Inhibitory Effect of PPARδAgonist GW501516 on Proliferation of Hypoxia-induced Pulmonary Arterial Smooth Muscle Cells by Regulating the mTOR Pathway

Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods PASMCs were incubated with different concentrations of GW501516(10,30,100 nmol/L)under the hypoxic condition.The proliferation was determined by a CCK-8 assay.The cell cycle progression was analyzed by flow cytometry.The expression of PPARδ,S phase kinase-associated protein 2(Skp2),and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting.Then PASMCs were treated with 100 nmol/L GW501516,100 nmol/L mammalian target of rapamycin(mTOR)inhibitor rapamycin and/or 2µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs.Results The presented data demonstrated that hypoxia reduced the expression of PPARδin an oxygen concentration-and time-dependent manner,and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle.In accordance with these findings,GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs.Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation,arresting the cell cycle,regulating the expression of Skp2 and p27,and inactivating mTOR in hypoxia-exposed PASMCs.Moreover,MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs.Conclusion GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.

NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1αexpression

Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4A1)activity alter the outcome of PAH.This study aimed to investigate the effects of NR4A1 on glycolysis in PAH and its underlying mechanisms.Methods:This study included twenty healthy volunteers and twenty-three PAH patients,and plasma samples were collected from the participants.To mimic the conditions of PAH in vitro,a hypoxia-induced model of pulmonary artery smooth muscle cell(PASMC)model was established.The proliferation of PASMCs was assessed using CCK8 assays.Results:Levels of NR4A1,hypoxia-inducible factor-1α(HIF-1α),and various glycolysis-related enzymes were measured.In addition,extracellular glucose and lactate production were assessed.The interaction between NR4A1 and HIF-1αwas evaluated by co-immunoprecipitation assays.Levels of NR4A1 and HIF-1αwas increased in PAH patients,and exposure to hypoxia resulted in increased levels of NR4A1 and HIF-1αin PASMCs.NR4A1 interacted with HIF-1α.NR4A1 overexpression enhanced hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,decreased glucose levels,increased lactate levels and promoted hypoxic PASMC viability.Conversely,silencing NR4A1 decreased hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,promoted glucose production,reduced lactate levels and inhibited hypoxic PASMC viability.Furthermore,overexpression of HIF-1αreversed the regulation of glycolysis caused by NR4A1 knockdown.Conclusion:NR4A1 enhances glycolysis in hypoxia-induced PASMCs by upregulating HIF-1α.Our findings indicate that the management of NR4A1 activity may be a promising strategy for PAH therapy.

Primary Ovarian Small Cell Carcinoma of Pulmonary Type: Analysis of 6 Cases and Review of 31 Cases in the Literatures

Objective Primary ovarian small cell carcinoma of pulmonary type(SCCOPT)is a rare ovarian tumor with a poor prognosis.The platinum-based chemotherapy is the standard treatment.However,there is little research on the clinical characteristics of SCCOPT and the potential benefits of other treatments due to its low incidence.The study aims to investigate clinicopathological characteristics and treatment of SCCOPT.Methods We summarized the clinical,imaging,laboratorical and pathological characteristics of 37 SCCOPT cases,in which 6 cases were admitted to the Gansu Provincial Hospital from the year of 2008 to 2022 and 31 cases reported in 17 English and 3 Chinese literatures.Results The median age of the studied SCCOPT cases(n=37)was 56.00(range,22-80)years.Almost 80%of them had a stageⅢorⅣtumor.All patients underwent an operation and postoperative chemotherapy.Nevertheless,all cases had a poor prognosis,with a median overall survival time of 12 months.Immunohistochemical y,the SCCOPT of all patients showed positive expressions of epithelial markers,such as CD56 and sex-determining region of Y chromosome-related high-mobility-group box 2(SOX-2),and negative expressions of estrogen receptor,progesterone receptor,vimentin,Leu-7,and somatostatin receptor 2.The tumor of above 80%cases expressed synaptophysin.Only a few cases expressed neuron-specific enolase,chromogranin A,and thyroid transcription factor-1.Conclusions SCCOPT had a poor prognosis.SOX-2 could be a biomarker to be used to diagnose SCCOPT.

Alleviation of acute pancreatitis-associated lung injury by inhibiting the p38 mitogen-activated protein kinase pathway in pulmonary microvascular endothelial cells

BACKGROUND Previous reports have suggested that the p38 mitogen-activated protein kinase signaling pathway is involved in the development of severe acute pancreatitis(SAP)-related acute lung injury(ALI).Inhibition of p38 by SB203580 blocked the inflammatory responses in SAP-ALI.However,the precise mechanism associated with p38 is unclear,particularly in pulmonary microvascular endothelial cell(PMVEC)injury.AIM To determine its role in the tumor necrosis factor-alpha(TNF-α)-induced inflammation and apoptosis of PMVECs in vitro.We then conducted in vivo experiments to confirm the effect of SB203580-mediated p38 inhibition on SAP-ALI.METHODS In vitro,PMVEC were transfected with mitogen-activated protein kinase kinase 6(Glu),which constitutively activates p38,and then stimulated with TNF-α.Flow cytometry and western blotting were performed to detect the cell apoptosis and inflammatory cytokine levels,respectively.In vivo,SAP-ALI was induced by 5%sodium taurocholate and three different doses of SB203580(2.5,5.0 or 10.0 mg/kg)were intraperitoneally injected prior to SAP induction.SAP-ALI was assessed by performing pulmonary histopathology assays,measuring myeloperoxidase activity,conducting arterial blood gas analyses and measuring TNF-α,interleukin(IL)-1βand IL-6 levels.Lung microvascular permeability was measured by determining bronchoalveolar lavage fluid protein concentration,Evans blue pulmonary cells was confirmed by performing a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis and examining the Bcl2,Bax,Bim and cle-caspase3 levels.The proteins levels of P-p38,NFκB,IκB,P-signal transducer and activator of transcription-3,nuclear factor erythroid 2-related factor 2,HO-1 and Myd88 were detected in the lungs to further evaluate the potential mechanism underlying the protective effect of SB203580.RESULTS In vitro,mitogen-activated protein kinase(Glu)transfection resulted in higher apoptotic rates and cytokine(IL-1βand IL-6)levels in TNF-α-treated PMVECs.In vivo,SB2035080 attenuated lung histopathological injury,decreased inflammatory activity(TNF-α,IL-1β,IL-6 and myeloperoxidase)and preserved pulmonary function.Furthermore,SB203580 significantly reversed changes in the bronchoalveolar lavage fluid protein concentration,Evans blue accumulation,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cell numbers,apoptosis-related proteins(cle-caspase3,Bim and Bax)and endothelial microstructure.Moreover,SB203580 significantly reduced the pulmonary P-p38,NFκB,P-signal transducer and activator of transcription-3 and Myd88 levels but increased the IκB and HO-1 levels.CONCLUSION p38 inhibition may protect against SAP-ALI by alleviating inflammation and the apoptotic death of PMVECs.

Iptakalim, a novel ATP-sensitive potassium channel opener, inhibits pulmonary arterial smooth muscle cell proliferation by downregulation of PKC-α

Iptakalim is a new ATP-sensitive potassium （KATp） channel opener, and it inhibits the proliferation of pulmonary arterial smooth muscle cells （PASMCs） and pulmonary vascular remodeling. However, the underlying mechanism remains unclear. In the present study, we found that iptakalim significantly decreased pulmonary artery pressure, inhibited pulmonary ariery remodeling and PKC-α overexpression in chronic hypoxia in a rat pulmonary hypertension model. Iptakalim reduced hypoxia-induced expression of PKC-α, and abolished the effect of hypoxia on PASMC proliferation significantly in a dose-dependent manner in vitro. Moreover, these effects were abol- ished by glibenclamide, a selective KArp channel antagonist. These results indicate that iptakalim inhibits PASMC proliferation and pulmonary vascular remodeling induced by hypoxia through downregulating the expression of PKC-α. Iptakalim can serve as a novel promising treatment for hypoxic pulmonary hypertension.

The Effect of Erigeron Breviscapus on Proliferation of Pulmonary Artery Smooth Muscle Cells in Hypoxic Porcines

In order to study the effect of Erigeron Breviscapus (EB) on proliferation of pulmonary artery smooth muscle cells (PASMC) in hypoxic porcines, immunohistochemical and MTT methods were employed to measure the proliferation of PASMC. It was found that the proliferation of PASMC in porcines was obvious, and the expression of proliferating cell nuclear antigen (PCNA) was significantly high within 48 h after exposure to hypoxia. The EB could inhibit the proliferation and the expression of PCNA in PASMC under hypoxia, but it had no effect on the proliferation and expression of PCNA in PASMC under normal condition. The EB could inhibit the proliferation and the expression of PCNA in PASMC induced by phorbol 12-myristate 13-acetate (PMA), an agonist of PKC in normal and hypoxic conditions. It was concluded that the hypoxia could enhance the proliferation and expression of PCNA in PASMC. The EB can inhibit the proliferation and expression of PCNA in PASMC under hypoxia through PKC-signal way. The EB may be used in treating the pulmonary hypertension by inhibiting the proliferation of PASMC and the pulmonary vascular remodeling.

Baicalin Induces IFN-α/β and IFN-γ Expressions in Cultured Mouse Pulmonary Microvascular Endothelial Cells

We studied the effect of baicalin,an extract from Radix Scutellariae（a traditional Chinese medicine） in inducing mouse pulmonary microvascular endothelial cells（MPMVECs） to produce interferons（IFNs）.MPMVECs were cultured in vitro in the presence of different concentrations of baicalin（10,20,and 30 μg mL-1）,and the cells and the culture media were harvested at various time intervals.The proteins and mRNA levels（relative to β-actin） of IFN-α/β and IFN-γ were analyzed by RT-PCR and enzyme-linked immunosorbent assay（ELISA）.It was observed that baicalin substantially up-regulated the expression of IFN-α/β and IFN-γ.In all baicalin-treated groups,the relative levels of IFN-α/β and IFN-γ mRNAs peaked after 12 h of culturing,and IFN-α/β and IFN-γ proteins peaked after 24 h of culturing.These results suggest that baicalin can effectively induce the expression of IFNs in pulmonary microvascular endothelial cells,and thus potentially act as an antiviral compound.This study may provide background information for developing new antiviral drugs based on baicalin.

Effects of calcium-activated chloride channels on proliferation of pulmonary artery smooth muscle cells in rats under chronic hypoxic condition

Objective：To investigate the effects of calcium-activated chloride （ClCa） channels on proliferation of pulmonary artery smooth muscle cells（PASMCs） in rats under chronic hypoxic condition. Methods：The cultured PASMCs were placed under normoxic and chronic hypoxic conditions：The cells were observed by light and electron microscope; The cell cycles were observed by flow-cytometry; Immunocytochemistry staining was used to detect the expressions of PCNA, c-fos and c-jun of PASMCs; Cytoplasmic free Ca^2＋ concentration （[Ca^2＋]i） in PASMCs was investigated by fluorescent quantitation using fluorospectrophotometer. Results：The PASMCs were contractile phenotype under normoxic conditions. Observation by transmission electron microscope： In kytoplasm of contractile phenotype cells, myofilament bundles were abundant and the content of cell organs such as Golgi＇s bodies were rare. The PASMCs were synthetic phenotype under chronic hypoxic condition. There were increased free ribosomes, dilated rough endoplasmic reticulums, highly developed Golgi complexes, decreased or disappeared thick filaments and dense body in kytoplasm of synthetic phenotype cells. After NFA and IAA-94, the situations were reversed The number of S ＋G2M PASMCs were significantly increased in chronic hypoxic condition; The NFA and IAA-94 were shown to significantly decrease them from （28.6±1.0）% to （16.0±1.6）% and the number of G0G1 PASMCs significantly increased from （71.4± 1.9）% to （83.9 ± 1.6）% （P〈 0.01）. In chronic hypoxic conditions, the expression of proliferating cell nucleus antigen was significantly increased; The NFA and IAA-94 were shown to significantly decrease it from （81 ± 6）% to （27 ± 7）%（P 〈 0.01）. The expression of c-fos and c-jun were significantly increased in＇chronic hypoxic conditions; The NFA and IAA-94 were shown to significantly decrease them from 0.15 ±0.02, 0.32 ± 0.05 to 0.05 ± 0.01, 0.12 ± 0.05, respectively （P〈 0.01）; Under chronic hypoxic conditions, [Ca^2＋]i was increased; The NFA and IAA-94 decreased it from （281.8±16,5）nmol/L to （117.7 ± 15.4）nmol/L（P 〈 0.01）. Conclusion：Hypoxia initiated the change of PASMCs from contractile to synthetic phenotype and increased proliferation of PASMCs. NFA and IAA-94 depressed cell proliferation by blocking ClCa channels in hypoxic condition. These may play an important role in proliferation of PASMCs under chronic hypoxic conditions.

Involvement of TRPC1 and Cyclin D1 in Human Pulmonary Artery Smooth Muscle Cells Proliferation Induced by Cigarette Smoke Extract

Cigarette smoking contributes to the development of pulmonary artery hypertension(PAH).As the basic pathological change of PAH,pulmonary vascular remodeling is considered to be related to the abnormal proliferation of pulmonary artery smooth muscle cells(PASMCs).However,the molecular mechanism underlying this process remains not exactly clear.The aim of this research was to study the molecular mechanism of PASMCs proliferation induced by smoking.Human PASMCs(HPASMCs)were divided into 6 groups:0%(control group),cigarette smoking extract(CSE)-treated groups at concentrations of 0.5%,1%,2%,5%,10%CSE respectively.HPASMCs proliferation was observed after 24 h.HPASMCs were divided into two groups:0(control group),0.5%CSE group.The mRNA and protein expression levels of transient receptor potential channel 1(TRPC1)and cyclin D1 in HPASMCs after CSE treatment were respectively detected by RT-PCR and Western blotting.The intracellular calcium ion concentration was measured by the calcium probe in each group.In the negative control group and TRPC1-siRNA transfection group,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein were detected.Data were compared with one-way ANOVA(for multiple-group comparison)and independent t-test(for two-group comparison)followed by the least significant difference(LSD)test with the computer software SPSS 17.0.It was found that 0.5%and 1%CSE could promote the proliferation of HPASMCs(P<0.05),and the former was more effective than the latter(P<0.05),while 3%and above CSE had inhibitory effect on HPASMCs(P<0.05).The mRNA and protein expression levels of TRPC1 and cyclin D1 in 0.5%and 1%CSE groups were significantly higher than those in the control group(P<0.05),while those in 3%CSE group were significantly decreased(P<0.05).Moreover,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein in TRPC1-siRNA transfection group were significantly reduced as compared with those in the negative control group(P<0.05).It was concluded that low concentration of CSE can promote the proliferation of HPASMCs,while high concentrations of CSE inhibit HPASMCs proliferation.These findings suggested that CSE induced proliferation of HPASMCs at least in part via TRPC1-mediated cyclin D1 expression.

DIFFERENTIAL EXPRESSION AND RESPONSE OF GROWTH FACTORS IN DIFFERENT METASTATIC VARIANTS OF HUMANPULMONARY GIANT CELL CARCINOMA

The cell lines PGbE1 and PGLH7, which have high and low metastatic potential respectively, were two different variants isolated from human pulmonary giant cell carcinoma cell line PG. This study compared the expression and response of several growth factors TGFa, TGFb, bFGF, IL 6, IL 8 and ANG in the two cell lines. By using RT PCR analysis and thymidine incor poration assay, it was found that IL 6, TGFa and their receptors IL 6R and EGFR were expressed at higher level in PGbE1 cells than in PGLH7 cells, while no significant differences were found in the expression of ANG, bFGF, IL 8, IL 8R and TGFβ. Recombinant IL 6 and TGFα stimulated the proliferation of both cells, while TGFβ had dual effects. These results suggest that ANG, bFGF IL 6, IL 8 and TGFα, β may be involved in the proliferation of pulmonary giant cell carcinoma via autocrine mechanism, and IL 6 and TGFa may play an important role in the metastasis of tumor cells.

DL0805 derivatives protect the pulmonary arterial cells via the RhoA/ROCK pathway

OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At present,several kinds of drugs have been used in the treatment of PAH.However,most of these drugs aim to relax pulmonary arteries and do not inhibit the injury of vessels.In other words,the drugs available for PAH treatment do not improve the survival rate of PAH patients and cannot satisfy the needs in clinic.To discover and develop novel candidate compounds effective on the treatment of pulmonary artery injury and remodeling will be very important.Based on these background,the present study aimed to study the protective effect of two novel Rho-kinases(Rho-associated coiledcoil forming protein serine/threonine kinase,ROCK)inhibitors,DL0805 derivatives(DL0805-1and DL0805-2),on pulmonary arterial cells and further evaluate the underlying mechanisms and the possibility of DL0805 derivatives become therapeutic drugs for PAH.METHODS The primary cultured pulmonary arterial cells including human pulmonary artery endothelium cells(HPAECs)and human pulmonary artery smooth muscle cells(HPASMCs)were used in this study.HPAECs were injured under hypoxia environment(1%O2)and treated with or without DL0805 derivatives.After 48 h,the proliferation and oxidative stress were observed.CCK8 was used to detect cell viability.DCFH-DA was used as probe for reactive oxygen species(ROS)under fluorescence imaging system.HPASMCs was stimulated by growth factors including platelet-derived growth factor-BB(PDGF-BB)and Fetal Bovine Serum(FBS).The proliferation was observed in the cells treated with or without DL0805 derivatives.HPASMCs treated with or without DL0805 derivatives were further incubated with endothelin(ET-1),the proliferation and cytoskeleton remodeling of cells were detected by immunofluorescence assay.At last,Western blotting(WB)and immunofluorescence assay were employed to analysis the underlying mechanisms in the above experiments.RESULTS 10μmol·L-1DL0805-2 could inhibit the proliferation of HPAECs induced by hypoxia.Each concentration of DL0805-1 and DL0805-2attenuated the production of ROS in HPAECs.Results from WB indicated that DL0805 derivatives decreased the injury of HPAECs induced by hypoxia through the inhibition of the expression of Rho A and the activity of ROCK.On HPASMCs,DL0805 derivatives reduced the proliferation induced by PDGF-BB and FBS and inhibited cytoskeleton remodeling induced by ET-1.Immunofluorescence assay showed that DL0805 derivatives inhibited ROCK activity and down regulated the phosphorylation levels of ROCK substrates.CONCLUSION DL0805derivatives exhibited protective effect on pulmonary arterial cells including endothelium cells and smooth muscle cells.Among the above experiments,DL0805-2 showed stronger potency than DL0805-1.These two compounds might protect the cells through the inhibition of Rho A/ROCK pathway and they probably have the potential in the treatment of PAH and deserve further evaluation.

Pulmonary Langerhans cell histiocytosis and multiple system involvement:A case report

BACKGROUND Pulmonary Langerhans cell histiocytosis(PLCH)is a relatively rare type of lung disease,common in middle-aged smoking men.It is characterized by proliferation and infiltration of Langerhans cells,and the formation of multiple parabronchial mesenchymal nodules in lung tissue,and may lead to organ dysfunction.There are no typical symptoms and signs,and it is easily misdiagnosed or missed,and therefore deserves clinical attention and further discussion.CASE SUMMARY We describe the case of a nonsmoking 46-year-old man with PLCH diagnosed based on clinical manifestations of fever and dry cough,with a history of hypothyroidism and diabetes insipidus for 9 years.Computed tomography(CT)-and CT-guided puncture examinations revealed no abnormalities,and he ultimately underwent thoracoscopic biopsy to confirm the diagnosis.The pathological diagnosis was PLCH.Thyroid function was maintained by medication.Pituitary magnetic resonance imaging showed that the pituitary stalk had become thinner.CONCLUSION LCH often involves multiple systems.Moreover,the pathogenesis is not clear,clinical manifestations lack specificity,and diagnosis requires special attention.Diagnosis of PLCH can significantly benefit from comprehensive multidisciplinary analysis.

Two smoking-related lesions in the same pulmonary lobe of squamous cell carcinoma and pulmonary Langerhans cell histiocytosis:A case report

BACKGROUND Pulmonary Langerhans cell histiocytosis(PLCH)is a rare cystic lung disease usually affecting young adults.It is predicted that PLCH is a lung tumor precursor associated with dysfunction of the myeloid dendritic cells in the lung.CASE SUMMARY A 70-year-old male patient presented with chronic cough and sputum.He had symptoms for 5 years and described shortness of breath on exertion for the previous 3 years.He had a 60 packs/year smoking history.Computerized tomography of the thorax revealed an 11-mm nodule in the right lung lower lobe superior segment and a 7-mm nodule in the right lung lower lobe poster basal segment.Those two nodules were resected by means of right thoracoscopic surgery.Pathological evaluation revealed a squamous cell carcinoma and PLCH.CONCLUSION Coexistent squamous cell carcinoma and PLCH suggest possible association between PLCH and lung cancer.

Hypoxia promotes cell proliferation by modulating E2F1 in chicken pulmonary arterial smooth muscle cells

In this study,we sought to investigate the expression of the transcription factor E2F1 in chicken pulmonary arterial smooth muscle cells upon hypoxia exposure,as well as the role that E2F1 played in the regulation of cell proliferation.Isolated chicken pulmonary arterial smooth muscle cells were subjected to hypoxia or normoxia for indicated time points.Cell viability,DNA synthesis,cell cycle profile,and expression of E2F1 were analyzed.The results showed that hypoxia promoted cell proliferation and DNA synthesis which was accompanied by an increased S phase entry and upregulation of E2F1 at mRNA and protein levels.Using siRNA technology,we demonstrated that gene inactivation of endogenous E2F1 abolished hypoxia-induced cell proliferation,DNA synthesis,and S phase entry compared with negative siRNA transfected cells.These results suggest that hypoxia-induced proliferation is mediated by inducing E2F1 in chicken pulmonary arterial smooth muscle cells.

Case report of a mixed pulmonary large cell neuroendocrine carcinoma

A 57 year-old male patient was found to have a lesion in the middle lobe of his right lung using chest computed tomography(CT).Tumor cells were detected,and surgical excision was performed.The patient was diagnosed with mixed large cell neuroendocrine carcinoma,and underwent six cycles of a chemotherapy regimen comprising etoposide combined with cisplatin.Genetic testing revealed an EGFR mutation,which prompted oxitinib-targeted therapy.To date,no signs of recurrence or metastasis have been reported.

Reconstruction of pulmonary artery by autologous pericardium or azygous venae for non-small cell lung cancer

Objective To review the clinical experience of reconstruction of pulmonary artery(PA) by a patch of autologus pericardium or azygous venae for non-small cell lung cancer. Methods Between March 1992 and August 2009,62 patients with locally advanced central lung cancer received sleeve resection and reconstruction of PA.

Bone Marrow Mesenchymal Stem Cells Ameliorate Lung Injury through Anti-inflammatory and Antibacterial Effect in COPD Mice

The anti-inflammatory and antibacterial mechanisms of bone marrow mesenchymal stem cells（MSCs） ameliorating lung injury in chronic obstructive pulmonary disease（COPD） mice induced by cigarette smoke and Haemophilus Parainfluenza（HPi） were studied. The experiment was divided into four groups in vivo： control group, COPD group, COPD＋HPi group, and COPD＋HPi＋MSCs group. The indexes of emphysematous changes, inflammatory reaction and lung injury score, and antibacterial effects were evaluated in all groups. As compared with control group, emphysematous changes were significantly aggravated in COPD group, COPD＋HPi group and COPD＋HPi＋MSCs group（P〈0.01）, the expression of necrosis factor-kappa B（NF-κB） signal pathway and proinflammatory cytokines in bronchoalveolar lavage fluid（BALF） were increased（P〈0.01）, and the phagocytic activity of alveolar macrophages was downregulated（P〈0.01）. As compared with COPD group, lung injury score, inflammatory cells and proinflammatory cytokines were significantly increased in the BALF of COPD＋HPi group and COPD＋HPi＋MSCs group（P〈0.01）. As compared with COPD＋HPi group, the expression of tumor necrosis factor-α stimulated protein/gene 6（TSG-6） was increased, the NF-κB signal pathway was depressed, proinflammatory cytokine was significantly reduced, the anti-inflammatory cytokine IL-10 was increased, and lung injury score was significantly reduced in COPD＋HPi＋MSCs group. Meanwhile, the phagocytic activity of alveolar macrophages was significantly enhanced and bacterial counts in the lung were decreased. The results indicated cigarette smoke caused emphysematous changes in mice and the phagocytic activity of alveolar macrophages was decreased. The lung injury of acute exacerbation of COPD mice induced by cigarette smoke and HPi was alleviated through MSCs transplantation, which may be attributed to the fact that MSCs could promote macrophages into anti-inflammatory phenotype through secreting TSG-6, inhibit NF-кB signaling pathway, and reduce inflammatory response through reducing proinflammatory cytokines and promoting the expression of the anti-inflammatory cytokine. Simultaneously, MSCs could enhance phagocytic activity of macrophages and bacterial clearance. Meanwhile, we detected anti-inflammatory and antibacterial activity of macrophages regulated by MSCs in vitro. As compared with RAW264.7＋HPi＋CSE group, the expression of NF-кB p65, IL-1β, IL-6 and TNF-α was significantly reduced, and the phagocytic activity of macrophages was significantly increased in RAW264.7＋HPi＋CSE＋MSCs group（P〈0.01）. The result indicated the macrophages co-cultured with MSCs may inhibit NF-кB signaling pathway and promote phagocytosis by paracrine mechanism.

Different Concentrations of Notoginsenoside Rg1 Attenuate Hypoxic and Hypercapnia Pulmonary Hypertension by Reducing the Expression of ERK in Rat PASMCs

Pulmonary arterial hypertension (PAH) is a serious disease which is characterized by increased vascular resistance and pressure. We have previously hypothesized that panax notoginseng saponins (PNS) might attenuate pulmonary vasoconstriction under hypoxia and hypercapnia condition. This study aims to investigate the effect of notoginsenoside Rg1, a main ingredient of PNS, with various concentrations (8, 40, 100 mg/L, respectively) on extracellular signal regulated kinase (ERK1/2) signaling pathway in pulmonary arterial smooth muscle cells (PASMCs). In addition, PASMCs were randomly divided into six groups: SD rat under normoxic condition as control group (N group), hypoxia hypercapnia group (H group), DMSO control group (HD group), Rg1-treatment groups (RgLRgM and RgH group). Western-blot and RT-PCR were used to test the expression of p-ERK protein and the expression of ERK1 mRNA and ERK2 mRNA. This study provided the evidence that the expression of p-ERK protein and the expression of ERK1 mRNA and ERK2 mRNA in HD group and H group were obviously higher than that in N group (P < 0.01), Whereas the level of ERK1/2 mRNA in Rg1-treatment groups was significantly lower than that in HD group and H group (P < 0.01), and the proper concentration of Rg1 is 40 mg/L. These results suggested that notoginsenoside Rg1 can attenuate pulmonary vasoconstriction which may lead to HHPV through reducing the expression of ERK1/2.

Calpain mediated pulmonary vascular remodeling in hypoxia induced pulmonary hypertension

OBJECTIVE To explore the role of calpain in in pulmonary vascular remodeling in hypoxia induced pulmonary hypertension and the underlying mechanism.METHODS Sprague-Dawley rats were randomly divided into hypoxia group and normoxia control group.Right ventricular systolic pressure(RVSP)and mean pulmonary artery pressure(m PAP)were monitored by the method of right external jugular vein cannula.Right ventricular hypertrophy index was expressed as the ratio of right ventricular weight to left ventricular weight(left ventricle plus septum weight).Level of calpain-1,calpain-2and calpain-4 m RNA in pulmonary artery trunk were determined by real-time PCR.Expression of calpain-1,calpain-2 and calpain-4 protein was determined by Western Blot.Primary rat pulmonary arterial smooth muscle cells(PASMCs)were divided into 4 groups:normoxia control group,normoxia+MDL28170 group,hypoxia group and hypoxia+MDL28170 group.Cell proliferation was detected by MTS and flow cytometry.Level of Ki-67 and PCNA m RNA were determined by real-time PCR.RESULTS RVSP,m PAP and right ventricular remodeling index were significantly higher in the hypoxia group than those in the normoxia group.In the hypoxia group,pulmonary vascular remodeling occurred,and the expression of calpain-1,calpain-2 and calpain-4 m RNA and protein expression was increased in the pulmonary artery.MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs accompanied with decreased Ki-67and PCNA m RNA expression.CONCLUSION Calpain mediated vascular remodeling via promoting proliferation of PASMCs in hypoxia induced pulmonary hypertension.

EPAS1 prevents telomeric damage-induced senescence by enhancing transcription of TRF1,TRF2,and RAD50

Telomeres are nucleoprotein structures located at the end of each chromosome,which function in terminal protection and genomic stability.Telomeric damage is closely related to replicative senescence in vitro and physical aging in vivo.As relatively long-lived mammals based on body size,bats display unique telomeric patterns,including the upregulation of genes involved in alternative lengthening of telomeres(ALT),DNA repair,and DNA replication.At present,however,the relevant molecular mechanisms remain unclear.In this study,we performed cross-species comparison and identified EPAS1,a well-defined oxygen response gene,as a key telomeric protector in bat fibroblasts.Bat fibroblasts showed high expression of EPAS1,which enhanced the transcription of shelterin components TRF1 and TRF2,as well as DNA repair factor RAD50,conferring bat fibroblasts with resistance to senescence during long-term consecutive expansion.Based on a human single-cell transcriptome atlas,we found that EPAS1 was predominantly expressed in the human pulmonary endothelial cell subpopulation.Using in vitro-cultured human pulmonary endothelial cells,we confirmed the functional and mechanistic conservation of EPAS1 in telomeric protection between bats and humans.In addition,the EPAS1 agonist M1001 was shown to be a protective compound against bleomycin-induced pulmonary telomeric damage and senescence.In conclusion,we identified a potential mechanism for regulating telomere stability in human pulmonary diseases associated with aging,drawing insights from the longevity of bats.