| Background and objective:Bronchial asthma is a common clinical respiratory disease,which is a kind of heterogeneous disease characterized by airway inflammation,airway remodeling and airway hyper-responsiveness,involving a variety of cells and cell components,such as airway inflammatory cells and structural cells.Hypoxia is believed to be a key factor in the progression of lung diseases,such as bronchial asthma,airway obstruction,and pulmonary hypertension.Hypoxia stimulates airway inflammation and airway remodeling,and induces the apoptosis of airway smooth muscle cell(ASMC)during airway remodeling.CD38 is a key enzyme for NAD~+degradation in vivo and plays an important role in regulating NAD~+levels and SIRT1 activity indispensable to NAD~+in cells.Evidences suggest that there are gender differences in the incidence and progression of lung diseases,and estrogen plays a crucial role in these pathological processes.Estrogen can affect smooth muscle contraction through CD38,but the specific molecular mechanism of its regulation is still unclear.This study aims to investigate the effect of CD38 deficiency on the apoptosis of mouse airway smooth muscle cells under hypoxic condition,and the regulation effect of estrogen on the CD38/SIRT1 signal axis,and to verify the protective effect of CD38deficiency on asthma in mice in an overall animal model,providing an important target and experimental basis for the treatment of asthma and other respiratory diseases.Methods:1.Estrogen(E2)regulation of CD38/SIRT1 expression in airway smooth muscle cells:isolation and in vitro culture of primary airway smooth muscle cells(ASMCs),treatment of ASMCs with different concentrations and different time of E2,detection of CD38 and SIRT1 by real-time PCR mRNA levels;Western blot analysis of CD38 and SIRT1 protein expression levels,clear dose-and time-dependent changes in E2 regulation of CD38 expression;comparing CD38,SIRT1,acetylated p53(Ac-p53)and total p53 expression levels of wild-type(WT)and CD38 knockout(CD38 KO)in ASMCs,as well as the intervention of E2 on their expression.2.Effect of CD38 on apoptosis of ASMCs under hypoxic conditions and intervention of E2:an in vitro hypoxic model was established,and the effect of E2 on CD38 and SIRT1 mRNA levels in ASMCs of WT and CD38 KO under hypoxic conditions was detected by real-time PCR;Western blot was used to detect the effect of E2 on CD38/SIRT1/p53 pathway after hypoxia stimulation;assayingapoptosis-related protein Bax and Bcl-2 expression analysis and Caspase-3 activity by Hoechst33258 staining;clarifying the protective effect of CD38 deletion on hypoxia-induced apoptosis of ASMCs and the intervention of E2.3.Molecular mechanism analysis of E2 regulation of apoptosis in ASMCs:observing of the effect of E2 on apoptosis of ASMCs in the presence of SIRT1-specific active agonist resveratrol(RSV);examining the effect of E2 on CD38 mRNA and protein expression in the presence of the estrogen receptor-specific inhibitor ICI182,780 to determine whether E2 regulates CD38 expression via estrogen receptors;elucidating through the above experiments the molecular mechanism of E2 affecting apoptosis of ASMCs by regulating CD38/SIRT1 signaling.4.Construction of the mouse asthma model induced by ovalbumin(OVA):comparison the degree of asthma in wild-type with smooth muscle CD38 KO mice;detecting of white blood cell count in alveolar lavage fluid of two groups of mice;HE staining was used to observe inflammatory cell infiltration and lung injury,and the protective effect of smooth muscle CD38 deletion on asthma in mice was verified at the overall animal level.Results:1.The results of dose-effect experiments showed that the expression of CD38mRNA and protein in ASMCs increased with the increase of E2 concentration,while the mRNA and protein expression of SIRT1 decreased.In time course experiments,CD38 mRNA and protein expression continued to increase within 48 h,SIRT1 mRNA and the expression level of protein decreased at 24 h and partially recovered at 48 h,and the protein levels of CD38 and SIRT1 showed a significant negative correlation at 24 and 48 h.Therefore,pre-treatment with 10 nM of E2 for 48h were selected for the subsequent experiments.2.E2 treatment can increase the expression of CD38 protein in wild-type ASMCs,decrease the expression of SIRT1 protein,and promote the expression of acetylated p53(Ac-p53).CD38 KO resulted in a significant increase in SIRT1 protein levels in ASMCs and a decrease in Ac-p53 protein levels.After E2 treatment,SIRT1and Ac-p53 expression levels in ASMCs were unchanged.3.Hypoxia treatment can significantly down-regulate the expression of CD38and SIRT1 mRNA in wild-type ASMCs,increase the level of CD38 protein,decrease the expression of SIRT1 and increase the expression of Ac-p53.E2 can inhibit the down-regulation of CD38 mRNA expression induced by hypoxia,and then promote the down-regulation of SIRT1 mRNA expression;E2 can further promote CD38protein expression,down-regulate SIRT1 protein level and promote p53 acetylation under hypoxia.CD38 KO can make the above effects of E2 disappear in ASMCs.4.E2 treatment significantly increased hypoxia-induced apoptosis of wild-type ASMCs,increased Caspase-3 activity,and significantly increased Bax/Bcl-2 ratio;CD38 KO significantly inhibited apoptosis of ASMCs induced by hypoxia;SIRT1agonist RSV can significantly inhibit E2-induced apoptosis of ASMCs.5.Estrogen receptor inhibitors(ICI182,780)significantly reduce the expression of CD38 mRNA and protein in ASMCs,and in the presence of ICI 182,780,counteract the role of E2 in promoting CD38 expression.6.In the overall animal model,smooth muscle-specific knockout CD38 mice had decreased sensitivity to asthma,decreased airway hyperresponsiveness,and decreased leukopenia in bronchoalveolar lavage fluid compared with control mice;significant improvement in lung histopathology and lung inflammation.Conclusions:E2 up-regulates CD38 gene expression through the estrogen receptor,acting on the downstream SIRT1/p53 signaling pathway,resulting in decreased expression of SIRT1,increased p53 acetylation,and promotion of hypoxia-induced apoptosis of ASMCs.This effect disappeared in ASMCs where CD38 deletion can eliminate E2.Smooth muscle-specific knockout of CD38 mice alleviates asthma symptoms and lung damage in mice.These results indicate that smooth muscle cell CD38 is a key target in the pathogenesis of asthma,and its regulation may provide new strategies for the treatment of respiratory diseases such as asthma.Background and objective:Acute lung injury(ALI)/Acute Respiratory Distress Syndrome(ARDS)is the damage of pulmonary capillary endothelial cells and alveolar epithelial cell damage caused by non-cardiac diseases,such as severe pneumonia,aspiration pneumonia,severe sepsis,severe trauma,and shock,which result in diffuse pulmonary interstitial edema and alveolar edema,leading to acute hypoxic respiratory insufficiency or respiratory failure with an uncontrolled systemic inflammatory response syndrome(SIRS)and the compensatory anti-inflammatory response(CARS).Due to its complex pathogenesis,ARDS has a high mortality rate,and there is no effective clinic treatment for this disease.In recent years,many studies have found that stem cells have multiple differentiation potentials and have achieved satisfactory results in difficult and complicated diseases that are difficult to solve with conventional treatment.At present,studies have suggested that the repair effect of human amniotic membrane-derived epithelial stem cells(h AESCs)on lung injury is achieved by immunomodulation,but the specific mechanism is still unclear.This study intends to establish a model of acute lung injury,confirming the repair effect of h AESCs on lung injury in vitro and in vivo,and try to find its related mechanism,providing scientific reference and experimental basis for the application of h AESCs in the treatment of this disease.Methods:1.Isolation,identification,and safety evaluation of h AESCs: h AESCs were isolated and cultured in vitro,and conditioned medium(CM)derived from h AESCs was obtained;h AESCs were identified by RT-PCR,immunofluorescence and flow cytometry,and their differentiation potential was identified,and the immunogenicity was evaluated;the tumorigenicity of h AESCs in vitro and in vivo was analyzed;the tissue distribution of h AESCs was observed using a small animal in vivo imager.2.Protective effect of h AESCs on acute lung injury in mice: human amniotic epithelial stem cells(h AESCs)were isolated and cultured in vitro to prepare cell suspensions and CMs derived from h AESCs;establish a model of acute lung injury induced by LPS in the airway,establish normal control group,LPS group,LPS+Cell group(tail vein injection h AESCs)and LPS+CM group(tail vein injection CM);the number of inflammatory cells in alveolar lavage fluid of each group was detected at 3and 7 days after model establishment;IL-6,TNF-α,IL-1β and IL-10 levels were detected by ELISA;histopathological changes and inflammatory cell infiltration were observed by HE staining;Sirius red staining to detect the degree of pulmonary fibrosis,evaluation of the protective effect of h AESCs or CM on acute lung injury in mice.3.In itro anti-inflammatory effect analysis of h AESCs or CM: human lung epithelial cells(BEAS-2B)were cultured in vitro,h AESCs or CM and BEAS-2B coculture system were established,LPS induced in vitro inflammatory response model,and real-time PCR was used to detect the changes of IL-6,TNF-α,IL-1βand IL-10 m RNA levels in BEAS-2B after LPS stimulation,and the anti-inflammatory effects of h AESCs or CM were verified in vitro.Results:1.h AESCs have multi-differentiation potential and low immunogenicity.h AESCs have no tumorigenicity in vivo and in vitro,mainly aggregating in the lung.2.Compared with the model group,tail vein injection of h AESCs and CM can significantly reduce the number of white blood cells in alveolar lavage fluid,reduce the release of pro-inflammatory factors IL-6,TNF-α,IL-1β,and increase the level of anti-inflammatory factor IL-10;h AESCs and CM injection can significantly improve LPS-induced lung inflammation and lung injury,lung injury scores are significantly reduced,and can effectively reduce lung fibrosis in mice.3.In the inflammatory response model,h AESCs or CM significantly inhibited the expression of pro-inflammatory genes IL-6,TNF-α and IL-1β in BEAS-2B.Conclusions:Human amniotic epithelial stem cells(h AESCs)have protective effects on LPS-induced acute lung injury in mice,and this protective effect may be related to h AESCs regulating lung inflammation.With multi-differentiation potential and low immunogenicity,h AESCs are highly safe,and easy to target in the lungs,showing good potential for use in the treatment of acute lung injury. |
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