The Relation Between 5-hydroxytryptamine Transporter And Apoptosis Of PASMCs In Rats With Left To Right Shunt Pulmonary Hypertension

Pulmonary hypertension(PAH) is a group of diseases characterized by elevation of pulmonary arterial pressure and pulmonary vascular resistence,leading to right ventrieular failure and death.PAH has a complex pathobiology involving many abnormal aspects in cellular structure, molecular signaling pathways , gene expression and so on.The causes are not entirely clear.Study found that vascular active substances and cytokines are generated unusually by inflammation, abnormal blood shearing force, hypoxia and focus on endothelial cells,PASMCs, causing the extracellular matrix accumulation, vasoconstriction, abnormal growth and remodeling and so on .They all contribute to pulmonary hypertension finally.Pulmonary vascular remodeling is the main pathological feature of pulmonary hypertension.Abnormal proliferation of cells and reducing of apoptotic cells was the main cause of the tissue remodeling. Apoptosis is a kind of orderly inflammatory death of nuclear cells. It is launched by intracellular specific program and implemented through activated DNA enzymes in the case of some physiology and pathology. It is also called programmed cell death. Apoptosis and proliferation are two opposites.The balance of them maintain the normal state of the tissue structure and physiological function. It has also been demonstrated that either increased proliferation or decreased apoptosis of PASMCs contribute to pulmonary vascular medial hypertrophy and vascular remodeling,which is an important mechanism of PAH. Under normal circumstances, hemal wall structure is stable via balance of apoptosis and proliferation. Various internal and external stimulating effects promot proliferation genes and/or apoptosis inhibition genes of pulmonary smooth muscle cells to express throught a series of signal transductic process, causing PASMCs proliferation and/or apoptotic reducing, migration.It contributs to pulmonary ascular wall thickening and pulmonary arterial reconstruction. Therefore, many scholars focus on promoting PASMCs apoptosis as therapeutic approach for patient with PAH in the future.Serotonin transporter (5-HTT) is a kind of membrane protein.It is expressed in neurons, platelets, PASMCs.The main function of serotonin transporter is transfering serotonin (5-HT) into cells to exert biological effects. 5 - HT is mediated into pulmonary smooth muscle cells by serotonin transporter and cause pulmonary arterial smooth muscle cell proliferation and hypertrophy through multiple signal transduction pathways.Then it cause pulmonary vascular reconstruction. Several study results indicate that serotonin transporter plays an important role in many types of pulmonary hypertension.Is there also a high expression of 5-HTT in pulmonary hypertension with left to right shunt? the relation between 5-HTT high expression and apoptosis of PASMCs has not been reported.Statins is inhibitors of HMG-CoA reductase, They have so many functions such as lipid lowering, improving endothelial function, stable atheromatous plaque, inhibiting platelet aggregation, anti-inflammatory, anti-oxidation, immune that they have been widely applied to Cardiology and Neurology. Recently, more and more scholars begin to pay close attention to "non- lipid lowering effect" of statins.Domestic and international studies have found that statins affect every aspect of pulmonary hypertension through many mechanisms. Statins reduce pulmonary artery pressure, improve pulmonary arterial diastolic function and restrain pulmonary vascular reconstruction through inducing pulmonary smooth muscle cells or vascular endothelial apoptosis and inhibiting their proliferation.It is a bright future to apply statins to treat PAH. Rosuvastatin is a new type of HMG-CoA reductase inhibitor.It is a new member of the statins family.Its role in pulmonary hypertension and mechanism has less been reported.There is even no report about its function in pulmonary hypertension with left to right shunt.In this study, we used abdominal aorta- inferior vena cava shunt method to establish the high blood flow rat model. Through observating 5-HTT expression and apoptosis of PASMCs,we explored whether these factors were involved in PAH with high blood flow and the possible mechanism of PAH with left to right shunt in rats.Meanwhile, we used rosuvastatin to intervene the model rats .We investigated whether rosuvastatin affected the expression of 5-HTT and apoptosis of PASMCs in rats to explore preliminarily the mechanism of rosuvastatin about pulmonary vascular remodeling of high blood flow rat.1. Forty SD rats used in the experiment were randomly divided into four groups: control group,4 weeks group, 8 weeks group and 11 weeks group. We established rat model with high lung blood flow through abdominal aorta- inferior vena cava shunt in 4 weeks group, 8 weeks group and 11 weeks group.The rats of controle group received laparotomy without shunt.2. Before executing rats, we measured pulmonary average pressure (mPAP) of rats with a mini polyethylene catheter.The ratio of RV weight to S﹢LV weight was calculated as index of the right ventricular hypertrophy.3.After paraffin embedding , the lung sections were stained with Hematoxyline-eosine(HE) and observed the chang of pulmonary vascular.We calculated vascular wall thickness/vascular diameter(WT % ) and vascular area/total vascular Area(WA % ) as the index about pulmonary vascular remodeling.4.Protein expression of 5-HTT in each rat was measured with immunohistochemical stain(SP). 5.Apoptosis of PASMCs in each rat was measured by TUNEL assay and apoptosis index(AI) was calculated.6.Protein expression and of 5-HTT and bcl-2 in each rat were measured by Western Blot.7.The expression of 5-HTTmRNA in each rat was measured by RT-PCR.8.According to above experiment thirty SD rats were randomly divided into three groups: controle group,shunt group, shunt+ rosuvastatin group. After operation the rats of shunt + rosuvastatin group were fed by rosuvastatin with dose of 10mg/kg/day for 11 weeks.Western Blot and TUNEL assay were applied to evaluate the affect of rosuvastatin on 5-HTT and bcl-2 protein expression and PASMCs apoptosis of rat with high lung blood flow.9.All data were expressed as mean士SD.Statistical analysis were performed by SPSS 13.0. Grouping t-test was used to compare the difference of two groups.ANOVA was used to compare the differences of many groups.A value of P<0.05 was statistically significant.1.With the increasing of shunt time, both mPAP and RVI of each shunt group were gradually elevated.Obvious elevation of pulmonary vascular pressure and right ventricular index appeared in model rats at the last 11 th week. Statistical analysis showed that there were significant differences between 11 weeks group and other groups(P<0.05).2.With the increasing of shunt time, pulmonary vascular remodelling appeared in rats of shunt group.The smooth muscle cells of pulmonary vascular proliferated and became hypertrophy.The intima became thicken and the lumen became stenosis. Meanwhile,both WT% and WA% were gradually elevated in shunt groups. Statistical analysis showed that there was a significant difference between each two groups(P<0.05).3.Immunohistochemistry indicated that with the increasing of shunt time, the expression of 5-HTT in shunt groups were gradually elevated. Statistical analysis showed that there was a significant difference between each two groups(P<0.05)4.TUNEL assay indicated that there are a certain proportion of the apoptotic cells in each group. With the increasing of shunt time, apoptosis of PASMCs in shunt groups gradually decreased with apoptosis index decreasing. Statistical analysis showed that there was a significant difference between each two groups(P<0.05)5.Western Blot showed that with the increasing of shunt time, the expression of 5-HTT and bcl-2 protein in shunt groups were gradually elevated.Statistical analysis showed that there were significant differences between 11 weeks group and other groups(P<0.05).6.RT-PCR indicated that with the increasing of shunt time, the expression of 5-HTTmRNA in shunt groups were gradually elevated.Statistical analysis showed that there were significant differences between 11 weeks group and other groups(P<0.05).7. With the increasing of shunt time, the level of 5-HTT gradually increased and apoptosis index of PASMCs gradually decreased.Correlation analysis showed that there was a significant negative correlation between level of 5-HTT and apoptosis index.8. Compared with shunt group, the expression of 5-HTT and bcl-2 protein in shunt + rosuvastatin group significantly decreased.The percentage of apoptotic PASMCs was obviously elevated. Statistical analysis showed that there was a significant difference between each two groups(P<0.05).1.Abdominal aorta - inferior vena cava shunt leads to obvious high pressure of pulmonary artery, right ventricular hypertrophy and pulmonary arterial reconstruction of rat2.The expressions of 5-HTT and bcl-2 protein dependence on shunt time are obviously enhanced in left to right shunt-induced PAH rats as well as the PASMCs apoptosis percentage is lower than normal percentage. The 5-HT signal transductic process via 5-HTT participates in pulmonary vascular remodeling through up-regulating expression of bcl-2 protein and suppressing apoptosis of PASMCs.3.Rosuvastatin protects rats with high lung blood flow against elevation of PAP and pulmonary vascular remodeling . It is considered that this function might relate to suppressing expression of 5-HTT and bcl-2 protein and promoting apoptosis of PASMCs.