Mechanism Of Tectorigenin Regulation Of Sirt1 To Reduce Oxidative Stress And Inhibit Airway Inflammation In Asthmatic Mice

Objective:Bronchial asthma is a chronic respiratory disease,a global public health problem and one of the most common chronic inflammatory diseases in children.Cough,chest tightness,difficulty breathing and wheezing are the main clinical symptoms,and inhaled corticosteroids(ICS)and long-acting beta agonists(LABA)are still used in combination to control asthma attacks.But for some people with asthma,ICS and LABA are not effective enough to control seizures.Long-term use of ICS has side effects,even resistance,resistance.Therefore,the search for complementary or alternative drugs to treat asthma remains an urgent issue.Continuous bronchitis is an important pathologic feature of asthma and is central to the progression of disease leading to airway remodeling and trachea stenosis.In the pathogenesis of asthma,oxidative stress can damage bronchial epithelial cells and activate inflammatory signals,which is an important driver of the progression of asthma.Therefore,relieving oxidative stress in the airway and thus reducing inflammation in the airway is a therapeutic approach worth exploring.Tectorigenin(Tectorigenin,TEC)is a dry shot of Belamcanda chinensis(L)from Iris.DC.flavonoids extracted.Various studies in modern medicine have shown that TEC has anti-proliferative,anti-inflammatory,antioxidant,anti-allergic and immunomodulatory properties.However,TEC interventions in mouse models of asthma have been poorly reported.Therefore,by establishing animal models of asthma and airway epithelial cell models,TEC used in vitro and in vivo to explore the mechanism of TEC’s role in inhibition of airway inflammation in asthma,with a view to informing future drug treatment of asthma.Methods:1.Effect of TEC on airway resistance and inflammation in asthma miceUsing OVA sensitized and stimulated female Balb/c mice,36 Balb/c mice were randomly assigned to 6 groups.Normal control group(Control),asthma group(OVA),TEC low dose group(OVA+25 mg/kg),TEC medium dose group(OVA+50 mg/kg),TEC high dose group(OVA+75 mg/kg),dexamethasone group(OVA+DXMS)were divided into control group(OVA+25 mg/kg).Within 24 hours of the last activation,the Buxco Fine Pointe-NAM assay system measured specific airway resistance to assess airway responsiveness in each group of mice.Inflammatory cell infiltration in the airways and perivascular of lung tissue of mice was observed by HE staining.Hyperiodate acid-sherf(PAS)staining was used to observe the proliferation and mucus secretion of lung airway cup cells in each group of mice.Inflammatory cells in bronchial alveolar lavage of mice were classified and counted by Wright-Giemsa staining.Serum levels of total Ig E,cytokines IL-4 and IFN-γwere measured by ELISA in each group of mice.2.Mechanisms of TEC regulation of Sirt1 to reduce oxidative stress and inflammation in asthmatic mice.A mouse model of asthma was constructed by reference to the first part.Twenty-four female Balb/c mice were randomly assigned to four groups of six.The control group was divided into control group(Control),asthma group(OVA),TEC+OVA group(optimal in vivo dose)and OVA+SRT1720(Sirt1 agonist)positive control group.Western blot,q RT-PCR and immunofluorescence were used to detect Sirt1expression in the lung and airway epithelium of asthmatic mice.Oxidative stress markers ROS,MDA,GSH and SOD activity were measured in lung tissue of each group of mice using the kit.Western blot was used to detect the MAPK signaling pathway p-ERK,ERK,p-JNK,JNK,p-P38 and P38 proteins,inflammatory factors TNF-α,IL-6,and IL-1βin mouse lung tissue.3.Effects of TEC on oxidative stress and inflammation in bronchial epithelial Beas-2B cells.(1)Effect of IL-4 stimulation on Sirt1 expression in human bronchial epithelial Beas-2B cells.Normal human bronchial epithelial Beas-2B cells were cultured in vitro and IL-4induced mimicry of the in vivo environment.Divided into Control and IL-4 groups.ROS levels in each cell group were measured by DCFH-DA probe method.Western blot detected Sirt1 protein in each cell line and q RT-PCR detected Sirt1 m RNA.Sirt1 is overexpressed in human bronchial epithelial Beas-2B cells.In human bronchial epithelial Beas-2B cells,Sirt1 was overexpressed and IL-4 induced,divided into Oe-Con,Oe-Con+IL-4,Oe-Sirt1,Oe-Sirt1+IL-4.DCFH-DA probe was used to measure ROS levels in each cell line,and the kit was used to measure MDA levels,SOD activity,GSH/GSSG ratio,NAD~+/NADH ratio in each cell line.MAPK signaling pathway p-ERK,ERK,p-JNK,JNK,p-P38,and P38 proteins were detected in each cell line by Western blot.The levels of inflammatory factors IL-6 and IL-1βwere measured by ELISA in each group.(2)Protective effects of TEC on inflammation and oxidative stress in human bronchial epithelial Beas-2B cells.First,the cell viability of human bronchial epithelial Beas-2B cells was measured using CCK-8 at different concentrations,and the best in vitro intervention concentrations were selected in the Control,IL-4,IL-4+TEC groups.DCFH-DA probe was used to detect ROS in each cell line.Western blot detected Sirt1 protein in each cell line and q RT-PCR detected Sirt1 m RNA.Knockdown of Sirt1 in human bronchial epithelial Beas-2B cells.They were divided into Sh-Con,Sh-Con+TEC,Sh-Sirt1,Sh-Sirt1+TEC.DCFH-DA probe was used to measure ROS levels in each cell line,and the kit was used to measure MDA levels,SOD activity,GSH/GSSG ratio,NAD~+/NADH ratio in each cell line.Western blot detected the MAPK signaling pathway p-ERK,ERK,p-JNK,JNK,p-P38,and P38 proteins.The levels of inflammatory factors IL-6 and IL-1βwere measured by ELISA in each group.Results:1.TEC can reduce airway reactivity and airway inflammation in OVA-induced asthma mice.(1)The s Raw values of OVA sensitized mice were significantly increased by Buxco Fine Pointe-NAM assay.Moderate doses of TEC reduced s Raw in asthmatic mice compared to the OVA sensitizing group.(2)HE and PAS staining of lung tissue sections showed significant increase in inflammatory cell infiltration,goblet cell proliferation and mucus secretion in the airways and perivascular of OVA mice.Moderate doses of TEC significantly reduced inflammatory cell infiltration,goblet cell proliferation and mucus secretion in the airway and perivascular.The number of leukocytes and eosinophils in BALF was significantly increased in OVA mice.The number of leukocytes and eosinophils in BALF of asthmatic mice was reduced by medium dose TEC.The ELISA assay showed that medium doses of TEC reduced total Ig E and IL-4 levels in the serum of asthmatic mice and increased IFN-γlevels compared to the OVA sensitizing group.2.TEC plays an anti-inflammatory role by modulating Sirt1 to reduce oxidative stress in OVA-induced asthma in mice.Western Blot,q RT-PCR and immunofluorescence assays showed significant downregulation of Sirt1 expression and significant increase in ROS and MDA in lung and airway of OVA sensitized mice.GSH content and SOD activity were significantly reduced.Protein expression levels of the inflammatory factors TNF-α,IL-6,IL-1βand the MAPK signaling pathway p-ERK,p-JNK and p-P38 were significantly upregulated.Moderate doses of TEC upregulated Sirt1 expression in lung tissue and airways of asthmatic mice.Decreased ROS and MDA in lung tissue of asthmatic mice and significantly increased GSH and SOD activity.Protein expression levels of the inflammatory factors TNF-α,IL-6,IL-1βand the protein expression levels of p-ERK,p-JNK,and p-P38 in the MAPK signaling pathway were downregulated in asthmatic mice.3.TEC plays an anti-inflammatory role by modulating Sirt1 to reduce oxidative stress in human bronchial epithelial Beas-2B cells.(i)IL-4 induced reduction of Sirt1 expression in Beas-2B cells and increased oxidative stress in Beas-2B cells.Sirt1 protein and m RNA expression levels were significantly reduced and ROS levels were significantly increased in IL-4-induced Beas-2B cells.In Beas-2B cells overexpressing Sirt1,overexpression of Sirt1 reduced ROS and MDA levels in IL-4-induced cells,while decreasing SOD activity,GSH/GSSG,NAD~+/NADH in cells.Downregulation of IL-4 induced increased levels of the inflammatory factors IL-6 and IL-1β-protein in the supernatant of Beas-2B cells,and increased expression of p-ERK,p-JNK,and p-P38 proteins in the MAPK signaling pathway.(2)TEC attenuates oxidative stress and suppresses cellular inflammation by upregulating Sirt1 expression in IL-4-induced human bronchial epithelial Beas-2B cells.The effects of different concentrations of TEC on the viability of Beas-2B cells were measured using CCK8.The results showed no significant effect on cell viability at 10μM.TEC interfered with Beas-2B cells and gave IL-4 induction.TEC intervention attenuated IL-4-induced downregulation of Sirt1 and increased ROS.In Beas-2B cells knockdown of Sirt1,ROS and MDA levels were significantly increased in IL-4-induced cells,and SOD activity and GSH/GSSG were decreased.Inflammatory factors IL-6 and IL-1βlevels were significantly elevated,and the expression of p-ERK,p-JNK and p-P38proteins in MAPK signaling pathway was significantly increased.TEC intervention reduced IL-4-induced increases in ROS and MDA,decreased SOD activity,decreased GSH/GSSG,and decreased NAD~+/NADH.Increased levels of IL-6,IL-1β,and expression of p-ERK,p-JNK,and p-P38 proteins in IL-4 induced MAPK signaling pathway were reduced.Conclusion:1.TEC reduced OVA-induced airway reactivity and airway inflammation in mice with asthma.2.TEC upregulated Sirt1 expression in lung tissues and airways of asthmatic mice,significantly improved lung oxidative stress,inhibited inflammatory factor expression,and activated MAPK signaling pathways.3.TEC was able to upregulate Sirt1 expression in vitro and inhibit IL-4-induced oxidative stress and cellular inflammation in Beas-2B cells.4.TEC inhibition of airway inflammation in asthma may be achieved by regulating Sirt1 to reduce oxidative stress.