| Objective:To explore the potential mechanism of PM2.5 induced lung injury in rats,astragaloside IV activated autophagy by regulating PI3K/AKT/m TOR signaling pathway,inhibited inflammatory response and oxidative stress injury,and thus protected the PM2.5-induced lung injury in rats,so as to provide a new strategy and experimental basis for the treatment of PM2.5-induced lung injury.Method:42 male SPF SD rats were randomly divided into 6 groups: control group(Con group),AS-Ⅳ control group(AS-Ⅳ group),model group(PM2.5 group),AS-Ⅳ IV low-Dose group(AS-Ⅳ 50 group),AS-Ⅳ high-dose group(AS-Ⅳ 100 group)and AS-Ⅳ+3-methyladenine group(AS-Ⅳ 100+3-MA group),each group has 7 animals.In this study,the lung injury model of rats was established by using the modeling method of inhalation of PM2.5.Except for the Con group and the PM2.5 group,the other groups were fully dissolved in AS-Ⅳ containing 1‰ DMSO sterile normal saline by intraperitoneal injection 3 days before modeling,and the doses were AS-Ⅳ group100 mg/kg,AS-Ⅳ 50 group 50 mg/kg,AS-Ⅳ 100 group 100 mg/kg,AS-Ⅳ 100+3-MA group 100 mg/kg,once a day for 3 consecutive days.Among them,AS-Ⅳ 100+3-MA group was intraperitoneally injected with AS-Ⅳ for 1 h,and then 3-methyladenine(15 mg/kg)fully dissolved in sterile normal saline was intraperitoneally injected once a day for 3 consecutive days.The Con group and PM2.5 group were intraperitoneally injected with sterile normal saline containing 1‰DMSO at the same volume.30 min after the last prophylactic administration,the modeling group(including PM2.5 group,AS-Ⅳ 50 group,AS-Ⅳ 100 group,and AS-Ⅳ 100+3-MA group)was started to model,and the modeling time was 24 hours.The modeling time was 2 times.The Con group and AS-Ⅳ group were instilled with an equal volume of normal saline in the airway according to the same method.The rats were sacrificed 12 h after the second modeling.Collecting bronchoalveolar lavage fluid and lung tissue.Then,the lung wet/dry mass ratio(W/D)was measured.Observing the pathological changes of lung tissue with hematoxylin-eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was used to detect tumors in alveolar lavage fluid Necrosis factor alpha(TNF-α),interleukin 6(IL-6)and C-reactive protein(CRP)inflammatory factor levels;detection of malondialdehyde(MDA)content,superoxide dismutase(SOD)and catalase(CAT)activity in lung tissue;autophagy immunofluorescence staining method to detect the fluorescence intensity expression of LC3 B protein in rat lung tissue;immunohistochemical method was used to detect distribution and expression of p-PI3 K,p-AKT and p-m TOR in rat lung tissue;observing the ultrastructure changes of rat lung tissue with transmission electron microscope;using Western blot to detect the expression of proteins LC3 B,p62,PI3 K,p-PI3 K,AKT,p-AKT,m TOR,p-m TOR,p65,p-p65,IKBα,p-IKBα in lung tissue.Result:(1)Effect of AS-Ⅳ on lung histopathology of PM2.5-induced lung injury in rats:lung tissue structure of rats in Con and AS-Ⅳ groups were regular;The pathological changes of lung tissue in the PM2.5 group were hemorrhage,alveolar structure was disordered,alveolar shape was changed,alveolar congestion was severe,a large amount of inflammatory exudate and other typical inflammatory changes can be seen in the interstitium;Compared with the PM2.5 group,the lung injury of rats with low and high doses of AS-Ⅳ and pretreatment with AS-Ⅳ+3-methyladenine were all various degrees of mitigation.(2)Effect of AS-Ⅳ on lung W/D ratio in rats with PM2.5-induced lung injury:lung dry-wet ratio in PM2.5 group was significantly increased compared with Con group.Compared with PM2.5 group,low and high doses of AS-Ⅳ could effectively reduce pulmonary edema,while 3-methyladenine reversed the effect of AS-Ⅳ.(3)Effect of AS-Ⅳ on the expression of inflammatory factors in BALF of PM2.5-induced lung injury rats: Compared with Con group,the expression levels of TNF-α,IL-6 and CRP in PM2.5 group were distinctly added.Compared with PM2.5 group,low and high doses of AS-Ⅳ could effectively reduce the expression levels of TNF-α,IL-6 and CRP,while 3-methyladenine reversed the effect of AS-Ⅳ.(4)Effects of AS-Ⅳ on oxidative stress indexes of lung tissue with PM2.5-induced lung injury in rats: Compared with the Con group,PM2.5 significantly increased MDA content,and decreased SOD and CAT activities;Compared with PM2.5 group,low and high doses of AS-Ⅳ can effectively reduce the content of MDA,and meanwhile increase the activities of SOD and CAT.However,this effect of AS-Ⅳ was reversed by 3-methyladenine.(5)Effects of AS-Ⅳ on autophagy immunofluorescence of PM2.5-induced lung injury in rats: PM2.5 significantly enhanced the expression of LC3 B relative fluorescence intensity,and AS-Ⅳ effectively discolored the expression of LC3 B relative fluorescence intensity.In addition,the number of autophagosomes in each group were analyzed.The results showed that PM2.5 obviously enhanced the fluorescence intensity and the number of autophagosomes in LC3 B compared with the Con group.Compared with PM2.5 group,AS-Ⅳ significantly weakened the fluorescence intensity of LC3 B and reduced the number of autophagosomes.(6)Ultrastructure analysis of lung tissue: Under transmission electron microscopy,alveolar macrophages in the Con group and AS-Ⅳ group were relatively complete in structure,with regular cell morphology,large and round cell nuclei,no enlargement of endoplasmic reticulum,and clear mitochondrial crest.In PM2.5 group,the number of alveolar macrophages added obviously,the cells got bigger,the cell structure was disturbed,the nucleus shape was irregular,the endoplasmic reticulum pool in the cytoplasm was enlarged,the mitochondria were seriously swollen.Lots of autophagosomes(Avi)and autophagosomes(Avd)were observed in alveolar macrophages,but Avi was the main autophagic structure.The number of alveolar macrophages decreased significantly in the low and high doses AS-Ⅳ groups,and the cell morphology changed and Avi was decreased compared with PM2.5 group.In ASIV 100+3-MA group,alveolar macrophages were arranged neatly,with clear edges and regular nuclear morphology.A few autophagy structures were observed,and Avd was the dominant one.(7)Immunohistochemical analysis of lung tissue: the positive expression of pPI3 K,p-Akt and p-m TOR protein in Con group and AS-Ⅳ groups was weak,and there were few brown yellow cells.The positive expressions of p-PI3 K,p-AKT and p-m TOR were markedly augmented in PM2.5 group,and the number of brown-yellow cells also were markedly augmented.The positive expressions of p-PI3 K,p-AKT and p-m TOR in low and high doses AS-Ⅳ groups were obviously decreased,and the brown-yellow cells were obviously decreased,while the effect of AS-Ⅳ was reversed by 3-methyladenine.(8)Expression of autophagy proteins LC3BII/I and p62: Compared with the Con group,the protein ratio of LC3-II/LC3-I and the expression of p62 in PM2.5 group were obviously added.Compared with the PM2.5 group,the protein ratio of LC3-II/LC3-I and the protein expression level of p62 were obviously reduced by low and high doses of AS-Ⅳ.However,this effect of AS-Ⅳ was reversed by 3-methyladenine.(9)Expression of PI3K/AKT/m TOR pathway proteins: Compared with the Con group,PM2.5 obviously elevated the protein ratio of p-PI3K/PI3 K,p-AKT/AKT and p-m TOR/m TOR.AS-Ⅳ pretreatment obviously reduced the protein ratio of pPI3K/PI3 K,p-AKT/AKT and p-m TOR/m TOR,while 3-methyladenine reversed the effect of AS-Ⅳ.(10)Expression of NF-κB signaling pathway proteins: Compared with the Con group,the protein ratios of p-IKBα/IKBα and p-p65/p65 in PM2.5 group were obviously elevated.Compared with PM2.5 group,AS-Ⅳ pretreatment significantly decreased the protein ratios of p-IKBα/IKBα and p-p65/p65,but 3-methyladenine reversed the effect of AS-Ⅳ.Conclusion:(1)AS-Ⅳ can protect against PM2.5-induced lung injury.(2)AS-Ⅳ may activate autophagy by regulating PI3K/AKT/m TOR signaling pathway,thereby inhibiting inflammatory response and oxidative stress injury,and protecting PM2.5-induced lung injury. |
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