the synthesis and expression of pulmonary vascular Rho kinase.(3) According to basic theory of TCM, the formula, compatibility and pharmacological analysis of Ma Xing Xiong Ting He Ji(mixture), we can declare that it can disperse the lung, astringe the lung, and reduce the lung, dissolve the phlegm, promote water, and activate the blood. This formula combine lung-releasing and blood stasis-removing and phlegm-dissolving all together, fcous on the TCM pathogenesis and syndrome differentiation of hypoxic pulmonary hypertension to achieve and exactly curative effect.