Effect And Mechanism Of Serum Exosomes In Beryllium Sulfate-induced Lung Injury Of SD Rats

Objective:To investigate the effect and mechanism of serum exosomes in beryllium sulfate-induced lung tissue oxidative stress and inflammation in SD rats.Methods:1.Forty SPF male SD rats were randomly divided into four groups,the control group and the BeSO₄ exposure group?dose of 4,8,and 12mg/kg BW?,which were exposed to sterilized ultrapure water?1 ml/kg?and BeSO₄ sterilized ultrapure water solution?4,8,12 mg/kg?according to non-exposed intratracheal instillation.Two weeks weighed once,after6 weeks,SD rats were anesthetized with ether and sacrificed using abdominal aortic phlebotomy for the collection of blood,preparation of serum.We counted the number of classification of peripheral blood leukocytes,and the expression of tumor necrosis factor-??TNF-??,interferon-??IFN-??,and interleukin-10?IL-10?.The lung was removed and weighed,and observe the macroscopic of the lung,then perform routine histopathological examination on the visible lesions.If there was no obvious lesions,the bronchoalveolar lavage fluid?BALF?was collected from the left lung to detected the total protein concentration and lactate dehydrogenase?LDH?activity.Take the right middle lung to preparation of lung homogenate,and we detected the content of hydrogen sulfide?H₂S?,malondialdehyde?MDA?,reactive oxygen species?ROS?,the activity of superoxide dismutase?SOD?,and glutathione peroxidase?GSH-PX?in lung tissue.We extracted the protein from the right lower lung,and detected the protein expression of cystathionine-?-lyase?CSE?,inducible nitric oxide synthase?iNOS?and cyclooxygenase-2?COX-2?by Western Blot.And the right upper lung tissue were taken for routine histopathological examination.We extracted exosomes from the serum of SD rats by ultra high speed centrifugation.Western blot was used to identify the protein of CD9 and TSG101,which are the marker protein of exosomes.The morphological structure of exosomes was observed by transmission electron microscopy?TEM?.The particle size and particle concentration was determined by nanoparticle tracking analysis?NTA?.The total protein concentration was detected by BCA protein quantitative.3.The RAW 264.7 cells were pretreated with 50 nmol/L rapamycin for 30 min,and then co-treated with 100?g/mL BeSO₄-Exo for 24 h.The activity of LDH,the expression of inflammation-related proteins COX-2and iNOS,the content of inflammatory factor TNF-?was detected by Western Blot and ELISA.Results:1.After exposure to BeSO₄,the SD rats in each BeSO₄ exposure group grown slower than the control group,and at the 6 weeks,the body weight of SD rats in each BeSO₄ exposure group was slightly lower than that of the control group.And the lung coefficient of SD rats in each BeSO₄ exposure group showed a downward trend.2.Compared with the control group,the total protein concentration and LDH activity in BALF were increased exposed to BeSO₄ group.The content of ROS and MDA of lung tissue were increased in each BeSO₄expose group,while the activity of SOD and GSH-PX were decreased.The protein expression of CSE and the content of H₂S were all down-regulated,and the protein expression of COX-2,iNOS were all up-regulated.Compared with the control group,the total number of peripheral blood leukocytes,neutrophils,lymphocytes and monocytes in each BeSO₄ expose group showed an upward trend,and the levels of TNF-?,IFN-?,IL-10 in serum were higher than those of control groups.The difference was statistically significant?P<0.05?,and it showed a dose-dependent effect.3.The results of histopathological examination showed that the lung tissue of the control group was normal,and the each BeSO₄ exposure group showed varying degrees of the thicken alveolar wall,alveolar space reduction,alveolar number reduction,shrinkage of alveolar cavity,interstitial inflammatory cell infiltration,collagen fibrosis and so on.With the increasing dose of BeSO₄,the pathological changes of lung tissue in SD rats were aggravated.4.We observed exosome with a typical membrane structure and a uniform size by TEM,and detected the expression of the CD9 and TSG101 proteins which are the characteristic of exosomes.From the result of NTA,we observed that the diameter of serum exosomes was103.00?129.90 nm.Compared with the control group,the particle concentration and total protein of serum exosomes were increased in each BeSO₄ exposed group,and the differences were statistically significant?P<0.05?.And the particle concentration and total protein of serum exosomes were increased with the higher BeSO₄ dose.5.Compared with the control group,the activity of LDH,the expression of inflammation-related proteins COX-2 and iNOS,the content of inflammatory factor TNF-?were increased in the BeSO₄-Exo group of RAW 264.7 cells.While the LDH activity,the COX-2 and iNOS expression,and the TNF-?content of the Rapa+BeSO₄-Exo group were lower than the BeSO₄-Exo group.And the differences were statistically significant?P<0.05?.Conclusions:1.BeSO₄ can induce lung tissue oxidative stress and inflammation in SD rats by non-exposed intratracheal instillation,and it has a dose-response relationship.2.Non-exposed intratracheal instillation of BeSO₄ can increase the release of serum exosomes in SD rats.The higher of the BeSO₄ dose,the more exacerbate of the lung injury,and the more release of serum exosomes.3.Serum exosomes may aggravate BeSO₄-induced pulmonary inflammation in SD rats through the mTOR signaling pathway.