Astragaloside Ⅳ Improves Inflammation And Vascular Remodeling In Pulmonary Arterial Hypertension Rats Via Calpain-1/NLRP-3 Signaling Pathway

ObjectiveIn this study,our aim was to study the protective effect of Astragaloside Ⅳ(ASⅣ)on monocrotaline(MCT)-induced rats with pulmonary arterial hypertension(PAH)and explore its potential mechanism.MethodsIn vivo,male SD rats were given a single intraperitoneal injection of 60mg/kg of MCT to establish PAH model.The right jugular vein intubation of the rat detected mean pulmonary arterial pressure(m PAP)≥ 25 mm Hg as a pulmonary hypertension model.After the successful establishment of the PAH model in rats,rats were given ASⅣ(40 and 80 mg/kg)by gavage for 28 days,and intraperitoneal injections of NLRP-3 inhibitor(MCC950)and calpain-1inhibitor(MDL-28170)(once a week).The rats were randomly divided into 6groups: blank control group;MCT group;MCT+MCC950(3 mg/kg);MCT+MDL-28170(20 mg/kg);MCT+ASⅣ(40 mg/kg)group and MCT+ASⅣ(80mg/kg)group.The morphological changes of rat cardiomyocytes was measured by Hematoxylin-Eosin(HE)staining.The fibrous collagen deposition of rat lung tissue was measured by Masson staining.The morphological changes of pulmonary arterioles in rat lung tissue was measured by HE staining and Sirius scarlet staining.2-Ethidium hydrogen(DHE)staining was used to observe the formation of reactive oxygen species(ROS)in rat lung tissue.ELisa method was used to detect the content of IL-6,TNF-α,IL-18,IL-1β,malondialdehyde(MDA)and Glutathione reductase(GSH-px).The expression of calpain-1 and NLRP-3 positive cells in rat lung tissue was measured by immunohistochemical.The expression of calpain-1and NLRP3-in rat lung tissue was measured by immunofluorescence.In vitro,human pulmonary artery endothelial cells(HPAECs)were incubated with monocrotaline pyrroline(MCTP)for 30 minutes,and MCC950(10 μmol/L),MDL-28170(10 μmol/L)and ASⅣ(50 μmol/L and 100 μmol/L)were added for 24 hours.The HPAECs were divided into six groups: blank control group;MCTP group: MCTP + MCC950 group;MCTP + MDL-28170group;MCTP+ASⅣ 50 μmol/L group;MCTP+ASⅣ 100 μmol/l L group.The proliferation of endothelial cells in HPAECs was detected by EDU proliferation kit.ROS formation in HPAECs was observed by DHE staining.The levels of IL-6,TNF-α,IL-18,IL-1β,MDA and GSH-px in HPAECs were detected by ELISA.The levels of calpain-1 and NLRP-3 were detected by Western blot.Calpain-1 knockout mice and C57 BL / 6N wild-type mice were injected with MCT(300 mg / kg)intraperitoneally to establish PAH model.The mice were divided into four groups: the wild type blank control group;the wild type MCTP group;calpain-1 knockout blank control group;calpain-1 knockout MCTP group.The right jugular vein catheterization was used to detect the m PAP of mice,and the success of the model was considered if the m PAP was more than 25 mm Hg.At the same time,RVSP and RV/LV+S ratio were measured by right jugular vein catheterization.The morphological changes of pulmonary arterioles in rat lung tissue was measured by HE staining.The levels of calpain-1,NLRP-3,caspase-1,IL-18 and IL-1β in lung tissue were detected by Western blot,and the expression of NLRP-3 in lung tissue was observed by immunofluorescence.ResultsIn vivo: compared with the control group,the hemodynamic index and cross-sectional area of cardiac cells increased significantly in MCT group;the deposition index of collagen and dry wet weight of lung tissue in MCT group were increased;the ratio of wall thickness(WT(%))and wall area(WA(%))of the lung tissue in MCT group were significantly increased;ROS in the lung tissue of MCT group was significantly increased;the content of MDA in rats was increased;the content of GSH-px was decreased in rat serum.The levels of IL-6,TNF-α,IL-18 and IL-1 β in the serum of MCT group were increased,and the levels of calpain-1 and NLRP-3 in the lung tissue of rats were increased.Compared with MCT group,the hemodynamic index and cross-sectional area of myocardial cells were significantly reduced in MCC950 group,MDL-28170 group and ASⅣ group;the deposition index of collagen and W/D index of rat lung tissue were significantly reduced;WT(%)and WA(%)in rat lung tissue were significantly reduced;the level of ROS decreased in rat lung tissue,the content of MDA in rats was decreased;the content of GSH-px was increased in rat serum.The levels of IL-6,TNF-α,IL-18 and IL-1βdecreased,and the levels of calpain-1 and NLRP-3 in the lung tissue of rats were decreased.In vitro: compared with the blank control group,the expression of EDU positive cells in HPAECs in MCTP group increased;the levels of IL-6,TNF-α,calpain-1,NLRP-3,IL-18 and IL-1β in HPAECs in MCTP group increased.Compared with the MCTP group,the expression of EDU-positive cells in HPAECs in the MCC950,MDL-28170 and ASⅣ groups decreased;the levels of IL-6,TNF-α,calpain-1,NLRP-3,IL-18 and IL-1β in HPAECs decreased.In calpain-1 knockout mice experiment: compared with the blank control group(wild type and knockout type),in wild-type MCT group mice,the ratio of RV/LV+S,RVSP,and m PAP were significantly increased;WA(%)and WT(%)in lungs tissues were significantly increased;calpain-1,NLRP-3,caspase-1,the levels of ASC,IL-18 and IL1-β in lung tissue were significantly increased.Compared with the wild-type MCT group,in calpain-1 knockout MCT group,the ratio of RV/LV+S,RVSP,and m PAP were significantly decreased;WA(%)and WT(%)in lungs tissues were significantly decreased;the levels of calpain-1,NLRP-3,caspase-1,ASC,IL-18 and IL-1β in lung tissue were significantly decreased.ConclusionsThe results show that ASⅣ has protective effect on MCT induced PAH in rats,and the mechanism may be through calpain-1 / NLRP-3 signaling pathway.(1)ASⅣ reduced the inflammatory response of PAH rats.(2)ASⅣ improved pulmonary arteriole remodeling in PAH rats.(3)ASⅣ may play a role through calpain-1 / NLRP-3 signaling pathway.