| Pulmonary hypertension(PH)is a life-threatening condition and a common cause of right cardiac failure.However,currently there are comparatively few medical methods for pulmonary hypertension.Epidemiological evidence suggests that the prevalence of PH is approximately 12-50/1,000,000 people.Sphingosine-1-phosphate(S1P)is a biologically active sphingolipid metabolite.Recent studies have focused on S1 P treatment and its important roles in many diseases,including cancer,atherosclerosis,diabetes and osteoporosis.In mammals,S1 P is produced by two sphingosine kinase(Sph K)isozymes,named Sph K1 and Sph K2.In recent years,studies have shown that S1 P can cause PH in rodents.In mammalian cells,sphingosine-1-phosphate lyase(SPL)is the only enzyme that causes irreversible degradation of S1 P.Recent publications indicated that the expression of Sph K1 and production of S1 P are significantly increased in lung and pulmonary artery smooth muscle cells separated from patients in vivo with PH.In addition,mice without Sph K1 do not have hypoxia-induced PH.Obviously,the above results indicate that the pathological S1 P signaling mediated by Sph K1 plays a key role in the pathogenesis of PH.However,It remains unknown whether SPL is able to prevent PH by reducing pulmonary S1 P.In a mouse model of hypoxia-induced PH,it is found that the expression of lung SPL significantly decreased and the level of S1 P in pulmonary tissue significantly increased.Suppression of SPL activity via drugs can increase pulmonary S1 P level,aggravate right-heart hypertrophy and pulmonary vascular remodeling induced by PH.In contrast,the overexpression of SPL can reduce the level of S1 P in pulmonary tissue of mice and delay the progression of hypoxia-induced PH.In consideration of the existing research results,extracellular regulated protein kinases 1/2(ERK1/2)plays the key pathogenic effect in the pulmonary vascular remodeling and the occurrence of PH.It is also found that inhibition of SPL via drugs can raise the phosphorylation level of hypoxia-induced ERK1/2,whereas the overexpression of SPL can reduce the phosphorylation level of ERK1/2 associated with PH.This study initially showed that the SPL may be able to delay the progression of PH by blocking pathological S1 P signal transduction and reducing the phosphorylation level of ERK1/2 related to S1 P.Therefore,SPL may be a promising new target for the treatment of PH.Study Objectives1.To observe the changes of S1 P and SPL level in tissue during hypoxia-induced PH2.To observe the effect of PH by inhibiting SPL3.To observe the effect of PH during the overexpression of SPL4.Preliminary investigation of the mechanism under the effect of SPL on PH Study methods Animal experiment1.PH mouse model was established via hyperbaric and hypoxic environment.In order to conduct the study of PH related experiments,firstly,a mouse model of PH was established.We took 8-week-old male C57BL/6J mice and divided them into two groups of 7 mice each.One group was fed normally as a Control group,and the other group was placed in a hyperbaric and hypoxic environment for 16 hours each day.The hyperbaric and hypoxic environment was 50 Kpa in atmospheric pressure,10% in oxygen content.After 8 weeks,a series of tests were performed.Cardiac functions of two groups of mice were tested by M-mode ultrasonography.Histological analysis was used to examine the thickness of pulmonary arterial wall in both groups.SPL and a-SMA level were detected by Western Blot.(α-SMA is a biomarker of smooth muscle cells,and if its expression level elevates,it means smooth muscle proliferation is obvious.)Pulmonary S1 P level was detected by ELISA kits.The activity of SPL was measured by using a fluorescence assay.Whether the PH model was successfully established was determined by the above experimental results.2.The effect of SPL on PH was observed by inhibiting its activityIn order to clearly observe the effects of inhibiting SPL activity on PH,we divided8-week-old male C57BL/6J mice into Control group and PH 8W group(exposed in hypoxia chamber for 8 weeks),then the two groups were administrated with THI(SPL specific inhibitor 25 mg/L,dissolved in 10 g/L glucose solution),and control solvent(Vehicle,10 g/L glucose solution)respectively.After 8 weeks of feeding,the heart function and structure of each group of mice were measured by using M-mode ultrasound;the pulmonary artery wall thickness was measured by histological analysis;the levels of SPL and a-SMA were detected by Western Blot;and the lung S1 P was detected by ELISA kits.Level;SPL activity was measured by fluorescence analysis experiment.3.The Effect of SPL on PH was observed via overexpression of SPL mice(SPL-TG mice)To further explore the relationship between SPL and PH,SPL-TG mice(carrying SPL transgenosis,and its SPL expression and activity were 3 times higher than WT mice)to observe the effects of SPL activity and overexpression on PH.We performed Control treatment and hypoxic chamber treatment on 8-week-old SPL-TG and WT C57BL/6J male mice.After 8 weeks of normal diet,M-mode ultrasonography was used to detect cardiac function in each group;pulmonary artery wall thickness was measured by histological analysis;SPL and α-SMA levels were detected by Western Blot;the pulmonary S1 P level was tested by ELISA kits;the activity of SPL was detected by fluorescence analysis experiment.4.The effect of SPL on ERK1/2 signal pathway was observed during the development of PHIn order to investigate the effect of SPL on the pathogenesis of PH,we divided8-week-old male C57BL/6J mice into Control group and PAH8 W group,administrated them with THI and control solvent respectively.The 8-week-old SPL-TG and WT C57BL/6J male mice were conducted Control treatment and PAH 8W treatment.After 8weeks,the pulmonary tissues of each group were extracted,ERK1/2 phosphorylation level in pulmonary tissue proteins of mice in each group were tested by Western Blot technique.Statistical AnalysisExperimental data were expressed as mean ± standard deviation(Mean±SEM).Statistical analysis was performed by using Graph Pad Prism-6.0 software.Data comparison between two groups was analyzed by using Student’s t-test.Data analyses of two or more groups were performed by using two-way ANOVA followed by post-hoc analysis.All data were statistically different at P < 0.05.Research Results1.The activity and expression of SPL was reduced in Pulmonary Tissue of PH Mice1)Pulmonary SPL level of mice with hypoxic-induced PH was reduced and their activity also was reduced.After the successful establishment of mouse model of hypoxia-induced PH,protein was extracted from pulmonary tissues of mice.The level of SPL was measured by Western Blot technique.Compared with the Control group,the level of SPL in PH group mice decreased.The activity of SPL was detected by fluorescence analysis.Compared with Control group,PH group had significantly lower SPL activity.2)Increased S1 P level in pulmonary tissue of mice with PH.The expression of S1 P in pulmonary tissue of mice was detected by ELISA.The level of S1 P was significantly higher in mice of PH group than in Control group.2.The inhibition of the activity of SPL aggravated the pathological changes on PH mice1)The use of THI aggravated the progression of right ventricular hypertrophy in mice.Echocardiography showed that PH 8W mice with for 8 weeks had significant right ventricular hypertrophy compared to the Control group.After administrating THI,the THI group had more severe right ventricular hypertrophy compared to the Vehicle group.2)THI reduced the activity of SPL and increased the level of S1 P.SPL level in pulmonary tissue of mice was measured by Western Blot technique.Pulmonary SPL expression in hypoxia-induced PH 8-week mice was significantly reduced,compared with the Control treatment.Expression of α-SMA increased significantly in PH8 W mice,compared with the Control group.On this basis,the trend of overexpression ofα-MSA in groups administrated THI was higher,compared with the Vehicle treatment.Fluorescence analysis experiment showed that the SPL activity of PH 8W mice was lower than that of the Control group.The results of ELISA showed that the level of S1 P was significantly higher.Compared to the Vehicle group,the above change was more significant in THI administration group.SPL activity reduction was more significant,and the trend of S1 P level increase was also more obvious.3.The pathological changes of PH were significantly reduced in SPL-TG mice1)Compared with WT mice,right ventricular hypertrophy caused by hypoxia-induced PH in SPL-TG mice were antagonized.Echocardiography showed that PH 8W mice had significant right ventricular hypertrophy compared with the Control group.And based on this,compared with WT group,right ventricular hypertrophy of SPL-TG group was relieved.2)SPL expression and activity in pulmonary tissue of SPL-TG mice raised,but their S1 P level was decreased.Analyzed by Western Blot technique and fluorescence analysis experiment,the expression and activity of SPL in pulmonary tissue were tested and the result showed that compared with the WT group,the activity and expression of SPL in SPL-TG mice were increased.Through ELISA experiment,it is found that compared with Control group,the S1 P level of PH 8W mice was increased significantly,but compared with WT group,the S1 P level increasing trend of SPL-TG mice was relieved.4.During the development of PH,the phosphorylation level of ERK1/2 signal pathway was inhabited by SPL in pulmonary tissue,which suggests that SPL may regulate the progression of PH through regulating ERK1/2 signal pathway.The phosphorylation level of ERK1/2 in pulmonary tissue of mice was detected by Western Blot technique,and phosphorylation level of ERK1/2 of PH 8W mice was significantly upregulated compared with the Control group.While after applying THI,and reducing the SPL activity of mice,the phosphorylation level of ERK1/2 of mice in THI group was higher upregulated compared with that of mice with Vehicle treatment..After the increasing of SPL expression and activity in SPL-TG group,its phosphorylation level increasing trend of ERK1/2 was relieved compared with mice in WT group.Conclusions1.We firstly observe the effect on PH after inhibiting SPL and find that inhibiting the activity of SPL would increase the level of S1 P in pulmonary tissue,thereby aggravate the development of PH.2.We use the SPL-TG mice to observe the effect of SPL on PH,and find that SPL caused the decrease of S1 P level in pulmonary tissue,so that the process of hypoxia-induced PH is antagonized.In conclusion,this experiment SPL plays an important role in the pathogenetic process of PH.The down regulated expression of SPL activity and the excitation of the S1 P signal pathway are both essential factors in the progression of PAH.This also suggested that SPL might be a novel and effective target for the treatment of PH. |
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