Involvement Of Inflammatory Cytokines And MMPs/TIMPs In SSRIs Produced Protective Effects Against Pulmonary Hypertension In Rats

IntroductionPulmonary arterial hypertension(PAH)is a common disease with a sustained elevation in pulmonary artery pressure(PAP),which may lead ultimately to right ventricular failure.Pathological features of PAH include pulmonary vasoconstriction, vascular remodeling,inflammation and microthrombosis.PAH is frequently caused by chronic obstructive pulmonary disease(COPD),hypoxia,chronic inflammation and portal hypertension.Several vasoactive factors are considered to be involved in the development of pulmonary hypertension,including as endothelin,prostacyclin, 5-hydroxytryptamine(5-HT),thromboxane A₂(TXA₂),platelet-derived growth factor and epidermal growth factor and cytokines(TNF-α,IL-1,IL-6,MCP-1 et al).Recently studies show 5-HT as a kind of endogenous vasoactive substance plays an important role in the development of PAH.It has been reported that the internalization of 5-HT into pulmonary artery smooth muscle cells(PASMCs)by a high-affinity serotonin transporter(SERT)promotes smooth muscle cell hyperplasia and hypertrophy.Furthermore,the expression of SERT in the pulmonary artery is increased in patients with pulmonary hypertension.Exposure of PASMC to hypoxia results in a rapid increase in the level of SERT mRNA and a 2.5-3.0-fold enhancement of 5-HT transporter activity.Familial pulmonary hypertension may be related to polymorphisms in the gene encoding SERT.A polymorphism with long and short forms affects SERT function with the long allele inducing an increased rate of SERT gene transcription.The SERT polymorphism also predicts the severity of pulmonary hypertension in patients with COPD.Therefore,5-HT and its transporter appear to be important players in the process of pulmonary hypertension.Smoller et al.suggested that there is a link between anxiety and respiratory symptoms,probably mediated by the serotonergic system.It has been found that a high proportion of patients with panic disorder suffer from COPD and the prevalence of panic disorder has been shown to be higher in patients with COPD.The selecetive serotonin reuptake inhibitors(SSRIs),such as fluoxetine,sertraline,paroxetine et al,are effective therapeutic drugs for patients with mood disorders and they act via inhibition of SEPT.Thus,SERT may be a molecular target for the treatment of COPD and pulmonary hypertension.However,in the lungs,it remains unknown whether SSRIs be the new target of therapy pulmonary arterial hypertension.Therefore,the aim of the present study was to investigate(1)the protective effect of the SSRIs against pulmonary arterial hypertension.(2)The mechanism of SSRIs protecting pulmonary arterial hypertensionâ‘ SSRIs inhibiting pulmonary arterial vascular remodeling.â‘¡SSRIs decreasing inflammation effect in lungs.MethodsMonocrotaline is used to establish the model of 'inflammatory' pulmonary hypertension in rats.Rats were treated with different dose of fluoxetine and sertraline by gavage for three weeks.Then,pulmonary haemodynamic measurement,right ventricular index,lung tissue morphological investigate,elastin and collagen staining were undertaken.SERT mRNA was assayed by reverse transcription-polymerase chain reaction(RT-PCR).Matrix metalloproteinases-2,9,tissue inhibitors of metalloproteinases-1,2,and cytokines IL-1β,MCP-1,TNF-αexpression were detected by Western blotting.Statistics were made to compare these indexes in different groups,and investigate the mechanism of SSRIs protecting against PAP. Results1.MCT induced pulmonary arterial hypertension in ratsThree weeks after MCT administration,pulmonary artery pressure and right ventricular index were measured.The chronic "inflammatory" PHT model in rats induced by MCT was successfully established.Mean pulmonary artery pressure was elevated in MCT group compared with control group from 15.3±2.2mmHg to 28.0±2.1 mmHg(P＜0.01).Right ventricular index was increased by MCT from 0.32±0.03 to 0.48±0.08(P＜0.01 vs control).The muscularization of lung tissues from the right lower lobe was investigated under light microscope.The rates of non-muscularization,partial muscularization and full muscularization were 60.9%, 25.5%and 13.6%,respectively,in control group;49.5%,17.5%and 33%,respectively, in MCT group.2.SSRI fluoxetine and sertraline protect against pulmonary arterial hypertensionFluoxetine and sertraline effectively decreased MCT-induced pulmonary arterial hypertension,compared with MCT group,PAP was decreased from 28.0±2.1 mmHg to 25.4±2.9 mmHg(P＜0.05)and 23.2±3.1mmHg(P＜0.05),respectively.Right ventricular index was reduced from 0.51±0.09 to 0.42±0.04 by sertraline(P＜0.05 vs MCT)and 0.48±0.08 by fluoxtine(P＜0.05 vs MCT).The rates of non-muscularization,partial muscularization and full muscularization were decreased from 49.5%,17.5%and 33%,respectively,in MCT group to 61.8%,24.6%,13.9%, respectively,in fluoxetine group and to 64.9%,23.6%,11.5%,respectively,in sertraline group.3.SERT mRNA expression in pulmonary arteriesThe levels of SERT mRNA in pulmonary arteries performed by RT-PCR were analyzed using house keeping geneβ-actin as control.The ratio of the PCR products of SERT gene overβ-actin gene was much higher in MCT rats than in control rats (MCT 0.99±0.06 vs control 0.86±0.08,P＜0.05),and it was decreased to 0.89±0.03 (P＜0.05 fluoxetine vs MCT)and 0.82±0.09(P＜0.05 sertraline vs MCT)4.fluoxetine decreased pulmonary hypertension and the thickness of pulmonary arterial wall in dose-related mannerFluoxetine decreased pulmonary pressure in dose-related,PAP in MCT+F10 and MCT+F2 groups were decreased from 29.8±7.5 to 26.9±5.7 mmHg(P＜0.05,vs MCT)and 24.5±3.3 mmHg,respectively.Compared with MCT group,the thickness in MCT+F2 and MCT+F10 groups were decreased from 53.5%±8.8%to 44.7%±9.7%and 38.5%±10.1%(P＜0.01,vs MCT),respectively.5.fluoxetine inhibited elastin and collagen content in lungsElastin content was stained by orcein staining.It was found that,in MCT group, the layer of elastin content thickened in pulomary arteries,and the structure appeared fragmentation compared with control.Fluoxetine low dose group and high dose group both kept integrity structure of elastin,and high dose group significantly decreased the elastin content compared with MCT group.Collagen deposition was visualized by Van Gieson stain in lungs.In MCT group,it found that there is lots of collagen deposition localized around pulmonary arteries and bronchioles.Fluoxetine decreased them in a dose-related manner compared with MCT group.6.fluoxetine inhibited the expression of MMP-2,9,TIMP-1,2 in dose-relatedProtein expression of MMPs and TIMPs were measured by Western blot. Compared with control group,the levels of MMP-2,MMP-9,TIMP-1,TIMP-2 in MCT group were significantly increased from 0.68±0.15,0.59±0.22,0.82±0.08 and 0.74±0.18 to 1.11±0.15,1.01±0.09,1.03±0.16 and 1.11±0.17,respectively. Fluoxetine inhibited MCT induced increase of these proteins in a concentration dependent manner.In MCT+F2 group,these levels were decreased to 0.95±0.20, 0.92±0.14,0.77±0.22 and 0.90±0.08 respectively.And in MCT+F10 group, these levels were significantly decreased to 0.80±0.27,0.73±0.16,0.67±0.18 and 0.87±0.10,respectively.7.Effect of fluoxetine on MCT-induced lung inflammationIn MCT group extensive morphological changes,including perivascular and peribronchiolar inflammatory cell infiltration and angiogenesis in lungs,were found and the thickness of alveolar walls was significantly increased.However,after treatment with fluoxetine,it was found that inflammation of lungs was not evident and decreased number of inflammation cells dispersed throughout the lungs dose dependently,8.Protein expression of inflammatory cytokines TNF-αand IL-1βProtein expression of inflammatory cytokines TNF-α,MCP-1 and IL-1βwere measured by Western blot.Compared with control group,the levels of TNF-α, MCP-1,IL-1βin MCT group were significantly increased from 0.58±0.24,0.64±0.11 and 0.74±0.19 to 1.00±0.22,0.92±0.12 and 1.16±0.22,respectively. Fluoxetine inhibited MCT induced increase of these cytokines in a concentration dependent manner.In MCT+F2 group the levels were decreased to 0.86 4±0.17,0.87±0.26 and 0.89±0.19,respectively.And in MCT+F10 group the levels were significantly decreased to 0.53±0.29,0.56±0.07 and 0.79±0.18,respectively.Conclusion1.SSRI sertraline and fluoxetine protects against MCT-induced pulmonary hypertension by decreasing pulmonary arterial pressure,right ventricular index and pulmonary artery remodeling,which might be related to a mechanism of SERT mRNA reduction.2.Fluoxetine dose-related decreased MCT-induce pulmonary hypertension and vascular remodeling.Fluoxetine not only inhibited elastin and collagen content in lungs,but also decreased MMP-2,9,TIMP-1,2 protein expression.It suggested that the effect of fluoxetine protecting against vascular remodeling may be related to the reduction of MMPs expression.3.Fluoxetine relieved inflammatory cell infiltration of lung tissues.In addition, fluoxetine inhibited protein expression of inflammatory cytokines,i.e.IL-1β,TNF-α, and MCP-1.The anti-inflammatory effects of fluoxetine contributed to inhibition of MCT-induced pulmonary hypertension in rats.