Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by limited therapeutic options and a poor prognosis. Trilobatin (TLB), a natural dihydrochalcone extracted from Lithocarpus polystachyus, possesses diverse bioactivities; however, its anti-fibrotic potential and underlying mechanisms remain poorly defined. Here, we demonstrate that TLB effectively mitigates bleomycin (BLM)-induced pulmonary fibrosis in vivo and suppresses transforming growth factor-beta 1 (TGF-β1)-driven epithelial-mesenchymal transition (EMT) in vitro. In fibrotic mice, TLB treatment significantly attenuated pro-inflammatory cytokine secretion, limited inflammatory cell infiltration, preserved alveolar architecture, and decreased collagen deposition, ultimately leading to improved survival rates. Through network pharmacology and molecular docking, we identified MEK1 as a primary molecular target of TLB, an interaction subsequently validated by the Cellular Thermal Shift Assay (CETSA). Mechanistically, TLB directly uncoupled the TGF-β-induced ERK signaling cascade by selectively inhibiting the phosphorylation of MEK1 and ERK1/2, without affecting the upstream activation of Raf1. Furthermore, pharmacological hyperactivation of MEK1 via forskolin abrogated the protective effects of TLB against EMT and fibrogenesis. Collectively, our findings reveal a previously unrecognized anti-fibrotic mechanism of TLB, suggesting that this natural small molecule is a promising therapeutic candidate for IPF by specifically targeting MEK1-dependent ERK signaling.

BACKGROUND:Osteopontin (OPN), a multifunctional bioactive protein, has been shown to be elevated in the serum and bronchoalveolar lavage fluid (BALF) of. individuals with asthma and modulate the airway smooth muscle cells (ASMCs) proliferation and migration, yet its underlying molecular mechanisms are not completely understood. The aim of the present study is to address this issue. METHODS:Primary rat ASMCs were cultured to verify the effects of OPN and focal adhesion kinase (FAK)/Src/yes-associated protein (YAP) axis on cell proliferation and migration. Cell proliferation was assessed by BrdU incorporation assay. Cell migration was assessed by Transwell assay. Immunofluorescence and immunoblotting analysis confirmed that OPN activated FAK/Src/YAP axis. Moreover, we established an ovalbumin (OVA)-induced asthma rat model to verify whether OPN/FAK/Src/YAP cascade axis was involved in airway remodeling and asthma progression. RESULTS:OPN induced YAP dephosphorylation and nuclear localization via the αVβ3 integrin mediated activation of FAK/Src pathway, and this in turn increased thrombospondin 1 (THBS1) expression leading to ASMCs proliferation and migration. Intriguingly, THBS1 further activated FAK/Src to form a positive feedback loop. Pre-treatment of cells with anti-αvβ3 neutralizing antibody, FAK inhibitor PF573228 or Src inhibitor Dasatinib blocked OPN-induced alterations of YAP, THBS1 and functions of ASMCs. In addition, OPN promotes airway remodeling in asthma by activating FAK/Src/YAP axis. CONCLUSIONS:Our study indicates that OPN stimulates ASMCs proliferation and migration by binding to αVβ3 and activating FAK/Src/YAP axis, suggesting that targeting this pathway might have potential value in the management of airway remodeling of asthma.

The continuous increase of nitrate (NO3-) level in rivers is a hot issue in the world. However, the driving mechanism of high NO3- level in large rivers is still lacking, which has limited the use of river water and increased the cost of water treatment. In this study, multiple isotopes and source resolution models are applied to identify the driving mechanism of high NO3- level and key processes of nitrogen cycling in the lower reaches of the Yellow River (LRYR). The major sources of NO3- were sewage and manure (SAM) in the low-flow season and soil nitrogen (SN) and chemical fertilizer (CF) in the high-flow season. Nitrification was the most key process of nitrogen cycling in the LRYR. However, in the biological removal processes, denitrification may not occur significantly. The temporal variation of contributions of NO3- sources were estimated by a source resolution model in the LRYR. The proportional contributions of SAM and CF to NO3- in the low-flow and high-flow season were 32.5%-52.3%, 44.2%-46.2% and 36.0%-40.8%, 54.9%-56.9%, respectively. The driving mechanisms of high NO3- level were unreasonable sewage discharge, intensity rainfall runoff, nitrification and lack of nitrate removal capacity. To control the NO3- concentration, targeted measures should be implemented to improve the capacity of sewage and wastewater treatment, increase the utilization efficiency of nitrogen fertilizer and construct ecological engineering. This study deepens the understanding of the driving mechanism of high nitrate level and provides a vital reference for nitrogen pollution control in rivers to other area of the world.

This study aims to clarify molecular mechanisms and tumor-associated functions of LINC00312 in lung cancer. GEO database was used to acquire lung cancer-related expression microarrays. Then, relevant databases were applied to predict the downstream miRNA for LINC00312 and the target mRNA for the potential miRNA, with their associations deeply confirmed through dual-luciferase and RIP assays. The expression levels of epithelial-mesenchymal transition-related proteins (N-cadherin, Vimentin, MMP-2, and MMP-9) were examined by Western blot. The prolifer-ation, migration, and invasion were evaluated through in vitro experiments including CCK-8 and Transwell assays and further validated by nude mouse xenograft tumor experiment. LINC00312, serving as a tumor suppressor, was down -regulated in lung cancer cells. RIP assay proved that miR-3175 bound LINC00312 and SEMA6A. The dual-luciferase assay showed that miR-3175 specifically targeted SEMA6A, suppressing the expression of SEMA6A. Overexpressing LINC00312 remarkably inhibited the binding between miR-3175 and SEMA6A. Overexpressing miR-3175 or silencing SEMA6A could hamper the effects of LINC00312 on lung cancer cells. LINC00312 inhibits lung cancer occurrence and progression via the miR-3175/SEMA6A axis.

Objective: To study the application effects of teach-back method combined with WeChat education in patients with chronic obstructive pulmonary disease (COPD). Methods: Convenience sampling was used to select 103 patients with COPD hospitalized in the department of respiratory medicine of a tertiary-level hospital from March to June 2021 as study subjects. Randomized grouping was carried out using the random number table method. The routine care was given in the control group, and the teach-back method combined with WeChat tutorials on the basis of routine care was applied in the intervention group. In the follow-up three months after discharge, the changes in pulmonary function, self-care ability, and quality of life of patients in the two groups were observed and compared. Results: After the intervention, the pulmonary function indexes of both groups improved significantly, and the improvement effect was more significant in the intervention group (P < 0.05); after the intervention, the self-care ability scores of the patients in the intervention group were significantly higher than those of the control group (P < 0.05); the quality-of-life scores of the patients in the two groups decreased at 3 months after discharge, and the scores of the patients in the intervention group were significantly lower than those of the patients in the control group (P < 0.05). Conclusion: The teach-back method combined with WeChat education can effectively improve the pulmonary function, self-care ability, and quality of life of patients with COPD.

River cascade development is one of the human activities that have the most significant impact on the water environment. However, the mechanism of cascade development affecting river hydrochemical components still needs to be further studied. In this study, water quality index(WQI), positive matrix factorization(PMF) model and multivariate statistical techniques were used to identify the mechanism of cascade development affecting river hydrochemical components in an typical cascade development Rivers, Lancang River, China. The results showed that the water quality of Lancang River is relatively good due to less affected by human activity. The spatial variation of river hydrochemistry is affected by the development of cascade reservoirs, and shows three patterns: irregular variation (pH and DO), fluctuating decreasing (Na+, Cl-, SO42- and HCO3-) and multi-peak variation (TN, TDN, NO3--N and NH4+-N). It's worth noting that the concentration of the most hydrochemical parameters is higher in the upper reaches (less human activities) than that in the middle and lower reaches of river due to the retention effect of the reservoir on the chemical composition. The PMF model outputs revealed that the rock weathering and internal source, sewage and soil nitrogen, and chemical fertilizer were primary material sources of Lancang River. Compared with the natural channel zone (41.0%), the interaction of water-rock has more influence on chemical component in the reservoir area (56.3%), while the contribution of fertilizer (11.2%) to the river hydrochemistry is less. The sites of downstream of the reservoir dam were affected by the retention of the reservoir and the disturbance of the bottom drainage, which leads to the weakening of the influence of the sewage (44.7%) on the river material and the increase of the contribution of fertilizer (25.0%). These results could provide valuable information in controlling the eutrophication of cascade reservoirs and the scientific construction of river cascade reservoirs.

目的 探讨和厚朴酚对慢性哮喘模型大鼠气道炎症和气道重塑的影响及其可能机制.方法 24 只雄性SD大鼠随机分为4 组(每组各6 只):对照组、卵清蛋白(ovalbumin,OVA)模型组、和厚朴酚组以及地塞米松组.OVA模型组第 1,7,14天腹腔注射新鲜配制OVA混悬液致敏,第21 天起接受 1%OVA溶液雾化吸入30 min/次,隔日 1 次,共雾化8 周.对照组PBS代替OVA腹腔注射及雾化吸入;和厚朴酚组和地塞米松组每次OVA雾化前 1h分别腹腔注射和厚朴酚溶液(5 mg/kg)和地塞米松(0.5 mg/kg).末次激发后 24h处死大鼠,观察各组大鼠肺组织病理变化;收集支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)进行炎性细胞计数及分类;ELISA法检测血清及BALF中IL-4、IL-6、IL-17 含量;免疫印迹法检测肺组织Yes相关蛋白(Yes-associated protein,YAP)水平变化.结果 与对照组相比,OVA模型组支气管管壁、平滑肌层增厚,气道及血管周围大量炎性细胞浸润;BALF炎性细胞总数、嗜酸性粒细胞及中性粒细胞数显著增加(P＜0.01),BALF中IL-4、IL-6、IL-17 水平显著增高(P＜0.01),大鼠肺组织内YAP表达增多(P＜0.01).与OVA模型组相比,和厚朴酚组及地塞米松组大鼠肺组织病理改变明显减轻,BAFL中炎性细胞总数、嗜酸性粒细胞及中性粒细胞数显著减少(P＜0.01),IL-4、IL-6、IL-17 水平降低(P＜0.01),而和厚朴酚组与地塞米松组间上述改变相似;与OVA模型组相比,和厚朴酚组大鼠肺组织内YAP表达减少(P＜0.01).结论 和厚朴酚可以抑制慢性哮喘模型大鼠气道炎症和气道重塑,推测其作用机制可能与和厚朴酚下调YAP表达有关.

The aims of the present study were to examine the signaling mechanisms for transforming growth factor-β1 (TGF-β1)-induced rat airway smooth muscle cells (ASMCs) proliferation and migration and to determine the effect of lipoxin A4 (LXA4) on TGF-β1-induced rat ASMCs proliferation and migration and its underlying mechanisms. TGF-β1 upregulated transcriptional coactivator Yes-associated protein (YAP) expression by activating Smad2/3 and then upregulated cyclin D1, leading to rat ASMCs proliferation and migration. This effect was reversed after treatment with the TGF-β1 receptor inhibitor SB431542. YAP is a critical mediator of TGF-β1-induced ASMCs proliferation and migration. Knockdown of YAP disrupted the pro-airway remodeling function of TGF-β1. Preincubation of rat ASMCs with LXA4 blocked TGF-β1-induced activation of Smad2/3 and changed its downstream targets, YAP and cyclin D1, resulting in the inhibition of rat ASMCs proliferation and migration. Our study suggests that LXA4 suppresses Smad/YAP signaling to inhibit rat ASMCs proliferation and migration and therefore has potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.

ObjectiveEarly life bronchiolitis has been hypothesised to be associated with the subsequent risk of persistent wheezing or asthma. However, the link remains controversial. The objective of our study was to evaluate the association between bronchiolitis before 2 years of age and the late-onset wheezing/asthma.DesignSystematic review and meta-analysis.MethodsPubMed, Embase and Web of Science databases were systematically searched for studies published between 1955 and January 2020. Meanwhile, we also checked through the reference lists of relevant articles to see whether these references included reports of other studies that might be eligible for the review. Cohort and case–control studies assessing the association between early-life bronchiolitis and late-onset wheezing/asthma were included in this meta-analysis. Data were extracted by two independent reviewers. Results were pooled using a random-effects model or fixed-effects model according to the heterogeneity among studies.Results32 original articles with 292 844 participants, which met the criteria, were included in this meta-analysis. Bronchiolitis before 2 years of age was associated with an increased risk of subsequent wheezing/asthma (relative risk=2.46, 95% CI 2.14 to 2.82, p<0.001). After categorising studies into different groups based on age at the end of follow-up, geographical region and study quality, the association still remained significant.ConclusionsThe meta-analysis indicates an association between bronchiolitis before 2 years of age and the wheezing/asthma in later life. Well-designed and highly standardised prospective studies that better address bias due to potential confounding factors are needed to validate the risk identified in our meta-analysis.PROSPERO registration numberCRD42018089453.

This meta-analysis was performed to determine the effects of continuous positive airway pressure (CPAP) on blood pressure (BP) in patients with systemic hypertension and obstructive sleep apnea (OSA). A systematic search was conducted using PubMed, Embase, Web of Science, Cochrane Library, and clinicaltrials.gov, without language restrictions. Randomized controlled trials on the treatment of hypertension and OSA with CPAP, compared with sham CPAP or no CPAP, were reviewed. Studies were pooled to obtain weighted mean differences (WMDs) with 95% confidence intervals (CIs). Nineteen trials (enrolling 1904 participants) met the inclusion criteria. CPAP had significant effects on 24-h systolic blood pressure (SBP) (WMD −5.01 mmHg, 95% CI −6.94 to −3.08; P < 0.00001), 24-h diastolic blood pressure (DBP) (WMD −3.30 mmHg, 95% CI −4.32 to −2.28; P < 0.00001), daytime SBP (WMD −4.34 mmHg, 95% CI −6.27 to −2.40; P < 0.0001), daytime DBP (WMD −2.97 mmHg, 95% CI −3.99 to −1.95; P < 0.00001), nighttime SBP (WMD −3.55 mmHg, 95% CI −5.08 to −2.03; P < 0.00001), nighttime DBP (WMD −2.33 mmHg, 95% CI −3.27 to −1.40; P < 0.00001), office SBP (WMD −3.67 mmHg, 95% CI −5.76 to −1.58; P = 0.0006), office DBP (WMD −2.61 mmHg, 95% CI −4.25 to −0.97; P = 0.002), and heart rate (WMD −2.79 beats/min, 95% CI −4.88 to −0.71; P = 0.009). CPAP treatment was associated with BP reduction in patients with systemic hypertension and OSA, except when the follow-up period was shorter than 3 months.

Pulmonary arterial hypertension is a devastating pulmonary vascular disease, in which the pathogenesis is complicated and unclear. Pulmonary arterial smooth muscle cells (PASMCs) proliferation is a key pathological feature of pulmonary arterial hypertension. It has been shown that ubiquitin-specific protease 7 (USP7) is involved in cancer cell proliferation via deubiquitinating and stabilizing E3 ubiquitin ligase mouse double minute 2 (MDM2). However, the effect of USP7 and MDM2 on platelet-derived growth factor (PDGF)-induced PASMCs proliferation is uncertain. This study aims to explore this issue. Our results indicated that PDGF up-regulated USP7 protein expression and stimulated PASMCs proliferation; this was accompanied with the increase of MDM2, forkhead box O4 (FoxO4) reduction and elevation of CyclinD1. While prior transfection of USP7 siRNA blocked PDGF-induced MDM2 up-regulation, FoxO4 down-regulation, increase of CyclinD1 and cell proliferation. Pre-depletion of MDM2 by siRNA transfection reversed PDGF-induced reduction of FoxO4, up-regulation of CyclinD1 and PASMCs proliferation. Furthermore, pre-treatment of cells with proteasome inhibitor MG-132 also abolished PDGF-induced FoxO4 reduction, CyclinD1 elevation and cell proliferation. Our study suggests that USP7 up-regulates MDM2, which facilitates FoxO4 ubiquitinated degradation, and subsequently increases the expression of CyclinD1 to mediate PDGF-induced PASMCs proliferation.

It has been shown that sphingosine-1-phosphate (S1P) is elevated in patients with pulmonary arterial hypertension (PAH) and promotes the proliferation of pulmonary artery smooth muscle cells (PASMCs). Meanwhile, S1P has been found to induce the activation of autophagy in several types of human diseases including cancers. However, it is still unclear whether activation of autophagy mediates S1P-induced PASMCs proliferation, and detailed mechanisms responsible for these processes are indefinite. The aims of this study are to address these issues. S1P dose- and time-dependently reduced the expression of E-cadherin/CDH1 and stimulated PASMCs proliferation; this was accompanied with the elevation of TNF receptor-associated factor 2 (TRAF2), up-regulation and ubiquitination of BECN1 and the activation of autophagy. Prior silencing TRAF2 or BECN1 using siRNA or pre-incubation of cells with autophagy inhibitor chloroquine phosphate (CQ) suppressed S1P-induced autophagy activation and subsequent CDH1 degradation and further PASMCs proliferation. Taken together, our study indicates that S1P promotes the activation of autophagy by accelerating TRAF2-mediated BECN1 up-regulation and ubiquitination, which in turn results in CDH1 reduction and contributes to PASMCs proliferation.

The aims of the present study were to investigate the signaling mechanisms for sphingosine-1-phosphate (S1P)-induced airway smooth muscle cells (ASMCs) proliferation and to explore the effect of activation of adenosine monophosphate-activated protein kinase (AMPK) on S1P-induced ASMCs proliferation and its underlying mechanisms. S1P phosphorylated signal transducer and activator of transcription 3 (STAT3) through binding to S1PR2/3, and this further sequentially up-regulated polo-like kinase 1 (PLK1) and inhibitor of differentiation 2 (ID2) protein expression. Pretreatment of cells with S1PR2 antagonist JTE-013, S1PR3 antagonist CAY-10444, knockdown of STAT3, PLK1 and ID2 attenuated S1P-triggered ASMCs proliferation. In addition, activation of AMPK by metformin inhibited S1P-induced ASMCs proliferation by suppressing STAT3 phosphorylation and therefore suppression of PLK1 and ID2 protein expression. Our study suggests that S1P promotes ASMCs proliferation by stimulating S1PR2/3/STAT3/PLK1/ID2 axis, and activation of AMPK suppresses ASMCs proliferation by targeting on STAT3 signaling pathway. Activation of AMPK might benefit asthma by inhibiting airway remodeling.

Potential effects of particulate matter with an aerodynamic diameter less than 2.5 μm (PM2.5) on innate immunity have raised concerns. As the first defense line, macrophages are able to induce inflammatory response. However, whether PM2.5 exposure affects macrophage polarizations and its underlying mechanisms remain unclear. THP-1 monocytic leukemia cells were differentiated into macrophages using PMA. Intracellular ROS level was measured by flow cytometry. The levels of phosphorylation of AMPK, mTOR and ULK1 (Ser555 and Ser757) and expression of several critical regulators of autophagy were detected by western blot. The expression levels of cell surface markers (CCR7 and CD200R) and cytokines (TNF-α and CCL17) were detected by real-time PCR and ELISA, respectively. Additionally, specific inhibitors were used to address the molecular mechanisms. PM2.5 exposure triggered autophagy in macrophages (up-regulation of Hsp90, ATG5, Beclin1 and ratio of LC3II/I), this was accompanied with increased ROS level and AMPK phosphorylation, inhibition of mTOR, up-regulation of p-ULK1 (Ser555) and reduction of p-ULK1 (Ser757). Furthermore, inhibition of ROS or AMPK activity abolished PM2.5-induced autophagy activation and changes of mTOR, ULK1, Hsp90, ATG5, Beclin1 and LC3II/I. PM2.5 stimulation up-regulated the expression of CCR7 and TNF-α, and down-regulated CD200R and CCL17. Pre-incubation of cells with NAC or compound C or chloroquine blocked PM2.5-induced changes of these markers and cytokines. This study indicates that PM2.5 exposure enhanced inflammatory M1 polarization through ROS/AMPK/mTOR/ULK1/autophagy axis, and suggests that targeting this pathway might have potential value in the management of inflammatory diseases.

The upregulation of osteopontin(OPN) has been found to contribute to the proliferation of pulmonary artery smooth muscle cells(PASMCs), and activation of PPARγ has been shown to suppress OPN expression in THP-1 cells. However, the molecular mechanisms underlying the upregulation of OPN expression and PPARγ agonist modulation of OPN expression in PASMCs remain largely unclear. Here we found that S1P stimulated PASMCs proliferation and up-regulated OPN expression in rat PASMCs, which was accompanied with the activation of phospholipase C(PLC), calcineurin and translocation of NFATc3 to nucleus. Further study showed that inhibition of PLC by U73122, suppression of calcineurin activity by cyclosporine A(CsA) or knockdown of NFATc3 using small interfering RNA suppressed S1P-induced OPN up-regulation. Activation of PPARγ by pioglitazone suppressed S1P-induced activation of calcineurin/NFATc3 signaling pathway and followed OPN up-regulation. Taken together, our study indicates that S1P stimulates OPN expression by activation of PLC/calcineurin/NFATc3 signaling pathway, and activation of PPARγ suppresses calcineurin/NFATc3-mediated OPN expression in PASMCs.

Background/Aims: The underlying molecular mechanisms involved in sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) mediation of platelet-derived growth factor (PDGF)-induced pulmonary arterial smooth muscle cell (PASMC) proliferation are still unclear, and the present study aims to address this issue. Methods: Small interfering RNA (siRNA) and microRNA inhibitor transfection was performed to block the expression of SphK1, bone morphogenetic protein receptor II (BMPRII) and microRNA-21 (miR-21). Gene expression levels of SphK1, BMPRII and inhibitor of DNA binding 1 (Id1) were detected by immunoblotting, miR-21 expression level was examined with qRT-PCR, and S1P production was measured by ELISA. Additionally, PASMC proliferation was determined by BrdU incorporation assay. Results: Our results indicated that PDGF increased the expression of SphK1 protein and S1P production, up-regulated miR-21 expression, reduced BMPRII and Id1 expression, and promoted PASMCs proliferation. Pre-silencing of SphK1 with siRNA reversed PDGF-induced S1P production, miR-21 up-regulation, BMPRII and Id1 down-regulation, as well as PASMC proliferation. Pre-inhibition of miR-21 also blocked BMPRII and Id1 down-regulation as well as PASMC proliferation caused by PDGF. Knockdown of BMPRII down-regulated Id1 expression in PASMCs. We further found that inhibition of PI3K/Akt and ERK signaling pathways, particularly ERK cascade, suppressed PDGF-induced above changes. Conclusion: Our study indicates that SphK1/S1P pathway plays an important role in PDGF-induced PASMC proliferation via miR-21/BMPRII/Id1 axis and targeting against SphK1/S1P axis might be a novel strategy in the prevention and treatment of pulmonary arterial hypertension (PAH).

The aims of the current study were to examine the signaling mechanisms for transforming growth factor‐β1 (TGF‐β1)‐induced rat airway smooth muscle cell (ASMC) proliferation and to determine the effect of activation of peroxisome proliferation–activated receptor‐γ (PPAR‐γ) on TGF‐β1‐induced rat ASMC proliferation and its underlying mechanisms. TGF‐β1 upregulated microRNA 21 (miR‐21) expression by activating Smad2/3, and this in turn downregulated forkhead box O1 (FOXO1) mRNA expression. In addition, TGF‐β1–Smad–miR‐21 signaling also downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and thus de‐repressed the PI3K–Akt pathway. Depletion of PTEN reduced the nuclear FOXO1 protein level without affecting its mRNA level. Inhibition of the PI3K–Akt pathway or proteasome function reversed PTEN knockdown‐induced nuclear FOXO1 protein reduction. Our study further showed that loss of FOXO1 increased cyclin D1 expression, leading to rat ASMC proliferation. Preincubation of rat ASMCs with pioglitazone, a PPAR‐γ activator, blocked TGF‐β1‐induced activation of Smad2/3 and its downstream targets changes of miR‐21, PTEN, Akt, FOXO1, and cyclin D1, resulting in the inhibition of rat ASMC proliferation. Our study suggests that the activation of PPAR‐γ inhibits rat ASMC proliferation by suppressing Smad–miR‐21 signaling and therefore has a potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.

The aims of the present study were to examine signaling mechanisms underlying transforming growth factor β1 (TGF-β1)-induced airway smooth muscle cells (ASMCs) proliferation and to determine the effect of adenosine monophosphate-activated protein kinase (AMPK) activation on TGF-β1-induced ASMCs proliferation and its potential mechanisms. TGF-β1 reduced microRNA-206 (miR-206) level by activating Smad2/3, and this in turn up-regulated histone deacetylase 4 (HDAC4) and consequently increased cyclin D1 protein leading to ASMCs proliferation. Prior incubation of ASMCs with metformin induced AMPK activation and blocked TGF-β1-induced cell proliferation. Activation of AMPK slightly attenuated TGF-β1-induced miR-206 suppression, but dramatically suppressed TGF-β1-caused HDAC4 up-expression and significantly increased HDAC4 phosphorylation finally leading to reduction of up-regulated cyclin D1 protein expression. Our study suggests that activation of AMPK modulates miR-206/HDAC4/cyclin D1 signaling pathway, particularly targeting on HDAC4, to suppress ASMCs proliferation and therefore has a potential value in the prevention and treatment of asthma by alleviating airway remodeling.

The upregulation of Sphingosine kinase 1 (SphK1) expression and accompanied sphingosine-1-phosphate (S1P) production have been reported to contribute to the proliferation of pulmonary artery smooth muscle cells (PASMC) and pulmonary arterial remodeling. However, the molecular mechanisms of SphK1/S1P upregulation in PASMC and the specific mechanisms of how SphK1/S1P pathway promotes PASMC proliferation remain largely unclear. This study aims to address these issues. Here, we demonstrated that TGF-β1 significantly upregulated SphK1 expression and S1P production by promoting the phosphorylation of Smad2/3 in PASMC. Further study indicated that SphK1/S1P pathway mediated TGF-β1-induced Notch3 activation in PASMC. In addition, we showed that TGF-β1 significantly induced proliferation of PASMC, while pre-inhibition of Smad2/3 phosphorylation with SB431542 or silencing SphK1 using small interfering RNA in advance, or pre-blocking Notch3 pathway with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), attenuated TGF-β1-induced PASMC proliferation. Taken together, our study indicates that Smad2/3/SphK1/S1P/Notch3 pathway mediates TGF-β1-induced PASMC proliferation and suggests this pathway as a potential therapeutic target in the prevention and treatment of pulmonary hypertension.