The Mechanism Of Yiqi And Jiedu Method Regulating P53/p21^CIP1/Rb And GATA4/NF-κB Cellular Senescence Signaling Pathway To Protect PM2.5-induced Lung Injury

ObjectiveThis experiment mainly collected PM2.5 native to Chengdu,and then established a lung injury mouse model with subacute exposure by non-invasive tracheal instillation,and revealed the dose-loss effect relationship between subacute PM2.5 exposure and lung injury.Screening the appropriate PM2.5 exposure concentration that is consistent with the“real world”medium-term,low-level PM2.5 exposure pattern to induce cell senescence phenotype.To study the efficacy of Yiqi and Jiedu method（with Ganduqing as the evaluation carrier）in the treatment of PM2.5 subacute exposure-induced lung injury and the molecular mechanism of regulating cell senescence phenotype.Focusing on the study of Yiqi and Jiedu method（using Ganduqing’s quality control ingredient Astragaloside Ⅳ as the evaluation vehicle）to protect PM2.5 subacute exposure-induced lung injury by regulating p53/p21^CIP1/Rb and GATA4/NF-κB cell senescence signaling pathways molecular mechanism.This study is expected to explore the molecular mechanism of Yiqi and Jiedu method in protecting lung injury from the perspective of"environmental aging"in a mouse model of PM2.5 connotation.MethodsPart 1:We have collected PM2.5 in Jinniu District,Chengdu,and obtained PM2.5freeze-dried powder through separation,purification,freeze-drying and other processes,and analyzed its metal element composition.Part 2:We Used the collected PM2.5 as a modeling tool.A PM2.5 subacute exposure lung injury mouse model was established by non-invasive tracheal instillation of PM2.5,and the suitable PM2.5 for inducing cell senescence phenotype was screened out.Fifty C57BL/6J mice were subjected to subacute exposure to PM2.5 for 4 weeks and were divided into blank control group（K group）,PM2.5 low-dose group（2.5mg/kg）,and PM2.5 medium-low dose group（5mg/kg）,PM2.5 medium dose group（10mg/kg）and PM2.5 high dose group（20mg/kg）.After the 4-week exposure,the survival of the mice and the degree of lung injury were observed,and the expression of key proteins of cell senescence phenotype in lung tissue and the staining of its markerβ-galactosidase were detected.To investigate the"effect relationship"between subacute exposure to PM2.5 and lung injury,determine the appropriate PM2.5 concentration to activate the cellular senescence phenotype.We will determine the appropriate PM2.5concentration to activate the cellular senescence phenotype based on the experimental results.Part 3:We took the mice with lung injury induced by subacute exposure to PM2.5as the observation object and Omega-3 as the positive control drug to study the treatment effect of Ganduqing on the mice with lung injury induced by subacute exposure to PM2.5,and its molecular mechanisms regulating cellular senescence phenotype.28 C57BL/6J mice were divided into 4 groups of 7 mice each as lank control group（K group）,PM2.5 model group,Ganduqing group and Omega-3 group.First,the therapeutic effect of Ganduqing on mice with PM2.5 subacute exposure-induced lung injury was explored by measuring the lung function of mice,detecting the edema index of lung tissue,and scoring the degree of lung injury and fibrosis injury.Western blotting was used to detect the expression of key proteins p21^CIP1 and p16^INK4a in cell senescence phenotype,and the staining of cell senescence phenotype markersβ-galactosidase and lipid melanin Sudan black was detected.Which will explore the molecular mechanism of Yiqi and Jiedu method（with Ganduqing as the evaluation carrier）regulating cell senescence phenotype.Part 4:We used PM2.5 subacute exposure-induced lung injury mice as the observation object,Nutlin-3a to activate the tumor suppressor gene p53 to study the molecular mechanism of regulating the cell senescence signaling pathway about p53/p21^CIP1/Rb and GATA4/NF-κB by astragaloside Ⅳ to protecting of PM2.5subacute exposure-induced lung injury.Forty-two C57BL/6J mice were divided into 6groups of 7 mice each.They were:blank control group（K group）,astragaloside Ⅳ negative control group（AS group）,PM2.5 model group,astragaloside Ⅳ low-dose group（ASL group）,and astragaloside Ⅳ high-dose group（ASH group）and Nutlin-3a group.First,the therapeutic effect of astragaloside Ⅳ on PM2.5 subacute exposure-induced lung injury in mice was studied by measuring the lung function,pulmonary edema index,and scoring the degree of lung tissue damage and fibrosis damage;Secondly,the expression of cell senescence pathway proteins about p53,p-p53,p21^CIP1,p-Rb,Rb,p16^INK4a,GATA4,p65,p-p65 and other proteins was detected by Western blotting,and the immunofluorescence staining ofγ-H2A.X（DNA double bond break marker protein）,detection of cell senescence phenotype markersβ-galactosidase and lipomelanin Sudan black staining,and observation of the ultrastructure of type II alveolar epithelial cells,demonstrating that Yiqi and Jiedu method（with astragaloside Ⅳ as the evaluation carrier）regulates the cell senescence signaling pathway of p53/p21^CIP1/Rb and GATA4/NF-κB to protect PM2.5-induced lung injury.ResultsPart 1:we analyzed the data of PM2.5 official monitoring points in Jinniu District,Chengdu in 2020 and 2021,and found that the annual average of PM2.5 in these two years was 55.46μg/m³ and 52.39μg/m³,respectively.This value is higher than the limit set by the World Health Organization of 35μg/m³.We used Laoying`s high-throughput air sampling machine to collect PM2.5 in the west third section of the First Ring Road,Jinniu District,Chengdu,and analyzed the metal element content of the collected PM2.5,and found that the content of Zn was the highest（6979.4 mg/kg）,followed by Al（5700 mg/kg）,the third is Fe（2860.8）,the lowest content is Ni（8.2mg/kg）,and the mean value of PM2.5 at the sampling point is 86.5μg/m³.Part 2:we found that PM2.5 with different concentration gradients（2.5mg/kg,5mg/kg,10mg/kg and 20mg/kg）could damage the lung tissue of C57BL/6J mice after4 weeks of subacute exposure.we also found that the weight of mice was reduced,and the degree of pulmonary edema was aggravated.We found that the HE scores of the PM2.5 concentration gradient groups induced lung injury were significantly different from those of the K group（P<0.001）,and the degree of lung injury was also aggravated with the increase of the concentration.By detecting the expression of key proteins(p21^CIP1 and p16^INK4a)and the markerβ-galactosidase staining of cell senescence phenotype,we found that low-dose PM2.5（2.5mg/kg）could not induce cell senescence phenotype,and the rest PM2.5 concentration can induce cell senescence phenotype.Medium-low dose PM2.5（5mg/kg）is safe for modeling,in line with the"real world"PM2.5 low-level,long-term exposure pattern,and can activate cell senescence phenotype.We chose medium-low dose（5mg/kg）as the PM2.5 exposure dose for subsequent experiments.Part 3:we found that the subacute exposure to PM2.5 at a concentration of 5mg/Kg increased the wet/dry ratio,HE staining pathological score and Ashcroft fibrosis score in lung tissue.These changes were consistent with subacute exposure to lung injury.We found that Ganduqing reduced the lung histopathological injury HE scores and Ashcroft fibrosis scores in mice with PM2.5 subacute exposure-induced lung injury.It also improved lung tissue edema and collagen fiber deposition,inhibited the expression andβ-galactosidase activity of key proteins of the cellular senescence phenotype(p21^CIP1 and p16^INK4a),and reduced cytokine production in the cellular senescence-associated secretory phenotype（SASP）pool,improved oxidative stress levels,and reduced lipomelanin deposition in lung tissue.Based on the above mechanism of action,Yiqi and Jiedu method played a protective role against lung injury.Part 4:We found that the success of the modeling method for establishing a mouse model of lung injury by subacute exposure to PM2.5 at a concentration of 5 mg/kg was verified again.Astragaloside Ⅳ can reduce lung tissue pathological damage HE scores and Ashcroft fibrosis scores in mice with PM2.5 subacute exposure-induced lung injury,improve lung tissue edema,inhibit the deposition of collagen fibers and lipomelanin in lung tissue,and inhibitβ-Galactosidase activity,improve oxidative stress levels.Astragaloside Ⅳ also reduces the degree of DNA double bond breakage,inhibite the expression of p53 and the phosphorylation level of p53,attenuated the activity of downstream p21^CIP1,turned Rb into a phosphorylated active state（Rb）,and partially restored the activity of Rb to promote the normal operation of the cell cycle.Astragaloside Ⅳ can also reduce the release of cytokines in the SASP pool by increasing the degradation of GATA4 in cells,inhibiting the phosphorylation level of p65,one of the downstream NF-κB components,and its nuclear transcription.Based on the above mechanism of action,AS Ⅳ has a protective effect on PM2.5 subacute exposure-induced lung injury.Conclusion（1）The average annual exposure level of PM2.5 in Chengdu in the past two years（2020 and 2021）is higher than the limit set by the World Health Organization.During the period of high PM2.5 exposure,citizens can take corresponding measures to reduce the harm of PM2.5 to the body.（2）In line with the"real world"medium and long-term,low-dose exposure mode,the survival rate of mice is high,and the PM2.5 concentration that can activate the cell senescence phenotype after subacute exposure（4 weeks）is5mg/kg.（3）Ganduqing,a representative formula of Yiqi and Jiedu method,can reduce lung inflammation and fibrosis damage,and inhibit the occurrence of senescence phenotype of lung tissue cells.This may be the underlying mechanism of Ganduqing’s protection from PM2.5 subacute exposure-induced lung injury in mice.（4）Astragaloside Ⅳ（a quality control component of Ganduqing）regulated the expression of senescence pathway-related proteins in p53/p21^CIP1/Rb and GATA4/NF-κB cells and cytokines in the SASP pool by reducing the degree of DNA double bond breakage and inhibiting DNA damage response release to play an important role in inhibiting lung injury.